Silk: Properties, Production and Uses : Properties, Production, and Uses [1 ed.] 9781621007234, 9781621006923

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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Silk: Properties, Production and Uses : Properties, Production, and Uses, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Silk: Properties, Production and Uses : Properties, Production, and Uses, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

MATERIALS SCIENCE AND TECHNOLOGIES

SILK

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

PROPERTIES, PRODUCTION AND USES

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

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MATERIALS SCIENCE AND TECHNOLOGIES

SILK

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

PROPERTIES, PRODUCTION AND USES

PORNANONG ARAMWIT EDITOR

Nova Science Publishers, Inc. New York Silk: Properties, Production and Uses : Properties, Production, and Uses, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Silk : properties, production, and uses / [edited by] Pornanong Aramwit. p. cm. Includes bibliographical references and index. ISBN 978-1-62100-723-4 (eBook) 1. Silk. I. Aramwit, Pornanong. TS1546.S55 2011 677'.39--dc23 2011037425

Published by Nova Science Publishers, Inc. † New York

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CONTENTS Preface

vii

About the Editor

ix

Chapter 1

Silk Use by Benthic Macroinvertebrates Jonathan Fingerut and Lindsay Simmen

Chapter 2

Muga Silk: Properties, Production and Uses Subrata Das

Chapter 3

Conservation and Diversification of Spider Silk: An Evolutionary Perspective Sara L. Goodacre

29

Evolution of Mulbery and Non-Mulberry Silk Fibers for Textile Applications Rungsima Chollakup and Wirasak Smitthipong

41

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Chapter 4

1 13

Chapter 5

Flavonoids and Carotenoids in Silkworm Cocoons Pornanong Aramwit

87

Chapter 6

A Novel Concept on Bioengineering-Silkworm Silk Fibre Karen Hoi-Yan Cheung

99

Chapter 7

Evaluating the Degradation of Silk Fabrics and Silk-Based Biomaterials Jolon M. Dyer and Caroline Solazzo

Chapter 8

Ecophysiological Influences on Spider Silk Properties and the Potential for Producing Adaptable, Degradation Resistant Biomaterials Sean J. Blamires and I-Min Tso

117

135

Chapter 9

Toward Eco-Friendly Composite Materials Based on Silk Fibroin Mariana Agostini de Moraes and Marisa Masumi Beppu

151

Chapter 10

Cellular Immunological Responses to Silk Materials Pornanong Aramwit

167

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vi

Contents

Chapter 11

Silk Fibroin in Medicine Antonella Motta, Michael Floren and Claudio Migliaresi

185

Chapter 12

Silk Materials for Drug Delivery Devices Pornanong Aramwit

219

Chapter 13

Application of Silk Sericin for the Controlled Release and Target-Specific Delivery of Biological Substances Ayumu Nishida

247

Chapter 14

Silk Proteins for Wound Healing Materials Pornanong Aramwit

265

Chapter 15

Use of Silk in Tendon/Ligament Tissue Engineering Thomas K. H. Teh, Siew-Lok Toh and James C. H. Goh

287

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Index

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309

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PREFACE Silkworms are fascinating creatures. Since ancient time, all parts of silkworm including its cocoon have been used for several applications especially in textile and medical areas. This bio-friendly animal has been paid much attention lately due to its unique fiber properties, its protein content as well as nutrition value from its body. This book is intended as a general introduction to the use of the silkworm and its products for the textile industry and in the human body for the purposes of aiding healing and correcting deformities. This text provides a familiarity with the uses of silk materials in textile and biological applications and also the rational basis for these applications. It covers such subjects as silkworm silk fiber composites, silk materials in medicine and cellular immunological responses to silk materials. We have also introduced a chapter on silk for tissue engineering, emphasizing the use of materials as scaffolds to guide cell growth and differentiation. A panel of international experts has contributed in comprising the chapters for the book. Each author gives their own view of the subject thereby rendering the book a very individual and diverse spectrum of the content. I would like to thank each of the authors for their diligent work and keen insight on the specific topics they have provided information on in each of the chapters. Throughout the work that has taken place in compiling the information presented, a large number of individuals have also contributed valuable time, assistance, and knowledge, I am cordially grateful. In editing this book, I have also received valuable assistance from Travis Hainstock, proofreading drafts of the whole text and providing helpful comments and corrections. Any errors of commission or omission that have remained in spite of my efforts at correction are my responsibility alone. Pornanong Aramwit Editor

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ABOUT THE EDITOR Pornanong Aramwit is an Associate Professor in the Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences at Chulalongkorn University, Bangkok, Thailand. She earned a B.Sc. in Pharmacy from Chulalongkorn University in 1992 and went on to the University of Illinois-Chicago, USA to earn Doctor of Pharmacy (Pharm.D.) in 1995 and a Ph.D. in Pharmaceutical Sciences from the University of Wisconsin-Madison, USA in 2001. Pornanong’s research and development experience has ranged from protein including silk proteins, biomaterials, tissue engineering, herbal substances such as mulberry leaf and fruit. She also did a lot of clinical researches especially in the area of nephrology. She is an expert in dermatology especially materials for wound healing application. She has received numerous research grants as the principal investigator to further her research in her areas of expertise. Numerous awards have recognized Pornanong’s achievements. For her project “Biocellulose with Sericin Mask” she won the Silver Award from the Design Innovation Contest 2010 Bangkok, Thailand. In 2009, she was choosen as International Health Professional of the Year by International Biographical Center, Cambridge, England. She won Best Poster Presentation Awards from Excellent Team Towards Excellent Medicine and The Thai Society of Burn Injury. She was Research Scholar from Pharmacia-UpJohn Corporation in Michigan, USA in 2001, and awarded Research Assistantship at the University of Wisconsin-Madison in 2000. Pornanong has written for more than forty international publications, contributed to at least six books, and has received six patents. She has been the advisor for numerous students at the Master, Ph.D., and Postdoctoral level.

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In: Silk: Properties, Production and Uses Editor: Pornanong Aramwit

ISBN: 978-1-62100-692-3 © 2012 Nova Science Publishers, Inc.

Chapter 1

SILK USE BY BENTHIC MACROINVERTEBRATES Jonathan Fingerut1 and Lindsay Simmen Department of Biology, Saint Joseph's University, Philadelphia, Pennsylvania, US

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ABSTRACT While most commonly associated with silkworms and spiders, silk also plays a significant role in the lives of many streambed-dwelling insect larvae. Activities such as nutrient acquisition, protection, dispersal, attachment, and pupation are all facilitated or, in some cases, rely on silk. Two of the most important members of freshwater stream ecosystems, caddisfly and blackfly larvae, provide excellent model systems in which to investigate these uses. Here we present an overview of how they use silk and the techniques and approaches that are necessary to discover the remarkable range of roles that silk plays.

INTRODUCTION Examples of animal silk use usually focus on representatives from two phyla: spiders (phylum Chelicerata) and silkworms (phylum Hexapoda). Spider silks have proven useful in medicine and in tissue engineering because of their great strength [1] and their excellent biocompatibility with human tissue [2]. The threads of silkworm moths, have also been used medicinally and have been used commercially to produce valuable textiles for centuries [3]. 1

Jonathan T. Fingerut has been an Assistant Professor at St. Joseph’s Universtiy since 2006. He earned a degree in Biology at Cornell University, Ithaca, New York in 1994. He became a teaching assistant at the University of California in 1997 where he would receive his Ph.D. in Biology in 2003. He then went on to the Academy of Natural Sciences of Philadelphia as a Research Associate for three years. After which, he became an Adjunct Professor at Philadelphia University for a year prior to taking his current post at St. Joseph’s University. Jonathan has received several grants and awards for his work in research not only from UCLA and St. Joseph’s where he studied and now works, but also from the National Science Foundation, Sea Grant National Biotechnology Initiative, National Sea Grant World Ocean Conference, and the Academy of Natural Sciences. Email address: [email protected]

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These are not, however, the only organisms that use silk (or silk-like materials), nor is the use of silk limited to the terrestrial realm. The aquatic larvae of midges (order Diptera) [4], some moths (order Lepidoptera) [4, 5] and most importantly caddisflies (order Trichoptera) and blackflies (order Diptera) which make up the majority of macrobenthic (large bottomdwelling) invertebrates in many streams [6, 7] all use silk for nutrient acquisition, protection, dispersal, attachment, and pupation. Though silk in aquatic environments plays many roles, and therefore may have interesting properties that could be of value to science and industry, there has been surprisingly little research on its exact material properties or biochemical makeup [3, 8]. One organism that is receiving some attention is the caddisfly, as they are closely related to Lepidoptera and are the most diverse insect order with exclusively aquatic members [7]. Comparative genetic analysis of Trichoptera and Lepidoptera has shown similarities in the silk genes of the two orders [9]. Both silks are comprised of a protein component called sericin, but Trichoptera’s sericin has been shown to differ from Lepidopteran proteins in that they are phosphorylated [8]. Because phosphorylated proteins have been found in bioadhesives of marine organisms, and because phosphates are commonly used to promote binding in paints and in dentistry, it is suggested that the phosphorylated proteins found in Trichoptera silk could have use as an underwater adhesive. Though practical application of macroinvertebrate silks has only begun to be investigated, how they are used by the organisms that produce them is better understood. One of most common uses for silk in aquatic systems is protection during larval growth and pupation. Caddisflies, for example, are divided into three suborders based on their silk constructions [10]. The first group, cocoon-makers (suborder Spicipalpia), creates rigid silk cocoons for pupation. The second group, case-makers (suborder Integripalpia), binds particles with silk to create rigid, mobile cases that both protect and camouflage the larvae. The final groups, retreat-makers (suborder Annulipalpia), use silk to bind particles in order to build stationary shelters for protection. Many of the retreat-makers also attach silk nets to their structures in order to capture food. A second common use of silk is for larval feeding. Three families of caddisflies (Philopotamidae, Polycentropidae, and Hydropsychidae) are known to have larvae that spin nets for nutrient acquisition [11]. Just as terrestrial spiders spin webs to capture prey, netspinning Trichoptera larvae spin silk nets to capture seston (suspended particles) from the water column. Shortly after hatching, larvae build nets attached to their permanent shelters [12] and as the larvae grow, so do their nets and the size of the nets’ webbing. Larvae graze on the contents of the net and will continue to acquire nutrients this way until they are ready for pupation. Caddisfly larvae have been shown to minimize the energetic costs of producing these nets by altering net dimensions in response to changes in water velocity and food concentration, such that net dimensions are inversely related to water velocity [12]. Larvae in slower flowing waters build larger nets with wider openings while larvae in faster flowing waters build smaller nets with narrower openings, thereby minimizing the energetic expense of spinning silk when possible. Net-spinning caddisfly larvae have been deemed “ecosystem engineers” because at high densities their silk structures bind benthic particles together, stabilizing stream channels by raising the velocity at which bed erosion is initiated by 10-30% [13]. It has also been shown that net-spinning Trichoptera can be affected by changes in water velocity. While larvae are well adapted to a range of velocities, frequent hydrologic disturbances have been shown to negatively affect Trichoptera larvae by destroying their nets and thus reducing feeding and fitness [14].

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Silk Use by Benthic Macroinvertebrates

3

A third but rarely studied use of silk is to control the organism’s dispersal both in terms of when it happens as well as where they end up [15]. The use of silk for both of these purposes is well illustrated by larvae of a second important member of the stream community, the black fly. As they rely on ambient flow to bring them food, remaining in place is of paramount importance. Black fly larvae are able to accomplish this, even in the face of fast flow, by forming silk pads to attach themselves to the substrate with their posterior hook circlet (Figure 1) [16] and feed by filtering a variety of small particles from the water column with their anterior labral fans [17]. These silk pads are quite durable, remaining on the substrate for up to 33 days, but larvae will often abandon pads much earlier to reduce the risk of being dislodged from the substrate [18]. Once larvae are attached to a silk pad via their posterior hook circlet, however, they are unlikely to be pulled off the substrate in normal field conditions [19] in part because the hooks hold the pad passively, and only require muscular action when disengaging. This is important to the survival and development of larvae, because as filter feeders, their nutrient acquisition depends on the surrounding flow, and larvae that are dislodged from the substrate might be deposited in unsuitably slow flowing water as discussed below.

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PCH

LF

TP

Figure 1.: Photo of an ~two-week old black fly larvae illustrating the posterior circlet of hooks (PCH), thoracic proleg (TP) and labral fans in folded position (LF).

If larvae are dislodged, or choose to leave, silk still has a role to play. As with many other small organisms that cannot swim or otherwise locomote fast enough to overcome advective flow, they often rely on what is known as fluid-mediated, or passive dispersal to move at scales larger than a few body lengths [20-22]. In heterogeneous habitats, however, purely passive dispersal can often deliver larvae into unsuitable conditions. For example, regions of

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slow flow can subject settlers to elevated predation pressures [23, 24], decreased food availability [25, 26] and limited opportunity to re-suspend and continue their dispersal [27]. Therefore, there may be significant fitness consequence for species that cannot control, or at least influence, their dispersal. For weak or non-swimmers, the ability to influence their vertical position in the water column is the most common form of control. Slow swimmers such as oyster or trematode larvae [28, 29] can often overcome the vertical flow component, which is on average an order of magnitude smaller than horizontal velocities. For those with no swimming ability, changes in density, size, or shape [30, 31] can influence the rate of deposition, positively or negatively, thus increasing or decreasing the organism’s time spent in the drift. Though best known in spiders (both in the popular and scientific literature) [32, 33], the use of silk or other filamentous structures to influence transport can be found in groups including cnidarians, molluscs, and as we discuss here, insects [15, 34]. Though a critical aspect of black fly biology, the study of how silk is used in its dispersal has, until recently, received relatively little attention. This can partly be attributed to the difficulty of experimenting with the silk itself. Silk threads are thin (~2-5 μm), transparent, colorless, and do not appear to easily auto-, or otherwise, fluoresce. In fact, a common question among black fly researchers when they meet is “have you figured out a way to visualize the silk yet?” to which the answer is so far a frustrating negative. Out of the water the silk can be seen easily (Figure 2), and though it can be seen in water under extremely strong light, such as produced by a laser, its thinness and the magnification necessary to see it makes laserillumination useless in realistic flowing water situations where the thread is buffeted about within and beyond the field of view.

Figure 2. Confocal microscopy of a single black fly dispersal thread. Silk: Properties, Production and Uses : Properties, Production, and Uses, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

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Silk Use by Benthic Macroinvertebrates

5

The most successful methods we have developed to investigate the use of threads are all indirect methods that track the position of the larval body and infers the presence of the silk. While such methods can be quite fruitful, it still leads many aspects of silk use, such as the exact method of production, how the larvae work the silk, how it behaves in flow, etc., unanswerable without the development of new techniques. What has been determined is that the silk can play a dual role in dispersal: allowing the larvae to either limit or extend their dispersal distance depending on the situation. Dispersal is most commonly initiated due to unsuitable filter-feeding opportunities caused by slow flow, or low food concentrations [35]. Additional reasons may be fleeing from predators or aggression from conspecifics over prime feeding locations [35]. In freshwater streams, their preferred fast flowing habitat type (riffles) are interspersed with regions of slow, unsuitable flow (pools). Remaining within the same riffle they leave from is therefore of paramount importance. The presence of silk facilitates this by increasing the affective size of the larvae and snagging on the ridges, protuberances and other topographic features of natural bedforms, thus helping the larvae settle quickly and stay within the riffle. In a laboratory test of this mechanism the interaction between the silk and a simple bed form (raised plate) showed an increase in settlement rate of 40 fold when compared to a smooth planar bed in a laboratory flume. Further, the majority of larvae that settled on the raised bed did so within the first cm of the leading edge indicating that it was an interaction with the edge that was facilitating settlement. A hydrodynamic explanation for this phenomena was ruled out based on fine-scale flow measurements that found no significant eddy structures that could explain the larvae’s delivery to the leading edge area [36]. While the ability of the larvae to come to a stop and settle so readily near the leading edge of the raised plate provides clear indication that larvae drift with silk, it is not the only evidence of its use in dispersal. The presence of a thread attached to larvae while they drift has been corroborated by filming them in real time as they snag a monofilament line set across the water column of a laboratory flume [36]. Larvae were observed coming to a halt directly behind the tripwire. Attachment via an unseen thread is the only explanation for how these non-swimming larvae could arrest their downstream movement. As each individual frame of the recording could be viewed independently, it was possible to infer when the filament made contact from the change in direction of the larvae. From this frame the distance from the tripwire could be determined, providing an estimate of silk length, as well as the relative position of the tripwire and larval body (Figure 3). Larvae were observed making contact, via threads, from both above and below the tripwire, but more often from below (2x) indicating that in the absence of strong turbulence (which was not present in this study) the larvae would more often ride below their threads. Further, the length of the thread compared to the length of the larval body maintained a ratio of ~6:1 regardless of the size of the larva. For the settlement plate study the distance from the leading edge where most larvae settled corresponded well with these measured lengths, further indicating that it was the presence of silk threads that was facilitating settlement. The correlation between larval size and dispersal thread length provides an interesting comparison to how the larvae use silk for a different purpose, namely the “lifelines” that allow them to remain connected to the substrate if disturbed or dislodged. For threads that are made during these emergencies, which can be made in 0.1-0.6 seconds [37], there is no correlation between the size of the larvae and the thread’s length.

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Figure 3. Diagram illustrating the data collection method for drifting larvae and their interaction with a cross-stream tripwire. Numbers represent frames starting from when the larvae entered the field of view. By frame 6 the larvae was stationary downstream of the tripwire. Silk length and orientation were determined by analyzing the first frame in which a change in direction or orientation (frame 5) was seen. Photo in the bottom left represents one actual frame in which two larvae can be seen, one still drifting downstream (A) and one stationary, downstream of the tripwire (B).

The mechanisms that are used to make these threads may therefore be different than those for dispersal. Due to the size and mass of the silk threads, the production and unfurling of dispersal threads in a viscous medium such as water would be problematic (think of pushing yarn through honey). Little is known about how they are produced, but there is some indication that a large bolus of silk is formed to act as a drag anchor, helping to draw out the line [38]. Further, there is evidence that the production of the thread can be facilitated by the use of the larvae’s single appendage, its prothoracic foreleg (Figure 1), which can be observed moving rhythmically towards and away from the body before larvae let go of the substrate [39]. The use of the proleg, which scales with the overall size of the larvae, might explain the allometric relationship seen in the tripwire study. The exact relation between the silk and the proleg, however, is difficult to discern due to the issues discussed earlier regarding visualization of the silk. The effect of silk on a larvae’s ability to settle within regions of fast flow are difficult to predict and to date have not been tested under field conditions. While the raised plate used in the laboratory experiment, with its knife-edge profile, represented a best-case scenario for capturing larvae via their threads, it is not much of a stretch to think that the topography of a natural stream bed element would provide multiple opportunities for settlement. Multiply the opportunities of a single bed element by the numerous elements that one larvae may pass over, and it becomes rather likely that the presence of silk would greatly increase the chances of a given larvae settling in a riffle, even if one lowers the chance of settlement an order of magnitude from that determined experimentally in the laboratory flume. Hydrodynamic theory predicts that without other factors (e.g. behavioral or morphologic adaptations) the regions with the fastest, and therefore best, flow conditions should also be the hardest to reach and settle in [40]. However, if one looks in a stream with a healthy blackfly population it can be difficult to find a single piece of gravel or cobble with no larvae, or even free space if the

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Silk Use by Benthic Macroinvertebrates

7

local population is large enough. The presence of the silk threads may help explain how such distributions are possible in light of the challenges facing drifting larvae. The presence of silk threads is not, however, a guarantee of success when drifting. Larvae may drift below their thread, or turbulence may move the entire body-thread complex away from the bed, both of which would eliminate any aid that the thread would provide. Therefore, some portion of the larvae that enter the drift within a given riffle should find themselves being washed downstream into the slow-flowing pool habitat that makes up the flipside of the alternating riffle-pool conditions found in most lotic systems. In fact, large numbers of larvae can be collected in the pool water column, with one study showing up to 100 larvae per m3 at the pools entrance (author unpub data). One would expect that threads might be considered a disadvantage in such habitat as anything that might decrease transport distances would increase the chances of being delivered to the pool bed. Once there, larvae would face increased predation pressures from organisms such as flatworms that are excluded from riffles by hydrodynamic forces [41], and a reduced flux of suspended organic matter to feed from [24, 40]. However, recent investigations have shown that the presence of silk threads can decrease transport distances in riffles while increasing their distances in pools. Thinking of larvae as inanimate particles, such as silt or sand, provides a null hypothesis against which to test the effects of silk threads on transport distances. The slower the fall velocity of a particle, the farther it will be carried under the same flow conditions, or the slower (less turbulent) the conditions under which it will be carried. This can be seen in the makeup of the bed in both the riffle and pool regions where the riffles are made up of large, heavy particles such as gravel, with the lighter, smaller particles forming sand and silt beds in pools. Decreasing the fall velocity of the larvae should, therefore, increase the distance they are transported [42, 43], and thus increase their chance of being carried all the way through a pool to the next downstream riffle before deposition. The ability of silk threads to reduce fall velocity was tested experimentally with live (with thread) and dead (without thread) larvae that were filmed falling through a column of still water [44]. The presence of silk was found to decrease fall velocity for all sizes (ages) of larvae, though the relative significance of this reduction decreased with larval size (36% reduction for 0.6 mm neonates compared to only 14% reduction for 7mm late-instar larvae). Translating these results to the real world where pools can be 100’s of meters long means that the main beneficiaries of silk’s parachute-like properties will be the smallest, youngest larvae. For a typical pool of 100 m in length, 25 cm in depth and an average mid-column water velocity of 15 cm/s, a neonate larvae without silk would be predicted to travel 58 m compared to only ~2 m for the larger age-class before hitting bottom. The presence of silk would add an additional 33 m to the neonate’s trip but only 0.3 m for the older larvae. This would provide the neonate with a good chance of making it to the next riffle before contact, but leave the older larvae just inside the entrance to the pool. For larvae that are unable to remain suspended long enough to make it through a pool, silk may still provide a chance for survival. Silk threads produced while on the bed should increase the drag force imparted on the larva while adding very little additional mass, thus increasing the chances of resuspension back into the water column by turbulent eddies [31, 45]. The best example of this is the “ballooning” behavior seen in spiders, but the same mechanism is also used by bivalve larvae [46]. The invisibility of the silk makes it impractical to directly measure the role threads would play in resuspending larvae in slow flow; the silk’s length, or even its presence could never be reliably determined. It is possible, however, to

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dynamically scale models of larvae so that drag forces with and without silk thread mimics can be measured. Doing so for different aged larvae, with their slightly different body plans, has shown that not only does the silk produce a significant increase in drag but that the less streamlined neonate larvae had higher measured drag forces than their sleeker older counterparts [44]. This should further increase the chances of resuspension for young larvae adding to their already sizeable advantage in dispersal distance due to lower fall velocities. The sleeker shape of the older larvae may be an adaptation to lower their drag, and thus minimize dislodgement, in preferred high-flow environments where they can competitively exclude younger larvae from the fastest, and therefore best, feeding spots. How the same physical adaptation can play the seemingly contradictory roles of both limiting and increasing dispersal distances when appropriate can be explained by the local hydrodynamic conditions. In fast flow, turbulence increases proportionally with velocity thus increasing the chance that the larval body-thread complex will be tumbled randomly with the silk sometimes above and sometimes below the body. When the silk rides below the body (Figure 4A), the effective size of the body is increased, thereby increasing the chances of making contact with the bed for at least some portion of the drift population. In slow flow, however, the denser body should ride below the silk (Figure 4B) thus increasing the drag force on the complex without presenting any greater surface area to make contact with the bed and thus interfere with resuspension. In fact, it is possible that the allometric ratio of silk thread to body length may be an adaptation to maximize the length of the silk without producing a thread that is so long that it has a good chance of falling back down to the bed and anchoring the larvae there like the aforementioned lifelines. Such a hypothesis is admittedly difficult to test considering the issues surrounding the visualization of the silk.

B A

Figure 4. Two possible orientations of the silk-body complex. (A) Under the turbulent conditions found in riffles, the silk would ride below the body at least some of the time, increasing the effective size of the larva. (B) In calmer pools, the body would most likely ride below the silk thus increasing drag but not chance of contact with the bed.

The examples provided here illustrate the important role that silk plays in the lives of macrobenthic organisms, and in particular two of the most important inhabitants of lotic ecosystems: caddis and black fly larvae. While the basic aspects of how larval Trichoptera Silk: Properties, Production and Uses : Properties, Production, and Uses, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

Silk Use by Benthic Macroinvertebrates

9

and Diptera use silk are now known, there is still a vast number of fascinating details that need to be elucidated and quantified before we can fully understand the role that silk plays at the population (e.g. demographics and size) and community (e.g. spatial distributions) level in these systems.

REFERENCES [1] [2]

[3] [4]

[5]

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[6] [7] [8] [9] [10] [11] [12] [13]

Fredriksson, C., M. Hedhammar, R. Feinstein, K. Nordling, Kratz, J. Johansson, F. Huss, and A. Rising, Tissue Response to Subcutaneously Implanted Recombinant Spider Silk: An in Vivo Study. Materials, 2009. 2: p. 1908-1922. Agapov, I., O. Pustovalova, M. Moisenovich, V. Bogush, O. Sokolova, V. Sevastyanov, V. Debabov, and M. Kirpichnikov, Three-Dimensional Scaffold Made from Recombinant Spider Silk Protein for Tissue Engineering. Doklady Biochemistry and Biophysics, 2009. 426: p. 127-130. Sehnal, F., Prospects of the practical use of silk sericins. Entomological Research, 2008. 38: p. S1-S8. Case, S., J. Powers, R. Hamilton, and M. Burton, Silk and Silk Proteins from Two Aquatic Insects, in Silk Polymers Materials Science and Biotechnology, D. Kaplan, W. Adams, B. Farmer, and C. Viney, Editors. 1994, American Chemical Society: Washington, DC. Solis, A., Aquatic and Semiaquatic Lepidoptera, in An Introduction to the Aquatic Insects of North America, R. Merritt, K. Cummings, and M. Berg, Editors. 2008, Kendall/ Hunt Publishing Company: Dubuque, IA. Adler, P.H. and J.W. McCreadie, The hidden ecology of black flies: sibling species and ecological scale. American Entomologist, 1997. 43: p. 153-161. Holzenthal, R., R. Blahnik, A. Prather, and K. Kjer. Trichoptera. The Tree of Life Web Project 2010 July, 2010 [cited 2011. Stewart, R. and C. Shuen-Wang, Adaptation of Caddisfly Larval Silks to Aquatic Habitats by Phosphorylation of H-Fibroid Serines. Biomacromolecules, 2010. 11: p. 969-974. Yonemura, N., M. Kazuei, T. Tamura, and F. Sehnal, Conservation of Silk Genes in Trichoptera and Lepidoptera. Journal of Molecular Evolution, 2009. 68: p. 641-653. Wiggins, G. and D. Currie, Trichoptera Families, in An Introduction to the Aquatic Insects of North America, R. Merritt, K. Cummings, and M. Berg, Editors. 2008, Kendall/Hunt Publishing Company: Dubuque, IA. Fowler, D. Net-spinning Caddisflies. Entomology Notes 2011 [cited 2011; Available from: http://insects.ummz.lsa.umich.edu/mes/notes/entnote14.html. Petersen, R., L. Petersen, and J. Wallace, Influence of velocity and food availability on catchnet dimencions of Neureclipsis bimaculata (Itichoptera: Polycentropodidae). Holarctic Ecology, 1984. 7: p. 380-389. Cardinale, B., E. Gelmann, and M. Palmer, Net spinning Caddisflies as stream ecosystem engineers: the influence of Hydropsyche on benthic substrate stability. Functional Ecology, 2004. 18: p. 381-387.

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[14] Beveridge, O. and J. Lancaster, Sub-lethal effects of disturbance on a predatory netspinning caddisfly. Freshwater Biology, 2007. 52: p. 491-499. [15] Abelson, A., D. Weihs, and Y. Loya, Hydrodynamic impediments to settlement of marine propagules, and adhesive-filament solutions. Limnology and Oceanography, 1994. 39(1): p. 164-169. [16] Reidelbach, J. and E. Kiel, Observations on the behavioral sequences of looping and drifting by black fly larvae (Diptera:Simuliidae). Aquatic Insects, 1990. 12: p. 49-60. [17] Adler, P.H., D.C. Currie, and D.M. Wood, The Black Flies (Simuliidae) of North America. 2004, Ithaca, NY: Cornell University Press. 941. [18] Kiel, E., Durability of Simuliid Silk Pads (Simuliidae, Diptera). Aquatic Insects, 1997. 19: p. 15-22. [19] Eymann, M., Drag on single larvae of the black fly Simulium vittatum (Diptera: Simuliidae) in a thin, growing boundary layer. Journal of the North American Benthological Society, 1988. 7: p. 109-116. [20] Palmer, M.A., J.D. Allan, and C.A. Butman, Dispersal as a regional process affecting the local dynamics of marine and stream benthic invertebrates. Trends in Ecology & Evolution, 1996. 11(8): p. 322-326. [21] Downes, B.J. and M.J. Keough, Scaling of colonization processes in streams: Parallels and lessons from marine hard substrata. Australian Journal of Ecology, 1998. 23(1): p. 8-26. [22] Shanks, A.L., B.A. Grantham, and M.H. Carr, Propagule Dispersal Distance and the Size and Spacing of Marine Reserves. Ecological Applications, 2003. 13(1): p. S159S169. [23] Malmqvist, B. and G. Sackmann, Changing risk of predation for a filter-feeding insect along a current velocity gradient. Oecologia, 1996. 108(3): p. 450-458. [24] Hart, D.D. and R. Merz, Predator-prey interactions in a benthic stream community: a field test of flow-mediated refuges. Oecologia, 1998. 114: p. 263-273. [25] Lesser, M.P., J.D. Witman, and K.P. Sebens, Effects of flow and seston availability on scope for growth of benthic suspension-feeding invertebrates from the Gulf of Maine. Biological Bulletin (Woods Hole), 1994. 187(3): p. 319-335. [26] Finelli, C.M., D.D. Hart, and R.A. Merz, Stream insects as passive suspension feeders: Effects of velocity and food concentration on feeding performance. Oecologia, 2002. 131(1): p. 145-153. [27] Schamel, L., Do Stream Pools Act as Sinks for Passively Transported Larvae?, in Biology. 2008, Saint Joseph's University: Philadelphia, PA. [28] Fingerut, J.T., C.A. Zimmer, and R.K. Zimmer, Larval swimming overpowers turbulent mixing and facilitates transmission of a marine parasite. Ecology, 2003. 84(9): p. 25022515. [29] Finelli, C.M. and D.S. Wethey, Behavior of oyster (Crassostrea virginica) larvae in flume boundary layer flows. Marine Biology, 2003. 143(4): p. 703-711. [30] Round, F.E., R.M. Crawford, and D.G. Mann, Diatoms: Biology and Morphology of the Genera. 1990: Cambridge University Press. 758. [31] Olivier, F. and C. Retière, How to leave or stay on the substratum when you can't swim? Ecidence of the role of mucus thread secretion by postlarvae of pectinaria koreni (Malmgren) in still water and flume experiments. Aquatic Ecology, 2006. 40: p. 503-519.

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[32] White, E.B., Charlotte's Web. 1952: Harper Collins. 184. [33] Humphrey, J.A.C., Fluid Mechanic constraints on spider ballooning. Oecologia, 1987. 73: p. 469-477. [34] Suter, R.B., Ballooning in spiders: results of a wind tunnel experiments. ethology, Ecology and Evolution, 1991. 3: p. 13-25. [35] Fonseca, D.M. and D.D. Hart, Density-dependant dispersal of black fly neonates is mediated by flow. Oikos, 1996. 75: p. 49-58. [36] Fingerut, J., D. Hart, and J. McNair, Silk filaments enhance the settlement of stream insect larvae. Oecologia, 2006. 150: p. 202-212. [37] Wotton, R.S., The use of silk life-lines by larvae of Simulium noelleri (Diptera). Aquatic Insects, 1986. 8(4): p. 255-261. [38] Kiel, E., Verhaltensbiologische studie an Simuliiden (Simuliidae, Diptera): Landen und ansiedeln auf einem cinema substrate. Angewandte Zoology, 1989. 76: p. 385-402. [39] Fonseca, D.M., Fluid-mediated dispersal in streams: Models of settlement from the drift. Oecologia, 1999. 121(2): p. 212-223. [40] Fonseca, D.M. and D.D. Hart, Colonization history masks habitat preferences in local distribution of stream insects. Ecology, 2001. 82(10): p. 2897-2910. [41] Hansen, R., D.D. Hart, and R. Merz, Flow mediates predator-prey interactions between triclad flatworms and larval black flies. Oikos, 1991. 60(187-196). [42] McNair, J., J. Newbold, and D.D. Hart, Turbulent Transport of Suspended Particles and Dispersing Benthic Organisms: How Long to Hit Bottom? Journal of Theoretical Biology, 1997. 188: p. 29-52. [43] Elliott, J., The distances by drifting invertebrates in a Lake District stream. Oecologia, 1971. 6: p. 350-379. [44] Fingerut, J.T., L. Schamel, A. Faugno, M. Mestrinaro, and H. P., Role of silk threads in the dispersal of larvae through stream pools. Journal of Zoology, 2009. 279(2): p. 137143. [45] Dey, S., Incipient Motion of Bivalve Shells on Sand Beds under Flowing Water. Journal of Engineering Mechanics, 2003. 129: p. 232-240. [46] Prezant, R. and K. Chalermwat, Flotation of the Bivalve Corbicula fluminea as a means of Dispersal. Science: News Series, 1984. 225: p. 1491-1493.

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In: Silk: Properties, Production and Uses Editor: Pornanong Aramwit

ISBN: 978-1-62100-692-3 © 2012 Nova Science Publishers, Inc.

Chapter 2

MUGA SILK: PROPERTIES, PRODUCTION AND USES Subrata Das* Li & Fung Private Limited, Bangalore, India

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ABSTRACT Muga is a rare non-mulberry silk and one of the costliest natural fibers, valued for its shimmering golden color, lusture and durability. Availability of this fiber is restricted to North-Eastern parts of India and its ever increasing demand has made this fiber of utmost importance. Although many studies have been carried out on the muga silkworm, very little is reported on the preparation, processing and properties of muga silk fibers. In this paper, an attempt has been made to assimilate the research findings on muga silk by various researchers along with in-house data and practical experience.

INTRODUCTION Silk, a fibrous protein secreted by several species of insects for building structures external to the body known as cocoons. Silk is mainly classified into two groups, as mulberry (Bombyx species) and non-mulberry (Antheraea species) varieties. Non-mulberry silks are also known as wild or vanya silks. Among the non-mulberry silks, muga is the most critical and important for an array of reasons. The golden strands of muga silk are recognized the world over as an exclusive silk and from the ancient time till date, India is the only producer *

Subrata Das has a Ph.D. from IIT New Dehli in Textile Technology. He has spent the major part of his professional career working for the Central Silk Technological Research Institute for the Government of India in Bangalore. He has also spent a large part of his professional career as manager of world-class laboratories for companies such as Merchandise Testing Laboratory, Consumer Testing Services in SBS Bangalore, and Consumer Testing Laboratory. His work has ranged from setting up laboratories, recruiting, training on US and EU testing standards, and to garment research, testing, and quality assurance. He is currently employed at Li & Fung (India) Pvt. Ltd., Bangalore, India as a Senior Manager (Technical). Subrata Das has achieved Senior Membership of American Association of Textile Chemists and Colourists (AATCC). E-mail address: [email protected]

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of this silk. “Muga” is derived from the Assamese word muga, meaning yellowish. Muga silk is famous for its natural golden color, lusture and durability. This silk is the product of the silkworm Antheraea assamensis Helfer endemic to Assam. The North-Eastern states of India hold a natural monopoly (99.86%) over the production of muga, albeit a small proportion of muga is also produced in Coochbehar, West Bengal State of India. Assam is the largest producer of muga silk in the country. The land area under muga plantations in these NE states is about 5362.11 ha (80% in Assam). Antherea assamensis Helfer [1] is the only worm which produces this unique variety of golden-yellow silk renowned for its color and texture. The muga silkworm is a holotabolous insect (Figure 1), passing through a complete metamorphosis from egg to adult stage through two intermediate stages of larva and pupa. The entire life cycle lasts for about 50 days in summer and 120 days in winter. The spinning of cocoons takes 3 to 7 days. Rearing of muga silkworms is an out-door process; on the other hand, spinning of cocoons is an in-door process. The host plants for the muga silkworm as identified are Som (Machilus bombycina Kost), Soalu (Litsae polyantha Juss) Bodakaki (Litsea glutinosa Lour), Tumri (Phoebe lanceolata Nees), Kaula (Persea duthei King), Bondasum (Persea glaucescens Nees), Gansarai (Cinamomum glanduliferum), and Lodh (Symplococos paniculata Wall) [1]. Out of them, Som (Machilus bombycina) and Soalu (Litsae polyantha) are the two most important host plants. The average life time of the muga silk moth (Figure 1) is about 5-7 days [1]. Muga cocoons are peduncle-less, elongated oval shaped (Figure 1) with length of about 5 centimeters and average diameter of about 2.5–3 centimeters.

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Meeting by JS(Silk), MoT

9/4/2009

16

Muga silkworms

Muga silk moths

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Muga Silk

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COMPOSITION OF MUGA SILK Like other silks, muga silk filament is also composed of proteins i.e., fibroin and sericin. These parts constitute up to 95% of the raw silk fiber, the remaining part consists of other proteins, waxes, fats, salts and ash. Fibroin is the main constituent of the silk filament and the sericin works as silk glue to protect the outer layer of the silk filament in the cocoon stage. An ample number of works have reported the amino acid composition of different silk fibers [2]. Now, it is a well known fact that fibroin consists of 20 different amino acids. In the early 1950s, acid hydrolysis, followed by ion exchange chromatography to separate and identify the amino acid compositions, was used. Amino acids such as glycine, alanine, and serine form the major components (about 80-90%) in mulberry as well as non-mulberry silk filaments, but specifically, in the case of muga silk the composition of those three amino acids is observed to be on the lower side at 68%. In a study on chemical composition and physical properties of muga silk, a high alanine content of 42.62% and very low arginine (2.83%) and cystine (0.34%) content is reported. Analyses of crystalline regions of muga silk estimates the composition of the glycine, alanine, and serine is of about 81–82%.

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PHYSICAL PROPERTIES OF MUGA SILK The density of muga silk is 1.31 gm/mL and its fineness is about 3-5 denier. It is reported that the denier of muga fibers decreases from the outer layer to the inner layer of the cocoon [2] and the finer fiber has higher density and higher crystallinity compared to its coarser counterpart. The denier and breaking extension of muga fiber reduces along the cocoon layer, whereas density, tenacity and initial modulus of the fiber reduces with the layer number. Also, the sericin content in the silk filament reduces from the outer to the inner part of the cocoon. The cross-sectional and longitudinal view of muga fiber has been analysed by Sen and Babu [2] and Gupta et al. [3] using Scanning Electron Microscopic (SEM) technique. SEM images of muga fiber have been given in Figure 2. Muga exhibits an elongated rectangular or a wedge-shape cross section. At higher magnification the presence of microvoid in muga fiber has also been noticed. The cross-sectional area of muga fiber reduces from layer to layer. The density of muga is lower than mulberry fibers, but it has the highest density comparative to the other two non-mulberry fibers. The tenacity of muga silk is about 4-5 gpd. The tensile property of coarse and fine muga fibers as obtained by Rajkhowa et al. [4] has been given in Table 1. Table 1. Tensile properties of muga fibers Muga fiber (3.24 denier) (1.90 denier)

Initial modulus (gf/den) 65.55 73.73

Tenacity (gf/den) 4.57 4.91

Breaking strain (%) 40.68 26.4

Toughness (gf/den) 1.06 0.83

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From Table 1 it is observed that compared to the coarser muga fibers, the finer fibers have higher strength and higher modulus, but lower percentage elongation-at-break [4], these correlate with their higher density and birefringence.

Longitudinal view

Cross-sectional view

Microvoids on muga silk fibers

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Figure 2. SEM images of muga silk fibers.

The tensile behavior of different silk fibers has been represented in Figure 3. From the comparative study of stress-strain behavior of different silk fibers, it has been concluded that muga has the highest elongation-at-break as well as the highest toughness (work of rupture), but the recovery of muga fiber is less than that of mulberry [4, 5]. Analyzing stress-strain curve, two yield plateaus have been observed during the extension of muga fiber. The occurrence of plateau in the stress strain curve has been analyzed by Rajkhowa et al. [4] with the reference of the presence of the amorphous phase. The amorphous phase, being more compliant than the crystalline phase, is the first to deform on application of load. The initial resistance to deformation, which is significant, arises from the interchain bonds that bind the molecules in the amorphous regions, the entanglements between the chains present in the amorphous regions, the constraints arising from the rigid crystallites between which the amorphous regions are sandwiched, and the restriction to free movement in the laterally ordered oriented amorphous regions. After the initial deformation, with increasing load and continued extension, the interchain bonds in the amorphous region of silk can break and make it easier for the molecular chains to extend, which results in higher elongation-at-break. But because of the randomly arranged chains in the amorphous regions, it continues to extend at almost a constant load or with slight increase in load, and give rise to another distinct yield and the yield plateau.

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Figure 3. Tensile behavior of silk fibers.

In a study on stress relaxation of Indian silks, it has been observed that the stress relaxation rate of muga is higher than that of mulberry silk. This is due to the presence of greater degree of viscoelastic component in muga fibers. X-ray crystalline diffraction and infrared crystalline index of muga fibers as reported by Rajkhowa et al. [4] are 0.43 and 0.5, respectively. The lower density of muga silk comparative to mulberry silk fiber may be because of its lower crystalline portion and the presence of bulky polar group in its structure. The lower crystallinity of muga has been reflected in birefringence result. The birefringence value of muga silk as obtained using Beckline method is 0.4 [4]. The presence of bulky polar group is more in the amorphous region which may inhibit the proper alignment of the protein chains and which results in the lower density of muga. In the studies on determining the chemical composition of different silks, it has been known that a substantial proportion of amino acids with bulky side groups are present in muga. It results in lower crystallinity of muga comparative to the mulberry silk. The X-ray diffraction curves of coarse and fine muga fibers have been given in Figure 4 (a) and 4 (b) [4]. The results obtained from wide angle X-ray scattering technique [6], in order to study the microcrystalline parameters, reveal information about the crystallite region of the fibers in muga silk variety that contributes to making these fibers stiffer than two other non-mulberry silks.

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(a)

(b)

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Figure 4. X-ray diffraction curves of muga fibers: (a) Coarse fiber (b) Fine fiber.

Dehydration in DSC thermogram was found to be higher in the case of muga comparative to mulberry and Eri in a DSC analysis conducted by Bora et al. [7]. Thermal stability of muga is higher than those of Eri and mulberry fibers. In DSC analysis, two endothermic peaks have been observed in the case of muga. The 1st peak represents the absorption of heat by the sample for dehydration, while the 2nd peak represents the decomposition of the fiber. The dehydration has been observed at 429K and decomposition at 655K. Maximum weight loss due to dehydration has been observed in the case of muga silk. It reveals the high moisture regain of muga fiber. Moisture regain of muga is 9.82%, whereas that of mulberry is 8.52% [2]. The higher absorption and desorption of atmospheric moisture by the muga fibers with the consequent evolution and absorption of heat add to the comfort of clothing. Also the higher thermal resistance of muga makes it suitable to use as a thermal insulator up to its decomposition limits.

MUGA SILK PRODUCTION In Assam, six overlapping muga cocoon crops are harvested in a year. The details of muga cocoon crops and their features have been given in the following Table 2 [8]. The average weight of one cocoon during two commercial seasons varies from 5 gm to 7.5 gm, shell weight from 0.35 gm to 0.6 gm, average filament length 300 meters to 500 meters with 3 to 5 denier fiber fineness.

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Table 2. Muga cocoon crops and its features Crop Name

Month

Nature of crop

Jarua Jethua Akharua Bhadia Kotia Aghania

February-March April-May June-July August-September October-November December-January

Seed Commercial-II Seed /Commercial Seed Commercial-I Seed /Commercial

Contribution in Yield 40%

60%

The state wide production of muga (in MT) has been reported in Table 3 [9]. Due to the growing demand of muga and fall in muga cocoon quantity, the price of muga silk has been observed to increase in the recent past. The recent prices of muga reeling cocoons and raw silk, as per the market of Guwahati, Assam are given in Table 4. Table 3. State wise production of muga (metric ton-MT)

1

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3 ) 5 6

2007-08 (April – March) 105.1 0.35 0.4 10.5 0.2 0.2 116.75

State 1) Assam 2) Arunachal Pradesh 3) Manipur 4) Meghalaya 5) Mizoram 6) Nagaland Total

2008-09 (April – March) 106 0.15 0.2 11 0.1 0.35 117.8

Table 4. Prices of muga reeling cocoons and raw silk Market

Quality

Unit

Feb. 2010 Min Max

Feb. 2009 Min Max

Feb. 2008 Min Max

Rs./1000 nos.

1200

1600

800

1200

600

700

Guwahati, Assam

Reeling cocoons Raw silk (Warp) Raw silk (Weft)

Rs./kg

7000

8000

6000

6200

4000

4000

Rs./kg

6000

6500

5000

5500

4500

4500

SEQUENCE OF MUGA SILK PREPARATION Commencing from the cocoons, the stages of operations involved in preparation of muga silk fabric can be categorized as yarn preparation, fabric preparation and chemical processing of muga silk.

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1. Muga Yarn Preparation Muga yarn preparation is as represented by the flow chart (Scheme 1). The cocoons are stifled to kill the muga pupa inside the cocoon so that emergence of moths from the cocoons is avoided keeping the continuity of the silk filament intact in the cocoon. There are many types of stifling methods adopted; they are sun drying, smoking, and hot air drying in a chamber [10]. Usually, stifling is carried out by exposing the cocoons to a higher temperature of about 140oC for 2 – 4 hours in a hot air oven. It is experienced that steam stifling is not suitable for muga cocoons [11].

Muga cocoons

Stifling

Sorting and Grading

Reelable cocoons

Non-reelable / Cut-Pierced cocoons

Cocoon cooking Cocoon cooking

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Deflossing

Reeling

Reeled yarn

Waste

Spinning

Spun yarn

Scheme 1. Muga yarn preparation.

After stifling, the cocoons are sorted into different grades (A, B and C). “A” grade cocoons are of superior quality and provides the highest yield (300-350 gms/1000 cocoons). “B” grade cocoons are of medium quality, although they are reelable, they produce lower quantity of muga silk after reeling (200-300 gms/1000 cocoons). Inferior quality “C” grade cocoons, along with cut-pierced cocoons, are non-reelable and hence they are used for silk spinning. During the reeling process, some of the A and B grade cocoons are identified to be non-reelable, they are also used for silk spinning. Methods used in muga cocoon cooking vary from one unit to another in the absence of a well optimized process. Reelers use their practical experience and skill to get the desired softness of cocoons. Duration of cooking also varies depending on the duration of cocoon storage prior to reeling. In one of the surveys in the Nalbari, Assam, a typical process has been observed which offers better productivity and quality; where cocoons are cooked in water

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containing 2.0 gm/L sodium carbonate with material to liquor ratio of 1:20 for 15-20 minutes at 95°C. Cooked cocoons are usually deflossed by hand before the commencement of reeling. Reeling of deflossed cocoons is done in luke warm water (50-60oC). Well cooked cocoons give raw silk recovery of 40-45% of the shell weight. Reeling of muga silk is carried out on a primitive machine called a Bhir as well as on a Motorised Reeling cum Twisting Machine (MRTM), developed by CSTRI, Central Silk Board, Bangalore, India. In general, this reeling cum twisting machine is power operated but also has a stand-by pedal driving arrangement [12]. Reeling yarn production in Bhir in gm/basin/8 hrs is around 95, whereas in case of MRTM it is around 155. Bhir yarn is preferred for weft in muga fabric weaving as it gives good lustre due to less twist in the yarn. Compared with the bhir yarn (60-80 denier), MRTM machine reeled yarn is of finer quality (40-50 denier), hence machine reeled yarn is profusely used for warp. For reeling warps, about 8 to 10 cocoons are reeled together while the weft requires 10 to 12 cocoons. Reeling of muga cocoons in MRTM has been shown in Figure 5.

Figure 5. Muga reeling in MRTM.

For spinning the non-reelable/cut-pierced cocoons, degumming of the cocoons is done. Degumming is done in water containing 10 gm/L sodium carbonate with material to liquor ratio of 1:10 for 35-40 minutes at 95°C. Muga spinning is done using Muga Spinning Machine (MSM), developed by CSTRI, Central Silk Board, Bangalore, India. The machine is power operated, but it can also be operated using a pedal driving arrangement. Muga spinning in MSM has been shown in Figure 6.

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Figure 6. Muga spinning in MSM.

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2. Muga Silk Weaving An earlier practice of muga silk weaving was to use the Assamese type loom (loin loom) which is still prevalent in some tribal settlements. In a loin loom, the warp is tied up in split bamboo, at the ends of which a leather strap is fastened which passes across the weaver [9]. Throw shuttle looms can still be seen in some rural parts of Assam but recently most of the weavers (98%) have started using the fly shuttle handlooms. Powerlooms, shuttle-less looms and such advanced looms are still not in use even for weaving expensive muga silk for external markets. The use of accessories is also rare in rural areas. Less than 10% of the rural weavers use the jacquard (120 hook, single lift, single cylinder) for textile designs and the rest of the weavers do not use any sort of accessories for muga silk weaving. Kamrup district is the centre of muga silk handloom weaving in Assam. The weaving activities of muga silk are concentrated in Sualkuchi and Bamundi sectors of Kamrup, known as one of the major silk weaving clusters in North-East India [9]. It is estimated that more than 75% of total muga cocoons produced in North-East India are concentrated in Kamrup district of Assam for cocoon trading, yarn production, fabric weaving and silk cloth marketing.

3. Chemical Processing of Muga Silk Muga silk is famous for its color and luster, which is often preserved for the manufacture of dress and other fabrics. In literature, very little information is available on the processing of Muga silk. The steps of chemical processing of muga silk have been presented by the flow chart in Scheme 2.

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Muga silk fabric

Degumming

H2O2

Soap-soda Acid Enzymatic

Bleaching*

Natural dyes Acid dyes Dyeing* Metal complex

Printing

Finishing * optional Chemical

Mechanical

Softner Anti-crease

Calendering Polishing

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Scheme 2. Chemical processing of muga.

3.1. Degumming The process of eliminating ‘gum‘ from raw silk is known as degumming of silk. In practice, degumming of muga silk is done in both the yarn as well as the fabric stage. The different ways of degumming silk are as follows [13]. a. Soap and Soda Method Soap and soda is a good degumming agent. Grey fabrics can be completely degummed by treating with soap-soda solution at close to boiling point for 1-2 hours. Neutral synthetic agents have no degumming properties. After degumming, the silk is thoroughly washed in water, with weak solutions of ammonium chloride or soda ash at 40-50C for 20 minutes. b. Acid Degumming Degumming of silk can also be done using organic acids, such as tartaric and oxalic, which hydrolyse the sericin at boil piont. But being costly, this method has never been used on an industrial scale [13]. c. Enzymatic Degumming Enzymatic degumming is emerging as an eco-friendly fiber gentle process. Proteolytic enzymes like trypsin and papain may be used for degumming. Large molecular weight enzymes do not penetrate into the interstices of the fabric and hence are suitable for yarn

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Subrata Das

degumming. In the case of fabric as total sericin is not removed by this treatment, a subsequent treatment with soap solution is necessary. The enzymes preferably hydrolyze peptide bonds formed by carboxyl groups of lysine and arginine of silk resulting in low molecular weight water soluble products which can be easily washed out. A critical control of pH and temperature is required to utilize the full potential of the enzymes.

Degumming Loss To determine the percentage of degumming loss, after the degumming process, the samples are washed thoroughly, dried and conditioned for 24 h under standard conditions of 27±2°C and 65% relative humidity. Degumming loss (%) is calculated as the percentage loss in weight. Degumming loss (%) =

Wt. of silk before degumming - Wt. of silk after degumming 100% Wt. of silk before degumming

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The degumming loss varies with the degumming methods, conditions and cocoon species. Indirectly degumming loss represents the amount of sericin present. At a standard processing condition, the degumming loss varies from the outer layer to inner layer. This may be due to the variation in the sericin content from layer to layer in the filament. The degumming loss in the case of muga (degummed with 25% Marseilles soap (w/w) at a boil for 90 minutes, at a liquor ratio of 1:40) was around 8.57 to 12.69%.

3.2. Bleaching Muga silk is rarely bleached to remove its shimmering natural golden color. However, when it is required to dye muga fabric, bleaching is done to lighten its natural color. Hydrogen peroxide bleaching is done at near neutral or mildly alkaline pH of 8-9 at 60-70C. 3.3. Dyeing The superiority of muga silk among all varieties of silk available in world lies in its natural gleaming golden color. The natural color of muga is highly preferred by customers. Hence, the dyeing of muga silk is not a common practice as is with other varieties of silk. Still for some traditional uses, muga silk is dyed using some natural dyes. Like in Manipur, muga phanek is produced by dyeing muga silk using natural dyes obtained from the products of different trees, such as Mallotus philippensis Muell, Solanum ferox Linn etc [14]. Acid dyes, metal complex and reactive dyes can also be used to dye muga silk. Acid dyes are cheap and give bright shades with medium to poor wash fastness, thereby necessitating the dry-cleaning of the dyed goods. On the other hand, metal-complex dyes give relatively better wash and light fastness. The use of reactive dyes on silk is also increasing as it provides bright shades with good wash fastness. In a study conducted by Somashekarappa et al. [15], the effect of bleaching and dyeing with acid dyes on the microstructural properties of muga has been studied. From the critical analysis of this study, it has been found that the crystal size value of muga fiber increases with bleaching and acid dye treatment. It is also to be noted that on treatment with acid dye, there is an increase in the values of crystal imperfect parameters and rearrangement of beta-pleated structure of protein molecules present in the

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Muga Silk

25

fiber, which results in an increase in percentage of elongation-at-break and reduction in tensile strength.

3.4. Printing Printing of muga silk is carried out mainly for sari and mekhla-chaddar. All styles of printing namely direct, discharge and resist are practiced. Acid, metal complex, reactive as well as some natural dyes are used for printing on muga fabric. Printing with reactive dyes is generally carried out with sodium alginate thickner. However, many reactive dyes can be fixed on silk under neutral conditions, hence it is also possible to print the silk with guar gum. Some fancy effects such as tone–in–tone, shear sucker or stone washed, can be imparted to silk fabrics by printing with sulphamic acid, shrinking agents such as calcium nitrate, or by first printing silk fabrics with polymers, printing with dyes, tucking them, crumpling in a rotary washer and finally drying in a tumbler [13].

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3.5. Finishing Muga silk being a noble fiber, apart from its color, is also valued for its sheen, feel and lustre. So care must be taken during its finishing so that its classic feel, scroopy handle and shimmering appearance are retained. Any mechanical and chemical finish that enhances these qualities is adopted for the finishing of muga silk. During finishing, muga silk is treated for obtaining the following functional properties to enhance its value which is required in niche market – Crease resistance Antistatic effect Spot resistance (water and oil drops) Flame retardancy Dimensional stability Wash and wear properties It is reported that after crease resistant finishing with silicone-containing epoxide, the silk’s dry and wet resilience greatly improves as well as it can withstand more than 20 washings. Many other types of new finishes based on UV absorbers, anti-microbial agents, anti-static agents and water as well as oil resistance finishes which have been developed for cotton and polyester can be successfully applied on muga silk. Some finishes have also been developed for silk fabrics to give a new look to the products such as stone washed, crumpled look or the peach finish, etc. So these finishes can also be effectively used for muga products, to achieve value addition.

3.6. Laudering Muga silk can be hand-washed and it has been popularly known that its lustre increases with every wash. Very often the silk outlives its owner. Saikia and Phukan [16] have studied the effect of repeated laundering on the physical properties of silk fabrics. Muga silk fabric demonstrated shrinkage after laundering. Abrasion resistance decreased with repeated laundering, indicating surface damage with loss of fiber from the yarn. The crease recovery of the sample was observed to improve after laundering and bending strength and flexural rigidity decreased, probably due to the removal of the surface finish [16].

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Subrata Das

MUGA PRODUCTS Muga has a very high traditional value in the culture of North Eastern India. Its products find places in the customary and traditional dresses used on the occasions of marriages, worship of God, etc. The key products of muga silks are mekhla (women’s lower wear designed with rows of flower diagram), chaddar (women’s upper wear designed with flower diagram on border, aachal and body) and saree, the traditional clothing made of muga silk. Fashion accessories like tie, scarf, kurta, shirt and coat are also woven from muga silk. Nowadays, muga threads are also being used in mulberry silk fabric in place of jari for producing some exclusive fabrics.

CONCLUSION The inherent natural golden color, durability and thermal properties are major factors influencing domestic and international creditability of muga silk, thereby enhancing its export potential. Focused research towards the augmentation of muga culture, technology improvement for better silk quality, increased production and product development will boost the muga silk sector. Muga silk should be processed with great care without weighing down its natural qualities. Hence, value added chemical processing would enhance the market acceptability of muga products. Scant and disseminated research in the muga silk arena has pointed towards the scope of its application in varied sectors in technical textiles.

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REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]

Brindroo, B.B. and R.K. Khatri, Feasibility of muga culture in Uttarakhand. Indian Silk, 2008. 47: p. 21-23. Sen, K. and K.M. Babu, Studies on Indian silk. I. macrocharacterization and analysis of amino acid composition. J Appl Polym Sci, 2004. 92(2): p. 1080-1097. Gupta, V.B., R. Rajkhowa, and V.K. Kothari, Physical characteristics and structure of Indian silk fibres. Ind J Fibre Text Res, 2000. 25(1): p. 14-19. Rajkhowa, R., V.B. Gupta, and V.K. Kothari, Tensile stress-strain and recovery behavior of Indian silk fibers and their structural dependence. J Appl Polym Sci, 2000. 77(11): p. 2418-2429. Sen, K. and M. Babu K, Studies on Indian silk. II. structure-property correlations. J Appl Polym Sci, 2004. 92(2): p. 1098-1115. Divakara, S., R. Somashekar, A. Raghu Yogesha, S. Ananthamurthy, and S. Roy, Correlation between microstructure and microrheological parameters of various silk fibres. Ind J Fibre Text Res, 2009. 34(2): p. 168-174. Bora, M.N., G.C. Baruah, and C.L. Talukdar, Investigation on the thermodynamical properties of some natural silk fibres with various physical methods. Thermochimica Acta, 1993. 218: p. 425-434. Mishra, S.N., Silk industry of Assam. Indian Silk, 1997: p. 22-26.

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Muga Silk [9] [10] [11] [12] [13] [14] [15]

Bajpeyi, C.M., N.V. Padaki, and S.N. Mishra, Review of silk handloom weaving in Assam. Textile Rev, 2010: p. 29-35. Ghosh, J. and R. Chakravorty, Muga cocoon dryer: An easy stifling alternative. Indian Silk, 2009: p. 24-30. Mishra, S.N., N.V. Padaki, D. Chattopadhyay, and R. Munshi, Training study material on "Reeling and spinning". 2009, Guwahati, India: RSTRS, Central Silk Board. Javali, U.C., S. Roy, and G.N. Ramaswamy, CSTRI teaser reeling cum twisting machine: Improvised for better performance. Indian Silk, 2009: p. 20-21. Gulrajani, M.L., Some recent developments in chemical processing of silk. Colourage Annu, 2004: p. 115-120. Sharma, H.M., A.R. Devi, and B.M. Sharma, Vegetable dyes used by the Meitei community of Manipur. Indian J Tradit Know, 2005. 4(1): p. 39-46. Somashekarappa, H., V. Annadurai, Sangappa, G. Subramanya, and R. Somashekar, Structure–property relation in varieties of acid dye processed silk fibers Mater Lett, 2002. 53(6): p. 415-420. Saikia, P. and A.R. Phukan, A study on some physical properties of silk after repeated laundering. Colourage, 1994. 41: p. 47-51.

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[16]

27

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In: Silk: Properties, Production and Uses Editor: Pornanong Aramwit

ISBN: 978-1-62100-692-3 © 2012 Nova Science Publishers, Inc.

Chapter 3

CONSERVATION AND DIVERSIFICATION OF SPIDER SILK: AN EVOLUTIONARY PERSPECTIVE Sara L. Goodacre1 School of Biology, University of Nottingham, UK

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ABSTRACT Analysis of the group of genes that encode spider silks shows that variants that code for proteins with highly divergent physical properties in fact share a common origin. The genetic signals of common ancestry are apparent in evolutionarily distant groups of species despite the divergence event estimated as having occurred as many as 350 million years ago. Analysis of the non-repetitive terminal ends of silk proteins shows that some elements of these are highly conserved across all silk types. These regions may be necessary for ubiquitous processes such as correct protein production but their presence in silks with divergent physical properties indicates that they are somehow decoupled from the process of determining the properties of the silk itself.

INTRODUCTION Spider silk has been the focus of a wide range of studies directed towards understanding its physical and chemical properties and the potential for biotechnological applications. In this 1

Sara Goodacre has been a RCUK Research Fellow at the School of Biology, University of Nottingham since 2006. Prior to this she was a Research Fellow at the University of East Anglia and at the University of Oxford for a year. She earned her Ph.D. at the University of Nottingham in 1999 where she has returned to further her professional career. Her work at Nottingham focusses on evolutionary, population and conservation genetic questions using spiders as model systems. Recently, work has begun on molecular genetic studies of spider silk focusing of a range of tarantula species. Other work includes studies of dispersal-strategy and the persistence of population differentiation in money spider meta-populations and studies of mating behaviour and sex ratio bias in tree dwelling spiders. E-mail address: [email protected]

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chapter we review the conservation and diversification of this gene family from an evolutionary perspective, focusing in particular upon the non-repetitive C and N termini. A fuller understanding of the properties of spider silk and how these can be manipulated can be achieved by taking an evolutionary standpoint and identifying the natural selective pressures that have driven the diversity observed in this group of proteins [1].

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EVOLUTIONARY DIVERSIFICATION OF SPIDERS AND THEIR SILK Spider silks are multimeric, modular fibers that are of considerable interest to biotechnologists because of their physical properties e.g. extreme extensibility and strength,which make them potentially useful in industrial applications. Spider silk is thought to have a different evolutionary origin to that produced by insects such as the silkworm Bombyx mori, and in its native form is stronger and more extensible than the silk of other arthropods. Most work in the field thus far has focused on silks produced by orb-weaving (araneoid) spiders, which come from the suborder Araneomorphae [2]. Less is known about the silks that are produced by spiders from the other major suborder, the Mygalomorphae. It seems likely that only a fraction of the molecular diversity that exists in silk proteins has been sampled to date and that our understanding of the physical properties of this group of potentially very useful proteins will benefit from taking a much broader view. The true spiders, (family: Araneae) diverged into the suborders Araneomorphae and Mygalo-morphae several million years ago. The divergence is estimated to be at least 240 million years ago based upon fossil evidence [3] and proposed to be as many as 390 million years ago when molecular dating methods are used [4]. Spiders within both suborders spin silk but the mygalomorphs are considered to possess more primitive characteristics than the araneomorphs both in terms of silk gland and spinneret morphology. Mygalomorphs extrude their silk through two pairs of spinnerets at the posterior end of their abdomen. Most species within the Araneomorphae extrude their silks directly though one of several pairs of spinnerets on their abdomen but some species have a structure known as a ‘cribellum’. This structure contains thousands of silk-extruding spiggots and acts as a spinning plate to produce large numbers of very fine silk strands that are combed out to produce a sheath of fibrils. The cribellum is believed to be the ancestral state within the Araneomorphae. It is believed to have arisen and been lost at least twice in this group of species, events that are proposed to have been accompanied by rapid changes in the number of species [5]. Mygalomorphs store their liquid silk within a single, undifferentiated silk gland whereas araneomorphs have many distinct silk glands, each of which is used to store one of up to 7 or 8 different types of silk. These include ampullate and flagelliform silks used for constructing webs, aciniform silks for wrapping prey and tubiliform silks for egg case construction. Mygalomorphs do not appear to be able to synthesize such a wide range of different silk types although very recent work indicates that some species may be able to extrude an additional silk from their feet [6, 7], an adaptation that is proposed to allow them to cling to vertical surfaces. The physical property of a spider’s silken structure is determined by several factors. The primary determinant is the chemical property of an individual silken fiber. This is governed both by the amino acid composition of the silk and by gene-environment interactions (such as

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Conservation and Diversification of Spider Silk

31

the rate of spinning, combing through a cribellum or by abiotic factors such as external humidity). A second determining factor is the combined properties of multiple silk types that are woven together to form a larger structure. Silks that are woven by spiders with a cribellum are extremely fine and achieve adhesiveness through van der Waals interactions and hydrophobic nodes in their protein sequence [8]. One remarkable property of cribellate silk is the ability for directional water collection through absorption onto the fibril sheath [9]. Silks spun by spiders without a cribellum (‘ecribellate’ silks) are typically thicker in diameter and coated with an aqueous silk protein that provides adhesiveness for prey capture [10]. All spider silks studied thus far consist of highly repetitive amino acid regions that are flanked by non-repetitive C and N-terminal domains. The N-terminus has a role in transport as it includes a signal peptide [11, 12]. It is also shown to be important in correct protein assembly through the formation of hydrogen bonds at alanine residues [13]. The function of the C-terminal domain remains uncertain, but there is a high degree of conservation at the amino acid level and it seems likely that it may have a role in protein transport, processing or both. It is present in the mature silk protein [14] and previous authors have proposed that it may be important in maintaining the aqueous state of silks prior to extrusion and in facilitating correct fiber formation [15-19]. Beckwitt & Arcidiacono [20] found the Cterminal sequence of spider silk to be highly conserved and proposed a further role in signaling within the cell. Roles in correct fiber formation and signaling are not incompatible and both could require sequence conservation. To date, most research has focused on the observed relationship between sequence motifs in the repeat region and the physical properties of the resulting silk strand [21]. This region typically contains poly-alanine and poly-(alanine-glycine) repeats that form β sheets. Interconnecting regions between the β-sheets act as nanosprings [22] that confer elasticity. Combinations of particular amino acids are associated with particular properties, for example GPGXX (where X is one of a small number of amino acids) appears to confer upon the fiber the ability to supercontract when wet. This feature thus far appears to be largely confined to the araneomorphs with supercontraction not observed to the same extent in mygalomorph species tested thus far [23].

CONSERVATION AND DIVERSIFICATION OF SILK GENES WITHIN THE ARANEAE The focus of most of the studies characterizing molecular and physical properties of silks thus far has been on spiders from the Araneomorphae where similarities among different silks suggest that they share a common ancestor [21]. The evolutionary relationships among functional homologues are unclear but it is thought likely that many of the genes in this family have evolved through duplication [20]. Determining the functional relationships is in itself not straightforward, being complicated by the existence of duplicate silk glands, spigots and spinnerets. Molecular reconstruction of evolutionary relationships amongst silk genes has largely been based upon studies of the non-repetitive C terminus because this region can be aligned reliably across species [24]. Sequence conservation in this region has been used to support the hypothesis that particular types of silk e.g. piriform [25] or aciniform [26] each likely has a single origin.

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Despite the evolutionary distance between the Araneomorphae and Mygalomorphae, recent work has identified a mygalomorph silk gene that appears to be homologous with the major ampullate silks [27]. Ampullate silk is used by spiders for constructing orb-webs and was previously thought to have evolved in the araneomorph lineage after the split with the mygalomorphs. The finding of a mygalomorph ampullate silk, however, indicates that an ancestral ampullate-like silk must predate the origin of the araneomorph clade. Recent studies of N and C termini of mygalomorph silks also suggests that they share a high degree of homology with their araneomorph counterparts [28-31] and therefore that these too must have their origins before the araneomorph/mygalomorph split. Together these findings imply that amino acid sequence conservation has been maintained over several hundred(s) of millions of years. Such extreme conservation of amino acid sequences appears to be a common feature throughout the silk gene family. As an illustrative example, the tubiliform repetitive units of orb-weaving spiders, a subgroup within the Araneomorphae, appear conserved in species that are believed to have diverged more than 125 million years ago [32].

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MODES OF EVOLUTION: EVIDENCE FOR DUPLICATION, RECOMBINATION AND DIVERSIFYING SELECTION The observation that many silks cluster according to type rather than according to species [25, 26, 29] suggests that their evolution involves a ‘birth and death’ process where gene duplication is followed by loss of function. Under such a process, genes are expected to cluster by gene or duplication order rather than by species, low levels of sequence homogeneity are expected between different genes (particularly at non-coding sites) and there will be evidence of gene loss or pseudo-gene formation. Consistent with this hypothesis is the recent finding of multiple copies of major ampullate type 1 (MaSp1) silks within a conserved gene cluster (estimated to be within an approximately 2-3 Mb segment) and a single copy of a second type of ampullate silk, MaSp2, at a separate location in two species of widow spider, Latrodectus hesperus and L. geometricus [33]. One of the MaSp1 copies was found to be a pseudogene, a finding that is consistent with a loss of function post duplication. The alternative hypothesis is that the dominant force is the homogenizing effect of recombination post-duplication. Whilst the ‘birth and death’ process described above likely involves recombination to create multiple closely-related copies, the distinction between this and the second hypothesis is that in the latter duplicated copies do not subsequently diverge independently from one another but are made similar through repeated recombination events. Under this scenario silks from within individual species are expected to be genetically similar to one another and to cluster together in phylogenetic analyses. This second hypothesis seems less likely to fully explain the observed data given that silks generally do not cluster according to species. However, there is evidence that recombination has contributed to the genetic diversity observed in this gene family, albeit not at a sufficient rate to completely prevent divergence of duplicated genes or gene regions. For example, studies of complete gene sequences or combinations of N and C termini have indicated a high – but not perfect degree of congruence between phylogenetic relationships estimated using the two different ends of the protein [29], an incongruence that is most simply explained by a recombination event.

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Conservation and Diversification of Spider Silk

33

The mechanism through which entire silk genes are duplicated is not known but silks contain many highly repetitive regions that are candidates for slippage during the process of genome replication, thus leading to replication of regions of the gene. Unequal recombination where similar, but non-homologous, regions align before cross over at meiosis, may further alter the number of such repeats. Evidence that such slippage occurs comes from identification of intra-individual variation in species such as Nephila clavipes [34] but this is proposed not to spread within a population because extreme length variants are removed by natural selection. A similar finding of evidence consistent with replication slippage followed by unequal recombination has been made in araneomorph tubiliform silks [32]. Studies of short sections of the C terminus of silks identified from a range of species found no evidence to suggest that the ends of ampullate silks were themselves recombinant [24], a finding that was not unexpected given the short length of sequence involved. Estimates were subsequently made of the non-synonymous: synonymous (dn/ds) substitution ratio, ω, for different amino acid positions within this non-recombining section. ω is expected to be less than 1 for most amino acids because selection acts to remove most non-synonymous substitutions. However in some cases selection against substitution may be relaxed (or even zero, in which case ω = 1). In the presence of positive (diversifying) selection ω is expected to significantly exceed 1. The results indicated that some amino acid sites within the major ampullate C terminus had a significant likelihood that ω>1 i.e. they were changing faster than expected even if there were no selective constraint on amino acid substitution. The rate of amino acid substitutions was therefore proposed in part to be driven by positive selection [24]. Since that study many more silk terminal sequences have become available. All have recognizable amino acid motifs that identify them as belonging to a silk gene, but one notable feature is that they are all extremely difficult to align at the DNA sequence level unless guided by the corresponding amino acid alignment. Insertions and deletions, which are common features within the region, further complicate accurate alignment and in practice, reliable alignment at the nucleotide level can only be made for closely related species. One such amino acid alignment is shown below for Latrodectus tubiliform silk N termini from L. hesperus, L. mactans, L. tredecimguttatus and L. geometricus that were published by Garb and Hayashi in 2005 [32] (genbank accession numbers are shown). 23 amino acid residue positions where at least one sequence differs from the reference, L. hesperus, are indicated. A test for recombination made (this study) using the DSS (difference in sum of squares) approach as implemented in the program TOPALI [35] indicates that there is no evidence for recombination having occurred within these 4 sequences. Estimates of ω, the parameter describing non-synonymous/synonymous (dn/ds) amino acid substitution ratios, are legitimate in the absence of recombination, which would otherwise obscure the evolutionary relationships by reducing linkage disequilibrium. Estimates of ω were made (this study) by maximum likelihood using the program codeml in the software package PAML [36] with three pairs of nested models are shown in table 1. In each case, we have compared the likelihoods of observing the actual data set under two models [37]. In the first model of each pair (M0, 1a, 7) we assumed that ω could not exceed 1. In the second model for each paired comparison (M3, M2a, M8) we allowed ω to exceed 1. Model M0 assumes all sites have the same ω and is compared with model M3 where one category of ω can exceed 1.

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Sara L. Goodacre

10 20 30 40 50 60 70 80 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..

AY953076 AY953077 AY953078 AY953079

L.hesperus L.mactans L.tredecimguttatus L.geometricus

FSSASSASAVGQVGYQIGLNAAQTLGISNAPAFADAVSQAVRTVGVGASPFQYANAVSNAFGQLLGEQGILTQENASSLASSVANAL .......................................S............................................... .Q.........................A...X.........................T..........................D.. ........S..............S...A......................................G..............A.....

110 120 130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..

AY953076 AY953077 AY953078 AY953079

L.hesperus L.mactans L.tredecimguttatus L.geometricus

GVPGLIVGPSIVSSLNAPIAGFAVPGVAQVIVPTAYSTLLAPVLSPAGLASTAATSRINDIAQSLSSTLSSGSQLAPDNVLPGLXQL ....................................................................................I.. ..........................E........F...................L.......G....I......T....I...I.. .....N...T.I..VS..F....T..LG...I..T..A................S....G............T........S..F.. 210 220 ....|....|....|....|....|

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AY953076 AY953077 AY953078 AY953079

L.hesperus L.mactans L.tredecimguttatus L.geometricus

AGVLIESLLEYTSALLALLQNAQIT ......................... ........F................ S.......F..............V.

Similarly, model M1a does not allow ω>1 and is compared with M2a where one category of ω can exceed 1. M7 assumes a continuous beta distribution of ω across sites, where the distribution can take a variety of shapes but where ω≤1. This is compared with M8, which assumes the same, continuous beta distribution of ω as M7, but with the addition of one extra site class that has a free ω ratio estimated from the data where ω can be greater than 1. Twice the difference between the likelihoods of these nested models is compared with the χ2 distribution in order to see whether we are significantly more likely to observe the actual data if we assume a model where ω can be >1.The power of taking the approach described above is that it gives the potential to detect a small number of sites for which ω > 1 in a region where the majority of sites are highly conserved. In the case of Latrodectus tubiliform silk we find that all models that allow ω to exceed 1 have higher likelihood scores than similar models where ω is constrained to be ≤1. The difference is statistically significant for a comparison involving one pair of models (M7 vs. M8, P1)

-1310.21

p(3)

dn/ds (1)

dn/ds (2)

dn/ds (3)

0.17

4.17

0.09

0.13

1

0.01

0.17

1

P

Q

-2x diff log l

Sig

22.46

8.42

NS

22.46

5.78

NS

6.25

2 weeks) in vivo from films. These results indicated that silk sericin is useful as an aqueous sustained-release material for large molecular weight drugs and the sustained-release will be dominant if the drugs are charged. Silk sericin with other polymers such as poloxamer has formed nanoparticles and has been used as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery [130]. Mandal and Kundu successfully loaded inulin-FITC (hydrophilic drug) and paclitaxel (hydrophobic drugs) in silk sericin/poloxamer nanoparticles, making them quite unique, being called universal drug carriers. The fabricated silk sericin/poloxamer nanoparticles are not only stable in aqueous solution and smaller in size but are also rapidly taken up by cells to achieve faster and prolonged delivery of drugs to the target site. Cytotoxicity assessment using MCF7, a breast cancer cell line, shows both efficiency and efficacy of these drug-loaded nanoparticles when compared with a free drug. Annexin V staining and Western blot analysis

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further confirm the induction of apoptosis and cell death. This data indicated a new aspect of silk sericin in the form of nanoparticles for its efficient and effective use as drug delivery vehicles in biomedical and tissue engineering applications.

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SPIDER SILK FIBROIN FOR DRUG DELIVERY The most widely studied spider silk in terms of structure and function is dragline silk from the spider Nephila clavipes [131, 132]. The dragline silk is secreted as a mixture of two proteins from specialized columnar epithelial cells of the major ampullate gland of orbweaver spinning spiders [131]. The molecular weight of these proteins range from 70 to 700 kDa depending on source. These silk proteins are characterized as block copolymer, composed of large hydrophobic blocks with highly conserved repetitive sequences consisting of short side-chain amino acids, such as glycine and alanine, with intervening small hydrophilic blocks with more complex sequences that consist of amino acids with bulkier side-chain and charged amino acids [25, 133]. Spider silk-based block copolymers have been designed via genetic engineering and used for the delivery of bioactive molecules, such as gene and drugs. In particular, recombinant silk proteins containing ligand molecules, selective delivery to target cells have been generated. For cancer treatment, spider silk fiber can be used as a drug delivery system by using nanoparticles or polyplexes targeting tumor cells containing tumor-homing peptides [23]. Other forms of target delivery systems using spider silk fibroin protein such as injectable hydrogels or implant materials to release drugs or genes are also investigated. The outstanding properties of spider silk fibers can be explained by the hierarchical and sophisticated composition of structural elements of two proteins which are ADF3 (Araneus diadematus fibroin) which is relatively hydrophilic and ADF4 which is relatively hydrophobic [134], their crystalline regions of different dimensions being embedded in an amorphous or pre-oriented matrix. The amorphous matrix is likely to be responsible for the extensibility of dragline silk while crystalline regions are responsible for their strength [135, 136]. The extent and type of structural features depend on the amino acid sequence of the underlying proteins (primary structure). Prolong released and constant drug levels during therapy were also designed using recombinant spider silk particles [137]. Due to the drawback of rapid re-solubilization in aqueous environments of natural polymers since they are often hydrophilic resulting in fast drug release profiles [138], natural hydrophobic biopolymer can be applied to diminish that problem [139]. Recombinantly produced engineered spider silk protein eADF4(C16) mimicking the known amino acid sequence of the natural spider silk protein ADF4 from the European garden spider Araneus diadematus was used [137]. The microspheres which have a smooth surface and are solid with high -sheet content of this recombinant spider silk can be formed easily by adding high concentrations of lyotropic salts which can be used for sustained controlled delivery of positively charged and sufficiently hydrophobic drug molecules. The mechanisms of drug release from spider silk particles are as followed: In the first step, drug molecules are attracted to a silk particle by electrostatic forces. After particle surface saturation, low molecular weight drugs start to diffuse into the biopolymer matrix. Drug molecules are bonded by attractive hydrophobic and electrostatic interactions. After

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complete loading and incubation in release media, drug molecules are transported to the particle surface due to concentration gradient driven transport processes. In time, drug molecules are slowly released from the surface leading to constant release rates. The results indicated that engineered spider silk particles have the potential for diverse applications where controlled release from mechanically tough and slowly biodegradable carriers is desirable. In contrast to particles which can be used as mobile carriers, films are more suitable for applications in which a stationary phase is needed. Recombinantly produced engineered spider silk proteins can be assembled into transparent and stable films. Control of material properties can be performed through the molecular structure of spider silk proteins. The solvent used such as hexafluoro-2-propanol or formic acid will determine the predominant secondary structure in the as-cast films. Spider silk films prepared from aqueous solution mainly consist of random coil structure [140, 141] whereas films prepared using hexafluoro2-propanol as the solvent typically yield -helix-rich structures [140, 142]. In contrast, films prepared from formic acid solutions are rich in -sheet structure [141, 143]. These structural conformations of the proteins determine the chemical stability and mechanical characteristics of this film which may affect its uses in biomedical applications.

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CONCLUSION Silk protein, both fibroin and sericin, can be successfully applied in controlled or targeted drug delivery. Silk fibroin has been widely investigated for its use in this area. Several forms of silk fibroin with or without any excipients has shown the sustained-release of drug. It has also been applied for gene or protein drug delivery. Further molecular modification of silk fibroin can change their physical and biological properties and make it suitable for different drug properties. Silk sericin formed as film, scaffold or nanoparticle exhibits similar property for prolonged-release of drugs. Since silk proteins are biocompatible, biodegradable and contain easily modified structure, they are good candidates as drug delivery materials.

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[91] Dinauer, N., S. Balthasar, C. Weber, J. Kreuter, K. Langer, and H. von Briesen, Selective targeting of antibody-conjugated nanoparticles to leukemic cells and primary T-lymphocytes. Biomaterials, 2005. 26(29): p. 5898-906. [92] Gupta, V., A. Aseh, C.N. Rios, B.B. Aggarwal, and A.B. Mathur, Fabrication and characterization of silk fibroin-derived curcumin nanoparticles for cancer therapy. Int J Nanomedicine, 2009. 4: p. 115-22. [93] Dinerman, A.A., J. Cappello, H. Ghandehari, and S.W. Hoag, Solute diffusion in genetically engineered silk-elastinlike protein polymer hydrogels. J Control Release, 2002. 82(2-3): p. 277-87. [94] Yoo, M.K., H.Y. Kweon, K.G. Lee, H.C. Lee, and C.S. Cho, Preparation of semiinterpenetrating polymer networks composed of silk fibroin and poloxamer macromer. Int J Biol Macromol, 2004. 34(4): p. 263-70. [95] Gil, E.S., D.J. Frankowski, R.J. Spontak, and S.M. Hudson, Swelling behavior and morphological evolution of mixed gelatin/silk fibroin hydrogels. Biomacromolecules, 2005. 6(6): p. 3079-87. [96] Yin, L., J. Ding, L. Fei, M. He, F. Cui, C. Tang, and C. Yin, Beneficial properties for insulin absorption using superporous hydrogel containing interpenetrating polymer network as oral delivery vehicles. Int J Pharm, 2008. 350(1-2): p. 220-9. [97] Mandal, B.B. and S.C. Kundu, Calcium alginate beads embedded in silk fibroin as 3D dual drug releasing scaffolds. Biomaterials, 2009. 30(28): p. 5170-7. [98] Datta, A., A.K. Ghosh, and S.C. Kundu, Purification and characterization of fibroin protein from tropical Saturniid silkworm, Antheraea mylitta. Insect Biochem Mol Biol, 2001. 31: p. 1013-1018. [99] Okhawilai, M., R. Rangkupan, S. Kanokpanont, and S. Damrongsakkul, Preparation of Thai silk fibroin/gelatin electrospun fiber mats for controlled release applications. Int J Biol Macromol, 2010. 46(5): p. 544-50. [100] Wang, X., T. Yucel, Q. Lu, X. Hu, and D.L. Kaplan, Silk nanospheres and microspheres from silk/pva blend films for drug delivery. Biomaterials, 2010. 31(6): p. 1025-35. [101] Kwon, T.K. and J.C. Kim, Complex coacervation-controlled release from monoolein cubic phase containing silk fibroin and alginate. Biomacromolecules, 2011. 12(2): p. 466-71. [102] Caboi, F., G.S. Amico, P. Pitzalis, M. Monduzzi, T. Nylander, and K. Larsson, Addition of hydrophilic and lipophilic compounds of biological relevance to the monoolein/water system. I. Phase behavior. Chem Phys Lipids, 2001. 109(1): p. 47-62. [103] Wasungu, L. and D. Hoekstra, Cationic lipids, lipoplexes and intracellular delivery of genes. J Control Release, 2006. 116(2): p. 255-64. [104] Worle, G., B. Siekmann, M.H. Koch, and H. Bunjes, Transformation of vesicular into cubic nanoparticles by autoclaving of aqueous monoolein/poloxamer dispersions. Eur J Pharm Sci, 2006. 27(1): p. 44-53. [105] Kuntsche, J., H. Bunjes, A. Fahr, S. Pappinen, S. Ronkko, M. Suhonen, and A. Urtti, Interaction of lipid nanoparticles with human epidermis and an organotypic cell culture model. Int J Pharm, 2008. 354(1-2): p. 180-95. [106] Kim, J.C., K.U. Lee, W.C. Shin, H.Y. Lee, J.D. Kim, Y.C. Kim, G. Tae, K.Y. Lee, and S.J. Lee, Monoolein cubic phases containing hydrogen peroxide. Colloids Surf B Biointerfaces, 2004. 36(3-4): p. 161-6.

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[107] Lopes, L.B., J.L. Lopes, D.C. Oliveira, J.A. Thomazini, M.T. Garcia, M.C. Fantini, J.H. Collett, and M.V. Bentley, Liquid crystalline phases of monoolein and water for topical delivery of cyclosporin A: characterization and study of in vitro and in vivo delivery. Eur J Pharm Biopharm, 2006. 63(2): p. 146-55. [108] Drummond, C.J. and C. Fong, Surfactant self-assembly objects as novel drug delivery vehicles. Curr Opin Colloid Interface Sci, 2000. 4(6): p. 449-456. [109] Choi, J.H., H.Y. Lee, J.C. Kim, and Y.C. Kinm, Monoolein cubic phases containing hydrophobically modified poly (N-isopropylacrylamide). J Ind Eng Chem, 2007. 13(3): p. 380-386. [110] Kwon, T.K. and J.C. Kim, Monoolein cubic phase containing acidic proteinoid: pHdependent release. Drug Dev Ind Pharm, 2011. 37(1): p. 56-61. [111] Benfenati, V., S. Toffanin, R. Capelli, L.M. Camassa, S. Ferroni, D.L. Kaplan, F.G. Omenetto, M. Muccini, and R. Zamboni, A silk platform that enables electrophysiology and targeted drug delivery in brain astroglial cells. Biomaterials, 2010. 31(31): p. 7883-91. [112] Wang, X., E. Wenk, X. Hu, G.R. Castro, L. Meinel, C. Li, H. Merkle, and D.L. Kaplan, Silk coatings on PLGA and alginate microspheres for protein delivery. Biomaterials, 2007. 28(28): p. 4161-9. [113] Piggee, C., Therapeutic antibodies coming through the pipeline. Anal Chem, 2008. 80(7): p. 2305-10. [114] Werner, R.G., Economic aspects of commercial manufacture of biopharmaceuticals. J Biotechnol, 2004. 113(1-3): p. 171-82. [115] Oh, K.S., S.K. Han, H.S. Lee, H.M. Koo, R.S. Kim, K.E. Lee, S.S. Han, S.H. Cho, and S.H. Yuk, Core/Shell nanoparticles with lecithin lipid cores for protein delivery. Biomacromolecules, 2006. 7(8): p. 2362-7. [116] Giteau, A., M.C. Venier-Julienne, A. Aubert-Pouessel, and J.P. Benoit, How to achieve sustained and complete protein release from PLGA-based microparticles? Int J Pharm, 2008. 350(1-2): p. 14-26. [117] Sinha, V.R. and A. Trehan, Biodegradable microspheres for protein delivery. J Control Release, 2003. 90(3): p. 261-80. [118] Guziewicz, N., A. Best, B. Perez-Ramirez, and D.L. Kaplan, Lyophilized silk fibroin hydrogels for the sustained local delivery of therapeutic monoclonal antibodies. Biomaterials, 2011. 32(10): p. 2642-50. [119] Gobin, A.S., R. Rhea, R.A. Newman, and A.B. Mathur, Silk-fibroin-coated liposomes for long-term and targeted drug delivery. Int J Nanomedicine, 2006. 1(1): p. 81-7. [120] Cheema, S.K., A.S. Gobin, R. Rhea, G. Lopez-Berestein, R.A. Newman, and A.B. Mathur, Silk fibroin mediated delivery of liposomal emodin to breast cancer cells. Int J Pharm, 2007. 341(1-2): p. 221-9. [121] Breitenkamp, R.B. and T. Emrick, Pentalysine-grafted ROMP polymers for DNA complexation and delivery. Biomacromolecules, 2008. 9(9): p. 2495-500. [122] Oupicky, D., C. Konak, P.R. Dash, L.W. Seymour, and K. Ulbrich, Effect of albumin and polyanion on the structure of DNA complexes with polycation containing hydrophilic nonionic block. Bioconjug Chem, 1999. 10(5): p. 764-72. [123] Ogris, M., S. Brunner, S. Schuller, R. Kircheis, and E. Wagner, PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended

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circulation in blood and potential for systemic gene delivery. Gene Ther, 1999. 6(4): p. 595-605. [124] Megeed, Z., M. Haider, D. Li, B.W. O'Malley, Jr., J. Cappello, and H. Ghandehari, In vitro and in vivo evaluation of recombinant silk-elastinlike hydrogels for cancer gene therapy. J Control Release, 2004. 94(2-3): p. 433-45. [125] Cappello, J., J. Crissman, M. Dorman, M. Mikolajczak, G. Textor, M. Marquet, and F. Ferrari, Genetic engineering of structural protein polymers. Biotechnol Prog, 1990. 6(3): p. 198-202. [126] Numata, K., B. Subramanian, H.A. Currie, and D.L. Kaplan, Bioengineered silk protein-based gene delivery systems. Biomaterials, 2009. 30(29): p. 5775-84. [127] Numata, K., J. Hamasaki, B. Subramanian, and D.L. Kaplan, Gene delivery mediated by recombinant silk proteins containing cationic and cell binding motifs. J Control Release, 2010. 146(1): p. 136-43. [128] Nishida, A., M. Yamada, T. Kanazawa, Y. Takashima, K. Ouchi, and H. Okada, Use of silk protein, sericin, as a sustained-release material in the form of a gel, sponge and film. Chem Pharm Bull (Tokyo), 2010. 58(11): p. 1480-6. [129] Nishida, A., T. Naganuma, T. Kanazawa, Y. Takashima, M. Yamada, and H. Okada, The characterization of protein release from sericin film in the presence of an enzyme: Towards fibroblast growth factor-2 delivery. Int J Pharm, 2011. [130] Mandal, B.B. and S.C. Kundu, Self-assembled silk sericin/poloxamer nanoparticles as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery. Nanotechnology, 2009. 20(35): p. 355101. [131] Vollrath, F. and D.P. Knight, Liquid crystalline spinning of spider silk. Nature, 2001. 410(6828): p. 541-8. [132] Jin, H.J. and D.L. Kaplan, Mechanism of silk processing in insects and spiders. Nature, 2003. 424(6952): p. 1057-61. [133] Winkler, S., D. Wilson, and D.L. Kaplan, Controlling beta-sheet assembly in genetically engineered silk by enzymatic phosphorylation/dephosphorylation. Biochemistry, 2000. 39(41): p. 12739-46. [134] Huemmerich, D., C.W. Helsen, S. Quedzuweit, J. Oschmann, R. Rudolph, and T. Scheibel, Primary structure elements of spider dragline silks and their contribution to protein solubility. Biochemistry, 2004. 43(42): p. 13604-12. [135] Hayashi, C.Y., N.H. Shipley, and R.V. Lewis, Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins. Int J Biol Macromol, 1999. 24(2-3): p. 271-5. [136] Gosline, J.M., P.A. Guerette, C.S. Ortlepp, and K.N. Savage, The mechanical design of spider silks: from fibroin sequence to mechanical function. J Exp Biol, 1999. 202(Pt 23): p. 3295-303. [137] Lammel, A., M. Schwab, M. Hofer, G. Winter, and T. Scheibel, Recombinant spider silk particles as drug delivery vehicles. Biomaterials, 2011. 32(8): p. 2233-40. [138] Soppimath, K.S., T.M. Aminabhavi, A.R. Kulkarni, and W.E. Rudzinski, Biodegradable polymeric nanoparticles as drug delivery devices. J Control Release, 2001. 70(1-2): p. 1-20. [139] Liu, X., Q. Sun, H. Wang, L. Zhang, and J.Y. Wang, Microspheres of corn protein, zein, for an ivermectin drug delivery system. Biomaterials, 2005. 26(1): p. 109-15.

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[140] Zhao, C., J. Yao, H. Masuda, R. Kishore, and T. Asakura, Structural characterization and artificial fiber formation of Bombyx mori silk fibroin in hexafluoro-iso-propanol solvent system. Biopolymers, 2003. 69(2): p. 253-9. [141] Vasconcelos, A., G. Freddi, and A. Cavaco-Paulo, Biodegradable materials based on silk fibroin and keratin. Biomacromolecules, 2008. 9(4): p. 1299-305. [142] Stephens, J.S., S.R. Fahnestock, R.S. Farmer, K.L. Kiick, D.B. Chase, and J.F. Rabolt, Effects of electrospinning and solution casting protocols on the secondary structure of a genetically engineered dragline spider silk analogue investigated via Fourier transform Raman spectroscopy. Biomacromolecules, 2005. 6(3): p. 1405-13. [143] Um, I.C., H.Y. Kweon, Y.H. Park, and S. Hudson, Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid. Int J Biol Macromol, 2001. 29(2): p. 91-7.

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Chapter 13

APPLICATION OF SILK SERICIN FOR THE CONTROLLED RELEASE AND TARGET-SPECIFIC DELIVERY OF BIOLOGICAL SUBSTANCES Ayumu Nishida* Pharmaceutical Technology, R&D, Kissei Pharmaceutical Co., Ltd. Nagano, Japan

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ABSTRACT Several biocompatible and biodegradable drug-release materials have been developed. Some of these materials, including synthetic polymers such as poly (lactic-coglycolic acid) (PLGA), natural polymers such as gelatin and chitosan, and combinations of these, are currently in medical use. Although these materials have excellent characteristics as biomaterials, their application has been limited due to low compatibility with certain drugs, the need to use large amounts of organic solvents or toxic crosslinkers during their preparation, and problems arising from bovine spongiform encephalopathy. There is therefore a need for new biocompatible and biodegradable drug-release materials. This chapter reviews the utility of silk sericin (SS) as an aqueous biomaterial for the controlled release and target-specific delivery of biologics.

*

Ayumu Nishida has worked in the Pharmaceutical Technology Research and Development Department at KISSEI Pharmaceutical Co., Ltd. in Nagano, Japan since 1995. He earned his degree from the Faculty of Agriculute in 1993 and his Master’s from the Graduate School of Agriculture at Shinshu University in Nagano, Japan in 1995. He went on to postgraduate work at the Graduate School of Pharmacy at the University of Pharmacy and Life Sciences in Tokyo, Japan. He continued his studies there and earned his Ph.D. in Pharmacy. In the last three years he has written publications on the properties of silk and their uses in the medical field. E-mail address: [email protected]

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INTRODUCTION Drug delivery systems (DDS) are used to ensure that drugs enter the body and reach the site where they are needed. DDS affect drug-release profiles, absorption, distribution, metabolism, and excretion, and thus affect both efficacy and drug safety. Several bio-compatible and biodegradable drug controlled release materials have been developed to date. Some materials, including synthetic polymers such as PLGA, natural polymers such as gelatin and chitosan, and combinations of such polymers [1-5], are already in use for medical purposes. It has been demonstrated that, through poly-ion complexation, gelatin hydrogels bind fibroblast growth factor [6], and cationized gelatin hydrogels bind plasmid DNA and epidermal growth factor [2, 7]. These gels sustain protein drug-release until the gels are degraded. Ionic interactions between a charged protein drug and charged drug-release material are useful in a drug delivery system. However, although such materials have several excellent characteristics as biomaterials, their application in DDS has been limited due to their low compatibility with certain drugs, the need to use large amounts of organic solvents or toxic cross-linker during their preparation, and problems arising from bovine spongiform encephalopathy. Therefore, the development of better drug-release materials with the required degree of biocompatibility is necessary. Silk sericin (SS) is a component of silk protein in the silkworm cocoon. Silk protein consists of fibroin as the fiber and SS as the glue. SS constitutes 25-30% of the silkworm (Bombyx mori) cocoon [8]. SS is composed of 18 different amino acids, and contains a high number of polar side chains with hydroxyl, carboxyl, and amino groups [8]. Isolation and characterization of SS components from the cocoon of Bombyx mori showed that SS primarily consists of three polypeptides with molecular weights of 150, 180-250, and 400 kDa [9, 10]. Analysis by phenylthiocarbamyl (PTC) amino acids method showed that the amino acid compositions of all three SS polypeptides are high in serine (Ser, 33.2-39.0%), glycine (Gly, 14.1-16.0%) and aspartic acid/asparagine (Asp/Asn, 11.3-15.7%) [9]. The structural analysis and cloning of the SS genes, Ser1 and Ser2 (Src-2), have been described [11-13]. Correspondence of the amino acid composition of SS with these genes suggested that the 150 and 400 kDa SSs correspond to Ser1 proteins (77-331 kDa) encoded by the Ser1 gene, and that the 250 kDa SS corresponds to S-2 protein (227 kDa) encoded by the Src-2 gene [9]. The most abundant component is the largest SS (400 kDa), which corresponds to the Ser1C protein (331 kDa) [9]. A repetitive 38-amino acid sequence rich in Ser (40%) dominates a large part of the Ser1C protein, and is predicted to have a strong tendency to form a β-sheet structure. Another part of the Ser1C protein is hydrophilic and has a high content of charged residues including acidic (glutamic acid (Glu) and Asp) and basic (lysine (Lys) and arginine (Arg)) amino acids [14]. In contrast, the 250 kDa SS polypeptide, which corresponds to the S2 protein (227 kDa), has less β-sheet forming propensity and higher hydrophilicity than the 150 and 400 kDa polypeptides [9]. Thus, SS is expected to form β-sheet matrices and bind charged proteins through its polar side chains. This chapter reviews the utility of SS as an aqueous biomaterial for DDS. SS has been prepared in the form of solutions, gels, creams, porous matrices, films, and micro- and nanospheres for uses such as drug-release dressing films, ointment bases, injectable gel and particulate preparations, and implants.

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SILK SERICIN AS A BIOLOGICAL SUBSTANCES

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During pretreatment of silk prior to spinning, SS is degraded into small molecular weight fragments and is removed as a waste product through a silk scouring process involving alkaline treatment with high-pressure steam. However, SS hydrolysate is reported to have various useful functions, for example as a cell attachment enhancer [15, 16], moisturizer [8], UV-resistant agent [17], antioxidant agent [18, 19], and enzyme carrier [20]. Moreover, previous studies reported improved oxygen permeability upon increasing the SS content of a poly (vinyl alcohol) (PVA) film [21], and effective induction of hydroxyapatite nucleation by a SS film in a biomimetic solution [22]. Recently, a serum-free medium using SS hydrolysate was developed with characteristics superior to commercial serum-free media for cell culture [23]. Miyamoto et al. [24] also reported that SS (average molecular weight: 30 kDa) is useful for cryopreservation of human adipose tissue derived stem/progenitor cells. Furthermore, consumption of SS suppressed colon tumorigenesis in 1,2-dimethylhydrazine-treated mice by reducing oxidative stress and cell proliferation [25, 26]. In a topical application, SS suppressed 7,12-dimethylbenz(a)anthracene (DMBA)/12-O- tetradecanoyl phorbol-13-acetate (TPA) -induced mouse skin tumorigenesis by reducing oxidative stress, inflammatory responses, and endogenous tumor promoter tumor necrosis factor-alpha (TNF-α) [27]. Taken together, these reports indicate that SS holds promise as a biologic for medical purposes. Recent studies using transgenic silkworm strains have resulted in the expression of recombinant proteins such as enhanced green fluorescent protein and human serum albumin, which were secreted in the SS layer of the cocoon [28, 29], and the expression of a cell attachment factor in the cocoon useful in the development of biocompatible and biodegradable artificial blood vessels [30]. As research on silk protein progresses, new directions in sericulture are expected. One of these directions is the use of SS as a biomaterial for DDS.

TECHNIQUES FOR PREPARING SERICIN-BASED DRUG DELIVERY DEVICES 1. Solutions To prepare various bio-pharmaceutical dosage forms, solubilization of raw materials is indispensable process. And aqueous process without organic solvent is one of advantages in developing drug products due to environmental care, recommended use of less toxic solvents to protect the safety of the patients, aseptic filtration and cost reduction. In a silk scouring process involving alkaline treatment with high-pressure steam, SS is hydrolyzed into small molecular weight fragments and is removed. SS hydrolysate is water-soluble. The molecular weight of SS tended to decrease as heating temperature and heating time increased [31, 32]. On the other hand, intact SS of cocoon exhibits insoluble property in water at room temperature because of taking a β-sheet in its protein secondly structure. Intact or slightlydegraded SS containing high molecular weight fragments (70-400 kDa) is obtained by dissolving SS in 8 M lithium bromide (LiBr) [10], 8 M lithium chloride (LiCl) [31], saturated lithium thiocyanate (LiSCN) [9], 8 M urea solution [33], or by heating for tens of minutes in

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water at above 85‐110 °C [31, 34, 35]. Recently, the physical and biological properties of SS solutions dissolved by various extraction methods were studied [36]. SS extracted by a high temperature and high-pressure degumming technique, citric acid, sodium carbonate, or urea solution, had different molecular weights (10 to > 225 kDa), mean particle size (4.62 to 824.42 nm), zeta potential (-68.36 to -15.87 mV) and amino acid content. It was speculated that urea-extracted SS had the smallest particle size may be in soluble form while SS from other methods had larger particle size may be present as hydrocolloids. And it was found that heat-degraded SS was the least toxic to cells and activated collagen production, while ureaextracted SS showed the lowest cell viability and collagen production [36]. It is important to regulate the physicochemical properties of SS solution to make SS preparations for DDS.

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2. Gels Gels have been used as drug delivery carriers for topical and oral applications, and for injection. Kundu et al. [37] reviewed the use of intact SS to prepare stable matrices, including hydrogels, porous materials, and films, all with good properties. A moldable gel has been prepared by applying an aging process to a SS solution. Zhu et al. [35] reported that the rate of gelation of a SS solution depended on the concentration of SS, and was fastest at 25 to 40 °C and pH 6.0 to 7.0. The mechanical strength of the SS gel increased and the surface tension decreased during gelation [35]. The gelation of SS molecules arises from a conformational change from random coil to β-sheet structure, and is facilitated by the formation of a three-dimensional network structure in the SS gel [10, 35]. The compressive strength of SS gel depends on the molecular weight of SS [32]. Teramoto et al. [10] reported that SS solution containing intact SS forms elastic hydrogels upon the addition of ethanol. This SS hydrogel can be prepared without the use of cross-linking chemicals or irradiation, and thus could be used as a naturally occurring biomaterial. The SS gel film was prepared by gelling SS solution with ethanol into a sheet, then drying [38]. Infrared analysis revealed that the SS gel film contained water-stable β-sheet networks formed during the gelation step, making the gel film morphologically stable towards swelling and giving it good handling properties in the wet state. The SS gel film rapidly absorbed water, equilibrating at about 80% water.

3. Cream A cream base is a useful carrier for transdermal drug delivery. Aramwit and Sangcakul [39] evaluated the usefulness of SS cream on wound healing in rats. To prepare the SS cream, SS powder was dissolved in warm water and then mixed with white petrolatum, mineral oil, lanolin, glycerin, bisabolol, propylparaben, and ethylparaben. In another study, gel formed from a SS solution was hydrolyzed by autoclaving at 110 °C for 10 min; the product was easily emulsified by mixing with oil to provide a firm cream [31]. On the other hand, foam mass was also created by mixing with oil before gelation. These processed SS formulations could be useful for use as a natural topical dosage form free of artificial additives.

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4. Porous Matrices Freeze drying is a useful approach for generating porous drug carriers. For the preparation of biological implants, many different scaffolds based on synthetic polymers such as PLGA, natural polymers such as gelatin or silk fibroin, or a combination of polymers, have been prepared by freeze drying methods [5, 6, 40-43]. Several studies have shown that scaffolds incorporating a drug exhibit excellent pharmacological effect due to controlled drug-release at the implantation site [5, 6, 41]. Freezing a SS solution induces ice nuclei to form and grow, resulting in dehydration of the SS phase and formation of an insolubilized porous matrix after freeze drying or thawing [44]. Tao et al. [44] prepared a porous sponge with pore radii ranging from 60 to 90 µm by freeze drying SS aqueous solutions. They also investigated the effects of SS concentration and the freezing temperature on the structure and properties of the products. They demonstrated that more concentrated solutions and a lower freezing temperature produced a smaller pore radius, higher porosity, and higher pore density. It was recently shown that sponge-like scaffolds of SS/gelatin and SS/PVA incorporating cross-linkers exhibit high physical strength and degrade only gradually in phosphate buffered saline (PBS) or water over several weeks [45, 46]. These SS scaffolds are therefore promising matrices for use in biodegradable implants for DDS.

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5. Films Casting is a general process for the preparation of films. Gelatin, chitosan, and silk fibroin films incorporating drugs such as fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) have been investigated for their ability to accelerate wound healing [7, 47, 48]. A SS film was prepared without the use of any cross-linker or organic solvent by casting either intact or slightly degraded SS in aqueous solution [38, 49]. Although the dried SS film was not flexible, the moistened SS film could be stretched. To understand the basis underlying these stretch properties, Teramoto et al. [49] used attenuated total reflection Fourier-transform infrared (ATR-FTIR) analysis to investigate the molecular orientation of SS films that had been stretched after moistening with various concentrations of aqueous ethanol. These analyses indicated that formation of aggregated strands among extended SS chains induced by ethanol treatment is key to generating the required molecular orientation. Strong intermolecular hydrogen bonds were inferred to allow the transmission of the stretching force between the aggregated strands, altering the molecular orientation. Dried SS films made of SS alone exhibited elastic distortion and were easily broken, showing that their use is not practical. Thus, to improve the film-forming properties and tensibility of SS film, the effect of the addition of plasticizer was examined [32]. The elongation of SS films increased between 170-400% with increasing glycerin or D-sorbitol content, but the tensile strength proportionally decreased. ATR-FTIR analysis showed that the secondary structure of SS in films containing plasticizer changed from random coil to β-sheet, similar to that observed for moistened SS film and gel. It was also demonstrated that the addition of glycerin or D-sorbitol enhanced moisture retention by SS film. Thus, to obtain a practical film with good film-forming characteristics and tensibility, the SS should be in a β-sheet conformation and the film should have sufficient moisture content. It was recently shown that a SS film with these characteristics is biodegradable [50].

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Cast films blending SS with other polymers such as PVA were prepared and characterized [21], and showed that SS molecules bind to the amorphous PVA molecules by intermolecular hydrogen bonds, resulting in the formation of molecular bundles. These films have high oxygen permeability. It was also shown that a blend with chitosan produces a SS film [51]. A mucoadhesive complex prepared by template polymerization of poly (acric acid) (PAA) in the presence of SS resulted in decreased water solubility of PAA and improved mucoadhesive properties of PAA, conducive for use as a transmucosal drug delivery system [52]. In addition, a cast blend film with SS and polyethylene glycol diglycidyl ether (PEGDE) used as a cross-linking agent showed good cytocompatibility [53]. Recently, SS film cross-linked with PVA showed promising results in animal cell culture by improving the adherence and spreading of cells on a poorly adhering PVA matrix surface [54]. These findings demonstrate the potential of SS film as a biodegradable and biocompatible carrier for DDS.

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6. Micro and Nanospheres Microsphere and nanosphere DDS using various biodegradable polymers such as poly (lactic acid) (PLA), PLGA, proteins (e.g., albumin, collagen, and gelatin), and polysaccharides such as alginic acid, hyaluronic acid, and chitosan have been studied and reviewed [55, 56]. The advantages of such micro- or nanospheres for incorporating drugs include controlled drug-release, enhancement of drug-absorption, the possibility of targeting drug delivery, and stabilization of the drug. Zhang et al. [20] prepared SS microspheres with an average particle size of about 10 µm by spray-drying SS solution (SS molecular weight: 50200 kDa). L-ASNase was covalently conjugated on the surface polar groups of the SS microspheres using a bifunctional reagent, glutaraldehyde. Self-assembled SS nanospheres 200-400 nm in diameter have been prepared by conjugating SS in aqueous solution with activated poly (ethylene glycol) (PEG) [57]. Recently, the use of self-assembled SS and poloxamer nanospheres as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery was reported [58]. SS was blended with the poloxamers, pluronic F-127 and F-87, in the presence of solvents to produce selfassembled micellar nanostructures capable of carrying both fluorescein isothiocyanate-inulin and the anticancer drug, paclitaxel. These nanospheres ranged between 100 and 110 nm in diameter. Thus, SS has promising potential as a particulate carrier in DDS.

DRUG CONTROLLED RELEASE 1. Fluorescein Isothiocyanate – Dextran and Fluorescein Isothiocyanate Albumin Release from Sericin Gel, Sponge and Film The release properties of three model drugs from SS gel, sponge, and film prepared from slightly degraded SS (150-400 kDa) were studied [32, 50]. Fluorescein isothiocyanate (FITC)-dextran (FD4, 4 kDa and FD70, 70 kDa) were used as two model drugs with significantly different molecular weights, and FITC-albumin (FA, 66 kDa) was used as a

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negatively charged drug. FD and FA are used widely as stable tracers in circulatory research, and as protein model drugs to evaluate drug-release properties from various dosage forms [59-63]. In each preparation, the release rate of the model drugs tended to be FA < FD70 < FD4. Large molecular weight FD70 (70 kDa) was released more slowly than small molecular weight FD4 (4 kDa) from SS film. FA (66 kDa) was released the slowest, for a period of 1 week or more from each preparation, even though the molecular weights of FA and FD70 are almost the same. The results suggest that SS is usable as an aqueous sustained-release material for high molecular weight drugs. Furthermore, if the drug is charged, its release can be sustained for an extended time. FD4 was released rapidly from each SS preparation. The release rate of FD70 followed the order film < gel < sponge. There was no significant difference in the release rate of FA from the three SS preparations. Of the three SS preparations, the sponge form disintegrated most easily during dissolution tests. SEM observation of SS film showed that the surfaces of SS films were smooth, and no pores were evident in film cross-sections [32]. On the other hand, many pores were observed in SS sponge. These observations suggest that drugs might easily diffuse from the pores in SS sponge into the dissolution medium. In SS gel, SS adopts a β-sheet structure that constructs the network structure of the hydrogel [10, 14]. This allows the gel to retain a large amount of water, possibly allowing larger molecular weight drugs like FD70 to be retained in the gel network structure. In the case of drugs that ionically interact with the carrier, the diffusion of the drug may be inhibited, judging from our observation that the initial release rate of FA from SS preparations was markedly slower compared to that of FD70 or FD4. The release of FA from SS gel, sponge, and film prepared from solutions containing two different concentrations of dissolved SS (1% and 2%, w/v) were tested [50]. For 2% SS preparations, all dosage forms sustained FA release for 2 weeks or more, which is markedly longer than the stained release by the 1% SS preparations. These results suggest that the release of a drug can be controlled by adjusting the concentration of SS.

2. Fibroblast Growth Factor-2 Controlled Release from Sericin Film Growth factors can be immobilized via ionic interactions to control their release. For example, fibroblast growth factor-2 (FGF-2) can be bound to gelatin hydrogels by poly-ion complexation, allowing its release to be controlled [6]. Wongpanit et al. [42] suggested that FGF-2 release from silk fibroin scaffolds is sustained by ionic interactions between fibroin (isoelectric point (pI): 3.8-4.5) and FGF-2 (pI 9.6). As described above, amino acid analysis of SS shows high amounts of serine (Ser, 33.2-39.0%), glycine (Gly, 14.1-16.0%), and aspartic acid/asparagine (Asp/Asn, 11.3-15.7%) [9]. The pI of SS is 5-6 in solution [64], so it is likely that FGF-2 (pI: 9.6) is immobilized on SS via ionic interactions. The release properties of FGF-2 from SS films were evaluated in dissolution medium in the absence of enzyme in vitro [65]. Small amounts of FGF-2 were immediately released, with the release gradually plateauing after 3 days then continuing for up to two weeks. Films containing a high concentration of SS exhibited a significantly smaller burst release of FGF-2 after 1 day than those containing a low concentration of SS. In a recent study, trypsin degradation of a SS preparation resulted in the accelerated release of FA from SS film in vitro, suggesting that FGF-2 would also be released gradually as the SS film is degraded [65].

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DRUG DELIVERY 1. L-Asparaginase Immobilized in Sericin Microspheres L-Asparaginase (L-ASNase)

has been extensively used in the treatment of acute lymphoblastic leukemia. In order to reduce immunological responses, prolong L-ASNase action time, and enhance the drug’s effect in blood, native L-ASNase is often chemically modified and immobilized in soluble biocompatible polymers such as albumin, dextran, PEG, and PVA [66-69] and in insoluble matrix-supports such as collagen, CM-cellulose, polyacrylamide, poly (2-hydroxyethyl methacrylate), and silk fibroin [70-74]. Zhang et al. [20] investigated SS as a biocompatible support for L-ASNase immobileization. SS microspheres were prepared from SS solution, and L-ASNase was immobilized to the microspheres as previously described. The immobilized L-ASNase retained over half the original activity of the free enzyme. Although the Km of conjugated SS was much lower than that of native L-ASNase, the affinity of L-ASNase for its substrate, L-asparagine, increased considerably when immobilized on SS microspheres. Furthermore, the bioconjugation of LASNase widened the optimum temperature range of the enzyme; the immobilized enzyme showed significantly higher thermostability and resistance to trypsin digestion compared with the native enzyme.

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2. Sericin–Insulin Bioconjugates Insulin is a hormone that regulates the level of blood glucose in the body. Although insulin is currently administered as an injected drug to treat insulin-dependent diabetes, this approach suffers from several drawbacks such as hydrolysis by proteases and a short half-life [75, 76]. In order to address these drawbacks, insulin has been modified chemically, for example by conjugation to polymers such as PEG, galactosyl, and dextran [77-80]. Zhang et al. [81] investigated the long-term effective insulin delivery of SS–insulin bioconjugates. SS–insulin bioconjugates prepared by cross-linking with glutaraldehyde at 4 °C for 5 h showed higher activity and greater stability in human serum than bovine serum albumin (BSA)–insulin bioconjugates in an in vitro study. The half-life of SS–insulin bioconjugate was also higher than that of BSA–insulin bioconjugates and intact insulin. Furthermore, no antigenicity of SS–insulin bioconjugates was observed in rabbits and mice, and their pharmacological activity lasted approximately 4 times longer in mice compared to soluble insulin.

3. Sericin–Poloxamer Nanospheres as Nanocarriers of Hydrophobic and Hydrophilic Drugs Paclitaxel is a mitotic inhibitor used in cancer chemotherapy. Paclitaxel formulations have a low therapeutic index due to the drug’s inability to selectively target tumor tissues, and to toxic side effects of the cremophor–ethanol diluent [82, 83]. To address these problems,

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several DDS have been evaluated such as nanospheres [84-88], liposomes [89, 90], emulsions [91-93], and micelles [94-97]. Mandal and Kundu [58] prepared self-assembled SS–poloxamer nanospheres as nanocarriers of hydrophobic (FITC-inulin) and hydrophilic (paclitaxel) drugs by blending with pluronic F-127 and F-87 in the presence of solvents. Rapid uptake of these nanospheres into cells was observed in vitro using breast cancer MCF-7 cells. In vitro cytotoxicity assays using paclitaxel-loaded nanospheres against breast cancer cells showed promising results comparable to free paclitaxel drugs. The induction of apoptosis in MCF-7 cells by drugencapsulated nanospheres was confirmed by fluorescence-activated cell sorter (FACS) and confocal microscopy using annexin V staining. Up-regulation of the pro-apoptotic protein Bax, down-regulation of the anti-apoptotic protein Bcl-2, and cleavage of the regulatory protein poly-ADP-ribose polymerase (PARP) were confirmed by Western blot analysis and suggested further drug-induced apoptosis in the cells. This study suggests that SS is an alternative natural biomaterial for the fabrication of self-assembled nanospheres in the presence of poloxamer, and holds promise for the delivery of both hydrophobic and hydrophilic drugs to target sites.

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4. Sericin Film Incorporating FGF-2 FGF-2 has a strong proliferative effect on endothelial cells, osteocytes, and chondrocytes [98, 99]. FGF-2 exerts its proangiogenic activity by interacting with various endothelial cell surface receptors. Also, cross-talk among FGFs, vascular endothelial growth factors (VEGFs), and inflammatory cytokines may play a role in the modulation of angiogenesis in different pathological conditions, including bone healing [100]. The administration of FGF-2 has shown therapeutic potential for tissue regeneration. However, the bioactivity of FGF-2 is unreliable when administrated in solution because of its short retention time at wound sites and short half-life caused by its susceptibility to enzymatic degradation in vivo. To enhance the therapeutic efficacy of FGF-2, a drug delivery system allowing sustained and localized release of FGF-2 is needed [101]. It was reported that the sustained release of FGF-2 from acidic gelatin hydrogel leads to prolonged vascularization [6]. Iwakura et al. [102] reported that a gelatin sheet incorporating FGF-2 accelerated sternal healing after bilateral internal thoracic artery removal in normal and diabetic rats. In another study, PLA and PLGA, amalgamated with gelatin sponge, released incorporated FGF-2 and bone morphogenic protein (BMP). As a result, skull bone damage in rats was diminished by enhanced calcification due to the osteoinductive activity of BMP, which was enhanced by FGF-2 [40]. Recently, SS film incorporating FGF-2 was prepared by a casting method, and the sustained release of FGF-2 from SS film was characterized in vitro [65]. In an in vivo study, SS film was implanted to cover a hole drilled in the skulls of rats, and the rate of tissue regeneration was evaluated [65]. Visual observation after 2 weeks showed that SS films incorporating 250 ng and 2500 ng of FGF-2 were markedly thickened due to tissue growth at the implanted site. In histological observations, larger amounts of collagen-like fiber tissue deposition were found, and the fiber tissue was more closely connected to the lip of the hole covered with SS film, when the film contained FGF-2, compared to holes not covered with SS film or covered with SS film not containing FGF-2. Although it was found no osteoid

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tissue formation in any of the holes, the implanted SS film containing FGF-2 seemed to release FGF-2 gradually and to stimulate the growth of collagen-like fiber tissue. It is expected that the growth of fiber tissue in defect holes is stimulated by the release of FGF-2 from the SS film covering the holes, accelerating bone formation. This conclusion is supported by the fact that collagen matrix works as a scaffold to promote the proliferation of mesenchymal stem cells, cartilage cells, and osteoid tissue formation [103]. The combination of SS film and FGF-2, and the prolonged localization of FGF-2 at the wound site, are effective at promoting wound healing. Thus, SS film may be useful in drug-release and biodegradable devices, and FGF-2 incorporated into film may accelerate bone remodeling and wound healing.

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BIOEROSION BEHAVIOR OF SERICIN PREPARATIONS Polymer biodegradation occurs by physical mechanisms such as sorption, swelling, dissolution, mineralization, crystallization, fatigue fracture, impact fracture, and wear, and by chemical mechanisms such as hydrolysis, enzymolysis, oxidation, and oxidative chain cleavage [104]. Thus, no single mechanism should be considered as causing biodegradation. During degumming of silk fabric, SS is hydrolyzed by proteases and removed from silk fibroin [105], and so enzymatic degradation may be a major degradation mechanism of SS protein. Drug release rate is affected by the biodegradation rate of the drug carrier. Yang et al. [106] investigated the release profiles of 10-hydroxycamptothecin (HCPT) encapsulated in BSA nanospheres in the absence and presence of trypsin. BSA nanospheres containing HCPT showed enhanced release upon exposure to trypsin. It was found that although the release of rose bengal as a model drug from human serum albumin nanospheres is likely too slow to be practical, the presence of trypsin accelerates the release rate of this encapsulated drug from nanospheres [107]. Wongpanit et al. [42] investigated the release of 125I-labeled FGF-2 from silk fibroin scaffolds under non-degradation and degradation conditions using protease type XIV from Streptomyces griseus (EC 3.4.24.31). In non-degradation conditions, FGF-2 immediately diffused from the scaffold and its release gradually plateaued. However, in the presence of degradative enzymes, the initial burst release was markedly increased. Aramwit et al. [46] reported that non-crosslinked SS hydrolysate/PVA scaffold completely dissolved in less than 30 min, while a high concentration of the cross-linker, genipin, inhibited the dissolution of SS in PBS. Cross-linked SS hydrolysate/gelatin scaffold degraded gradually over a period of 28 days or more [45]. Takeoka et al. [108] reported that the interperitoneal injection of high molecular SC solution into a rat model was dehydrated and gradually degraded into small particles. Intact SS forms a β-sheet structure [14], resulting in a slower degradation rate. Degradation of intact SS is reportedly caused by hydrolysis of its hydrophilic region [109] by macrophages and peptidases [55]. Recently, films incorporating a model protein drug, FA, were prepared using high molecular weight SS. To evaluate the biodegradation of this film, its enzymatic degradation and drug-release properties were evaluated in vitro using trypsin [65]. In the absence of trypsin, SS film swelled rapidly, kept its shape, and remained intact for 28 days or longer due to its β-sheet structure. In the presence of trypsin, SS film gradually degraded, with the rate of degradation depending on

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the concentration of trypsin, indicating that SS likely underwent hydrolysis and is thus biodegradable. SS film incorporating a model protein drug, FA, also gradually degraded in the presence of trypsin, and released FA in a sustained manner for 2 weeks or longer. In the absence of trypsin, the release of FA was markedly slower. In an in vivo study, the degradation of SS film and gel containing FA as a model protein drug was evaluated by implanting the film or gel under the skin of rats, and the amounts of FA remaining and the weight of the films and gels were determined [50]. The SS film and gel was encapsulated in collagen-like material from connective tissue and gradually decreased in size and weight over time. The degradation rate of the SS gel was faster than that of the SS film. The drug was retained for more than 6 weeks in the SS film and for 3 weeks in the SS gel, with the amount of FA at the implantation site decreasing as the SS preparation biodegraded. Thus, the in vivo release of a protein drug from SS preparations may be slower than its release under in vitro enzymatic conditions.

CONCLUSION

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The silk protein SS can be used in various dosage forms such as gel, cream, porous matrix, film, and micro- and nanospheres. SS hydrolysate is water-soluble and easy to bind to modified drugs or other materials. Intact or partially degraded SS from cocoons can be used to prepare water-based matrices due to the β-sheet structure of SS, without the need for any toxic crosslinker or organic solvent. It has been demonstrated that these biocompatible and biodegradable preparations are useful as carriers for the controlled or targeted delivery of biologics. Furthermore, the study of modified silk proteins from transgenic silkworm strains may also have significant potential for use in DDS in the future.

REVIEWED BY Hiroaki Okada, Ph. D., Tokyo University of Pharmacy and Life Sciences.

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[36] Aramwit, P., S. Kanokpanont, T. Nakpheng, and T. Srichana, The effect of sericin from various extraction methods on cell viability and collagen production. Int J Mol Sci, 2010. 11(5): p. 2200-11. [37] Kundu, S.C., B.C. Dash, R. Dash, and D.L. Kaplan, Natural protective glue protein, sericin bioengineered by silkworms: Potential for biomedical and biotechnological applications Prog Polym Sci, 2008. 33(10): p. 998-1012. [38] Teramoto, H., T. Kameda, and Y. Tamada, Preparation of gel film from Bombyx mori silk sericin and its characterization as a wound dressing. Biosci Biotechnol Biochem, 2008. 72(12): p. 3189-96. [39] Aramwit, P. and A. Sangcakul, The effects of sericin cream on wound healing in rats. Biosci Biotechnol Biochem, 2007. 71(10): p. 2473-7. [40] Tanaka, E., Y. Ishino, A. Sasaki, T. Hasegawa, M. Watanabe, D.A. Dalla-Bona, E. Yamano, T.M. van Eijden, and K. Tanne, Fibroblast growth factor-2 augments recombinant human bone morphogenetic protein-2-induced osteoinductive activity. Ann Biomed Eng, 2006. 34(5): p. 717-25. [41] Rohman, G., S.C. Baker, J. Southgate, and N.R. Cameron, Heparin functionalisation of porous PLGA scaffolds for controlled, biologically relevant delivery of growth factors for soft tissue engineering. J Mater Chem, 2009. 19(48): p. 9265-9273. [42] Wongpanit, P., H. Ueda, Y. Tabata, and R. Rujiravanit, In vitro and in vivo release of basic fibroblast growth factor using a silk fibroin scaffold as delivery carrier. J Biomater Sci Polym Ed, 2010. 21(11): p. 1403-19. [43] Sahoo, S., S.L. Toh, and J.C. Goh, A bFGF-releasing silk/PLGA-based biohybrid scaffold for ligament/tendon tissue engineering using mesenchymal progenitor cells. Biomaterials, 2010. 31(11): p. 2990-8. [44] Tao, W., M. Li, and R. Xie, Preparation and structure of porous silk sericin materials. Macromol Mater Eng, 2005. 290(3): p. 188-194. [45] Mandal, B.B., A.S. Priya, and S.C. Kundu, Novel silk sericin/gelatin 3-D scaffolds and 2-D films: fabrication and characterization for potential tissue engineering applications. Acta Biomater, 2009. 5(8): p. 3007-20. [46] Aramwit, P., T. Siritientong, S. Kanokpanont, and T. Srichana, Formulation and characterization of silk sericin-PVA scaffold crosslinked with genipin. Int J Biol Macromol, 2010. 47(5): p. 668-75. [47] Fedakar-Senyucel, M., M. Bingol-Kologlu, R. Vargun, C. Akbay, F.N. Sarac, N. Renda, N. Hasirci, G. Gollu, and H. Dindar, The effects of local and sustained release of fibroblast growth factor on wound healing in esophageal anastomoses. J Pediatr Surg, 2008. 43(2): p. 290-5. [48] Tanaka, A., T. Nagate, and H. Matsuda, Acceleration of wound healing by gelatin film dressings with epidermal growth factor. J Vet Med Sci, 2005. 67(9): p. 909-13. [49] Teramoto, H. and M. Miyazawa, Molecular orientation behavior of silk sericin film as revealed by ATR infrared spectroscopy. Biomacromolecules, 2005. 6(4): p. 2049-57. [50] Nishida, A., M. Yamada, T. Kanazawa, Y. Takashima, K. Ouchi, and H. Okada, Sustained-release of protein from biodegradable sericin film, gel and sponge. Int J Pharm, 2011. 407(1-2): p. 44-52. [51] Srihanam, P., W. Simcheur, and Y. Srisuwan, Study on silk sericin and chitosan blend film: morphology and secondary structure characterizations. Pak J Biol Sci, 2009. 12(22): p. 1487-90.

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[70] Jefferies, S.R., R. Richards, and F.R. Bernath, Preliminary studies with L-asparaginase bound to implantable bovine collagen heterografts: a potential long-term, sustained dosage, antitumor enzyme therapy system. Biomater Med Devices Artif Organs, 1977. 5(4): p. 337-54. [71] Hasselberger, F.X., H.D. Brown, S.K. Chattopadhyay, A.N. Mather, R.O. Stasiw, A.B. Patel, and S.N. Pennington, The preparation of insoluble, matrix-supported derivatives of asparaginase for use in cancer therapy. Cancer Res, 1970. 30(11): p. 2736-8. [72] Mori, T., T. Tosa, and I. Chibata, Preparation and properties of asparaginase entrapped in the lattice of polyacrylamide gel. Cancer Res, 1974. 34(11): p. 3066-8. [73] O'Driscoll, K.F., R.A. Korus, T. Ohnuma, and I.M. Walczack, Gel entrapped Lasparaginase: kinetic behavior and antitumor activity. J Pharmacol Exp Ther, 1975. 195(2): p. 382-388. [74] Zhang, Y.Q., W.L. Zhou, W.D. Shen, Y.H. Chen, X.M. Zha, K. Shirai, and K. Kiguchi, Synthesis, characterization and immunogenicity of silk fibroin-L-asparaginase bioconjugates. J Biotechnol, 2005. 120(3): p. 315-26. [75] Siddiqui, O., Y. Sun, J.C. Liu, and Y.W. Chien, Facilitated transdermal transport of insulin. J Pharm Sci, 1987. 76(4): p. 341-5. [76] Liu, J.C., Y. Sun, S. Ovais, C. Y.W., W.M. Shi, and J. Li, Blood glucose control in diabetic rats by transdermal iontophoretic delivery of insulin. Int J Pharm, 1988. 44(13): p. 197-204. [77] Soriano, I., C. Evora, and M. Llabrés, Preparation and evaluation of insulin-loaded poly-lactide) microspheres using an experimental design. Int J Pharm, 1996. 142(2): p. 135-142. [78] Myhre, D.V. and W.B. Geho, Galactosyl-insulin conjugates useful in treating diabetics, E. Patent, Editor. 1984, Procter & Gamble (US). [79] Hinds, K.D. and S.W. Kim, Effects of PEG conjugation on insulin properties. Adv Drug Deliv Rev, 2002. 54(4): p. 505-30. [80] Baudys, M., D. Letourneur, F. Liu, D. Mix, J. Jozefonvicz, and S.W. Kim, Extending insulin action in vivo by conjugation to carboxymethyl dextran. Bioconjug Chem, 1998. 9(2): p. 176-83. [81] Zhang, Y.Q., Y. Ma, Y.Y. Xia, W.D. Shen, J.P. Mao, and R.Y. Xue, Silk sericin-insulin bioconjugates: synthesis, characterization and biological activity. J Control Release, 2006. 115(3): p. 307-15. [82] Weiss, R.B., R.C. Donehower, P.H. Wiernik, T. Ohnuma, R.J. Gralla, D.L. Trump, J.R. Baker, Jr., D.A. Van Echo, D.D. Von Hoff, and B. Leyland-Jones, Hypersensitivity reactions from taxol. J Clin Oncol, 1990. 8(7): p. 1263-8. [83] Terwogt, J.M., B. Nuijen, W.W. Huinink, and J.H. Beijnen, Alternative formulations of paclitaxel. Cancer Treat Rev, 1997. 23(2): p. 87-95. [84] Damascelli, B., G.L. Patelli, R. Lanocita, G. Di Tolla, L.F. Frigerio, A. Marchiano, F. Garbagnati, C. Spreafico, V. Ticha, C.R. Gladin, M. Palazzi, F. Crippa, C. Oldini, S. Calo, A. Bonaccorsi, F. Mattavelli, L. Costa, L. Mariani, and G. Cantu, A novel intraarterial chemotherapy using paclitaxel in albumin nanoparticles to treat advanced squamous cell carcinoma of the tongue: preliminary findings. AJR Am J Roentgenol, 2003. 181(1): p. 253-60.

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[85] Mitra, A. and S. Lin, Effect of surfactant on fabrication and characterization of paclitaxel-loaded polybutylcyanoacrylate nanoparticulate delivery systems. J Pharm Pharmacol, 2003. 55(7): p. 895-902. [86] Potineni, A., D.M. Lynn, R. Langer, and M.M. Amiji, Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pH-sensitive biodegradable system for paclitaxel delivery. J Control Release, 2003. 86(2-3): p. 223-34. [87] Lee, M.K., S.J. Lim, and C.K. Kim, Preparation, characterization and in vitro cytotoxicity of paclitaxel-loaded sterically stabilized solid lipid nanoparticles. Biomaterials, 2007. 28(12): p. 2137-46. [88] Niu, G., C.H. Castro, N. Nguyen, S.M. Sullivan, and J.A. Hughes, In vitro cytotoxic activity of cationic paclitaxel nanoparticles on MDR-3T3 cells. J Drug Target, 2010. 18(6): p. 468-76. [89] Kunstfeld, R., G. Wickenhauser, U. Michaelis, M. Teifel, W. Umek, K. Naujoks, K. Wolff, and P. Petzelbauer, Paclitaxel encapsulated in cationic liposomes diminishes tumor angiogenesis and melanoma growth in a "humanized" SCID mouse model. J Invest Dermatol, 2003. 120(3): p. 476-82. [90] Yang, T., F.D. Cui, M.K. Choi, J.W. Cho, S.J. Chung, C.K. Shim, and D.D. Kim, Enhanced solubility and stability of PEGylated liposomal paclitaxel: in vitro and in vivo evaluation. Int J Pharm, 2007. 338(1-2): p. 317-26. [91] Kan, P., Z.B. Chen, C.J. Lee, and I.M. Chu, Development of nonionic surfactant/phospholipid o/w emulsion as a paclitaxel delivery system. J Control Release, 1999. 58(3): p. 271-8. [92] Constantinides, P.P., K.J. Lambert, A.K. Tustian, B. Schneider, S. Lalji, W. Ma, B. Wentzel, D. Kessler, D. Worah, and S.C. Quay, Formulation development and antitumor activity of a filter-sterilizable emulsion of paclitaxel. Pharm Res, 2000. 17(2): p. 175-82. [93] Rodrigues, D.G., C.C. Covolan, S.T. Coradi, R. Barboza, and R.C. Maranhao, Use of a cholesterol-rich emulsion that binds to low-density lipoprotein receptors as a vehicle for paclitaxel. J Pharm Pharmacol, 2002. 54(6): p. 765-72. [94] Liggins, R.T. and H.M. Burt, Polyether-polyester diblock copolymers for the preparation of paclitaxel loaded polymeric micelle formulations. Adv Drug Deliv Rev, 2002. 54(2): p. 191-202. [95] Krishnadas, A., I. Rubinstein, and H. Onyuksel, Sterically stabilized phospholipid mixed micelles: in vitro evaluation as a novel carrier for water-insoluble drugs. Pharm Res, 2003. 20(2): p. 297-302. [96] Lukyanov, A.N., Z. Gao, and V.P. Torchilin, Micelles from polyethylene glycol/phosphatidylethanolamine conjugates for tumor drug delivery. J Control Release, 2003. 91(1-2): p. 97-102. [97] Seow, W.Y., J.M. Xue, and Y.Y. Yang, Targeted and intracellular delivery of paclitaxel using multi-functional polymeric micelles. Biomaterials, 2007. 28(9): p. 1730-40. [98] Connolly, D.T., B.L. Stoddard, N.K. Harakas, and J. Feder, Human fibroblast-derived growth factor is a mitogen and chemoattractant for endothelial cells. Biochem Biophys Res Commun, 1987. 144(2): p. 705-12.

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[99] Gospodarowicz, D., N. Ferrara, L. Schweigerer, and G. Neufeld, Structural characterization and biological functions of fibroblast growth factor. Endocr Rev, 1987. 8(2): p. 95-114. [100] Presta, M., P. Dell'Era, S. Mitola, E. Moroni, R. Ronca, and M. Rusnati, Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev, 2005. 16(2): p. 159-78. [101] Tabata, Y., Significance of release technology in tissue engineering. Drug Discov Today, 2005. 10(23-24): p. 1639-46. [102] Iwakura, A., Y. Tabata, T. Koyama, K. Doi, K. Nishimura, K. Kataoka, M. Fujita, and M. Komeda, Gelatin sheet incorporating basic fibroblast growth factor enhances sternal healing after harvesting bilateral internal thoracic arteries. J Thorac Cardiovasc Surg, 2003. 126(4): p. 1113-20. [103] Reddi, A.H., Cell biology and biochemistry of endochondral bone development. Coll Relat Res, 1981. 1(2): p. 209-26. [104] Kopecek, J. and K. Ulbrich, Biodegradation of biomedical polymers. Prog Polym Sci, 1983. 9: p. 1-58. [105] Freddi, G., R. Mossotti, and R. Innocenti, Degumming of silk fabric with several proteases. J Biotechnol, 2003. 106(1): p. 101-12. [106] Yang, L., F. Cui, D. Cun, A. Tao, K. Shi, and W. Lin, Preparation, characterization and biodistribution of the lactone form of 10-hydroxycamptothecin (HCPT)-loaded bovine serum albumin (BSA) nanoparticles. Int J Pharm, 2007. 340(1-2): p. 163-72. [107] Lin, W., M.C. Garnett, S.S. Davis, E. Schacht, P. Ferruti, and L. Illum, Preparation and characterisation of rose Bengal-loaded surface-modified albumin nanoparticles. J Control Release, 2001. 71(1): p. 117-26. [108] Takeoka, M., S. Taniguchi, and H. Miyake. Evaluation of safety and physiological effect of sericin. in Sericin Symposium on Northeastern Industrial Research Center of Shiga Prefecture. 2004. Nagahama, Japan. [109] Teramoto, H., A. Kakazu, and T. Asakura, Native structure and degradation pattern of silk sericin studied by 13C NMR spectroscopy Macromolecules, 2006. 39(1): p. 6-8.

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Chapter 14

SILK PROTEINS FOR WOUND HEALING MATERIALS Pornanong Aramwit1 Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand

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ABSTRACT Silk materials have been shown to promote wound healing since the 1990s. Both sericin and fibroin have been found to be an effective substrate for the proliferation of adherent animal cells and can be used as a substitute for collagen. Because of their excellent physical and biological properties, silk sericin and fibroin were widely investigated for their use in biomedical applications including wound healing materials.

INTRODUCTION Human skin is considered one of the most important organs of the body, providing a multitude of structural and functional benefits, ensuring perfect homeostasis [1]. It is a complex organ made of 2 layers of dermis and epidermis. The loss of the integrity of the protective barrier served by skin through injury or illness may result in infection, dehydration, and necrosis, which may ultimately lead to severe trauma and shock, with morbid consequences [2]. Because of that, a number of research groups have been working on strategies to promote the process of wound healing for several decades. Indeed, the wound healing process is complex and involves the interactions of many different types of cells and matrix components to establish a provisional tissue and eventually a complete regenerated epidermis 1

Pornanong Aramwit is an Associate Professor in the Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences at Chulalongkorn University, Bangkok, Thailand. She earned a B.Sc. in Pharmacy from Chulalongkorn University in 1992 and went on to the University of Illinois-Chicago, USA to earn Doctor of Pharmacy (Pharm.D.) in 1995 and a Ph.D. in Pharmaceutical Sciences from the University of WisconsinMadison, USA in 2001.

Email: [email protected]

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[3, 4]. Among the different steps involved in wound healing, the closure of the wounded area by the epithelial cells is one of the most important as it restores an intact epidermal barrier and protects the underlying tissue [5]. Reepithelialization involves the controlled migration of keratinocytes after their division at the wound edges (epithelial tongue) [6]. In some pathological conditions, such as diabetes, reepithelialization never occurs, leading to the development of acute or chronic non-healing wounds. To promote wound healing, wound dressings should meet several criteria: (1) biocompatibility, (2) prevent dehydration of the wound and retain a favorable moist environment, (3) physically protect the wound against dust and bacteria, (4) allow gas exchange and (5) promote epithelialization by delivering specific, active molecules [5]. Silk materials have been shown to promote wound healing since the 1990s. Tsubouchi developed a silk fibroin-based wound dressing that could accelerate healing and could be peeled off without damaging the newly formed skin [7]. The non-crystalline fibroin film of the wound dressing had a water content of 3-16% and a thickness of 10-100 m. Subsequently, the wound dressing was made with a mixture of both fibroin and sericin [8]. The non-crystalline fibroin-sericin film had a degree of crystallization of less than 10%. The occlusive dressing had a 10% or greater solubility in water at room temperature and a water absorptivity of 100% or more at room temperature. A membrane composed of sericin and fibroin is an effective substrate for the proliferation of adherent animal cells and can be used as a substitute for collagen. Minoura et al. and Tsukada et al. investigated the attachment and growth of animal cells on films made of sericin and fibroin [9, 10]. Cell attachment and growth were dependent on maintaining a minimum of around 90% sericin in the composition membrane. Films of pure component protein (fibroin or sericin) permitted cell attachment and growth comparable to that on collagen, a widely used substrate for mammalian cell culture.

SILK FIBROIN FOR WOUND DRESSING MATERIAL Non-woven Silk Fibroin Mats Silk fibroin nanofibers can be successfully prepared by electrospinning [11]. Electrospinning has been widely explored recently because electrospun fibers are good candidates for a wide variety of applications, including high performance filters, delivery carriers [12] as well as biomaterial scaffold for tissue engineering and wound dressings [13]. These applications benefit from the large specific surface area and high porosity of the electrospun mat [14, 15]. The porous nanofibrous structured electrospun membranes create favorable properties as wound dressings including: controlled evaporative water loss, excellent oxygen permeability, promotion of fluid drainage, and inhibition of exogenous microorganism invasion due to their ultra-fine pores [5]. While a significant number of natural and synthetic materials have been electrospun to form wound dressings, challenges remain in terms of biocompatibility, mechanical properties and overall functional performance. Nonwoven silk fibroin membranes fabricated by electrospinning have gained much attention due to the ability to produce polymer nanofibers with diameters in the range of several micrometers down to tens of nanometers [14]. Researchers have investigated the effects of nonwoven silk

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fibroin microfibrous nets on the culture of a wide variety of human cell lines including osteoblasts, fibroblasts, keratinocytes, and endothelial cells. These studies have shown that these microfibrous nets support the adhesion, proliferation, and cell-cell interactions [16]. In addition, nonwoven silk fibroin nanofibrous mats were also found to support attachment, spreading and proliferation of human bone marrow stromal cells, keratinocytes and fibroblasts in vitro [14, 17]. This outcome improved with a coating of collagen type I or fibronectin, most likely due to the presence of Arg-Gly-Asp (RGD) sequences available for cell binding. The biocompatibility of nonwoven microfibrous membranes has been shown to be composed of partially dissolved native silk fibroin fibers [18]. There was no infiltration of the lymphocytes present in the tissue even after 6 months of subcutaneous implantation, which indicates a good biocompatibility. In addition, the implanted silk fibroin membranes were integrated with the surrounding tissue within 6 months and no obvious degradation observed. Previous in vivo studies have demonstrated silk fibroin-based membranes as promising materials for skin regeneration [18, 19]. During electrospinning of silk fibroin, harsh solvents such as hexafluoro-2-propanol, hexafluoroacetone and formic acid have been previously employed [14]. It is well known that harsh solvents should be avoided in bioapplications, therefore, Cao et al. developed a simple, aqueous process for silk electrospinning with higher molecular weight [11]. Chen and Wang et al. also investigated the allaqueous processes for electrospinning but the optimal concentrations of silk fibroin employed were around 30 wt% which could be considered as “protein fragment” of fibroin [20, 21]. The silk fibroin nanofiber using aqueous solution developed by Cao et al. was controllable morphology and thickness which mainly depends on the fibroin concentration and electrical conductivity of the solution [11]. It also displayed satisfactory mechanical properties with apparent stress and stain at break being approximately 11.1 MPa and 10.2, respectively. They concluded that the procedure for the preparation of silk fibroin aqueous solution, which degrades the molecular weight of silk protein much less, makes an important contribution to the mechanical properties of the silk fibroin mats obtained. Schneider et al. presented the functionalization of silk fibroin mats to enhance wound healing using epidermal growth factor (EGF) [5]. Since EGF plays a significant role in the wound healing process, especially the stimulation of proliferation and migration of keratinocytes [22-24]. EGF has high affinity receptors expressed in both fibroblasts and kerainocytes and has been shown to accelerate wound healing in vivo [25, 26]. It has been demonstrated that the first 5 days after injury are the most critical during which maximal differences are seen between EGF treated and untreated wounds. EGF application after this period produces no significant improvement over controls, since by this time reepithelialization has already occurred in both groups [27]. Due to its relatively short half life of about 1 h, loss of occupied receptors through turnover, and a lag time of 8-12 h to commit cells to DNA synthesis [25], it is necessary to apply EGF frequently to a wound to maintain effective local concentration during the critical period of initial wound healing [27]. Therefore, topical applications of this molecule such as a dressing applied on top of the wound, could be an easy but powerful way to locally deliver EGF and to protect the tissue during the reconstruction phase [5]. Based on this idea, Schneider et al. used electrospun silk mats that were functionalized by adding EGF during the electrospinning process. They found that the release rate of EGF from electrospun silk mats was over a 6 day period. The use of EGF-charged silk mats increased the rate of wound closure by more than 3.5-fold compared to the silk dressing without EGF. Moreover, the material integrity was conserved during the

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complete healing process. They concluded that this material has tremendous potential for novel therapeutic bioapplications especially since these silk mats present many properties that would be ideal for creating a new generation of biologically active wound dressings for the acceleration of non-healing chronic wounds. Not only silk fibroin from mulberry silkworm has been applied for electrospinning, nonmulberry silkworm such as Antheraea mylitta, a wild non-mulberry tropical tasar silkworm, was also used for biospinning [28]. The amino acid composition of A. mylitta fibroin indicates that it is rich in glycine and alanine as the major amino acids [29]. During natural spinning the silk I-like conformation is thought to be converted into silk II, which is predominantly formed of crystalline -sheet [30]. These -sheets are orientated along the axis of the fibers, giving it strength and stiffness comparable with or superior to that of high performance synthetic materials [31]. Non-mulberry silk fibroins from A. mylitta also have sequences similar to RGD which help in cell attachment [32]. However, due to large fiber diameters of 25-30 m which is similar in size to a single cell, the cells have to rest on the fibers to adhere to the surface and proliferate, without passing through [28]. Thus, for cell adherence to fibrous matrices, the mesh-like network is an important parameter for cell support in the initial phase of attachment, holding the cells onto it. Once cells attach to the fibers they proliferate normally. After the biospinning process, the silk fibers obtained were aligned into linear, mixed or random patterns forming interconnected, macroporous threedimensional matrices. Drawn silk fibers showed enhanced stability to protease treatment in comparison with naturally occurring native gland fibroin protein. A viability assay suggested biocompatibility of these matrices in vitro. Fluorescence and confocal microscopy indicated normal cell attachment, spreading and proliferation on these biospun silk matrices. The results provided evidence for the use of biospun silk matrices as natural, inexpensive and alternative substrate for both wound healing and tissue engineering applications. Non-woven direct compression of silk fibroin indicated for use as wound dressing material was also reported. It has been claimed to promote healing without adhesion to the wound bed resulting in less destruction of new epithelial tissues. Figure 1 and 2 show the non-woven direct compression silk fibroin dressing.

Figure 1. Non-Woven Silk Fibroin Dressing by Direct Compression.

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Figure 2. Non-Woven Silk Fibroin Dressing by Direct Compression.

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Silk Fibroin Films Film made of sericin and fibroin has excellent oxygen permeability and is similar to human cornea in its functional properties. Because of that, silk film was not only used as wound dressing material but has also been hoped to form artificial corneas [33]. Silk fibroin films can be prepared from aqueous or organic solvent systems, as well as after blending with other polymers. Silk films prepared from aqueous silk fibroin solution had oxygen and water vapor permeability dependent on the content of silk I (crystallization) and silk II (-sheet secondary structure) structures [34]. The silk I structure is the water-soluble state and upon exposure to heat or physical spinning easily converts to a silk II structure. Alteration of silk structure was induced by treatment with 50% methanol with varying times. Changes in silk structure resulted in differing mechanical and degradability properties of the films [35]. Nanoscale silk fibroin films can also be formed from aqueous solution using a layer-by-layer technique [36]. Fibroblast attachment to silk films has been shown to be as high as for collagen films [9, 32]. Other mammalian and insect cells also showed good attachment on silk fibroin films when compared with collagen films [37]. Silk films, employed for healing full thickness skin wounds in rats, healed in 7 days faster with a lower inflammatory response than traditional procine-based wound dressings [19]. Transparent films cast from a blend of silk and cellulose showed increased mechanical strength compared with silk films alone [38]. Films cast from blends of silk fibroin and recombinant human-like collagen were seeded with hepatocytes and showed higher cell viability than silk fibroin films alone [39]. Silk fibroin solution, when coated on polyurethane and poly (carbonate) urethane films and scaffolds, increased the adhesion and proliferation of human fibroblasts [40, 41].

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Silk Fibroin Porous Sponges Porous sponge scaffolds are important for tissue engineering applications for cell attachment, proliferation and migration as well as for nutrient and waste transport. Regenerated silk fibroin solutions, both aqueous and solvent, have been utilized in the preparation of porous sponges. Sponges have been formed using porogens, gas foaming and lyophilized [42]. Aqueous silk fibroin sponges demonstrated improved cell attachment than the solvent-based porous sponges, likely due to these rougher surfaces. Sponges formed from a blend of poly (vinyl alcohol), chitosan and silk fibroin showed the best healing of the epidermis and dermis of rats when compared to the paired and single polymers [43]. Porous semi-interpenetrating networks (SIPNs) hydrogel from silk fibroin was also designed to utilize as a matrix for wound healing [44]. Since the porous sponge from silk fibroin itself is brittle, whereas its application to biomedical materials also requires sufficient mechanical strengths. Improvements in mechanical properties of silk fibroin-based materials have been sought by blending with other synthetic or natural polymers including poly(sodium glutamate), poly(ethylene glycol), poly(vinyl alcohol), sodium alginate, chitosan and cellulose. Yoo et al. prepared SIPNs composed of silk fibroin and poloxamer macromer [44]. Interpenetrating polymer networks (IPNs) are the blending system of two independently cross-linked polymers which can be formed by independently crosslinking a second component, a hydrophilic or hydrophobic polymer, within the crosslinked hydrophilic network [45, 46]. If the second component is a linear polymer, the formed network is called SIPNs. The advantage of this system is the formation of a mechanically stronger hydrogel, increasing the compatibility of the polymer blends, which exhibit favorable properties of phase separated materials [47]. Schmolka reported that poloxamer gels satisfied the major characteristics of an optimal dressing material for early management of skin burns [48]. Poloxamer 407 cleanses the wound of tissue detritus due to its surfactant nature and can significantly increase the rate of wound healing possibly by stimulation of endogenous production of epidermal growth factor [49]. Therefore, silk fibroin/poloxamer SIPNS composed of silk fibroin and poloxamer macromer are expected to have greater enhanced mechanical strength and the ability of wound healing than those of silk fibroin. Yoo et al. found that porous SIPNs hydrogel composed of silk fibroin and poloxamer macromer terminated with acrylate groups can be prepared for wound healing application by freezedrying after UV irradiation [44]. The SIPN hydrogels obtained were porous and their pores were well interconnected throughout the scaffold matrix indicating its good oxygen and water vapor permeability. The resistance of SIPN hydrogels against compression was much higher than that of silk fibroin itself or of a silk fibroin/poloxamer blend, and increased with the poloxamer content. The morphology of SIPN hydrogels was also affected by the SIPN hydrogel composition and the freezing temperature. From their results, it indicated that silk fibroin/poloxamer SIPN hydrogels will be expected to be useful for wound dressing applications. Dal Pra et al. prepared silk fibroin-based formic acid-crosslinked three-dimensional nonwoven devices using anisotropic silk fibroin fibers which were enclosed within an isotropic matrix of silk fibroin in film form [50]. They found that both fibers and films were firmly crosslinked by formic acid treatment and water-insoluble owning to their -sheet crystalline structure. After testing with cells, normal adult human epidermal keratinocytes (HEKs) and adult human dermal fibroblasts (HDFs) could be successfully co-cultured on

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such nonwovens for up to 75-95 days in vitro, thus forming a novel kind of dermo-epidermal equivalent, in which the cells were able to cohabit with reciprocal benefits as they remained viable, metabolically active and performed specific functions (e.g. the de novo production and assembly of collagen fibers) for lengthy terms, but never released urea nitrogen (an index of protein catabolism) or proinflammatory interleukin-1. These scaffolds, once implanted in the animals in vivo, induced the engineering of a novel, well-vascularized reticular connective tissue without eliciting, even six moths after their implantation [18]. The subcutaneous tissues of mice after implantation by silk fibroin-based nonwovens not only are biologically well tolerated but even guide the de novo production of a vascularized reticular connective tissue. The newly formed vascular tree guarantees the viability, i.e. the durability, of the engineered reticular connective tissue, while only a quite mild foreign body response with no fibrosis happened [18]. After 6 months in vivo, the silk fibroin nonwovens did not appear to have undergone any significant biodegradation, having in the meanwhile been smoothly integrated into the murine subcutaneous tissue. Clearly, while a major in vivo biodegradation of the implanted silk fibroin nonwovens remained possible on lengthier terms, the present results imply the notion that biocompatible silk fibroin nonwovens are to be integrated, at least provisionally, into the host’s tissue to act there as effective guides for tissue regeneration. Their findings supported the view that three-dimensional silk fibroin-based nonwovens may be excellent candidates for beneficial applications in the field of human tissue engineering, regeneration and repair. It is conceivable that three-dimensional silk fibroin nonwovens may be of use even at sites of implantation other than the skin (such as bone, cartilage, tendons). The matrix texture, as well as the nature of the biomaterial, was also reported to control cell adhesion, proliferation, shape and function [51, 52]. The effects of the scaffold’s surface microstructure on modulating the spatial organization and functions of cells have been investigated, specifically for endothelial cells [51, 53], nerve cells [51], hepatocytes [54, 55] and skin fibroblasts [56]. However, little is known about the textural effects of the fibrous matrix on tissue engineering, although the three-dimensional structure has profound effects on cell morphology, proliferation, migration, differentiation, and function [57]. The highly specific surface area and highly porous three-dimensional structure of woven or non-woven fabrics places them among the most promising material forms used in tissue engineering applications and are quite desirable for high-density cell and tissue cultures. Fibrous materials offer a potentially wide range of supra-structures, created by changing the fiber diameter, orientation, porosity and fabric (woven, knitting and non-woven) characteristics. Since silk fibroin can be formed in different structures such as woven (microfiber), non-woven (nanofiber) and film, the cytocompatibility and cell behavior on the different textures of silk fibroin were investigated in order to find the best matrices for biomedical applications [58]. Silk fibroin microfiber matrix was obtained by degumming the natural grey silk while silk fibroin film and nanofiber matrices were prepared by casting electrospinning the formic acid solutions of the regenerated silk fibroin, respectively. The cell attachment and spreading of normal human oral keratinocytes that was seeded onto the silk fibroin matrices indicated that the silk fibroin nanofiber matrix was more preferable than silk fibroin film and silk fibroin microfiber matrices for wound dressings and scaffolds for tissue engineering applications. Silk fibroin nanofiber matrix promoted cell adhesion and spreading of type I collagen better than silk fibroin film matrix due to its higher level of surface area for cells to attach and high porosity as well as surface area-to-volume ratio.

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Modification of Silk Fibroin Structures for Wound Dressing Surface modification can be used to alter cell attachment and impact cell proliferation. Surface modification with the integrin recognition sequence RGD can increase cell attachment [59]. Modification of silk fibroin with poly(ethylene glycol) showed decreased attachment of fibroblasts [60]. Silk fibroin can be functionalized using the amino acid side chain chemistry. The limitation to using silk fibroin for chemical modification is the limited total content of modifiable amino acid side chain groups: 3.3% of the amino acids contain carboxyl side groups [61] compared with 9.5% found in bovine collagen [62]. Silk has been used for a carrier of antibiotics and antimicrobial peptide for wound dressing as well. Since the bacterial contamination of wounds is an important global health care issue and is associated with many wounds, ranging from traumatic skin tears and burns to chronic ulcers and complications following surgery or device implantations. This can lead to impaired wound healing, resulting in (1) rising treatment costs and (2) a traumatic and potentially life-threatening condition for the patients [63]. Currently, wound infections caused by multidrug-resistant bacteria are a major issue in wound care. The advantages and disadvantages of systemically delivered antibiotics versus topical application to treat infected cutaneous wounds is part of a controversial discussion but still needs clarification by further research. At present, topical delivery of antibiotics is carried out by ointments, creams or gels but future treatment may consist of drug-device combination therapy and wound dressing. There are currently some tools on the market dealing with the combination of common antimicrobials incorporated into wound dressings for topical delivery. Some of these antimicrobials such as povidone-iodine and silver which are used with many modern dressings, actively affect the wound healing process [64]. These drug-device combination systems should locally release the drug continuously over a long period without exceeding the therapeutically effective concentration [65]. Because of this idea, colistin loaded ST-silk membrane was investigated for its activity against Pseudomonas aeruginosa both in vitro and in vivo [64]. The in vitro study demonstrated a concentration-dependent antimicrobial effect against P. aeruginosa with complete elimination at the highest loading concentrations (2.7, 27 and 270 mg/mL). All colistin membranes demonstrated lower colony-forming unit counts compared with the corresponding phosphate-buffered saline or carrier controls. The in vivo testing using rat burn infection model demonstrated a colony-forming unit reduction of greater than 3 log-scales for the colistin-loaded ST-silk membranes after 3 days. This study demonstrated that occlusive ST-silk membranes loaded with an antimicrobial agent may be an effective dressing for infected wounds. Bai et al. also modified the surface of Bombyx mori silk fibroin films by covalently couple antimicrobial peptide, Cecropin B (CB) [66]. The investigation of modification conditions showed that water-unsolvable silk fibroin films should be activated by 1-ethyl-3(dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl)/N-hydroxysuccinimide (NHS) solution followed by reacting in CB peptide phosphate buffer solution at ambient temperature for 2 h. The surface-modified silk fibroin films had the satisfied antimicrobial activity and durability. After the surface modification by the antimicrobial peptide, the contact angle was decreased, accompanied by a considerable increase of surface roughness. The elemental composition analysis suggested that the peptides were undoubtedly coupled to the surface of silk fibroin films. This approach may provide a new option to engineer the

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surface-modified implanted materials preventing the biomaterial-centered infection or use it as wound dressing material. Carbonized silk materials exhibit a highly antibacterial property and possess multiple numbers of dimples on their surfaces by heating and are used as supports for catalysts which are suitable for adsorption and decomposition of harmful substances and for deodorization. By utilizing the antibacterial properties of carbonized silk, many products have been developed or proposed to develop biomedical applications such as carbonized silk products for external skin preparations, wound dressing, hazardous substance decomposer, mask, carbonized cocoons for gas adsorbention [67]. Higher carbon yield and better performance of silk based carbon fiber has been developed by Khan et al. [67]. They produced carbon fibers from natural biopolymer, B. mori silk fibroin fibers treated with iodine vapor at 100C 12 h.. The carbonization process was carried out by heating to 800C in argon atmosphere at a single or multiple step carbonization process. In the case of the single step process, the obtained carbon fiber was fragile and was difficult to handle. On the other hand, both strength and the carbon yield of the carbon fibers prepared under multi-step heating were considerably increased. Besides, the highest carbon yield (ca. 36 wt%) was achieved from iodinated silk fibroin fibers under multi-step process which can be applied for many applications including wound dressing material.

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Biomaterial Films/Scaffolds by Blending Silk Fibroin with other Natural or Synthetic Polymers The structure and properties of silk fibroin films and scaffolds can be further modified by blending with other natural and synthetic polymers such as cellulose [38], chitosan [68, 69], poly(ethylene oxide) [70], polyacrylamide [71], poly(ethylene glycol) [72-74], poly(vinyl alcohol) [75], poly(-caprolactone-co-D,L-lactide) [76], collagen [39], polyallylamide [77], Scarboxymethyl keratin [78, 79] and other systems. Although most of these materials have not been fully tested in vivo for biocompatibility and degradability, a few reports have shown that silk fibroin films and some blend/composite materials promote in vivo healing when used as a wound dressing [19, 43]. Blending silk fibroin with other polymers such as chitosan resulting in the improvement of the tensile strength and water/moisture sensitivity [80]. Niamsa et al. reported that the nanocomposite blend films containing methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (MPEG-b-PDLL) nanoparticles with different chitosan/silk fibroin blend ratios were successfully prepared by film casting. The MPEG-b-PDLL nanoparticles, dispersed throughout the film matrices were approximately less than 1 m in sizes with spherical shape. The nano-composite blend films showed nanoporous structure. Because of its physical properties, these biodegradable nanocomposite blend films have potential for use in drug delivery, wound dressing as well as tissue engineering applications. Not only nanocomposite blend film has been prepared by chitosan with silk fibroin, polyelectrolyte complex porous scaffolds from both biomaterials have been studied for its application in tissue engineering. Bhardwaj and Kundu found that polyelectrolyte complex scaffolds of silk fibroin and amino polysaccharide chitosan showed pore sizes in the range of 100-160 m with good interconnectivity and high porosity [81]. The addition of silk fibroin reduced the degradation of

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chitosan containing scaffolds in lysozyme solution. The blended scaffolds showed a higher compressive strength and modulus than the individual components. Chitosan incorporation also had an antibacterial effect when incorporated at the higher levels in the blends. In vitro cytocompatiblity results demonstrated that the blended scaffolds supported the growth and adhesion of feline fibroblasts which is good indication for its use as material for skin or cartilage tissue engineering. Hyaluronic acid, one composition of the extracellular matrix which plays a vital role in tissue regeneration and angiogenesis, was added into silk fibroin solution to prepare fibroinbased porous composite scaffolds after the freeze-drying process [82]. The results indicated that hyaluronic acid exhibited important effects on pore formation and hydrophilicity of the fibroin-based scaffold. The aqueous-fibroin/hyaluronic acid scaffolds had highly homogeneous and interconnected pores with porosity of above 90% and controllable pore size ranging from 123-253 m. The water uptake of fibroin/hyaluronic acid scaffolds increased significantly with the increase of hyaluronic acid content. Containing hyaluronic acid at a defined content range, such as 3-6%, fibroin-based scaffolds’ affinity to primary neural cells was improved. They found that in 6% hyaluronic acid/fibroin scaffolds, neurosphere-forming cell migrated from their original aggregate and adhered tightly to the surface of scaffolds. This composite should be an important option for neural tissue engineering.

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GUMMING SILK PROTEIN, SERICIN, FOR WOUND DRESSING MATERIAL Various forms of silk sericin such as cream [83], gel film or film [84, 85] and scaffolds [86] has been studied for promoting its wound healing property due to the fact that it can promote cell proliferation, growth, attachment and collagen production [83, 87-89]. Sericin cream was shown to promote wound-size reduction compared to cream base [83, 90]. Both two-dimensional (films) (Figure 3) and three-dimensional matrices (hydrogel and porous scaffolds, Figure 4) from sericin have been reported. Silk sericin can be easily formed into a gel film without using any chemical modifications [84]. Silk sericin solution was gelled with ethanol into a sheet shape and then dried. This structural feature rendered the gel film morphologically stable against swelling and had food handling properties in the wet state. The silk sericin gel film rapidly absorbed water, equilibrating at a water content of about 80%, the value is as high as those of previously reported natural polymer-based wound dressings [9195], and exhibited elastic deformation up to a strain of about 25% in the wet state. The flexibility of silk sericin gel film and its ability to attach to curved surfaces are significant advantages for its application as wound dressing. Such highly elastic film material is useful for covering wounds at moving parts, such as joints. It is noteworthy that these properties of silk sericin gel film were achieved by physical intermolecular interactions among silk sericin molecules alone, whereas previously reported natural polymer-based materials generally employ chemical modifications such as cross-linking or grafting [91-95]. This feature of silk sericin gel film has great significance in its application as a wound dressing because the toxicity problem caused by residual chemicals does not need to be considered. A culture of mouse fibroblasts on the silk sericin gel film indicated that it had low cell adhesion property

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which is suitable for wound dressing material because strong cell adhesion might cause destruction of regenerated tissues when the dressing is peeled off and it also shows no cytotoxicity. This silk sericin gel film also exhibited optical transparency which might be an additional advantage since it enables direct observation of wounds.

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Figure 3. Silk Sericin Film.

Figure 4. Silk Sericin Scaffold.

Due to its high oxygen permeability, high surface area (as shown by Scanning Electron Microscope (SEM) in Figure 5) and moisture absorption compared to film or cream, silk sericin scaffold seems to be a better candidate for wound healing material. However, membranes of silk sericin itself are fragile in the dry state. Blending silk sericin with watersoluble polymers such as polyvinyl alcohol for making film or scaffold has been investigated [96, 97]. Sericin hydrogels blended with polyvinyl alcohol by irradiation at 40 kGy have been

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reported [98]. Blended hydrogels have excellent moisture adsorbing, desorbing and elastic properties and have potential applications ranging from soil conditioners to materials for medical use including wound dressings [99].

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Figure 5. Scanning Electron Microscope (SEM) of Sericin Scaffold.

Hydrogels are also prepared using silk sericin from sericin-hope cocoons [100], which consist of mostly sericin (>98%). Sericin from this mutant sericin-hope cocoon contains intact sericin and forms elastic hydrogels with the addition of ethanol, without any cross-linking by chemicals or irradiation. Films prepared from sericin-hope cocoons behave like fibrous material when hydrated and can be used for drug delivery and tissue engineering purposes without any toxicity [101, 102]. Silk sericin membranes also can be prepared from native silk sericin protein extracted from the middle silk gland of non-mulberry Antheraea mylitta [86]. Silk sericin protein extracted from the gland contained a higher amount of -sheets, which increased upon treatment with ethanol. Gland source not only allows extraction of native silk sericin but also problems related to high brittleness and difficulty in malleability of silk sericin membrane were overcome. The membranes did show robustness, good mechanical strength and high temperature stability. Cytocompatibility of the membranes on feline fibroblast cells indicated normal spreading and proliferation on the silk sericin membranes. Moreover, this membrane showed low inflammatory response which seems to be a good candidate for biomedical applications. Interpenetrating polymer networks (IPNs) from silk sericin and poly(N-isopropyl-acrylamide) using cross-linking agents can be formed successfully which exhibited channels homogeneously distributed throughout the network membrane [103]. Similar structure has been found in silk sericin with polyvinyl alcohol scaffolds [97]. As mentioned earlier, silk sericin itself formed a fragile scaffold. Adding other polymers such as polyvinyl alcohol and a plasticizer such as glycerin can improve its properties. Uniform pore distribution, stable structures with good compressive strength, high swellability and the desired level of silk sericin released from scaffold can be achieved by varying the concentration of the crosslinking agent. Other polymers have been used to blend with silk sericin in order to form stable scaffolds as well. Gelatin is of great interest because of its abundance, cost effective-

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ness and excellent functional properties. It is used widely in the medical industry as a plasma expander, wound dressing and as a controlled drug-release agent [104-106]. In addition, its use has been reported in matrices for release of bioactive components and growth factor enhancers, corneal-wound healing, neural-cell transplantation, and as scaffolds for tissue engineering [107, 108]. Mandal et al. prepared three dimensional silk sericin/gelatin scaffolds using non-mulberry A. mylitta silk cocoon sericin protein [109]. Blended silk sericin/gelatin three dimensional scaffolds were highly porous with an optimum pore size of 170  20 m. The scaffolds were robust with enhanced mechanical strength and showed high compressibility. Swelling studies showed high swellability along with complete degradation in the presence of phosphate-buffered saline. Cell cycle of feline fibroblasts analysis showed cytocompatibility without any cell cycle arrests. Low immunogenicity of the matrices as observed through tumor necrosis factor- release revealed its potential as future biopolymeric graft material. Not only skin wounds have been healed using silk sericin as biomedical material, corneal wounds in rats showed a significantly higher healing rate when treated with silk sericin [110, 111]. Cornea is a highly specialized and unique organ, it is continually subjected to abrasive forces and occasional mechanical trauma due to its anatomic location. Damage to the cornea can result in scarring or opacification, causing visual defects that compromise transparency and that can even lead to a complete vision loss. The corneal wound repair process involves cell adhesion, migration, proliferation, matrix deposition and tissue remodeling [112]. Many of these biological processes are mediated by growth factors, cytokines and other mediators released in injured tissues or cells [113]. Since silk sericin has been reported to promote healing of skin wounds, Nagai et al. hypothesized that silk sericin may enhance corneal wound healing in the debrided corneal epithelium of rats [111]. They removed the epithelium from the corneas of rats and corneal wounds were monitored using a fundus camera equipped with a digital camera. The results showed that corneal wounds of rats instilled with saline was approximately 10% healing at 12 h, and approximately 65% healing at 24 h after corneal epithelial abrasion. The corneal wounds of rats instilled with saline showed almost complete healing by 36 h after corneal epithelial abrasion while healing rate of the one instilled with the sericin solution was higher than the saline-treated one. The centripetal movement and proliferation of corneal epithelial cells are important for the healing of corneal wounds in rat exfoliated corneal epithelium. In general, it is known that epithelial cells from the surface reduce and eventually cover the wound surface, with cell proliferation providing cells to rebuild the tissue and tissue remodeling to restore the stratified epithelium [114-117]. Normally, cell proliferation starts approximately 12 h after corneal epithelial abrasion [118]. Therefore, the reduction in the size of the wound may be due to cell movement during the 12 h after corneal epithelial abrasion, followed by cell proliferation. They also found that the corneal healing rate constant increased with increasing sericin concentration. In addition, the adhesion and proliferation of human cornea epithelial cell line treated with 0.01-0.5% sericin solutions were enhanced, reaching a maximum at treatments with 0.2 and 0.1% sericin solution, respectively. They concluded that silk sericin solution has a potent effect in promoting corneal wound healing in rats, probably due to increased cell movement and proliferation. These findings provide significant information for designing further studies to develop potent corneal wound-healing drugs. They further investigated this effect in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model for human type 2 diabetes [110]. Since

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diabetic keratopathy is a well-known cause of ocular complications secondary to type 2 diabetes mellitus, it is an entity that includes slow healing or loose adhesion of the corneal epithelium after wounding in diabetic patients [119]. Clinically, the damage to the corneal epithelium during vitreous surgery and retinal photocoagulation sometimes induces visionthreatening corneal complications, such as persistent epithelial defects in diabetic patients [120]. The increase of glucose level in the cornea and tears are important mechanisms underlying the delay in corneal wound healing in type 2 diabetic mellitus. High glucose levels suppress the proliferation of human corneal epithelial cells [121]. Using silk sericin solutions, 5 and 10%, to treat corneal wound in OLETF rats showed that it increased the healing rate within 12 h after corneal epithelial abrasion without effect on glucose level. This result is similar to their results reported earlier that silk sericin solution can promote corneal wound healing. They concluded that silk sericin is safe and can be used in diabetic patients to treat corneal wounds.

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CONCLUSION The wide range of molecular structures, remarkable mechanical properties, morphology control, versatile processability and surface modification options make silk protein an attractive polymeric biomaterial for design, engineering and processing into film, hydrogel, and scaffolds for several applications including materials for wound healing for skin, the largest organ in the human body. Three-dimensional porousity of silk fibroin and sericin with or without other polymers or structural modifications possess favorable surface morphology, useful mechanical features, biocompatibility, and the ability to support cell adhesion, proliferation, and differentiation have expanded silk-based biomaterials as promising scaffolds for tissue engineering. The further advantages of silk materials are their generally slow rates of degradation of silk fibroin in vivo, both silk fibroin and sericin can be coupled with the versatile control of structure, morphology and surface chemistry, offer a range of utility for this family of protein polymers in many needs in biomaterials. To date, most of the impact with silk-based biomaterials has been with only one source of silk, from B. mori silkworm. As new sources of silk proteins become available, such as from spiders and via genetic engineering and modification of native silk sequence chemistries, the range of material properties can be generated and utilized for biomaterials for further expand options and lead to additional medical impact.

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[119] Schultz, R.O., D.L. Van Horn, M.A. Peters, K.M. Klewin, and W.H. Schutten, Diabetic keratopathy. Trans Am Ophthalmol Soc, 1981. 79: p. 180-99. [120] Perry, H.D., G.N. Foulks, R.A. Thoft, and F.I. Tolentino, Corneal complications after closed vitrectomy through the pars plana. Arch Ophthalmol, 1978. 96(8): p. 1401-3. [121] Fujita, H., I. Morita, H. Takase, K. Ohno-Matsui, and M. Mochizuki, Prolonged exposure to high glucose impaired cellular behavior of normal human corneal epithelial cells. Curr Eye Res, 2003. 27(4): p. 197-203.

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ISBN: 978-1-62100-692-3 © 2012 Nova Science Publishers, Inc.

Chapter 15

USE OF SILK IN TENDON/LIGAMENT TISSUE ENGINEERING Thomas K.H. Teh1, 21, Siew-Lok Toh,1,3 and James C.H. Goh1,2 1

Departments of Bioengineering, National University of Singapore, Singapore 2 Orthopaedic Surgery, National University of Singapore, Singapore 3 Mechanical Engineering, National University of Singapore, Singapore

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ABSTRACT In this chapter, we will focus on the application of silk fibroin (SF) as material for tendon/ligament tissue engineering scaffolds and contrast this treatment method with other existing modalities for ruptured tendon/ligament. Upon demonstrating the benefits of using SF as material for tendon/ligament tissue engineering scaffold, the various scaffold architectures will also be compared, with detailed works on the knitted structure being presented, highlighting the state of the art in tendon/ligament tissue engineering 1

Teh Kok Hiong Thomas is a Research Fellow in the Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore. His areas of research encompass tissue engineering, biomaterials and biomechanics with particular interest in the biomechanical and biochemical cues in tendon/ligament regeneration. He is an active member of the Biomedical Engineering Society, Singapore as well as a member of TERMIS-SYIS Asia Pacific and Stem Cell Society, Singapore Toh Siew Lok is an Associate Professor and the Deputy Head of the Department of Bioengineering, National University of Singapore. He is the Vice President of the Biomedical Engineering Society (Singapore), the Secretary of the International Federation of Medical and Biological Engineering Asia Pacific Working Group. He is a Chartered Engineer with the Institute of Mechanical Engineers (UK) (IMechE), a member of the American Society of Mechanical Engineers (MASME) and a member of the Institute of Engineers Singapore (MIES). He was the Treasurer of the World Congress in Biomechanics held in Singapore in 2010. His areas of research include Experimental Mechanics, Biomechanics, Functional Tissue Engineering with a particular interest in the use of silk in tissue engineering of ligaments. Goh Cho Hong, James is Professor and Head of the Department of Bioengineering, National University of Singapore. He is the President of the Biomedical Engineering Society (Singapore), the Secretary-General of the International Federation of Medical and Biological Engineering. He is also a Member of the World Council of Biomechanics, the Secretary-General of the Asia-Pacific Association for Biomechanics and the Treasurer of the Executive Council for the World Association for Chinese Biomedical Engineers. His areas of research include musculoskeletal tissue engineering and orthopaedic biomechanics with particular interest in the use of silk in tissue engineering of ligaments. E-mail address: [email protected], [email protected], [email protected]

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research and its future trends. Prior to this, however, it is necessary to gain an understanding of the function and microstructure of the tissue of interest, and the prevalence of its injury in order to appreciate the significance of the matter.

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INTRODUCTION Silk fibroin (SF) of the Bombyx mori is a valuable material that has been used diversely in applications ranging from textile to the biomedical industries. It is often sought for in biomedical applications that demands mechanical strength and elasticity [1-3], such as in the case of sutures and surgical meshes. The material has also been commonly regarded to be suitable for application in permanent implants as it was regarded to be non-degradable in vivo, with high retention of its initial tensile strength beyond 60 days of implant [1]. Silk fibroin protein primarily exists in two different structural conformations as Silk I and Silk II [4, 5]. While Silk II is defined as anti-parallel β-sheets with polypeptide main chains that are aligned and adjacent chains connected by hydrogen bonds, Silk I composes of α-helix and random coil structures [6, 7]. The transition from metastable Silk I to the stable and mechanically viable Silk II form occurs upon external stimulation such as heating and shearing [8-10]. It is the Silk II protein structural arrangement that the superior mechanical properties of SF is attributed to. Despite the excellent mechanical properties, there was generally an initial reluctance in applying B. mori silk for tissue engineering applications due to concerns raised in the past with regards to inflammatory responses invoked by the material in several biomedical applications [11-19]. Over the years there has been better understanding of the material, as it is realized that SF will not likely induce hypersensitivity if the wax-like sericin coating in raw silk is removed via the degumming process [1, 3, 16, 20-22]. Moreover, with long term in vivo studies, it is clear that SF does degrade over time via proteolytic degradation in vivo and is absorbed by the body, though a longer period of time is required. It is the extensive hydrogen bonding and significant crystallinity in the SF protein structure that are responsible for the long degradation period of SF in vivo. The combination of these two characteristics thus made SF a unique material suitable for application in tissue engineering scaffolds where mechanical robustness and long term degradation is required. Consequently, SF is typically used in musculoskeletal and orthopaedic tissue engineering applications, such as tendon/ligament repairs [23-26], where a very gradual transfer of load from the scaffold to the growing tissue is desired.

FUNCTION AND MICROSTRUCTURE OF THE TENDON/LIGAMENT Tendons and ligaments are short bundles of tough fibrous connective tissues, which function to translate motion between muscles and bones or to provide stability in the musculoskeletal system by providing bone to bone attachment at joints [27-31]. During motion, the contraction of a muscle results in load transfer from the muscle, via its tendon, to a bone across a joint, which results in movement of the bone around the joint. This motion, at the same time, strains the ligaments between the bones. Through this sequence of events, it is

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clearly illustrated that tendons operate to bring about movements around the joints while the ligaments prevent excessive motion of the joints and thereby provide stability [27-31]. Microscopically, the tendons/ligaments are composed of parallel collagenous fibrils, consisting of triple helix tropocollagen molecules, arranged in a multi-level hierarchy ranging from submicron fibrils to micron level fibers and to larger entities called fascicles. Such an organization provided the tissues excellent axial tensile load bearing capacity [32-34]. In fact, the collagen fiber bundles are arranged in the direction of functional need and act in conjunction with elastic and reticular fibers, with ground substance that are composed of glycosaminoglycans (GAG) and tissue fluid, to give tendons/ligaments their mechanical characteristics, constituting characteristic features of fiber reinforced composites [35]. In unstressed tendons/ligaments, collagen fibers take on a sinusoidal pattern, often referred to as a "crimp" pattern, and it is believed to be created by the cross-linking or binding of collagen fibers with elastic and reticular fibers. A structure as such optimizes axial tensile load bearing capacity by imparting great strengths with limited extensibility and is essential for tendons/ligaments to perform their function [32, 33, 36].

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INJURY PREVALENCE AND HEALING OF THE TENDON/LIGAMENT Of the various tendon/ligament tissues, the anterior cruciate ligament (ACL) is one of the most highly stressed. It is essential for maintaining physiological knee mechanics and joint stability by resisting the anterior tibial translation and rotational loads [37-39]. Although the ACL functions optimally under normal physiological loading, it is one of the most frequently injured structures [40]. It is common for the ACL to rupture or tear due to shocks sustained through contact sports, as it takes up approximately 75% of the anterior shock load at the knee. It has been estimated recently that 11 in 1000 people, out of the general population, suffer knee ligament injuries per year [41]. The ACL is the most commonly injured of the knee ligaments, contributing to 80% of total knee ligament injuries, with 65% of the operated injured ACLs predominantly associated with sports and recreational activities [41]. The rupture or tear of ACL can cause significant knee joint instability, which can lead to injuries of other ligaments and development of degenerative joint diseases, transitioning from knee instability, to meniscus tears and leading to the eventual osteoarthritis [42, 43]. Nevertheless, the injured tendon/ligament has poor ability to heal intrinsically. This is largely due to the lack of vasculatures in the tissues [44, 45]. Factors that can moderate the rate of healing include age, systemic factors and local factors such as blood supply, synovial environment (for ligaments within synovial capsules), mechanical stresses and inflammatory cellular response [46]. Despite these factors, the healing process generally encompasses three phases of varying duration and rates: the acute inflammatory or reactive response phase, the regenerative or repair phase, and the tissue remodeling or maturation phase [27, 31, 40, 4751]. Intrinsic healing as such often resulted in healed tissues with persistent disorganization and abnormalities, resulting in the inability of the healed tissue to regain its original properties. It is therefore necessary to surgically intervene the reparative process to both expedite and improve the quality of tissue healing. With an estimated 200,000 patients in America who required reconstructive surgery of the ligaments in 2002, billing over five billion dollars [52], there is a clear need for cost and time effective treatment methodologies.

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TISSUE ENGINEERING AS THE FUTURE TREATMENT STANDARD FOR RUPTURED TENDON/LIGAMENT In the field of sports medicine, and especially that for ruptured tendon/ligament treatment, surgical reconstruction has been the standard treatment modality [53], which very often sacrifices effective tissue self regeneration over the long run for the short term goal of accelerated functional restoration. Since there is limited tissue self regeneration, these methods very often fail with time. The use of autografts, allografts or synthetic grafts has been typically practiced for the restoration of knee joint function. However, several disadvantages and risks persist in these methods, which include ligament laxity, donor site morbidity, pathogen transfer, mechanical mismatch, poor tissue integration and foreign body inflammation [54-56]. These complications often necessitate revision surgeries, which further interrupt site recovery and burden not only the patients financially but also the medical services of its resources. Consequently, there is an increased need to research for more effective treatment solutions [57, 58], of which tissue engineering has evoked much interest as it offers the potential of regenerating autologous functional tissues [23, 24, 33, 42, 59, 60]. For the purpose of tissue engineering tendon/ligament tissues, the goal will be to generate neotissue from autologous cells grown on biocompatible and biodegradable scaffolds. The scaffold is an important component of a tissue engineered tendon/ligament as it translates two-dimensional (2D) cell culture to three-dimensional (3D) structure and assume the role of mimicking the extra cellular matrix (ECM) to support the development of functional neotissue. Specific to the tendon/ligament tissue, it should be designed for immediate functional mechanical demands of the reconstructed knee, while being able to subsequently degrade at a rate similar to tissue ingrowth. In other words, the tendon/ligament scaffold should lose its mechanical integrity gradually and allow the remodeled tissue to regenerate and gain strength by gradually loading it. An ideal scaffold typically possesses the following characteristics: 1) 2) 3) 4)

Biocompatible and biodegradable material that is suited for distinctive applications Suitable porosity that allows cell infiltration and medium perfusion into the scaffold Sufficient surface area for cell attachment, growth and proliferation Architecture or geometry that facilitates tissue attachment and regeneration, while imparting the required mechanical properties at various stages of tissue regeneration.

SUITABILITY OF THE KNITTED SILK FIBROIN SCAFFOLD FOR TENDON/LIGAMENT TISSUE ENGINEERING To achieve these requirements, the material and the architectural aspects of tendon/ligament tissue engineered scaffolds have been extensively studied by our group. A variety of scaffold materials that are biodegradable have been explored, with popular choices ranging from the synthetic poly(α-hydroxyester) family to natural polymers such as collagen and SF. The intention of utilizing biodegradable materials in scaffolds for tendon/ligament tissue engineering is motivated by the disadvantages of previous non-degradable materials. These non-degradable materials have caused inflammatory responses and rendered the need

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for revision surgeries due to the release of wear particles and other materials into the surrounding tissue structure. Synthetic biodegradable polymers have been popularly used for orthopaedic applications. They include poly(α-hydroxy) acids, poly(ε-caprolactone), poly(orthoester), copoly(etherester), poly(carbonate), poly(iminocarbonate) and poly(dioxanone) [61, 62]. The advantages of these materials include controllable chemical uniformity and physical properties, and modifiable degradation and mechanical properties through chemical manipulations [63, 64] to make them suitable for a variety of applications. In particular, the poly(α-hydroxyester) family is often used for tendon/ligament tissue engineering applications since they are FDA approved and commercially available in fibrous form (Table 1). Table 1. Physical and mechanical properties of the poly(α-hydroxyester) family and silk fibroin. [1, 61, 65-70] Natural Polymer

Synthetic Polymer Copolymer

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Polymer

(10:90)

DLPLAGA (50:50)

DLPLAGA (85:15)

[C2H2O2]y

[C3H4O2]10 [C2H2O2]90

[C3H4O2]50 [C2H2O2]50

[C3H4O2]85 [C2H2O2]15

[C3H4O2]x

339-394 (fibers)

570-910 (fibers)

41.4-55.2

41.4-55.2

24

Complete resorption: 12-24

PGA

PLAGA

Composition

100

Chemical Structure Tensile Strength (MPa) Maximum Strain (%)

Approximate Resorption Duration (months)

15-35 (fibers) Loss of mech. prop.: 1 month Full resorption: 50-75 days

PLLA

SF

100

NA (Gly-SerGly-AlaGly-Ala)n

The family is made up of poly(glycolide) (PGA), poly(L-lactide) (PLLA) and their copolymers of poly(lactide-co-glycolide) (PLAGA), which can be synthesized in a variety of methods [66, 71]. The poly(α-hydroxyester) family degrades via hydrolysis of the ester bonds and bulk erosion, with a loss of mechanical strength over a period of 2-4 weeks for PGA to 24 weeks for PLLA in pH 7 fluid at 37°C [68, 70, 72]. Lactic acid is released during the course of degradation for PLLA and its copolymers. Specifically for PLAGA polymers, the pH of the degradation solution (mainly lactic acid) can cause autocatalysis, whereby the lowered pH in the local environment can cause increase in degradation rate [61, 73]. Although cellular

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and tissue biocompatibility of polylactides have been shown in several toxicological studies and the American Gentox program [61], problems persist to limit their application as biodegradable polymers. These problems include issues due to the progress of resorption process in vivo, control of mechanical properties with degradation in vivo, effect of local pH decrease, burst phenomenon and the possibility of mutagenicity due to the degradation products [61, 66, 67, 69, 74-78]. On the other hand, collagen tendon/ligament implants have been used experimentally and was found to degrade by a sequential attack from lysosomal enzymes [79]. The degradation rate can be controlled via the extent of cross-linking involved. However, limitations such as allogenicity of the collagen, batch-to-batch variability and the lack of flexibility in fabrication and modification persist with the use of this material. Unlike these materials which have exhibited poor mechanical strengths and short degradation periods in the context of tendon/ligament tissue engineering, SF has been shown to be a promising candidate for this application upon removal of the hyper-allergenic sericin component from raw silk [23-25, 33, 80, 81]. The material also has compatible degradation rate that involves a gradual loss of tensile strength over 1 year in vivo due to proteolytic actions [1, 33]. More importantly, SF has outstanding and customizable mechanical properties, with superior strength and elasticity, making it suitable for use in constructs with high porosity without compromising the overall mechanical robustness of the construct [1, 25, 33, 81, 82]. As a natural protein, SF has been shown to act as a suitable structural template and bears equivalence to collagen for cell attachment and growth [1, 34, 83]. To further mimic the ECM structure, SF has been successfully electrospun to form sub-micron nonwoven meshes, which is found to enhance cell adhesion and spreading of type I collagen due to its high surface to volume ratio [80, 84, 85]. From the architectural perspective, tendon/ligament tissue engineered scaffolds need to be mechanically sound and possess similar loading responses to the native tissue such that mechanical cues that resemble the native environment can be transferred to the developing neo-tissue. Textile technologies have been used to fabricate woven or non-woven scaffolds [86, 87]. Some of the textile technique that have been used for tendon/ligament tissue engineering scaffolds include axial fiber structures, woven structures, 2-D braids and knitted structures. Studies performed using the braided and woven structures have shown incompetence of these architectures in supporting uniform tissue regeneration resulting from poor nutrient transmission, cell attachment, infiltration and matrix production, especially in the early tissue regeneration phases [88-90]. On the other hand, the knitted fibrous structures are made by interlocking a series of loops, involving one or more yarns, to create a porous fabric for tissue ingrowth and have been used in LARS and Stryker ligaments [67, 74, 91-93]. The knitted structures have also been made with PLGA and have been shown to have high porosity (>50%) and more interconnected voids compared to braided structures, especially at their tensioned states [94]. These spaces allow effective cell adhesion and medium perfusion, thereby stimulating uniform ECM formation, which is critical during the repair process and helps functional integration of the engineered tissue into the surrounding tissues. Although it may not have as high a loading capacity as the braided structures, the use of a mechanically superior material such as SF can compensate for this limitation.

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OPTIMIZING DEGUMMING CONDITIONS FOR RETENTION OF SILK FIBROIN MECHANICAL PROPERTIES FOR TENDON/LIGAMENT SCAFFOLD APPLICATION One of the fundamental reasons of choosing SF as scaffold material for tendon/ligament tissue engineering is its excellent mechanical properties. However, this is very much dependent on the process of removing sericin from raw silk. Typically the thermo-chemical treatment approach is being used in degumming of raw silk to attain SF, which will generally affect the structural state of SF. Consequently, there will be changes in the SF microstructure, which will inevitably alter the mechanical and degradation properties of SF. This has been reported by Pe’rez-Rigueiro et al. who observed adverse effects of degumming with distilled water on the tensile strength of forcibly reeled silkworm silk fibers [95, 96]. Jiang et al. have also investigated the tensile behavior and morphology of SF upon degumming using different solution types and observed varying extent of reduction in tensile properties of SF with different degumming solutions [97]. The various degumming chemical solution types can be largely classified into two categories: alkaline and enzymatic. Both of these types of chemical degumming uses a combination of sericin removal approach, which includes dispersion, solubilization and hydrolysis of the various sericin polypeptides [98]. The primary mechanism utilized by alkaline degumming is hydrolysis. Since hydrolytic agents are used, moderation of the conditions used is important. Although recent studies have looked into using proteolytic enzymes for degumming purpose, there exist several limitations to the method [99-102]. These limitations include higher shear and bending rigidity of resultant SF, the presence of residual sericin at the overlap points and core of processed silk structures [103] and the higher cost of enzymes as compared to chemicals used for alkaline degumming. Even though it has been shown by investigators that amongst the various degumming methods, alkaline degumming using Na2CO3 causes significant structural and mechanical changes [97, 104], it remains as a popular method due to the high effectiveness of sericin removal within a relatively short duration [1, 3]. Moreover, it is often necessary to degum silk of a processed form, such as knits or braids. These architectures are generating much interest in recent load-bearing tissue engineering development where SF is used as the scaffold material [1, 3, 23-26]. Although fabricating scaffold from raw silk instead of degummed silk can ease fabrication process and better protect SF by the sericin coating during the fabrication process, it poses new challenges. Challenges arise as it is difficult to remove sericin from the core of these structures, where raw silk is not exposed to the degumming solution. In these cases, degumming using the Na2CO3 alkaline method proves to be effective and produces scaffolds with negligible hypersensitivity and inflammatory reactions [24-26]. To maximize the benefits of using knitted SF as scaffold, we have optimized the degumming method and identified a set of conditions, such that SF hydrolytic damage, together with the resultant mechanical and structural deterioration, is reduced [81]. This is essential for the production of functional scaffolds for tendon/ligament tissue regeneration where the mechanical aspect is crucial. In this study, Teh et al. degummed knitted silk scaffolds for various durations (5–90 min) and temperatures (60–100 °C). Mechanical agitation and use of refreshed solution during degumming were further included as part of the degumming process to investigate how these factors contributed to degumming efficiency and

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fibroin preservation. Characterizations of SF morphology, mechanical properties and protein components were determined by scanning electron microscopy (SEM), single fiber tensile tests and gel electrophoresis (SDS–PAGE), respectively. Sericin removal was ascertained via SEM imaging and a protein fractionation method involving SDS–PAGE. Results showed that fibroin fibrillation, leading to reduced mechanical integrity, was mainly correlated with prolonged degumming duration. This resulted in a decrease of fracture point, elastic modulus and yield point with increasing degumming duration. Specifically, there was a significant drop in mechanical properties observed in the samples that were degummed for more than 30 min. This phenomenon was consistently observed regardless of variations in other degumming conditions. Refreshing of the degumming solution and mechanical agitation also aggravated the decline in degummed SF mechanical properties. These observations suggested that harsher degumming conditions as such disrupted the secondary structure and weakened the non-covalent interaction of fibroin chains, such as van der Waal’s and hydrogen bonds. This was further positively indicated by the presence of fibrillations observed in these groups. Although the rate of mechanical deterioration was reduced when degumming was conducted at lower temperatures, sericin was observed to be present even after prolonged degumming, rendering the conditions non feasible. Through the series of optimization, knitted SF scaffolds were observed to be optimally degummed and experienced negligible mechanical and structural degradation when subjected to alkaline degumming with mechanical agitation for 30 min at 100 °C.

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IN VITRO CHARACTERIZATION OF THE HYBRID SILK FIBROIN SCAFFOLD FOR TENDON/LIGAMENT TISSUE ENGINEERING APPLICATION With the main knitted SF undertaking the bulk physiological loading during the early tissue development stages, it is necessary to integrate a substrate component to close up the pores of the knitted structure to facilitate initial cell seeding and form the hybrid SF scaffold. Our group has investigated the feasibility of two different types of such substrate: the electrospun SF mesh and the SF sponge. The electrospun SF mesh mimicked more closely the ECM structure, as it was composed of nonwoven meshes of sub-micron diameter fibers and was found to enhance cell adhesion and spreading of Type I collagen due to its high surface to volume ratio [3, 34, 105]. The mesh thus complemented the SF knit that had SF fibers ranging from 8-14 µm in diameter; a range comparable to that of collagen fibers (1-20 µm) seen in natural tendon/ligament tissues [24]. Furthermore, it is possible to customize the alignment of the electrospun SF mesh to provide topographical cues for more effective tendon/ligament tissue development (Figure 1). In yet another study, aligned SF electrospun fibrous meshes were produced and integrated with the SF knit to form the aligned SF hybrid scaffold (AL) [106]. Mesenchymal stem cells that were seeded on the AL scaffolds were shown to be proliferative and aligned along the electrospun silk fibers’ direction of alignment, forming oriented spindle-shaped morphology and produced an aligned ECM network. Such aligned morphology was not observed in the group with random electrospun SF (RD, Figure 2).

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B

S: Knitted SF, E: Electrospun SF. Magnification: 64 ×. Scale bar: 200 µm.

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Figure 1. Phase contrast micrograph illustrating knitted SF integrated with (A) randomly arranged electrospun SF and (B) aligned electrospun SF.

Magnification: 400 ×. Scale bar: 20 µm. Figure 2. Confocal micrograph illustrating actin fibers (red) and nuclei (blue) of fluorescent-stained MSCs seeded on (A) RD and (B) AL scaffolds, and cultured for 3 days.

Gene expression and production of ligament-related proteins were also increased as compared to hybrid SF scaffolds with randomly arranged electrospun SF fibers, indicating that the aligned SF topography acted as differentiative cues towards forming ligament fibroblasts. Consequently, the cultured aligned constructs were significantly stiffer and stronger as compared to the randomly arranged counterpart. These results thus indicated that the fibrous alignment provided favorable topographical cues, which worked synergistically with the SF material that provided suitable surface chemistry to promote cellular and ECM alignment. The aligned hybrid SF scaffold system is thus promising for the enhancing cell proliferation, differentiation, and thre function for tendon - ligament tissue engineering applications.

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We have also explored the use of SF sponge as seeding substrate for the hybrid SF scaffold system. In this scaffold system, weblike micro-porous SF sponges were formed in the pores of the knitted structure (Figure 3) to function as an ECM-like substrate to support initial cell attachment and growth. The system was designed such that the knitted structure held the micro-porous SF sponges together and provided structural strength, while the microporous structure of the SF sponges mimicked the ECM to promote cell proliferation, function, and differentiation. The hybrid scaffold was fabricated via a lyophilization process of the knitted SF soaked in aqueous SF solution (2% w/v). Upon completing the lyophilization process, SF sponge would remain from the aqueous SF solution and would be integrated with the SF knitted mesh.

KS: Knitted silk, SS: SF sponge. Magnification: 50 ×. Scale bar: 500 µm. Figure 3. Scanning electron micrograph of weblike micro-porous SF sponge integrated with knitted silk.

In an in vitro study conducted using adult human bone marrow-derived mesenchymal stem cells (hMSCs), it was found that the hMSCs adhered and grew well on the hybrid SF sponge scaffold [23]. Specifically, it was demonstrated that MSCs on the hybrid SF sponge scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of ligament-related genes (e.g., type I, III collagen and tenascin-C) also increased with time. When compared with the group that used the fibrin gel system for cell seeding instead of the SF sponge, the hMSCs exhibited more active cellular function as evident from the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ligament-related gene markers, immunohistochemical and Western blot evaluations of ligament-related ECM components. Based on these results combined with that obtained from 3-(4,5-dimethylthiazol2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay (Figure 4) and Glycosaminoglycan (GAG) production (Figure 5), the hybrid SF sponge scaffold was shown to promote proliferation, function, and differentiation of hMSCs at both gene and protein levels.

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* Significant difference between two groups at p