Sponsored by

(TERMIS)

(Elsevier)

Tissue Engineering & Regenerative Medicine International Society

Biomaterials

Department of Science & Technology

Workshop on

16-17 March 2011

New Visions for Biomaterialsand Regenerative Medicine

Organized by

Professor David Williams, FREngEditor-in-Chief, Biomaterials

Biomaterials

Sree Chitra Tirunal Institute for Medical Sciences & Technology Thiruvananthapuram, Kerala

  

Workshop on

New Visions for Biomaterials and Regenerative Medicine

16-17 March 2011

Organized by

Professor David Williams, FREng Editor-in-Chief, Biomaterials

President-elect, Tissue Engineering & Regenerative Medicine International Society (TERMIS)

Director of International Affairs Wake Forest Institute of Regenerative Medicine, North Carolina, USA

Visiting Professor Sree Chitra Tirunal Institute for Medical Sciences & Technology, Kerala, India

Emeritus Professor, University of Liverpool, UK

Sponsored by

Tissue Engineering & Regenerative Medicine International Society (TERMIS)

Biomaterials (Elsevier)

Department of Science & Technology, Govt. of India, New Delhi

Sree Chitra Tirunal Institute for Medical Sciences & Technology

Thiruvananthapuram, Kerala

Endorsed by

Society for Biomaterials and Artificial Organs, India (SBAOI) Society for Tissue Engineering and Regenerative Medicine, India (STERMI)

 

New Visions for Biomaterials and Regenerative Medicine 16th ‐ 17th March 2011 

 Preface 

 Professor  David Williams  has  been  appointed  as  a  Visiting  Professor  at  the  Sree Chitra  Tirunal  Institute  for Medical  Sciences  &  Technology,  Thiruvananthapuram, India. He has traveled to, and lectured in, India for the last 30 years.  In 2010 he was elected as President‐elect of TERMIS and will take up the role of President in 2013, when  will  have  responsibility  for  global  developments  in  tissue  engineering  and regenerative medicine. He is also Editor‐in‐Chief of the journal Biomaterials.  Although  some very  good work  is  performed  in  India  in  biomaterials  science  and tissue  engineering,  scientists  from  India  are  currently  under‐represented  at International  conferences  and  publish  less  frequently  in  the  major  journals  than many competing countries.  It  was  proposed,  therefore  that,  during  his  March  2011  visit  to Thiruvananthapuram, Professor Williams will organize and deliver a Workshop that will  be  devoted  to  instructional  presentations  on  the  directions  that  biomaterials science are currently taking, and to guidance on research and publication strategies that will help Indian scientists in this very competitive area.  With this perspective, a two day workshop is organized by Prof. DF Williams. This workshop  is  sponsored  by  Biomaterials  journal,  Tissue  Engineering  and Regenerative  Medicine  International  Society,  Department  of  Science  and Technology,  New  Delhi  and  Sree  Chitra  Tirunal  Institute  for  Medical  Sciences  & Technology,  Thiruvananthapuram.  This  Workshop  is  endorsed  by  Society  for Biomaterials  and  Artificial  Organs  (India)  and  Society  for  Tissue  Engineering  and Regenerative Medicine (India).  On  behalf  of  Prof.  David  F  Williams  we  welcome  you  all  to  this  Workshop  at SCTIMST, Thiruvananthapuram. The encouragement and support we received from Prof. K. Radhakrishnan, Director, SCTIMST and Dr. G.S. Bhuvaneshwar, Head, BMT Wing, has been the inspiration in the development of this workshop. We appreciate the  speakers  for  sparing  their  time,  Mr.  Willi  Paul  and  Dr.  Rekha  MR  for  overall efforts,  institute  faculty  and  staff members of  Facility  for Advanced Drug Delivery Systems  (FADDS)  and Biosurface Technology Division  for  their  help  in  organizing this workshop. 

Chandra P. Sharma Coordinator NVBRM 2011 BMT Wing, SCTIMST,  Thiruvananthapuram 

New Visions for Biomaterials and Regenerative Medicine

March 16-17, 2011

Patron Dr. K. Radhakrishnan Director, SCTIMST

Advisor Dr. GS Bhuvaneshwar

Head BMT Wing, SCTIMST

Coordinator Dr. Chandra P. Sharma

Senior Scientist G & Associate Dean, SCTIMST

Treasurer Mr. Willi Paul

Organizing Committee Dr. Anilkumar PR Dr. Anilkumar TV

Dr. Annie John Dr. Anoop Kumar T

Dr. Anugya Bhatt Mr. Arun Anirudhan

Mr. Balram S Dr. Harikrishna Varma PR

Dr. Harikrishnan VS Dr. Jayabalan M Dr. Jayasree RS

Dr. Kalliyanakrishnan V Dr. Kumary TV

Dr. Leena Joseph Dr. Lissy K. Krishnan

Dr. Lizzymol PP Dr. Manoj Komath

Dr. Maya A. Nandakumar Dr. Mira Mohanty Dr. Mohanan PV

Mr. Muraleedharan CV Mr. Nagesh DS Mr. Neelakantan Nair OS Dr. Niranjan D Khambete Dr. Prabha D Nair Mr. Rajkrishna Rajan Mr. Ramesh Babu V Dr. Ramesh P Mr. Ranjit D Mr. Ranjith G Dr. Rekha MR Dr. Roy Joseph Dr. Sabareeswaran A Dr. Sachin J. Shenoy Mr. Sajithlal MK Dr. Sandhya CG Dr. Sreenivasan K Mr. Sujesh Sreedharan Dr. Umashankar PR Mr. Vinod Kumar V

New Visions for Biomaterials and Regenerative Medicine

Programme

Day 1: March 16, 2011 Session I: 'Publishing in High Impact Factor Journals' 13:30 – 14:00 Registration 14:00 – 14:10 Welcome and Introduction Prof. K. Radhakrishnan

14:10 – 15:30 Publishing in Biomaterials Prof. DF Williams & Ms. Peggy O”Donnell

15:30 – 16:00 Meet Prof. David Williams Prof. David Williams 16:00 – 16:30 Tea

Day 2: March 17, 2011

Session II: Principles of Regenerative Medicine Chairperson: Dr. G.S. Bhuvaneshwar 9:00 – 10:00 Silk proteins as biomaterial for tissue

engineering and regenerative medicine Prof. SC Kundu

10:00 – 10:30 Tea 10:30 – 11:30 Biomaterials in Regenerative Medicine Prof. DF Williams 11:30 – 12:30 The Essential Materials Paradigms

for Regenerative Medicine Prof. DF Williams

12:30 – 13:30 Lunch

Session III: 'The Way Forward for Regenerative Medicine and Biomaterials Research in India' Chairperson: Dr. Mira Mohanty 13:30 – 14:30 Scaffold free tissue constructs for ocular

surface regeneration Dr. TV Kumary

14:30 – 15:30 Design of Biomimetic Matrix for in vitro Tissue Regeneration with Adult Stem Cells

Dr. Lissy Krishnan

15:30 – 16:00 Tea 16:00 – 17:00 Stem cells and Ceramics for the repair of

bony defects in Orthopaedics Dr. Annie John

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Publishing in BiomaterialsgProfessor David Williams, Editor-in-Chief

Peggy O’Donnell, Managing Editorbiomaterials@online.be

India March 2011India March 2011

Publishing in High Impact Factor JournalsWhy publish scientific papers

Why publish in scientific journalsWhat is an Impact Factor

-Who decides what papers are accepted for

publishingTh b i l f i tifi itiThe basic rules of scientific writing

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Why Publish Scientific PapersThere is no point in carrying out research if the results are never

analysed, conclusions properly drawn and appropriately disseminated.

Unless constrained by confidentiality (military productUnless constrained by confidentiality (military, product development, industrial know-how, economic forecasting for commercial reasons), the outputs of research should be subjected to review and comment by other scientists and disseminated as widely as possible.

The best indicators of the scientific quality of individuals are their output of scientific papers - importance of CVs.

The quality of research teams and their corporate institutions areThe quality of research teams and their corporate institutions are judged primarily by their outputs, including scientific papers, -also patents, strategic reviews etc.

Why Publish in Scientific JournalsThe main forms of published outputs are edited books, conference

proceedings, magazines and peer-reviewed scientific journals.Edited books are rarely peer-reviewed and the choice of author and

bj ll d b f h li f h ib i isubject usually made before the quality of the contribution is known.

Abstracts of conference proceedings are usually reviewed before acceptance of the paper for the conference, but the full papers are rarely reviewed and the quality is very variable.

Magazines are of variable quality, content determined by editorial policy, commercial considerations and topicality of subjects.

Scientific journals also vary in quality but they should always be peer-reviewed, are subject to independent editorial control and are generally widely available and widely read.

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What is an Impact FactorThe value of a journal is judged by the impact which its papers have on

society.It is impossible to assess how many people read any one particular paper,

although it is possible with some publishers to know how many timesalthough it is possible with some publishers to know how many times particular papers are downloaded from on-line versions of journals.

We can, however, measure exactly how many times readers quote a particular paper in their own subsequent publications.

It is convention in scientific writing for authors to cite any previous work which is directly relevant to their paper, where they have drawn upon the previously published work in support of their work or where they seek to contradict previous data or theory.

U ll t th d f i tifi th ill b li t f hUsually at the end of a scientific paper there will be a list of such ‘references’ to previous work, these generally being known as ‘citations’

The number of citations to a particular paper is a mark of the quality and impact of that paper.

What is an Impact FactorThe impact of a journal on society is judged by the collective impact of its

papers.This is assessed through the Impact Factor of the journal, which is

calculated annually by the Institute of Scientific Information USA andcalculated annually by the Institute of Scientific Information, USA, and published each June.

The Impact Factor of a journal for 2010 will be published in June 2011 and is calculated as the total number of citations that had appearedin 2010to all papers published in that journal during the years 2008 and 2009, divided by the total number of papers published in the journal during those two years.

Impact Factors tend to vary considerably with the broad subject matter of a journal Many engineering journals have IFs less than 1 0 and an IF ofjournal. Many engineering journals have IFs less than 1.0 and an IF of 2-3 is considered quite high. In some areas of biology, IFs are in excess of 20, and some as high as 30. IFs should be normalised to the subject area.

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Background• Established in 1980• Previous editors, Garth Hastings, Nick Peppas, Bob

Langerg• David Williams appointed editor in 1996• Editorial process, hard copy, Liverpool• David Williams appointed sole Editor-in-Chief in 2001• Peggy O’Donnell appointed to manage editorial

process from office in Brussels in 2001, migration to electronic submission

• Web submission and review process initiated in 2003• Journal continues to improve, IF of 7.365 in 2009

Statistics• Aim is to complete review by 2 months,

with most substantially less, but this y ,depends on availability of referees

• Less than 10% take a little longer than 2 months

• Production time has progressively decreased during recent years.

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Statistics –Submissions

2002 7002002 7002004 16002006 21002008 25002009 29002010 36002010 360010per day, every day of the year

Statistics –December 2010• Average time from submission to first decision

16 days• Average time from submission to final decisionAverage time from submission to final decision

22 days• Average time for author revision

5 days• Average time from acceptance to publication on-line

20 days• Average time from acceptance to publication in hard• Average time from acceptance to publication in hard

copy 9.5 weeks

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Statistics –Manuscript Origin, 2010

USA 661(44% accept)China598(24% accept)South Korea 200 (34% accept)Japan 149 (41% accept)Japan 149 (41% accept)Germany 131 (29% accept)UK99 (26% accept)France98 (27% accept)Italy 85(17% accept)Singapore 69 (49% accept)

India 119 (8 % accept)

Current Procedures• Submission through EES• Mandatory Author Declaration• Editor-in-Chief reads every submission,

decides on either rejection (50%) or review process (50%)

• Four referees selected, cascade process (7,000 name – keywordprocess (7,000 name keyword combinations in database)

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Current Procedures• Editor-in-Chief makes decision based on

referees reports (1-4) and his own opinionA f th 25% j t d t thi t• A further25% are rejected at this stage

• Reports sent to corresponding author together with Mandatory Editor’s Requirements

• Authors given limited time (1 week) to revise manuscriptI t f f di ( h t ti• Importance of proof reading (short time, no new material)

Best Practice• Content• Style and length• Title, authors and key-words

Ab• Abstract• Introduction• Materials and Methods• Results• Discussion• Conclusions• Acknowledgements• References• Figures and Tables• Figures and Tables• Revising papers

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Best Practice : ContentOriginal research only – hypothesis driven not just collecting data.No unsolicited review papers, no short technical notes, no techniques

oriented papers.C it i f i i lit l t th i d b t ti l dCriteria of originality, relevance to the aims and scope, substantial and

significant new data.No synthesis and characterisation of materials unless biologically relevantGenerally no in vitro tests on materials that are already in clinical use.No comparisons of commercial products unless they are of clinical

relevance.No experiments that confirm established facts.No papers that add incrementally to author’s recent papers.

f ( )No sequences of papers (dividing work into separate parts),

Best Practice : Style and lengthRead recent issues of the journal to assess acceptable style. Read

‘Writing Papers for Biomaterials’ given in the Editorial Manager home page.

The Editor determines that all papers follow a similar style of presentationThe Editor determines that all papers follow a similar style of presentation and this must be observed. The Managing Editor will send papers back to authors for correction if the style is clearly inappropriate. Continued failure to comply will result in rejection.

The style should be conventional English text, without formats that involve excessive use of notes, bullet points, lists etc.

If English is not the mother tongue of any of the authors, use the services of a translator / scientific writer.

There is no specified length. The manuscript must be substantial but should be focused and concise and not too verbose or repetitive.

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Best Practice : Title, Authors and Keywords

TitleConcise but informative, avoid multiple parts to titles.Title should be readily identified by search engines, therefore avoid

redundant words or phrases that will not contribute to visibility.Avoid sentences or definitive statements as titles such as ‘titanium causes

the over-expression of gene x but not gene y’.Avoid ‘novel’, ‘new’, innovative or ‘unique’.Do not use uncommon abbreviations or trade names.AuthorsInclude all authors who significantly contributed to the work, but not any

one else (e.g. for political or funding reasons). The number of authors should be consistent with the amount of work done

KeywordsIdeally should not be repetitive of the words in the title; use list provided in y p ; p

the journal where possible, adding new words where essential.

Best Practice : Abstract

The Abstract is often the first thing that will be read – it is available free of charge with most databases and will determine whether the full version will be accessed or not – therefore take great care.

Avoid self-centred claims –’this is the first paper to’, ‘we present an entirely novel approach’ etc, and focus on facts.

Abstract should be a single paragraph, without sub-headings.Put the work into perspective but only very briefly. Do not over-

elaborate experimental methods, concentrate on the data and its significance.

Do not include references and avoid abbreviations except where pthey are inevitable or obvious.

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Best Practice : Introduction

The best introductions are about two manuscript pages longThe readership of Biomaterials is sophisticated and does not need

lengthy explanations of disease states, demographic trends, the size of markets etc.

A very brief summary of the background to the work is sufficient,A very brief summary of the background to the work is sufficient, with citations to the many body of recent work on the relevant topic. Do not include too many references, but make sure you cite all the relevant references of the last couple of years.

Explain the rationale of the project and of the methodology used. Ideally the Introduction should focus on the hypothesis that is being tested in the experiments.

Do not describe the results obtained and do not speculate on the significance of your findings in this section.

Explain abbreviations when first usedExplain abbreviations when first used

Best Practice : Materials and Methods

This should have sufficient detail to allow the reader to repeat the experiments.

All materials, supplies, equipment etc should be described as completely as possible, with manufacturers / distributers details.

Arrange the section with a logical number of sub-sections – avoid using tooArrange the section with a logical number of sub sections avoid using too many sub-sections of a few lines length. Most sub-sections should be formatted as single paragraphs, with clear succinct titles.

Frequently, some of the methods will have been used by the author in other recently published manuscripts. This is acceptable, giving a reference to previous descriptions. Include, if necessary a brief summary of the technique.

Always include a section describing how the data will be handled statistically.

Thi ti h ld b itt i th t tThis section should be written in the past tense.

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Best Practice : Results / Discussion

It is acceptable to have Results and Discussions as separate or combined sections.

Avoid repetition of sections of Introduction or Materials.The results should mention all figures and tables.Avoid excessive numbers of sub headings again avoid sentences asAvoid excessive numbers of sub-headings – again avoid sentences as

sub-headings in these sections. It is better to refer to results in the present tense.The data should be presented according to the statistical rules set out in

the Methods. If the data show no statistical significance this should be clearly stated and not re-interpreted as a ‘trend’.

The Discussion should place the results in the context of the current state of knowledge and how this new data advances that knowledge. It is permissible to speculate on the future significance of the findings, but always within the bounds of reasonableness.

Best Practice : Conclusions

The manuscript must have a separate Conclusions section. This should be a single paragraph summary of the main findings andshould be a single paragraph summary of the main findings and their immediate significance. This should not be an extended discussion nor should it be speculative. It should not include any references

It should be factual and not self-congratulatory – no ‘novel, ‘for the first time’ etc. Never end the Conclusions with ‘this work shows that material x is biocompatible and is a promising candidate for application y’.

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Best Practice : References

Use the minimum number of references that are necessary to support statements of fact, to describe methodology, or to discuss competing or supporting evidence and theories. Do not use multiple references to support single simple un-contentious issues. Typically a manuscript should have 30-35 references.

Follow the correct style precisely – this is given in the Instructions for Authors but is the largest problem we have with non-compliance. Do not use software, such as Reference Manager, without checking that it delivers the modified form of Vancouver that we use.

Be generous with references to the work of competitors and avoid t h lf it titoo much self-citation.

Best Practice : Figures and Tables

Use the minimum number of tables and figures to represent the data accurately.

Avoid large charts that give minimal data – bar charts that compare one property for two materials- simple data should just be given in the text.

Avoid excessively large panels of images that do not contribute to the d t di f th d tunderstanding of the data.

Always include statistical data (e.g. confidence limits). Avoid simplistic schemes of experimental set-ups ( e.g. electrospinning,

micro-patterning).Figure captions should be sufficiently long to explain the data, but should

not be repetitive of the text. Note that Elsevier does not publish colour images in the printed version

anymore (unless author pays, or work was commissioned by the Editor).

Additional data may be included as ‘Supplementary Material’ which isAdditional data may be included as Supplementary Material which is included in on-line version but not printed copy.

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Best Practice : Revising Papers

The majority of accepted manuscripts have to be revised after the review process.

Carry out the revision process quickly –if not resubmitted in the timeframe given, the paper could be rejected.

Consider all comments made by the referees. Make appropriate changes in the revision Occasionally the Editor will agree to a rebuttal from thein the revision. Occasionally the Editor will agree to a rebuttal from the author which conveys a scientific argument (not a personal view) why a change should not be made.

Most requests for revision come with one or more MANDATORY EDITOR’S REQUIREMENTS. These obviously should be followed.

The resubmission should include an explanation of what the author has done to comply with the requested changes.

No changes in authors are allowed at this stage unless with permission of the Editor-in-Chief following a written explanation of why this is necessary and the submission of signed statements from new authors y gor authors that have been deleted.

Best Practice :Journal Sections

As from January 2011, Sections have been revised. They are:

• Biomaterials Design and Medical Device Performance • Biomaterials and the Stem Cell Niche • Biomaterials and Regenerative Medicine• Biomaterials and Cancer• Biomaterials at the Nanoscale for Diagnostic Systems• Biomaterials for the Delivery of Drugs, Genes, Vaccines and Active

Molecules

Author recommends section but Editor-in-Chief makes final decision

As from March 2011, will have occasional Debate Sections

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Significant IssuesCommunication with the office.Referees.Multiple submissions.Multiple submissions.Correction of errors after acceptance.What to do after a manuscript is rejected.Advice on areas of research, alternative

publication options.Archiving and definitive versions.

Silk Proteins as Biomaterial for TissueEngineering and Regenerative Medicine

Nandana Bhardwaj, Sunita Nayak and S. C. Kundu*

Recent developments in the field of tissueengineering and regenerative medicine haveaccelerated the demand for the functionalbiomaterials which are natural in origin,biodegradable and biocompatible withenhanced mechanical properties. Silkproteins from cocoons and glands of bothmulberry silkworms Bombyx mori(Bombycidae, domesticated) and non-mulberry Antheraea mylitta (Saturniidae,wild) are exploited considerably as textilematerial since decades now finds newapplications as natural biopolymer in tissueengineering. Silk fibers are composed of afibrous protein (fibroin) core and a glueprotein (sericin) surrounding it. The majorbiomedical applications of silk revolvearound B. mori fibroin protein. Limitedknowledge about the wild non-mulberrytropical tasar silkworm A. mylitta as beingendemic to India forms the basis of our study.Silk proteins based matrices such as 2D films,3D scaffolds, biospun scaffolds, micro beads,hydrogels, micromolded matrices andnanoparticles are engineered andcharacterized as biomaterial system for tissueengineering, 3D tumor model, developmentof immobilization substrates and drugdelivery vehicles for controlled release ofdrugs and bioactive molecules. Further, silkfibroin matrices are used as substrates forstem cell culture, osteogenic, adipogenic andchondrogenic differentiations, subcutaneousfibroblast adhesion and non-differentiationinto myofibroblasts. Potential sericinmatrices in the form of 3D scaffolds, 2D filmsand nanoparticles are also fabricated andcharacterized for various tissue engineeringapplications and silk sericin from A. mylittais found to have potential anti-oxidant effectand anti-apoptotic properties.

INTRODUCTION

Tissue engineering and regenerativemedicine are an up comingmultidisciplinary field with greaterimpact on health-care technology. Thisarea employs new therapeutic strategyand biomedical technologies, whichsupport and accelerate the regeneration

and repair of defective and damagedtissues. Biomaterial technologycombining cells with scaffoldingmaterials plays an important role in thisfield. The scaffolds provide the cells alocal environment, enhance and regulatecell proliferation and differentiation.This is very crucial point for the cell-based tissue regeneration. Restorationand maintenance are expected by thenatural healing potentials of the bodyitself.

In this context the importance of silkprotein material has increased with itsproven potential as a natural biopolymerfor tissue engineering and biomedicalapplications. Silks are highly expressedprotein polymer produced by variousspecies of spiders of class Arachnida andsilkworms of the order Lepidoptera forvarious structural roles like webformation, safety lines, egg protection,and cocoon formation (Kaplan et al.,1998; Altman et al., 2003). The fibroussilk protein is synthesized inside the silkglands of these organisms withinspecialized epithelial cells. Theseproteins are stored in the middle silkgland and at the end of the fifth instar;they are expelled through the anteriorduct and spinneret (Sutherland et al.,2010). The silk fiber obtained from thesilkworm cocoons comprises of twomajor protein components. The fibroin,core protein, contributes 70-80 % ofcocoons silkworm. It is a fibrous,glycoprotein linked by disulfide bridgesand is composed of heavy (H) chain,Light (L) chain (Zhou et al., 2000;Kikuchi et al., 1992) with hydrophobicnature. Silk fibroin, extracted fromsilkworms cocoon has found diverseapplication in the biomedical field asnatural biopolymers due to its distinctivebiophysical and mechanical properties.These properties include high tensile

strength, controlled biodegradability andbiocompatibility with low antigenicityand non-inflammatory responses(Altman et al 2003; Vepari and Kaplan2007). Silk-based biomaterial offers thestrongest and toughest naturalbiopolymer and provides manyprospects for its processing,functionalization, and biologicalintegration (Lawrence et al., 2008). Thesecond silk protein sericin forms theouter hydrophilic adhesive coating thatunites fibroin and constitutes 20-30%silk cocoons of silkworm (Kundu et al2008a). Commercially, silk obtainedfrom domesticated mulberry, Bombyxmori and wild non-mulberry; Antheraeamylitta, Antheraea assama andAntheraea pernyi silkworms have beenused in the textile industry for its lusterand mechanical properties.

Although for centuries, clinically silk hasbeen used as sutures, it is presentlyappreciated much as biomaterial invarious forms like 2D films, 3Dscaffolds, hydrogels, nanofibers,membranes, sponges and nanoparticlesand is being exploited in vitro and in vivofor cell culture and tissue engineering(Altman et al 2003). Recently tissueengineering based on stem cells and 3Dsilk fibroin scaffolds has escalatedinterest in a variety of applications suchas skeletal tissue engineering like bone,ligament, and cartilage, connectivetissues like skin etc. (Wang et al 2006,Bhumiratana et al., 2011). B. morifibroin has been utilized as a matrix forgrowth and proliferation studies ofvarious cell types including osteoblast,fibroblast, hepatocyte and keratinocyte(Gotoh et al., 2004; Min et al., 2004;Unger et al., 2004; Chiarini et al., 2003).However, stronger cell adhesion hasbeen observed on films formed by silkfibroins from A. pernyi, the wild-type

16 N. Bhardwaj, S. Nayak and S.C. Kundu

silkworm comparative to those from B.mori domestic silkworms (Minoura etal., 1995). This suggests that due to thepresence of tripeptide sequence Arg(R)-Gly (G)-Asp (D), a recognition site forintegrin-mediated cell adhesionsequence, exclusively present in wildsilkworm’s silk fibroin, increases the celladhesion property (Pierschbacher andRuoslahti, 1984). Three-dimensional silkfibroin porous materials have shownhuman cell-compatibility and haveoffered their application for thevascularization of the newly formedtissue (Wang et al., 2006) suggestingtheir use as a 3D model to monitor cellproliferation and migration. Thedifferent methods to prepare silk fibroinporous materials are using porogens, gasforming, and freeze-drying, freeze-drying/foaming, and electrospun fibers(Park et al., 2008).

Silk hydrogels formed from regeneratedsilk fibroin by a sol-gel transition in thepresence of acid, ions, or other additivesfor drug delivery in vitro, in vivo and forcell culture and regenerative medicinepurpose as porous matrixes have beeninvestigated by several investigators (seeMandal et al, 2009a). Recentlylyophilized silk fibroin hydrogel matrix(lyogel) has been used for the sustainedrelease of monoclonal antibodies(Guziewicz et al., 2011).

Silk fibroin nanoparticles for sustainedand long-term release, with minimal tono toxicity to healthy tissues have beeninvestigated. It has been reported thatsilk fibroin-derived particles due tosustained release augments intracellularuptake and retention of the entrappeddrug with the down modulation of morethan one pathway (Mathur and Gupta,

2010). Sustained release of growth factorfrom the silk nanoparticles demonstratesits potential application for growth factordelivery (Kundu et al 2010; Numata andKaplan 2010).

The work evolved from our laboratory,mainly emphasize on silk proteins (bothsericin and fibroin) obtained from Indianorigin tropical tasar silkworm Antheraeamylitta. The possibility of using wildnon-mulberry silk protein as abiopolymer remains unexploredcompared to domesticated mulberry silkprotein. One of the main reasons thatthere has not been any suitable methodof extraction of silk protein fibroin fromcocoons and silk glands. We havedeveloped a novel and ecofriendlymethod for dissolution of gland silkprotein fibroin of non-mulberrysilkworm, A. mylitta. Silk gland fibroin

Mulberry and non-mulberry silkworms, sources of proteins and different matrices

Silk Proteins as Biomaterial 17

is found to be optimally soluble in 1%(w/v) anionic surfactant sodium dodecylsulfate (SDS) because of enhanceddissolution via internalization ofhydrophobic amino groups inside ahydrophilic amino acid core in the formof micelles. The new technique isimportant and unique because it uses amild surfactant for fibroin dissolutionand is advantageous over otherpreviously reported techniques usingchaotropic salts (Mandal and Kundu,2008 a). We have fabricated and utilizeddifferent types of matrices like films,scaffolds, hydrogels, and nanoparticlesfor various tissue engineeringapplications. Pure silk proteins and theirblends with different polymers such aschitosan, carboxy methyl cellulose,gelatin, hydroxyl propyl methylcellulose (HPMC), polyethylene glycol,lactose have been fabricated andcharacterized biophysically andbiochemically for variety of cell basedtissue engineering applications.

We have evaluated the macrophageresponses elicited by non-mulberrysilkworm Antheraea mylitta fibroinprotein and biocompatibility of the silkfibroin (SF) films by adherence of L929murine fibroblasts as well as comparedthem with mulberry silkworm Bombyxmori fibroin protein and collagenmatrices. Fibroin and collagen matriceshave also been subjected to a study oncomparative kinetics of adhesion ofL929 dermal fibroblasts to help informulation of dermal coverings ormatrices which support a highcellularization potential with dermalcells at the time of wound recovery(Acharya et al., 2008 a).

We have fabricated SF-based scaffoldsand films conjugated with lactose byusing a cyanuric chloride (CY) linker(Acharya et al., 2008 b). We can adjustthe properties of this material to supportattachment of fibroblastic cells. At thesame time, development of a fibrogenicphenotype is suppressed even inpresence of the pro-fibrotic cytokineTGF â1; growth on this material canreverse fibrogenic myofibroblasts intonormal fibroblasts The effects ofstructurally different alcohols on solventstability and biocompatibility of pureand blended silk protein fibroin matriceshave also investigated with silk fibroinfilms of A. mylitta (Acharya et al., 2009a). The thermal, mechanical and surfaceproperties of pure and blended filmshave also been evaluated. They haverevealed that methanol and isopropanol-

treated pure and blended films supportcell adhesion, proliferation, and viability.Later on we have studied the mechanicaland surface properties of fibroin filmfrom the non-mulberry tropicalsilkworm A. mylitta to determine thesuitability of non- mulberry fibroin as apotential substratum for supporting celladhesion and proliferation (Acharya etal., 2009 b). As A. mylitta silk fibroinfilms are non-toxic to dermal fibroblastcells, non-mulberry fibroin might be auseful alternative substrate to the morecommon B. mori fibroin for a variety ofbiomedical applications. We have testedthe suitability of a surfactant solventsystem for dissolution and regenerationof non-mulberry silk gland fibroinprotein and its related biophysical andcell proliferating properties, thusexploring new avenues for its use asnatural biopolymeric material (Mandaland Kundu 2008 b). Other polymers likehydroxyl propyl methyl cellulose(HPMC) and sodium carboxy methylcellulose have also been used forblending (Kundu et al., 2008 b, Kunduet al., 2011). The effect of blended filmson cell viability and adhesion has beenevaluated.

After the investigation of SF films, wehave focused on fabrication andcharacterization of 3-D scaffolds of A.mylitta. This wild non-mulberry tropicaltasar silk gland fibroin protein in its non-bioengineered form has been processedwithout using organic solvents for thefabrication of functional 3D scaffolds forintended biotechnological and tissueengineering applications. Thesescaffolds indicate non-mulberry 3D silkgland fibroin protein as an inexpensive,high strength, slow biodegradable,biocompatible, and alternative naturalbiomaterial (Mandal and Kundu, 2008c). Freeze-drying techniques underdifferent temperature regimes have beenused to fabricate functional 3D silkfibroin protein scaffolds (Mandal andkundu 2009a). We have investigated therole of pore size, porosity andinterconnectivities in cell proliferationand migration of fibroblasts. Recently,we have fabricated polyelectrolytecomplex porous scaffolds of silk fibroinand chitosan and evaluated its suitabilityfor cartilage tissue engineering(Bhardwaj and Kundu, 2011). The effectof blending on stability, degradation,mechanical, antimicrobial properties,cell attachment and adhesion has beenstudied. The results indicate suitabilityof these scaffolds for various tissueengineering applications. A. mylitta silk

fibroin protein have showncytocompatibility with wide variety ofcell types such as feline fibroblasts,human mesenchymal stem cells,chondrocytes, rat bone marrow stromalcells etc. We have investigated the 3-Dscaffolds as substrate for osteogenic andadipogenic differentiation of rat bonemarrow cells (Mandal and Kundu, 2009b and c). The scaffolds have showedmechanically robust and have shownhomogenous pore distribution.Histological analysis has shownosteogenic differentiation andadipogenic differentiation within silkscaffolds resulting in extensivemineralization in the form of depositednodules and by the presence of lipiddroplets as observed through intenseAlizarin Red S staining and Oil Red O.Real-time PCR studies revealed highertranscript levels for osteogenic andadipogenic genes.

Apart from the cell based tissueengineering applications, silk fibroinmatrices such as beads, hydrogels andnanoparticles have been utilized for drugdelivery applications. Silk fibroin (SF)and polyacrylamide (PAAm) semiinterpenetrating network (semi-IPN)hydrogels have also been fabricated andcharacterized to investigate their use asa matrix for sustained drug release. Thematrices are synthesized by redoxreaction using N, N2- Methylenebis(acrylamide) (N, N2-MBAAm) ascrosslinker, ammonium persulfate (APS)as initiator and N, N, N’, N2-tetramethylenediamine (TEMED) asaccelerator. The physico-chemicalproperties including morphology,stability, swelling, mechanical,rheological and compatibility have alsobeen investigated. Further, the releasekinetics of the semi-IPNs are evaluatedusing two model compounds i.e. trypanblue dye and FITC labeled inulin for itspotential use in tissue engineering anddrug delivery applications (Mandal et al.,2009a). Silk fibroin beads have beenused for controlled and sustained releaseof two different model compounds i.e.BSA (66 kDa) and FITC–Inulin (3.9kDa) from predefined porous silkscaffolds in an independent fashion(Mandal and Kundu, 2009 d). Theproteins have been selected for theirdifferences in their molecular weights toevaluate the ability of the deliverysystem to modulate the independentrelease of the two compounds. We havealso investigated silk protein fibroinspherical nanoparticles from bothmulberry and non-mulberry silk utilizing

18 N. Bhardwaj, S. Nayak and S.C. Kundu

dimethyl sulfoxide as desolvating agent(Kundu et al., 2010). The investigationoutlines for their morphology, surfaceproperties, conjugation withfluorescence isothiocyanate and thecellular uptake of these nanoparticles bymurine squamous cell carcinoma as wellas VEGF release from the nanoparticlesto explore their use as potentialtherapeutic drug delivery system.

Surface topology and roughness presentpowerful cues for cells. The surface canstrongly influence cell morphology,adhesion, and proliferation. Themechanisms mediating these phenomenaare still unclear. The understanding ofcell responses to material surfaces isrequired in order to design and fabricatenext-generation tissue engineeringmaterials. We have investigated theeffects of nanoroughness assemblies ofsilk fibroin protein membranes oncytoskeletal organization, proliferation,and viability using primary rat bonemarrow cells (Mandal et al., 2010). Thesilk fibroin films treated with 50 %ethanol to 100 % ethanol have been usedfor nanoroughness study.

Recently, we have initiated work usingA. mylitta silk fibroin scaffolds for invitro model system. The model maymimic the in vivo microenvironment andhas escalated much interest in cancerresearch. Similarly A. mylitta silk fibroinprotein matrices have been used aspotential biomaterial for in vitro tumormodeling using MDA-MB-231 andLNCaP prostate cancer cancer cells(Talukdar et al., 2011). For control wehave used B. mori scaffolds, matrigelsand tissue culture plates. The cells haveattached and proliferated well on A.mylitta scaffolds and films. The cellsgrown in all 3D cultures have shownmore MMP-9 activity indicating a moreinvasive potential. Yield coefficient ofglucose consumed to lactate produced bycells on 3D fibroin is found to be similarto that of cancer cells in vivo. Theseresults suggest that this silk fibroin 3Dmatrix will provide flexibility in termsof manipulation of microenvironmentsystem for tumor model. This can beused for investigation of individualfactors such as growth factors andsignaling peptides, as well as evaluationof anticancer drugs.

Apart from silk fibroin protein of A.mylitta, silk protein sericin has also beenutilized for various applications in ourlaboratory. Sericin protein has beenbiophysically characterized from

peduncles and cocoons (Dash et al.,2006, Dash et al., 2007). Later oninvestigations of silk sericin protein forinhibition of UV induced apoptosis andantioxidant activities have been carriedout (Dash et al., 2008 a and b). Silksericin proteins as biomaterial have alsobeen studied with films and scaffolds.This study indicates that silk sericinprotein are also suitable for tissueengineering applications (Mandal et al.,2009 b, Dash et al., 2009). In vitroimmunogenicity of sericin has beeninvestigated with TNF-á release usinglipopolysaccharide as positive controland has been found less immunogenicas compared to the control (Dash et al.,2009). Silk sericin nanoparticles havebeen utilized for delivery of hydrophilicand hydrophobic drugs (Mandal andKundu, 2009e).

Currently we are working with pure silkand blended silk fibroin scaffolds for invitro cartilage tissue engineering. Thebovine chondrocytes are used to studythe cell viability, proliferation,biomechanical properties andbiochemical properties of the 3Dcartilage construct. The effect of initialcell seeding is very important in cartilagetissue engineering. The effect of differentseeding density of chondrocytes isimportant for the extracellular matrixproduction, biochemical andbiomechanical properties also.Chondrocytes and mesenchymal stemcells are two important sources of cellsfor cartilage tissue engineering. The ratbone marrow cells are being used forstudying the differentiation intochondrocytes in silk fibroin blendedscaffolds to observe the effect on cellviability and proliferation andextracellular matrices production. Thedirected chondrogenesis of humanembryonic stem cell-derived neural crestcells in non-mulberry silk 3D micro-environment are also being investigated.

Breast cancer commonly spreads to boneat a frequency of approximately 70% inpatients having distant metastasis.However, the mechanism of bonemetastasis is not well understood. Henceit is very important to have an in vitromodel to study the various interactionsbetween breast cancer and bone tissue.Co-culture studies of cancer cells andosteoblast are being attempted to studythe interactions between these cells. Anappropriate model will not only thrownew light on the area but also help indesigning newer and effectivetreatments. Other work are being carried

out include fabrication of silk proteinsand blended nanoparticles for release ofbioactive molecules, sericin basedmatrices such as films and scaffolds forvariety of applications. The pHresponsive silk sericin protein hydrogelsfor drug delivery applications and asbiomaterials are also being investigated.The biomaterial database is beingcreated to maintain the huge amount ofdata and retrieval of information. Thedatabase is prepared by the use ofMySQL, HTML, PHP and java script. Itprovides the information related tohomologous query sequences against thebiomaterials database, based on BLASTsearch tool and retrieve the usefulinformation from it.

Acknowledgements

SCK wishes to thank to his under- andpostgraduate students and collaboratorswithout them this write up would havenot been possible. This work isfinancially supported by Indo-AustraliaBiotechnology Fund, Department ofBiotechnology and its BioinformaticsCentre, Indo-Russia BiotechnologyProgramme, Department of Science andTechnology, Council of Scientific andIndustrial Research, Government ofIndia, and Indo-US Science andTechnology Forum, New Delhi.

REFERENCES

Acharya C, Ghosh SK, Kundu SC (2009a).Silk fibroin film from non-mulberry tropicaltasar silkworms: a novel substrate for in vitrofibroblast culture. Acta Biomaterialia, 5: 429-437.Acharya C, Ghosh SK, Kundu SC (2008a).Silk fibroin protein from mulberry and non-mulberry silkworms: cytotoxicity,biocompatibility and kinetics of L929 murinefibroblast adhesion, Journal of MaterialScience: Materials in Medicine, 19:2827-36.Acharya C, Hinz B, Kundu SC (2008b). Theeffect of lactose-conjugated silk biomaterialson the development of fibrogenic fibroblast.Biomaterials 29, 4665-4675.Acharya C, Kumary TV, Ghosh SK, KunduSC (2009b). Characterization of fibroin andPEG blended fibroin matrices for in vitroadhesion and proliferation of osteoblasts.Journal of Biomterial Science, 20: 543-565.Altman GH, Diaz F, Jakuba C, Calabro T,Horan RL, Chen J, Lu H, Richmond J, KaplanDL (2003). Silk-based biomaterials.Biomaterials 24: 401–416.Bhumiratana S, Grayson WL, Castaneda A,Rockwood DN, Gil ES, Kaplan DL, Vunjak-Novakovic G (2011). Nucleation and growthof mineralized bone matrix on silk-hydroxyapatite composite scaffolds.Biomaterials, 32: 2812-2820 .

Silk Proteins as Biomaterial 19

Chiarini A, Petrini P, Bozzini S, Pra IDArmato U (2003). Silk fibroin/poly(carbonate)-urethane as a substrate forcell growth: in vitro interactions with humancells, Biomaterials 24: 789–799.Dash BC, Mandal BB and Kundu SC (2009).Silk gland sericin protein membranes:fabrication and characterization for potentialbiotechnological applications. Journal ofBiotechnology, 144: 321-329.Dash R, Acharya C, Bindu PC and Kundu SC(2008a). Antioxidant potential of silk proteinsagainst hydrogen peroxide-induced oxidativestress in skin fibroblasts. BMB Reports, 41:236-241.Dash R, M. Mandal, SK. Ghosh, Kundu S C(2008b). Silk sericin protein of tropical tasarsilkworm inhibits UVB-induced apoptosis onhuman skin keratinocytes. Molecular CellularBiochemistry, 311(1-2) 111-119.Dash R, Mukherjee S, Kundu SC (2006).Isolation, purification and characterization ofsilk protein sericin from cocoon peduncles oftropical tasar silkworm, Antheraea mylitta.International Journal of BiologicalMacromolecules, 38: 255-258.Dash R, Sudip K. Ghosh, David L. Kaplan,Kundu, SC (2007). Purification andbiochemical characterization of 70 kDasericin from tropical tasar silkwormAntheraea mylitta. ComparativeBiochemistry and Physiology Part-B,147:129-134.Gotoh Y, Niimi S, Hayakawa T, Miyashita T(2004). Preparation of lactose-silk fibroinconjugates and their application as a scaffoldfor hepatocyte attachment. Biomaterials, 25:1131–1140.Gupta V, Aseh A, Ríos CN, Aggarwal BB,Mathur AB (2009). Fabrication andcharacterization of silk fibroin-derivedcurcumin nanoparticles for cancer therapy,International Journal of Nanomedicine,4:115-22.Guziewicz N, Best A, Perez-Ramirez B,Kaplan DL (2011). Lyophilized silk fibroinhydrogels for the sustained local delivery oftherapeutic monoclonal antibodies,Biomaterials. 32: 2642-50.Kaplan DL (1998). Fibrous proteins - Silk asa model system. Polymer Degradation andStability, 59: 25 –32.Ki CS, Park SY, Kim HJ, Jung HM, Woo KM,Lee JW, Park YH (2008). Development of 3-D nanofibrous fibroin scaffold with highporosity by electrospinning: Implications forbone regeneration. Biotechnology Letters,30:405–410.Kikuchi Y, Mori K, Suzuki S, Yamaguchi K,Mizuno S (1992). Structure of the Bombyxmori fibroin light-chain-encoding gene:upstream sequence elements common to thelight and heavy chain. Gene, 110: 51-158.Kundu J, Chung YI, Kim YH, Tae G, KunduSC (2010). Silk fibroin nanoparticles for

cellular uptake and control release.International Journal of Pharmaceutics, 388:242-250.Kundu J, Mohapatra R, Kundu S C (2011)Silk fibroin protein-sodium carboxymethylcellulose blend films for biotechnologicalapplications. Journal of Biomaterials Science,Polymer Edition, 22, 519-539.Kundu J, Patra C, Kundu SC (2008b) Design,fabrication and characterization of silkfibroin- HPMC-PEG blended films as avehicle for transmucosal delivery. MaterialScience and Engineering C 28, 1376-1380.Kundu SC, Dash BC, Dash R, Kaplan DL(2008a). Natural protective glue proteinsericin, bioengineered by silkworms:Potential for biomedical and biotechnologicalapplications. Progress in Polymer Science,33: 998-1012.Lawrence BD, Cronin-Golomb M,Georgakoudi I, Kaplan DL, Omenetto FG(2008). Bioactive Silk protein biomaterialsystems for optical devices.Biomacromolecules, 9:1214–1220.Mandal BB, Kapoor S, Kundu SC (2009a).Silk fibroin/polyacrylamide semi-interpenetrating network hydrogels forcontrolled drug release. Biomaterials, 30:2826-2836.Mandal BB, Kundu SC (2009b). Non-mulberry silk gland fibroin protein 3Dscaffold for enhanced differentiation ofhuman mesenchymal stem cells intoosteocytes. Acta Biomaterialia, 5: 2575-2590.Mandal BB, Kundu SC (2009a). Cellproliferation and migration in silk fibroin 3Dscaffolds. Biomaterials, 30: 2956-65.Mandal BB, Priya AS, Kundu S C (2009b).Novel silk sericin-gelatin 3D scaffolds and2D films: Fabrication and characterization forpotential tissue engineering applications. ActaBiomaterialia, 5: 3007-3020.Mandal BB, Kundu S C (2009c). Osteogenicand adipogenic differentiation of rat bonemarrow cells on non-mulberry silk glandfibroin 3D scaffolds Biomaterials, 30: 5019-5030.Mandal BB, Kundu S C (2009d). Calciumalginate beads embedded in silk fibroin as 3Ddual drug releasing scaffolds. Biomaterials,30: 5170-5177.Mandal BB and Kundu S C (2009e). Selfassembled silk sericin/poloxamernanoparticles as nanocarriers of hydrophobicand hydrophilic drugs for targeted delivery.Nanotechnology, 20: 355101-15.Mandal BB, Kundu SC (2008a). A novelmethod for dissolution and stabilization ofnon-mulberry silk gland protein fibroin usinganionic surfactant sodium dodecyl sulfate.Biotechnology & Bioengineering, 99: 1482-1488.Mandal BB, Kundu SC (2008b). Non-bioengineered silk gland fibroin protein:

characterization and evaluation of matricesfor potential tissue engineering applications.Biotechnology and Bioengineering, 100:1237- 50.Mandal BB, Kundu S C (2008c). Non-bioengineered high strength three-dimensional gland fibroin scaffolds fromtropical non-mulberry silkworm for potentialtissue engineering applications.Macromolecular Bioscience, 8: 807-818.Mandal BB, Das S, Choudhury K, Kundu SC(2010). Implication of silk film RGDavailability and surface roughness oncytoskeletal organization and proliferation ofprimary rat bone marrow cells. TissueEngineering Part-A, 16:2391-403.Min BM, Lee G, Kim SH, Nam YS, Lee TS,Park WH (2004). Electrospinning of silkfibroin nanofibers and its effect on theadhesion and spreading of normal humankeratinocytes and fibroblasts in vitro,Biomaterials 25: 1289–1297.Minoura N, Aiba S, Gotoh Y, Tsukada M,Imai Y (1995). Attachment and growth ofcultured fibroblast cells on silk proteinmatrices. Journal of Biomedical MaterialsResearch, 29:1215–1221.Bhardwaj N, Kundu SC (2011) Silk proteinfibroin and chitosan polyelectrolyte complexporous scaffolds for tissue engineeringapplications. Carbohydrate Polymer (inpress) doi:10.1016/j.carbpol.2011.02.027.Numata K, Kaplan DL (2010). Silk-basedgene carriers with cell membranedestabilizing peptides. Biomacromolecules,11: 3189–3195.Pierschbacher MD, Ruoslahti E (1984). Cellattachment activity of fibronectin can beduplicated by small synthetic fragments ofthe molecule. Nature 309:30-33.Sutherland TD, Young JH, Weisman S,Hayashi CY, Merritt DJ (2010). Insect silk:one name, many materials. Annual Reviewof Entomology, 55:171-88.Talukdar S, Mandal M, Hutmacher D,Russell P, Soekmadji C, Kundu SC (2011).Engineered silk fibroin protein 3-D for invitro tumor model. Biomaterials, 32: 2149-59.Unger RE, Wolf M, Peters K, Motta A,Migliaresi C, Kirkpatrick CJ (2004). Growthof human cells on a non-woven silk fibroinnet: a potential for use in tissue engineering.Biomaterials, 25: 1069–1075.Vepari C, Kaplan DL (2007). Silk as abiomaterial. Progress in Polymer Science,32: 991-1007.Wang Y, Kim HJ, Vunjak-Novakovic G,Kaplan DL (2006). Stem cell-based tissueengineering with silk biomaterials.Biomaterials, 27: 6064-82.Zhang YQ (2002). Applications of naturalsilk protein sericin in biomaterials.Biotechnology Advances, 20: 91–100.

SC Kundu is currently Professor, Department of Biotechnology, Indian Institute of Technology, Kharagpur-721302* kundu@hijli.iitkgp.ernet.in Fax. 03222 278433

JOM • April 201148 www.tms.org/jom.html

The Essential Materials Paradigms for Regenerative Medicine*

David Williams

Medical technology is changing rapidly. Several disease states can now be treated very effectively by implant-able devices that restore mechanical and physical functionality, such as replacement of hip joints or restora-tion of heart rhythms by pacemakers. These techniques, however, are rather limited, and no biological functional-ity can be restored through the use of inert materials and devices. This paper explores the role of new types of bio-materials within the emerging area of regenerative medicine, where they are able to play a powerful role in per-suading the human body to regenerate itself.

INTRODUCTION

We must place the paradigm for ma-terials in regenerative medicine within the context of the objectives of regen-erative medicine and the new role for biomaterials in this area. We have been using biomaterials in medical technol-ogy for a variety of purposes for well over half a century. Most of these ap-plications have involved implantable medical devices that have been used for the replacement of diseased or damaged tissues or organs, or for the replacement of the function of such organs, or for assisting in the repair of traumatized tissues. The most obvious examples of these are the prostheses for total replacement of joints such as hips and knees that suffer from osteo-arthritis, intraocular lenses for patients with cataracts, grafts that bypass ath-erosclerotic arteries, pacemakers and defibrillators for patients with defec-tive heart rhythms, deep brain stimula-tors for Parkinson’s disease sufferers, and plates and other devices to repair broken bones. Clearly, these medical devices have

How would you……describe the overall significance of this paper?

…describe this work to a ma-terials science and engineering professional with no experience in your technical specialty?.

…describe this work to a layper-son?

to provide for a specific function, and the materials used for their construc-tion have to meet the specifications for these functions, including some highly specific mechanical and physical prop-erties. In addition, these materials have to remain in the body for protracted pe-riods of time, often for the remaining natural lifetime of the patients, and in doing so must not be damaged by the environment of the body nor cause any

significant and harmful effect on that body. The latter two aspects are sub-sumed within the phenomena of bio-compatibility. As I have argued recently,1 we have had a fairly good idea of what con-stitutes biocompatibility under these conditions, even if we have not fully understood all of the mechanisms by which biomaterials interact with the environment of the human body. In the vast majority of circumstances, we re-quire that the biomaterials used in long-term implantable devices should suffer no significant corrosion, degradation or other form of deterioration within the body, nor should they exhibit any form of toxicity or irritation to the tis-sues and organs of that body. Thus, my current definition of biocompatibility in this context is ‘The biocompatibility of a long term implantable medical de-vice refers to the ability of the device to perform its intended function, with the desired degree of incorporation in the host, without eliciting any undesirable local or systemic effects in that host.’ Accordingly, we now use a small group of thoroughly stable and biologically inert materials, which we may describe as conventional biomaterials, for these devices, including some titanium al-loys, platinum group metals and their alloys, cobalt-chromium based alloys, certain forms of carbon, oxide ceram-ics such as alumina, polytetrafluoro-ethylene, polyethylene, Polyethylene terephthalate textiles, some acrylic polymers, polyether ether ketone, and silicone elastomers.

THE LIMITATIONS OF CONVENTIONAL MEDICAL DEVICES

So far, so good. With these basic con-ventional biomaterials, and with some

OverviewBiomaterials in Regenerative Medicine

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very well designed and tested products, and good quality surgical procedures, we should get excellent results in the majority of patients; over 90% of pa-tients with total hip or knee prostheses will still have intact, functioning, pain-free, devices 15 or more years later. Similar stories will be found with most types of implantable device. However, these medical devices are limited in their function; indeed they all perform a physical or mechanical function, such as transmission of me-chanical forces, channeling of blood flow, providing electrical signals, al-lowing light transmission, and so on, but not any biological function. They are replacing aged, diseased, or dam-aged tissues with synthetic or man-made materials that are dead rather than living. There is, and always will be, a serious limitation to the useful-ness of such medical devices. There are many diseases, and many human conditions, that these medical devices cannot help; included here are diabetes, Alzheimer’s disease, liver failure, and cystic fibrosis. One possible alternative to inert medical devices involves the trans-plantation of living tissues to the pa-tient from some donor site. This may be achieved in situations where the do-nor is also the patient, taking a bone or skin graft from some undamaged part of the body and transposing it, as viable tissue, to an injured part. Alternatively we may use donors who are different to the patient; this may be achieved with

a live donor, but is mostly done with those who have died and from whom the required tissue (e.g., corneas) or or-gans (e.g., heart, kidney) can be quick-ly and atraumatically removed. A big advantage is that the patient now has a living replacement, and there is not the same limitation of function as that associated with synthetic biomaterials. The main disadvantages are that donor organs and tissues are scarce and that they are likely to induce an immune response in the patient, a phenomenon which has to be treated by the use of immunosuppressants, not without its own problems. Since the two main methods of tis-sue replacement—implantable bioma-terials and transplanted tissues—both have their limitations, alternative therapies are being sought. It is intui-tively obvious that it would be better if we were able to regenerate new tissue rather than try to replace it artificially. The problem with humans, and indeed mammals in general, is that there is very limited ability to regenerate tis-sues. A few species, such as newts and salamanders, are able to regenerate sig-nificant parts of their bodies; mecha-nisms vary from species to species but usually involve either the retention of stem cells in the adult body that are able to migrate to a site of healing, or the de-differentiation of cells into embryonic-like stem cells, forming a blastema, a mass of proliferating de-differentiated cells capable of forming new tissue. However, the evolution of

mammals has left them with very lim-ited capacity to do this. We are able to heal skin to a limited extent, and can regenerate bone in order to heal frac-tures, but we do not spontaneously re-generate our bodies when they become diseased or damaged. Since we had the capacity to generate tissues during the development of a fetus and in infancy, it would appear that generation of new tissue is not impossible in humans, but rather that the mechanisms have been switched off in adulthood. Regenerative medicine, in which attempts are made to recapitulate that capacity under controlled conditions, provides one potential alternative to the replacement of diseased and dam-aged tissue.2,3

BIOMATERIALS IN REGENERATIVE MEDICINE

If regenerative medicine provides a possibility of recreating new tissues in-stead of replacing them with synthetic structures, it might be concluded that biomaterials have little place in the fu-ture of medicine. It is indeed possible that biomaterials will play little or no role in some forms of regenerative medicine. However, if we now con-sider how the ability to generate new tissue can be switched back on, it will become obvious that biomaterials will probably have a crucial role. The key to any regenerative process is the cell. If we need to generate new bone, we need the active function of bone-producing cells, the osteoblasts. For cartilage it will be the chondro-cytes, for skin the keratinocytes, for nerves the neurons, and so on. These cannot start to generate new tissue spontaneously; they have to be given some signals. In general these have to include both molecular signals and mechanical signals; that is, we need a variety of biochemicals such as growth factors, and mechanical stress to initi-ate and maintain the cell function. In the case of mechanical stress, the phe-nomenon mechanotransduction, medi-ated both by fluid shear stresses and structural stresses within the material, which affect the attachment of inte-grins on cell surfaces to the material, is very important. Biomaterials will play a significant role in the delivery of these signals. Equally importantly, any

TISSUE ENGINEERING: CENTRAL PARADIGM AND VARIATIONS

Central Paradigm

● Cell sourcing● Cell expansion and manipulation● Cell seeding and extracellular matrix expression● Mechanical and molecular signalling● Implantation of construct● Full incorporation into host

Variations on Central Paradigm

● Cell sourcing: Fully differentiated, embryonic stem cells, adult stem cells, iPS cells, autologous or allogeneic

● Cell expansion, manipulation and seeding: Growth factors, Gene transfer● Implantation of construct: Preformed scaffold plus cells or injectable self curing gel

incorporating cells● Mechanical and molecular signaling: In vivo● Extracellular matrix expression: In vivo● Full development within the host

JOM • April 201150 www.tms.org/jom.html

tissue that is grown in this way has to have shape, and it is biomaterials tem-plates that will give it this shape. We normally describe such a template as a ‘scaffold,’ which is often a degradable porous structure or alternatively, a hy-drogel.4,5

Before discussing the specifications for such materials, we must briefly de-scribe the situations in which they may be used. Most of the early attempts to generate tissue in this way involved the techniques of in vitro (or ex vivo) tissue engineering6 (see the sidebar). This is concerned with the sourcing of the appropriate cells, expanding the cell colony into a sufficiently large volume, seeding these cells into an ap-propriate scaffold within some form of in vitro bioreactor, feeding these cells with a relevant cocktail of stimulating molecules and nutrients, perfusing the bioreactor with fluid so that fluid shear stresses provide mechanical stimuli to the cells, harvesting the tissue that forms in the scaffold, and implanting this new construct into the patient. In recent years, it has been recognized that although this is an acceptable con-cept, and can indeed result in relevant regenerated tissue under some cir-cumstances,7,8 it does not necessarily provide the most effective way to do this. Alternatives in which much of the cell signaling occurs within the patient rather than within an external bioreac-tor have been described9 (see the side-bar). Under these circumstances, it may well be only cells and the stimulating molecules are used, perhaps by injec-tion into the patient, without any bio-material support, in processes known as cell therapy. However, it is becom-ing clear that the majority of situations will require a scaffold, so now we must turn to the optimal nature of such a ma-terial; in other words, as the title of this paper suggests, what are the essential materials paradigms for regenerative medicine.

MATERIALS SPECIFICATIONS

We should perhaps start this discus-sion of what should be the specifica-tions for scaffold materials in regen-erative medicine by saying what they should not be. It was noted above that a small group of materials now exists

from which biomaterials for implant-able medical devices are naturally selected. To the examples mentioned earlier we must add a few materials that have been developed for use in biodegradable devices, in particular, a group of polymers that are used for transient function in the body, such as resorbable surgical sutures. Since all of these materials, both biostable and biodegradable, are used in clini-cal devices, they have been required to meet certain requirements of regu-latory bodies, especially the U.S. Food and Drug Administration (FDA). The tests that the materials have to undergo have been designed to confirm that the materials comply with the conditions of the definition of biocompatibility given before, which essentially means

that they do the patient no harm. This implies that it will not interact biologi-cally with the patient. In order to obtain FDA approval for a new biomaterial to be used in an implantable medical de-vice, companies have to spend millions of dollars, and the process may take many years. It is of no surprise, therefore, that when seeking biomaterials for new tis-sue engineering scaffolds, most eyes turned toward materials that had prior approval for use in implantable devices in the hope that regulators would con-cede that if they did no harm in such devices, they would do no harm in these scaffolds. It was largely the syn-thetic biodegradable polymers used in sutures and similar devices that tissue engineering companies used in their first products. Whilst of course it is es-sential that these scaffolds do not harm to patients, it is nowhere near sufficient to imply that they must not interact

biologically with the tissues that are involved, since that is precisely what we want them to do in their stimulatory role. Thus, prior regulatory approval in other medical technology scenarios is definitely not a proper specification. Thus a similar definition to that giv-en before for biomaterials in implant-able medical devices may derived for these new products, for example “the biocompatibility of a scaffold or matrix for a tissue engineering product refers to the ability to perform as a substrate that will support the appropriate cel-lular activity, including the facilitation of molecular and mechanical signaling systems, in order to optimize tissue re-generation, without eliciting any unde-sirable local or systemic responses in the eventual host.” Let us consider, therefore, how we can develop better specifications for such materials. We can use as an exam-ple a porous polymeric scaffold and we wish to develop specifications for that scaffold in order to generate, de novo, tissue such as a nerve or a blood vessel. Following the paradigm of ex vivo tissue engineering mentioned before, we start with a cell source. There are several alternatives, primarily includ-ing stem cells, which may be embryon-ic stem cells, induced pluripotent stem cells, amniotic fluid derived stem cells, or autologous adult stem cells, or they may be fully differentiated adult cells, such as chondrocytes or cardiomyo-cytes. The nature of these cells must have some impact on the materials we use for their support, since we require them to be manipulated to varying ex-tents and with varying objectives. It has become quite clear in recent years that the precise environment of stem cells in culture, including the material substrate (often now referred to as the stem cell niche) has a powerful role in determining their performance. Once we have seeded cells into a scaffold, in-stead of the scaffold material ignoring the cells, we require them to facilitate the desired biological processes, which may mean that the cells should be ad-herent to their surface, so that control of hydrophilicity and molecular mobil-ity become very important. The molecular signals and nutri-ents may be simply added to the cell culture medium, but we may require

In order to obtain FDA approval for a new bio-material to be used in an implantable medical device, companies have to spend millions of dollars, and the process may take many years.

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greater control, in which case these ac-tive molecules may be attached to the materials surfaces or incorporated into the scaffold material for controlled re-lease over time. For example, specific interactions with cell receptors may be induced by the attachment of specific peptide sequences to the material. The delivery of the appropriate mechanical signals requires effective design of the bioreactor, but the stresses have to be converted into biological signals with-in the cells, in the processes of mecha-notransduction,10 the stress transfer being controlled by elastic modulus of the substrate. Assuming that the required quality of tissues is being generated, the scaf-fold material then has to degrade. Its initial presence will have given form to the developing structure and it should have facilitated the signaling as just discussed, but then it is no longer re-quired and should give way to the new tissue. This is not a trivial process, since the degradation of a material implies the generation and release of degrada-tion products. If a polymer degrades, the degradation products may include physical fragments as the material dis-integrates, and also smaller molecules such as oligomers, side groups, and monomers. Many of the products of degradation are pro-inflammatory, that is they can initiate an inflammatory re-action, the aggressive cells of which can destroy the tissue that has just been generated. In addition, if the scaffolds are derived from natural polymers or natural tissues, this may be very advan-tageous, but some of the products may also stimulate an immune response, which is definitely not required. We can therefore see that the de-velopment of material specifications for scaffolds in regenerative medicine is complex, and there may be many competing requirements. The subject is fast moving and there is some data in the world’s literature concerning the physical and biological character-istics of potential biomaterials and the tissues that can be generated within them. These materials include proteins such as collagen,11 elastin,12 keratin,13 fibrin,14 and silk.15 They also include polysaccharides such as heparin,16 chi-tosan,17 hyaluronan,18 and alginate.19 They may be biopolyesters, such as

one of the polyhydroxybutyric acid group or other polyalkanoates20 or syn-thetic polymers such as the aliphatic polyesters,21 polyethylene glycol,22 or polyurethanes.23 Their intrinsic charac-teristics involve structural and chemi-cal parameters, their materials-science oriented functional properties, and biological functionality. These include features of pore architecture, includ-ing pore size,24 shape,25 and distribu-tion,26 elasticity, including moduli27 and time-dependent deformation,28 the surface energetics parameters such as hydrophobic–hydrophilic balance and molecular mobility.29 In addition, the chemical functionality,30 environmen-tal responsiveness with respect to pH,31 stress32 and temperature,33 the surface topography at micro- and nano-scale,34

and biodegradation mechanisms such as hydrolysis and enzymatic degrada-tion35 and the rates of degradation36 and metabolism of degradation prod-ucts37 will all play a role. Biological functionalization may be imparted by, for example, growth factors (e.g., nerve growth factor,38 proteins such as laminin,39 peptide motifs,40 cellular antigens (CD molecules), genes/tran-scription factors or oxygen generating species). The tissue generation will be achieved through the activity of cells, including stem cells from a variety of sources41,42 induced pluripotent stem cells43 or adult cells.44 The attempted tissue generation may take place ex vivo using bioreactors,45 cell printing,46 or microfluidics.47

PERSPECTIVES

The above paragraph summarizes just a few of the options open to those working on the development of regen-

erative medicine techniques. Although the nature of the cells that are used is critical, and without molecular sig-nals the whole process will not start, it is clear that biomaterials will play a powerful role. As we move away from conventional biomaterials for the pur-poses of scaffold and matrix design, the need for radically new concepts in biomaterials has become very obvious. Indeed, I have recently re-examined the basic ideas of biomaterials sci-ence48 and concluded that new para-digms are required, both in regenera-tive medicine and other new forms of medical technology. Our conventional biomaterials have usually been solid, macroscopic metals, polymers, ceram-ics, and composites, designed with ro-bustness in mind, and manufactured by conventional engineering routes. Our new biomaterials are more likely self-assembled nanoscale structures derived from natural sources and manipulated at a molecular level in order to give the optimal properties, with a greater em-phasis on the biological events. The new paradigms have not yet led to clear, unequivocal specifications, and only when they do so will the role of the biomaterials scientist within the new era of medical technology become obvious. There are, indeed, many ma-terials opportunities here.

References

1. D.F. Williams, Biomaterials, 29 (2009), pp. 2941–2953.2. A. Atala, R. Lanzo, J.A Thomson, and R. Nerem, edi-tors, Principles of Regenerative Medicine, 2nd Edition (London: Academic Press, 2011).3. E.A. Phelps and A.J. Garcia, Current Opinions in Biotechnology, 21 (2010), pp. 704–709.4. N. Huebsch and D.J. Mooney, Nature, 462 (7272) (2009), pp. 426–432.5. J. Kopecek and J. Yong, Polymer International, 56 (2007), pp. 1078–1098. 6. D.F. Williams, Trends in Biotechnology, 24 (2006), pp. 4–6.7. A. Atala, S.B. Bauer, S. Soker, J.J. Yoo, and A.D. Re-tik, Lancet, 367 (9518) (2006), pp. 1241–1246.8. N. di Maggio, F. Piccinini, M. Jaworski, A. Trump, D. Wendt, and I. Martin, Biomaterials, 32 (2011), pp. 321–329. 9. P.H. Warnke et al., Biomaterials, 27 (2006), pp. 3163–3167.10. M.R. Caplan and M.M. Shah, Cell Biochemistry and Biophysics, 54 (2009), pp. 1–10.11. L. Cen, W. Liu, L. Cui, W. Zhang, and Y. Cao, Pedi-atric Research, 63 (2008), pp. 492–496.12. N. Annabi, S.M. Mithieux, A.S. Weiss, and F. Deh-ghani, Biomaterials, 31 (2010 ), pp. 1655–1665.13. P. Sierpinski et al., Biomaterials, 29 (2008), pp. 118–128. 14. A. Breen, T. O’Brien, and A. Pandit, Tissue Engi-

As we move away from conventional biomate-rials for the purposes of scaffold and matrix design, the need for radically new concepts in biomaterials has be-come very obvious.

JOM • April 201152 www.tms.org/jom.html

neering Part, 15 (2009) pp. 2010–2014.15. J.A. Klunge, O.S Rabotyagova, G.C. Leisk, and D. Kaplan, Trends in Biotechnology, 26 (2008), pp. 244–251.16. T. Nie, R.E. Akins, and K.K. Klick, Acta Biomateria-lia, 5 (2009), pp. 865–875.17. R.A.A Muzzarelli, Carbohydrate Polymers, 76 (2009), pp. 167–182.18. G.D. Prestwich and J.W. Kuo, Current Pharmaceu-tical Biotechnology, 9 (2008), pp. 242–245.19. O. Jeon, K.H. Bouhadir, J.M. Mansour, and E Als-berg, Biomaterials, 29 (2009), pp. 2724–2734.20. G Q. Chen and Q. Wu, Biomaterials, 26 (2005), pp. 6565–6578.21. E. Bible, D.Y.S. Chau, M.R. Alexander, J. Price, K.M. Shakesheff, and M. Modo, Biomaterials, 30 (2009), pp. 2985–2994.22. R.M. Namba, A.A. Cole, K.B. Blugstad, and M.J. Mahoney, Acta Biomaterialia, 5 (2009), pp. 1884–1897.23. C.A. Bashur, R.D. Shaffer, L.A. Dahlgren, S.A. Guelcher, and A.S. Goldstein, Tissue Engineering Part A, 15 (2009), pp. 2435–2445.24. S.M. Lien, L.J. Ko, and T.J. Huang, Acta Biomateria-lia, 5 (2009), pp. 670–679.25. W. Bian and N. Bursac, Biomaterials, 30 (2009), pp. 1401–1412.26. F.P. W. Melchels, A.M.C. Barradas, C.A. van Blit-terswijk, J. de Boer, J. Feijen, and D.W. Grijpma, Acta Biomaterialia, 6 (2010), in press. Can this be

updated?27. N.D. Leipiz and M.S. Shoichet, Biomaterials, 30 (2009), pp. 6867–6878.28. D.O. Freytes, J. Martin, S.S. Velankar, A.S. Lee and S. F. Badylak, Biomaterials, 29 (2008), pp. 1630–1637.29. L. Li et al., J. Materials Chemistry, 19 (2009), pp. 2789–2796. 30. G. Rohman, S.C. Baker, J. Southgate, and N.R. Cameron, J. Materials Chemistry, 19 (2009), pp. 9265–9273. 31. A.W. Chan and R.J. Neufeld, Biomaterials, 30 (2009), pp. 6119–6129.32. C. Candrian et al., Arthritis and Rheumatism, 58 (2008), pp. 197–208.33. S. Masuda, T. Shimizu, and T. Okano, Advanced Drug Delivery Reviews, 60 (2008), pp. 277–285.34. C.J. Bettinger, R. Langer, and J.T. Borenstein, Angewandt Chemie International, 48 (2009), pp. 5406–5415. 35. A.M. Martins et al., Tissue Engineering Part A, 15 (2009), pp. 2940–2945.36. A.L. Becker, A.N. Zelikin, A.P.R. Johnston, and F. Caruso. Langmuir, 25 (2009), pp. 14079–14085.37. J.E. Reing et al., Tissue Engineering Part A, 15 (2009), pp. 605–614.38. M.D. Wood, G.H. Borschel, and S.E. Sakiyama-Elbert, J. Biomedical Materials Research, Part A, 89 (2009), pp. 909–918.39. C.C. Tate, D.A. Shear, M.C. Tate, D.R. Archer, D.G.

Stein, and M.C. LaPlaca, J. Tissue Engineering and Regenerative Medicine, 3 (2009), pp. 208–217.40. S.J. Todd, D.J. Scurr, J.E. Gough, M.R. Alexander, and R.V. Ulijn, Langmuir, 25 (2009), pp. 7533–7539.41. F. Guilak, D.M. Cohen, B.T. Estes, J.M. Gimble, W. Liedtke, and C.S. Chen, Cell Stem Cell, 5 (2009), pp. 17–26.42. J.A. Burdick and G. Vuniak-Novakovic, Tissue Engi-neering Part A, 15 (2009), pp. 205–219.43. T.J. Nelson, A. Martinez-Fernandez, S. Yamada, C Perez-Terzoc, Y. Ikeda, and A. Terzic, Circulation, 120 (2009), pp. 408–416.44. N.D. Hsieh-Bonassera et al., Tissue Engineering Part A, 15 (2009), pp. 3513–3523. 45. J.W. Bjork and R.T. Tranquillo, Biotechnology and Bioengineering, 104 (2009), pp. 1224–1234.46. C. Norotte, F.S. Marga, L.E. Niklason, and G. For-gacs, Biomaterials 30, (2009) pp. 5910–5917.47. C.P. Huang et al., Lab on a Chip, 9 (2009), pp. 1740–1748.48. D.F. Williams, Biomaterials, 30 (2009), pp. 5897–5909.

David F. Williams is currently Professor of Biomaterials and Director of International Affairs at Wake Forest Institute of Regen-erative Medicine, Winston-Salem, North Carolina, e-mail, dfwillia@wfubmc. He is Editor-in-Chief of the journal Biomaterials.

Scaffold Free Tissue Constructs for OcularSurface Regeneration

T.V. Kumary

INTRODUCTION

Elements of Tissue engineering forreconstruction of tissues and organs arecells, scaffolds and growth factors/supplements. Although the scaffolds areusually biodegradable, use of scaffoldresults in the implantation of a foreignmaterial into human body. Moreover,some applications of tissue engineeringlike skin and cornea do not require theuse of scaffold. In order to eliminate theuse of scaffolds in constructing threedimensional tissue structures and organs,thermoresponsive, smart culture surfaceshave been studied [1–3]. These surfacescan be used for detachment of cell layerswith intact cell–cell and cell—extracellular matrix [4], avoiding thedamages caused by enzymatic ormechanical treatment. The reversiblethermal switching property ofthermoresponsive polymers enable it toundergo phase transition betweenhydrophilic and hydrophobic stages [5].Above the lower critical solutiontemperature (LCST) the polymer will behydrophobic resulting in the attachmentand growth of cells, while below theLCST it turns hydrophilic enabling thedetachment of cells. Culture surface forcorneal cell growth should becytocompatible and support limbal cellculture as regeneration and repair of thecorneal epithelium in the eye is ensuredby adult stem cells that reside in limbus,a region in the corneoscleral rim (6).Deficiency of stem cells, due to eitherinherited or acquired reasons, lead toseveral complications (7) and can resultin gradual opacity and blindness. Limbalstem cell transplantation after ex vivoexpansion of autologous limbal stemcells (LSC) (8) is ideal for treating limbalstem cell deficiency. The use of 3T3feeder layer is a prerequisite for thecultivation and expansion of LSC (9).

However, this murine feeder layer leavesopen the possibility of xeno-contamination of limbal cells. Inaddition, the cultured cells can take upsialic acid (Neu5Gc) from mouse feedercells that can raise circulating antibody, leading to in vivo cell killing aftertransplantation. Moreover, Food anddrug administration (FDA, USA), hasclassified tissues regenerated using 3T3feeder layer as xenogeneic . These factsunderscore the demand to develop feederfree culture systems for transplantationpurpose

Objective of this study was to preparethermoresponsive polymers to act as cellculture substrate and for preparation ofin vitro scaffold and feeder free tissueconstructs. As scaffold free tissueconstructs are ideal for ocular surfaceregeneration, attempt was made tocreate scaffold free corneal constructusing feeder free culture of limbal stemcells and to assess the efficacy ofscaffold free constructs bytransplantation to rabbit limbal stem celldeficient model.

METHODOLOGY

Different PIPAAm based polymericformulations were prepared that can beused to obtain cultured intact cornealepithelial cell sheets which can findapplication for ocular surfaceregeneration. Polymeric formulationswere characterized physico chemicallyby different techniques like Fouriertransform infrared spectroscopy (FTIR),Differential scanning calorimetry,Profilometry, Atomic force microscopy.Any material intended for medicalapplication should be safe for the humanbody or should be provencytocompatible before in vivo use. Thethermoresponsive copolymer coatedsurfaces were evaluated for

cytocompatibility by analyzing its abilityin supporting viability, metabolicactivity, attachment, spreading andproliferation of fibroblast cells incomparison to tissue culture polystyrene(TCPS). Xeno feeder free culture oflimbal stem cells was standardized bymanipulation of the microenvironment.Thermoresponsiveness or temperatureinduced cell detachment to formdetachable cell layers which is requiredfor generation of scaffold free tissueconstruct was also looked into. Tissueconstructs were analysed for theirspecific cell characteristics.

To prove the concept of ocular surfaceregeneration, a limbal stem celldeficiency model was created in rabbitsby ablation of the limbal area followedby debridement of the cornealepithelium. Confirming the generatedmodel, tissue construct was transplantedand the regenerative process wasmonitored.

RESULTS & DISCUSSION

Different formulations ofthermoresponsive polymers showed thespecific characteristics uponphysicochemical chatracterisation andwas cytocompatible (10, 11, 12). TheLCST of PNIPAAm can be tuned tospecific applications bycopolymerization with other comonomerhaving either higher hydrophilic orhydrophobic nature Moreove the culturesurface supported limbal stem cellsgrowth.. A combination of Iscove’smodified Dulbecco’s medium andPanserin 801 resulted in formation ofautofeeder layer with maintenance ofprogenitor characteristics, thusmimicking natural tissuearchitecture(13).Thermoresponsivenesswas confirmed by detachment of tissueconstruct(14). Generated tissue also

28 T.V. Kumary

showed the specific markers of stemcells as well as epithelial cells (15). Thespecific explant culture systemmimicked natural tissue architecture andniche closely by enabling formation ofautofeeder layer and thereby eliminating

the need of xeno-feeder layers for exvivo expansion of epithelial progenitorcells. Other sources of stem cells likebone marrow messenchymal stem cellscan also be differentiated to cornealepithelial lineage. This will bespecifically useful in the of bilaterallimbal stem cell deficiency.

Limbal stem cell deficiency modelrabbits showed the main hall marks ofLSCD like neo vascularization andcorneal opacity. Pre clinical evaluationby transplantation of tissue constructshowed very promising results of ocularsurface regeneration.

References

1. Hirose M, Kwon OH, Yamato M, KikuchiA, Okano T. Creation of designed shape cellsheets that are noninvasively harvested andmoved onto another surface.Biomacromolecules. 2000;1:377–81.2. Okano T, Yamada N, Sakai H, Sakurai Y. Anovel recovery system for cultured cells usingplasma-treated polystyrene dishes graftedwith poly(n-isopropylacrylamide). J BiomedMater Res.1993;27:1243–51.

3. von Recum H, Kikuchi A, Okuhara M,Sakurai Y, Okano T, Kim SW. Retinalpigmented epithelium cultures on thermallyresponsive polymer porous substrates. JBiomater Sci Polym Ed.1998;9:1241–53.4. Kushida A, Yamato M, Konno C, KikuchiA, Sakurai Y, Okano T. Decrease in culturetemperature releases monolayer endothelialcell sheets together with deposited fibronectinmatrix from temperature-responsive culturesurfaces. J Biomed Mater Res. 1999;45:355–62.5. Takezawa T, Mori Y, Yoshizato K. Cellculture on a thermoresponsive polymersurface. Biotechnology (N Y). 1990;8:854–6.6. Dua HS, Azuara-Blanco A.. Limbal stemcells of the corneal epithelium. SurvOphthalmol 2000, 44:415–425.7. Ang LP, Tan DT. Ocular surface stem cellsand disease: Current concepts and clinicalapplications. Ann Acad Med Singapore 2004,33:576–5808. Meller D, Pires RT, Tseng SC.. Ex vivopreservation and expansion of human limbalepithelial stem cells on amniotic membranecultures. Br J Ophthalmol 2002, 6:463–4719. Nishida K, Yamato M, Hayashida Y,Watanabe K, Maeda N, Watanabe H,Yamamoto K, Nagai S, Kikuchi A, Tano Y,Okano T.. Functional bioengineered cornealepithelial sheet grafts from corneal stem cellsexpanded ex vivo on a temperature-responsive cell culture surface.2004,Transplantation 77:379–385.10. P. R. Anil Kumar, K. Sreenivasan, T. V.Kumary, Alternate Method for GraftingThermoresponsive Polymer for Transferring

In Vitro Cell Sheet Structures. Journal ofApplied Polymer Science, 2007, 105, 2245–225111. Thomas N. Abraham, Vidya Raj, TilakPrasad, P. R. Anil Kumar, K. Sreenivasan, T.V. Kumary, A Novel Thermoresponsive GraftCopolymer Containing PhosphorylatedHEMA for Generating Detachable CellLayers, Journal of Applied Polymer Science,2010, 115, 52–6212. Viji Mary Varghese, Vidya Raj, K.Sreenivasan, T. V. Kumary In vitrocytocompatibility evaluation of athermoresponsive NIPAAm-MMAcopolymeric surface using L929 cells J MaterSci: Mater Med.2010, 21:1631–163913. Viji Mary Vargheese, Tilak Prasad, T.V.Kumary, Optimization of Culture Conditionsfor an Efficient Xeno-Feeder Free LimbalCell Culture System Towards Ocular SurfaceRegeneration. Microscopy Research AndTechnique 2010, 73:1045–105214. P.R. Anil Kumar a, H.K. Varma b, T.V.Kumary, Cell patch seeding and functionalanalysis of cellularized scaffolds for tissueengineering Biomed. Mater. 2 (2007) 48–5415. Nithya Joseph, Tilak Prasad, Vidya Raj,P. R. Anil Kumar, K. Sreenivasan, T. V.Kumary, A Cytocompatible Poly(N-isopropylacrylamide-coglycidylmethacrylate)Coated Surface as New Substrate for CornealTissue Engineering, Journal of Bioactive AndCompatible Polymers, 2010,. 25:58-74

Dr. TV Kumary is Scientist G & SIC, Tissue Culture Laboratory, Biomedical Technology Wing ofSree Chitra Tirunal Institute for Medical Sciences & Technology tvkumari@sctimst.ac.in

Design of Biomimetic Matrix for in vitroTissue Regeneration with Adult Stem Cells

Lissy Krishnan

Once the differentiation of adult stemcells can be controlled in the laboratory,these cells may become the basis oftransplantation-based therapies. Themicroenvironments in which stem cellsreside is termed as “niche” whichregulate stem cell fate. Replication of thein vivo niche conditions for in vitrostandardization of cell expansion mayturn out useful for regenerative therapies.The stem cells and niche may supporteach other during development andreciprocally signal to maintain each otherduring adulthood. Simple location ofstem cells is not sufficient to define aniche; the niche must have both anatomicand functional dimensions (1). For invitro culture of adult stem cells it is oftendifficult to define the niche because thefactors present in vivo in themicroenvironment and the type ofsignaling available for celldifferentiation and tissue homeostasis isnot clearly understood. Fibrin is a goodreservoir that binds various ECMcomponents such as adhesive proteins(eg. fibronectin and gelatin) and growthfactors (eg, fibroblast growth factor[FGF] and vascular endothelial growthfactor [VEGF]), making it a matrix withcell adhesion and signaling potential [2].

The concept of using a fibrin basedmatrix for growing differentiatedendothelial cell was tested before it wasmodified and used for growingcirculating endothelial progenitor cells(EPC) and differentiating it intoendothelial cells (EC). We havedesigned a fibrin composite thatmaintains endothelial cell culturethrough several passages withoutaltering its phenotype [3]. In addition,HSVEC isolated from diseased arterywas found to grow on the fibrin matrixforming more non thrombogenicphenotype after 2 to 3 passages on

designed matrix (4). It was found thataddition of more angiogenic andmitogenic growth factors andglycosaminoglycans to the fibrin matrixhas promoted deposition of ECMespecially the components of basementmembrane by EC (5-7). It was shownthat the matrix can be coated on variousbiomaterial surfaces for improvedHUVEC attachment, proliferation andsurvival (8-11).

Depending on the cell that has to begrown, the composition of niche needsto be different. The concept of usingspecific matrices for differentiation ofcirculating progenitors from humanperipheral blood mononuclear cells(PBMNC) fraction has beendemonstrated earlier (12,13).

METHODS

Tissue culture polystyrene (TCPS)(NUNC, Rakslide, Denmark) werecoated with fibrin composite asdescribed earlier (3) with somemodification to create specific matrix forendothelial progenitor cells, smoothmuscle cells or neuronal progenitor cells.Morphological analysis of the cells inculture was done periodically. Specificmarkers were used for identifying cellphenotype.

RESULTS

Use of specifically designed matricescan generate endothelial cells, smoothmuscle cells and neuronal cells fromunselected population of PBMNCisolated from human blood. Thedifferentiation was demonstrated usingspecific markers for each cell types.Many of the components of the matrixin all 3 types of niche are common suchas fibrin, gelatin, fibronectin and certaingrowth factors. Cell differentiation was

confirmed in each case using specificmarkers. The designed matrices allowedcell survival and growth in a cell specificmanner.

References

1. David T. Scadden, The stem-cell niche asan entity of action, Nature, 441 (7097), 1075-1079 (29 June 20062. Clark RA. Fibrin glue for wound repair:facts and fancy. Thromb Haemost2003;90:1003-1006.3. Prasad CK, Krishnan LK. Effect of passagenumber and matrix characteristics ondifferentiation of endothelial cells cultured fortissue engineering. Biomaterials2005;26:5658-5667.4. Prasad CK. Jayakumar K. and KrishnanLK (2006) Phenotype Gradation of HumanSaphenous Vein Endothelial Cell (HSVEC)from Cardiovascular Disease Subjects.Endothelium: 13; 341 -352.5. Divya P., Sreerekha P.R. and Krishnan LK(2007) Growth Factors Up regulateDeposition and Remodeling of ECM byEndothelial Cells Cultured for TissueEngineering Applications. BiomolecularEngineering 24: 593-602.6. Divya P, and Krishnan LK (2009), Designof fibrin matrix composition to enhanceendothelial cell growth and extra cellularmatrix deposition for in vitro tissueengineering Artificial Organs 33 :16-25.7. Divya P. and Krishnan LK. 2009Glycosaminoglycans restrained in fibrinmatrix improve ECM remodeling byendothelial cells grown for vascular tissueengineering Tissue Engineering &Regenerative Medicine 3:377-388.8. Prasad CK, Muraleedharan CV, KrishnanLK. Bio-mimetic composite matrix thatpromotes endothelial cell growth formodification of biomaterial surface. JBiomed Mater Res A 2007;80:644-654.9. Krishna Prasad C. and Krishnan LK. 2008Regulation of Endothelial Cell Phenotype byBiomimetic Matrix Coated on Biomaterialsfor Cardiovascular Tissue Engineering. ActaBiomaterialia .4:182-191.10. Divya Pankajakshan, Lizymol PPhilipose, Minshiya Palakkal, Kalliyana

30 Lissy Krishnan

Krishnan and Krishnan LK (2008)Development of a fibrin composite coatedpoly (e-caprolactone) scaffold for potentialvascular tissue engineering applications. JBiomed Mater Res Part B. 87B; 570-579.11. Divya P. Kalliyana Krishnan V. andKrishnan LK 2007 Vascular TissueGeneration in Response to SignallingMolecules Integrated with a Novel poly(e-

caprolactone)- Fibrin Hybrid Scaffold. TissueEngineering & Regenerative Medicine 1:389-397 published online doi:10.1002/term.48.12. Sreerekha P.R. Divya P and Krishnan LK(2006) Adult Stem Cell Homing andDifferentiation in vitro on Composite FibrinMatrix. Cell Proliferation: 36;301-312.

Dr. Lissy Krishnan is Scientist G & SIC, Thrombosis Research Unit, Biomedical Technology Wingof Sree Chitra Tirunal Institute for Medical Sciences & Technology. lissykk@sctimst.ac.in

13. Anumol Jose and Krishnan LK. Effectof Matrix Composition on Differentiation ofNestin-positive Neural Progenitors fromCirculation into Neurons J. Neural Eng. 7(2010, 10p) doi:10.1088/1741-2560/7/3/036009

Stem cells and Ceramics for the Repair ofBony Defects in Orthopaedics

Annie John

In this era of Regenerative Medicine, bio-functional aspects of bioactive ceramicsintended for the repair of bony defects willbecome a relevant technology in Orthopaedicapplications. This is because bone defectscaused by tumor resection, trauma andskeletal abnormalities still remain a majorclinical problem in attaining functional boneafter the treatment. Stem cell-based tissueengineering is a promising technology in theeffort to create functional tissue of choice.Successful bone tissue engineering relies notonly on the differentiation of cells in contactwith the implant material but also on theproliferation capacity of the cells to obtain aproper colonization of the 3-D matrix to makea ‘hybrid bone’ in vitro. In the developmentof such ‘combination products’, the utility ofincorporating cells have been initiated intoceramic scaffolds.

Sree Chitra Tirunal Institute for MedicalSciences & Technology, Trivandrum, India,is an Institute of National Importance in thefield of Biomedical Technology -biomaterials, biomedical devices and TissueEngineering. Research in the area ofbioceramics, is one of the major activities inthe Institute where novel bioceramiccompounds have been developed in theBioceramics Laboratory to form a broadrange of goal oriented indigenous, costeffective materials for bone grafts in thehealth care system. The 3-D lattice structureof the bioactive ceramic offers a moreappropriate niche where cell-cell/cell-material interactions could be favored andbe closer to their in vivo situationcounterpart.

In this context, the focus is on the isolation,expansion and characterization of bonemarrow-derived mesenchymal stem cells(BMMSCs) in vitro and its ability to support,express bone markers and maintainosteogenesis on bioactive ceramic scaffoldsin the cell culture system. To create stable butdegradable implants for hard tissueregeneration, a material that mimics themineral phase of bone is the first choice as asubstrate. Ceramics have a chemicalcomposition similar to bone and are known

to bond with bone. They are pervaded byadequate pores larger than 100 µm for thecells to dwell within and to facilitate oxygenand nutrient supply. For tissue engineering,these highly cell loaded scaffolds arefabricated as implants. The living partdemands for an appropriate architecture andabove all the material needs to bebiocompatible. These cells adhered, attachedand displayed a well spread-out cellmorphology making focal contacts on thematerial surface, eventually formingmultilayer of aligned cells fabricating a cellsheet-like covering, probably influenced bythe physical and chemical stimuli emanatedfrom the underlying 3-D ceramic scaffold.Cells invaded and migrated into the 3-Dporous ceramic. Optimal functionality of atissue depends on its appropriate histologicalorganization and the alignment of cells in theECM is an essential indicator of tissueintegrity to improve the functionality of tissueconstructs.

Subsequently, transplantation of thefabricated cell-seeded ceramic scaffolds tosegmental bony defects bridged the defect atan early period where healing was uneventful.Implanted ‘bio-constructs’ encouragedosteoinduction, osteoconduction,osteointegration and biodegradation withoutfibrous tissue formation. The newly formedbone organized, mineralized and attained thecontour of the original bone with time in vivo.In short, the research is dedicated on thepossibility of sculpting and designing bio-functional ceramics for the prospect of repairof large segmental defects in clinicalsituations which is a formidable challenge inorthopaedic reconstructive surgery.

INTRODUCTION

In this era of Regenerative Medicine,bio-functional aspects of bioactiveceramics intended for the repair of bonydefects is posed as a relevant technologyin Orthopaedic applications. This isbecause bone defects caused by tumorresection, trauma and other skeletalabnormalities still remain a major

clinical problem in attaining functionalbone after the treatment. Bone has theremarkable capacity to heal without scarformation, but this regenerative processfails in patients with large bone lesionsor impaired wound healing, requiringclinical intervention. Such critical-sizedsegmental bone defects do not healspontaneously during the lifespan of theorganism (Reichert JC et al., 2009).

Stem cell-based tissue engineering is apromising technology in the effort tocreate functional tissue of choice.Successful bone tissue engineering reliesnot only on the differentiation of cellsin contact with the implant material butalso on the proliferation capacity of thecells to obtain a proper colonization ofthe 3-D matrix to make a ‘hybrid bone’in vitro. Thus the triad of tissueengineering consists of (a) 3D scaffold(b) cells and (c) growth factor, each ofthese can be used independently or incombination. In the development of such‘combination products’, the utility ofincorporating cells have been performedinto ceramic scaffolds with and withoutgrowth factors. Research in the area ofbioceramics, is one of the majoractivities in the Institute where novelbioceramic compounds have beendeveloped to form a broad range of goaloriented indigenous, cost effectivematerials for bone grafts in the healthcare system.

So the focus of the research is on –

1. The isolation, expansion andcharacterization of bone marrow-derivedmesenchymal stem cells in vitro and

2. Its ability to support, express bonemarkers and maintain osteogenesis onbioactive ceramic scaffolds in the cellculture system and its preclinicaltranslations.

32 Annie John

METHODOLOGY

Material

In-house developed bioactive ceramics(Bioceramics Laboratory, SCTIMST,Trivandrum) – triphasic ceramiccomposite coated hydroxyapattie (HASi)was the material of choice for the in vitroand in vivo validation as bonesubstitutes. The 3-D surface topographywas assessed by Scanning electronmicroscopy.

In vitro - Cell culture Procedures

MSCs were isolated from goat bonemarrow, expanded and differentiated inan osteogenic medium and characterisedfor their osteogenic markers. Scaffoldswith 1 x 105 cells/cm2 seeded onto themare fabricated as implants and thesehighly cell loaded scaffolds are used fortissue engineering. Morphological andfunctional evaluation of such TEconstructs was done by SEM and usingother biochemical markers.

In vivo – Animal surgical Procedures

Segmental bone defects were created inanaesthetized goats under the approvaland guidelines of the IAEC andCPCSEA. This bone loss was replacedwith in-house developed bioactiveceramics by a press fit method/with thesupport of internal or external fixators.Bare implants and cell-seeded implantswith and without PRP were also used.The implant sites were assessed post-implantation, by radiography and furtherevaluated by histology of Stevenals Blueand Van-Gieson Picrofuschin stainedPolymethyl Methacrylate sections.

RESULTS

In vitro

Bioactive ceramic showed a 3D roughsurface topography having pores of 50– 500 um size with interconnections.Cells adhered, attached and displayed awell spread-out cell morphology makingfocal contacts on the material surface,eventually forming multilayer of alignedcells fabricating a cell sheet-likecovering, probably influenced by thephysical and chemical stimuli emanatedfrom the underlying 3-D ceramicscaffold. Cells even invaded andmigrated into the 3-D porous ceramic –in vitro and displayed their functionalmarkers like ALP and Osteocalcin.

In vivo

Upon implantation, the bioactiveceramic uneventfully osteointegrated atthe defect site with the in growth of denovo bone tissue in par with materialdegradation assessed by radiography.The bioactive ceramic uneventfullyosteointegrated at the defect site by theingrowth of de novo trabeculae bone.Implanted ‘bio-constructs’ therebyencouraged osteoinduction,osteoconduction, osteointegration andbiodegradation without fibrous tissueformation. Subsequently, transplantationof such fabricated cell-seeded ceramicscaffolds to bony defects bridged andhealed the defect at an early period wherehealing was uneventful. The newlyformed bone eventually organized,mineralized and attained the contour ofthe original bone with time in vivo.

DISCUSSION

To create stable but degradable implantsfor hard tissue regeneration, a materialthat mimics the mineral phase of bone isthe first choice as a substrate. Ceramics(Hydroxyapatite) have a chemicalcomposition similar to bone andbioactive. Preparation of the scaffoldswith adequate pores larger than 100 µmfor the cells to dwell within and tofacilitate oxygen and nutrient supplyensures optimal cell colonization. The3-D lattice structure of the bioactiveceramic offers a more appropriate nichewhere cell-cell/cell-material interactionscould be favored and be closer to theirin vivo counterpart. This satisfies theneed for an appropriate architecture atthe cellular level and ensuresbiocompatibility. Optimal functionalityof a tissue depends on its appropriatehistological organization and thealignment of cells in the ECM is anessential indicator of tissue integrity toimprove the functionality of tissueconstructs.

Mesenchymal stem cells (MSCs) showextensive capacity for expansion in vitro,so these cells have been considered ascandidates for cell therapy. These cellscan differentiate into multiple celllineages including bone, cartilage,muscle, bone marrow stroma, tendon /ligament, fat, dermis, and otherconnective tissues (Caplan AI 2005).MSC population must express CD105,CD73 and CD90 and they themselves,secrete a broad spectrum of bioactivemacromolecules that are bothimmunoregulatory and serve to structure

a regenerative microenvironment in thearena of tissue injury.

Through the addition of osteogenicsupplements including dexamethasone,ascorbic acid and â-glycerophosphate,purified culture-expanded MSCs can bedifferentiated into osteogenic lineage invitro, as envisaged by the production ofcollagen-rich matrix, increasedexpression of non-collagenous proteinslike ALP, osteocalcin and finally by theformation of mineralized matrix (KernS et al., 2006. (Son E et al., 2007).

Therefore, the current state of art withinbone tissue engineering consists of thecombination of culture-expanded MSCswith 3D porous biomaterials.Although the implantation of MSC-seeded biomaterials has shownsignificant increase in skeletal repair(Kadyala et al., 1997, Bruder SP et al.,1998; Li Z et al., 2005) additional in vitroculture methods have been developedthat promote osteogenic differentiationof MSCs in vitro in lieu of in vivoincorporation. These cell-loadedceramics have succeeded in creatingbone formation in animal models (LiuG et al., 2008; Nair et al., 2009c; Nair etal., 2009d). The possibility ofengineering bone in a goat model hasbeen tested using osteogenic inducedbone marrow-derived MSCs and HASi.The cells were cultured on the bioactiveceramic for 1 week before thetransplantation. In the surgicalprocedure, a 2 cm segmental defect wascreated in the femur of the goat modeland the defect was treated with tissue-engineered HASi (HASi+C) and HASialone. The materials were stabilized withtitanium intramedullay nails and screws.In bare HASi groups, lamellar pattern ofbone formation was seen only in theperipheral region, while the mid regionwas filled with woven bone at 4 months.The mid region was later organized intolamellar pattern at 6 months and acomplete bone remodeling at 12 months.Interestingly, in HASi+C groups, amature lamellar pattern of newly formedbone was observed throughout the defectat 4 months. Thus, whatever the qualitiesattained in the tissue-engineered groupsat 4 months was achievable only at 6months in bare HASi groups. This maybe due to the combined osteoinductiveand osteoconductive effect of HASi+Cgroups (Nair et al., 2009d; Nair MB etal., 2010).

It was observed that the culture of cellson HASi could not only enhance bone

Stem cells and Ceramics for the Repair of Bony Defects in Orthopaedics 33

regeneration, but also mediate materialdegradation (HASi degraded faster inHASi+C groups than HASi alonegroups), which is one of the mainproblems encountered in the category oftissue-ingrowth. It is hypothesised thatosteogenic induced MSCs cultured onHASi create an ECM that can favor thesuccessive migration of osteoclasts.Among many ECM proteins,osteopontin has an important role inpromoting the adhesion of osteoclasts tothe scaffold surface through theirarginine-glycine-aspartic acid sequence(Mastrogiacomo M et al., 2006). In vitrostudies have shown that the expressionof osteopontin was significantlyenhanced on HASi than HA (Nair MBet al., 2009b). Thus the combined effectsof the characteristics of the material(porosity and silica content) and theability of cells on HASi to attractosteoclasts have improved cell-mediatedresorption in HASi+C groups. Thesecells can also secrete inductive factorsthat recruit indigenous mesenchymalstem cells (MSCs) into osteogeniclineage and produce mature bone matrixquickly. Thus the combined contributionfrom cells on the scaffolds and host-derived MSCs together withbiomolecules can advance osteogenesisat the defect site.

The cost of almost all geneticallyengineered growth factors isprohibitively high, which limits its usefor large defects where large amount ofgrowth factors are needed. Platelet-richplasma is a proven source of manygrowth factors like platelet-derivedgrowth factor (PDGF), transforminggrowth factor-beta (TGF-â), vascularendothelial growth factor (VEGF) andinsulin-like growth factor (IGF-I)(Anitua E et al., 2007). In this study, theplatelets adhered and were activated onthese materials without the use oftriggering agents (Nair et al., 2009). Theactivation was significantly enhanced onHASi than HA where the chemicalcomposition, particularly silica mayhave played an important role (NemmarA et al., 2005). Nevertheless,HASi+CP groups showed a betterresponse qualitatively where the lamellarbone was more organized (Nair et al.,2009c). In general, it is understood thatthat PRP does not have any strongosteogenic effect so that filling a defectwith PRP alone will not allowosteogenesis to occur in larger defects.Instead, PRP has an osteopromotiveeffect and can enhance bone regeneration

in the presence of osteogenic precursorcells by encouraging their proliferationand differentiation (Schlegel KA et al.,2004). Additionally, MSCs associatedwith PRP are potent angiogenic inducersproven to release VEGF leading to fasterbone regeneration and remodeling(Yamada Y et al., 2004). Kitoh et al.,(2007) observed that transplantation ofbone marrow cells and PRP shortenedtreatment period and reduced associatedcomplications by accelerating new boneformation in distraction osteogenesis

CONCLUSION

Interestingly, the research focuses on thepossibility of sculpting and designingbio-functional ceramics for the prospectof repair of large segmental defects inclinical situations which is a formidablechallenge in orthopaedic reconstructivesurgery. Segmental bone defects canresult either from acute trauma orchronic infection requiring boneresection or chronic non-unions due toosteoporosis or osteomyletis,osteogenesis imperfecta orosteosarcoma. The current treatmentslike autograft, allograft or distractionosteogenesis are associated with severallimitations. Therefore the focus is on thenew concept called “Tissue engineering”in which 3D porous biodegradablescaffolds are incorporated with cells orgrowth factors to regenerate tissuewithin the body. It is apparent from thestudy that the tissue-engineered groupsshowed enhanced regenerationcompared to the bare material groups inthe healing of the bony defect. Althoughsignificant results have been achieved,there are challenges that are to beaddressed before the strategy canbecome a reality in the future.

Acknowledgments

The author acknowledges The Director& The Head, SCTIMST for the facilitiesprovided to carry out the work; Dr. H.K. Varma for the bioceramics; Dr. K.V.Menon and Dr. Sachin J Shenoy forsurgical procedures; Dr. P.R.Umashankar for the Large Animalfacility; DRDO for financial assistance;and CSIR for Dr M.B.N.

References

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2. Bruder SP, Kraus KH, Goldberg VM,Kadiyala S. The effect of implants loaded withautologous mesenchymal stem cells on thehealing of canine segmental bone defects.Bone Joint Surg Am. 1998; 80(7): 985-96.3. Caplan AI. Review: Mesenchymal stemcells: Cell-based reconstructive therapy inorthopedics. Tissue Eng 2005; 11: 1198–1211.4. Kadyala S, Jaiswal N, Bruder SP. Culture-expanded, bone marrow-derivedmesenchymal stem cells can regenerate acritical-sized segmental bone defect. TissueEng. 1997, 3(2): 173-85.5. Kern S, Eichler H, Stoeve J, Klüter H,Bieback K. Comparative analysis ofmesenchymal stem cells from bone marrow,umbilical cord blood, or adipose tissue. StemCells. 2006; 24(5): 1294-301.6. Kitoh H, Kitakoji T, Tsuchiya H, Katoh M,Ishiguro N. Transplantation of cultureexpanded bone marrow cells and platelet richplasma in distraction osteogenesis of the longbones. Bone 2007; 40(2): 522-8.7. Li Z, Yang Y, Wang C, Xia R, Zhang Y,Zhao Q, Liao W, Wang Y, Lu J. Repair ofsheep metatarsus defects by using tissue-engineering technique. J Huazhong Univ SciTechnolog Med Sci. 2005; 25(1): 62-7.8. Liu G, Zhao L, Zhang W, Cui L, Liu W,Cao Y. Repair of goat tibial defects with bonemarrow stromal cells and beta-tricalciumphosphate. J Mater Sci Mater Med. 2008;19(6): 2367-76.9. Mastrogiacomo M, Corsi A, Francioso E,Di Comite M, Monetti F, Scaglione S, FaviaA, Crovace A, Bianco P, Cancedda R.Reconstruction of extensive long bone defectsin sheep using bioresorbable bioceramicsbased on silicon stabilized Tricalciumphosphate. Tissue Eng. 2006; 12(5): 1261-73.10. Nair MB, Varma H, Shenoy SJ, John A.Treatment of goat femur segmental defectswith silica-coated hydroxyapatite—one-yearfollow-up. Tissue Eng Part A. 2010;16(2):385-91.11. Nair MB, Varma HK, John A. Platelet-richplasma and fibrin glue coated bioactiveceramics enhances growth and differentiationof goat bone marrow-derived stem cells.Tissue Eng Part A. 2009a; 15(7):1619-31.12. Nair MB, Varma HK, John A. Triphasicceramic coated hydroxyapatite as a niche forgoat stem cell-derived osteoblasts for boneregeneration and repair. J Mater Sci MaterMed. 2009b; 20 Suppl 1:S251-8.13. Nair MB, Varma HK, Menon KV, ShenoySJ, John A. The reconstruction of goat femursegmental defect using triphasic ceramiccoated hydroxyapatite together withautologous cells and platelet-rich plasma. ActaBiomater. 2009c; 5(5):1742-55.14. Nair MB, Varma HK, Menon KV, ShenoySJ, John A. Tissue regeneration and repair ofgoat segmental femur defect with bioactivetriphasic ceramic-coated hydroxyapatitescaffold. J Biomed Mater Res A. 2009d;91(3):855-65.15. Nemmar A, Nemery B, Hoet PH, VanRooijen N, Hoylaerts MF. Silica particlesenhance peripheral thrombosis: key role of

34 Annie John

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Dr. Annie John PhD is Scientist E & SIC, Transmission Electron Microscope Facility, BiomedicalTechnology Wing of Sree Chitra Tirunal Institute for Medical Sciences & Technology.ajkari@sctimst.ac.in

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Brief Biodata Professor David F. Williams FREng

David Williams was trained as a materials scientist at the University of Birmingham, UK (B.Sc. 1965, Ph.D. 1969, D.Sc. 1982). In 1968 he took up a faculty position in the School of Medicine at the University of Liverpool, UK, where he remained for 40 years, writing, researching and teaching on the science of biomaterials. He created the Department of Clinical Engineering in the University and was its Head for 20 years. He was granted a Personal Chair in the University in 1984, then the youngest person in the history of the University to receive such an honour. Towards the end of his career in Liverpool, he became the Pro-Vice-Chancellor (equivalent to Academic Vice-President) of the University, and then won major funding from the British Government to form, in collaboration with the University of Manchester, the UK Centre for Tissue Engineering, of which he became Director. He was instrumental in obtaining over 25 million euro for a European Union Integrated Project, involving 25 partners across Europe, for a systems engineering approach to tissue engineering (STEPS). Professor Williams has extensive experience with medical device and regenerative medicine companies, and significant experience in both patent and product liability litigation. He is President-elect of Tissue Engineering and Regenerative Medicine International Society (TERMIS).

Professor Williams is the Editor-in-Chief of Biomaterials, now the leading journal in the field of biomaterials science. During his research career he has published over 30 books, including the first text book in this area, Implants in Surgery, and the Williams Dictionary of Biomaterials, and around 400 papers. He has presented keynote and plenary lectures at conferences in over 30 countries over the last 30 years. He has received the major awards from the US (Clemson Award 1982, Founders Award 2007), European (George Winter Award, 1996), UK (Presidents Award 2004, Chapman Medal of the Institute of Materials, 2007) and Indian (Sharma Award 2008) societies of biomaterials. He was a scientific adviser to the European Commission and wrote many Opinions on which European laws in health technology and nanotechnology are based. In 1999 he was elected as a Fellow of the Royal Academy of Engineering in recognition of his contributions to engineering in medicine.

Professor Williams left the University of Liverpool, and the UK, at the end of 2007. While retaining the title of Emeritus Professor at Liverpool, he is currently Professor of Biomaterials and Director of International Affairs, Wake Forest Institute of Regenerative Medicine, North Carolina, USA., where he has responsibilities for establishing and managing international collaborations. He is also a Visiting Professor in the Christiaan Barnard Department of Cardiothoracic Surgery, Cape Town, South Africa, a Visiting Professorial Fellow at the Graduate School of Biomedical Engineering, University of New South Wales, Australia, and a Guest Professor, Tsinghua University, Beijing and Visiting Professor, Shanghai Jiao Tong University, China. He has been appointed as Visiting Professor at Sree Chitra Tirunal Institute for Medical Sciences & Technology. He spends approximately 5 months a year in the USA, 1 in Europe, 2 in South Africa, 2 in Australasia and 2 in Asia.

Ms. Peggy O”Donnell

Peggy O”Donnell is Managing Editor of the journal Biomaterials and had played a significant role in making Biomaterials journal the number one in Materials Science with an impact factor of 7.365 (2009).

Dr. Subhas C Kundu

SC Kundu, Ph.D. is Professor and the formerly Founder-Head of Department of Biotechnology of Indian Institute of Technology (IIT) - Kharagpur since July 1994. Before joining IIT Kharagpur he was also an Assistant Professor to a Full Professor including Head of Department at Manipur University (erstwhile Jawaharlal Nehru University Centre, Imphal, India from June 1976 to June 1994). He teaches Genetics, cell and molecular biology and recombinant DNA technology to under graduates and pre-doctoral students at IIT. Dr. Kundu received his post doctoral training at Institute of Molecular Biology, Moscow; Department of Biology, York University, Canada; Medical University, Lubeck, Germany; Department of Biology & Biochemistry, Brunel University, UK. His main area of interest is molecular genetics and silk biomaterials. He has published over 75 research articles in Journals such as Chromosoma, Experimental Cell Research, Genetical Research, Cytogenetics and Cell Genetics, and Molecular Biology, J. General Virology, Biomaterials, Macromolecular Biosciences, Progress in Polymer Science, Biotech Advances, Soft Matter, Tissue Engineering etc. He is associated with several national and international research project work (like Indo-Australia Biotechnology Fund, Indo-Russia Biotechnology Programme, Indo-Us Science and Technology Forum, New Delhi) focusing mainly on silk protein based biomaterials and cell based tissue engineering and regenerative medicine.

Dr. T.V. Kumary Dr. T.V. Kumary, presently Scientist ‘G & In Charge of the Tissue Culture Laboratory in the BMT wing has a doctorate in Biochemistry from Medical Faculty of University of Kerala. She joined our Institute in 1984. She has set up the Tissue Culture Laboratory for evaluation of cytocompatibility of Materials for Medical Devices. In keeping with the recent elevation of testing laboratories she along with her team has brought the laboratory to international standards by implementing Quality system based on ISO, accredited by the French Agency. In addition to routine testing of materials for cytocompatibility, the morphological, biochemical and functional aspects of cell-material interaction are also carried out in the lab. Dr. Kumary has been a recipient of Leverhulme Trust Fellowship, which she did at University of Liverpool. Her interests today include development of in vitro systems for testing of materials and Tissue Engineering.

Dr. Lissy K. Krishnan

Lissy K. Krishnan started her pre-doctoral research on platelet biology in 1980. She obtained her Ph.D from the Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST), Trivandrum in Platelet Biochemistry. From 1990 to 1992 she was at the University of

Minnesota, and worked as a Post Doctoral Research Associate. She transferred back to SCTIMST in 1992 and took charge as the Leader of Thrombosis Research Unit in 1995.

She has contributed to product development for clinical use by developing Fibrin Glue and its standardization of viral inactivation to meet EP and WHO requirements. The product is now clinically used. Another biological product under evaluation is anti viper venom antibodies. Her major contribution in tissue engineering is towards development of small diameter vascular grafts. Her approach to use fibrin matrix as an in vitro homing site for human adult stem cells has resulted in differentiation of circulating progenitors into endothelial cells, smooth muscle cells, keratinocytes and neurons to be used for tissue engineering and for regenerative medicine.

She teaches a course on Stem Cells and Regenerative Medicine and blood compatibility of Biomaterials to post graduate students in her Institution and guides graduate students enrolled for both Ph.D and M. Phil programs. She has several peer reviewed publications, text book chapters, conference proceedings and patents to her credit and is active in various academic societies.

Dr. Annie John Annie John Ph.D, Scientist E & in-charge -Transmission Electron Microscopy (TEM) Laboratory, Div. of Implant Biology and the Division of Laboratory Animal Science to date at the Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojapura, Trivandrum -695012, Kerala, India. Her area of research includes; Biomaterials & Bone Tissue Engineering - Ceramics as bone substitutes and Adult Stem Cell Research in various National & International research projects involving analytical & microscopy studies of cells and tissues and its response to materials in different animal models. Graduated from University of Kerala; A Postdoctoral Fellow of The Japan Society for the Promotion of Science (JSPS) for foreign research in Japan (Long Term in 1999- 2001 & Short Term in 2006), at Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; A recognized Ph.D guide of the Institute; have attended many National and International Conferences and has several publications to credit.

Dr. Chandra P. Sharma FBAO, FBSE

Chandra P. Sharma is Head, Biosurface Technology Division, Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST), Thiruvananthapuram since January 1980. He has been Associate Head, Biomedical Technology Wing, SCTIMST and presently he is Associate Dean, PhD Affairs. He is also Adjunct Professor, Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal University. He is basically a Solid State Physicist from IIT Delhi and received his training in Biomaterials area in the University of Utah (USA) with Prof. D.J. Lyman as a graduate student and in the University of Liverpool, England with Prof. D.F. Williams as a Post Doctoral Research Associate. Dr. Sharma has been awarded

FBSE (Fellow Biomaterials Science & Engineering) by The International Union of Societies for Biomaterials Science & Engineering (IUS-BSE) in 2008 and FBAO (Fellow Biomaterials and Artificial Organs) by Society for Biomaterials & Artificial Organs (India) (SBAOI) in 2011. He has been recognized with various awards and honors such as MRSI medal (1994) and MRSI-ICSC Superconductivity & Materials Science Annual Prize Award 2009 - Materials Research Society of India, Distinguished Scientist award - Society for Biomaterials and Artificial Organs, India (SBAOI) and shares Whitaker and National Science Foundation Award - International Society for Artificial Organs (ISAO) USA. He is the founder of the SBAOI and the Society for Tissue Engineering and Regenerative Medicine, India (STERMI). Dr. Sharma has published over 300 research papers and has processed 32 patents including in Canada, European Union, Japan and USA. He has edited several special issues of International Journals and two books. He is also the Founder editor of Trends in Biomaterials and Artificial Organs (journal published by SBAOI). His basic activities relate understanding of blood/tissue - material interactions at the interface. His laboratory has completed prestigious programmes under NMITLI-CSIR New Delhi on oral delivery of insulin and has been awarded the FADDS project (Facilities for Micro/Nanoparticles based Advanced Drug Delivery Systems) under Drugs and Pharmaceuticals Research Programme, Department of Science & Technology, New Delhi, with a funding of over US$ 1 million. It is being planned to convert FADDS as a National Facility with a funding of about US$ 3 million.

Notes

Sponsored by

(TERMIS)

(Elsevier)

Tissue Engineering & Regenerative Medicine International Society

Biomaterials

Department of Science & Technology

Workshop on

16-17 March 2011

New Visions for Biomaterialsand Regenerative Medicine

Organized by

Professor David Williams, FREngEditor-in-Chief, Biomaterials

Biomaterials

Sree Chitra Tirunal Institute for Medical Sciences & Technology Thiruvananthapuram, Kerala