TISSUE ENGINEERING OF AN OSTEOCHONDRAL TRANSPLANT BY USING A CELL / SCAFFOLD CONSTRUCT HO SAEY TUAN, BARNABAS (B.eng Hons) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOENGINEERING YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2009 Preface This thesis is submitted for the degree of Doctorate of Philosophy in the Graduate Program of Bioengineering (NUS Graduate School for Integrative Sciences and Engineering) at the National University of Singapore. No part of this thesis has been submitted for any other degree or equivalent to another university or institution. All the work in this thesis is original unless references are made to other works. Parts of this thesis had been published or presented in the following : International Refereed Journal Publication Cover pages of some of the following papers are found in the appendix. 1. Ho STB and Hutmacher DW. Application of micro CT and computational modeling in tissue engineering applications. Computer-aided design, 37(11), pp. 1151 – 1161. 2005. 2. Shao XX, Hutmacher DW, Ho STB, Goh JCH and Lee EH. Evaluation of a hybrid scaffold / cell construct in repair of high loading-bearing osteochondral defects in rabbits. Biomaterials, 27(7), pp 1071 – 1080. 2006. 3. Ho STB and Hutmacher DW. Review journal : A comparison of Micro CT with other techniques used in the characterization of scaffolds. Biomaterials, 27(8), pp. 1362 – 1376. 2006. 4. Ho STB and Dietmar W. Hutmacher. Combining Micro CT and Computer Aided Analysis For Bone Engineering Applications. Engineering research (National University of Singapore), 20 (3), Oct issue, pp. 20. 2005. 5. Swieszkowski W, Ho STB, Kurzydlowski KJ and Hutmacher DW. Repair and regeneration of osteochondral defects in the articular joints. Biomolecular engineering, 24, pp. 489 – 495. 2007. 6. Ho STB, Cool SM, Hui JH, Hutmacher DW. The influence of fibrin based hydrogels on the chondrogenic differentiation of human mesenchymal stem cells. Manuscript in preparation. i 7. Ho STB, Ekaputra AK, Hui JH and Hutmacher DW. An electrospun membrane for the resurfacing of cartilage defects. Manuscript in preparation. 8. Ho STB, Hutmacher DW, Ekaputra AK, Hitendra KD and Hui JH. The evaluation of a biphasic osteochondral implant coupled with an electrospun membrane in a large animal model. Manuscript in preparation. Book Chapters 1. Ho STB, Duvall C, Gulberg RE and Hutmacher DW. Micro Computed Tomography in the biomedical sciences. Techniques in microscopy for biomedical applications, edited by Dokland T, Hutmacher DW, Ng MML and Schantz JT. World Scientific. 2006. Intellectual Competition 1. Ho STB and Hutmacher DW. Mimics innovation awards 2005. Winner in category 1: Innovative implant design system. 5000 Euros awarded. Oral presentation on 4th June 2005, Leuven, Belgium. International and Local Conferences and Awards 1. Ho STB, Hutmacher DW. Tissue engineering of an osteochondral transplant by using a cell / scaffold construct. Poster presentation. Joint meeting of the Tissue Engineering Society International and the European Tissue Engineering Society. Lausanne. 2004. 2. Ho STB, Shao XX and Hutmacher DW. Tissue engineering of an osteochondral transplant by using a cell / scaffold construct. Oral presentation. International Conference on Materials for Advanced Technologies. 3rd – 8th July 2005 Singapore. Symposium A, Advanced biomaterials. 3. Ho STB, Hutmacher DW. Invited speaker at the Mimics user conference, held in conjunction with ICBME, Singapore. Oral presentation entitled “The evaluation of an osteochondral implant by using mimics.” 2005. 4. Ho STB and Hutmacher DW. AO (Arbeitsgemeinschaft für Osteosynthese Association for the Study of Osteosynthesis) resorbable workshop seminar organized by Synthes and AO foundadtion, Singapore. Oral presentation entitled “Micro CT evaluation of osteochondral implants”. 6th Dec 2005. ii 5. Ho STB, Shao XX and Hutmacher DW. 3rd international workshop on biomodeling and bioprinting. 10th – 11th May 2006, Singapore. Poster presentation entitled “Articular osteochondral defect repair with biphasic scaffold and bone marrow mesenchymal stem cells.” 6. Hutmacher DW, Ho STB, Banas K, Chen A, Cholewa M, Jian LK, Li ZJ, Liu G, Maniam S, Moser HO, Gureyev TE and Wilkins SW. Characterization of composite scaffolds for bone engineering. Oral presentation. International Conference on Materials for Advanced Technologies. 1st – 6th July 2007 Singapore. Symposium N, Synchrotron radiation for making and measuring materials. 7. Hitendra KD, Ho STB, Hutmacher DW and Hui JH. Repair of large osteochondral defects using hybrid scaffolds with bone marrow-derived mesenchymal stem cells ( BMSCs ) and nanofibre mesh. Oral presentation. 30th Annual Scientific Metting of Singapore Orthopaedic Association. 13 – 17th Nov 2007. Young Orthopaedic Investigator’s Award Symposium. Ho Saey Tuan, Barnabas Singapore, June 2009 iii Acknowledgements “Trust in the Lord with all your heart and lean not on your own understanding” Proverbs 3, verse 5. The Holy Bible. The work presented here started out as an ambition for human insight and understanding, but it has caused me to acknowledge God and his unfathomable ways, for it was not by mere coincidence that he has appointed people to direct me in this arduous journey. First and foremost, I would like to thank Professor James Goh. It is an honor to work under an undisputed pioneer who had contributed significantly to the field of bioengineering. You could have declined in accepting me as your student given the transition that I was in, and yet it is because of your mentoring that I am able to accomplish this academic pursue. Professor Dietmar Hutmacher, I am grateful for your guidance all these years. Even amidst the repeated failures, you have always been patient with my mistakes and it was through those trying moments that I seek to emulate not just your quest for excellence but also your outstanding character. Furthermore you have inspired not just me but the entire lab group with your excitement and passion in science. Associate Professor James Hui, I would always recall of your supervision especially during the regular Monday meetings despite of your numerous hospital obligations. I would have been deprived not just of the generous funding but also the crucial input of a respected clinician if not for your interest in research. Dr Simon Cool, I thank you for those discussions that we have and it was through your critic that I was able to gain from your experience to surmount challenges. Moreover your generosity in time and lab resources has made this work possible. Furthermore I would also like to thank Professor Robert Guldberg for his encouragement and advice especially during the time when he visited Singapore. There are many colleagues, seniors and superiors who have assisted me in the lab either technically, administratively or just by being a friend when I was in need. They would include Professor Teoh who provided the assess to the micro CT in Biomat, those from NUSSTEP : Wanping, Julee and Kwee Hua. Dr Yang, a selfless mentor on the ground. Those from ODC : Chong Sue Wee, Grace Lee, Siew Leng and Sing Chik. Those from LAC : James low and Yong Soon Chiong. Those from the engineering lab: Kee Woei, Monique and Andrew. Dr Evelyn Yip who has avail her lab despite the pressing constraints. Chris Lam, an intelligent friend who has gone out of his way to help me. Mere words would not suffice to express my gratitude towards those who were “behind the scenes”. The prayers of my cell group leader Quek Wee Hiang and fellow cell members have kept me going. My parents, Tan Chiew Hong and the late Ho Khoon Khin who have given me their blessings, support and have affirmed me throughout the years. iv Table of Contents Page Preface i Acknowledgments iv Table of Contents v Summary x List of Tables xii List of Figures xiii List of Abbreviations xv Chapter 1. Introduction 1.1. Clinical background 1.2. Tissue engineering Chapter 2. Literature Review Osteochondral biology 2.1.1. Articular cartilage 2.1.2. Bone 2.2. Osteochondral defects 2.3. Conventional therapies 2.4. Tissue engineering approach 10 2.4.1 Cell based therapies 2.4.2 Scaffold based techniques : Biphasic and monophasic 2.4.3 Classes of scaffolds and fabrication techniques 2.4.4 Scaffolding materials 2.4.5 Material selection 12 14 24 31 45 2.1. v 2.5. Micro Computed Tomography (CT) 47 Research Program 49 3.1. Overview 49 3.2. Four research stages 50 3.2.1. An optimum cell encapsulation matrix that supports cartilage growth. 3.2.2. Tissue engineering of an osteochondral implant in a rabbit model. 3.2.3. A synthetic substitute for the periosteal flap. 3.2.4. Evaluation of the developed osteochondral construct in a preclinical animal model. 50 Chapter 3. Chapter 4. 51 52 53 Optimization of fibrin based hydrogels for the design of cartilage implants 4.1. Abstract 54 4.2. Introduction 55 4.3. Materials and Method 59 4.3.1. 4.3.2. 4.3.3. 4.3.4. 4.3.5. 4.3.6. 4.3.7. 4.3.8. 4.3.9. 59 59 60 61 62 62 63 64 65 4.4. Reagents and chemicals MSC isolation and expansion Hydrogel encapsulation and chondrogenic induction Biphasic osteochondral construct FDA - PI staining RNA extraction and real time PCR Histology and immunohistochemistry Quantitative assays Statistical analysis Results 4.4.1. 4.4.2. 4.4.3. 4.4.4. 4.4.5. 66 Cell seeding and viability Chondrogenic differentiation in the hydrogels Cell seeding of the biphasic osteochondral construct Tissue development in the biphasic environment DNA, GAG and collagen II content 66 67 71 72 75 vi 4.5. Discussion 77 4.6. Conclusion 82 Chapter 5. The in vivo evaluation of the biphasic osteochondral implant. 5.1. Abstract 83 5.2. Introduction 84 5.3. Materials and Method 87 5.3.1. Reagents and chemicals 5.3.2. Scaffold fabrication 5.3.3. Scaffold characterization 5.3.4. Scanning Electron Microscopy (SEM) 5.3.5. Bone marrow aspiration, MSC isolation and culturing 5.3.6. Implant preparation with fibrin encapsulation 5.3.7. Surgical implantation 5.3.8. Histology 5.3.9. Micro CT analysis of in vivo samples 5.3.10. Indentation of the repaired cartilage 5.3.11. Statistical analysis 87 87 88 89 89 89 90 90 90 91 92 Results 93 5.4.1. Mechanical and architectural properties of scaffolds 5.4.2. Bone repair 5.4.3. Cartilage repair 93 95 99 5.5. Discussion 103 5.6. Conclusion 110 5.4. Chapter 6. Resurfacing of the cartilage defect with a PCL – collagen electrospun mesh. 6.1. Abstract 111 vii 6.2. Introduction 112 6.3. Materials and Method 114 6.3.1. Reagents and chemicals 6.3.2. Fabrication of the PCL – Collagen electrospun meshes 6.3.3. Collagen retention analysis 6.3.4. Tensile test and porosity measurement 6.3.5. MSC isolation and expansion 6.3.6. Cell cultures 6.3.7. FDA - PI staining 6.3.8. Scanning Electron Microscopy (SEM) 6.3.9. Real time PCR 6.3.10. Histology and immunostaining 6.3.11. Statistical analysis 114 114 115 115 116 116 117 117 117 118 118 6.4. Results 119 6.5. Discussion 129 6.6. Conclusion 134 Chapter 7. The evaluation of the biphasic osteochondral construct in the pig model. 7.1. Abstract 135 7.2. Introduction 136 7.3. Materials and Method 139 7.3.1. Reagents and chemicals 7.3.2. Scaffold fabrication 7.3.3. Fabrication of the PCL – Collagen 20% electrospun mesh 7.3.4. Bone marrow aspiration, MSC isolation and culturing 7.3.5. Fibrin encapsulation of MSC within the biphasic construct 7.3.6. Surgical implantation 7.3.7. Gross morphology and histology 7.3.8. Micro CT 7.3.9. Indentation of the repaired cartilage 7.3.10. Statistical analysis 139 139 140 140 140 140 141 143 144 144 Results 145 7.4. viii 7.4.1. Cartilage repair 7.4.2. Bone repair 145 155 7.5. Discussion 161 7.6. Conclusion 170 Chapter 8. Conclusions and Recommendations 8.1. Conclusions 171 8.2. Recommendations for Future Research 178 References 179 Appendix ix References 143. 144. 145. 146. 147. 148. 149. . 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. Johnson DL, Urban WP, Jr., Caborn DN, Vanarthos WJ and Carlson CS. Articular cartilage changes seen with magnetic resonance imaging-detected bone bruises associated with acute anterior cruciate ligament rupture. American Journal of Sports Medicine, 26(3), p.409-414. 1998. Rangger C, Kathrein A, Freund MC, Klestil T and Kreczy A. Bone bruise of the knee: histology and cryosections in cases. Acta Orthopaedica Scandinavica, 69(3), p.291-294. 1998. Frenkel SR, Clancy RM, Ricci JL, Di Cesare PE, Rediske JJ and Abramson SB. Effects of nitric oxide on chondrocyte migration, adhesion, and cytoskeletal assembly. Arthritis and Rheumatism, 39(11), p.1905-1912. 1996. Laurencin CT, Ambrosio AM, Borden MD and Cooper JA, Jr. Tissue engineering: orthopedic applications. Annu Rev Biomed Eng, 1, p.19-46. 1999. Jackson DW, Lalor PA, Aberman HM and Simon TM. Spontaneous repair of full-thickness defects of articular cartilage in a goat model. A preliminary study. Journal of Bone and Joint Surgery, 83A(1), p.53-64. 2001. Convery FR, Akeson WH and Keown GH. The repair of large osteochondral defects. An experimental study in horses. Clin Orthop Relat Res, 82, p.253-262. 1972. Cartilage repair registry report, volume 4, Genzyme tissue repair, Cambridge, February 1998. Kimura T, Yasui N, Ohsawa S and Ono K. Chondrocytes embedded in collagen gels maintain cartilage phenotype during long-term cultures. Clin Orthop Relat Res(186), p.231-239. 1984. Buckwalter JA. Evaluating methods of restoring cartilaginous articular surfaces. Clin Orthop Relat Res(367 Suppl), p.S224-238. 1999. www. Articular cartilage injury com. van Susante JL, Buma P, Homminga GN, van den Berg WB and Veth RP. Chondrocyte-seeded hydroxyapatite for repair of large articular cartilage defects. A pilot study in the goat. Biomaterials, 19(24), p.2367-2374. 1998. Zhou G, Liu W, Cui L, Wang X, Liu T and Cao Y. Repair of porcine articular osteochondral defects in non-weightbearing areas with autologous bone marrow stromal cells. Tissue Engineering, 12(11), p.3209-3221. 2006. Sellers RS, Peluso D and Morris EA. The effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage. Journal of Bone and Joint Surgery, 79(10), p.1452-1463. 1997. Kolettas E, Muir HI, Barrett JC and Hardingham TE. Chondrocyte phenotype and cell survival are regulated by culture conditions and by specific cytokines through the expression of Sox-9 transcription factor. Rheumatology, 40(10), p.1146-1156. 2001. Cotran RS, Kumar V and Collins T. Cellular pathology. II. Adaptions, intracellular accumulations and cell aging, 6th edition, Robbins pathological basis of disease, ed. Kumar V and Abbas AK. Philadelphia: WB Saunders. 46 - 47. 1999. Im GI, Kim DY, Shin JH, Hyun CW and Cho WH. Repair of cartilage defect in the rabbit with cultured mesenchymal stem cells from bone marrow. Journal of Bone and Joint Surgery. British Volume, 83(2), p.289-294. 2001. Wakitani S, Goto T, Pineda SJ, Young RG, Mansour JM, Caplan AI and Goldberg VM. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. Journal of Bone and Joint Surgery, 76(4), p.579-592. 1994. Donovan PJ and Gearhart J. The end of the beginning for pluripotent stem cells. Nature, 414(6859), p.92-97. 2001. Lee EH and Hui JH. The potential of stem cells in orthopaedic surgery. Journal of Bone and Joint Surgery. British Volume, 88(7), p.841-851. 2006. Friedenstein AJ, Petrakova KV, Kurolesova AI and Frolova GP. Heterotopic of bone marrow.Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 6(2), p.230-247. 1968. Worster AA, Brower-Toland BD, Fortier LA, Bent SJ, Williams J and Nixon AJ. Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factorbeta1 in monolayer and insulin-like growth factor-I in a three-dimensional matrix. Journal of Orthopaedic Research, 19(4), p.738-749. 2001. 186 References 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S and Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science, 284(5411), p.143-147. 1999. Service RF. Tissue engineers build new bone. Science, 289(5484), p.1498-1500. 2000. Li WJ, Chiang H, Kuo TF, Lee HS, Jiang CC and Tuan RS. Evaluation of articular cartilage repair using biodegradable nanofibrous scaffolds in a swine model: a pilot study. J Tissue Eng Regen Med, 3(1), p.1-10. 2009. Crabb ID, O'Keefe RJ, Puzas JE and Rosier RN. Synergistic effect of transforming growth factor beta and fibroblast growth factor on DNA synthesis in chick growth plate chondrocytes. Journal of Bone and Mineral Research, 5(11), p.1105-1112. 1990. Morales TI and Roberts AB. Transforming growth factor beta regulates the metabolism of proteoglycans in bovine cartilage organ cultures. Journal of Biological Chemistry, 263(26), p.12828-12831. 1988. Wakitani S, Kimura T, Hirooka A, Ochi T, Yoneda M, Yasui N, Owaki H and Ono K. Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel. Journal of Bone and Joint Surgery. British Volume, 71(1), p.74-80. 1989. Caplan AI. Mesenchymal stem cells. Journal of Orthopaedic Research, 9(5), p.641-650. 1991. Tatebe M, Nakamura R, Kagami H, Okada K and Ueda M. Differentiation of transplanted mesenchymal stem cells in a large osteochondral defect in rabbit. Cytotherapy, 7(6), p.520-530. 2005. Lee KB, Hui JH, Song IC, Ardany L and Lee EH. Injectable mesenchymal stem cell therapy for large cartilage defects--a porcine model. Stem Cells, 25(11), p.2964-2971. 2007. Slivka MA Leatherbury NC, Kieswetter K and Niederauer GG. In vitro compression testing of fiber reinforced, bioabsorbable, porous implants. Synthetic bioabsorbable polymers for implants, ASTM STP 1396, ed. P.J. Agrawal CM, Lin ST. 2000, West Conshohocken: American society for testing and materials. Stiehl JB. A clinical overview patellofemoral joint and application to total knee arthroplasty. Journal of Biomechanics, 38(2), p.209-214. 2005. Hinman RS and Crossley KM. Patellofemoral joint osteoarthritis: an important subgroup of knee osteoarthritis. Rheumatology, 46(7), p.1057-1062. 2007. Eggli PS, Muller W and Schenk RK. Porous hydroxyapatite and tricalcium phosphate cylinders with two different pore size ranges implanted in the cancellous bone of rabbits. A comparative histomorphometric and histologic study of bony ingrowth and implant substitution. Clin Orthop Relat Res(232), p.127-138. 1988. Wei X, Rasanen T and Messner K. Maturation-related compressive properties of rabbit knee articular cartilage and volume fraction of subchondral tissue. Osteoarthritis and Cartilage, 6(6), p.400-409. 1998. Teo JC, Si-Hoe KM, Keh JE and Teoh SH. Relationship between CT intensity, micro-architecture and mechanical properties of porcine vertebral cancellous bone. Clin Biomech (Bristol, Avon), 21(3), p.235-244. 2006. Orr TE, Villars PA, Mitchell SL, Hsu HP and Spector M. Compressive properties of cancellous bone defects in a rabbit model treated with particles of natural bone mineral and synthetic hydroxyapatite. Biomaterials, 22(14), p.1953-1959. 2001. Spahn G and Wittig R. [Biomechanical properties (compressive strength and compressive pressure at break) of hyaline cartilage under axial load]. Zentralblatt fur Chirurgie, 128(1), p.78-82. 2003. Harley BA, Lynn AK, Wissner-Gross Z, Bonfield W, Yannas IV and Gibson LJ. Design of a multiphase osteochondral scaffold III: Fabrication of layered scaffolds with continuous interfaces. J Biomed Mater Res A. 2009. Mow VC, Kuei SC, Lai WM and Armstrong CG. Biphasic creep and stress relaxation of articular cartilage in compression? Theory and experiments. Journal of Biomechanical Engineering, 102(1), p.73-84. 1980. Mow VC, Gibbs MC, Lai WM, Zhu WB and Athanasiou KA. Biphasic indentation of articular cartilage--II. A numerical algorithm and an experimental study. Journal of Biomechanics, 22(8-9), p.853-861. 1989. Mak AF, Lai WM and Mow VC. Biphasic indentation of articular cartilage--I. Theoretical analysis. Journal of Biomechanics, 20(7), p.703-714. 1987. 187 References 185. 186. 187. 188. 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. DiSilvestro MR and Suh JK. A cross-validation of the biphasic poroviscoelastic model of articular cartilage in unconfined compression, indentation, and confined compression. Journal of Biomechanics, 34(4), p.519-525. 2001. Barbero A, Grogan SP, Mainil-Varlet P and Martin I. Expansion on specific substrates regulates the phenotype and differentiation capacity of human articular chondrocytes. Journal of Cellular Biochemistry, 98(5), p.1140-1149. 2006. Schlichting K, Schell H, Kleemann RU, Schill A, Weiler A, Duda GN and Epari DR. Influence of scaffold stiffness on subchondral bone and subsequent cartilage regeneration in an ovine model of osteochondral defect healing. American Journal of Sports Medicine, 36(12), p.2379-2391. 2008. Lee CR, Grodzinsky AJ, Hsu HP and Spector M. Effects of a cultured autologous chondrocyteseeded type II collagen scaffold on the healing of a chondral defect in a canine model. Journal of Orthopaedic Research, 21(2), p.272-281. 2003. Kelly DJ and Prendergast PJ. Mechano-regulation of stem cell differentiation and tissue regeneration in osteochondral defects. Journal of Biomechanics, 38(7), p.1413-1422. 2005. Yang Y, Magnay J, Cooling L, Cooper JJ and El Haj AJ. Effects of substrate characteristics on bone cell response to the mechanical environment. Medical & Biological Engineering & Computing, 42(1), p.22-29. 2004. Lee CR, Grodzinsky AJ and Spector M. Biosynthetic response of passaged chondrocytes in a type II collagen scaffold to mechanical compression. J Biomed Mater Res A, 64(3), p.560-569. 2003. Roche S, Ronziere MC, Herbage D and Freyria AM. Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering. Biomaterials, 22(1), p.9-18. 2001. Catelas I, Dwyer JF and Helgerson S. Controlled release of bioactive transforming growth factor beta-1 from fibrin gels in vitro. Tissue Eng Part C Methods, 14(2), p.119-128. 2008. Risbud MV and Sittinger M. Tissue engineering: advances in in vitro cartilage generation. Trends in Biotechnology, 20(8), p.351-356. 2002. Bryant SJ and Anseth KS. The effects of scaffold thickness on tissue engineered cartilage in photocrosslinked poly(ethylene oxide) hydrogels. Biomaterials, 22(6), p.619-626. 2001. Ma P. Scaffolds for tissue fabrication. Materials today, 7(5), p.30 - 40. 2004. Bhattarai N, Edmondson D, Veiseh O, Matsen FA and Zhang M. Electrospun chitosan-based nanofibers and their cellular compatibility. Biomaterials, 26(31), p.6176-6184. 2005. Li WJ, Danielson KG, Alexander PG and Tuan RS. Biological response of chondrocytes cultured in three-dimensional nanofibrous poly(epsilon-caprolactone) scaffolds. J Biomed Mater Res A, 67(4), p.1105-1114. 2003. Li WJ, Jiang YJ and Tuan RS. Chondrocyte phenotype in engineered fibrous matrix is regulated by fiber size. Tissue Engineering, 12(7), p.1775-1785. 2006. Li WJ, Tuli R, Okafor C, Derfoul A, Danielson KG, Hall DJ and Tuan RS. A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials, 26(6), p.599-609. 2005. Freed LE, Vunjak-Novakovic G, Biron RJ, Eagles DB, Lesnoy DC, Barlow SK and Langer R. Biodegradable polymer scaffolds for tissue engineering. Bio/Technology, 12(7), p.689-693. 1994. Vacanti CA, Langer R, Schloo B and Vacanti JP. Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation. Plastic and Reconstructive Surgery, 88(5), p.753759. 1991. Shinoka T, Shum-Tim D, Ma PX, Tanel RE, Isogai N, Langer R, Vacanti JP and Mayer JE, Jr. Creation of viable pulmonary artery autografts through tissue engineering. Journal of Thoracic and Cardiovascular Surgery, 115(3), p.536-545; discussion 545-536. 1998. Leong KF, Cheah CM and Chua CK. Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs. Biomaterials, 24(13), p.2363-2378. 2003. Lu L and Mikos AG. The importance of new processing techniques in tissue engineering. MRS Bull, 21(11), p.28-32. 1996. Boyan BD, Hummert TW, Dean DD and Schwartz Z. Role of material surfaces in regulating bone and cartilage cell response. Biomaterials, 17(2), p.137-146. 1996. Kurashina K, Kurita H, Kotani A, Kobayashi S, Kyoshima K and Hirano M. Experimental cranioplasty and skeletal augmentation using an alpha-tricalcium phosphate/dicalcium phosphate 188 References 208. 209. 210. 211. 212. 213. 214. 215. 216. 217. 218. 219. 220. 221. 222. 223. 224. 225. 226. 227. 228. dibasic/tetracalcium phosphate monoxide cement: a preliminary short-term experiment in rabbits. Biomaterials, 19(7-9), p.701-706. 1998. Lin FH, Liao CJ, Chen KS, Sun JS and Liu HC. Degradation behaviour of a new bioceramic: Ca2P2O7 with addition of Na4P2O7.10H2O. Biomaterials, 18(13), p.915-921. 1997. Lieberman JR, Le LQ, Wu L, Finerman GA, Berk A, Witte ON and Stevenson S. Regional gene therapy with a BMP-2-producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents. Journal of Orthopaedic Research, 16(3), p.330-339. 1998. Kujawa MJ, Carrino DA and Caplan AI. Substrate-bonded hyaluronic acid exhibits a sizedependent stimulation of chondrogenic differentiation of stage 24 limb mesenchymal cells in culture. Developmental Biology, 114(2), p.519-528. 1986. Kujawa MJ and Caplan AI. Hyaluronic acid bonded to cell-culture surfaces stimulates chondrogenesis in stage 24 limb mesenchyme cell cultures. Developmental Biology, 114(2), p.504518. 1986. West DC, Hampson IN, Arnold F and Kumar S. Angiogenesis induced by degradation products of hyaluronic acid. Science, 228(4705), p.1324-1326. 1985. West DC and Kumar S. The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity. Experimental Cell Research, 183(1), p.179-196. 1989. Mauck RL Wang CCB, Chen FH, Lu HH, Ateshian GA and Hung CT. Dynamic deformational loading of chondrocyte-seeded agarose hydrogels modulates deposition and structural organization of matrix constituents. Summer bioengineering conference. 2003. Benya PD and Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell, 30(1), p.215-224. 1982. Rahfoth B, Weisser J, Sternkopf F, Aigner T, von der Mark K and Brauer R. Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits. Osteoarthritis and Cartilage, 6(1), p.50-65. 1998. Okamoto Y, Minami S, Matsuhashi A, Sashiwa H, Saimoto H, Shigemasa Y, Tanigawa T, Tanaka Y and Tokura S. Application of polymeric N-acetyl-D-glucosamine (chitin) to veterinary practice. Journal of Veterinary Medical Science, 55(5), p.743-747. 1993. Wozniak G. Fibrin sealants in supporting surgical techniques: The importance of individual components. Cardiovascular Surgery, 11 Suppl 1, p.17-21. 2003. Neidert MR, Lee ES, Oegema TR and Tranquillo RT. Enhanced fibrin remodeling in vitro with TGF-beta1, insulin and plasmin for improved tissue-equivalents. Biomaterials, 23(17), p.37173731. 2002. Albala DM. Topics for discussion--clinical questions and answers. Cardiovascular Surgery, 11 Suppl 1, p.29-32. 2003. Bensaid W, Triffitt JT, Blanchat C, Oudina K, Sedel L and Petite H. A biodegradable fibrin scaffold for mesenchymal stem cell transplantation. Biomaterials, 24(14), p.2497-2502. 2003. Cox S, Cole M and Tawil B. Behavior of human dermal fibroblasts in three-dimensional fibrin clots: dependence on fibrinogen and thrombin concentration. Tissue Engineering, 10(5-6), p.942954. 2004. Tonnesen HH and Karlsen J. Alginate in drug delivery systems. Drug Development and Industrial Pharmacy, 28(6), p.621-630. 2002. Perka C, Spitzer RS, Lindenhayn K, Sittinger M and Schultz O. Matrix-mixed culture: new methodology for chondrocyte culture and preparation of cartilage transplants. Journal of Biomedical Materials Research, 49(3), p.305-311. 2000. Cohen SB, Meirisch CM, Wilson HA and Diduch DR. The use of absorbable co-polymer pads with alginate and cells for articular cartilage repair in rabbits. Biomaterials, 24(15), p.2653-2660. 2003. Martinsen A, Skjak-Braek G and Smidsrod O. Alginate as immobilization material: I. Correlation between chemical and physical properties of alginate gel beads. Biotechnology and Bioengineering, 33(1), p.79-89. 1989. Smidsrod O and Skjak-Braek G. Alginate as immobilization matrix for cells. Trends in Biotechnology, 8(3), p.71-78. 1990. Marijnissen WJ, van Osch GJ, Aigner J, Verwoerd-Verhoef HL and Verhaar JA. Tissue-engineered cartilage using serially passaged articular chondrocytes. Chondrocytes in alginate, combined in vivo with a synthetic (E210) or biologic biodegradable carrier (DBM). Biomaterials, 21(6), p.571-580. 2000. 189 References 229. 230. 231. 232. 233. 234. 235. 236. 237. 238. 239. 240. 241. 242. 243. 244. 245. 246. 247. 248. Lawson MA, Barralet JE, Wang L, Shelton RM and Triffitt JT. Adhesion and growth of bone marrow stromal cells on modified alginate hydrogels. Tissue Engineering, 10(9-10), p.1480-1491. 2004. Mosahebi A, Wiberg M and Terenghi G. Addition of fibronectin to alginate matrix improves peripheral nerve regeneration in tissue-engineered conduits. Tissue Engineering, 9(2), p.209-218. 2003. Temenoff JS and Mikos AG. Review: tissue engineering for regeneration of articular cartilage. Biomaterials, 21(5), p.431-440. 2000. Ishaug-Riley SL, Okun LE, Prado G, Applegate MA and Ratcliffe A. Human articular chondrocyte adhesion and proliferation on synthetic biodegradable polymer films. Biomaterials, 20(23-24), p.2245-2256. 1999. Corden TJ, Jones IA, Rudd CD, Christian P, Downes S and McDougall KE. Physical and biocompatibility properties of poly-epsilon-caprolactone produced using in situ polymerisation: a novel manufacturing technique for long-fibre composite materials. Biomaterials, 21(7), p.713-724. 2000. Hennink WE and van Nostrum CF. Novel crosslinking methods to design hydrogels. Adv Drug Deliv Rev, 54(1), p.13-36. 2002. Sawhney AS, Pathak CP and Hubbell JA. Bioerodible hydrogels based on photopolymerized poly(ethylene glycol)-co-poly(alpha-hydroxy acid) diacrylate macromers Macromolecules, 26(4), p.581-587. 1993. Elisseeff J, Puleo C, Yang F and Sharma B. Advances in skeletal tissue engineering with hydrogels. Orthod Craniofac Res, 8(3), p.150-161. 2005. Salinas CN and Anseth KS. The influence of the RGD peptide motif and its contextual presentation in PEG gels on human mesenchymal stem cell viability. J Tissue Eng Regen Med, 2(5), p.296-304. 2008. El-Amin SF, Lu HH, Khan Y, Burems J, Mitchell J, Tuan RS and Laurencin CT. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering. Biomaterials, 24(7), p.1213-1221. 2003. Pitt CG, Chasalow FI, Hibionada YM, Kilimas DM and Schinder A. Aliphatic polyester. I. The degradation of poly (ε - caprolactone) in vivo. Journal of application science, 26, p.3779. 1981. Kweon H, Yoo MK, Park IK, Kim TH, Lee HC, Lee HS, Oh JS, Akaike T and Cho CS. A novel degradable polycaprolactone networks for tissue engineering. Biomaterials, 24(5), p.801-808. 2003. Hutmacher DW, Ng KW, Kaps C, Sittinger M and Klaring S. Elastic cartilage engineering using novel scaffold architectures in combination with a biomimetic cell carrier. Biomaterials, 24(24), p.4445-4458. 2003. Wang F SL, Darling A, Khalil S, Sun W, Guceri S, Lau A. . Precision extruding deposition and characterization of cellular poly-ε-caprolactone tissue scaffolds. Rapid prototyping journal, 110(1), p.42 - 49. 2004. Heidemann W, Jeschkeit S, Ruffieux K, Fischer JH, Wagner M, Kruger G, Wintermantel E and Gerlach KL. Degradation of poly(D,L)lactide implants with or without addition of calciumphosphates in vivo. Biomaterials, 22(17), p.2371-2381. 2001. Schantz JT, Teoh SH, Lim TC, Endres M, Lam CX and Hutmacher DW. Repair of calvarial defects with customized tissue-engineered bone grafts I. Evaluation of osteogenesis in a three-dimensional culture system. Tissue Engineering, Suppl 1, p.S113-126. 2003. Bolder SB, Verdonschot N, Schreurs BW and Buma P. Acetabular defect reconstruction with impacted morsellized bone grafts or TCP/HA particles. A study on the mechanical stability of cemented cups in an artificial acetabulum model. Biomaterials, 23(3), p.659-666. 2002. Nery EB, LeGeros RZ, Lynch KL and Lee K. Tissue response to biphasic calcium phosphate ceramic with different ratios of HA/beta TCP in periodontal osseous defects. Journal of Periodontology, 63(9), p.729-735. 1992. Yang Z, Yuan H, Tong W, Zou P, Chen W and Zhang X. Osteogenesis in extraskeletally implanted porous calcium phosphate ceramics: variability among different kinds of animals. Biomaterials, 17(22), p.2131-2137. 1996. Arnold U, Lindenhayn K and Perka C. In vitro-cultivation of human periosteum derived cells in bioresorbable polymer-TCP-composites. Biomaterials, 23(11), p.2303-2310. 2002. 190 References 249. 250. 251. 252. 253. 254. 255. 256. 257. 258. 259. 260. 261. 262. 263. 264. 265. 266. Zhou YF, Sae-Lim V, Chou AM, Hutmacher DW and Lim TM. Does seeding density affect in vitro mineral nodules formation in novel composite scaffolds? J Biomed Mater Res A, 78(1), p.183-193. 2006. Zhou Y, Chen F, Ho ST, Woodruff MA, Lim TM and Hutmacher DW. Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts. Biomaterials, 28(5), p.814-824. 2007. Xynos ID, Hukkanen MV, Batten JJ, Buttery LD, Hench LL and Polak JM. Bioglass 45S5 stimulates osteoblast turnover and enhances bone formation In vitro: implications and applications for bone tissue engineering. Calcified Tissue International, 67(4), p.321-329. 2000. Yuan H, de Bruijn JD, Zhang X, van Blitterswijk CA and de Groot K. Bone induction by porous glass ceramic made from Bioglass (45S5). Journal of Biomedical Materials Research, 58(3), p.270276. 2001. McLaughlin F, Mackintosh J, Hayes BP, McLaren A, Uings IJ, Salmon P, Humphreys J, Meldrum E and Farrow SN. Glucocorticoid-induced osteopenia in the mouse as assessed by histomorphometry, microcomputed tomography, and biochemical markers. Bone, 30(6), p.924-930. 2002. Muller R, Hahn M, Vogel M, Delling G and Ruegsegger P. Morphometric analysis of noninvasively assessed bone biopsies: comparison of high-resolution computed tomography and histologic sections. Bone, 18(3), p.215-220. 1996. Goulet RW, Goldstein SA, Ciarelli MJ, Kuhn JL, Brown MB and Feldkamp LA. The relationship between the structural and orthogonal compressive properties of trabecular bone. Journal of Biomechanics, 27(4), p.375-389. 1994. Muller R, Hildebrand T and Ruegsegger P. Non-invasive bone biopsy: a new method to analyse and display the three-dimensional structure of trabecular bone. Physics in Medicine and Biology, 39(1), p.145-164. 1994. Showalter C, Clymer BD, Richmond B and Powell K. Three-dimensional texture analysis of cancellous bone cores evaluated at clinical CT resolutions. Osteoporosis International, 17(2), p.259-266. 2006. DW. HSaH. Application of micro CT and computation modeling in bone tissue engineering. . Computer-aided design, 37, p.1151 - 1161. 2005. Shao XX, Hutmacher DW, Ho ST, Goh JC and Lee EH. Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits. Biomaterials, 27(7), p.1071-1080. 2006. Verna C, Dalstra M, Wikesjo UM and Trombelli L. Healing patterns in calvarial bone defects following guided bone regeneration in rats. A micro-CT scan analysis. Journal of Clinical Periodontology, 29(9), p.865-870. 2002. Lin AS, Barrows TH, Cartmell SH and Guldberg RE. Microarchitectural and mechanical characterization of oriented porous polymer scaffolds. Biomaterials, 24(3), p.481-489. 2003. Watz H, Breithecker A, Rau WS and Kriete A. Micro-CT of the human lung: imaging of alveoli and virtual endoscopy of an alveolar duct in a normal lung and in a lung with centrilobular emphysema--initial observations. Radiology, 236(3), p.1053-1058. 2005. Simopoulos DN, Gibbons SJ, Malysz J, Szurszewski JH, Farrugia G, Ritman EL, Moreland RB and Nehra A. Corporeal structural and vascular micro architecture with X-ray micro computerized tomography in normal and diabetic rabbits: histopathological correlation. Journal of Urology, 165(5), p.1776-1782. 2001. Jorgensen SM, Demirkaya O and Ritman EL. Three-dimensional imaging of vasculature and parenchyma in intact rodent organs with X-ray micro-CT. American Journal of Physiology, 275(3 Pt 2), p.H1103-1114. 1998. Jones AC, Sakellariou A, Limaye A, Arns CH, Senden TJ, Sawkins T, Knackstedt MA, Rohner D, Hutmacher DW, Brandwood A and Milthorpe BK. Investigation of microstructural features in regenerating bone using micro computed tomography. J Mater Sci Mater Med, 15(4), p.529-532. 2004. Landers R, Hubner U, Schmelzeisen R and Mulhaupt R. Rapid prototyping of scaffolds derived from thermoreversible hydrogels and tailored for applications in tissue engineering. Biomaterials, 23(23), p.4437-4447. 2002. 191 References 267. 268. 269. 270. 271. 272. 273. 274. 275. 276. 277. 278. 279. 280. 281. 282. 283. 284. 285. 286. Catelas I, Sese N, Wu BM, Dunn JC, Helgerson S and Tawil B. Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro. Tissue Engineering, 12(8), p.2385-2396. 2006. Ragan PM, Chin VI, Hung HH, Masuda K, Thonar EJ, Arner EC, Grodzinsky AJ and Sandy JD. Chondrocyte extracellular matrix synthesis and turnover are influenced by static compression in a new alginate disk culture system. Archives of Biochemistry and Biophysics, 383(2), p.256-264. 2000. Hunter CJ, Mouw JK and Levenston ME. Dynamic compression of chondrocyte-seeded fibrin gels: effects on matrix accumulation and mechanical stiffness. Osteoarthritis and Cartilage, 12(2), p.117130. 2004. Soon-Shiong P, Feldman E, Nelson R, Heintz R, Yao Q, Yao Z, Zheng T, Merideth N, Skjak-Braek G, Espevik T and et al. Long-term reversal of diabetes by the injection of immunoprotected islets. Proceedings of the National Academy of Sciences of the United States of America, 90(12), p.58435847. 1993. Hall BK and Miyake T. Divide, accumulate, differentiate: cell condensation in skeletal development revisited. International Journal of Developmental Biology, 39(6), p.881-893. 1995. Hall BK and Miyake T. All for one and one for all: condensations and the initiation of skeletal development. Bioessays, 22(2), p.138-147. 2000. Tuan RS. Biology of developmental and regenerative skeletogenesis. Clin Orthop Relat Res(427 Suppl), p.S105-117. 2004. Tacchetti C, Tavella S, Dozin B, Quarto R, Robino G and Cancedda R. Cell condensation in chondrogenic differentiation. Experimental Cell Research, 200(1), p.26-33. 1992. Goldring MB, Tsuchimochi K and Ijiri K. The control of chondrogenesis. Journal of Cellular Biochemistry, 97(1), p.33-44. 2006. Brown LF, Lanir N, McDonagh J, Tognazzi K, Dvorak AM and Dvorak HF. Fibroblast migration in fibrin gel matrices. American Journal of Pathology, 142(1), p.273-283. 1993. Clark RA, Tonnesen MG, Gailit J and Cheresh DA. Transient functional expression of alphaVbeta on vascular cells during wound repair. American Journal of Pathology, 148(5), p.1407-1421. 1996. Gorodetsky R, Vexler A, Shamir M, An J, Levdansky L, Shimeliovich I and Marx G. New cell attachment peptide sequences from conserved epitopes in the carboxy termini of fibrinogen. Experimental Cell Research, 287(1), p.116-129. 2003. De Ceuninck F, Lesur C, Pastoureau P, Caliez A and Sabatini M. Culture of chondrocytes in alginate beads. Methods Mol Med, 100, p.15-22. 2004. Hauselmann HJ, Fernandes RJ, Mok SS, Schmid TM, Block JA, Aydelotte MB, Kuettner KE and Thonar EJ. Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. Journal of Cell Science, 107 ( Pt 1), p.17-27. 1994. Gagne TA, Chappell-Afonso K, Johnson JL, McPherson JM, Oldham CA, Tubo RA, Vaccaro C and Vasios GW. Enhanced proliferation and differentiation of human articular chondrocytes when seeded at low cell densities in alginate in vitro. Journal of Orthopaedic Research, 18(6), p.882-890. 2000. Lin YJ, Yen CN, Hu YC, Wu YC, Liao CJ and Chu IM. Chondrocytes culture in three-dimensional porous alginate scaffolds enhanced cell proliferation, matrix synthesis and gene expression. J Biomed Mater Res A, 88(1), p.23-33. 2009. Rowley JA, Madlambayan G and Mooney DJ. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials, 20(1), p.45-53. 1999. Noth U, Tuli R, Osyczka AM, Danielson KG and Tuan RS. In vitro engineered cartilage constructs produced by press-coating biodegradable polymer with human mesenchymal stem cells. Tissue Engineering, 8(1), p.131-144. 2002. Mwale F, Stachura D, Roughley P and Antoniou J. Limitations of using aggrecan and type X collagen as markers of chondrogenesis in mesenchymal stem cell differentiation. Journal of Orthopaedic Research, 24(8), p.1791-1798. 2006. Rucklidge GJ, Milne G and Robins SP. Collagen type X: a component of the surface of normal human, pig, and rat articular cartilage. Biochemical and Biophysical Research Communications, 224(2), p.297-302. 1996. 192 References 287. 288. 289. 290. 291. 292. 293. 294. 295. 296. 297. 298. 299. 300. 301. 302. 303. 304. 305. 306. 307. 308. Shapiro F, Koide S and Glimcher MJ. Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. Journal of Bone and Joint Surgery, 75(4), p.532-553. 1993. Lietman SA, Miyamoto S, Brown PR, Inoue N and Reddi AH. The temporal sequence of spontaneous repair of osteochondral defects in the knees of rabbits is dependent on the geometry of the defect. Journal of Bone and Joint Surgery. British Volume, 84(4), p.600-606. 2002. Kuhne JH, Bartl R, Frisch B, Hammer C, Jansson V and Zimmer M. Bone formation in coralline hydroxyapatite. Effects of pore size studied in rabbits. Acta Orthopaedica Scandinavica, 65(3), p.246-252. 1994. Hunziker EB. Articular cartilage repair: are the intrinsic biological constraints undermining this process insuperable? Osteoarthritis and Cartilage, 7(1), p.15-28. 1999. Brown TD, Radin EL, Martin RB and Burr DB. Finite element studies of some juxtarticular stress changes due to localized subchondral stiffening. Journal of Biomechanics, 17(1), p.11-24. 1984. Serink MT, Nachemson A and Hansson G. The effect of impact loading on rabbit knee joints. Acta Orthopaedica Scandinavica, 48(3), p.250-262. 1977. Chu CR, Coutts RD, Yoshioka M, Harwood FL, Monosov AZ and Amiel D. Articular cartilage repair using allogeneic perichondrocyte-seeded biodegradable porous polylactic acid (PLA): a tissue-engineering study. Journal of Biomedical Materials Research, 29(9), p.1147-1154. 1995. Martin I, Miot S, Barbero A, Jakob M and Wendt D. Osteochondral tissue engineering. Journal of Biomechanics, 40(4), p.750-765. 2007. Obradovic B, Martin I, Padera RF, Treppo S, Freed LE and Vunjak-Novakovic G. Integration of engineered cartilage. Journal of Orthopaedic Research, 19(6), p.1089-1097. 2001. Beris AE, Lykissas MG, Papageorgiou CD and Georgoulis AD. Advances in articular cartilage repair. Injury, 36 Suppl 4, p.S14-23. 2005. Bentley G, Biant LC, Carrington RW, Akmal M, Goldberg A, Williams AM, Skinner JA and Pringle J. A prospective, randomised comparison of autologous chondrocyte implantation versus mosaicplasty for osteochondral defects in the knee. Journal of Bone and Joint Surgery. British Volume, 85(2), p.223-230. 2003. Peterson L, Minas T, Brittberg M, Nilsson A, Sjogren-Jansson E and Lindahl A. Two- to 9-year outcome after autologous chondrocyte transplantation of the knee. Clin Orthop Relat Res(374), p.212-234. 2000. Lam CX, Savalani MM, Teoh SH and Hutmacher DW. Dynamics of in vitro polymer degradation of polycaprolactone-based scaffolds: accelerated versus simulated physiological conditions. Biomed Mater, 3(3), p.034108. 2008. Ostrander RV, Goomer RS, Tontz WL, Khatod M, Harwood FL, Maris TM and Amiel D. Donor cell fate in tissue engineering for articular cartilage repair. Clin Orthop Relat Res(389), p.228-237. 2001. Wakitani S, Imoto K, Kimura T, Ochi T, Matsumoto K and Nakamura T. Hepatocyte growth factor facilitates cartilage repair. Full thickness articular cartilage defect studied in rabbit knees. Acta Orthopaedica Scandinavica, 68(5), p.474-480. 1997. Wei X, Gao J and Messner K. Maturation-dependent repair of untreated osteochondral defects in the rabbit knee joint. Journal of Biomedical Materials Research, 34(1), p.63-72. 1997. O'Driscoll SW and Fitzsimmons JS. The role of periosteum in cartilage repair. Clin Orthop Relat Res(391 Suppl), p.S190-207. 2001. Ueno T, Kagawa T, Kanou M, Fujii T, Fukunaga J, Mizukawa N, Sugahara T and Yamamoto T. Immunolocalization of vascular endothelial growth factor during heterotopic bone formation induced from grafted periosteum. Annals of Plastic Surgery, 53(2), p.150-154. 2004. Li ST. Biological biomaterials: Tissue - derived biomaterials (collagen). Biomaterials. Principles and applications, ed. Park JB and Bronzino JD. NW USA: CRC press. 117 -141. 2003. Kempson GE. Mechanical properties of articular cartilage, second edition, Adult articular cartilage, ed. Freeman MAR. Tunbridge wells: Pitman medical. 333 - 415. 1979. Williamson AK, Chen AC, Masuda K, Thonar EJ and Sah RL. Tensile mechanical properties of bovine articular cartilage: variations with growth and relationships to collagen network components. Journal of Orthopaedic Research, 21(5), p.872-880. 2003. Thomas V, Jose MV, Chowdhury S, Sullivan JF, Dean DR and Vohra YK. Mechanomorphological studies of aligned nanofibrous scaffolds of polycaprolactone fabricated by electrospinning. Journal of Biomaterials Science, Polymer Edition, 17(9), p.969-984. 2006. 193 References 309. 310. 311. 312. 313. 314. 315. 316. 317. 318. 319. 320. 321. 322. 323. 324. 325. 326. 327. 328. Weightman B and Kempson GE . Load carriage, second edition, Adult articular cartilage, ed. Freeman MAR. Tunbridge wells: Pitman medical. 291 - 332. 1979. Kajitani K, Ochi M, Uchio Y, Adachi N, Kawasaki K, Katsube K, Maniwa S, Furukawa S and Kataoka H. Role of the periosteal flap in chondrocyte transplantation: an experimental study in rabbits. Tissue Engineering, 10(3-4), p.331-342. 2004. O'Driscoll SW and Fitzsimmons JS. The importance of procedure specific training in harvesting periosteum for chondrogenesis. Clin Orthop Relat Res(380), p.269-278. 2000. Haddo O, Mahroof S, Higgs D, David L, Pringle J, Bayliss M, Cannon SR and Briggs TW. The use of chondrogide membrane in autologous chondrocyte implantation. Knee, 11(1), p.51-55. 2004. Chia SL, Gorna K, Gogolewski S and Alini M. Biodegradable elastomeric polyurethane membranes as chondrocyte carriers for cartilage repair. Tissue Engineering, 12(7), p.1945-1953. 2006. Telemeco TA, Ayres C, Bowlin GL, Wnek GE, Boland ED, Cohen N, Baumgarten CM, Mathews J and Simpson DG. Regulation of cellular infiltration into tissue engineering scaffolds composed of submicron diameter fibrils produced by electrospinning. Acta Biomater, 1(4), p.377-385. 2005. Rho KS, Jeong L, Lee G, Seo BM, Park YJ, Hong SD, Roh S, Cho JJ, Park WH and Min BM. Electrospinning of collagen nanofibers: effects on the behavior of normal human keratinocytes and early-stage wound healing. Biomaterials, 27(8), p.1452-1461. 2006. Matthews JA BE, Wnek GE, Simpson DG and Bowlin GL. Electrospinning of collagen type II: A feasibility study. . Journal of bioactive and compatible polymers, 18, p.125 - 134. 2003. Glaser C and Putz R. Functional anatomy of articular cartilage under compressive loading Quantitative aspects of global, local and zonal reactions of the collagenous network with respect to the surface integrity. Osteoarthritis and Cartilage, 10(2), p.83-99. 2002. Guilak F, Ratcliffe A, Lane N, Rosenwasser MP and Mow VC. Mechanical and biochemical changes in the superficial zone of articular cartilage in canine experimental osteoarthritis. Journal of Orthopaedic Research, 12(4), p.474-484. 1994. Fujihara K, Kotaki M and Ramakrishna S. Guided bone regeneration membrane made of polycaprolactone/calcium carbonate composite nano-fibers. Biomaterials, 26(19), p.4139-4147. 2005. Shin HJ, Lee CH, Cho IH, Kim YJ, Lee YJ, Kim IA, Park KD, Yui N and Shin JW. Electrospun PLGA nanofiber scaffolds for articular cartilage reconstruction: mechanical stability, degradation and cellular responses under mechanical stimulation in vitro. Journal of Biomaterials Science, Polymer Edition, 17(1-2), p.103-119. 2006. Chen ZC, Ekaputra AK, Gauthaman K, Adaikan PG, Yu H and Hutmacher DW. In vitro and in vivo analysis of co-electrospun scaffolds made of medical grade poly(epsilon-caprolactone) and porcine collagen. Journal of Biomaterials Science, Polymer Edition, 19(5), p.693-707. 2008. Zong X, Ran S, Kim KS, Fang D, Hsiao BS and Chu B. Structure and morphology changes during in vitro degradation of electrospun poly(glycolide-co-lactide) nanofiber membrane. Biomacromolecules, 4(2), p.416-423. 2003. Bolgen N, Menceloglu YZ, Acatay K, Vargel I and Piskin E. In vitro and in vivo degradation of non-woven materials made of poly(epsilon-caprolactone) nanofibers prepared by electrospinning under different conditions. Journal of Biomaterials Science, Polymer Edition, 16(12), p.1537-1555. 2005. Shin M, Yoshimoto H and Vacanti JP. In vivo bone tissue engineering using mesenchymal stem cells on a novel electrospun nanofibrous scaffold. Tissue Engineering, 10(1-2), p.33-41. 2004. Shin M, Ishii O, Sueda T and Vacanti JP. Contractile cardiac grafts using a novel nanofibrous mesh. Biomaterials, 25(17), p.3717-3723. 2004. Venugopal J and Ramakrishna S. Biocompatible nanofiber matrices for the engineering of a dermal substitute for skin regeneration. Tissue Engineering, 11(5-6), p.847-854. 2005. Tibesku CO, Szuwart T, Kleffner TO, Schlegel PM, Jahn UR, Van Aken H and Fuchs S. Hyaline cartilage degenerates after autologous osteochondral transplantation. Journal of Orthopaedic Research, 22(6), p.1210-1214. 2004. Peretti GM, Gill TJ, Xu JW, Randolph MA, Morse KR and Zaleske DJ. Cell-based therapy for meniscal repair: a large animal study. American Journal of Sports Medicine, 32(1), p.146-158. 2004. 194 References 329. 330. 331. 332. 333. 334. 335. 336. 337. 338. 339. 340. 341. 342. 343. 344. 345. 346. 347. 348. 349. 350. Solchaga LA, Yoo JU, Lundberg M, Dennis JE, Huibregtse BA, Goldberg VM and Caplan AI. Hyaluronan-based polymers in the treatment of osteochondral defects. Journal of Orthopaedic Research, 18(5), p.773-780. 2000. Camplejohn KL and Allard SA. Limitations of safranin 'O' staining in proteoglycan-depleted cartilage demonstrated with monoclonal antibodies. Histochemistry, 89(2), p.185-188. 1988. Kangarlu A and Gahunia HK. Magnetic resonance imaging characterization of osteochondral defect repair in a goat model at T. Osteoarthritis and Cartilage, 14(1), p.52-62. 2006. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science, 276(5309), p.71-74. 1997. Gratz KR, Wong BL, Bae WC and Sah RL. The effects of focal articular defects on cartilage contact mechanics. Journal of Orthopaedic Research, 27(5), p.584-592. 2009. Brown TD, Pope DF, Hale JE, Buckwalter JA and Brand RA. Effects of osteochondral defect size on cartilage contact stress. Journal of Orthopaedic Research, 9(4), p.559-567. 1991. Hunziker EB and Rosenberg LC. Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane. Journal of Bone and Joint Surgery, 78(5), p.721-733. 1996. Mochizuki Y GVaCA. Enzymatic digestion for the repair of superficial articular cartilage lesions Transactions of the orthopaedic research society, 18, p.728. 1994. Rudert M. Histological evaluation of osteochondral defects: consideration of animal models with emphasis on the rabbit, experimental setup, follow-up and applied methods. Cells Tissues Organs, 171(4), p.229-240. 2002. Ripamonti U. Osteoinduction in porous hydroxyapatite implanted in heterotopic sites of different animal models. Biomaterials, 17(1), p.31-35. 1996. Gopferich A. Mechanisms of polymer degradation and erosion. Biomaterials, 17(2), p.103-114. 1996. Muschler GF, Nakamoto C and Griffith LG. Engineering principles of clinical cell-based tissue engineering. Journal of Bone and Joint Surgery, 86-A(7), p.1541-1558. 2004. Rai B, Ho KH, Lei Y, Si-Hoe KM, Jeremy Teo CM, Yacob KB, Chen F, Ng FC and Teoh SH. Polycaprolactone-20% tricalcium phosphate scaffolds in combination with platelet-rich plasma for the treatment of critical-sized defects of the mandible: a pilot study. Journal of Oral and Maxillofacial Surgery, 65(11), p.2195-2205. 2007. Lam CX, Hutmacher DW, Schantz JT, Woodruff MA and Teoh SH. Evaluation of polycaprolactone scaffold degradation for months in vitro and in vivo. J Biomed Mater Res A. 2008. Namba RS, Meuli M, Sullivan KM, Le AX and Adzick NS. Spontaneous repair of superficial defects in articular cartilage in a fetal lamb model. Journal of Bone and Joint Surgery, 80(1), p.410. 1998. Siegling J. Growth of the epiphyses. . The journal of bone and joint surgery, America., 23, p.23 36. 1941. Smidt GL. Biomechanical analysis of knee flexion and extension. Journal of Biomechanics, 6(1), p.79-92. 1973. Mundermann A, Dyrby CO, D'Lima DD, Colwell CW, Jr. and Andriacchi TP. In vivo knee loading characteristics during activities of daily living as measured by an instrumented total knee replacement. Journal of Orthopaedic Research, 26(9), p.1167-1172. 2008. Pauwels F, A new theory concerning the influence of mechanical stimuli on the differentiation of the supporting tissues. Biomechanics of the Locomotor Apparatus, ed. Furlong PMaR. 1980, Berlin: Springer. 75-407. Carter DR. Mechanical loading history and skeletal biology. Journal of Biomechanics, 20(11-12), p.1095-1109. 1987. Jung M, Breusch S, Daecke W and Gotterbarm T. The effect of defect localization on spontaneous repair of osteochondral defects in a Gottingen minipig model: a retrospective analysis of the medial patellar groove versus the medial femoral condyle. Laboratory Animals, 43(2), p.191-197. 2009. Buckwalter JA, Martin JA, Olmstead M, Athanasiou KA, Rosenwasser MP and Mow VC. Osteochondral repair of primate knee femoral and patellar articular surfaces: implications for preventing post-traumatic osteoarthritis. Iowa Orthopaedic Journal, 23, p.66-74. 2003. 195 References 351. 352. 353. 354. 355. Wakitani S, Goto T, Young RG, Mansour JM, Goldberg VM and Caplan AI. Repair of large fullthickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel. Tissue Engineering, 4(4), p.429-444. 1998. Bail H, Klein P, Kolbeck S, Krummrey G, Weiler A, Schmidmaier G, Haas NP and Raschke MJ. Systemic application of growth hormone enhances the early healing phase of osteochondral defects--a preliminary study in micropigs. Bone, 32(5), p.457-467. 2003. Nelson BH, Anderson DD, Brand RA and Brown TD. Effect of osteochondral defects on articular cartilage. Contact pressures studied in dog knees. Acta Orthopaedica Scandinavica, 59(5), p.574579. 1988. Raynaud S, Champion E, Lafon JP and Bernache-Assollant D. Calcium phosphate apatites with variable Ca/P atomic ratio III. Mechanical properties and degradation in solution of hot pressed ceramics. Biomaterials, 23(4), p.1081-1089. 2002. Kassis I, Zangi L, Rivkin R, Levdansky L, Samuel S, Marx G and Gorodetsky R. Isolation of mesenchymal stem cells from G-CSF-mobilized human peripheral blood using fibrin microbeads. Bone Marrow Transplantation, 37(10), p.967-976. 2006. 196 Appendix Computer-Aided Design 37 (2005) 1151–1161 www.elsevier.com/locate/cad Application of micro CT and computation modeling in bone tissue engineering Ho Saey Tuana, Dietmar W. Hutmachera,b,* b a Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore 119260 Department of orthopaedic Surgery, Faculty of Medicine, National University of Singapore, Singapore 119260 Accepted February 2005 Abstract Computer aided technologies, medical imaging, and rapid prototyping has created new possibilities in biomedical engineering. The systematic variation of scaffold architecture as well as the mineralization inside a scaffold/bone construct can be studied using computer imaging technology and CAD/CAM and micro computed tomography (CT). In this paper, the potential of combining these technologies has been exploited in the study of scaffolds and osteochondral repair. Porosity, surface area per unit volume and the degree of interconnectivity were evaluated through imaging and computer aided manipulation of the scaffold scan data. For the osteochondral model, the spatial distribution and the degree of bone regeneration were evaluated. In this study the versatility of two softwares Mimics (Materialize), CTan and 3D realistic visualization (Skyscan) were assessed, too. q 2005 Elsevier Ltd. All rights reserved. Keywords: Computer aided design; Medical imaging; Scaffolds; Bone engineering; Micro CT 1. Introduction Tissue engineering is the application of that knowledge to the building or repairing of tissues. Generally, engineered tissue is a combination of living cells and a support structure called scaffolds. The scaffold, depending on the tissue or organ in production, can be anything from a matrix of collagen, a structural protein, to synthetic biodegradable plastic laced with chemicals that stimulate cell growth and multiplication. The ‘seeded’ cells that initiate regeneration come from laboratory cultures or from the patient’s own body. The utilization of computer aided technologies in tissue engineering has evolved over time and were termed by Sun et al. as ‘computer aided tissue engineering (CATE)’ [1]. Combining computer assisted design (CAD) with computer assisted manufacturing (CAM) is of particular * Corresponding author. Tel.: C65 874 5105; fax: C65 777 3537. E-mail address: biedwh@nus.edu.sg (D.W. Hutmacher). 0010-4485//$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cad.2005.02.006 interest to tissue engineers to reproduce complex scaffold architectures without requiring the use of moulds. While the engineering potential of various scaffold architectures is considerable, the ability to design and optimise structures is still very much ad hoc since local structure and mechanical/transport properties have not been measurable during tissue growth in vitro or in vivo. Hence, computer aided design allows to design different scaffold architectures systemically. Previous studies have primarily used existing computer aided design (CAD) techniques to create a specific design. Hutmacher et al. [2] used Stratasys QuicksliceTM (QS) software to lay down alternating material patterns that produced triangular and polygonal pores in Polycaprolactone (PCL) scaffolds. Chu et al. [3] created hydroxyapatite (HA) scaffolds with interconnecting square pores using Unigraphics TM CAD software. Traditional methods for evaluating osseointegration of tissue engineered scaffold/cell constructs are based on 2D histological and radiographical techniques and in rare cases mechanical testing. To further the development of optimal scaffold architectures and to characterise accurately the growth of bone into scaffolds a fast and non-destructive ARTICLE IN PRESS Biomaterials 27 (2006) 1071–1080 www.elsevier.com/locate/biomaterials Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits Xin Xin Shaoa,1, Dietmar W. Hutmachera,b,Ã, Saey Tuan Hob, James C.H. Goha,b, Eng Hin Leea,c a Department of Orthopaedic Surgery, National University of Singapore, 10 Lower Kent Ridge Road, Singapore 119260, Singapore b Division of Bioengineering, National University of Singapore, Engineering Drive 1, Singapore 117576, Singapore c Division of Graduate Medical Studies, National University of Singapore,12 Medical Drive, Singapore 117598, Singapore Received June 2005; accepted 21 July 2005 Available online 29 August 2005 Abstract The objective of this study was to evaluate the feasibility and potential of a hybrid scaffold system in large- and high-load-bearing osteochondral defects repair. The implants were made of medical-grade PCL (mPCL) for the bone compartment whereas fibrin glue was used for the cartilage part. Both matrices were seeded with allogenic bone marrow-derived mesenchymal cells (BMSC) and implanted in the defect (4 mm diameter  mm depth) on medial femoral condyle of adult New Zealand White rabbits. Empty scaffolds were used at the control side. Cell survival was tracked via fluorescent labeling. The regeneration process was evaluated by several techniques at and months post-implantation. Mature trabecular bone regularly formed in the mPCL scaffold at both and months post-operation. Micro-Computed Tomography showed progression of mineralization from the host–tissue interface towards the inner region of the grafts. At months time point, the specimens showed good cartilage repair. In contrast, the majority of months specimens revealed poor remodeling and fissured integration with host cartilage while other samples could maintain good cartilage appearance. In vivo viability of the transplanted cells was demonstrated for the duration of weeks. The results demonstrated that mPCL scaffold is a potential matrix for osteochondral bone regeneration and that fibrin glue does not inherit the physical properties to allow for cartilage regeneration in a large and high-load-bearing defect site. r 2005 Elsevier Ltd. All rights reserved. Keywords: Osteochondral tissue engineering; Scaffold; Bone marrow-derived precursor cells; Fibrin glue 1. Introduction Nine percent of the United States population aged 30 and older has clinical osteoarthritis (OA) of the hip or knee, with total direct cost was estimated at $28.6 billion dollars per year [1]. Hence in the 21st century, articular ÃCorresponding author. Division of Bioengineering, Faculty of Engineering, National University of Singapore, Engineering Drive 1, Singapore 117576, Singapore. Tel.: + 65 6874 1036; fax: +1 65 6777 3537. E-mail addresses: shaoxin@ucalgary.ca (X.X. Shao), biedwh@nus.edu.sg (D.W. Hutmacher). Current address: Heritage Medical Research Building, Room 435, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alb., Canada T2N 4N1. 0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.07.040 osteochondral repair remains a clinical challenge because of the increasing morbidity of traumatic injury and arthritis while only a few of current treatments were able to yield satisfactory clinical results from a longterm point of view [1,2]. Although autografts or allografts of osteochondral, periosteal and perichondrial tissue as well as bone prosthetic replacement are the common treatments for clinical osteochondral reconstruction, many shortcomings limit the repair procedure and compromise long-term results [3,4]. Hence, these limitations have led to the development of alternative treatment methodologies [5,6]. Tissue engineering of osteochondral constructs might be regarded as one with the biggest potential to make a significant clinical impact in the future. ARTICLE IN PRESS Biomaterials 27 (2006) 1362–1376 www.elsevier.com/locate/biomaterials Review A comparison of micro CT with other techniques used in the characterization of scaffolds Saey Tuan Hoa, Dietmar W. Hutmachera,b,à a Faculty of Engineering, Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119280, Singapore Faculty of Medicine, Department of Orthopaedic Surgery, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119280, Singapore b Received 13 July 2005; accepted 18 August 2005 Available online 19 September 2005 Abstract The structure and architecture of scaffolds are crucial factors in scaffold-based tissue engineering as they affect the functionality of the tissue engineered constructs and the eventual application in health care. Therefore, effective scaffold assessment techniques are required right at the initial stages of research and development so as to select or design scaffolds with suitable properties. Various techniques have been developed in evaluating these important features and the outcome of the assessment is the eventual improvement on the subsequent design of the scaffold. An effective evaluation approach should be fast, accurate and non-destructive, while providing a comprehensive overview of the various morphological and architectural characteristics. Current assessment techniques would include theoretical calculation, scanning electron microscopy (SEM), mercury and flow porosimetry, gas pycnometry, gas adsorption and micro computed tomography (CT). Micro CT is a more recent method of examining the characteristics of scaffolds and this review aims to highlight this current approach while comparing it with other techniques. r 2005 Elsevier Ltd. All rights reserved. Keywords: Scaffold characterization; Micro CT Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . 2. Architectural and structural parameters . 3. Theoretical method and SEM analysis . . 4. Mercury porosimetry . . . . . . . . . . . . . . 5. Gas pycnometry . . . . . . . . . . . . . . . . . 6. Gas adsorption . . . . . . . . . . . . . . . . . . 7. Flow porosimetry . . . . . . . . . . . . . . . . 8. Micro CT . . . . . . . . . . . . . . . . . . . . . . 9. A micro CT study . . . . . . . . . . . . . . . . 10. Conclusion . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1363 1363 1364 1367 1368 1368 1369 1370 1371 1373 1374 ÃCorresponding author. Faculty of Engineering, Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119280, Singapore. Tel.: +65 94309696. E-mail addresses: biedwh@nus.edu.sg, g0201956@nus.edu.sg (D.W. Hutmacher). 0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.08.035 Biomolecular Engineering 24 (2007) 489–495 www.elsevier.com/locate/geneanabioeng Repair and regeneration of osteochondral defects in the articular joints Wojciech Swieszkowski a,*, Barnabas Ho Saey Tuan b, Krzysztof J. Kurzydlowski a, Dietmar W. Hutmacher c a Warsaw University of Technology, Faculty of Materials Science and Engineering, Woloska 141, Warszawa 02-507, Poland b Division of Bioengineering, National University of Singapore, Engineering Drive1, Singapore 117675, Singapore c Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove QLD 4059, Australia Abstract People suffering from pain due to osteoarthritic or rheumatoidal changes in the joints are still waiting for a better treatment. Although some studies have achieved success in repairing small cartilage defects, there is no widely accepted method for complete repair of osteochondral defects. Also joint replacements have not yet succeeded in replacing of natural cartilage without complications. Therefore, there is room for a new medical approach, which outperforms currently used methods. The aim of this study is to show potential of using a tissue engineering approach for regeneration of osteochondral defects. The critical review of currently used methods for treatment of osteochondral defects is also provided. In this study, two kinds of hybrid scaffolds developed in Hutmacher’s group have been analysed. The first biphasic scaffold consists of fibrin and PCL. The fibrin serves as a cartilage phase while the porous PCL scaffold acts as the subchondral phase. The second system comprises of PCL and PCL-TCP. The scaffolds were fabricated via fused deposition modeling which is a rapid prototyping system. Bone marrow-derived mesenchymal cells were isolated from New Zealand White rabbits, cultured in vitro and seeded into the scaffolds. Bone regenerations of the subchondral phases were quantified via micro CT analysis and the results demonstrated the potential of the porous PCL and PCL-TCP scaffolds in promoting bone healing. Fibrin was found to be lacking in this aspect as it degrades rapidly. On the other hand, the porous PCL scaffold degrades slowly hence it provides an effective mechanical support. This study shows that in the field of cartilage repair or replacement, tissue engineering may have big impact in the future. In vivo bone and cartilage engineering via combining a novel composite, biphasic scaffold technology with a MSC has been shown a high potential in the knee defect regeneration in the animal models. However, the clinical application of tissue engineering requires the future research work due to several problems, such as scaffold design, cellular delivery and implantation strategies. # 2007 Elsevier B.V. All rights reserved. Keywords: Osteochondral defects; Cartilage; Subchondral bone; Repair; Tissue engineering 1. Introduction In the natural joint articular cartilage and subchondral bone form the load-bearing system that provides a large range of joint motion with excellent lubrication, stability and uniform distribution of high acting loads. Articular cartilage (AC) together with subchondral bone plays a very important role in the natural joints (Mow et al., 1993). Cartilage protects the subchondral bone from high stresses, increases joint congruence thereby reducing nominal contact pressure. Articular * Corresponding author at: Division of Materials Design, Faculty of Materials Science and Engineering, Warsaw University of Technology, Woloska 141, 02507 Warsaw, Poland. Tel.: +48 222348792; fax: +48 222348750. E-mail address: Wojciech.Swieszkowski@materials.pl (W. Swieszkowski). 1389-0344/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bioeng.2007.07.014 cartilage also allows for low-friction movements within the joint (Little, 1969). Very often, articular cartilage and subchondral bone undergoes degeneration as the result of osteoarthritis and related disorders leading to severe pain, joint deformity and loss of joint motion, thus requiring surgical procedures for treatment of osteochondral defects (Redman et al., 2005). Annually, over million surgical procedures involving cartilage replacement are performed in Europe. The origin of the cartilage and subchondral bone degradation is still unknown. One of the scenarios could be that the subchondral bone becomes weaker—less dense and is not able to support cartilage in transmitting loads to the cancellous and cortical bones. As a result of that the cartilage fracture arises. Although some studies have achieved success in repairing small cartilage defects, no accepted method for complete repair [...]...Summary of thesis Traditional clinical remedies are unable to address osteochondral defects adequately Given the paucity of available alternatives, the author aims to harness the advances in stem cell and biomaterial research to create a biphasic osteochondral implant that caters to both cartilage and bone regeneration The endeavor was driven by the hypothesis that a biomechanically competent biphasic... distinct phases of composite materials as described in the biomechanical modeling of cartilage by Mow et al, Mak et al and Disilvestro et al [182-185] A biphasic osteochondral scaffold can comprise of 2 separate units which are combined at the point of application An example would be the 16 Chapter 2 bilayered scaffold that Schaefer et al devised using a PGA cartilage mesh and a PolyLactic-co-Glycolic Acid... omission of the cartilage matrix during the cell transplantation procedure [188] Biphasic vs Monophasic The biphasic approach in designing an osteochondral scaffold excels over that of monophasic as the cartilage and bone phases can be individually customized in terms of the material, structural and mechanical aspects so as to assist the regeneration of the specific tissues The monophasic scaffold can be... was an intermediate zone with a gradual transition in porosity [26] The inherent advantage of the biphasic design given the flexibility in varying the material, structural and mechanical properties of the 2 phases so as to assist cartilage and bone repair is an appealing strategy which many investigators have exploited (table 2.1) Monophasic approach A monophasic osteochondral scaffold is defined as... accomplishments, there is yet to be a clinically viable tissue engineering approach that aids osteochondral regeneration This is because most of the proposed implants cannot be directly translated into medical products due to material, mechanical, structural and biological limitations Biological compatibility stems from the material composition of the implant Materials such as chitosan and hydroxyapatite... mechanical properties are fabricated so as to mimic the physiological biomechanics of the osteochondral tissue Oliveira et al used this approach in the development of a chitosan hydroxyapatite bilayer scaffold [9] The chitosan matrix served as the cartilage phase and it has a Young’s modulus of 2.9 MPa while the stiff hydroxyapatite bone scaffold has a higher modulus of 153 MPa [9] In addition to that,... preclinical model xi List of Tables Table 2.1 Biphasic and monophasic osteochondral scaffolds Table 2.2 Rapid prototyping techniques Table 2.3 The 5 categories of scaffolds Table 2.4 Biomaterials used in osteochondral tissue engineering Table 2.5 Material evaluation for the design of the present osteochondral implant Table 4.1 Real time PCR Primer sequences Table 5.1 Architectural characterization of PCL and... these aspects, careful thought is given to the design concept, material selection and scaffold fabrication technique 15 Chapter 2 Biphasic approach Osteochondral functional restoration is dependent on the biological and biomechanical properties of the implanted scaffold These should mimic the native characteristics of cartilage and bone Bone is a well vasculatized tissue while cartilage is avascular,... the foam based scaffolds may not be fully interconnected and this hampers tissue repair These material, mechanical, structural and biological constraints have confounded researchers in their quest for a feasible tissue engineered osteochondral construct Hence the author is mindful of these challenges and initiates an investigation guided by the hypothesis that a combination of MSC loaded hydrogel and... with the expression of collagen II and GAG can be promoted by plating the chondrocytes onto collagen coated surfaces [186] Hence chondrogenic and osteogenic differentiations can be modulated through cell substrate interactions In line with these findings, Tanaka et al attempted to repair an osteochondral lesion by implanting collagen and Tri Calcium Phosphate (TCP) into the cartilage and bone defects . Tissue engineering of an osteochondral transplant by using a cell / scaffold construct. Poster presentation. Joint meeting of the Tissue Engineering Society International and the European Tissue. Tissue Engineering Society. Lausanne. 2004. 2. Ho STB , Shao XX and Hutmacher DW. Tissue engineering of an osteochondral transplant by using a cell / scaffold construct. Oral presentation to material, mechanical, structural and biological limitations. Biological compatibility stems from the material composition of the implant. Materials such as chitosan and hydroxyapatite are