DEVELOPMENT OF A BIORESORBABLE BONE GRAFT ALTERNATIVE FOR BONE ENGINEERING APPLICATIONS CHRISTOPHER LAM XU FU (B.Eng (Hons), M.Eng; NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DIVISION OF BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2010 Preface This thesis is submitted for the degree of Doctorate of Philosophy in the Division of Bioengineering at the National University of Singapore. No part of this thesis has been submitted for any other degree or equivalent at another university or institution. All the work in this thesis is original unless reference is made to other works. Parts of this thesis have been published or presented in the following: International Refereed Journal Publications 1. Hutmacher DW, Schantz JT, Lam CXF, KC Tan, TC Lim, State of the art and future directions of scaffold-based bone engineering from a biomaterials perspective. Journal of Tissue Engineering and Regenerative Medicine. 1(4), 2007, pp245-260. 2. Lam CXF, Hutmacher DW, Schantz JT, Woodruff MA, Teoh SH. Evaluation of Polycaprolactone Scaffold Degradation for months In Vitro and In Vivo. Journal of Biomedical Materials Research A. 90, 2009. pp.906-919. (epub:21 Jul 2008) 3. Lam CXF, Savalani MM, Teoh SH, Hutmacher DW. Dynamics of In Vitro Polymer Degradation of Polycaprolactone-based Scaffolds: Accelerated versus Simulated Physiological Conditions. Biomedical Materials. 3(3), 2008. 4. Sawyer AA, Song SJ, Susanto E, Chuan P, Lam CXF, Woodruff MA, Hutmacher DW, Cool SC. The stimulation of healing within a rat calvarial defect by mPCL–TCP/collagen scaffolds loaded with rhBMP2. Biomaterials. 30, 2009, pp. 2479–248. 5. Abbah SA, Lam CXF, Hutmacher DW, Goh JCH, Wong HK. Biological performance of a polycaprolactone-based scaffold used as fusion cage device in a large animal model of spinal reconstructive surgery. Biomaterials. 30, 2009. pp. 5086–5093. 6. Lam CXF, Abbah SA, Ramruttun KA, Hutmacher DW, Goh JCH, Wong HK. Autogenous Bone Marrow Stromal Cell Sheets Loaded mPCL/TCP Scaffolds Induced Osteogenesis in a Porcine Model of Spinal Interbody Fusion. Tissue Engineering (submitted) i Book Chapters 1. Schumann D, Ekaputra AK, Lam CXF, Hutmacher DW. (2007) Biomaterials/Scaffold - Design of bioactive, multiphasic PCL/collagen type I and type II – PCL-TCP/collagen composite scaffolds for functional tissue engineering of osteochondral repair tissue by using electrospinning and FDM techniques, in Tissue Engineering (Methods in Molecular Medicine). H. Hauser and M. Fussenegger. New Jersey, Humana Press: 101-124. 2. Hutmacher DW, Lam CXF (2008). Scaffold and Implant Design: Considerations relating to Characterization of Biodegradibility and Bioresorbability, in Degradation Rate of Bioresorbable Materials: Prediction and Evaluation. Buchanan FJ. Woodhead Publishing Ltd International Conference Presentations 1. Lam CXF, Tan KC, Teoh SH, Hutmacher DW. Evaluation of Longterm In Vitro Degradation of Polycaprolactone and Polycaprolactonebased Scaffolds, 3rd World Congress on Regenerative Medicine, Leipzig, Germany, 18-20 October, 2007. 2. Lam CXF, Hutmacher DW, Woodruff MA, Jones AC, Knackstedt M, Schantz JT. Evaluation of 2-year Calvarial Reconstruction with Polycaprolactone and Polycaprolactone-based Scaffolds in a Rabbit Model, 3rd World Congress on Regenerative Medicine, Leipzig, Germany, 18-20 October, 2007. (Oral) 3. Lam CXF, Hutmacher DW, Teoh SH. Long-term In Vitro Degradation of Polycaprolactone Scaffolds, TERMIS-AP, Tokyo, Japan, 3-5 December, 2007. (Oral) 4. Lam CXF, Schantz JT, Teoh SH, Hutmacher DW. Evaluation of In Vitro and In Vivo Degradation of Polycaprolactone Composite Scaffolds, TERMIS-AP, Tokyo, Japan, 3-5 December, 2007. 5. Lam CXF, Hutmacher DW, Woodruff MA, Jones AC, Knackstedt M, Schantz JT. Evaluation of 2-year Calvarial Reconstruction with Polycaprolactone and Polycaprolactone-based Scaffolds in a Rabbit Model, TERMIS-AP, Tokyo, Japan, 3-5 December, 2007. (Oral) 6. Lam CXF, Hutmacher DW, Teoh SH. Long-term In Vitro Degradation of Polycaprolactone Scaffolds. International Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering – From Research to Clinical Applications, Singapore, 5-6 January, 2008. ii 7. Lam CXF, Abbah SA, Goh JCH, Hutmacher DW, Wong HK. PCLTCP Composite Scaffolds with Marrow Derived Cell-Sheets in a Porcine Spinal Fusion Model: Preliminary Evaluation. 8th World Biomaterials Congress, Amsterdam, The Netherlands, 28 May - June, 2008. (Oral - Student Award) 8. Abbah SA, Lam CXF, Yang K, Goh JCH, Hutmacher DW, Wong HK. A Bioresorbable Device in Combination with Bone Morphogenetic Protein-2 for Anterior Lumbar Interbody Fusion in Porcine Model. TERMIS-EU, Porto, Portugal, 22 – 26 June, 2008. (Oral) 9. Abbah SA, Lam CXF, Yang K, Goh JCH, Hutmacher DW, Wong HK. Fusion Performance of a Novel Bioresorbable Cage Used in Anterior Lumbar Interbody Fusion. 31st Annual Scientific Meeting, Singapore Orthopaedic Association (SOA); Singapore, 12-15 Nov, 2008. (Oral N Balachandran Best Paper Award) 10. Lam CXF, Abbah SA, Yang K, Goh JCH, Hutmacher DW, Wong HK. Fusion Performance of a Novel Bioresorbable Cage in Anterior Lumbar Interbody Fusion. 13th International Conference on Biomedical Engineering (ICBME), Singapore, – December 2008. (Oral – Outstanding Paper Award ) 11. Lam CXF, Abbah AS, Goh JCH, Hutmacher DW, Wong HK. Evaluation of a Polycaprolactone Based Bioresorbable Scaffold for Bone Regeneration at Load Bearing Sites. 5th International Conference on Materials for Advanced Technologies (ICMAT) Symposium A: Advanced Biomaterials and Regenerative Medicine in conjunction with 2nd Asian Biomaterials Congress (ABMC), Singapore, 28 June - July 2009. (Oral) 12. Lam CXF, Abbah SA, Yang K, Goh JCH, Hutmacher DW, Wong HK. Evaluation of Bioresorbable PCL/TCP Cage for Interbody Spinal Fusion Applications in a Porcine Model. 2nd TERMIS World Congress in conjunction with 2009 Seoul Stem Cell Symposium, S. Korea, 31 Aug – Sep 2009. (Oral) iii Acknowledgements The author would like to take this opportunity to express his heart-felt gratitude to the following people who had in one way or another helped towards the successful completion of this research project, and for making the project a truly memorable learning experience. ƒ Firstly, God Almighty which has made all things possible and given the following wonderful mentors and beautiful helpers to journey with me. ƒ Professors Dietmar W. Hutmacher, James Goh, Wong Hee Kit and Asst. Professor Jan-Throsten Schantz, my key supervisors, for their invaluable advice, patience, guidance, encouragement and support throughout this project; ƒ Professor Teoh Swee Hin, who is the pioneer in the FDM scaffolds, for his invaluable mentorship and support; ƒ Drs. Abbah SA, Yang Kai, Ni GX, Sim CS and Song SJ for their aid, advice and skillful surgical talent; ƒ Assoc. Professor Ian Gibson, who has been supportive of my research commitments in providing invaluable advice and resources; ƒ Dr. Simon Cool (IMB) for his invaluable support and expert advice; ƒ This project would also not have been possible without the unconditional support and friendship of all my colleagues, friends and lab mates who have, in ways more than one helped me progress; Andrew KE, Clarice Chen, Barney Ho, Subha Rath, Dinah Tan, Ng KW, Amy Chou, Nimoe, David Leong, Khor HL, Maik B, Harmeet S, Jean Lim, Monique M, Mohan A, Anurag G, Earnest M, Anand K, Anthony Jones, Evelyn S, Zhou YF, Detlef S, Bai HF, Gajadar B, Mia W, Amber S, Monica S, iv Sambit S, Amit K, Radek and Joanna, Sang Joon A, Tarik A, Anand L, staff of Bioengineering, Orthopaedic Surgery, DES SGH and NUSTEP. For all the technical, administrative support rendered, great company and friendship, encouragement and tolerance in the sharing of equipment, that helped made this project enjoyable and meaningful; ƒ Last but not least, my loving family who has supported and endured my long hours away from home. My lovely wife, Pauline, children Joanne and Joel, and my parents. v Table of Contents Preface i Acknowledgement iv Table of Contents vi Summary viii List of Tables xi List of Figures xii List of Abbreviations xx CHAPTER – INTRODUCTION 1.1 Background 1.2 Bone Tissue Engineering 1.2.1 The Scaffold 1.2.2 Cells 1.2.3 Biomolecules (Growth Factors) 1.3 Hypothesis & Objectives 1 10 13 CHAPTER – BACKGROUND AND SIGNIFICANCE 2.1 The Human Bone 2.1.1 Function of Bone 2.1.2 Bone Physiology and Structure 2.1.3 Mechanical Properties of Bone 2.1.4 The Ossification Process 2.2 Bone Regeneration, Repair and Healing 2.2.1. Bone Remodeling and Fracture Healing 2.2.2. Bone Grafts 2.3 State of the Art in Bone Tissue Engineering 2.3.1 Requirements of a Bone Graft Alternative 2.3.2. Bone Engineering Scaffolds – Role in Tissue Engineering 2.3.3 Scaffold Fabrication Techniques – Role in Tissue Engineering 2.3.4 Cells – Role in Tissue Engineering 2.3.5 Biomolecules - Growth Factors 2.4 Degradation and Bioresorption 2.4.1 Calcium Phosphate Bioceramics 2.4.2 Polycaprolactone 2.5 Spinal Fusion 2.5.1 Biology and Function of the Spine 2.5.2 Lower Back Pain and Spine Pathologies 2.5.3 Spinal Fusion Technique: Anterior Lumbar Interbody Fusion (ALIF) 16 16 16 17 22 23 25 25 28 33 34 38 57 62 72 78 78 79 93 93 94 95 vi CHAPTER 3.1 3.2 3.3 CHAPTER – CHARACTERISATION OF COMPOSITE PCL/TCP SCAFFOLDS FOR BONE ENGINEERING Experimental Setup: Materials and Methods Results and Discussions Conclusions 98 98 103 114 – SCAFFOLD DEGRADATION: LONG-TERM IN VITRO & IN 115 VIVO 4.1 4.2 4.3 Experimental Setup: Materials and Methods Results and Discussions Conclusions CHAPTER – PERFORMANCE OF MPCL/TCP SCAFFOLD SYSTEM IN A SMALL ANIMAL MODEL 5.1 Experimental Setup: Materials and Methods 5.2 Results and Discussions 5.3 Conclusions 115 124 147 148 148 152 167 CHAPTER – PERFORMANCE OF SCAFFOLD SYSTEM IN A CLINICAL RELEVANT SPINAL FUSION MODEL 6.1 Experimental Setup: Materials and Methods 6.2 Results and Discussions 6.3 Conclusions 169 177 202 CHAPTER – CONCLUSIONS AND RECOMMENDATIONS 7.1 Conclusions 7.2 Recommendations 204 204 207 References 168 208 vii Summary Tissue engineered bone has become a relevant need for the unmet clinical needs for regenerating bone, wither in orthopaedic or maxillofacial area. However, despite the number of such limited products and bone grafts available today, which also extends to grafts of allogenic sources (eg. DBM). Today, autografts remain the gold standard despite its limited availability and disadvantages. This research was aimed at developing a novel bone graft alternative and strategy using engineering techniques and enhanced with commercially available biomolecules. It was hypothesised that the combination of rhBMP-2 and a 3D relevant bone complementing porous scaffold could be a suitable bone graft alternative for load bearing applications. For this purpose, the bioresorbable PCL and enhanced PCL/TCP scaffold systems designed and fabricated for bone regeneration. In the first study, the basic scaffold biomaterial and structure was found to be capable of supporting the attachment, proliferation and differentiation of primary mammalian bone marrow stromal cells. Cells remained viable and metabolically active through the 28-day study. As degradation and resorption are the key characteristics for the bioresorbable scaffold systems, yet this is an extremely dynamic process, the second experiment analysed thoroughly the degradation profile and mechanism the scaffold system. It was established that the scaffold system had functional structural stability up to months, and complemented bone regeneration, transfer of load in a timely manner and did not release toxic byproducts beyond threshold limits. viii In the third experiment, mPCL/TCP bone scaffold system was used to reconstruct a critical-sized rat calvarial defect, with rhBMP-2, to assess the feasibility and performance of such a tissue engineering strategy. The scaffold structure on its own was able to restore the shape of the cranium, as well as functional stability and excellent integration to the host bone, tested via mechanical analyse. While its porous structure, revealed by histology, complemented the ingrowth of neo-bone and tissue, to achieve successful repair of the defect. In the forth experiment, scaffold system (a biocage) with rhBMP-2 was used, as a bone graft alternative, in a large animal load-bearing site for lumbar spinal fusion using the anterior lumbar interbody fusion (ALIF) technique. The clinical outcome showed functional fusion, with stiffness (resistance to motion) and histological neo-bone formation comparable to the autograft control. Semi-quantification by radiographic means revealed more mineralised bone. Results indicated that the biocages with lower than clinical rhBMP-2 doses, achieved functional fusion, in a safe manner. The bioresorbable scaffold systems that are designed to encourage rapid bone ingrowth and thus, promptly transfer of the load-bearing/sharing dynamics to new tissues, even at highly demanding sites of bone regeneration like the lumbar interbody fusion site. As there is an imperative need for “off the shelf” tissue engineering products to meet the current unmet clinical needs, the strategy of using the commercial mPCL/TCP biocage with rhBMP-2 could provide an immediate viable option as a bone graft alternative for a variety of bone engineering situations. Thus, this strategy of tissue engineering by stimulating host tissue and bone regeneration clearly surpasses other permanent implant strategies, ix References 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. Ambrosio, A.M., et al., Degradable polyphosphazene/poly(alphahydroxyester) blends: degradation studies. Biomaterials, 2002. 23(7): p. 1667-72. Stella, B., et al., Encapsulation of gemcitabine lipophilic derivatives into polycyanoacrylate nanospheres and nanocapsules. Int J Pharm, 2007. 344(1-2): p. 71-7. Smith, M.J., et al., Suture-reinforced electrospun polydioxanoneelastin small-diameter tubes for use in vascular tissue engineering: a feasibility study. Acta Biomater, 2008. 4(1): p. 58-66. Guelcher, S.A., Biodegradable polyurethanes: synthesis and applications in regenerative medicine. Tissue Eng Part B Rev, 2008. 14(1): p. 3-17. Kimura, Y., Biodegradable Polymers in Biomedical Applications of Polymeric Materials T. Tsuruta, et al., Editors. 1993, CRC Press. p. 164-190. Engelberg, I. and J. Kohn, Physico-mechanical properties of degradable polymers used in medical applications: a comparative study. Biomaterials, 1991. 12(3): p. 292-304. Hakkarainen, M., Aliphatic Polyesters: Abiotic and Biotic Degradation and Degradation Products, in Degradable Aliphatic Polyesters, A.C. Albertsson, Editor. 2002, Springer: Berlin. p. 113-138. Pitt, C.G., et al., Aliphatic Polyesters .1. the Degradation of Poly(Epsilon-Caprolactone) Invivo. Journal of Applied Polymer Science, 1981. 26(11): p. 3779-3787. Kweon, H., et al., A novel degradable polycaprolactone networks for tissue engineering. Biomaterials, 2003. 24(5): p. 801-8. Pitt, C., G. , Polycaprolactone and its Copolymers, in Biodegradable Polymers as Drug Delivery Systems, M. Chasin and R. Langer, Editors. 1990, Marcel Dekker New York. p. 71-119. Woodward, S.C., et al., The intracellular degradation of poly(epsiloncaprolactone). J Biomed Mater Res, 1985. 19(4): p. 437-44. Pitt, C.G., Non-microbial Degradation of Polyesters: Mechanisms and Modifications, in Biodegradable polymers and plastics, M. Vert, et al., Editors. 1992, Royal Society of Chemistry: Cambridge [England]. p. 719. Pitt, C.G., et al., Sustained drug delivery systems II: Factors affecting release rates from poly(epsilon-caprolactone) and related biodegradable polyesters. J Pharm Sci, 1979. 68(12): p. 1534-8. Darney, P.D., et al., Clinical evaluation of the Capronor contraceptive implant: preliminary report. Am J Obstet Gynecol, 1989. 160(5 Pt 2): p. 1292-5. Ory, S.J., et al., The effect of a biodegradable contraceptive capsule (Capronor) containing levonorgestrel on gonadotropin, estrogen, and progesterone levels. Am J Obstet Gynecol, 1983. 145(5): p. 600-5. Bezwada, R.S., et al., Monocryl suture, a new ultra-pliable absorbable monofilament suture. Biomaterials, 1995. 16(15): p. 1141-8. Tomihata, K., M. Suzuki, and Y. Ikada, The pH dependence of monofilament sutures on hydrolytic degradation. J Biomed Mater Res, 2001. 58(5): p. 511-8. 215 References 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. Shalaby, S.W. and R.A. Johnson, Synthetic Absorbable Polyesters, in Biomedical Polymers: Designed-to-Degrade Systems, S.W. Shalaby, Editor. 1994, Hanser Publishers. p. - 34. Middleton, J.C. and A.J. Tipton, Synthetic biodegradable polymers as orthopedic devices. Biomaterials, 2000. 21(23): p. 2335-46. Nimni, M.E., et al., Chemically modified collagen: a natural biomaterial for tissue replacement. J Biomed Mater Res, 1987. 21(6): p. 741-71. Boyd, C.D., et al., Mammalian tropoelastin: multiple domains of the protein define an evolutionarily divergent amino acid sequence. Matrix, 1991. 11(4): p. 235-41. Brennan, E.P., et al., Antibacterial activity within degradation products of biological scaffolds composed of extracellular matrix. Tissue Eng, 2006. 12(10): p. 2949-55. Friess, W., et al., Characterization of absorbable collagen sponges as rhBMP-2 carriers. International Journal of Pharmaceutics, 1999. 187(1): p. 91-99. Nimni, M.E., Collagen: structure, function, and metabolism in normal and fibrotic tissues. Semin Arthritis Rheum, 1983. 13(1): p. 1-86. Burgeson, R.E. and M.E. Nimni, Collagen types. Molecular structure and tissue distribution. Clin Orthop Relat Res, 1992(282): p. 250-72. Hak, D.J., The use of osteoconductive bone graft substitutes in orthopaedic trauma. J Am Acad Orthop Surg, 2007. 15(9): p. 525-36. Triplett, R.G., et al., Pivotal, randomized, parallel evaluation of recombinant human bone morphogenetic protein-2/absorbable collagen sponge and autogenous bone graft for maxillary sinus floor augmentation. J Oral Maxillofac Surg, 2009. 67(9): p. 1947-60. Yefang, Z., et al., Comparison of human alveolar osteoblasts cultured on polymer-ceramic composite scaffolds and tissue culture plates. Int J Oral Maxillofac Surg, 2007. 36(2): p. 137-45. Schumann, D., et al., Biomaterials/scaffolds. Design of bioactive, multiphasic PCL/collagen type I and type II-PCL-TCP/collagen composite scaffolds for functional tissue engineering of osteochondral repair tissue by using electrospinning and FDM techniques. Methods Mol Med, 2007. 140: p. 101-24. Hao, W., et al., Collagen I gel can facilitate homogenous bone formation of adipose-derived stem cells in PLGA-beta-TCP scaffold. Cells Tissues Organs, 2008. 187(2): p. 89-102. Yang, C., et al., The application of recombinant human collagen in tissue engineering. BioDrugs, 2004. 18(2): p. 103-19. Hench, L.L. and J. Wilson, An Introduction to bioceramics. Advanced series in ceramics. 1993, Singapore: : World Scientific. x, 386 p. Hench, L.L., Biomaterials: a forecast for the future. Biomaterials, 1998. 19(16): p. 1419-23. Bohner, M., Calcium orthophosphates in medicine: from ceramics to calcium phosphate cements. Injury, 2000. 31 Suppl 4: p. 37-47. Fernandez, E., et al., Calcium phosphate bone cements for clinical applications. Part I: solution chemistry. J Mater Sci Mater Med, 1999. 10(3): p. 169-76. 216 References 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. Leeuwenburgh, S.C., et al., Mechanical properties of porous, electrosprayed calcium phosphate coatings. J Biomed Mater Res A, 2006. 78(3): p. 558-69. Zyman, Z., et al., Porous calcium phosphate ceramic granules and their behaviour in differently loaded areas of skeleton. J Mater Sci Mater Med, 2008. 19(5): p. 2197-205. Eggli, P.S., W. Muller, and R.K. Schenk, 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, 1988(232): p. 127-38. Vaccaro, A.R., The role of the osteoconductive scaffold in synthetic bone graft. Orthopedics, 2002. 25(5 Suppl): p. s571-8. Kuhne, J.H., et al., Bone formation in coralline hydroxyapatite. Effects of pore size studied in rabbits. Acta Orthop Scand, 1994. 65(3): p. 24652. Elliott, J.C., P.E. Mackie, and R.A. Young, Monoclinic Hydroxyapatite. Science, 1973. 180(4090): p. 1055-1057. Williams, D.F., Medical and dental materials. 1992, Weinheim ; Cambridge: VCH. 469 p. Ravaglioli, A., et al., Interface between hydroxyapatite and mandibular human bone tissue. Biomaterials, 1992. 13(3): p. 162-7. Famery, R., N. Richard, and P. Boch, Preparation of Alpha-Tricalcium and Beta-Tricalcium Phosphate Ceramics, with and without Magnesium Addition. Ceramics International, 1994. 20(5): p. 327-336. Umegaki, T., M. Shiomi, and T. Kanazawa, Preparation of Sintered Beta-Tricalcium Phosphate from Dehydrated Amorphous CalciumPhosphate. Yogyo-Kyokai-Shi, 1987. 95(8): p. 770-774. Yoshii, T., et al., Fresh bone marrow introduction into porous scaffolds using a simple low-pressure loading method for effective osteogenesis in a rabbit model. J Orthop Res, 2009. 27(1): p. 1-7. Abbah, S.A., et al., Biological performance of a polycaprolactonebased scaffold used as fusion cage device in a large animal model of spinal reconstructive surgery. Biomaterials, 2009. 30(28): p. 5086-93. Oi, Y., et al., Beta-tricalcium phosphate and basic fibroblast growth factor combination enhances periodontal regeneration in intrabony defects in dogs. Dent Mater J, 2009. 28(2): p. 162-9. Kinoshita, Y., et al., Alveolar bone regeneration using absorbable poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and gelatin sponge incorporating basic fibroblast growth factor. Int J Oral Maxillofac Surg, 2008. 37(3): p. 275-81. Linovitz, R.J. and T.A. Peppers, Use of an advanced formulation of beta-tricalcium phosphate as a bone extender in interbody lumbar fusion. Orthopedics, 2002. 25(5 Suppl): p. s585-9. Jarcho, M., Calcium phosphate ceramics as hard tissue prosthetics. Clin Orthop Relat Res, 1981(157): p. 259-78. Ratner, B.D., Biomaterials science : an introduction to materials in medicine. 1996, San Diego: Academic Press. xi, 484 p. 217 References 186. 187. 188. 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. Holmes, R.E., R.W. Wardrop, and L.M. Wolford, Hydroxylapatite as a bone graft substitute in orthognathic surgery: histologic and histometric findings. J Oral Maxillofac Surg, 1988. 46(8): p. 661-71. Holmes, R.E. and H.K. Hagler, Porous hydroxyapatite as a bone graft substitute in cranial reconstruction: a histometric study. Plast Reconstr Surg, 1988. 81(5): p. 662-71. Goto, T., et al., Resorption of synthetic porous hydroxyapatite and replacement by newly formed bone. J Orthop Sci, 2001. 6(5): p. 444-7. Hench, L. and S. Best, Ceramics, Glasses, and Glass-Ceramics, in Biomaterials science : an introduction to materials in medicine, B.D. Ratner, Editor. 2004, Elsevier Academic Press: San Diego, Calif. p. 153-169. Yuan, H., et al., Bone formation induced by calcium phosphate ceramics in soft tissue of dogs: a comparative study between porous alpha-TCP and beta-TCP. J Mater Sci Mater Med, 2001. 12(1): p. 713. Epstein, N.E., An analysis of noninstrumented posterolateral lumbar fusions performed in predominantly geriatric patients using lamina autograft and beta tricalcium phosphate. Spine J, 2008. 8(6): p. 882-7. Orthovita, Vitoss™ Synthetic Cancellous Bone Void Filler Remodeling (Resorption). Internal White Paper, 2000. Bonfield, W., et al., Hydroxyapatite reinforced polyethylene--a mechanically compatible implant material for bone replacement. Biomaterials, 1981. 2(3): p. 185-6. Tanner, K.E., R.N. Downes, and W. Bonfield, CLINICALAPPLICATIONS OF HYDROXYAPATITE REINFORCED MATERIALS. British Ceramic Transactions, 1994. 93(3): p. 104-107. Bonfield, W. and K.E. Tanner, Biomaterials - A new generation. Materials World, 1997. 5(1): p. 18-20. Pankajakshan, D., V.K. Krishnan, and L.K. Krishnan, Vascular tissue generation in response to signaling molecules integrated with a novel poly(epsilon-caprolactone)-fibrin hybrid scaffold. J Tissue Eng Regen Med, 2007. 1(5): p. 389-97. Wu, Y.C., et al., Bone tissue engineering evaluation based on rat calvaria stromal cells cultured on modified PLGA scaffolds. Biomaterials, 2006. 27(6): p. 896-904. Nakanishi, Y., et al., Tissue-engineered urinary bladder wall using PLGA mesh-collagen hybrid scaffolds: a comparison study of collagen sponge and gel as a scaffold. J Pediatr Surg, 2003. 38(12): p. 1781-4. Jung, Y., et al., A poly(lactic acid)/calcium metaphosphate composite for bone tissue engineering. Biomaterials, 2005. 26(32): p. 6314-22. Kim, S.S., et al., Poly(lactide-co-glycolide)/hydroxyapatite composite scaffolds for bone tissue engineering. Biomaterials, 2006. 27(8): p. 1399-409. Lin, F.H., et al., The merit of sintered PDLLA/TCP composites in management of bone fracture internal fixation. Artif Organs, 1999. 23(2): p. 186-94. Schiller, C. and M. Epple, Carbonated calcium phosphates are suitable pH-stabilising fillers for biodegradable polyesters. Biomaterials, 2003. 24(12): p. 2037-43. 218 References 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 215. 216. 217. 218. De Groot, J.H., H.W. Kuijper, and A.J. Pennings, A novel method for fabrication of biodegradable scaffolds with high compression moduli. Journal of Materials Science-Materials in Medicine, 1997. 8(11): p. 707-712. Mikos, A.G., et al., Preparation and Characterization of Poly(L-Lactic Acid) Foams. Polymer, 1994. 35(5): p. 1068-1077. Wake, M.C., P.K. Gupta, and A.G. Mikos, Fabrication of pliable biodegradable polymer foams to engineer soft tissues. Cell Transplant, 1996. 5(4): p. 465-73. Mikos, A.G., et al., Preparation of poly(glycolic acid) bonded fiber structures for cell attachment and transplantation. J Biomed Mater Res, 1993. 27(2): p. 183-9. Hutmacher, D.W., M. Sittinger, and M.V. Risbud, Scaffold-based tissue engineering: rationale for computer-aided design and solid freeform fabrication systems. Trends Biotechnol, 2004. 22(7): p. 354-62. Hutmacher, D.W. and S. Cool, Concepts of scaffold-based tissue engineering--the rationale to use solid free-form fabrication techniques. J Cell Mol Med, 2007. 11(4): p. 654-69. Ciardelli, G., et al., Blends of poly-(epsilon-caprolactone) and polysaccharides in tissue engineering applications. Biomacromolecules, 2005. 6(4): p. 1961-1976. Williams, J.M., et al., Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sintering. Biomaterials, 2005. 26(23): p. 4817-4827. Bibb, R. and G. Sisias, Bone structure models using stereolithography: a technical note. Rapid Prototyping Journal, 2002. 8(1): p. 25-29. Cooke, M.N., et al., Use of stereolithography to manufacture criticalsized 3D biodegradable scaffolds for bone ingrowth. Journal of Biomedical Materials Research Part B-Applied Biomaterials, 2003. 64B(2): p. 65-69. Wu, W.Z., et al., Fabrication of Repairing Skull Bone Defects Based on the Rapid Prototyping. Journal of Bioactive and Compatible Polymers, 2009. 24: p. 125-136. Zhou, W.Y., et al., Selective laser sintering of porous tissue engineering scaffolds from poly(L)/carbonated hydroxyapatite nanocomposite microspheres. Journal of Materials Science-Materials in Medicine, 2008. 19(7): p. 2535-2540. Kim, S.S., et al., Survival and function of hepatocytes on a novel threedimensional synthetic biodegradable polymer scaffold with an intrinsic network of channels. Ann Surg, 1998. 228(1): p. 8-13. Hutmacher, D.W., Scaffold design and fabrication technologies for engineering tissues--state of the art and future perspectives. J Biomater Sci Polym Ed, 2001. 12(1): p. 107-24. Lam, C.X., et al., Evaluation of polycaprolactone scaffold degradation for months in vitro and in vivo. J Biomed Mater Res A, 2009. 90(3): p. 906-19. Taboas, J.M., et al., Indirect solid free form fabrication of local and global porous, biomimetic and composite 3D polymer-ceramic scaffolds. Biomaterials, 2003. 24(1): p. 181-194. 219 References 219. 220. 221. 222. 223. 224. 225. 226. 227. 228. 229. 230. 231. 232. 233. 234. 235. 236. Zhong, W.H., et al., Short fiber reinforced composites for fused deposition modeling. Materials Science and Engineering a-Structural Materials Properties Microstructure and Processing, 2001. 301(2): p. 125-130. Osteopore(TM). www.osteopore.com.sg. Caplan, A.I., D. Reuben, and S.E. Haynesworth, Cell-based tissue engineering therapies: the influence of whole body physiology. Adv Drug Deliv Rev, 1998. 33(1-2): p. 3-14. Li, R.K., et al., Cell therapy to repair broken hearts. Can J Cardiol, 1998. 14(5): p. 735-44. Przyborski, S.A., Differentiation of human embryonic stem cells after transplantation in immune-deficient mice. Stem Cells, 2005. 23(9): p. 1242-50. Bruder, S.P., et al., Mesenchymal stem cells in osteobiology and applied bone regeneration. Clin Orthop Relat Res, 1998(355 Suppl): p. S247-56. Kimelman, N., et al., Review: gene- and stem cell-based therapeutics for bone regeneration and repair. Tissue Eng, 2007. 13(6): p. 1135-50. Caplan, A.I., Mesenchymal stem cells. J Orthop Res, 1991. 9(5): p. 641-50. Sodek, J., B. Ganss, and M.D. McKee, Osteopontin. Crit Rev Oral Biol Med, 2000. 11(3): p. 279-303. Ritter, N.M., M.C. Farachcarson, and W.T. Butler, EVIDENCE FOR THE FORMATION OF A COMPLEX BETWEEN OSTEOPONTIN AND OSTEOCALCIN. Journal of Bone and Mineral Research, 1992. 7(8): p. 877-885. Romberg, R.W., et al., Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium-binding proteins. Biochemistry, 1986. 25(5): p. 1176-80. Fujisawa, R. and Y. Kuboki, Preferential adsorption of dentin and bone acidic proteins on the (100) face of hydroxyapatite crystals. Biochim Biophys Acta, 1991. 1075(1): p. 56-60. Karaoz, E., et al., Characterization of mesenchymal stem cells from rat bone marrow: ultrastructural properties, differentiation potential and immunophenotypic markers. Histochem Cell Biol, 2009. 132(5): p. 533-46. Ducy, P., et al., Increased bone formation in osteocalcin-deficient mice. Nature, 1996. 382(6590): p. 448-52. Lodie, T.A., et al., Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction. Tissue Eng, 2002. 8(5): p. 739-51. Bruder, S.P., et al., Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells. J Orthop Res, 1998. 16(2): p. 155-62. Gnecchi, M., et al., Evidence supporting paracrine hypothesis for Aktmodified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J, 2006. 20(6): p. 661-9. Hess, D., et al., Bone marrow-derived stem cells initiate pancreatic regeneration. Nat Biotechnol, 2003. 21(7): p. 763-70. 220 References 237. 238. 239. 240. 241. 242. 243. 244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254. Zorina, T.D., et al., Recovery of the endogenous beta cell function in the NOD model of autoimmune diabetes. Stem Cells, 2003. 21(4): p. 377-88. Miyahara, Y., et al., Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med, 2006. 12(4): p. 459-65. Kocher, A.A., et al., Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med, 2001. 7(4): p. 430-6. Rafii, S. and D. Lyden, Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med, 2003. 9(6): p. 702-12. Bartholomew, A., et al., Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Experimental Hematology, 2002. 30(1): p. 42-48. Parenteau, N., Skin: the first tissue-engineered products. Sci Am, 1999. 280(4): p. 83-4. Szczypka, M.S., et al., Rare incorporation of bone marrow-derived cells into kidney after folic acid-induced injury. Stem Cells, 2005. 23(1): p. 44-54. Ryan, E.A., et al., Five-year follow-up after clinical islet transplantation. Diabetes, 2005. 54(7): p. 2060-9. Devine, S.M. and R. Hoffman, Role of mesenchymal stem cells in hematopoietic stem cell transplantation. Curr Opin Hematol, 2000. 7(6): p. 358-63. Di Nicola, M., et al., Human bone marrow stromal cells suppress Tlymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood, 2002. 99(10): p. 3838-3843. Tse, W.T., et al., Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation, 2003. 75(3): p. 389-97. Aggarwal, S. and M.F. Pittenger, Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 2005. 105(4): p. 1815-1822. Potian, J.A., et al., Veto-like activity of mesenchymal stem cells: functional discrimination between cellular responses to alloantigens and recall antigens. J Immunol, 2003. 171(7): p. 3426-34. Cool, S.M. and V. Nurcombe, Heparan sulfate regulation of progenitor cell fate. J Cell Biochem, 2006. 99(4): p. 1040-51. Fiedler, J., et al., BMP-2, BMP-4, and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells. J Cell Biochem, 2002. 87(3): p. 305-12. Nakamura, Y., et al., Low dose fibroblast growth factor-2 (FGF-2) enhances bone morphogenetic protein-2 (BMP-2)-induced ectopic bone formation in mice. Bone, 2005. 36(3): p. 399-407. Ornitz, D.M., FGFs, heparan sulfate and FGFRs: complex interactions essential for development. Bioessays, 2000. 22(2): p. 108-12. Centrella, M., et al., Transforming growth factor-beta gene family members and bone. Endocr Rev, 1994. 15(1): p. 27-39. 221 References 255. 256. 257. 258. 259. 260. 261. 262. 263. 264. 265. 266. 267. 268. 269. 270. 271. Zellin, G., Growth factors and bone regeneration. Implications of barrier membranes. Swed Dent J Suppl, 1998. 129: p. 7-65. Zellin, G., et al., Opposite effects of recombinant human transforming growth factor-beta on bone regeneration in vivo: effects of exclusion of periosteal cells by microporous membrane. Bone, 1998. 22(6): p. 613-20. Si, X., et al., Expression of BMP-2 and TGF-beta mRNA during healing of the rabbit mandible. Eur J Oral Sci, 1997. 105(4): p. 325-30. Linkhart, T.A., S. Mohan, and D.J. Baylink, Growth factors for bone growth and repair: IGF, TGF beta and BMP. Bone, 1996. 19(1 Suppl): p. 1S-12S. Bak, B., P.H. Jorgensen, and T.T. Andreassen, Dose response of growth hormone on fracture healing in the rat. Acta Orthop Scand, 1990. 61(1): p. 54-7. Mitlak, B.H., et al., The effect of systemically administered PDGF-BB on the rodent skeleton. J Bone Miner Res, 1996. 11(2): p. 238-47. Jackson, R.A., V. Nurcombe, and S.M. Cool, Coordinated fibroblast growth factor and heparan sulfate regulation of osteogenesis. Gene, 2006. 379: p. 79-91. Schliephake, H., et al., The use of basic fibroblast growth factor (bFGF) for enhancement of bone ingrowth into pyrolized bovine bone. Int J Oral Maxillofac Surg, 1995. 24(2): p. 181-6. Quarto, N., D.C. Wan, and M.T. Longaker, Molecular mechanisms of FGF-2 inhibitory activity in the osteogenic context of mouse adiposederived stem cells (mASCs). Bone, 2008. 42(6): p. 1040-52. Quarto, N. and M.T. Longaker, FGF-2 inhibits osteogenesis in mouse adipose tissue-derived stromal cells and sustains their proliferative and osteogenic potential state. Tissue Eng, 2006. 12(6): p. 1405-18. Einhorn, T.A., Clinical applications of recombinant human BMPs: early experience and future development. J Bone Joint Surg Am, 2003. 85-A Suppl 3: p. 82-8. Cool, S.M. and V. Nurcombe, The osteoblast-heparan sulfate axis: control of the bone cell lineage. Int J Biochem Cell Biol, 2005. 37(9): p. 1739-45. Matthews, S.J., Biological activity of bone morphogenetic proteins (BMP's). Injury, 2005. 36 Suppl 3: p. S34-7. Cunningham, N.S., V. Paralkar, and A.H. Reddi, Osteogenin and recombinant bone morphogenetic protein 2B are chemotactic for human monocytes and stimulate transforming growth factor beta mRNA expression. Proc Natl Acad Sci U S A, 1992. 89(24): p. 117404. Carlson, B.M., Principles of regenerative biology. 2007, Amsterdam ; London: Elsevier/Academic. xix, 379 p. Boden, S.D., et al., Posterolateral lumbar intertransverse process spine arthrodesis with recombinant human bone morphogenetic protein 2/hydroxyapatite-tricalcium phosphate after laminectomy in the nonhuman primate. Spine (Phila Pa 1976), 1999. 24(12): p. 117985. Haidar, Z.S., R.C. Hamdy, and M. Tabrizian, Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part 222 References 272. 273. 274. 275. 276. 277. 278. 279. 280. 281. 282. 283. 284. 285. 286. 287. B: Delivery systems for BMPs in orthopaedic and craniofacial tissue engineering. Biotechnol Lett, 2009. 31(12): p. 1825-35. Sandhu, H.S., et al., BMPs and gene therapy for spinal fusion: summary statement. Spine, 2003. 28(15 Suppl): p. S85. Cowan, C.M., et al., MicroCT evaluation of three-dimensional mineralization in response to BMP-2 doses in vitro and in critical sized rat calvarial defects. Tissue Eng, 2007. 13(3): p. 501-12. Edwards, R.B., 3rd, et al., Percutaneous injection of recombinant human bone morphogenetic protein-2 in a calcium phosphate paste accelerates healing of a canine tibial osteotomy. J Bone Joint Surg Am, 2004. 86-A(7): p. 1425-38. Bai, B., et al., Histological changes of an injectable rhBMP-2/calcium phosphate cement in vertebroplasty of rhesus monkey. Spine (Phila Pa 1976), 2009. 34(18): p. 1887-92. Hollinger, J.O., et al., Recombinant human bone morphogenetic protein-2 and collagen for bone regeneration. J Biomed Mater Res, 1998. 43(4): p. 356-64. Jeon, O., et al., Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-conjugated poly(Llactic-co-glycolic acid) scaffold. Biomaterials, 2007. 28(17): p. 276371. Kandziora, F., et al., Bone morphogenetic protein-2 application by a poly(D,L-lactide)-coated interbody cage: in vivo results of a new carrier for growth factors. J Neurosurg, 2002. 97(1 Suppl): p. 40-8. Liao, S.S., et al., Lumbar spinal fusion with a mineralized collagen matrix and rhBMP-2 in a rabbit model. Spine, 2003. 28(17): p. 195460. Hwang, C.J., et al., Immunogenicity of bone morphogenetic proteins. J Neurosurg Spine, 2009. 10(5): p. 443-51. Perri, B., et al., Adverse swelling associated with use of rh-BMP-2 in anterior cervical discectomy and fusion: a case study. Spine J, 2007. 7(2): p. 235-9. Sheehan, J.P., et al., The safety and utility of recombinant human bone morphogenetic protein-2 for cranial procedures in a nonhuman primate model. J Neurosurg, 2003. 98(1): p. 125-30. Crawford, C.H. and J.M. Buchowski, Bone Morphogenetic Protein Use in Anterior Cervical Spine Surgery: A Review of Current Literature Concerning Indications, Safety, and Efficacy. Neurosurgery Quarterly, 2009. 19(4): p. 283-287. Gopferich, A., Mechanisms of polymer degradation and erosion. Biomaterials, 1996. 17(2): p. 103-114. Li, S. and M. Vert, Biodegradation of Aliphatic Polyesters, in Degradable Polymers: Principles and Applications, G. Scott and D. Gilead, Editors. 1995, Chapman & Hall p. 43-87. Ottenbrite, R.M., Discussion of Degradation Terminology in Biodegradable polymers and plastics, M. Vert, et al., Editors. 1992, Royal Society of Chemistry: Cambridge [England]. p. 73-92. Vert, M., Biodegradable polymers and plastics. 1992, Cambridge [England]: : Royal Society of Chemistry. xii, 302 p. 223 References 288. 289. 290. 291. 292. 293. 294. 295. 296. 297. 298. 299. 300. 301. 302. Vert, M., et al., Bioresorbability and Biocompatibility of Aliphatic Polyesters. Journal of Materials Science-Materials in Medicine, 1992. 3(6): p. 432-446. Li, S., Hydrolytic degradation characteristics of aliphatic polyesters derived from lactic and glycolic acids. J Biomed Mater Res, 1999. 48(3): p. 342-53. Li, S.M., H. Garreau, and M. Vert, Structure Property Relationships in the Case of the Degradation of Massive Aliphatic Poly-(AlphaHydroxy Acids) in Aqueous-Media .1. Poly(Dl-Lactic Acid). Journal of Materials Science-Materials in Medicine, 1990. 1(3): p. 123-130. Grizzi, I., et al., Hydrolytic degradation of devices based on poly(DLlactic acid) size-dependence. Biomaterials, 1995. 16(4): p. 305-11. Ginde, R.M. and R.K. Gupta, Invitro Chemical Degradation of Poly(Glycolic Acid) Pellets and Fibers. Journal of Applied Polymer Science, 1987. 33(7): p. 2411-2429. Gopferich, A., D. Karydas, and R. Langer, Predicting Drug-Release from Cylindric Polyanhydride Matrix Discs. European Journal of Pharmaceutics and Biopharmaceutics, 1995. 41(2): p. 81-87. Li, S.M., H. Garreau, and M. Vert, Structure Property Relationships in the Case of the Degradation of Massive Poly(Alpha-Hydroxy Acids) in Aqueous-Media .2. Degradation of Lactide-Glycolide Copolymers Pla37.5ga25 and Pla75ga25. Journal of Materials Science-Materials in Medicine, 1990. 1(3): p. 131-139. Shih, C., Chain-End Scission in Acid-Catalyzed Hydrolysis of Poly(D,L-Lactide) in Solution. Journal of Controlled Release, 1995. 34(1): p. 9-15. Li, S.M., H. Garreau, and M. Vert, Structure-Property Relationships in the Case of the Degradation of Massive Poly(Alpha-Hydroxy Acids) in Aqueous-Media .3. Influence of the Morphology of Poly(L-Lactic Acid). Journal of Materials Science-Materials in Medicine, 1990. 1(4): p. 198-206. Bostman, O.M., Intense granulomatous inflammatory lesions associated with absorbable internal fixation devices made of polyglycolide in ankle fractures. Clin Orthop Relat Res, 1992(278): p. 193-9. Bergsma, E.J., et al., Foreign body reactions to resorbable poly(Llactide) bone plates and screws used for the fixation of unstable zygomatic fractures. J Oral Maxillofac Surg, 1993. 51(6): p. 666-70. Bergsma, J.E., et al., Late degradation tissue response to poly(Llactide) bone plates and screws. Biomaterials, 1995. 16(1): p. 25-31. Therin, M., et al., Invivo Degradation of Massive Poly(Alpha-Hydroxy Acids) - Validation of Invitro Findings. Biomaterials, 1992. 13(9): p. 594-600. Vert, M., S. Li, and H. Garreau, More About the Degradation of La/Ga-Derived Matrices in Aqueous-Media. Journal of Controlled Release, 1991. 16(1-2): p. 15-26. Li, S.M. and M. Vert, Crystalline Oligomeric Stereocomplex as an Intermediate Compound in Racemic Poly(Dl-Lactic Acid) Degradation. Polymer International, 1994. 33(1): p. 37-41. 224 References 303. 304. 305. 306. 307. 308. 309. 310. 311. 312. 313. 314. 315. 316. 317. 318. 319. Pitt, C.G., et al., Aliphatic polyesters II. The degradation of poly (DLlactide), poly (epsilon-caprolactone), and their copolymers in vivo. Biomaterials, 1981. 2(4): p. 215-20. Li, S., et al., Enzymatic degradation of block copolymers prepared from epsilon-caprolactone and poly(ethylene glycol). Biomacromolecules, 2002. 3(3): p. 525-30. Eldsater, C., et al., The biodegradation of amorphous and crystalline regions in film-blown poly(epsilon-caprolactone). Polymer, 2000. 41(4): p. 1297-1304. Chu, C.C., The in-vitro degradation of poly(glycolic acid) sutures-effect of pH. J Biomed Mater Res, 1981. 15(6): p. 795-804. Chu, C.C., An in-vitro study of the effect of buffer on the degradation of poly(glycolic acid) sutures. J Biomed Mater Res, 1981. 15(1): p. 1927. Makino, K., H. Ohshima, and T. Kondo, Mechanism of hydrolytic degradation of poly(L-lactide) microcapsules: effects of pH, ionic strength and buffer concentration. J Microencapsul, 1986. 3(3): p. 20312. Reed, A.M. and D.K. Gilding, Biodegradable Polymers for Use In Surgery - Poly(Glycolic)-Poly(Lactic Acid) Homo and Co-Polymers .2. In vitro Degradation. Polymer, 1981. 22(4): p. 494-498. McBeth, J. and K. Jones, Epidemiology of chronic musculoskeletal pain. Best Practice & Research in Clinical Rheumatology, 2007. 21(3): p. 403-425. Ludwig, S.C. and S.D. Boden, Osteoinductive bone graft substitutes for spinal fusion: a basic science summary. Orthop Clin North Am, 1999. 30(4): p. 635-45. Weinstein, J.N., et al., Surgical versus nonsurgical therapy for lumbar spinal stenosis. N Engl J Med, 2008. 358(8): p. 794-810. Chou, R., et al., Diagnosis and treatment of low back pain: a joint clinical practice guideline from the American College of Physicians and the American Pain Society. Ann Intern Med, 2007. 147(7): p. 47891. Boswell, M.V., et al., Interventional techniques: evidence-based practice guidelines in the management of chronic spinal pain. Pain Physician, 2007. 10(1): p. 7-111. Joseph, V. and Y.R. Rampersaud, Heterotopic bone formation with the use of rhBMP2 in posterior minimal access interbody fusion: a CT analysis. Spine (Phila Pa 1976), 2007. 32(25): p. 2885-90. Sandhu, H.S., Anterior lumbar interbody fusion with osteoinductive growth factors. Clin Orthop Relat Res, 2000(371): p. 56-60. Margaritondo, G. and R. Meuli, Synchrotron radiation in radiology: novel X-ray sources. Eur Radiol, 2003. 13(12): p. 2633-41. Thomlinson, W., et al., Research at the European Synchrotron Radiation Facility medical beamline. Cell Mol Biol (Noisy-le-grand), 2000. 46(6): p. 1053-63. Jones, A.C., et al., Analysis of 3D bone ingrowth into polymer scaffolds via micro-computed tomography imaging. Biomaterials, 2004. 25(20): p. 4947-4954. 225 References 320. 321. 322. 323. 324. 325. 326. 327. 328. 329. 330. 331. 332. 333. 334. 335. Jones, A.C., et al., Investigation of microstructural features in regenerating bone using micro computed tomography. J Mater Sci Mater Med, 2004. 15(4): p. 529-32. Schantz, J.T., et al., Repair of calvarial defects with customized tissueengineered bone grafts - I. Evaluation of osteogenesis in a threedimensional culture system. Tissue Engineering, 2003. 9: p. S113S126. Hutmacher, D.W. and C.X.F. Lam, Scaffold and Implant Design: Considerations relating to Characterization of Biodegradibility and Bioresorbability, in Degradation rate of bioresorbable materials : prediction and evaluation, F. Buchanan, Editor. 2008, CRC Press; Woodhead Publishing: Boca Raton, Fla. Cambridge. Perrin, D. and J. English, Polyglycolide and Polylactide, in Handbook of Biodegradable Polymers, A. Domb, J. Kost, and D. Wiseman, Editors. 1998, Harwood Academic Publishers. p. 3-27. Albertsson, A. and I. Varma, Aliphatic Polyesters: Synthesis, Properties and Applications, in Degradable Aliphatic Polyesters, A. Albertsson, Editor. 2002, Springer. p. 1–40. Sun, H., et al., The in vivo degradation, absorption and excretion of PCL-based implant. Biomaterials, 2006. 27(9): p. 1735-40. Dhert, W.J., et al., A mechanical investigation of fluorapatite, magnesiumwhitlockite, and hydroxylapatite plasma-sprayed coatings in goats. J Biomed Mater Res, 1991. 25(10): p. 1183-200. Dhert, W.J., et al., A finite element analysis of the push-out test: influence of test conditions. J Biomed Mater Res, 1992. 26(1): p. 11930. Williams, J.M., et al., Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sintering. Biomaterials, 2005. 26(23): p. 4817-27. Hollister, S.J., et al., Engineering craniofacial scaffolds. Orthod Craniofac Res, 2005. 8(3): p. 162-73. Alt, V., et al., [Health-economic considerations for the use of BMP-2 for spinal surgery in Germany]. Z Orthop Ihre Grenzgeb, 2006. 144(6): p. 577-82. Ruhe, P.Q., et al., Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits. Biomaterials, 2004. 25(11): p. 2123-32. Zhou, Y., et al., Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts. Biomaterials, 2007. 28(5): p. 814-24. Singh, K., et al., Use of recombinant human bone morphogenetic protein-2 as an adjunct in posterolateral lumbar spine fusion: a prospective CT-scan analysis at one and two years. J Spinal Disord Tech, 2006. 19(6): p. 416-23. Haidar, Z.S., R.C. Hamdy, and M. Tabrizian, Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part A: Current challenges in BMP delivery. Biotechnol Lett, 2009. 31(12): p. 1817-24. Zhang, H., D.J. Sucato, and R.D. Welch, Recombinant human bone morphogenic protein-2-enhanced anterior spine fusion without bone 226 References 336. 337. 338. 339. 340. 341. 342. 343. 344. 345. 346. 347. 348. 349. 350. encroachment into the spinal canal: a histomorphometric study in a thoracoscopically instrumented porcine model. Spine (Phila Pa 1976), 2005. 30(5): p. 512-8. Panjabi, M.M., Biomechanical evaluation of spinal fixation devices: I. A conceptual framework. Spine (Phila Pa 1976), 1988. 13(10): p. 112934. Lysack, J.T., D. Yen, and G.A. Dumas, In vitro flexibility of an experimental pedicle screw and plate instrumentation system on the porcine lumbar spine. Med Eng Phys, 2000. 22(7): p. 461-8. Ahn, Y.H., et al., Comparison of the load-sharing characteristics between pedicle-based dynamic and rigid rod devices. Biomed Mater, 2008. 3(4): p. 44101. Hasegawa, S., et al., A 5-7 year in vivo study of high-strength hydroxyapatite/poly(L-lactide) composite rods for the internal fixation of bone fractures. Biomaterials, 2006. 27(8): p. 1327-32. Lam, C., et al., Composite PLDLLA/TCP Scaffolds for Bone Engineering: In Vitro Cell Response and Degradation. . (manuscript in preparation), 2010. Teo, J.C., et al., Relationship between CT intensity, micro-architecture and mechanical properties of porcine vertebral cancellous bone. Clin Biomech (Bristol, Avon), 2006. 21(3): p. 235-44. Morgan, E.F. and T.M. Keaveny, Dependence of yield strain of human trabecular bone on anatomic site. J Biomech, 2001. 34(5): p. 569-77. Hobatho, M.C., J.Y. Rho, and R.B. Ashman, Anatomical variation of human cancellous bone mechanical properties in vitro. Stud Health Technol Inform, 1997. 40: p. 157-73. Hou, F.J., et al., Human vertebral body apparent and hard tissue stiffness. J Biomech, 1998. 31(11): p. 1009-15. Misch, C.E., Z. Qu, and M.W. Bidez, Mechanical properties of trabecular bone in the human mandible: implications for dental implant treatment planning and surgical placement. J Oral Maxillofac Surg, 1999. 57(6): p. 700-6; discussion 706-8. Wolfinbarger, L., Jr., et al., A comprehensive study of physical parameters, biomechanical properties, and statistical correlations of iliac crest bone wedges used in spinal fusion surgery. I. Physical parameters and their correlations. Spine (Phila Pa 1976), 1994. 19(3): p. 277-83. Nouda, S., et al., Adjacent vertebral body fracture following vertebroplasty with polymethylmethacrylate or calcium phosphate cement: biomechanical evaluation of the cadaveric spine. Spine (Phila Pa 1976), 2009. 34(24): p. 2613-8. Steffen, T., A. Tsantrizos, and M. Aebi, Effect of implant design and endplate preparation on the compressive strength of interbody fusion constructs. Spine (Phila Pa 1976), 2000. 25(9): p. 1077-84. Lowe, T.G., et al., A biomechanical study of regional endplate strength and cage morphology as it relates to structural interbody support. Spine (Phila Pa 1976), 2004. 29(21): p. 2389-94. Hollinger, J.O., H. Uludag, and S.R. Winn, Sustained release emphasizing recombinant human bone morphogenetic protein-2. Adv Drug Deliv Rev, 1998. 31(3): p. 303-318. 227 References 351. 352. 353. 354. 355. 356. 357. 358. 359. 360. 361. 362. 363. 364. 365. Akamaru, T., et al., Simple carrier matrix modifications can enhance delivery of recombinant human bone morphogenetic protein-2 for posterolateral spine fusion. Spine, 2003. 28(5): p. 429-434. Seeherman, H.J., et al., Recombinant human bone morphogenetic protein-2 delivered in an injectable calcium phosphate paste accelerates osteotomy-site healing in a nonhuman primate model. J Bone Joint Surg Am, 2004. 86-A(9): p. 1961-72. Blumenthal, N.M., et al., Effect of surgical implantation of recombinant human bone morphogenetic protein-2 in a bioabsorbable collagen sponge or calcium phosphate putty carrier in intrabony periodontal defects in the baboon. J Periodontol, 2002. 73(12): p. 1494-506. Seeherman, H., et al., Injectable rhBMP-2/CPM paste for closed fracture and minimally invasive orthopaedic repairs. J Musculoskelet Neuronal Interact, 2003. 3(4): p. 317-9; discussion 320-1. Jiya, T., et al., Posterior lumbar interbody fusion using nonresorbable poly-ether-ether-ketone versus resorbable poly-L-lactide-co-D,Llactide fusion devices: a prospective, randomized study to assess fusion and clinical outcome. Spine, 2009. 34(3): p. 233-7. Bruckner-Tuderman, L. and P. Bruckner, Genetic diseases of the extracellular matrix: more than just connective tissue disorders. J Mol Med, 1998. 76(3-4): p. 226-37. Aumailley, M. and B. Gayraud, Structure and biological activity of the extracellular matrix. J Mol Med, 1998. 76(3-4): p. 253-65. Jackson, R.A., et al., The use of heparan sulfate to augment fracture repair in a rat fracture model. J Orthop Res, 2006. 24(4): p. 636-44. Lyon, M., et al., Interaction of hepatocyte growth factor with heparan sulfate. Elucidation of the major heparan sulfate structural determinants. J Biol Chem, 1994. 269(15): p. 11216-23. Raines, E.W., et al., The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF)-AB and -BB and inhibits the binding of PDGF to its receptors. Proc Natl Acad Sci U S A, 1992. 89(4): p. 1281-5. Park, J.E., G.A. Keller, and N. Ferrara, The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell, 1993. 4(12): p. 1317-26. Park, Y.S. and Y.C. Lin, Effect of epidermal growth factor (EGF) and defined simple media on in vitro bovine oocyte maturation and early embryonic development. Theriogenology, 1993. 39(2): p. 475-84. Raab, G. and M. Klagsbrun, Heparin-binding EGF-like growth factor. Biochim Biophys Acta, 1997. 1333(3): p. F179-99. Freeman, M.R., et al., Heparin-binding EGF-like growth factor is an autocrine growth factor for human urothelial cells and is synthesized by epithelial and smooth muscle cells in the human bladder. J Clin Invest, 1997. 99(5): p. 1028-36. Roberts, A.B. and M.B. Sporn, Transforming growth factor beta. Adv Cancer Res, 1988. 51: p. 107-45. 228 References 366. 367. 368. 369. 370. 371. 372. 373. 374. 375. 376. 377. 378. 379. 380. 381. 382. Butzow, R., et al., A 60-kD protein mediates the binding of transforming growth factor-beta to cell surface and extracellular matrix proteoglycans. J Cell Biol, 1993. 122(3): p. 721-7. Saksela, O., et al., Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation. J Cell Biol, 1988. 107(2): p. 743-51. Blanquaert, F., D. Barritault, and J.P. Caruelle, Effects of heparan-like polymers associated with growth factors on osteoblast proliferation and phenotype expression. J Biomed Mater Res, 1999. 44(1): p. 63-72. Bilic-Curcic, I., et al., Origins of endothelial and osteogenic cells in the subcutaneous collagen gel implant. Bone, 2005. 37(5): p. 678-87. Kruyt, M.C., et al., Viable osteogenic cells are obligatory for tissueengineered ectopic bone formation in goats. Tissue Eng, 2003. 9(2): p. 327-36. Qian, Y., et al., Natural bone collagen scaffold combined with autologous enriched bone marrow cells for induction of osteogenesis in an ovine spinal fusion model. Tissue Eng Part A, 2009. 15(11): p. 3547-58. Zaky, S.H. and R. Cancedda, Engineering craniofacial structures: facing the challenge. J Dent Res, 2009. 88(12): p. 1077-91. Luginbuehl, V., et al., Localized delivery of growth factors for bone repair. Eur J Pharm Biopharm, 2004. 58(2): p. 197-208. Rider, C.C., Heparin/heparan sulphate binding in the TGF-beta cytokine superfamily. Biochem Soc Trans, 2006. 34(Pt 3): p. 458-60. Woodruff, M.A., et al., Sustained release and osteogenic potential of heparan sulfate-doped fibrin glue scaffolds within a rat cranial model. J Mol Histol, 2007. 38(5): p. 425-33. Zhao, B., et al., Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2. J Biol Chem, 2006. 281(32): p. 23246-53. Raiche, A.T. and D.A. Puleo, Cell responses to BMP-2 and IGF-I released with different time-dependent profiles. J Biomed Mater Res A, 2004. 69(2): p. 342-50. Westerhuis, R.J., R.L. van Bezooijen, and P. Kloen, Use of bone morphogenetic proteins in traumatology. Injury, 2005. 36(12): p. 1405-12. Vonau, R.L., et al., Combination of growth factors inhibits bone ingrowth in the bone harvest chamber. Clin Orthop Relat Res, 2001(386): p. 243-51. Polly, D.W., Jr., et al., A cost analysis of bone morphogenetic protein versus autogenous iliac crest bone graft in single-level anterior lumbar fusion. Orthopedics, 2003. 26(10): p. 1027-37. Schantz, J.T., et al., Repair of calvarial defects with customised tissueengineered bone grafts II. Evaluation of cellular efficiency and efficacy in vivo. Tissue Eng, 2003. Suppl 1: p. S127-39. Weinand, C., et al., Human shaped thumb bone tissue engineered by hydrogel-beta-tricalciumphosphate/poly-epsilon-caprolactone scaffolds and magnetically sorted stem cells. Ann Plast Surg, 2007. 59(1): p. 46-52; discussion 52. 229 References 383. Ho, S.T., et al., The evaluation of a biphasic osteochondral implant coupled with an electrospun membrane in a large animal model. Tissue Eng Part A, 2009. 230 [...]... organs or tissues can be managed or treated and annually, this alone, costs the USA an estimated $400 billion [1] In severe medical cases, treatments can range from removal, repair and replacement with graft transplants, combined with tailored rehabilitative and preventive therapies However, availability of donor grafts, for bone as well as other soft tissues for tissue and organ treatments remains... a small animal model The proof of concept, for the effective scaffold system delivery of rhBMP-2, for the bone engineering approach would be evaluated via the calvarial reconstruction of a critical-sized rat cranial defect, with and without growth factors (rhBMP-2) The key objective is to assess its feasibility and performance when incorporated as a part of a tissue engineering strategy 4) Performance... permanent implant could cause further material, stress or load mismatch over the individual’s lifetime x List of Tables Table 2.1 Mechanical properties of bone [79, 80] Table 2.2 Commercial bone graft alternatives Table 2.3 Comparison of the properties of the Poly(hydroxy esters) [151, 152] Table 3.1 List of materials used for fabrication of mPCL-based and PCLbased scaffolds Table 4.1 Scaffold groups and... Crystallinity behaviour of PCL, mPCL and mPCL/TCP scaffolds throughout the in vitro degradation period Figure 4.9 Gross overview and µCT analysis of the same rabbit calvarial explanted after 2 years in a calvarial defect (a) Gross overview of explanted rabbit calvarial Implanted scaffolds observed to be intact with portions being replaced by calcified matrix (b) Micro-CT images of rabbit calvarial with scaffolds... osteoblasts Hence, living cells would be a constituent of the transplanted bone graft or alternative Additionally, in situations where structural support is required, cortical autografts could be harvested as a block However, vascularised grafts and cortico-cancellous bone chips have been reported to have shorter healing time 3 Chapter 1 Introduction compared with a nonvascularised grafts or a massive... PCL-based scaffolds are stable and safe for long-term implantation iii) Porous PCL-based scaffolds with collagen mesh, is capable of efficient delivery of rhBMP-2 for bone engineering applications iv) PCL-based scaffolds together with rhBMP-2 are capable of engineering bone in a pre-clinical large animal model Based on this hypothesis, the specific objectives were identified: 1) Characterisation of composite... cortico-cancellous bone block [10, 11] Despite autografts possessing osteoinductivity, osteoconductivity and osteogenicity; the success of bone grafting also depends on how adequately the graft is incorporated into the defect site and host, these are influenced by many factors, such as type of graft (chips, block, vascular or avascular), site of transplantation, quality of transplanted bone and host bone, ... the implantation site for gross examination and biocage material retrieval Image shows all sample groups at 6 months (A) Autograft: uniform bone trabeculae observed at the defect cross section but with some fibrous tissue and irregular non-mineralised tissue regions, indicative of possible graft resorption which could lead to pseudoarthrosis due to lack of adequate fusion (B) Biocage alone: scaffold... being a major component of the musculoskeletal system, composed of a network of muscles, bones, joints, tendons, and ligaments that provide us with the ability to perform daily tasks It acts as a rigid support and protective framework for the organs and soft tissues, and also a system of mechanical levers for full mobility Musculoskeletal disorders and diseases significantly impact the quality of life... immunological and pathological risks associated with allografts and xenografts [12, 13] Alternatives to bone grafts have long been considered for treating moderate to large defects in orthopaedic applications, especially for the two most frequent procedures: long bone nonunions and spinal fusions Furthermore, based on the growing numbers of research and publications over the decade, there is an impending . products and bone grafts available today, which also extends to grafts of allogenic sources (eg. DBM). Today, autografts remain the gold standard despite its limited availability and disadvantages Bai HF, Gajadar B, Mia W, Amber S, Monica S, iv Sambit S, Amit K, Radek and Joanna, Sang Joon A, Tarik A, Anand L, staff of Bioengineering, Orthopaedic Surgery, DES SGH and NUSTEP. For all. – Outstanding Paper Award ) 11. Lam CXF, Abbah AS, Goh JCH, Hutmacher DW, Wong HK. Evaluation of a Polycaprolactone Based Bioresorbable Scaffold for Bone Regeneration at Load Bearing Sites.