THE EVALUATION OF BIOACTIVE POLYCAPROLACTONE SCAFFOLDS AS PROTEIN DELIVERY SYSTEMS FOR BONE ENGINEERING APPLICATIONS BINA RAI (B. Sci. (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ORAL & MAXILLOFACIAL SURGERY NATIONAL UNIVERSITY OF SINGAPORE 2006 PREFACE The thesis is submitted for the degree of Doctor of Philosophy in the Department of Oral & Maxillofacial Surgery at the National University of Singapore under the supervision of Associate Professor Ho Kee Hai and Professor Teoh Swee Hin. No part of this thesis has been submitted for other degree at other university or institution. To the author’s best knowledge, all the work presented 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 JOURNAL PUBLICATIONS 1. B. Rai, S.H. Teoh, D.W. Hutmacher, T. Cao, F. Chen, K. Yacob and K.H. Ho. The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds. Biomaterials 2004; 25(24): 5499-5506. 2. B. Rai, S.H. Teoh, D.W. Hutmacher, T. Cao and K.H. Ho. Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP-2. Biomaterials 2005; 26(17): 3739-3748. 3. B. Rai, S.H. Teoh and K.H. Ho. An in vitro evaluation of PCL-TCP composites as delivery systems for platelet-rich plasma. Journal of Controlled Release 2005; 107(2): 330-342. 4. Y. Lei, B. Rai, K.H. Ho and S.H. Teoh. In vitro degradation of novel bioactive polycaprolactone-20 % tricalcium phosphate composites for bone engineering. Accepted with minor revision by Journal of Material Science and Engineering. 5. B. Rai, M.E. Oest, K.M. Dupont, K.H. Ho, S.H. Teoh, R.E. Guldberg. Platelet-rich plasma delivery on 3D polycaprolactone-tricalcium phosphate scaffolds promotes early vascularization during segmental bone repair. Submitted to Journal of Material Research. 6. B. Rai, Y. Lei, K.M. Si-Hoe, K.B. Yacob, F. Chen, S.H. Teoh, K.H. Ho. Three dimensional polycaprolactone- tricalcium phosphate scaffolds loaded with platelet-rich plasma facilitates the placement of dental implants and induces mandibular bone regeneration simultaneously. Submitted to Journal of Oral and Maxillofacial Surgery. CONFERENCE PAPERS 1. B. Rai, S.H. Teoh, D.W. Hutmacher, F. Chen, K. Yacob, C. Tong and K.H. Ho. The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds. 7th World Biomaterials Congress, Symposium 30: Developing New Biomaterials: the Composite Approach, 17-21 May 2004, Sydney, Australia. 2. B. Rai, S.H. Teoh, D.W. Hutmacher, T. Cao and K.H. Ho. Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP-2. Joint Meeting of Tissue Engineering Society International & European Tissue Engineering Society, 10-13 October 2004, Lausanne, Switzerland. 3. B. Rai, S.H. Teoh and K.H. Ho. An evaluation of PCL-TCP composites as delivery systems for platelet-rich plasma. Regenerate Conference, 1-3 June 2005, Georgia, USA. 4. Y. Lei, B. Rai, K.H. Ho and S.H. Teoh. In vitro degradation of novel bioactive polycaprolactone-20 % tricalcium phosphate composites for bone engineering. International Conference on Materials for Advanced Technologies, 4-8 July 2005, Singapore. 5. B. Rai, M.E. Oest, K.M. Dupont, K.H. Ho, S.H. Teoh, R.E. Guldberg. Platelet-rich plasma delivery on 3D polycaprolactone-tricalcium phosphate scaffolds promotes early vascularization during segmental bone repair. 8th TESI Annual Meeting, 22-25 October 2005, Shanghai, China. 6. B. Rai, M.E. Oest, K.M. Dupont, K.H. Ho, S.H. Teoh, R.E. Guldberg. Platelet-rich plasma delivery on 3D polycaprolactone-tricalcium phosphate scaffolds promotes early vascularization during segmental bone repair. The 12th International Conference on Biomedical Engineering, 7-10 December 2005, Singapore. ACKNOWLEDGEMENTS The author would like to thank Professor Teoh Swee Hin and Associate Professor Ho Kee Hai for all their guidance, complete trust and belief in her capabilities. She especially appreciates the freedom entrusted to her in making major decisions and in steering the direction of her project. She hopes she has fulfilled Professor Teoh’s criteria for a true researcher, one who has “content, character and contact”. She is extremely grateful to Associate Professor Cao Tong and his team from the Dentistry Research Lab and Associate Professor Dietmar Hutmacher and his group from the Tissue Engineering Lab. She also immensely thanks Associate Professor Robert Guldberg and his team over at the Georgia Institute of Technology for providing her the opportunity of an enriching overseas experience. She acknowledges her family, in particular her mother, Madam Pushpa Devi, for all her sacrifices, for always being there and for pushing her to strive for excellence. She thanks her bosom friends for being so kind and understanding during this stressful period. Most importantly, she thanks god for blessing her with this amazing accomplishment. “Thank you God for the ocean, when all I asked for was some rain”. SUMMARY The research scope encompasses the creation of novel bioactive composite scaffolds consisting of polycaprolactone (PCL) physically blended with 20 % tricalcium phosphate (TCP) particles. The supposition was for these scaffolds to be superior bone substitutes than the first generation pure PCL scaffolds due to its likeness to the living bone in terms of its composition. An additional perception was for these scaffolds to serve simultaneously as protein delivery systems to further augment its bone regenerative capacity. After the formulation of the concept and fabrication process, the scaffolds were subjected to both in vitro and in vivo experiments to test their efficacy. The scope of this thesis ended with animal studies, a stage just before preclinical trials. PCL-TCP scaffolds loaded with osteoblasts sustained osteogenic expression in vitro. The osteoblasts readily colonized the surfaces, rods and pores of the scaffolds while maintaining their osteogenic phenotype for four weeks. The addition of recombinant human bone morphogenetic protein-2 (rhBMP-2) enhanced the differentiated function of these osteoblasts that resulted in accelerated mineralization, followed by their death as they underwent terminal differentiation. Hence, the scaffolds were (1) capable of facilitating the process from cellular attachment to differentiation to mineral, (2) not toxic to the cells and (3) its 3D-architecture and porosity allowed for the infiltration of cells and loading of proteins. The protein release profile is a function of the degradation of the scaffolds. The objective was to characterize the in vitro degradation behavior of PCL-TCP scaffolds, paying special attention to how the inclusion of TCP would affect the degradation properties. Based on weight loss, water uptake and pH measurements, it was found that PCL-TCP scaffolds were degraded slowly in phosphate buffered saline (PBS). A calcium-rich layer was nucleated on the scaffold’s surface that finally resulted in hydroxyapatite precipitation after weeks of immersion in simulated body fluid (SBF) as verified by Xray diffraction, scanning electron microscopy and biochemical analysis. The effectiveness of PCL-TCP scaffolds as protein delivery systems for a single osteoinductive factor was evaluated. Pure PCL scaffolds of similar architecture were adopted as controls to investigate if the addition of TCP resulted in disparate release profiles. Protein retention was 49.1 % ±  for PCL-TCP scaffolds. The scaffolds were loaded with rhBMP-2 in fibrin sealant and immersed in PBS for weeks. The rhBMP-2 particles were distributed uniformly on the rods’ surfaces of PCL-TCP scaffolds. Bi- and tri-phasic burst-like release profiles were observed for scaffolds loaded with 10 and 20 µg/ml rhBMP-2 respectively. PCL-TCP scaffolds retained rhBMP-2 longer than pure PCL scaffolds. The stability and bioactivity of eluted proteins were verified as well to ensure that the released growth factor could still execute its primary function of stimulating tissue regeneration. To evaluate the efficacy of the scaffolds as delivery systems for multiple growth factors, platelet-rich plasma (PRP) was used. The buffers sufficed as important determinants of the release profiles obtained for transforming growth factor and platelet derived growth factor. PBS-soaked scaffolds manifested a tri-phasic burstlike profile that was absent in SBF. SBF-soaked scaffolds showed sustained release of the growth factors and total release was not achieved, whereas total release was realized for PBS-soaked scaffolds. Only release profiles for SBF-soaked scaffolds were growth factor mediated in terms of their amounts and sizes. The ultimate goal of obtaining the release profile of growth factors was to correlate the protein release to the stages of bone regeneration observed from subsequent in vivo studies. We hypothesized that delivery of autologous PRP within a structural scaffold would promote more rapid early revascularization of the defect region and enhance longer-term functional bone repair. To test this hypothesis, we quantified the effects of PRP delivery within PCL-TCP scaffolds on vascularization, mineralization, and mechanical properties of large segmental defects in the rat femur. The in vivo study demonstrated that (1) PCLTCP scaffolds were effective at promoting bone formation within critically-sized femoral defects, (2) PRP delivery promoted early vascularization within bone repair constructs, and (3) micro-CT imaging techniques may be used to evaluate both vascularization and mineralization in a challenging in vivo test bed of bone regeneration strategies. The application of our bone regenerative strategy to a canine model and for a longer-term period was investigated. The research showed that PRP loaded PCL-TCP scaffolds could facilitate the placement of dental implants, shorten wound healing time and stimulate mandibular bone regeneration simultaneously in mongrels. PRP-treated defects had 98.3 and 58.3 % higher bone volume than controls at and months respectively. New bone trabeculae were observed in close apposition to the dental implants and penetrated the defect site. The scaffolds experienced 33 % degradation from to months, finally occupying only 46.9 % of the cross-sectional area. In conclusion, the work presented in the thesis showed for the first time that threedimensional, bioactive polycaprolactone scaffolds can serve effectively as protein delivery systems for bone regeneration and has the potential for clinical applications. Table of Contents PREFACE i ACKNOWLEDGEMENTS iii SUMMARY iv TABLE OF CONTENTS vii LIST OF FIGURES xv LIST OF TABLES xxii CHAPTER INTRODUCTION 1.1 BACKGROUND 1.1.1 Current treatments for bone defects 1.1.2 Bone tissue engineering strategies 1.2 RESEARCH OBJECTIVES 1.3 RESEARCH SCOPE CHAPTER LITERATURE REVIEW 2.1 BONE PHYSIOLOGY 2.2 BIOMATERIALS SELECTION 12 2.2.1 Polycaprolactone (PCL)-based scaffolds 12 2.2.2 Rational for composite biomaterials 17 2.2.3 Fibrin tisseel sealant biomatrix technology 19 2.2.4 Recombinant bone morphogenetic protein-2 21 2.2.5 Platelet-rich plasma 24 2.2.6 Dental implants 28 CHAPTER PROPOSED RESEARCH PROGRAM 3.1 RESEARCH OBJECTIVES 30 3.2 MILESTONE CHART 34 CHAPTER CYTOCOMPATIBILITY AND BIODEGRADATION OF PCL-TCP SCAFFOLDS 4.1 INTRODUCTION 36 4.2 MATERIALS AND METHODS 37 4.2.1 Scaffold design and fabrication 37 4.2.2 Porosity calculation 38 4.2.3 Bone morphogenetic protein-2 38 4.2.4 Cytocompatibility study 40 Dog osteoblast culture and cell seeding on PCL-TCP scaffold 40 Cellular viability and proliferation assays 41 Cellular adhesion assay 42 Extracellular matrix production 42 4.2.5 Biodegradation study 43 Water uptake 43 Weight loss 44 pH measurements 44 4.2.6 Bioactivity study 44 Scanning electron microscopy 44 X-ray diffraction analysis 44 Von Kossa staining 45 Ionised calcium and phosphate concentrations 45 4.2.7 Statistical analysis 45 4.3 RESULTS 45 4.3.1 Scaffold morphology 45 4.3.2 Cytocompatibility study 45 Cellular viability and proliferation 45 Cellular adhesion 49 Extracellular matrix production 50 4.3.3 Biodegradation study 52 Water uptake 52 Weight Loss 53 Ph measurements 54 Bioactivity study 54 4.4 DISCUSSION 56 4.4.1 Cytocompatibility 56 Effect of rhBMP-2 on cell proliferation 56 Effect of rhBMP-2 on cell differentiation 60 10 facilitates the absorption of fluid molecules which result in stable blood clot formation, hence aiding in the initial wound healing processes at the defect site. The fabrication method of polycaprolactone nanofibers by electrospinning is simple yet interesting. The PCL polymer solution is delivered at a constant flow rate (Q = 0.1 ml/min) to a metal capillary (1.6 mm OD, mm ID, 50 mm length) connected to a highvoltage power supply. Upon applying a high voltage such as 13 kV, a fluid jet is ejected from the capillary. As the jet accelerates towards a grounded collector, the solvent evaporates and a charged polymer fiber is deposited on the collector in the form of a nonwoven fabric. The fabric is stored in a dessicator for several days, and then cut into squares of desired dimensions. For sterilization, the scaffolds are placed in 70 % ethanol for three hours. These nanofiber scaffolds may very well represent the next generation of synthetic biomaterials used as protein delivery systems. The extent of nanofiber deposition as well as its porosity can be tailored to achieve the best protein loading and release profile. 201 REFERENCES • Aghaloo TL, Moy PK, Freymiller EG. Evaluation of platelet-rich plasma in combination with freeze-dried bone in the rabbit cranium. Clin Oral Impl Res 2005; 16: 250-257. • American Academy of Orthopaedic Surgeons (2003). Facts about fractures. In: American Academy of Orthopaedic Surgeons website http://www.aaos.org/wordhtml/ research/stats/factshtm. Cited 18 November 2003. • American Cancer Society (2003). What are the key statistics for bone cancer? In: American Cancer Society website http://www.cancer.org. Cited 18 November 2003. • Apornmaeklong P, Kochel M, Depprich R, Kubler NR, Wurzler KK. Influence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study. Int J Oral Maxillofac Surg 2004; 33: 60-70. • Babensee JE, Anderson JM, Melntire LV and Mikos AG. Host response to tissue engineered devices. Adv Drug Deliv Rev 1998; 33: 111-139. • Bancroft GN, Mikos (2001) Bone tissue engineering by cell transplantation. In: Ikada Y, Oshima N (eds) Tissue engineering for therapeutic use. Elsevier, New York, USA, p151. • Basu S, Marini CP, Bauman FG, Shirazian D, Damiani P, Robertazzi R. Comparative study of biological glues: cryoprecipitate glue, two-component fibrin sealant and “French” glue. Ann Thorac Surg 1995; 60: 1255-1262. • Bentley MD, Ortiz MC, Ritman EL, Romero JC. The use of microcomputed tomography to study microvasculature in small rodents. Am J Physiol Regulatory Integrative Comp Physiol 2002; 282: R1267-R1279. • Bessho K, Carnes DL, Cavin R, Ong JL. Experimental studies on bone induction using low molecular weight poly (DL-lactide-co-glycolide) as a carrier for recombinant human bone morphogenetic protein-2. J Biomed Mater Res 2002; 61:61-65. 202 • Blaker JJ, Gough JE, Maquet V, Notingher I, Boccaccini AR. In vitro evaluation of novel bioactive composites based on Bioglass-filled polylactide foams for bone tissue engineering scaffolds. J Biomed Mater Res 2003; 67A: 1401-1411. • Boccaccini AR, Roether JA, Hench LL, Maquet V and Jerome R. A composites approach to tissue engineering. Ceram Eng Sci Proc 2002; 23: 805-816. • Boden SD. Bioactive factors for bone tissue engineering. Clin Orthop 1999; 367:S8494. • Bonewald LF, Mundy GR. Role of transforming growth factor-beta in bone remodeling. Clin Orthop Rel Res 1990; 250: 261-272. • Bostrom RD, Mikos AG (1997). Tissue engineering of bone. In: Atala A, Mooney DJ (eds) Synthetic biodegradable polymer scaffolds. Birkhauser, Berlin, Germany, p215. • Brown KL, Cruess RL. Bone and cartilage transplantation in orthopaedic surgery. A review. J Bone Joint Surg Am 1982; 64: 270-279. • Canalis E, Economides AN and Gazzerro E. Bone morphogenetic proteins, their antagonists and the skeleton. Endo Rev 2003; 24: 218-235. • Cao T, Ho KH, Teoh SW. Scaffold design and in vitro study of osteochondral coculture in a three-dimensional porous polycaprolactone scaffold fabricated by fused deposition modeling. Tissue Eng 2003; 9: S103–112. • Cartmell S, Huynh K, Lin A, Nagaraja S, Guldberg RE. Quantitative microcomputed tomography analysis of mineralization within three-dimensional scaffolds in vitro. J Biomed Mater Res 2004; 69A: 97-104. • Choi BH, Im CJ, Huh JY, Suh JJ, Lee SH. Effect of platelet rich plasma on bone regeneration in autogenous bonegraft. Int J Oral Maxillofac Surg 2004; 33: 56-59. • Ciapetti G, Ambrosio L, Savarino L, Granchi D, Cenni E, Baldini N, Pagani S, Guizzardi S, Causa F, Giunti A. Osteoblast growth and function in porous poly caprolactone matrices for bone repair: a preliminary study. Biomaterials 2003; 24: 3815-3824. • Coombes AGA, Rizzi RC, Williamson M, Barralet JE, Downes S, Wallace WA. Precipitation casting of polycaprolactone for applications in tissue engineering and drug delivery, Biomaterials 2004; 25:315-325. 203 • Darney PJ, Monroe SE, Klaisle CM, Alvarado A. Clinical evaluation of the Capranor contraceptive implant: preliminary report. Am J Obstet Gynecol 1989; 160: 1292-1295. • Doillon CJ (2002). Modification of natural polymers: fibrinogen-fibrin. In: Atala A, Lanza RP (eds) Methods of tissue engineering. San Diego, California. Academic Press. p555-565. • Du C, Cui FZ, Zhang W, Feng Q L, Zhu XD, Groot KD. Formation of calcium phosphate/collagen composites through mineralization of collagen matrix. J Biomed Mater Res 2000; 50: 518-527. • Duvall CL, Taylor WR, Weiss D, Guldberg RE. Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury. Am J Physiol Heart Circ Physiol 2004; 287: H302-H310. • Elbert SES, Hubbel JA. Development of fibrin derivatives for controlled release of heparin binding growth factors. J Cont Rel 2000; 65: 389-402. • Endres M, Hutmacher DW, Salgado AJ, Kaps C, Ringe J, Reis RL, Sittinger M, Brandwood A, Schantz JT. Osteogenic induction of human bone marrow derived mesenchymal progenitor cells in novel synthetic polymer hydrogel matrices. TE 2003; 9: 689-701. • Enneking WF, Eady JL, Burchardt H. Autogenous cortical bone grafts in the reconstruction of segmental skeletal defects. J Bone Joint Surg Am 1980; 62:1039-1058. • Erbe EM, Marx JG, Clineff TD, Bellincampi LD. Potential of an ultraporous beta-tricalcium phosphate synthetic cancellous bone void filler and bone marrow aspirate composite graft. Eur Spine J 2001; 10: S141-146. • Feinberg, S.E., Aghaloo, T.L., Cunningbam, L.L. Role of tissue engineering in oral and maxillofacial reconstruction: findings of the 2005 AAOMS Research Summit. J Oral Maxillofac Surg 2005; 63: 1418-1425. • Fennis JPM, Stoelinga PJW, Jansen JA. Reconstruction of the mandible with an autogenous irradiated cortical scaffold, autogenous corticocancellous bone-graft and autogenous platelet-rich-plasma: an animal experiment. Int J Oral Maxillofac Surg 2005; 34: 158-166. 204 • Ferreira, C.F., Gomes, M.C.C., Filho, J.S., Granjeiro, J.M., Simoes, C.M.O., Magini, R.D.S. Platelet-rich plasma influence on human osteoblasts growth. Clin Oral Impl Res 2005; 16: 456-460. • Frank A, Rath SK, Boey F, Venkatraman S. Study of the initial stages of drug release from a degradable matrix of poly (d, l-lactide-co-glycolide). Biomaterials 2004; 25: 813-821. • Fratzl P, Gupta HS, Paschalis EP, Roschger P. Structure and mechanical quality of the collagen-mineral nano-composite in bone. J Mater Chem 2004; 14: 2115-2123. • Freymiller EG, Aghaloo TL. Platelet-rich plasma: Ready or not. J Oral Maxillofac Surg 2004; 62: 484-488. • Gauthier O, Muller R, Stechow DV, Lamy B, Weiss P, Bouler JM, Aguado E, Daculsi G. In vivo bone regeneration with injectable calcium phosphate biomaterial: A threedimensional micro-computed tomographic, biomechanical and SEM study. Biomaterials 2005; 26: 5444-5453. • Gitelis S, Saiz P. What's new in orthopaedic surgery. J Am Coll Surg 2002; 194:788-791. • Guehennec LL, Goyenvalle E, Aguado E, Cuny MH, Enkel B, Pilet P, Daculsi G, Layrolle P. Small animal models for testing macroporous ceramic bone substitutes. J Biomed Mater Res Part B: Appl Biomater 2005; 72: 69-78. • Hayashi T, Nakayama K, Mochizuki M, Matsuda T. Studies on biodegrdable polycaprolactone fibers. Part 3. Enzymatic degradation in vitro. Pure Appl Chem 2002; 74: 869-880. • Hedberg EL, Shih CK, Lemoine JJ, Timmer MD, Liebschner MAK, Jansen JA, Mikos AG. In vitro degradation of porous poly (propylene fumarate)/poly (DL-lactic-coglycolic acid) composite scaffolds. Biomaterials 2005; 26: 3215-3225. • Hench LL, Splinter RJ, Allen WC, Greelee TK. Bonding mechanisms at the interface of ceramic prosthetic materials. J Biomed Mater Res 1971; 2:117-141. • Hock JM, Canalis E. Platelet derived growth factor enhances bone cell replication, but not differentiated function of osteoblasts. Endocrinology 1994; 134; 1423-1428. • Holdsworth DW, Thornton MM. Micro-ct in small animal specimen imaging. Trends in Biotech 2002; 20: S34-39. 205 • Huang YC, Kaigler D, Rice KG, Krebsbach PH, Mooney DJ. Combined angiogenic and osteogenic factor delivery enhances bone marrow stromal cell-driven bone regeneration. J Bone Miner Res 2005; 20: 848-857. • Hutmacher DW, Schantz T, Zein I, Ng KW, Teoh SH, Tan KC. Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling. J Biomed Mater Res 2001; 55:203-216. • Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000; 21: 2529-2543. • Ito K, Yamada Y, Nagasaka T, Baba S, Ueda M. Osteogenic potential of injectable tissue-engineered bone: A comparison among autogenous bone, bone substitute (Biooss), platelet-rich plasma, and tissue-engineered bone with respect to their mechanical properties and histological findings. J Biomed Mater Res 2005; 73A: 63-72. • Jeong JC, Lee J, Cho K. Effects of crystalline microstructure on drug release behavior of poly (e-caprolactone) microspheres. J Control Rel 2003; 92: 249–58. • Jin QM, Takita H, Kohgo T, Atsumi K, Itoh H, Kuboki Y. Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation. J Biomed Mater Res 2000; 51: 491–499. • Jinguishi S, Urabe K, Okazaki K, Hirata G, Sakai A, Ikenoue T. Intramuscular bone induction by recombinant bone morphogenetic protein-2 with beta-tricalcium phosphate as a carrier: in vivo bone banking for muscle-pedicle autograft. J Orthop Sci 2002; 7: 490–494. • Jones AC, Milthorpe B, Averdunk H, Limaye A, Senden TJ, Sakellariou A, Sheppard AP, Sok RM, Knackstedt MA, Brandwood A, Rohner D, Hutmacher DW. Analysis of 3D bone ingrowth into polymer scaffolds via micro-computed tomography imaging. Biomaterials 2004; 25: 4947-4954. • Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 2005; 26: 5474-5491. • Kasten P, Luginbuhl R, Griensven MV, Barkhausen T, Krettek C, Bohner M, Bosch U. Comparison of human bone marrow stromal cells seeded on calcium deficient hydroxyapatite, β-tricalcium phosphate and demineralized bone matrix. Biomaterials 2003; 24: 2593–2603. 206 • Kawabata M, 1998 Kawabata M, Imamura T, Miyazono K. Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev 1998; 9:49-61. • Kawamura M, Urist MR. Human fibrin is a physiologic delivery system for bone morphogenetic protein. Clin Orthop 1988; 235: 302-310. • Kawase T, Okuda K, Wolff LF, Yoshie H. Platelet-rich plasma derived fibrin clot formation stimulates collagen synthesis in periodontal ligament and osteoblastic cells in vitro. J Periodontol 2003; 74: 858-864. • Kim HD, Valentini RF. Retention and activity of BMP-2 in hyaluronic acid-based scaffolds in vitro. J Biomed Mater Res 2002; 59: 573–584. • Kim HW, Knowles JC, Kim HE. Development of hydroxyapatite bone scaffold for controlled drug release via polycaprolactone and hydroxyapatite hybrid coatings. J Biomed Mater Res Part B: Applied Biomater 2004; 70B: 240-249. • Kim SG, Chung CH, Kim YK, Park JC, Lim SC. Use of particulate dentin-plaster of paris combination with/without platelet-rich plasma in the treatment of bone defects around implants. Int J Oral Maxillofac Implants 2002; 17: 86-94. • Kim SG, Kim WK, Park JC, Kim HJ. A comparative study osseointegration of Avana implants in a demineralized freeze-dried bone alone or with platelet rich plasma. J Oral Maxillofacial Surg 2002; 60: 1018–1025. • Koefoed M, Ito H, Gromov K, Reynolds DG, Awad HA, Rubery PT, Vinther MU, Soballe K, Guldberg RE, Lin ASP, O’Keefe RJ, Zhang X, Schwarz EM. Biological effects of rAAV-caAlk2 coating on structural allograft healing. Mol Ther 2005; 12: 212-218. • Kokubo T, Hak DJ, Hazelwood SJ, Reddi AH. Development of an atrophic non-union model and comparison to a closed healing fracture in rat femur. J Ortho Res 2003; 21: 503-510. • Kokubo T, Kim HM, Kawashita M. Novel bioactive materials with different mechanical properties. Biomaterials 2003; 24: 2161-2175. • Kokubo T, Kushitani H, Sakka S, Kitsugi T and Yamamuro T. Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W3. J Biomed Mater Res 1990; 24: 721-734. 207 • Laurencin CT and Lu HH. Polymer-ceramic composites for bone tissue engineering. In: Davies JE, editor. Bone engineering. (Toronto, Canada: em squared incorporated, 2000). pp. 462-472. • Lee SC, Shea M, Battle MA, Kozitza K, Ron E, Turek T, Schaub RG, Hays WC. Healing of large segmental defects in rat femurs is aided by rhBMP-2 in PLGA matrix. J Biomed Mater Res 1994; 28: 1149–1156. • Lin ASP, Barrows TH, Cartmell SH, Guldberg RE. Microarchitectural and mechanical characterization of oriented porous polymer scaffolds. Biomaterials 2003; 24: 481-489. • Linkhart TA, Mohan S, Baylink DJ. Growth factors for bone growth and repair: IGF, TGFB and BMP. Bone 1996; 19: 1S-12S. • Liu Y, Groot DK, Hunziker EB. BMP-2 liberated from biomimetic implant coatings induces and sustains direct ossification in an ectopic rat model. Bone 2005; 36: 745757. • Liu Y, Hunziker EB, Layrolle P, Bruijn JDD, Groot KD. Bone morphogenetic protein-2 incorporated into biomimetic coatings retains its biological activity. TE 2004; 10:101108. • Lowry KJ, Hamson KR, Peng LB, Calaluce R, Evans ML, Anglen JO, Allen WC. Polycaprolactone/glass bioabsorbable implant in a rabbit humerus fracture model. J Biomed Mater Res 1997; 36: 536-541. • Lucarelli E, Beccheroni A, Donati D, Sangiorgi L, Cenacchi A, Vento AMD, Meotti C, Bertoja AZ, Giardino R, Fornasari PM, Mercuri M, Picci P. Platelet-derived factors enhance proliferation of human stromal cells. Biomaterials 2003; 24: 3095-3100. • Maquet V, Boccaccini AR, Pravata L, Notingher I, Jerome R. Porous polyhydroxyacid/ bioglass composite scaffolds for bone tissue engineering. I: preparation and in vitro characterization. Biomaterials 2004; 25: 4185-4194. • Marx RE, Carlson ER, Eichstaedt RM. Growth factor enhancement for bone grafts. Oral Surg Med Oral Pathol Oral Radiol Endod 1998; 85: 638-646. • Marx RE. Platelet-rich plasma: Evidence to support its use. J Oral Maxillofac Surg 2004; 62: 489-496. 208 • Matin K, Senpuku H, Hanada N, Ozawa H, Ejiri S. Bone regeneration by recombination human bone morphogenetic protein-2 around immediate implants: A pilot study in rats. Int J Oral Maxillofac Implants 2003; 18: 211-217. • Matsuda N, Lin WL, Kumar NM, Cho MI, Genco RJ. Mitogenic, chemotactic and synthetic responses of rat periontal ligament fibroblastic cells to polypeptide growth factors in vitro. J Periodontol 1992; 63: 515-525. • Matsumoto T, Okazaki M, Inoue M, Yamaguchi S, Kusunose T, Toyonaga T, Hamada Y, Takahashi J. Hydrozyapatite particles as controlled release carrier of protein. Biomaterials 2004; 25: 3807-3812. • Matsuo T, Sugita T, Kubo T, Yasunaga Y, Ochi M, Murakami T. Injectable, magnetic liposomes as a novel carrier of recombinant human BMP-2 for bone formation in a rat bone-defect model. J Biomed Mater Res 2003; 66: 747–754. • Miller NA, Bene MC, Penaud J (2000). Periodontal applications. In: Lanza RP, Langer R, Vacanti J (eds) Principles of tissue engineering. San Diego, California. Academic Press, p821-833. • Mistry AS, Mikos AG. Tissue engineering strategies for bone regeneration. Adv Biochem Engin/Biotechnol 2005; 94: 1-22. • Mooney D, Mikos A. The promise of tissue engineering. Sci Am 1999; April: 37-43. • Mori M, Isobe M, Yamazaki Y, Ishihara K, Nakabayashi N. Restoration of segmental bone defects in rabbit radius by biodegradable capsules containing recombinant human bone morphogenetic protein-2. J Biomed Mater Res 2000; 50:191–198. • Murphy WL, Peters MC, Kohn DH, Mooney DJ. Sustained release of vascular endothelial growth factor from mineralized poly(lactide-co-glycolide) scaffolds for tissue engineering. Biomaterials 2000; 21:2521-2527. • Murphy WL, Simmons CA, Kaigler D, Mooney DJ. Bone regeneration via a mineral substrate and induced angiogenesis. J Dent Res 2004; 83: 204-210. • Nabeshima Y, Kurosaka M, Yoshiya S, Mizuno K. Effect of fibrin glue and endothelial cell growth factor on the early healing response of the transplanted allogenic meniscus: a pilot study. Knee Surg Sports Traumatol Arthrosc 1995; 3: 34-38. • National Osteoporosis Foundation (2003). Fast facts on osteoporosis. In: National Osteoporosis Foundation website 209 http://www.nof.org/osteoporosis/diseasefacts.html. Cited 18 November 2003. • Niedhart C, Maus U, Redmann E, Rohlfing BS, Niethard FU, Herbert CH. Stimulation of bone formation with an in situ setting tricalcium phosphate/rhBMP-2 composite in rats. J Biomed Mater Res 2003; 65A: 17-23. • Oates TW, Rouse CA, Cochran DL. Mitogenic effects of growth factors on human periodontal ligament cells in vitro. J Periodontol 1993; 64: 142-148. • Oest ME, Dupont KM, Guldberg RE. The evaluation of mineralization and vascularisation in rat segmental defects. Submitted to J Ortho Res. • Ogino, Y., Ayukawa, Y., Tsukiyama, Y., Koyano, K. The effect of platelet-rich plasma on the cellular response of rat bone marrow stromal cells in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005; 100: 302-307. • Orban JM, Marra KG, Hollinger JO. Composition options for tissue engineered bone. Tissue Eng 2002; 8: 529-539. • Oyama T, Nishimoto S, Tsugawa T, Shimizu F. Efficacy of platelet-rich plasma in alveolar bone grafting. J Oral Maxillofacial Surg 2004; 62: 555-558. • Partridge K, Yang X, Clarke NMP, Okubo YP, Bessho K, Sebald W, Howdle SM, Shakesheff KM, Oreffo RO. Adenoviral BMP-2 gene transfer in mesenchymal stem cells, in vitro and in vivo bone formation on biodegradable polymer scaffolds. Biochem Biophys Res Com 2002; 292: 144–152. • Perninand DE, English JP (1997). In: Dome AJ, Kost J, Wiseman DM (eds) Handbook of biodegradable polymers. Harwood Academic, Amsterdam, Holland, p63. • Pfeilschifter J, Oechsner M, Naumann A, Gronwald rGK, Minned HW, Ziegler R. Stimulation of bone matrix apposition in vitro by local growth factors, a comparison between insulin like growth factor 1, platelet derived growth factor and transforming growth factor. Endocrinology 1990; 127: 69-75. • Pitt, C.G., Chasalow, F.I., Hibionada, Y.M., Klimas, D.M., Schindler, A. Aliphatic polyesters 1. The degradation of poly (ε-caprolactone) in vivo. J Appl Polym Sci 1981; 26: 3779. 210 • Pittsburg Tissue Engineering Initiative (2005). Chapter Tissue Engineering. In: website http://www.ptei.org/stuff/chapter 4_4_bone_te.pdf. Cited 20th September 2005. • Raghoebar, G.M., Schortinghuis, J., Liem, R.S.B., Ruben, J.L., van der Wal, J.E., Vissink, A. Does platelet-rich plasma promote remodeling of autologous bone grafts used for augmentation of the maxillary sinus floor? Clin Oral Impl Res 2005; 16: 349356. • Rai B, Oest ME, Dupont KM, Ho KH, Teoh SH, Guldberg RE. Platelet-rich plasma delivery on 3D polycaprolactone-tricalcium phosphate scaffolds promotes early vascularisation during segmental bone repair. Submitted to Biomaterials. • Rai B, Teoh SH, Ho KH, ChenF, Yacob K, Hutmacher DW, Tong C. The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds. Biomaterials 2004; 25: 5499–5506. • Rai B, Teoh SH, Hutmacher DW, Tong C and Ho KH. Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP-2. Biomaterials 2005; 26(17): 37393748. • Rai B, Teoh SH, Ho KH. An evaluation of PCL-TCP composite scaffolds as delivery systems for platelet rich plasma. J Control Rel 2005: 107; 330-342. • Raiche AT, Puleo DA. Cell responses to BMP-2 and IGF-1 released with different time-dependent profiles. J Biomed Mater Res 2004; 69A: 342-350. • Ratner BD, Bryant SJ. Biomaterials: Where we have been and where we are going. Annu Rev Biomed Eng 2004; 6: 41-75. • Reddi AH. Bone morphogenetic proteins: an unconventional approach to isolation of first mammalian morphogens. Cytokine Growth Factor Rev 1997; 8:11-20. • Rodriguez A, Anastassov G, Lee H, Buchbinder D, Wettan H. Maxillofacial sinus augmentation with deproteinated bovine bone and platelet rich plasma with stimultaneous insertion of endosseous implants. J Oral Maxillofacial Surg 2003; 61:157–163. • Rose FRAJ, Oreffo ROC. Bone tissue engineering: Hope vs hype. Biochem Biophys Res Com 2002; 292: 1-7. 211 • Rubin JP, Yaremchuk MJ. Complications and toxicities of implantable biomaterials used in facial reconstructive and aesthetic surgery: a comprehensive review of the literature. Plast Reconstr Surg 1997; 100: 1336–1353. • Ruhe PQ, Deutman HCK, Wolke JGC, Spauwen PHM, Jansen JA. Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits. Biomaterials 2004; 25: 2123-2132. • Sahni A, Odrljin T, Francis CW. Binding of basic fibroblast growth factor to fibrinogen and fibrin. J Biol Chem 1998; 273:7554-7559. • Salgado AJ, Gomes ME, Chou A, Coutinho OP, Reis Rl, Hutmacher DW. Preliminary study on the adhesion and proliferation of human osteoblasts on starch-based scaffolds. Mater Sci Eng 2002; 20: 27–33. • Saoto N, Takaoka K. New synthetic biodegradable polymers as BMP carriers for bone tissue engineering. Biomaterials 2003; 24: 2287–2293. • Schantz JT, Hutmacher DW, Lam CXF, Brinkmann M, Wong KM, Lim TC, Chou N, Guldberg RE, Teoh SH. Repair of calvarial defects with customized tissue engineered bone grafts, evaluation of cellular efficiency and efficacy in vivo. TE 2003; 9: S127S139. • Schantz JT, Hutmacher DW, Ng KW, Teoh SH, Chim H, Lim TC. Induction of ectopic bone formation by using human periosteal cells in combination with a novel scaffold technology. Cell Transplant 2002; 11: 125-138. • Schliephake H. Bone growth factors in maxillofacial skeletal reconstruction. Int J Oral Maxillofac Surg 2002; 31: 469-484. • Schmidmaier G, Wildemann B, Lubberstedt M, Haas NP, Raschke M. IGF-1 and. TGFB1 incorporated in a polylactide implant coating stimulates osteoblasts differentiation and collagen-1 production but reduces osteoblast proliferation in cell culture. J Biomed Mater Res 2003; 65B: 157-162. • Schmitt JM, Hwang K, Winn SR, Hollinger JO. Bone morphogenetic proteins: an update on basic biology and clinical relevance. J Orthop Res 1999; 17: 269–278. • Schmoekel H, Schense JC, Weber FE, Gratz KW, Gnagi D, Muller R, Hubbel JA. Bone healing in the rat and dog with non-glycosylated BMP-2 demonstrating low solubility in fibrin matrices. J Orthop Res 2004; 22: 376-381. 212 • Seelich T. Tissucol, biochemistry and methods of application. J Head Neck Pathol 1982; 3: 65-69. • Shanaman R, Filstein MR, Meyer MJD. Localized ridge augmentation using BGR and platelet rich plasma: Case reports. Int J Periodont Restor Dent 2001; 21:345-355. • Sheridan MH, Shea LD, Peters MC, Mooney DJ. Bioabsorbable polymer scaffolds for tissue engineering capable of sustained growth factor delivery. J Cont Rel 2000; 64: 91102. • Smith, K.L., Schimpf, M.E., Thompson, K.E. Bioerodible polymers for delivery of macromolecules. Adv Drug Deliv Res 1990; 4: 343. • Stark G, Kaiser H, Horch R, Kopp J, Spilker G. Cultured autologous keratinocytes suspended in fibrin glue with allogenic overgraft for definitive burn wound coverage. Eur J Plast Surg 1995; 18: 267-271. • Suba Z, Takacs D, Gaal SG, Kovacs K. Facilitation of β-Tricalcium phosphate induced alveolar bone regeneration by platelet-rich plasma in beagle dogs: a histologic and histomorphometric study. Int J Oral Maxillofac Implants 2004; 19: 832-838. • Sun Q, Chen RR, Shen Y, Mooney DJ, Rajagopalan S, Grossman PM. Sustained vascular endothelial growth factor delivery enhances angiogenesis and perfusion in ischemic hind limb. Pharm Res 2005; 22: 1110-1116. • Sykaras N, Triplett RG, Nunn ME, Iacopino AM, Opperman LA. Effect of recombinant human bone morphogenetic protein-2 on bone regeneration and osseointegration of dental implants. Clin Oral Implant Res 2001; 12: 339–343. • Tabata Y. Tissue regeneration based on growth factor release. TE 2003; 9:S5-S15. • Tanahashi M and Matsuda T. Surface functional group dependence on apatite formation on self-assembled monolayers in a simulated body fluid. J Biomed Mater Res 1997; 34: 305-315. • Thomson RC, Shung KC, Yaszemski MJ, Mikos AG (2000). Polymer scaffold processing. In: Lanza RP, Langer R (eds) Principles of tissue engineering. 2nd edition. San Diego, California. Academic Press, p251263. 213 • Thorn JJ, Sorensen H, Fogh UW, Andersen M. Autologous fibrin glue with growth factors in reconstructive maxillofacial surgery. Int J Oral Maxillofac Surg 2004; 33: 95–100. • Tozum TF, Demiralp B. Platelet-rich plasma: A promising innovation in dentistry. J Can Dent Assoc 2003; 69:664-664h. • Tsay RC, Vo J, Burke A, Eisig SB, Lu HL, Landesberg R. Differential growth factor retention by platelet rich plasma composites. J Oral Maxillofac Surg 2005; 63: 521-528. • Urist MR. Bone: formation by autoinduction. Science 1965; 150:893-899. • van Luyn MJ, van Wachem PB, Damink LO, Dijkstra PJ, Feijen J, Nieuwenhuis P. Relations between in vitro cytotoxicity and crosslinked dermal sheep collagens. J Biomed Mater Res 1992; 26: 1091–1110. • Vehof JWM, Ruijter AE, Spauwen PHM, Jansen JA. Influence of rhBMP-2 on rat bone marow stromal cells cultured on titanium fiber mesh. TE 2001; 7: 373–383. • Vert M, Li MS, Spenlehauer G, Guerin P. Bioresorbability and biocompatibility of aliphatic polyesters. J Mater Sci 1992; 3: 432-446. • Wang M. (2004) Bioactive ceramic-polymer composites for tissue replacement. In: Teoh SH (ed) Engineering materials for biomedical applications. World Scientific Publishing Pte Ltd, Singapore, p8-1. • Wei G, Pettway GJ, McCauley LK, Ma PX. The release profiles and bioactivity of parathyroid hormone from polylactic-co-glycolic acid microspheres. Biomaterials 2004; 25: 345-352. • Weibrich G, Kleis WKG, Hafner G. Growth factor levels in the platelet rich plasma produced by different methods, Curasan type PRP kit versus PCCS PRP kit. Int J Oral Maxillofac Implants 2002; 17: 184-190. • Whitaker MJ, Quirk RA, Howdle SM, Shakesheff KM. Growth factor release from tissue engineering scaffolds. JPP 2001; 53:1427-1437. • Wiltfang J, Schlegel KA, Mosgau SS, Nkenke E, Zimmermann R, Kessler P. Effects of platelet-rich plasma on bone healing in combination with autogenous bone and bone substitutes in critical size defects. Clin Oral Impl Res 2003; 14: 213–218. • Winn SR, Uludag H, Hollinger JO. Carrier systems for bone morphogenetic proteins. Clin Orthop 1999; 367: S95–106. 214 • Wong C, Inman E, Spaethe R, Helgerson S. Fibrin based biomaterials to deliver human growth factors. Thromb Haemost 2003; 89: 573–582. • Woodard SC, Brewer PS, Moatmed F, Schindler A, Pitt CG. The intracellular degradation of poly (ε-caprolactone). J Biomed Mater Res 1985; 19: 1437-1444. • Wozney JM and Li RH. Engineering what comes naturally. Nature Biotechnology 2003; 21: 506-508. • Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA. Novel regulators of bone formation: molecular clones and activities. Science 1988; 242: 1528–1534. • Wu L and Ding J. In vitro degradation of three-dimensional porous poly (D, L-lactidecoglycolide) scaffolds for tissue engineering. Biomaterials 2004; 25: 5821-5830. • Yamaguchi A, Komori T, Suda T. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs and Cbfa1. Endocr Rev2000; 21: 393–411. • Yamamuro T, Hench L, Wilson J. Handbook of bioactive ceramics. Vol II. Calcium phosphate and hydroxyapatite ceramics. CRC Press Inc. 1990. • Yang D, Chen J, Jing Z, Jin D. Platelet-derived growth factor (PDGF)-AA: A selfimposed cytokine in the proliferation of human fetal osteoblasts. Cytokine 2000; 12: 1271-1274. • Yang L, Rai B, Teoh SH. In vitro degradation study of novel 3D polycaprolactonetricalcium phosphate scaffolds for bone tissue engineering. Submitted to J Mater Sci. • Younger EM, Chapman MW. Morbidity at bone graft donor sites. J Orthop Trauma 1989; 3: 192-195. • Yuan H, Bruijn JD, Zhang X, Blitterswijk CAV, Groot K. Use of an osteoinductive biomaterial as a bone morphogenetic protein carrier. J Mater Sci 2001; 12: 761–766. • Zechner, W., Tangi, S., Tepper, G., Furst, G., Bernhart, T., Haas, R., Mailath, G., Watzek, G. Influence of platelet-rich plasma on osseous healing of dental implants: A histologic and histomorphometric study in minipigs. Int J Oral Maxillofac Implants 2003; 18: 15-22. • Zein I, Hutmacher DW, Tan KC and Teoh SH. Fused deposition modeling of novel scaffold architectures for tissue engineering applications. Biomaterials 2002; 23: 11691185. 215 • Zhang X, Xie C, Lin AS, Ito H, Awad H, Lieberman JR, Rubery PT, Schwarz EM, O’Keefe RJ, Guldberg RE. Periosteal progenitor cell fate in segmental cortical bone graft transplantation: implications for functional tissue engineering. J Bone Miner Res 2005; 20: 2124-2137. • Ziegler J, Wohlfart UM, Kessler S, Breitig D, Gunther KP. Adsorption and release properties of growth factors from biodegradable implants. J Biomed Mater Res 2002; 59: 422-428. 216 [...]... delivery systems for the repair 28 of bone tissue The merit of the work was that the PCL-based scaffolds could serve not only as a template for bone growth but also as a delivery vehicle for osteoinductive factors so as to eventually improve the quality of the bone regenerated in terms of the amount/volume of bone formed, infiltration of vascular networks and bone unions 1.3 RESEARCH SCOPE ',6&29(5 . porosity allowed for the infiltration of cells and loading of proteins. The protein release profile is a function of the degradation of the scaffolds. The objective was to characterize the in vitro. evaluate the efficacy of the scaffolds as delivery systems for multiple growth factors, platelet-rich plasma (PRP) was used. The buffers sufficed as important determinants of the release profiles. 1 THE EVALUATION OF BIOACTIVE POLYCAPROLACTONE SCAFFOLDS AS PROTEIN DELIVERY SYSTEMS FOR BONE ENGINEERING APPLICATIONS BINA RAI (B. Sci. (Hons), NUS) A THESIS SUBMITTED