The evaluation of bioactive polycaprolactone scaffolds as protein delivery systems for bone engineering applications

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. 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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