Development of a bioresorbable bone graft alternative for bone engineering applications

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. 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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.