Tissue engineering of a vascularized bone graft

TISSUE E GI EERI G OF A VASCULARIZED BO E GRAFT SUBHA ARAYA RATH (MBBS, MMST) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DIVISIO OF BIOE GI EERI G ATIO AL U IVERSITY OF SI GAPORE 2009 Acknowledgements It is a pleasure to thank those who made this thesis possible and the list cannot be completed with few names. I understood at the end of my PhD work that the journey of few years was possible only with the help of the innumerable number of friends, teachers and mentors whose unique help was absolutely important in maintaining proper direction. Therefore, first of all, I would like to thank all those persons who came across in my PhD journey and their contribution was very important, however insignificant it may look. I would like to express my special thanks to Prof. Michael Raghunath, who not only taught us basic methods of research as a mentor and teacher but also helped me beyond limits for my PhD completion. This thesis would not have been possible without his supportive lessons throughout my PhD carrier and his special role for my thesis completion. I owe my deepest gratitude to Prof Dietmar W. Hutmacher, who constantly guided me from the beginning until the very end of this PhD journey. I am very grateful to work with such a scientific and fatherly figure. His supportive words through skype web chatting and his magical emails made me feel his presence in each step of this journey even without his physical presence. I would like to express my sincere and overwhelming gratitude to Dr. med. Ulrich Kneser, and Dr. J. Thorsten Schantz for their guidance and invaluable advice imparted throughout this project work. I remain very much indebted to all my supervisors for their magnanimous and unflinching inspiration instilled in me. The knowledge I gained from each of them in different areas of my work will be helpful to dedicate my life to science. i The support and guidance I received from the Lab of Tissue Engineering in NUS and DSO NUSTEP, Singapore, were exemplary and made my research experience memorable. I also take this opportunity to thank Lam Xu Fu Chirstopher, PhD student in Lab. of Tissue Engineering, who guided and helped me in all scaffold related work throughout my research work. I am indebted to him to support right until the end of my thesis work. I express my sincere thanks to Barney for his microCT lessons; Andrew for his works related to Extracel-HP. I am grateful to Clarice, Dr. Gajadhar Bhakta, Evelyne, Anurag, David, Monique, Anand and Dr. Kee Woei for their timely advice and knowledge in every aspect of my PhD work. I am indeed fortunate to have worked alongside such committed and hardworking people. I would like to show my special gratitude to Dr. Sambit Sahoo and Dr. Sampurna Sattar for their moral support as well as scientific inputs in every new experiment. The support for this work by my seniors, especially Dr. Sriram Vedula, Dr Dev Chatterjee, and Dr. Karthik Harve are gratefully acknowledged. I humbly express my sincere gratitude to Dr. Galyna Pryymachuk for her invaluable guidance, and advice imparted during this investigation. I remain very much beholden to her to teach me the microsurgical techniques for the orderly and successful execution of the experiments for the completion of this thesis. I would like to thank Dr Andreas Arkudas for his wise thoughts and kind words of advice at every point in my endeavour without which it would not have been possible to realize the thesis work. My special thanks go to Dr. Elias Polykandroitis for contributing many ideas during the initial phase of my project. I would also like to thank Dr. Oliver Bleizifer, Dr. Saskia Schnabl, and Dr. Justus Bier to be helpful with friendly advices throughout my work. ii I am also grateful to many people who have helped and contributed, in one way or another, to my research project. It has been a great pleasure to have worked with Stefan Fleischer, Katja Schubert, and Dorothee Klummp, who have been indispensable in the smooth completion of the project work. I would like to thank all staff in division of bioengineering, NUS and department of plastic surgery, University Hospital, Erlangen for their official and financial support. Last, but not the least, I would also like to thank my family, especially my parents; my wife, Bagmi; my brothers Bibhu and Prabhu for being very encouraging and supportive throughout my PhD career. July 2009 (Dr. Subha Narayan Rath) National University of Singapore iii List of published work • Leong DT, Abraham MC, Rath SN, Lim TC, Chew FT, Hutmacher DW. Investigating the effects of preinduction on human adipose-derived precursor cells in an athymic rat model. Differentiation. 2006 Dec; 74(9-10):519-29. • Rath SN, Woodruff MA, Susanto E, Haupt LM, Hutmacher DW, Nurcombe V, Cool SM. Sustained release and osteogenic potential of heparan sulfate-doped fibrin glue scaffolds within a rat cranial model. J Mol Histol. 2007 Sep 12. • Rath SN, Cohn D, Hutmacher DW. Comparison of Chondrogenesis in Static and Dynamic Environment Using PCL-PEO Scaffold. Accepted in Journal of Virtual and Physical Prototyping. • Fiegel HC , Pryymachuk G , Rath S , Bleiziffer O , Beier JP , Bruns H , Kluth D , Metzger R , Horch RE , Till H , Kneser U . Fetal Hepatocyte Transplantation in a Vascularized AV-Loop Transplantation Model in the Rat. Journal of cellular and molecular medicine. 2008 May 24. H H H H H H H H H H H H H H H H H H H H H H • Rath SN, Arkudas A, Pryymachuk G, Polykandroitis E, Christopher LXF, Bier JP, Horch RE, Hutmacher DW, Kneser U. Development of a Pre-vascularized 3D Composite ScaffoldHydrogel System Using an Artery-Venous Loop for Tissue Engineering Applications; submitted to Biomaterials. • Rath SN, Pryymachuk G, Bleiziffer OA, Lam CXF, Arkudas A, Ho STB, Bier JP, Horch RE, Hutmacher DW, Kneser U. Hyaluronan-based heparin-incorporated hydrogels for generation of axially vascularized bioartificial bone tissues: in vitro and in vivo evaluation in a PLDLLATCP-PCL-composite system; submitted to Tissue Engineering. iv Table of contents Acknowledgements . i List of published work iv Table of contents v Summary . x List of tables xiii List of figures . xiv List of symbols xvii CHAPTER 1. Introduction 1.1. Background 1.2. Tissue engineering . 1.2.1 Importance of vascularization in tissue engineering 1.2.2 Making a graft vascularized . 1.3. Aims and hypotheses . 1.3.1 Specific aim 1: To establish the scaffold architecture and optimize the artery-venous (A-V) loop model in rat using the scaffold and to standardize the explantation procedure. . 1.3.2 Specific aim 2: To develop and evaluate pre-vascularized 3D composite scaffold-hydrogel systems using an A-V loop for a possible graft. . 1.3.3 Specific aim 3: Application of bone morphogenetic protein-2 (BMP-2) or osteoblasts in the vascularized composite scaffold-modified hyaluronan hydrogel system for development of a vascularized bone graft. . 1.4. Organization of the Thesis . CHAPTER 2. Background to the Research . 10 2.1. Angiogenesis and vascularization 10 2.2. Importance of vascularization in tissue-specific tissue engineering 13 2.2.1 Bone tissue engineering 13 2.2.2 Skin tissue engineering . 15 v 2.2.3 Genito-urinary tissue engineering 16 2.2.4 Limitations of current status of tissue engineering . 16 2.3. Methods of generating vascularized grafts 18 2.3.1 Cell Source . 18 2.3.2 Scaffold Material and design 20 2.3.3 Use of growth factors . 21 2.3.4 Gene therapy for angiogenesis . 22 2.3.5 Extracellular Matrix Components 23 2.4. Surgical approach with artery-venous loop . 25 2.4.1 Cell free method: using body as bioreactor 30 CHAPTER 3. Experiment I: To establish the scaffold architecture and optimize the artery-venous (A-V) loop model in rat using the scaffold and to standardize the explantation procedure. 32 3.1. Introduction 32 3.2. Materials and methods . 35 3.2.1 Scaffold fabrication 35 3.2.2 Porosity and pore interconnectivity calculation . 37 3.2.3 Mechanical testing 38 3.2.4 A-V loop model 39 3.2.5 Explantation 40 3.2.6 Micro CT analysis 40 3.2.7 Histology 41 3.2.8 Statistical analysis 42 3.3. Results 43 3.3.1 Scaffold gross morphology . 43 3.3.2 Porosity and interconnectivity 43 3.3.3 Mechanical testing 44 3.3.4 Explantation 44 3.3.5 Histology 46 vi 3.4. Discussion 46 3.5. Conclusion . 51 CHAPTER 4. Experiment II: Development and characterization of a pre- vascularized 3D composite scaffold-hydrogel system using an artery-venous loop for tissue engineering applications. 53 4.1. Introduction 53 4.2. Materials & methods 56 4.2.1 Scaffold fabrication 56 4.2.2 Experimental design . 57 4.2.3 Hydrogels . 58 4.2.4 Surgical procedures 58 4.2.5 Explantation 59 4.2.6 Micro CT analysis 59 4.2.7 Histology 60 4.2.8 Histomorphometric analysis . 60 4.2.9 Immunohistochemistry . 61 4.2.10 Vascular corrosion cast preparation . 61 4.2.11 Scanning electron microscopy 62 4.2.12 Statistical analysis 62 4.3. Results: 63 4.3.1 Surgery and animals: 63 4.3.2 Micro CT analysis: . 63 4.3.3 Histology & Corrosion casting: 64 4.3.4 Histomorphometry 67 4.3.5 Immunohistochemistry . 69 4.4. Discussion: . 70 4.5. Conclusion: 74 vii CHAPTER 5. Experiment III: Applying bone morphogenetic protein-2 (BMP-2) or osteoblasts in the composite scaffold-modified hyaluronan hydrogel system along with A-V loop for a vascularized bone graft formation. 76 5.1. Introduction 76 5.2. Materials and methods . 78 5.2.1 Scaffold fabrication 78 5.2.2 Osteoblasts isolation and expansion . 78 5.2.3 Osteoblast culture in modified hyaluronan hydrogel . 81 5.2.4 AlamarBlue metabolic assay 82 5.2.5 PicoGreen DNA Quantification Assay . 83 5.2.6 FDA/PI fluorescent staining of osteoblasts 83 5.2.7 Release kinetics of rhBMP2 from hyaluronan hydrogel (Extracel-HP) . 84 5.2.8 Experimental design . 85 5.2.9 Surgical procedures and explantation . 86 5.2.10 RNA isolation and quantitative RT-PCR . 86 5.2.11 Micro CT analysis 87 5.2.12 Histology 88 5.2.13 Histomorphometric analysis . 88 5.2.14 Immunohistochemistry . 88 5.2.15 Corrosion cast technique 88 5.2.16 Statistical analysis 88 5.3. Results 89 5.3.1 Osteoblasts in Hyaluronan hydrogel 89 5.3.2 Release kinetics 91 5.3.3 Surgery and animals . 92 5.3.4 Micro CT analysis 92 5.3.5 Histology 95 5.3.6 Histomorphometry 97 5.3.7 Corrosion casting 98 viii 5.3.8 Immunohistochemistry . 99 5.3.9 Real time PCR 101 5.4. Discussion 103 5.5. Conclusion . 111 CHAPTER 6. CO CLUSIO S A D RECOMME DATIO S . 113 6.1. Conclusions 113 6.2. Recommendations for future research . 116 Reference: . 118 ix Chapter 6. Conclusions and recommendations CHAPTER 6. CO CLUSIO S A D RECOMME DATIO S 6.1. Conclusions Starting from metal alloys, the cells and growth-factor based biological constructs impact a drastic change for bone tissue engineering applications. Although scientists still attempt to understand the basic bone physiology and the role of different transcription factors for bone development, a number of composite biomaterials are practiced for bone fracture and bone loss treatments. The attempts and research publications to mimic the anatomy of bone by using a combination of composite materials, growth factors, mesenchymal stem cells or osteoblasts provide a strong hope for a realistically fulfillment of a suitable tissue engineered product in near future. With the advent of bioreactors and well established differentiation methods for stem cells, a highly suitable bone product in the laboratory seems feasible. However, a number of important issues still need to be addressed. Despite some success, tissue engineered bone products have not become widely in use, indicating some unresolved issues for in vivo use. The autograft from iliac crest continues to be the gold standard in bone loss cases. In this PhD research, a novel ceramic polymer composite biomaterial for bone tissue engineering approach was evaluated, in order to sustain the mechanical load of bone in spite of highly porous structure for effective tissue in-growth. A systematic approach was attempted to develop a vascularized bone graft with PLDLLA-TCPPCL scaffolds and hyaluronan hydrogel along with additives for proper differentiation of the tissue construct. The biomaterial needs to be biologically well compatible for viable bone in-growth. Therefore, the biomaterial with a specific shape and along with 113 Chapter 6. Conclusions and recommendations a hydrogel was evaluated with microsurgically created A-V loop for possible fibrovascular tissue development and new sprouting angiogenesis from the loop. This technique has produced an established A-V loop supplied composite scaffold hydrogel system for further exploration for successful bone tissue engineering application. The experiments conducted were focused on the development of a novel vascularized biomaterial for prolonged survival of cells or growth factors to develop a well-differentiated tissue. In this PhD thesis, The PLDLLA-TCP-PCL scaffold shape was attempted to provide the basic shape of the final construct, while all the extra additives can be helpful for changing the progress towards bone and angiogenesis growth deep into the construct. The experiments show that the mechanical strength and shape will be provided by the PLDLLA-TCP-PCL scaffold; but the A-V loop will provide angiogenesis and make the whole construct viable. The hydrogel will be helpful not only for allowing the ingrowth of angiogenesis but also the additions of growth factors or cells for proper differentiation of the construct. This is biologically more relevant and similar to bone anatomy and function. Furthermore, the physical properties of the scaffold can be changed with its two different parts namely, the ceramic part providing the strength and bioactivity, while the polymer part providing relatively quick degradability and shear stress sustainability. However, the microsurgically constructed A-V loop provides the main nutrition for the survival of the whole construct and later can make it act like a vascularized autograft. Based on the above strategy, the vascularized scaffold hydrogel system can keep the osteoblasts survived. Although a well defined bone was absent histologically, still there is favourable gene expression for bone markers. Assessment of angiogenesis pattern and progression in those two different hydrogels indicated that there were 114 Chapter 6. Conclusions and recommendations significant differences between them. The vascular growth in fibrin glue is related to the quick degradation of the hydrogel in vivo; however, the density and pattern after eight weeks are not comparatively more relative to four weeks. Moreover, the growth in the hyaluronan matrix is slow and progressive in relation to its slow degradation in vivo and after eight-week time point the fibro-vascular growth was very much similar to that of fibrin glue hydrogel. The A-V loop based scaffold has also been shown to be well suited for further exploration of growth factors or cells. In conclusion, this project has devised and demonstrated a method that can precisely engineer a highly vascularized scaffold for bone tissue engineering application, and yet, is versatile enough to be readily adapted into further experiment for different tissue growth by applying extra additives. When we analyze the specific objectives postulated in Chapter 1, the following conclusions were drawn from the experimental results: • A synthetic PLDLLA-TCP-PCL scaffold with mechanical properties similar to a cancellous bone was fabricated and for thorough vascularization of such a biomaterial the artery-venous loop was established and analyzed after Microfil injection and explantation. • The PLDLLA-TCP-PCL scaffold along with two different hydrogels namely, fibrin glue and hyaluronan were evaluated along with the surgically constructed A-V loop for possible support of angiogenesis and the rate of angiogenesis was analyzed with fast degradation of fibrin glue and slow degradation of the modified hyaluronan matrix. • The modified hyaluronan hydrogel sustains implanted osteoblasts well with alkaline phosphatase positive osteoblasts and high percentage of live cells. • The release kinetic of BMP-2 was slow and the degradation of the modified hyaluronan matrix was prolonged with some intact matrix left even after eight weeks. 115 Chapter 6. Conclusions and recommendations • The scaffold hyaluronan hydrogel system can be used to incorporate BMP2 to support the development of extra-osseous bone for possible bone graft application. • Although bone histology is lacking from the construct in the analyzed eightweek time point, the gene expression shows a well-favored tissue construct for bone graft and highly vascularized tissue for implanted cells to be viable. This technique of combining PLDLLA-TCP-PCL scaffold with modified hyaluronan matrix has the potential to be developed into a clinically viable tissue engineered bone construct. 6.2. Recommendations for future research The results in this PhD research showed that using PLDLLA-TCP-PCL scaffold with a suitable dose of BMP-2 can make a useful well-vascularized bone construct to replace the present gold standard iliac crest autograft for bone loss states. However, for clinically useful application of this bone-construct product, further evaluation of a number of aspects will need to be carried out, as recommended below: 1. In this research, degradation of the PLDLLA-TCP-PCL was not observed in vivo in the observed period of weeks at all. But for clinical application the degradation time and kinetics of this defined product must be studied in vivo. 2. In spite of highly satisfactory angiogenesis growth from A-V loop in the observed eight-week time period, a long term study is desirable for demonstration of the permanent nature of the blood vessels as in many research articles it was shown some seemingly permanent blood vessels actually degenerate with time in a longer duration study [62]. It is therefore, recommended that the A-V loop with the scaffold hydrogel system were studied for almost 6-12 months with proper measurement of angiogenesis. 116 Chapter 6. Conclusions and recommendations 3. With the adopted experimental set-up in this thesis, only the osteoblast implanted scaffolds have intense angiogenesis throughout the scaffold. However, even with growth factors, some areas of central parts still lack new blood vessel growth. It may be due to unsuitable porosity of the scaffolds or the short time period of study. These variations need to be tested to have intense angiogenesis similar to natural tissue. 4. In spite of the fact that a higher percentage of cells or a greater amount of growth factors can be active due to supplied A-V loop, there still needs a lag period of few days in which the growth of new blood vessels occurs. We can postulate another hypothesis that the cells or growth factors applied only after complete vascularization will make more active tissue-engineered product compared to the present status. This hypothesis needs to be tested compared to the present product. 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Prefabrication of vascularized bone grafts using recombinant human osteogenic protein-1--part 3: dosage of rhOP-1, the use of external and internal scaffolds. Int J Oral Maxillofac Surg 2004 Mar;33(2):164-172. 128 [...]... surgery are based upon the principle of replacing defective tissues with viable, functional alternatives The two main types of grafts currently in use are autografts and allografts An autograft or autogenous graft is a section of tissue taken from the patient's own body, whereas an allograft is taken from a cadaver In spite of many available treatment modalities for tissue loss, currently, the gold standard... flaps for skin; because the related blood vessels are constant and reasonably suitable for micro surgical manipulations [57] The advantage of vascularized graft is that it can effectively resist infection, can repair the loss of large segmental defects even in a diseased bed, and it does not undergo graft failure [58] In tissue engineering field, many ways are manipulated to make the graft vascularized. .. maintain, or improve tissue functions [3] In the following time, there were a lot of changes in research from the original views given by them to fabricate tissue engineered substances Although all tissues were probable candidates of tissue engineering approach, few tissues such as skin, cartilage, and bone are in advanced stage because of their potential need and relative ease of application 1 Chapter... wound healing in adults [29] The overall rate of vascular in-growth can be stimulated more, if the feeding parent vessels passed through the graft Many researchers have accomplished this by making an artery-venous (A- V) loop to supply blood to the graft [15, 30-33] In most of the cases, a contralateral artery/vein graft is made anastomosis between an artery and a vein for an A- V loop which can readily... make a viable graft Optimization of the interplay of cells and growth factors in the scaffolds might eventually allow generation of different axially vascularized grafts for application in reconstructive surgery This research project makes a promising approach for a vascularized graft for further exploration xii List of tables Table 3-1: The individual components of the composite scaffold and their... from an accident, bone transplants may provide an appropriate solution When there is a necessity of bone transplantation, two options are there: bone from the patient himself may be utilized, which is called autograft or donor bone tissue may be arranged in different forms Although autografts are still considered the best graft, there are limitations to its supply and associated morbidity to already... Numerous studies have been published in an attempt of 17 Chapter 2 Background to the research vascularization of tissues with pedicle graft with varying degrees of success [50-52] At the same time studies are being carried out to test the pre-vascularization of implant before it can be actually used as a transplant in the defect site [53-55] 2.3 Methods of generating vascularized grafts A free graft does not... especially for the central portions of the graft Keeping this view as the main target, a number of research approaches were directed to make a graft viable The cells and factors which are necessary for development of a normal vasculature during embryonic development are recapitulated during situations of neoangiogenesis in adults A number of factors involved in neoangiogenesis can be used such as Vascular... shorter healing time and a graft which is resistance to infection and extrusion This method is called pre-vascularization, which is a process of making neovascularization in tissues by implanting a vascular pedicle into them that can later be transferred to defect site as a graft by micro vascular anastomosis [49] This method has long been applied to skin and other soft tissues in plastic and reconstructive... survival of the graft is dependent upon an intact vascular supply, without which the graft functionality is hampered [13] A bone loss site may be treated by direct application of autografts or osteogenic growth factors for bone formation However, in many cases the local site is compromised by huge bone mass loss, scarring, or irradiation for cancer therapy, without having any healthy tissue If we apply . of a vascularized bone graft. 8 1.4. Organization of the Thesis 9 CHAPTER 2. Background to the Research 10 2.1. Angiogenesis and vascularization 10 2.2. Importance of vascularization in tissue- specific. eventually allow generation of different axially vascularized grafts for application in reconstructive surgery. This research project makes a promising approach for a vascularized graft for. bone are in advanced stage because of their potential need and relative ease of application. Chapter 1. Introduction 2 1.2. Tissue engineering The acute shortage of human tissues and organs