Co culture based osteochondral tissue engineering

CO-CULTURE-BASED OSTEOCHONDRAL TISSUE ENGINEERING CHEN KELEI (B. Eng. Sichuan University of China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 Acknowledgments I would like to express my deepest gratitude to my supervisors, Associate Professor Toh Siew Lok and Professor James Goh, who have led and inspired me towards research in the exciting and multidisciplinary field of tissue engineering. I am deeply appreciative of Dr. Sambit Sahoo, Dr. Teh Kok Hiong Thomas and Dr Shi Pujiang, who as my post-docs and also labmates, assisted and made my initiation into research smooth and easy. I wish to thank all my colleagues at the Tissue Repair Lab and NUSTEP Lab. Special thanks have to be given to our Laboratory Technologists, Ms Lee Yee Wei and Ms Serene Goh, who have conscientiously ensured that the lab is always in order and have supported efficiently in the logistics of this study. I would like to thank my fellow labmates, Eugene, Kian Siang, Peng Fei, Pamela, Yuwei, Sujata and Puay Yong for their support through both the exhilarating and challenging times of my research pursuit. Acknowledgement is also due to undergraduate student, Weixiong, who have assisted in parts of this research during his final year project. I would like to thank Ms. Kay En from the Electron Microscopy Unit for her kind help in SEM characterizations, Hock Wei for his help in the biomechanical characterizations, Dr. Li Ang from the Nano Biomechanics lab for his help in AFM characterizations, Dr. Yeow Chen-Hua for his help in micro-CT analysis, Yangxiao for her help in PQ- CT test. The greatest thanks are due to my parents who have supported and nurtured me through my life. Together with all my family members and my girlfriend Jiajing, they have trusted and supported my decisions. Lastly, I would like to thank all my friends in Singapore, who truly made this place my home! Table of Content Table of Content i Summary .vii List of figures x List of Tables . xvi List of Abbreviations .xvii Chapter Introduction 1.1. Background and Significance . 1.2. Objectives and research program 1.2.1. Stage 1: 1.2.2 Stage 2: Development of a 3D-3D co-culture model for osteochondral interface and multilayered constructs generation in vitro 1.2.3. Stage 3: Design and fabricate an appropriate co-culture system and use it in osteochondral tissue engineering . 1.2.4. Stage 4: Design and fabricate a co-culture bioreactor and the effectiveness of hypertrophic chondrogenic stimulation medium . 10 1.3. Scope of Thesis . 10 i Chapter Literature Review 12 2.1. Introduction . 12 2.2. Osteochondral tissue Anatomy and Bioproperties 13 2.3. Osteochondral Defects 17 2.4. Conventional Treatment 18 2.5. Tissue Engineered osteochondral Grafts . 20 2.5.1 Cells 23 2.5.2 Scaffolds . 25 2.5.3 Biochemical cues 30 2.5.4 Cell-to-cell interaction and co-culture approach 31 2.6. Summary . 33 Chapter Stage1. Silk/RADA scaffold fabrication and 2D-3D co-culture model for osteochondral interface generation 35 3.1. Introduction . 35 3.2. Materials and Methods 35 3.2.1. Scaffold preparation . 35 3.2.2. silk/RADA scaffold bio-analysis . 37 3.2.3. Cell culture and in vitro 2D-3D interface co-culture model design . 38 3.2.4. Cell proliferation 41 3.2.5. Total RNA extraction, cDNA synthesis and real-time PCR analysis . 41 3.2.6. Biochemical test . 43 3.2.7. Morphological characterization . 44 3.2.8. Histology and immunohistochemistry . 45 3.2.9. Statistical analysis 45 3.3. Result 46 3.3.1. Scaffold Characterization . 46 ii 3.3.2. Silk/RADA bio-analysis 47 3.3.2. BMSC proliferation on scaffolds and cell morphology . 48 3.3.3. Effects of co-culture on collagen and GAG production 50 3.3.4. q-PCR analysis for gene expression 51 3.3.5. 2D-3D interface co-culture effect on GAG, mineralization and collagen deposition in ECM . 54 3.3.6. Calcium content . 56 3.4. Discussion . 57 3.4.1 Fabrication silk/RADA scaffold for osteochondral tissue engineering 57 3.4.2 2D-3D co-culture model for osteochondral interface regeneration 59 3.5. Conclusion 63 Chapter Stage 2: Development of a 3D-3D co-culture model for osteochondral interface and multilayered constructs generation in vitro 64 4.1. Introduction . 64 4.2. Materials and Methods 65 4.2.1. Scaffold preparation . 65 4.2.2. Cell culture and in vitro 3D co-culture model . 65 4.2.3. Total RNA extraction, cDNA synthesis and real-time PCR analysis . 66 4.2.4. Total GAG assays 68 4.2.5. Morphological characterization . 68 4.2.6. Histology 69 4.2.7. Statistical analysis 69 4.3. Result 70 4.3.1. Osteochondral Co-culture Construct . 70 4.3.2. Total GAG deposition 71 4.3.3. Effect of Osteogeneic-chondrogenic Co-Cultures on BMSCs different iii iation 71 4.3.4. Osteochondral multilayered constructs with interface generation . 73 4.3.5. Histology and SEM 75 4.4 Discussion 77 4.4.1. Osteogenic/chondrogenic BMSCs co-culture system introduced the hypertrophic chondgenic differentiation 78 4.4.2. Generation of multilayered osteochondral construct with osteochondral interface. . 79 4. 5. Conclusion . 81 Chapter Stage 3. Design and fabricate an appropriate co-culture system and use it in osteochondral tissue engineering 82 5.1. Introduction . 82 5.2. Materials and Methods 83 5.2.1. Scaffold preparation . 83 5.2.2. Two-chambered co-culture well fabrication and co-culture system design . 83 5.2.3. Cell culture and co-culture in two chambered wells 84 5.2.4. Scaffold diffusion analysis . 85 5.2.5. Total RNA extraction, cDNA synthesis, quantitative real-time PCR analysis and DNA electrophoresis . 86 5.2.6. Histology and Immunohistochemistry . 87 5.2.7. Morphological characterization and Mineralization analysis 88 5.2.8. Statistical analysis 89 5.3 Results 89 5.3.1. Scaffold diffusion analysis . 89 5.3.2. q-RT-PCR analysis for gene expression . 90 5.3.3. Cell morphology 93 iv 5.3.4. Mineralization, GAG and Collagen deposition in multilayered ECM by using two chambered co-culture well . 94 5.4 Discussion 97 5.5 Conclusion . 101 Chapter Stage 4: Design and fabricate a three-chambered co-culture bioreactor and the effectiveness of hypertrophic chondrogenic stimulation medium . 103 6.1. Introduction . 103 6.2. Materials and methods 104 6.2.1. Scaffold preparation . 104 6.2.2. Effectiveness of hypertrophic chondrogenic medium (HCM) for generation of the osteochondral interface 105 6.2.3. Fabrication of three-chambered Bioreactor . 108 6.2.4. Scaffold diffusion analysis . 111 6.2.5. Cell seeding and culture in three-chambered bioreactor . 111 6.2.6. Total RNA extraction, cDNA synthesis, quantitative real-time PCR analysis and DNA electrophoresis . 112 6.2.7. Histology for bioreactor cultured samples . 113 6.2.8. SEM analysis . 113 6.2.9. Mechanical analysis . 114 6.2.10 Statistical analysis . 114 6.3. Result 115 6.3.1. Effectiveness of hypertrophic chondrogenic medium (HCM) . 115 6.3.2. Scaffold diffusion analysis by using three-chambered wells . 119 6.3.3. q-RT-PCR analysis for gene expression . 120 6.3.4. Cell morphology 122 6.3.5. Mineralization, GAG deposition in multilayered ECM by using three-chambered bioreactor . 123 v 6.3.6. Mechanical properties 127 6.4. Discussion . 129 6.4.1. Effectiveness of HCM for osteochondral interface generation by BMSCs . 129 6.4.2. Effectiveness of three-chambered bioreactor for osteochondral interface and multilayered osteochondral tissue formation . 132 6.5. Conclusion 135 Chapter Conclusions and Recommendations . 136 7.1 Conclusions 136 7.2 Recommendations and future work . 139 Reference 142 Appendix . 163 A. List of Publication . 163 B-1. Design of two-chambered co-culture well . 165 B-2. Design of three-chambered co-culture well . 168 vi Summary The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. An optimal method that can regenerate multilayered tissue structure is needed. In this study, several co-culture methods were designed and investigated their effectiveness for generating the osteochondral interface and multilayered structure in vitro. Rabbit bone marrow stromal cells (BMSCs) and silk/RADA (Ac-RADARADARADARADA-CONH2) peptide scaffold were used in this study. The study was grouped into four stages: (i) design and development of the scaffold and osteochondral interface formation by 2D-3D co-culture system, (ii) development 3D-3D co-culture system, (iii) the development of static co-culture wells, and (iv) three-chamber bioreactor. The first stage involved the design and fabrication of the silk/RADA scaffold. 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Toh WS, Lee EH, Guo XM, Chan JK, Yeow CH, Choo AB, et al. Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-derived chondrogenic cells. Biomaterials 2010 Sep;31(27):6968-6980. 162 Appendix A. List of Publication International Journal Publications 1. Chen KL, Sambit S, He PF, Ng KS,Toh SL, Goh JCH. A Hybrid Silk/RADA-based Fibrous Scaffold with Triple Hierarchy for Ligament Regeneration. Tissue engineering, part A. 2012 Jul. 2. Chen KL, Teh TK, Sujata R, Toh SL, Goh JCH. Osteochondral Interface Generation by Rabbit Bone Marrow Stromal Cells and Osteoblasts Co-culture. Tissue engineering, part A. 2012 Sep. 3. Chen KL, Shi PJ, Teh TK , Toh SL, Goh JCH. In vitro generation of a multilayered osteochondral construct with the osteochondral interface using rabbit bone marrow stromal cells and a silk/peptide based scaffold. Journal of Tissue Engineering and Regenerative Medicine. 2013 Feb. 4. Chen KL, Ng KS, Sujata R, Goh JCH, Toh SL. In vitro generation of whole osteochondral constructs using rabbit bone marrow stromal cells by employing a two-chambered co-culture well design. Journal of Tissue Engineering and Regenerative Medicine. 203 March. 5. Shi PJ, Chen KL, Goh JCH. Efficacy of BMP-2 Delivery from Natural Protein Based Polymeric Particles. Advanced Healthcare Materials. 2013 Jan. 6. He P, Sahoo S, Ng KS, Chen KL, Toh SL, Goh JC. Enhanced osteoinductivity and osteoconductivity through hydroxyapatite coating of silk-based 163 tissue-engineered ligament scaffold. J Biomed Mater Res A. 2013 Feb. Conference 1. Chen KL, He PF, Ng KS, Goh JCH, Toh SL. Combined Scaffold formed from Silk and Self-assembly Peptide(RADA-I) and the Use in Ligament Regeneration. 6th World congress of Biomechanics. Aug 1-6, 2010, Singapore. 2. Chen KL, Toh SL, Goh JCH. Co-culture-based Osteochondral Interface Regeneration by Using Bone Mesenchymal Stem Cells. TERMIS-NA 2010 Conference & Exposition. December 5-8, 2010, Orlando, US. 3. Chen KL, Toh SL , Goh JCH. In Vitro Osteochondral Multilayer Regeneration from Bone Marrow Stromal Cells with Silk/Peptide Matrix. TERMIS-AP 2011. August 3-5, Singapore. 4. Chen KL, Toh SL , Goh JCH. Co-culture-based Osteochondral Interface Regeneration by Bone Marrow Stromal Stem Cells. TERMIS-AP 2011. August 3-5, Singapore. 5. Chen KL, Toh SL , Goh JCH. The generation of osteochondral multilayered constructs by bone marrow stromal cells in vitro. 243rd ACS National Meeting. March 25 – 29, 2012, San Diego, US. 6. Chen KL, Toh SL , Goh JCH. In vitro generation of osteochondral constructs from bone marrow stromal cells on silk/peptide scaffold in co-culture well. 9th World Biomaterials Congress. Jule 1-5, Chengdu, China. 164 B-1. Design of two-chambered co-culture well Co-culture plate with dimensions 150mm x 100mm x 20mm (depth) were designed (Figure B-1 – B-2) and fabricated from transparent Polycarbonate blocks. Each plate contains wells. At the same time, Polycarbonate septum of dimensions 14mm x 10mm x 2mm with a 2mm radius hole in the middle (Figure B-4) and Polycarbonate lids of dimensions 150mm x 100mm x 5mm (Figure B-3) were made. Septum was cemented to the groove in the well of the plate using silicone. Fig. B-1 Design drawing of co-culture plate (without septum) 165 Fig. B-2 Section view of co-culture plate (without septum) Fig. B-3 Design drawing of septum 166 Fig. B-4 Design drawing of lid 167 B-2. Design of three-chambered co-culture well This three-chambered well was assembled by parts: main part with dimensions 28mm x 60mm x 20mm were designed (Figure B-5) and fabricated from transparent Polycarbonate blocks. Each main part was covered by two pieces of side covers: 5mm x 60mm x 2mm with a groove in middle for O’ring (Figure B-6) and Polycarbonate cover of dimensions 64mm x 4mm x 2mm (Figure B-7) were made. Fig. B-5 Design of main part. 168 Fig. B-6 Design of side cover. 169 Fig. B-7 Design of top cover. 170 [...]... proliferated during the 4 week culture period (B) DNA content analysis by PicoGreen test (C) GAG production decreased by co- culture at Week 2 and 4 (C-1) Alcian blue staining for control group at Week 4, (C-2) Alcian blue staining for co- culture group at week 4 Control group had significantly more GAG staining than the co- culture group (D) Collagen production increased by co- culture at week 4 (*, p< 0.05)... chondrogenic and osteogenic differentiated progenies, a whole osteochondral construct with osteochondral interface could be generated in vitro 1.2.3 Stage 3: Design and fabrication an appropriate co- culture system and use it in osteochondral tissue engineering To generate a whole osteochondral construct by using a static two-chambered co- culture well that could simultaneously provide osteogenic and chondrogenic... in vivo [49, 51, 52] For the complex osteochondral tissue engineering, the regeneration of the osteochondral interface is considered a challenge in both research and clinic contexts Co- culture method provides a possible solution to induce the formation of interface [29, 53] Moreover, to achieve the requirement for multilayered osteochondral tissue generation, special co- culture system should be designed,... samples (A, C) Control group, (B, D) Co- culture group Scale bar=200 µ 55 m Fig 3-11 Immuno-staining for Type II Collagen and Type X Collagen (A, C, E) Control group, (B, D, F, G) Co- culture group (A, B) H&E staining (C, D) Type II Collagen staining (E, F, G) Type X Collagen staining, (G) Vertical section of the co- culture group scaffold Scale bar= 500 µm 56 Fig 3-12 Calcium content analysis... after 2 weeks of co- culture 122 Fig 6-9 H&E histology analysis of three regions of osteochondral constructs cultured after 2 weeks showing that different cell morphologies in three regions on co- cultured osteochondral constructs could also be observed (Scale bar=100 µm); (A) Osteogenic region; (B) Middle region; (C) Chondrogenic region; (D) Osteogenic control; (E) Chondrogenic control ... region; (D) Osteogenic control; (E) Chondrogenic control 126 xiv Fig 6-13 Mechanical testing of BMSCs-seeded scaffolds after 2 weeks’ of co- culture (A, B) Instron 3345 Tester system, sample was indicated by arrow (C) Max compressive loads were compared among three regions of co- cultured samples and cell-free scaffold control 128 Fig B-1 Design drawing of co- culture plate (without... Osteochondral constructs are defined as tissues that are composed primarily of bone and cartilage, specially the articular cartilage that is found in all joints in our bodies The osteochondral tissue consists of multiple tissue layers with different structures, such as cartilage layer, osteochondral interface, subchondral bone layer and bone [1, 2] Osteochondral tissues provide important connective and... bottom harf (conatcted with osteoblasts) for co- culture group (*,p< 0.05) 57 Fig 4-1 Osteochondral co- culture system: rBMSCs are mixed with RADA peptide solution then seeded on each silk scaffold, and are cultured for two weeks in chondrogenic medium and osteogenic medium for two weeks Then these culture scaffolds in different medium were combined by using RADA self-assembly peptide and co- cultured... Osteogenic control; (B, D) Chrogenic control m); 125 Fig 6-11-3 SEM images for mineralized particulars and collagen fibers in osteogenic region 126 Fig 6-12 Histology analysis of the osteochondral constructs co- cultured for 2 weeks showing that Alcian blue positive staining could be observed in Middle and Chondrogenic region in co- culture samples and chondrogenic control (Scale... chondrogenic control group than co- culture group and osteogenic control xi group in week 2 (B) The GAG amount was decreased by co- culture in chondrogenic part compared with chondrogenic control group (*p< 0.05) 71 Fig.4-4 Gene expression analysis study one: normalized expression levels of chondrogenic, osteogenic and hypertrophy-related genes in three groups after 7 and 14 days of co- culture (* p . co- cultured for another 2 weeks by using cocktail culture medium. 66 Fig. 4-2 Macroscopic image of the construct and histology analysis of the two layered osteochondral constructs co- cultured. chondrogenic region from co- culture group compared with control group after 2 weeks of co- culture. 122 Fig. 6-9 H&E histology analysis of three regions of osteochondral constructs cultured after. plates, and subsequently placed in contact and co- cultured. By co- culture, specific regulatory stimulations from osteoblasts in the 2D-3D interface co- culture system could induce the formation of