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Feasibility of cartilage tissue engineering using cellulose membrane in vivo bioreactor

Authors
LI, XUEGUANG
Department
대학원 의학과
Degree
Doctor (2020)
Table Of Contents
Background 1
1.1 Cartilage tissue engineering 1
1.2 In vivo bioreactor 1
1.3 Cellulose membrane 2
1.4 Aims of study 3
Chapter I 5
2.1 Introduction 5
2.2 Materials and Methods 7
2.2.1 Cell isolation and culture 7
2.2.2 Pellet culture 7
2.2.3 Preparation of cellulose membrane chamber 7
2.2.4 Ectopic chondrogenesis in the subcutaneous and IV bioreactor environment 8
2.2.5 Permeability assay 9
2.2.6 In vivo bioreactor fluid component analysis 9
2.2.7 Gross observation and size measurement of the pellets 10
2.2.8 Cell viability assay 10
2.2.9 Histology and Immunohistochemistry 10
2.2.10 Biochemical assay 11
2.2.11 Mechanical test 12
2.2.12 Statistical analysis 13
2.3 Results 14
2.3.1 Set up the in vivo bioreactor 14
2.3.2 IV bioreactor fluid characteristics and Confirm the fluid liquidity 16
2.3.3 Gross observation, size measurement and cell viability of the pellets 20
2.3.4 Histological observation of the pellets 22
2.3.5 Immunohistochemical observation of the pellets 23
2.3.6 Calcification of the pellets 25
2.3.7 Biochemical analysis for the content of DNA, GAGs and collagen 27
2.3.8 Compressive strength 29
2.4 Discussion 31
Chapter II 35
3.1 Introduction 35
3.2 Materials and Methods 38
3.2.1 Cell isolation and culture 38
3.2.2 Pellet culture 38
3.2.3 Preparation of cellulose membrane chamber 39
3.2.4 Ectopic chondrogenesis in the subcutaneous and IV bioreactor environment 39
3.2.5 Cartilage defect repair 40
3.2.6 Measurement of transmittance 40
3.2.7 Permeability assay 41
3.2.8 In vivo bioreactor fluid component analysis 41
3.2.9 Gross observation and size measurement of the pellets 42
3.2.10 Cell viability assay 42
3.2.11 Histology and Immunohistochemistry 42
3.2.12 Biochemical assay 47
3.2.13 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 47
3.2.14 Western Blot analysis 48
3.2.15 Statistical analysis 49
3.3 Results 51
3.3.1 Set up the in vivo bioreactor 51
3.3.2 IV bioreactor fluid characteristics and Confirm the fluid liquidity 54
3.3.3 Gross observation, size measurement and cell viability of the pellets 57
3.3.4 Histological observation of the pellets 60
3.3.5 Immunohistochemical observation of the pellets 61
3.3.6 Calcification of the pellets 62
3.3.7 Biochemical analysis for the content of DNA, GAGs and collagen 67
3.3.8 Molecular analysis of immunogenicity 69
3.3.9 Macroscopic and histological observation of the ectopic engineered cartilage for cartilage repair in vivo 73
3.3.10 Immunohistochemical observation of the ectopic engineered cartilage for cartilage repair in vivo 75
3.3.11 Histological observation of the synovium 76
3.4 Discussion 80
Conclusion 84
References 85
Abstract
Articular cartilage is the connective tissue of diarthrodial joints that function is to provide a smooth, lubricated surface for articulation and to distributes loads. Articular cartilage degenerates due to multiple factors, such as trauma, bone malalignment, overweight, osteoarthritis and inflammatory arthritis. Articular cartilage is devoid blood vessels, lymphatics and nerves, and once damaged, it is difficult to heal itself. Current treatments include marrow tapping techniques, osteochondral auto/allo-grafting and cell-based techniques. But the result is generally a fibrocartilage, and the treatment is not satisfactory. Although cartilage tissue engineering as a promising new treatment method is being widely studied, there are still many hurdles to be solved. For example, degradation of the cartilage matrix and immune problems. This study aims to devise a new in vivo bioreactor for better culture of cartilage tissue. First, we evaluated whether the diffusion chamber made of cellulose membrane and silicone tube under the skin of nude mice was similar to the joint cavity environment, and whether it supported chondrogenesis. Secondly, we cultured cartilage tissue from xenogeneic cells in rabbit subcutaneous diffusion chamber and implanted it into the cartilage defect. Focusing on chondrogenesis, immunogenicity and cartilage healing, thereby establishing a in vivo bioreactor.

CHAPTER I : Nude mouse subcutaneous model, known as heterotopic chondrogenesis, is popularly used for cartilage tissue engineering. Yet this model frequently has not been interpreted real bioreactor for chondrogenesis by different environment to joint, such as adhesion of implanted tissue to surrounding tissue allowing vascular invasion, with resulting in osteogenic differentiation. The purpose of this study was to create an insulated subcutaneous cavity to reduce such limitations. Study design included two groups; the free cavity (subcutaneous) and insulated cavity (in vivo bioreactor) on subcutaneous transplantation. Rabbit chondrocytes were pellet-cultured at an initial cell count of 3 × 105 for 2 weeks. For the subcutaneous group, the cell pellets were surgically inserted subcutaneously. For the in vivo (IV) bioreactor, pellets were inserted in the Cellulose membrane chamber was composed of a 8 mm × 8 mm silicone tube and cellulose membrane. First evaluated the IV bioreactor fluid appearance, component and liquidity, and then evaluate chondrogenesis of the pellets using gross observation, cell viability, histology, biochemical analysis and mechanical test. The fluid color and transparency of IV bioreactor were similar to synovial fluid (SF) and the component was also close to SF compared to the serum. The IV bioreactor group showed more hyaline like tissue with less osteogenic differentiation. The IV bioreactor group offers a novel way to achieve heterotopic chondrogenesis, similar to the synovial joint environment.
CHAPTER II : To regenerate tissue engineered cartilage as a source for the restoration of cartilage defects, we used a human fetal cartilage progenitor cell (hFCPC) pellet for improve the chondrogenesis and modulation the immune response with a in vivo (IV) bioreactor system, that was buried subcutaneously in the host and then implanted into a cartilage defect. In vivo bioreactor (IVB) was composed of silicone tube and cellulose nanoporesize membrane. FCPC pellets were first cultured in vitro for 3 days, and then cultured in vitro, subcutaneous and IV bioreactor for 3 weeks. First evaluated the IV bioreactor fluid appearance, component and liquidity, and then evaluate chondrogenesis and immunogenicity of the pellets using gross observation, cell viability, histology, biochemical analysis, RT-PCR, and Western Blot, finally evaluates the cartilage repair and synovial inflammation using histology. The fluid color and transparency of IV bioreactor were similar to synovial fluid (SF) and the component was also close to SF compared to the serum. IV bioreactor system not only promotes the synthesis of cartilage matrix and maintains cartilage phenotype, but also delays the occurrence of calcification compared with subcutaneous. A IV bioreactor, which has been predominantly adopted to study cell differentiation, was effective in preventing host immune rejection.
Keywords
Cartilage tissue engineeringIn vivo bioreactorCellulose membraneFetal cartilage progenitor cellsPellet culture
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LI, XUEGUANG
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