Feasibility of cartilage tissue engineering using cellulose membrane in vivo bioreactor

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.