Articular cartilage is a resilient load-bearing tissue that forms the articulating surface of diarthrodial joints and provides low friction, lubrication, and wearless characteristics necessary for repetitive gliding motion. However, because articular cartilage has neither vascular nor lymphatic channel supply and hence difficult access to stem cells, its self-healing potential is restricted. If left untreated, cartilage damage results in osteoarthritis (OA) and its endstage from, can only be managed by joint replacement surgery. We hereby provide means to repair cartilage using extracellular matrix (ECM) membrane on bone marrow stimulation (BMS) techic and development of a novel analytic method for detection of engineered cartilage.
Chapter Ⅰ: BMS has been regarded as a first line procedure for repair of articular cartilage. However, repaired cartilage from BMS is known to be unlike that of hyaline cartilage and its inner endurance is not guaranteed. The reason presumably came from a shortage of cartilage forming cells in blood clots derived by BMS. In order to increase repairable cellularity, the feasibility of autologous bone marrow-derived buffy coat transplantation in repair of large full-thickness cartilage defects was investigated in this study. Rabbits were divided into four groups: the defect remained untreated as a negative control; performance of BMS only (BMS group); BMS followed by supplementation of autologous bone marrow buffy coat (Buffy coat group); transplantation of autologous osteochondral transplantation (AOTS) as a positive control. Repair of cartilage defects in the Buffy coat group in a rabbit model was more effective than BMS alone and similar to AOTS. Gross findings, histological analysis, histological scoring, immunohistochemistry, and chemical assay demonstrated that supplementation of autologous bone marrow buffy coat after BMS arthroplasty effectively repaired cartilage defects in a rabbit model, and was more effective than BMS arthroplasty alone. Supplementation of autologous bone marrow-derived buffy coat in cases of BMS could be a useful clinical protocol for cartilage repair.
Chapter Ⅱ: The recombinant human transforming growth factor beta-3 (rhTGF-β3) is known as a key regulator of chondrogenesis of stem cells and cartilage formation. We have developed a novel drug delivery system that continuously releases rhTGF-β3 using an extracellular matrix (ECM) membrane fabricated from cultured porcine chondrocytes. We hypothesized that rhTGF-β3-loaded ECM membrane could significantly enhance cartilage regeneration, when it was applied on the osteochondral defect of rabbit articular cartilage treated with the bone marrow stimulation (BMS) technique. New Zealand white rabbits of 16 weeks old (an average weight of 3.0-3.5kg, n=78) were subjected to osteochondral defect on right knee joints and divided into four groups: The defect was (1) left untreated as a negative control (Control group), (2) covered with ECM membrane after BMS (Control-m), (3) treated with direct injection of rhTGF-β3, and covered with the ECM membrane after BMS (TGF-i) and (4) covered with rhTGF-β3-loaded ECM membrane after BMS (TGF-m). The results showed that repair of osteochondral defects was more efficient in the TGF-m group than the other groups including that with direct injection of rhTGF-β3 in gross findings, histological analysis, histological scoring and chemical assays for GAGs, collagen and DNA contents. We speculate that the sustained release of rhTGF-β3 using the ECM membrane after BMS could be a useful clinical protocol for cartilage repair.
Chapter Ⅲ: In cartilage tissue engineering, it is very important to evaluate specifically and non-destructively the quality of engineered tissues in terms of matrix content and their three dimensional (3D) distribution. The objective of this study was to evaluate the feasibility of using microfocal computed tomography (µCT) with Hexabrix, a contrast agent, in examining the quality of engineered cartilage. Chondrocytes were isolated from the knee articular cartilage of 2- to 3-week-old rabbits. Cells at passage 2 (5.0 x 105/ml) were loaded dynamically on polyglycolic acid (PGA) scaffolds (2 mm in diameter and 2 mm in thickness), and cultivated in a chondrogenic defined medium for 7, 14, 21and 28 days in vitro. The engineered cartilages were incubated with undiluted Hexabrix 320 for 20 min and analyzed by µCT scanning. The scanning data were visualized by 2D and 3D images and quantified by counting the number of voxels. The results were validated by histological images of engineered cartilages by Safranin-O staining and proteoglycan content by biochemical analysis. The optimal threshold value for quantification was determined by regression analysis with the total contents of sulfated glycosaminoglycans (GAGs) from 14 individual samples. The biological effect of uCT scanning on the engineered cartilage was also examined for cell viability and total contents of sulfated GAGs and DNA. The 2D µCT image of an engineered cartilage was matching well with the histological image of corresponding section by Safranin-O staining. Quantitative data obtained by 3D µCT images of 14 engineered cartilages showed a strong correlation with sulfated GAGs contents obtained by biochemical analysis (R2=0.883, P<0.001). Repeated exposure of engineered cartilages to Hexabrix 320 and µCT scanning did not affect significantly cell viability, total DNA content and total sulfated GAGs content. µCT imaging using Hexabrix 320 provides high spatial resolution and sensitivity to assess sulfated GAGs content and 3D distribution in engineering cartilage. Therefore it could be a valuable tool to evaluate the quality of engineered cartilage and greatly benefit its developmental process.