| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 106 === Background
Cartilage injuries are common problem in orthopedic clinical practice with an estimated 60% of prevalence as revealed by routine knee arthroscopic procedures. Since cartilage is an avascular tissue with limited capacity of self-repair, these chondral lesions are likely to continuously progress that results in osteoarthritis. To prevent further degeneration of articular surface, many treatments have been developed to promote cartilage healing by applying cells or tissues, such as bone marrow stimulation therapy, osteochondral autograft transplantation (OAT), and autologous chondrocyte implantation (ACI).
Although bone marrow stimulation through microfractures is a simple approach, the outcome of repair is often unpredictable. Moreover, the tissue generated for repair is fibrocartilage. OAT is a single-stage approach, in which defects are filled with autologous osteochondral plugs. The disadvantages of this technique include donor-site morbidity, technical difficulties in matching the lesion contour, limitation by the size of defect, and the risk of cartilage and bone collapse. ACI can yield hyaline-like tissues more similar to native cartilage in histological, mechanical and clinical aspects than that produced by microfractures. However, it requires multiple surgical procedures and a costly process of in vitro chondrocyte expansion, which may cause chondrocytes dedifferentiation. To avoid complicated procedures required in in vitro chondrocytes expansion for cartilage repair, development of a culture-free, one-stage approach combining Platelet-rich fibrin (PRF) and autologous cartilage graft may be the solution. Secondly, to solve the cell source problem in cartilage tissue repair, it is important to explore the feasibility of using heterotopic chondrocyte embedded in type II collagen (Col II) matrix in articular (AR) cartilage defect regeneration.
Methods: The chemotactic effects of PRF on chondrocytes harvested from the primary culture of rabbit cartilage were evaluated both in vitro and ex vivo. The rabbit chondrocytes were cultured with different concentrations of PRF medium, and were evaluated for their ability of cell proliferation, chondrogenic gene expression, cell viability, and extracellular matrix syntheses. In second part of study, auricular chondrocytes (AU) were cultured with Col II supplement and analyzed for their articular chondrogenesis. The regulatory effects of Col II on differentiation of AU chondrocytes as having the characteristics of articular cartilage will be carefully determined. Methods of assessment includes glycosaminoglycan deposition and the expression of articular cartilage-specific mRNA (such as type II collagen mRNA; aggrecan, Lubricin, and SOX 9). To further enhance the possibility of its future clinical applications, the auricular chondrocytes will be embedded in Col II matrix to form a three-dimensional ‘tissue-engineered cartilage’. Histological analysis and physical characteristics analysis (such as elastic modulus) of this tissue will also be checked. For in vivo study, the chondral defects were created on the established animal models of rabbit and porcine. The results were evaluated by gross anatomy, histology, and objective scores to validate the treatment results.
Results: PRF improved the chemotaxis, proliferation, and viability of cultured chondrocytes. Gene expression of chondrogenic markers including type II collagen and aggrecan revealed that PRF induced the chondrogenic differentiation of cultured chondrocytes. The efficacy of PRF on cell viability was comparable with fetal bovine serum. In the animal disease models, the morphological, histological, and objectively quantitative evaluation demonstrated that PRF combined with cartilage granules is feasible in facilitating chondral repair. In AU chondrocytes conversion study, the AU chondrocytes fabricated collagen constructs were implanted into rabbit osteochondral defect and their repair was evaluated by histological analysis.
Conclusions: PRF enhances migration, proliferation, viability, and differentiation of chondrocytes, thus showing appealing capacity for cartilage repair. The data altogether provide evidences to confirm the feasibility of the one-stage, culture-free method of combining PRF and autologous cartilage for repairing articular chondral defects. On the other hand, chondrocytes harvested from AU cartilage can be converted to become similar to cells of AR cartilage exclusively by extracellular matrices. The results confirmed the feasibility of engineering AU chondrocytes to mitigate the drawbacks of applying heterotopic chondrocytes for cartilage repair. In this study, we propose a feasible and efficient methodology for the application of autologous cartilage transplantation using heterotopic chondrocytes, which is expected to expedite the potential clinical application of cartilage repair.
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