| Summary: | 碩士 === 臺灣大學 === 醫學工程學研究所 === 95 === The skeleton is composed of cartilage and bone which provide the functions such as support, protection, and movement in daily life. Articular cartilage tissue is composed of chondrocytes and extracellular matrix, where the chondrocytes only make up less than 10% of the total volume of cartilage. In healthy cartilage, the matrix is composed of collagens, especially type II collagen, proteoglycans, and noncollagenous proteins, and is filled with water because of the hydrophilic property of the framework.
Cartilage possesses limited ability to achieve spontaneous repair due to its dense extracellular matrix and lacking of blood vessels, lymphatics and innervation. When lesions occur in articular cartilage, there is no bleeding and thus no mechanism for the replacement of lost or damaged tissue. Neighboring chondrocytes may respond by local proliferation; however, because they are sequestered in the dense matrix, they do not migrate into the damaged region to fill the void. If injury extends through the chondral layer to the subchondral bone and underlying vasculature, a repair response can hence occur, but the newly formed cartilage will be gradually populated with type I collagen and degenerate to a fibrocartilaginous scar tissue after 6–8 months.
Circumstances that impair chondrocytes function hence disrupt the balance of synthesis and catabolism and lead to the development of osteoarthritis. In the progression, osteoarthritis eventually impairs the function of a whole joint, including the cartilage, the subchondral bone, the synovium and the periarticular connective and muscular tissues. The current treatment options are fairly limited which include only symptomatic treatment of limited efficacy with analgesics, non-steroidal anti-inflammatory agents or intra-articular administration of steroids or hyaluronic acid, and if the joint destruction can not be halted, the ultimate measure is joint replacement.
Tissue engineering has been a feasible way to regenerate cartilage in vitro, which combines cells, scaffolds, and signals to mimic the original environment of tissues in vitro. In addition, there are some Chinese herbal medicines have been used to treat the degeneration of the cartilage for thousands of years, but the precise mechanism of their potent chondrogenesis effects have not been fully elucidated. Therefore, in this study, we investigated the involved mechanism of two single chemical compounds: aucubin and betulin which were separately extracted from Plantago asiatic and Ampelopsis brevipedunculata (Maxim.) Trautv. by focusing on their proliferation and antioxidant activity.
The experiment was divided into two parts. One was two-dimensional chondrocytes culture; the other was three-dimensional scaffold culture. After treating two-dimensional chondrocytes or three-dimensional seeded chondrocytes with optimal concentration of single chemical compounds, the proliferation and matrix productivity of them were evaluated by real-time reverse-transcriptase polymerase chain reaction, ELISA assays, and immuno-histochemical staining. The important role in scavenging free radicals by those extracted chemical compounds was detected by the chemiluminescence method.
The results showed that, in two-dimensional chondrocytes culture, both aucubin and betulin could effectively promote the mRNA expression of ECM and inhibit the mRNA expression related with ECM degradation at appropriate concentration, and the ability of O2•‾ scavenging made aucubin and betulin as protectants of chondrocytes, which would stimulate chondrocyte proliferation and maintain the basic chondrocyte activities. In three-dimensional scaffold culture environment, betulin can significantly stimulate chondrocyte proliferation and maintain the basic chondrocyte activities until four-week cultivation, which suggested that in the future application the in vitro three-dimensional cultivation of chondrocyte-scaffold hybrids should be maintained more than four weeks, and of course the addition of 0.32μg/ml of betulin into the cultured environment possessed positive effects toward chondrocytes and the whole cartilage-mimic tissue.
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