| Tendon and ligament are tough connective tissue. It plays an important regulating role in the sports by the changes of its structure, mechanical properties and form to respond to external mechanical stimulation. However, some excessive, repetitive mechanical strains will result in tendon/ligament wound or rupture, which is harmful to human health. The management of damaged tendon/ligament is still one of the most difficult and challenging clinical issues in orthopaedics, and many questions remain unsolved such as enhancing the rate, quality, and completeness of healing.Marrow mesenchymal stem cells (MSCs) are the special cells that have proliferation and multi-differentiation ability. By the special conditions, MSCs can be induced to proliferate and differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells and so on. This multiple plasticity of MSCs enhances the chance for healing and even tissue-engineered tendon/ligament. However, insofar little is known about the extracellular matrix (ECM) regulate proliferation and selective differentiation to tendon/ligament fibroblasts by MSCs. Thus, this paper focuses on the regulation of selective differentiation of rat bone marrow mesenchymal stem cells (rMSCs) into tendon/ ligament fibroblasts by collagen I and elastin, which are the main extracellular martix components of tendon/ligament cells. The main research works and results are as follows:1. Isolation and culture of rMSCs in vitroThe rMSCs were isolated by centrifugation with 1.073g/ml percoll solution as the result indicated: The isolated cells are homogeneous histoleucocyte. Cultured in DMEM with 15% FBS for 24 hours, cells adhered, divided and grew into colony. After about 2 weeks they achieved confluence, and show fibroblast-like morphology. The nonadherent cells were removed by changing the medium. The passage cells, whose caryoplasm were a little big, grew and adhered so fast that after about 1week they achieved confluence and as the fusiform. Gimsa stained nuclear were round or ellipse and several nucleoli were thus clear meanwhile cytoplasm was lilaceous.2. Identification and cell cycle analysis of rMSCsUsing the immunostaining to detect the cell surface antigens, and using flow Cytometry to analyze the cell cycle, we detected that the surface antigen CD34 negative while CD44 and CD29 were positive, and G0/G1 phase cells were (89.74±3.87)%, S phase were (2.49±2.2)% and G2/M phase were (7.7±3.7)%.3. Differentiation of rMSCs induced by collagen ITo investigate the ability of different concentration of Collagen I to induce differentiation of MSCs, RT-PCR quantitate was applied to analyze the expression of Collagen I, Collagen III and Scleraxis, which are the special markers of tendon cells, in 7day 14day and 21day during the process. It demonstrated that:After 7 days'induction of Collagen I with different concentration , Collagen I and Collagen III expression were detected while Scleraxis expression not obvious. After 14 days, the expression of the three makers all increased rapidly and reached the maximum, especially at the concentration of 100μg/ml. After 21 days, the expressions decreased. It was suggestd that 100g/ml Collagen I with 14days'induction was the best condition to induce MSCs differentiation.4. Differentiation of rMSCs induced by elastinWith the same method to investigate the ability of Elastin induce differentiation of rMSCs with different concentration.It demonstrated that: it is the same as Collagen I induce condition, after 7days Collagen I, Collagen III expression were detected, but different from the Collagen I inducetion effect, the Scleraxis expression were detected after 7 days'induction . 14 days later, the expression of the three makers all increased rapidly and reached the maximum, especially at the concentration of 20μg/ml and after 21 days, the expressions decreased. It was suggestd that 20g/ml Elastin with 14days'induction was the best condition to induce MSCs differentiation.The result of our research indicated that Collagen I and Elastin take an important effect in MSCs differentiation and under suitable conditions MSCs could differentiated into tendon fibroblasts. These researches were not only significant to know self-renewal of MSCs and the rule of its differentiation by molecule regulation but also take the information to clinic therapy of tendon or ligament injured and the tissue engineering of the tendon and ligament. |
