Effects Of Tenocytes Microenvironment On Proliferation And Tendon/ligament-associated Gene Expression In Bone Marrow-derived Mesenchymal Stem Cells

Tendon/ligament is tenacious connective tissue which responds to external mechanical stimulation by changing its structure, mechanical properties and form, and plays an important role in the sports. However, inappropriate physical training or excessive repetitive stretching often leads to tendon/ligament overuse injuries. The management of damaged tendons/ligaments is still one of the most challenging problems in orthopedics. There are many problems remain unsolved, such as reducing curing time, enhancing healing quality and so on.Mesenchymal stem cells (MSCs) are an attractive cell source for a wide variety of tissue engineering strategies, because they are a pluripotent population of cells capable of differentiating along multiple mesenchymal cell lineages, including osteoblasts, chondrocytes, and adipocytes, and do not pose the ethical concerns inherent in the use of embryonic or fetal tissue. This plasticity offers a promising strategy for improved wound healing in injured tendons/ligaments, and even tissue-engineered tendons/ligaments.The ability of self-renewal and differentiation of stem cells mainly depends on their located microenvironment. It has been reported that various physical and chemical factors or special microenvironments play an important role in regulating proliferation and differentiation of MSCs. But little is known about proliferation and differentiation behaviors of MSCs under tenocytes microenvironment. Thus, using transwell system in this paper, we construct a co-culture model to simulate tenocytes microenvironment, and study the effect of this microenvironment on proliferation and tendon/ligament-associated gene expression of MSCs by biochemical and molecular biology methods.The main research works and results are as follows:1. Isolation and cultivation of rat MSCs and tenocyte in vitroMSCs were isolated by centrifugation with 1.073 g/mL percoll solution. Results showed that the isolated cells were homogeneous histoleucocyte and adhered, divided and grew into colony after cultivation in DMEM with 10% FBS for 24 hours. About 2 weeks later, they achieved confluence, and showed fibroblast-like morphology. Nonadhesive cells were removed by changing the medium. The cells were released with 0.25% trypsin containing 0.02% EDTA for subculture. Tenocytes were isolated by tissue fragment adherent method. Rat forefoot tendon tissue was isolated under sterile environment, cut into pieces and cultured in DMEM. Primary tenocytes could be found around the tissue and presented a typical long fusiform and homogeneous morphology. Cell surface is very velvet and the nucleolus is spindly. About 2 weeks later, tenocytes arounding tissue fragment grew into a gyrate colony.2. Characterization of rat MSCsNucleoli of cultured cells, stained by Gimsa, were round or ellipse. Several dark purple nucleoli were thus clear meanwhile cytoplasm was lilaceous. Immunostaining showed that the surface antigen CD34 was negative while CD44 and CD29 were positive. Also, we detected cell cycle distribution by flow cytometry, result showed that G0/G1 phase cells were (89.74±3.87) %, S phase were (2.49±2.20) % and G2/M phase were (7.70±3.70) %. It suggests that most of cells were in quiescent stage and this is consistent with the characteristic of stem cells.3. Effects of tenocytes microenvironment on proliferation of MSCsIn this study, we measured growth curve of MSCs, and then investigated proliferation of MSCs after co-culture with tenocytes by MTT assay and RT-PCR method. Our results indicated that growth latency of MSCs was at day1 and day2, the growth period was from 3 to 7 days. After 7 days, the cells turned into the period of growth platform. At the first 2 days of co-culture, there was no significant difference in growth between control and co-cultue group. Since 3rd day, MSCs in co-culture group grew faster than control and a significant difference was found. From cell morphology we can see that co-culture group has more cell number than control. RT-PCR products analysis demonstrate that after co-culture with tenocyte for 4 and 7 days respectively, expression of c-fos gene was up-regulated evidently and there was a statistically significant difference compared with control. These results show that tenocyte microenvironment can promote the proliferation of MSCs.4. Effects of tenocyte microenvironment on differentiation of MSCsThis paper characters on the effect of tenocyte microenvironment on differentiation of MSCs. The expression of tendon/ligament-associated gene collagen typeⅠandⅢ( Col I and Col III), Tenascin(TNC)and Scleraxis(SCX) of MSCs were analyzed by RT-PCR after co-culture with tenocyte for 7, 14 and 21days. The results showed that: after co-culture with tenocyte for 7 days, the relative expression of ColⅠ,ColⅢ,TNC and SCX was up-regulated compared with control,but no significant difference was observed. However, expression of those genes was up-regulated continually after co-culture with tenocyte for 14 and 21 days and a statistically significant difference was observed compared with control. Especially at 21 day, the relative expression of these 4 associated genes was very close to the expression in tenocyte. Also, after co-culture with tenocyte, morphology of MSCs began to change from spindle into rhombic, which is similar to tenocyte. Our results indicated that tenocytes microenvironment can promote the expression of tendon/ligament-associated gene in MSCs and have a tendency to induce the differentiation of MSCs towards tenocytes.Our results show that microenvironment plays an important role in proliferation and differentiation of MSCs. Co-culture with tenocytes can not only promote the proliferation but also up-regulate the expression of tendon/ligament-associated gene in MSCs, which suggest the differentiation of MSCs towards tenocytes. These results provide a theoretical and methodological reference for the management of damaged tendons/ligaments and tissue-engineered tendons/ligaments.