Experimental Studies On The Directed Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Tendon Fibroblasts By Mechanical Stretch

Tendon and ligament are tenacious connective tissues which respond to external mechanical stimulation by changes of its structure, mechanical properties and form, and play an important role in the sports. However, some excessive, repetitive mechanical strains will result in tendon/ligament wound or rupture, which is harmful to human health. The therapy of damaged tendon/ligament is still one of the most difficult and challenging clinical issues in orthopaedics. There are many problems unsolved, such as reducing curing time, enhancing healing quality and so on.Rat bone marrow mesenchymal stem cells (rMSCs) are a certain kind of cells that have proliferation and multi-differentiation ability. Under some special condition, rMSCs can differentiate into osteoblasts, chondrocytes, adipocytes, muscle cells and so on. This multiple plasticity of rMSCs gives us a new chance for healing and even tissue-engineered tendon/ligament. However, little is known about tendon fibroblasts differentiation of rMSCs regulated by mechanical stretch. Thus, this paper focuses on the directed differentiation of rat bone marrow mesenchymal stem cells into tendon fibroblasts by mechanical stretch. The main research works and results are as follows:①Isolation and culture of rMSCs in vitroThe rMSCs were isolated by centrifugation with 1.073g/ml percoll solution. The results indicated the isolated cells are homogeneous histoleucocyte. Cultured in DMEM with 10% FBS for 24 hours, the cells adhered, divided and grew into colony. After about 2 weeks they achieved confluence, and showed fibroblast-like morphology. The nonadhesive cells were removed by changing the medium. The passage cells, whose caryoplasm were a little big, grew and adhered so fast that after about 1 week they achieved confluence and as the spindly. The rMSCs cytoskeleton is investigated by coomassie brilliant blue staining. The cytoskeleton is blue bunchy, and parallel to cell long axis.②Identification and cell cycle analysis of rMSCsUsing the Immunohistochemistry 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)%.③Processing of cell stretch apparatus We developed a single axis cell stretch apparatus, according to the principle of basement membrane deformation, which has five components: control component, mechanical component, electromotor component, cooling component and culture component. The stretch fashion of this apparatus is stretch-keep-contract- keep. The comprehensive evaluation of this system shows that the system operates stably, the temperature can be controlled, the frequency and deformation(frequency:0.01-1.5HZ, strain 0-50%) can be regulated easily, the duration can be controlled and aseptic operation is simple. It can be used to different frequency, strain and duration stretch experiment.④Directed differentiation of rMSCs by mechanical stretchThis paper characters the response of rMSCs to mechanical stretch by the change of morphology and relative genes. The influence of morphology and relative genes of rMSCs by single axis mechanical stretch (10%, 0.1HZ, 24h and 48h) was investigated. The results show that: the alignment of the rMSCs began to change after loading for 24 h, while the rMSCs became more slightness and the angles of cell alignment and stretch direction were from 60o to 90o after loading for 48 h and most of the rMSCs aligned vertically to the stretch direction. The relative expression of ColⅠ, ColⅢ, TNC and SCX toβ-actin was analyzed by RT-PCR. The results showed that after loading for 24h, the relative expression of ColⅠand ColⅢof stretch group was up regulated compared to control(static culture in same condition), and all groups didn't express TNC and SCX. However, the relative expression of ColⅠand ColⅢcame back to control level and the stretch group expressed TNC and SCX after loading for 48 h while the static control didn't express. It was suggested that the synthesis of ColⅠand ColⅢbegan to increase after loading for 24 h, the special markers of tendon fibroblast were expressed after loading for 48 h, and the rMSCs tend to differentiation to tendon fibroblast.Meanwhile, the relative genes of tendon fibroblast were investigated in this paper. Tendon fibroblast was isolated by tissue fragment adherent method. The morphology of primary tendon fibroblast is typical long fusiform and homogeneous. The surface is very velvet and the nucleolus is spindly. ColⅠand ColⅢwere expressed largely, while TNC and SCX were expressed a little in tendon fibroblast investigated by RT-PCR.This paper plays an useful role in the study of the differentiation of rMSCs into tendon/ligand fibroblast by mechanical stretch and supplies a reference for the cure and tissue of tendon/ligand in clinic.