Effects Of Shock Waves On Tenocyte Proliferation And Extracellular Matrix Metabolism

Objective: The mechanism of tendon healing has been widely reported,because of the vivo studies are subject to many factors, which many studies have been carried out in vitro.So far on the tendon cells and tendon fibroblasts to participate in the healing mechanisms are not fully understood.After the chick embryo tendon cells were cultrued by Dem at 1971,the study on tenocyte was gradually in depth,and its biological characteristics are also gradually being recognized. The shock wave is an effective noninvasive modality for resolving various tendon pathologies.However, scientific rationale and mechanism of shock wave therapy remains limited. This study aims to investigate the effects of shock waves and their biochemical mechanisms on tenocyte proliferation and matabolism.Methods: Tenocytes harvested from Hindlimb flexor tendon of New Zealand white rabbits were used in this study. According to the principle of random implanted into A group (ESW intervention group), B group (non-intervention group); After the optimal energy density (0.36mj/mm2/200frequency) interference,Cell viability was assayed by MTT methods..Synthesis of collagen,nitric oxide (NO),matrix metalloproteinase-1(MMP-1) and transforming growth factor-₁ (TGF-β) were determined by ELISA,the Col-1 gene exppress was studied by RT-PCR.Results:1. The tendon tissue was observated by the inverted microscope,As early as the third day, the tenocytes round in shape with refractive index,climbed from the tissue, radial to the edge of the tissue , and adherented growth to the distant. No later than five days the latest cells climbed out.The tenocytes of the digestion method group began to adherent to the petri dish when cultured 48 h, and gradually extended to spindle and polygonal. The cells come from the tissue began to adherent at about the 6th day that the integration into a single- monolayer, cell-cell gap is very small, and at this time the primary cells can be passaged; The nuclear is intensitv obseved by confocal microscope after staining with fluorescence, some cells have superimposed place; It can be clearly seen the cell's morphology was spindle observed from the high power field. Intervention group cells after subcutrued growed faster than the control group, the cell nuclear is intensive staining with hoechst33342 observed by LSCM. 72 hours later, cells gradually extended pseudopodia, and develomented to a polygon size.2. The MTT colorimetric assay was determined to evaluate the mitochondria activity of the tenocytes after shock wave (0.36mj/mm2,200times)exposure at 24h,48h,72h and 1 week. A group OD value were higher than B group except at 1week, there was a significant difference (P<0.05). A group OD value gradually increased at the 24h, it reached the peak at the 48h and has begun to enter the platform at 72th d; B group OD values increased significantly at the 3th d, appearance the peak at the 5th d and has begun to enter the platform at the 7th d.3. The concentration of TGF-β,iNOs,MMP-1,and Col-1 in conditioned medium were evaluated by enzyme-linked immunosorbent assay (ELISA), A group value were higher than B group except at 1week, there was a significant difference (P<0.05),when they were detected at 24h,48h,72h and 1 week after interventing by ESW.5. PCR products revealed that Col-1mRNA expression of two groups can be detected at the 48h after ESW intervention, A group was significantly stronger than the B group. Col-1mRNA sustained expression in the whole process and appears peak at the 48h, Apart from the 1week were no significant differences (P <0.05).Conclusion:1. Appropriate intensity ESW can promote and induct tenocytes proliferation;2. Selecting the appropriate energy and density is the key step of ESW promote tenocytes differentiation;3. The production of TGF-β,iNOs and Col-1 exppressing enhanced Markedly after ESW intervented.So it implied that the tendon healing is relevent with these matabolisms which secreting enhanced markbaly at the early stagy. But the exppression of MMP-1 may inhibit the tenocytes proliferation at the early stagy,which is important to the remolding of the tenocytes.4. The results of this study show the causes that extracorporeal shock wave promote of tendon cell proliferation may be the expression of NO and other media were enhanced, which started the other cytokines, such as accelerated collagen matrix synthesis, the results may provide a theoretical basis for clinical treatment of tendon terminal disease with ESW.