Effects Of Mechanical Stretching Frequency On Tendon Stem Cells Proliferation And Differentiation

BackgroudTendinopathy is one of the major diseases of sports medicine and orthopedics. Almost16.4million people suffered from tendon and ligament diseases and at least100,000ofthem are turned into tendinopathy in the USA every year.The causes of tendinopathy may varied from the internal and external factors,but therepeatedly and excessive stress load is the main pathogenic factors during the process oftendinopathy development. Stress load includes three kinds factors of intensity, frequencyand time. The high-frequent sports are the mostly relevant pathogenic factors totendinopathy. Kujala et al put forward achilles tendon tendinopathy cumulative incidencewas higher in the middle-distance race athletes and achilles tendon rupture cumulativeincidence was higher in the sprinters(middle-distance race athletes movement frequency is3.0Hz, sprinters movement frequency is4.5Hz); Wu Ziying et al put forward lowfrequency stretching could repair the damaged achilles tendon, on the contrary, highfrequency stretching may cause tendinopathy occurrence.Excessive stress load is probably one of the main causes of TSCs abnormaldifferentiation. Zhang and Shi put forward that different stretching intensity and time haddifferent influence on the TSCs differentiation, but the different frequency of TSCsproliferation differentiation effect is not clear? Among cyclic stretching load models invitro, uniaxial cyclic stretching model can simulate TSCs stress better in vivo and whichwere used to study the effects on tendinopathy under the condition of stress loads. Uniaxialcyclic stretching models in vitro cell at present basically has STREX series, Wang and CaoHonghui prophase development model of our team. But the above models of frequencyparameters are low and cannot satisfy the experimental conditions of this topic, and thereare some improvements in other ways.ObjectiveTherefore, this topic main research purpose of this study is to establish a multiple frequency uniaxial cyclic stretching model; to establish the appropriate and excessivestrecth frequency threshold; and to preliminary discuss the effects of different stretchingfrequency on tendon stem cells proliferation and differentiation, which provideexperimental basis for further study the effect of exercise load on tendinopathy.MethodsⅠ establishment of a multiple frequency uniaxial cyclic stretching model1．Dishes were made of silicone and analyzed with the finite element analysisThe microgrooved dishes were made of PDMS; Finite element software ABAQUSanalyzed silicone cell cultures of the new design and preliminary design.2.Mechanical parts running stability and effective stretching performance were testedMechanical parts running stability of the stretching model were observed; Siliconedishes actual running frequency and displacement distance of stretching before and afterwere measured; F-actin in cytoskeleton were observed by laser confocal scanningmicroscope.Ⅱ Effects of different stretching frequency on proliferation and differentiation oftendon stem cells in vitro1. isolation, culture and identification of rat TSCs in vitro.①Flow cytometry instrument tested TSCs proliferation ability;②Flow cytometry instrument and laser confocal observed stem cell markersexpression;③Chemistry induction methods observed multi-directional differentiation ability ofTSCs.2. To observe and test stretching effects of frequency on TSCs deformation andproliferation3. real-time PCR and Western blot tested stretching frequency on TSCs differentiationResultsⅠ New dishes strain distributions were uniformity and stretching model was easy tooperate with wide range of design parameters1. TSCs proliferation had no significant difference in standard cell culture dishes andin silicone dishes (p﹥0.05); when the microgrooves were deeply3microns, TSCs weresuitable for growth orientation; stress, displacement and strain distribution of new design dishes were better than that of the early stage design dishes.2. Stretching model ran stability; the elasticity of dishes had no obvious change beforeand after stretching; TSCs were no obvious Falling off and floating; silicone dishes actualrunning frequency and strain displacement distance showed no significant differencecomparing with set frequency and theoretical strain displacement distance (P﹥0.05);F-actin filaments seemed much thinner and broke partly of stretching groups.Ⅱ Different stretching frequency had effects on TSCs proliferation and differentiation1. TSCs were characteristic of stem cells①TSCs were clone growth.②Flow cytometry instrument tested CD90and CD44(+), CD106and CD11b (-);Immunofluorescence tested Ⅰ type collagen (+), Ⅲ type collagen (-), SSEA-4(+),Nucleostemin (+).③Cell contained more calcium nodule formation after alizarin red staining;cytoplasm were a large number of red dye lipid drops after oil red staining; there were aizenacid glycosaminoglycans after toluidine blue staining.2、Stretching frequency had effects on TSCs morphology growth direction andproliferation①In the basilar membrane slick silicone dishes, TSCs appearance of stretchinggroups became from pebbles into long fusiform and the growth directions are same asstretching axis; In silicone dishes with microgrooves, TSCs growed as fusiform along themicro grooves;②There was no effect on TSCs proliferation under low stretching frequency (P﹥0.05)；high stretching frequency decreased TSCs proliferation (P﹤0.05).3、Stretching frequency had effects on TSCs differentiationThere was no effect on TSCs differentiation into tenocyte、osteogenic lineages underlow stretching frequency (P﹥0.05)； high stretching frequency decreased TSCsdifferentiation into tenocyte lineages,while promoted TSCs differentiation into osteogeniclineages (P﹤0.05)；stretching frequency had no effect on TSCs differentiation intoadipogenic and chondrogenic lineages (P﹥0.05). Conclusion1.New designed silicone dishes strain distribution were uniform, which were a kind ofideal dishes for adherent cells stretching experiment; stretching model was easy to operatewith wide range of design parameters and suitable for adherent cells biomechanical loadexperiment study in vitro.2. Rat derived TSCs were characteristic of stem cells; There was no effect on TSCsproliferation and differentiation under low stretching frequency, while high stretchingfrequency decreased TSCs proliferation and promoted differentiation into osteogenic andlineages but decreased TSCs differentiation into tenocyte lineages.