| BackgroundTendon micro injury is a widespread tissue injury in athletes and common laborers.It often occurs after repeatedly,excessive stretching exersice.Local pain,swelling and limited movement are main symptoms.There are also tendon fiber disorders and fracture in histology.Tendon micro injury which can’t be effectively repaired is considered to be the main reason of tendon degeneration and tendinopathy.It has been reported that the incidence of rotator tendon disease in the general population is 2.4 to 20%,and the incidence of Achilles tendinopathy in high-level athletes is as high as 52%,which seriously impact people’s daily life and the competitive level of athletes.Therefore,to study the factors that have effect on micro injured tendon,and to look for ways promoting the micro injured tendon repairing,are still needed currently.Tendons are mechanical transmission tissue between bones and muscles.Stretch loading plays an important role on the maintain of tendon physiological functions.Some scholars believe that long-term excessive stretch loading on tendon making it injury beyond repair is one of the important reasons leading to tendinopathy.At the same time,other studies have found that eccentric training as one type of stretch loadings can alleviate or even cure tendinopathy.Therefore,stretch loading,like a double-edged sword,can not only lead to tendon injury but also promote tendon repair.Therefore,it’s important to study the mechanism of stretch loading on tendon,give play to the role of its advantage and avoid the adverse impact.Objective1.To explore the influences of different stretch loading conditions on micro injured Achilles tendon in rats,confirm the ideal stretch loading condition that promote tendon repair,and clarify the differences on TSCs biological characteristics after the ideal stretch loading.2.To clarify the early effects of stretch loading on the immediate early genes(IEG)expression of TSCs.Methods1.The preparation of micro injured Achilles tendon model in ratsAfter anesthesia,six 8-week old male SD rats were injected with collagenase solution into the right side Achilles tendon as the experimental group,and with PBS solution into the left side Achilles tendon as the control group.After 1 week of injection,the gross observation,the histopathology and the expression of differentiation marker gene were compared between the groups.2.The effects of stretch loading on the repair of micro injured Achilles tendon in ratsAfter adapted treadmill training,both Achilles tendons of 72 SD rats were injected with type I collagenase solution.Then randomly,rats were divided into 3 groups: the control group(cage breeding),the low intensity group(13 m/min,20 min/day)and the high intensity group(17 m/min,1 h/day).At the beginning of the treadmill running,1 week and 4 weeks after running respectively,the gross observation,histopathology,biomechanics and differentiation marker gene expression of Achilles tendon were compared among the groups to confirm the ideal condition of stretch loading.And then with the ideal stretch loading condition,the TSCs extracted from nine 8-week male SD rats were divided into 3 groups: the control group(Achilles tendons injected with PBS solution),the collagenase induced group(CI group: Achilles tendons injected with type I collagenase solution),and the CI + treadmill group(CI+TR group: Achilles tendon injected with type I collagenase solution + ideal intensity treadmill running).To observe the intervention effect on TSCs biology with the following methods: to compare colony numbers with crystal violet staining;to compare colony morphology with inverted phase contrast microscope;to compare the proliferation ability with CCK8 method;to compare the apoptosis and cell surface markers with fluid cytology;to compare cell stem markers with immunofluorescence;to compare cell differentiation ability with cells staining and RT-PCR method.3.The effects of stretch loading on the early gene expressions of TSCs from rat Achilles tendon in vitroTSCs were isolated and cultured in vitro,then stretched by using the cell uniaxial circulation stretch device.The cells were grouped with 2% ~ 12% stretch intensities and different stretch time respectively.RT-PCR was used to observe the expression of c-fos mRNA and differentiation marker genes in the early stage of stretching.Result1.Compared with the control group,the tendons from CI group had a paratendon tissue hyperplasia,and a lack of luster.The histological analysis showed there were abnormal increases in the number of cells,disordered arrangement of collagen fibers,fiber damage and so on.The expression of tendinous differentiation marker genes(Col I,TNMD)was significantly reduced(P < 0.05),and the expression of osteogenic differentiation marker gene(Runx2)was increased(P < 0.05).2.After treadmill running for 1 week,there was no difference in the gross observation,histological observation and biomechanical properties of the Achilles tendon in each group(P>0.05).However,the relative expressions of tendinous differentiation marker genes(Col I,TNMD)in the Achilles tendons of the low intensity group were higher than that of the control group and the high intensity group(P < 0.05).After 4 weeks running,the low-intensity group paratendon hyperplasia significantly reduced when compared with the control group,and the high intensity group’s paratendon connective tissue was still more,and there was a large number of neovascularization.The low intensity group’s semi-quantitative histological score was significantly better than the control group(P < 0.05),while the high intensity group’s score showed significantly worse than that of the control group(P < 0.05).The ultimate force and the tensile strength of the low intensity group’s tendons was higher than that in the control group(P < 0.05),while there was no difference between the high intensity group and the control group.Compared with the control group and the high intensity group,the expression of Col I gene in the low intensity group was significantly higher(P < 0.05),and there was no significant difference in the relative expression of TNMD gene among the groups(P > 0.05).3.There were more TSCs colonies in the CI group compared with the CI+TR group.The cells in the colony were more compact in the CI+TR group.Using CCK-8 tests after culturing TSCs for 24 h and 48 h respectively,the OD values of the CI+TR group increased significantly more than that of the CI group(P < 0.05).There was no significant difference among the experimental groups after cell apoptosis assay.The expression of Nanog in TSCs from the CI+TR group was higher than that in the CI group(P < 0.05).CD44 and CD73 in the CI+TR group expressed morecompared with the CI groupor the control group.The expressions of osteogenetic differentiation marker genes(Runx2,Dlx5)and chondrogenic differentiation marker gene(Col II)were obviously reduced in the CI+TR group when compared with the CI group(P < 0.05).However the expressions of tendinous differentiation marker genes(Col I,TNMD)and adipogenic differentiation marker genes(ap2,PPARγ)in the CI+TR group were significantly increased compared with that in the CI group(P < 0.05).After osteogenic induction,the expressions of osteogenic differentiation marker genes(Runx2,Dlx5)from TSCs were higher in the CI+TR group than control group orthe CI group(P < 0.05).The CI group relative expression of Runx2 was lower than the control group(P < 0.05).After adipogenic induction,the relative expressionsof adipogenic differentiation marker genes(ap2,PPARγ)from the CI+TR group TSCs were significantly higher than that in the CI group(P < 0.05),and the relative expression of PPARγwas higher than control group(P < 0.05).The CI group TSCs’ relative expression of PPARγwas lower than the control group(P < 0.05).After chondrogenic induction,the relative expressions of chondrogenic differentiation marker genes(Col II,Sox9)from the CI+TR group’s TSCs were lower than that of the CI group(P < 0.05),and the relative expressions of Col II and Sox9 from TSCs in the CI group were significantly increased compared with the control group and the CI+TR group(P < 0.05).4.The relative expression of c-fos mRNA from TSCs peaked at 30 min under the stretch intensity of 4% or 8% compared with the control group(P < 0.05).Even under the stretch intensity of 2%,the relative expression of c-fos m RNA from TSCs could increase(P < 0.05),and the relative expressioncould be further increased under the stretch intensities of 6%,8% or 12%(P < 0.05).The relative expression of c-fos m RNA could increase under the stretch loading for just 5 min(P < 0.05).The relative expressions of Col I,TNMD,Dlx5 and Runx2 increased at 120 min under the stretch intensity of 8%(P < 0.05).Conclusions1.After the injection of type I collagenase solution into the SD rat Achilles tendons,they showed the similar charecters to that in the clinical micro injured tendon s in gross observation and histopathological performance.It means this model is an ideal animal model which could be used to simulate the micro injury of the tendons.2.There was no significant different influence under different stretch loading intensities on the repair of micro injured tendons for 1 week.However,after 4 weeks training,the low intensity(13 m/min,20 min/day)treadmill training can promote the repair of rat micro injured Achilles tendon,and the high intensity(17 m/min,1 h/day)treadmill training block the repair.Therefore,for the repair of Achilles tendon micro injury in rats,the stretch loading of 13 m/min,20 min/day running for 4 weeks is an ideal load condition.3.The effect of ideal stretch loading on rat Achilles tendon TSCs were manifested in the proliferation ability,cell colony morphology,cell stem,the expression of CD73 and CD44,tendinous differentiation and adipogenic differentiation ability and so on.The results show that stretch loading has significant effects on the proliferation and differentiation of TSCs.4.In vitro uniaxial cyclic stretch TSCs experiment,2% stretch intensity for 5 min could cause the change of c-fos mRNA gene expression of TSCs.The relative expression of c-fos m RNA reached peak value at the time of 30 min.The differentiation marker gene expressions were different under differenttime points or loading intensities.It is suggesting that the mechanical stimulation with different time or intensity may have affected the differentiation of TSCs in early stage. |
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