| BackgroundTendons transfer tensile loads from muscle to bone, which enable joint motions and stabilize joints. Tendons sustain large mechanical loads in vivo and as a result, tendons were frequently injured. Aging has been confirmed as a predisposing factor of tendinopathy and bad recovery quality following tendon repair. Current treatment methods are generally not effective and involve either symptomatic relief with non-steroidal anti-inflammatory drugs and physical therapy or surgery when conservative treatments failed. The limitation in treatment options is due to our incomplete knowledge of age-related tendinopathy. Studies over the past decades have uncovered a number of important mechanical and cellular changes of aging tendon. However, the basis of aging as a major risk factor for tendon injury and impaired tendon healing remained poorly understood. The objectives of this study are to characterize the aging tendon stem cells,identify the response of tendon stem cells on the moderate stretch in vitro and moderate treadmill running in vivo; and evaluate the effects of tendon defect healing respond to the moderate treadmill running.MATERIALS AND METHODSA total of 24 aging Fisher female rats(20 months old) and 6 young Fisher female rats(2 months old) obtained from the national institute of aging(NIA) were divided into two groups: moderate treadmill running group(MTR) and cage control group, with 9 rats in each group. The rats in control group were allowed to move freely in cages during treadmill running experiments. All 9 rats in the MTR group were trained for one week by running at the speed of 13 meters/min, 15 min/day, and 5 days/week on an Exer-6M Open Treadmill(Columbus Instruments, Columbus, OH). Following this training period, rats in the MTR group ran at the same speed for 30 min/day and 5 days/week for 4 additional weeks. After the end of treadmill running, 3 rats from each group were sacrificed for isolation of TSCs and in vitro culture. The remaining 6 rats in each group were used for the patella tendon window defect model study.RESULTS1.Verification of isolated aging rat TSCsTSCs isolated from aging rats were first verified by expression of stem cell markers using flow cytometry and immunostaining. The isolated rat TSCs were positive for the fibroblast surface marker, CD90, and stem cell markers, Stro-1 and SSEA-1 revealed by the flow cytometry analysis. These cells were also negative for hematopoietic cell surfa ce markers(CD34, CD45), mesenchymal stem cell surface marker(CD44), and endothelial cell surface marker(CD31). In addition, immunostaining was positive for the stem cell markers, OCT-4, NS, and Nanog in the isolated aging TSCs.2.Aging impair the ability of proliferation and self-renewalAfter cuture for 10-16 days, TSCs derived from both adult and aging rats’ patella tendon formed colonies. However, the numbers of colony is lower and the diameter of colony is smaller in aging rats group, compared with adult rats group. TSCs derived aging rats is more slender, multivariable and organized in a more disorder style. The results of flow cytometry showed that TSCs of aging rats expressed less CD90, compared with adult rats. And TSCs of both of aging and adult rats expressed stem cells’ markers: OCT-4, Nanog, NS. The PDT of aging and young rats is 34.9±1.4h, 23.9±0.8h respectively,the PDT of aging rat is significantly longer then the PDTof young rats(p < 0.01). The value of OD in aging and adult TSCs when cuture for 24 hours were 0.62±0.04 and 0.47±0.05, respectively. The value of OD in aging rats is significantly lower compared with the value of OD in young rats,(P=0.002). The value of OD in aging and adult TSCs when cuture for 48 hours were 0.73±0.03 and 0.54±0.03, respectively. The value of OD in aging rats is significantly lower compared with the value of OD in young rats,(P=0.004). the results of RT-PCRshowed that the expresions of P1 ColI, LPL, Col II, Osteocalcin in aging TSCswere 0.4-fold, 2.1-fold, 1.5-fold, 5.6-fold.3. Moderate stretch and treadmill running enhanced clonogenicity and proliferation of aging TSCsThe expressions of OCT-4, Nanog, and NS in aging TSCs were increased under 4% stretch; in addion, the proliferation of TSCs was enhanced significantly. The results of RT-PCR showed that the expressions of COI I, COL II, LPL, and Osteocalcin in in aging TSCs under 4% stretch were 4.14-fold,0.27-fold,0.23-fold,0.70-fold, respectively. TSCs isolated from the MTR and cage control groups formed colonies after 10-16 days in culture. However, in the MTR group the numbers of colonies formed were higher and the diameter of TSC colonies were bigger in size than the control group. Further, morphology of aging TSCs from rat patellar tendons without treadmill running were slender shaped and those in the MTR group were round and cobble-stone shaped.We also tested the effects of MTR on the proliferation capability of aging TSCs by performing PDT and CCK assays. The results showed that MTR significantly enhanced(31 %) the proliferation of TSCs when compared to the cage control(p < 0.01). Consistent with the decreased PDT, significant increase in OD values(10 %) was also observed for aging TSCs subjected to MTR in the CCK test(p < 0.036).Expressions of stem cell markers in aging TSCs were evaluated by flow cytometry and RT-PCR. Flow cytometry analysis showed that MTR significantly enhanced the expressio n of SSEA-1 and Stro-1 in aging rat TSCs when compared to the control group. Quantitation of these results revealed that the increase was 2-fold for SSEA-1 and 6-fold for Stro-1. In addition, q RT-PCR showed a significant increase in the expression of stem cell markers, OCT-4(4-fold) and Nanog(3-fold) in the MTR group, and the tenocyte marker Col I(3.5-fold). However, the expression of non-tenocyte gene(Sox-9-0.09-fold, PPAR?-0.27-fold, and Runx-2-0.13-fold) in the MTR group was significantly downregulated when compared to the cage control.4. Moderate treadmill running improved the healing of rat patella tendon window defectThe gross appearance of rat patella tendons in the cage control group 4 weeks after creating the window defect indicated high vascularization and unhealed incision that appeared to be closing after 8 weeks although the wound defect was still visible. However, MTR for 5 weeks prior to creating the wound improved healing of the patellar tendon defect as evidenced by the smaller wound size when compared to the cage control at the 4 week time point. After 8 weeks however, the window defect in the MTR group nearly disappeared. These results were corroborated by HE staining, which indicated filling of the hollow wound defect in the cage control with cells/collagen fibers in the MTR group. These results further showed better organization and alignment of the collagen fibers at 8th weeks postoperatively in the MTR group compared to the cage control. Immunohistochemical staining further verified these results and revealed higher Col III expression at 8th weeks postoperatively indicating tenogenesis in the MTR group than the cage control, however there are no obvious tenogenesis at 4th weeks postoperatively in both control and MTR groups.ConclusionAging attenuate the stemness of tendon stem cells. The TSCs derived the patella tendon of aging rats have a decreased the proliferation, colony ability and prone to differentiate into non-tenocyte. Moderate stretch in vitro and moderate treadmill running in vivo enhanced the proliferation of aging TSCs and induces differentiation of TSCs into tenocytes. Finally, we found that moderate treadmill running improved the quality of tenden healing. These findings of this study shed new insights into the mechanisms by which pre-exercise causes beneficial effects of on tendons. Our results may highlight causes and therapies for age-related tendinopathy and be used to design clinical rehabilitation protocols to treat tendon injuries in aging patients. |
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