Objective To investigate whether mechanical stretch stimulation affects the expression of the immediate early gene c-fos mRNA in rat Achilles-derived tendon stem cells (TSCs)in vitro. Methods TSCs were isolated from the Achilles tendons of 8 weeks old male Sprague Dawley rats by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated by a uniaxial cyclic stretching loading system under the condition of 1 Hz, respectively with 4% or 8% stretch intensity for 0, 5, 15, 30, 60, and 120 minutes. At each time point, TSCs were collected to detect c-fos mRNA expressions and to find the best time-point Tmax by real-time fluorescence quantitative PCR. Then, TSCs were simulated with 2%, 4%, 6%, 8%, or 12% stretch intensity for Tmax to observe the relative expressions of c-fos mRNA under different stretch intensities. Next, TSCs were stretched for 0, 5, or 15 minutes respectively and followed by incubation at relax status up to Tmax to observe the changes of c-fos mRNA expressions after short period stimulation. Finally, TSCs were stimulated with 4% or 8% stretch intensity respectively for 0, Tmax, or 120 minutes to detect the expressions of the tenogenic differentiation related genes [collagen type I, tenomodulin (TNMD)], the osteogenic differentiation related genes [runt related transcription factor 2 (Runx2), distal-less homeobox 5 (Dlx5)], and the adipogenic differentiation related gene [fatty acid binding protein 4 (FABP4)]. Results Under 4% or 8% stretch intensity, the relative expressions of c-fos mRNA significantly increased at 15 minutes (P<0.05), reached the maximum at 30 minutes (P<0.05), and returned to baseline at 60 minutes (P>0.05) when compared with expression at 0 minute. Therefore, Tmax was 30 minutes. The stretch intensity of 2% was enough to cause the expression of c-fos mRNA at 30 minutes, and the expression was significantly higher under the stretch intensity of 6%, 8%, and 12% than 2% and 4% (P<0.05). Even for a short period stimulation of 5 minutes, c-fos mRNA expression could still significantly increase at 30 minutes (P<0.05). The relative expressions of differentiation related genes at 30 and 120 minutes showed no significant difference when compared with the expression at 0 minute under 4% stretch intensity (P>0.05); but the relative expression of Runx2 gene significantly increased at 30 minutes, and the relative expressions of collagen type I, TNMD, Dlx5, and Runx2 increased at 120 minutes under 8% stretch intensity (P<0.05). Conclusion Mechanical stretch stimulation can affect the relative expression of the immediate early gene c-fos mRNA of rat Achilles-derived tendon stem cellsin vitro, and there is time- and intensity-dependence. It is suggested that the mechanical stimulation with different time or intensity may affect the differentiation of TSCs at early stage. This study is meaningful for the further study on TSCs intracellular mechanical signal transfer mechanism.
Objective To explore the effect of different intensity treadmill training on the repair of micro-injured Achilles tendon induced by collagenase in rats. Methods Seventy-two 8-week-old male Sprague Dawley rats (weighing, 200-250 g) were selected. After adaptive treadmill training for 1 week, rats were injected with 30 μL type I collagenase solution (10 mg/mL) into both Achilles tendons to make micro-injured Achilles tendon models. After 1 week of cage feeding, the rats were randomly divided into 3 groups: the control group, the low-intensity group, and the high-intensity group, 24 rats each group. The rats in control group could move freely, and the rats underwent daily treadmill training at the intensity of 13 m/min and 20 min/d in the low-intensity group and at the intensity of 17 m/min and 60 min/d in the high-intensity group. At immediate, 1 week, and 4 weeks after training, bilateral Achilles tendons were collected from 8 rats of each group for gross observation, histological analysis, and mechanical testing. Results At immediate after training, there was no significant difference in the gross observation, histological observation, and biomechanical properties of the Achilles tendon between groups (P>0.05). The gross observation showed connective tissue hyperplasia near Achilles tendon and lackluster tendon in each group at 1 week; hyperplasia significantly reduced in the low-intensity group when compared with the control group, and there were more connective tissue and a large number of neovascularization in the high-intensity group at 4 weeks. At 1 week, there was no significant difference in the semi-quantitative histological total score between groups (P>0.05), but there were significant differences in vascularity between low-intensity group or high-intensity group and control group (P<0.05). At 4 weeks, the semi-quantitative histological total score was significantly higher in high-intensity group than control group and low-intensity group (P<0.05), and in control group than low-intensity group (P<0.05). There were significant differences in collagen arrangement, cell morphology, abnormal cells, and vascularity between low-intensity group and high-intensity group or control group (P<0.05). And there was significant difference in abnormal cells between high-intensity group and control group (P<0.05). The mechanical testing showed that there was no significant difference in cross-sectional area of the Achilles tendon, the ultimate force, tensile strength, and elastic modulus between groups at 1 week (P>0.05); the low-intensity group was significantly higher than the control group in the ultimate force and the tensile strength (P<0.05), and than high-intensity group in the ultimate force and elastic modulus (P<0.05), but no significant difference was found in the other indexes between groups (P>0.05) at 4 weeks. Conclusion Low-intensity treadmill training can promote the repair of rat micro-injured Achilles tendon induced by collagenase.