| Tendons are dense, fibrous connective tissues that connect bone to muscle and are responsible for transmitting mechanical loading from skeletal muscle to bone. Mechanical loading in the form of exercise from skeletal muscle on tendons muscle is increased. It is well recognized that the change of tendon loading as a result of repetitive tendon overuse is the primary etiological factor of tendinopathy. Damaged tendon tissue heals very slowly and rarely attains the structural integrity and mechanical strength of normal, undamaged tendon due to poor vascular distribution, few cells and slow metabolism. Therefore, tendinopathy has been widely concerned in the medical field.Tendinopathy is common in orthopedic and sports medicine, rehabilitation medicine and geriatric medicine. It is a common disorder in runners, jumpers and excessive users. This injury has been claimed to account for 30-50% of all sports-related injuries and half of all occupational illnesses in the USA. With the improved quality of life and more exercise but incorrect way and unhealthy lifestyle, an increasing number of people are suffering from tendinopathy in China. People with tendinopathy have become poor in the term of life quality and exercise performance, especially for athletic, even fail to return to the stadium. Therefore, the prevention and treatment of tendinopathy are widely concerned by professers at home and abroad and become an urgent problem.There are various treatments for tendinopathy, including conservative and surgical treatments. Conservative treatments include rest, changes in exercise, physical therapy and rehabilitation training, non-steroidal anti-inflammatory analgesic drugs (NSAIDs) and local injections, etc. These methods are often effective in short term. Although surgery is the last resort in those failing conservative management, it is still unclear how the removal of adhesions and excision of degenerate areas affects healing, vascularity or resolves pain. Therapeutics has been largely ineffective because fundamental mechanisms that underlie pathogenesis remain unknown. Therefore, it is vital for us to investigate the pathogenesis of tendinopathy in order to seek more effective ways of prevention and treatment.At present, a lot of hypothesis and theories on the pathogenesis of tendinopathy are reported, including the micro-injury hypothesis, the inflammatory mediators hypothesis, the mechanics-biology coupling hypothesis, the ischemia-reperfusion theory and the abnormal differentiation hypothesis theory. Each one seems to be sound and scientific, but fail to full explain the pathologic changes of tendinopathy. Thus, it may be reasonable to combine with all hypothesis and theories and comprehensive consideration to explore the underlying pathogenesis of tendinopathy.Tendons transmit mechanical loading from skeletal muscle in vivo. In turn, tendons like bones and muscles can reconstruct under mechanical loading, which may influence the mechanical property, structure, and composition of tendon. Training is an effective approach for observation the process of reconstruction. Generally, moderate training can perfect the structure, synthesize collagen, improve the biomechanical performance, and even repair micro-injury. While, strenuous training may damage the structure, degrade of collagen, worsen the biomechanical performance, fail to repair, and lead to rupture.However, there is a vast difference in the response of tendon to training, depending on various factors such as aging, sex as well as the type, and duration intensity of mechanical loading in which it is applied, both in humans and in animals. Especially, the intensity of mechanical loading on tendons in the form of exercise can result in tendinopathy.In this study, we will establish four kinds of intensity through the treadmill running model. Subsequently, the alterations in mechanical property, structure, and composition of rat Achilles tendon will be examined. Further, the patho-physiologic changes will be understood at the molecular level. At last, the proliferation and differentiation of tendon stem cells will be measured to explain the physiological and pathological changes in tendons.Part One:The effect of treadmill running with different intensities on structure and mechanical property of Achilles tendonsObjectives1. To investigate the effect of treadmill running with different intensities on mechanical property of tendons;2. To investigate the effect of treadmill running with different intensities on structure of tendons.Materials and methods1. Experimental animals and exercise protocolsA total of 24 male SD rats (8 weeks old, weight 200-250 g) were randomly and evenly assigned to one of four groups as follows:(1) sedentary control (CON, n=6), (2) low-intensity running (LIR, n=6), (3) medium-intensity running (MIR, n=6), and (4) high-intensity running (HIR, n=6). Animals in LIR, MIR, and HIR groups were regularly trained for 8 weeks.2. HarvestAt the end of 8 weeks’running program, all animals were sacrificed. Then, Achilles tendons were collected. A randomly selected hind limb (left or right) of each animal was used for biomechanical testing. The contralateral Achilles tendon was assigned for histological assays.3. Index detectionBody weight; Biomechanical testing; Cross sectional area (CSA); Transmission electron microscopy (TEM); Hematoxylin-eosin (H&E) staining; Picrosirius red staining4. Statistical methodsResults were expressed as the mean ± standard deviation. Statistical analysis was carried out using one-way analysis of variance (ANOVA) and Tukey’s test for post hoc analysis. The data analysis was done using SPSS 16.0 (Chicago, IL, USA) with significance set at P≤0.05.ResultsBody weight:Differences were less marked between CON, LIR, MIR and HIR groups (P>0.05).Biomechanical testing:The mechanical property was less changed in LIR group (P>0.05), but significantly improved in MIR group (P<0.05), and decreased in HIR group (P<0.05).CSA:Less changes was recorded between CON, LIR, MIR and HIR groups (P >0.05).TEM:The average collagen fibril diameter was less changed in LIR group (P> 0.05), but significantly increased in MIR group (P<0.05), and decreased in HIR. group (P<0.05).H&E staining:Collagen fibers from CON group were lightly eosinophilic and arranged in parallel with occasional elongated basophilic nuclei. And, Achilles tendons sections from LIR group looked similar to those in CON. Further, Achilles tendons sections from MIR group had the uniform appearance of well-aligned, parallel and dense collagen bundles. However, disorganized collagen fibers with more space were found in the HIR group.Picrosirius red staining:Collagen fibers were organized parallelly and straightly with crimps in CON and LIR groups. Whereas, regularly and densely organized collagen fibrils were revealed in MIR group. On the contrary, irregularly and loosely organized collagen fibrils were shown in HIR group.ConclusionIn this study, a marked intensity-specific effect of running on the mechanical property and structure of rat Achilles tendons is demonstrated. Less changes under low-intensity training is found in mechanical property and structure of Achilles tendons. Further, medium-intensity training could perfect the structure, improve the biomechanical performance, and be beneficial to Achilles tendons. However, high-intensity training might damage the structure, worsen the biomechanical performance, and be detrimental to Achilles tendons.Part Two:The effect of treadmill running with different intensities on biochemical composition in Achilles tendonsObjectives1. To investigate the effect of treadmill running with different intensities on collagen content in tendons;2. To investigate the effect of treadmill running with different intensities on the expression of MMP-13 and TIMP-1 in tendons;3. To investigate the effect of treadmill running with different intensities on the expression of decorin, biglycan and aggrecan in tendons. Materials and methods1. Experimental animals and exercise protocolsA total of 24 male SD rats (8 weeks old, weight 200-250 g) were randomly and evenly assigned to one of four groups as follows:(1) sedentary control (CON, n=6), (2) low-intensity running (LIR, n=6), (3) medium-intensity running (MIR, n=6), and (4) high-intensity running (HIR, n=6). Animals in LIR, MIR, and HIR groups were regularly trained for 8 weeks.2. HarvestAt the end of 8 weeks’ running program, all animals were sacrificed. Then, Achilles tendons were collected. A randomly selected hind limb (left or right) of each animal was frozen in liquid nitrogen and stored for mRNA and protein expression analysis at -80℃. The contralateral Achilles tendon was fixed with 4% neutral formaldehyde and assigned for histological assays.3. Index detectionImmunohistochemistry (IHC); Enzyme-linked immunosorbent assay (ELISA); Quantitative real-time polymerase chain reaction (qPCR)4. Statistical methodsResults were expressed as the mean ± standard deviation. Statistical analysis was carried out using one-way analysis of variance (ANOVA) and Tukey’s test for post hoc analysis. The data analysis was done using SPSS 16.0 (Chicago, IL, USA) with significance set at P≤0.05.ResultsIHC:The content of Col I was less changed in LIR group (P>0.05), but significantly increased in MIR group (,P<0.05), and decreased in HIR group (P< 0.05). The content of Col Ⅲ was less changed in LIR and MIR groups (P>0.05), and increased in HIR group (P<0.05). The content of decorin was less changed in LIR group (P>0.05), but significantly increased in MIR group (P<0.05), and decreased in HIR group (P<0.05).ELISA:The expression level of MMP-13 and TIMP-1 was less changed in LIR group (P>0.05), but significantly increased in MIR group (P<0.05). Meanwhile, the expression of MMP-13 was significantly increased (P< 0.05), however, the expression of TIMP-1 was significantly decreased (P<0.05) in HIR group.qPCR:The expression level of Col I was less changed in LIR group (P>0.05), but significantly increased in MIR group (P<0.05), and decreased in HIR group (P< 0.05). The expression level of Col Ⅲ was less changed in LIR and MIR groups (P> 0.05), and increased in HIR group (P<0.05). The expression level of decorin, biglycan and aggrecan was less changed in LIR group (P>0.05), but higher level of decorin and lower level of biglycan were observed in MIR group (P<0.05). Conversely, lower level of decorin and higher level of biglycan and aggrecan were found in HIR group (P<0.05).ConclusionIn this study, a marked intensity-specific effect of running on the biochemical composition in rat Achilles tendons is demonstrated. Less changes under low-intensity training is found in the biochemical composition in Achilles tendons. Further, medium-intensity training could synthesize Col I, regulate fibrillogenesis, and be beneficial to Achilles tendons. However, high-intensity training might degrade Col I, destroy fibrillogenesis, and be detrimental to Achilles tendons.Part Three:The effect of treadmill running with different intensities on tendon stem cells in Achilles tendonsObjectives1. To investigate the effect of treadmill running with different intensities on the proliferation of tendon stem cells in tendons;2. To investigate the effect of treadmill running with different intensities on the differentiation of tendon stem cells in tendons.Materials and methods1. Experimental animals and exercise protocolsA total of 24 male SD rats (8 weeks old, weight 200-250 g) were randomly and evenly assigned to one of four groups as follows:(1) sedentary control (CON, n=6), (2) low-intensity running (LIR, n=6), (3) medium-intensity running (MIR, n=6), and (4) high-intensity running (HIR, n=6). Animals in LIR, MIR, and HIR groups were regularly trained for 8 weeks.2. Harvest and cultivationAt the end of 8 weeks’running program, all animals were sacrificed. Then, Achilles tendons were collected. Tendon stem cells were isolated and cultured to passage 3-5.3. Index detectionMTT assay; Quantitative real-time polymerase chain reaction (qPCR); Western blotting assay4. Statistical methodsResults were expressed as the mean+standard deviation. Statistical analysis was carried out using one-way analysis of variance (ANOVA) and Tukey’s test for post hoc analysis. The data analysis was done using SPSS 16.0 (Chicago, IL, USA) with significance set at P≤0.05.ResultsMTT assay:Cell proliferation of tendon stem cells increased significantly in MIR group (P>0.05), but not in groups LIR and HIR (P<0.05).qPCR:The expression level of Col II and Sox 9 was less changed in LIR and MIR groups (P>0.05), and increased in HIR group (P<0.05). The expression level of Runx 2 was less changed in LIR, MIR or HIR group (P>0.05).Western Blotting assay:The expression level of BMP-2 was less changed in LIR and MIR groups (P>0.05), and increased in HIR group (P<0.05).ConclusionIn this study, a marked intensity-specific effect of running on tendon stem cells in rat Achilles tendons is demonstrated. Less changes under low-intensity training is found in proliferation and differentiation of tendon stem cells in Achilles tendons. Further, medium-intensity training could promote proliferation of tendon stem cells, differentiate into tenocytes, and be beneficial to Achilles tendons. However, high-intensity training might induce chondrogenic differentiation of tendon stem cells (TSCs) though BMP-2 signaling, and be detrimental to Achilles tendons. |
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