Role Of TGF-β3 In Mechanical Reduced Differentiation Of Tendon Stem Cells

Tendinopathy is caused by tiny lesion after overuse, ossification and steatosis can be seen in the tendon with tendinopathy. Tenocytes are the functional cells in tendon, which have no ablity of differentiation, thus couldn’t be involved in ossification or steatosis. Tendon stem ceells(TSCs), which are progenitor to tenocytes and have mulriple differentiation potential, are key to repair tiny lesion and might be the cause of ossification and steatosis in tendons.It was found mechanical loading on TSCs could lead the cell differentiat ioto tenocytes, but over-loading might bring osteogenic, chondrogenic or adipogenic differentiation which could lead to failed recovery. TGF-β3 is a menber of TGF-βfamily, which is widely expressed in any tissue. Members of this family have defferent roles in differentiation, and are mechanical sensitive. But wether TGF-β3 is nvolved in mechanic reduced TSCs differentiationremained unknown.In our study, we exploreeffects of TGF-β3 on TSCs differentiationand it’s responce to mechanicalloading, and find out the role of TGF-β3 in mechanic reduced TSCs differentiation.Section one: Effects of Mechanical Loading on TSCs Differentiation and on TGF-β3 Expression.TSCs have mulriple differentiation potential, and could started differentiation by mechanical loading. In this section, we test the response of TDCs to mechanical loading for both differentiation and TGF-β3 expression, and look for the right mechanical loading for tendogenic and non- tendogenic differentiations.MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats(aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. The cells were exposed to repeated mechanical stretching of 4% and 8%, at the frequency of 1Hz. Cells were harvested at the time points of 12 hours, 24 hours, 36 hours, and 48 hours for further test.The mRNA expressions of tendogenic differentiation related genes Col I,TNC,TNMD,Scx,and non-tendogenic differentiation related genes,Runx2 for osteogenic differentiation,Sox9 for chondrogenic differentiation, PPARγ for adipogenic differentiation were measured by real-time quantitative PCR(qRT-PCR) together with the expression of TGF-β3. The cells were not intervened as control group.Results1. Mechanical loading on TSCs differentiationRepeated mechanical stretching of 4%, at the frequency of 1Hz for 24 to 36 hours, mRNA expression of tendogenic differentiation related genes Col I, TNC, TNMD and Scx up-regulated 2.50±10.7,4.12±1.46,2.56±0.07,1.41±0.34 folds compared with control group. mRNA expression of Sox9 and PPARγ down-regulated to 0.49±0.25,0.53±0.19 folds compared with control group.Repeated mechanical stretching of 4%, at the frequency of 1Hz for 24 hours, mRNA expression of tendogenic differentiation related genes Col I, TNMD up-regulated 1.84±0.53,2.22±0.45 folds compared with control group, and then dropped. At 24 to 36 hours, mRNA expression of Runx2 Sox9 and PPARγ down-regulated to 2.42±0.43,2.78±0.85,3.33±1.17 folds compared with control group. TGF-β3 up-regulated 2.53 to 4.71 folds compared with control group from 12 hours to 48 hours.2.TGF-β3’s response to mechanical loadingTGF-β3 up-regulated 1.69 to 3.24 folds compared with control group from 12 hours to 48 hours at mechanical stretching of 4%, at the frequency of 1Hz.TGF-β3 up-regulated 2.53 to 4.71 folds compared with control group from 12 hours to 48 hoursat mechanical stretching of 4%, at the frequency of 1Hz.SummaryRepeated mechanical stretching of 4%, at the frequency of 1Hz for 36 hours can reduce the TSCs different into tenocytes and repress the adipogenic differentiation. Repeated mechanical stretching of 8%, at the frequency of 1Hz for 24 to 36 hours led TSCs’ non-tendogenic differentiation. Espression of TGF-β3 up-regulated in response to repeated mechanical stretching of both 4% and 8%, and it was higher and earlier in response to 8%stretching than 4%.Section two: Effects of TGF-β3 on Differentiation of TSCs at Differert Concentration.Knowing that mechanical loading could up-regulate the expression of TGF-β3. We treated TSCs with TGF-β3 in this section to see it`s effect on TSCs’ differentiation.MethodsTSCs of passages 3 were stimulated with TGF-β3 at the concentration of 1ng/ml, 2.5ng/ml and 5ng/ml. Cells were harvested at the time points of day 1,day 3, and day 5,the mRNA expressions of tendogenic differentiation related genes were measured by real-time quantitative PCR(qRT-PCR). The cells were not intervened as control group.Results:1.Tendogenic differentiationrelated genes Col I,TNC, TNMD and Scx up-regulated 1.49 to 1.85, 1.57 to 3.03, 1.53 to 3.65 and 1.65 to 2.29 foldseparately in response to TGF-β3 of 2.5 ng/ml to 5ng/ml at 3 to 5 days.2. Chondrogenic differentiationrelated genes Sox9 down-regulated 0.34 to 0.73 folds, in response to TGF-β3 of 1ng/ml to 2.5ng/ml at day 1, and Sox9 and Col II up-regulated 1.38 to 1.40, and 1.44 folds in response to TGF-β3 of 2.5ng/ml to 5ng/ml at day 5.3. Osteogenicdifferentiationrelated genes Rux-2 down-regulated 0.54 to 0.56 folds, in response to TGF-β3 of 1ng/ml to 2.5ng/ml at day 1, and Rux-2and ALP up-regulated 2.60, 1.93 to 2.42 folds in response to TGF-β3 of 2.5ng/ml to 5ng/ml at day 5.4. Adipogenic differentiationrelated genes PPARγdown-regulated 0.36 to 0.68 foldsseparately in response to TGF-β3 of 1ng/ml to 5ng/ml at 1 to 5 days, and at the 5th day, AP2up-regulated 1.59 to 2.63 folds than control group.SummaryEffects of TGF-β3 on TSCs were complicated and depended on the concentration and time of duration, which shares the same pattern of effects of mechanical loading. TGF-β3 was able to lead TSCs differentiated to tenocytes, which was shown related to the concentration and time of duration buy statistical method. Treated with low concentration of TGF-β3 in short time inhibit non-tendogenic differentiation of TSCs,but when the conversation of TGF-β3 went high, TSCs tended to be non-tendogenic and differentiated into osteocytes or chondrocytes.TGF-β3 could be a key factor in mechanic reduced TSCs differentiation.Section three: Role of TGF-β3 in Mechanical Reduced Differentiation of TSCs.To investigate the role of TGF-β3 in mechanic reduced TSCs differentiation. We use TGF-β receptor blockers SB431542 and LY2109761 to block the single, and test the TSCs’ response on mechanical loading.MethodsTSCs of passages 3 were treated with SB431542 and LY2109761 in concentration of 2mM separately when exposed to the mechanical loading of 4% and 8%, 1Hz, for 30 hours. Expressions of differentiation related genes were tested by qRT-PCR and WB.ResultsUp –regulate of tendogenic differentiation related genes Col I,TNC and Scx in response to mechanical loading of 4% was blocked by TGF-β receptor blockers along with the expression of TNC and Scx protein.For the 8% stretching group, up –regulate of osteogenic differentiation ralated genes runx2 and ALP were not repressd, but the expression of runx2 protein shown to be repressd.The mRNA of chondrogenic differentiation gene Sox9 was repressd compared with the stretching group,but it’s level on WB remained the same. For adipogenic differentiation ralated genes AP2, block of TGF-β receptor shown significant effects on their expression for both mRNA and protein level.SummaryTGF-β3 involves in tendogenic differentiation of TSCs in response to mechanical loading, and inhibits osteogenic differentiation. TGF-β3 do not work alone mechanical reduced adipogenic differentiation of TSCs. We found no involvement of TGF-β3 inchondrogenic differentiation of TSCs in response to mechanical loading.Conclusion1.TGF-β3 up-regulated in response to mechanical loading.2. TGF-β3 can reduce both tendogenicand non-tendogenic differentiations of TSCs.3. TGF-β3 involve in tendogenic differentiation of TSCs in response to mechanical loading, and inhibits osteogenic differentiation. It’s might be the key factor between the tendogenic and osteogenic differentiation reduced by mechanical loading.