Project description:Tendons play a critical role in the transmission of forces between muscles and bones, and chronic tendon injuries and diseases are among the leading causes of musculoskeletal disability. For many types of tendinopathies, women have worse clinical outcomes than men. It is possible that sex-based differences in tendon morphology, composition and mechanical properties may explain the greater susceptibility of women to develop tendinopathies. Our objective was to evaluate the mechanical properties, biochemical composition, transcriptome, and cellular activity of plantarflexor tendons from four month old male and female C57Bl/6 mice using in vitro biomechanics, mass spectrometry-based proteomics, genome-wide expression profiling, and cell culture techniques. Differences between groups were tested using t-tests (Î±=0.05). While the Achilles tendons of male mice were approximately 6% larger than female mice (P<0.05), the cell density of female mice was around 19% larger than males (P<0.05). No significant differences in the length (P=0.34), peak force (P=0.86), peak stress (P=0.52) or energy loss during stretch (P=0.94) of plantaris tendons were observed. Mass spectrometry proteomics analysis revealed no significant difference between sexes in the abundance of major extracellular matrix (ECM) proteins like collagen types I (P=0.30) and III (P=0.68), but female mice had approximately two-fold elevations (P<0.05) in different minor ECM proteins such as fibronectin, periostin, and tenascin. Using microarray analysis, there was no significant differences (P>0.05) in the expression of most major and minor ECM genes, and in the expression of genes involved in tendon fibroblast specification or proliferation. Whole tendon qPCR analysis showed significant expression differences in elastin, scleraxis, and tenomodulin. Cell culture techniques to test the effects of sex-specific extracellular environment on cellular activity show significant differences in gene expression of type I and III collagen, Ki67, scleraxis, and tenomodulin. Histologic analysis demonstrated that males have larger tendon cross-sectional area and lower cell density when compared to females. However, there were no differences between the sexes in the mechanical properties of tendons or in the majority of primary structural extracellular matrix proteins, although elevations were observed in some minor ECM proteins. Microarray analysis also showed no significant sex-based differences in the expression of major genes associated with collagen composition, extracellular components and turnover, and fibroblast proliferation. Cell culture tests of non-autonomous cellular activity show no major signals for ECM synthesis nor fibroblast proliferation. Our results indicate that while male mice expectedly had larger tendons, male and female mice have very similar mechanical properties and biochemical composition, with small increases in minor ECM proteins and proteoglycans in female tendons. The role that these minor ECM proteins and proteoglycans play in tendon repair should be evaluated in future studies. No treatment administered, study done to evaluate normal expression profiles of male and female tenocytes. This analysis is based on a Mouse Gene ST 2.1 strip that was processed in the microarray facility in May 2015 using the wt-pico kit.

Project description:Tendons play a critical role in the transmission of forces between muscles and bones, and chronic tendon injuries and diseases are among the leading causes of musculoskeletal disability. For many types of tendinopathies, women have worse clinical outcomes than men. It is possible that sex-based differences in tendon morphology, composition and mechanical properties may explain the greater susceptibility of women to develop tendinopathies. Our objective was to evaluate the mechanical properties, biochemical composition, transcriptome, and cellular activity of plantarflexor tendons from four month old male and female C57Bl/6 mice using in vitro biomechanics, mass spectrometry-based proteomics, genome-wide expression profiling, and cell culture techniques. Differences between groups were tested using t-tests (α=0.05). While the Achilles tendons of male mice were approximately 6% larger than female mice (P<0.05), the cell density of female mice was around 19% larger than males (P<0.05). No significant differences in the length (P=0.34), peak force (P=0.86), peak stress (P=0.52) or energy loss during stretch (P=0.94) of plantaris tendons were observed. Mass spectrometry proteomics analysis revealed no significant difference between sexes in the abundance of major extracellular matrix (ECM) proteins like collagen types I (P=0.30) and III (P=0.68), but female mice had approximately two-fold elevations (P<0.05) in different minor ECM proteins such as fibronectin, periostin, and tenascin. Using microarray analysis, there was no significant differences (P>0.05) in the expression of most major and minor ECM genes, and in the expression of genes involved in tendon fibroblast specification or proliferation. Whole tendon qPCR analysis showed significant expression differences in elastin, scleraxis, and tenomodulin. Cell culture techniques to test the effects of sex-specific extracellular environment on cellular activity show significant differences in gene expression of type I and III collagen, Ki67, scleraxis, and tenomodulin. Histologic analysis demonstrated that males have larger tendon cross-sectional area and lower cell density when compared to females. However, there were no differences between the sexes in the mechanical properties of tendons or in the majority of primary structural extracellular matrix proteins, although elevations were observed in some minor ECM proteins. Microarray analysis also showed no significant sex-based differences in the expression of major genes associated with collagen composition, extracellular components and turnover, and fibroblast proliferation. Cell culture tests of non-autonomous cellular activity show no major signals for ECM synthesis nor fibroblast proliferation. Our results indicate that while male mice expectedly had larger tendons, male and female mice have very similar mechanical properties and biochemical composition, with small increases in minor ECM proteins and proteoglycans in female tendons. The role that these minor ECM proteins and proteoglycans play in tendon repair should be evaluated in future studies.

Project description:Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. It is hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing. To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis. Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with microarray data. Groups of assays that are related as part of a time series. Computed

Project description:Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. It is hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing. To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis. Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with microarray data.

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Regional transcriptomics and mechanical properties of rat Achilles tendons

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