Project description:Little is understood about the roles of tendon cells during flexor tendon healing. To better understand tendon cell functions, the Scx-Cre mouse was crossed to the DTR mouse model to facilitate scleraxis lineage cell depletion prior to acute flexor tendon injury and repair. WT (cre-) and experimental (cre+) mice underwent complete transection and repair of the flexor digitorum longus tendon. Repaired tendons were harvested at 14 and 28 days post-repair for bulk RNA-Seq analysis to examine possible mechanisms driving differential healing due to Scx lineage cell depletion.
Project description:An Infinium microarray platform (GPL28271, HorvathMammalMethylChip40) was used to generate DNA methylation data from many tissues from horses We generated DNA methylation data from n=333 horse tissue samples representing tissues. Blood samples were collected via venipuncture into EDTA tubes from across 24 different horse breeds (buffy coat). The other tissues were collected at necropsy. The tissue atlas was generated from two Thoroughbred mares as part of the FAANG initiative 37, with the following tissues profiled: adipose (gluteal), adrenal cortex, blood (PBMCs; only n=1 mare), cartilage (only n=1 mare), cecum, cerebellum (2 samples each from lateral hemisphere and vermis), frontal cortex, duodenum, fibroblast, heart (2 samples each from the right atrium, left atrium, right ventricle, left ventricle), hypothalamus, ileum, jejunum, keratinocyte, kidney (kidney cortex and medulla), lamina, larynx (i.e. cricoarytenoideus dorsalis muscle), liver, lung, mammary gland, mitral valve of the heart, skeletal muscle (gluteal muscle and longissimus muscle), occipital cortex, ovary, parietal cortex, pituitary, sacrocaudalis dorsalis muscle, skin, spinal cord (C1 and T8), spleen, suspensory ligament, temporal cortex, tendon (deep digital flexor tendon and superficial digital flexor tendon), uterus.
Project description:Purpose: The goal of this study is to compare transcriptional profiles of flexor tendon healing in wild-type (WT, C57Bl/6J) to superhealer (MRL/MpJ) miceto gain insights in the biological drivers of the tendon injury response between the C57 and MRL mice. Methods: RNA was isolated from patially lacerated or uninjured flexor tendon 7 days post-injury. Results: Transcriptional analysis of biological drivers showed positive enrichment of TGFB1 in both C57 and MRL healing tendons. only MRL tendons exhibited downstream transcriptional effects of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and ECM organization pathways in the C57 tendons. Conclusions: There is altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair.
Project description:Injuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Adhesions have been associated with PAI-1, a master suppressor of protease activity including matrix metalloproteinases (MMP). In the present study, we used next generation RNA sequencing (RNA-seq) to assess genome-wide differences in mRNA expression due to PAI-1 deficiency after zone II flexor tendon injury. Ingenuity pathway analysis was used to characterize molecular pathways and biological drivers associated with differentially expressed genes. Analysis of hundreds of overlapping and differentially-expressed genes in PAI-1 knockout and C57Bl/6J mice during tendon healing revealed common and distinct biological processes associated with the regulation of matrix organization, cell cycle, and immune response. Most importantly, we identified the activation of PTEN signaling and the inhibition of FOXO1-associated biological processes as a unique transcriptional signature of the healing tendon in the PAI-1 KO mice. The differences in transcriptomics between the two mouse strains are transcriptionally related to complex cross-talk between PI3K/Akt/mTOR, PKC, and MAPK signaling cascades that drive differences in transcriptional regulation of cell proliferation, survival and senescence, and chronic inflammation as potential drivers of fibrotic healing and adhesions in the C57Bl/6J injured tendons. These transcriptional observations should guide future studies to develop an improved understanding of the biological limitations of tendon healing as the basis for rational design of targeted therapeutics for scar-free regenerative healing of tendon.
Project description:Tendon is a highly organized, dense connective tissue that has been demonstrated to have very little turnover. In spite of the low turnover, tendon can grow in response to loading, which may take place primarily at the periphery. Tendon injuries and recurrence of injuries are common in both human and animal in sports. It is unclear why some areas of the tendon are more susceptible to such injury and whether this is due to intrinsic regional differences in extracellular matrix (ECM) production or tissue turnover. This study aimed to compare populations of tenocytes derived from the tendon core and periphery. Tenocytes were isolated from equine superficial digital flexor tendons (SDFT), and the proliferation capacity was determined. ECM production was characterized by immuno- and histological staining and by liquid chromatography-mass spectrometry-based proteomics. Core and periphery SDFT cultures exhibited comparable proliferation rates and had very similar proteome profiles, but showed biological variation in collagen type I deposition. In conclusion, the intrinsic properties of tenocytes from different regions of the tendon are very similar and other factors in the tissue may contribute to how specific areas respond to loading or injury.
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.
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:Tendon is a highly aligned connective tissue, in which the macro-structure consists of collagen-rich fascicles surrounded by interfascicular matrix (IFM). In a series of recent studies in equine tissue, we have demonstrated specialisation of tendon composition, structure and mechanics to achieve the tendon’s functional requirements, specifically reporting extensive specialisation of the IFM region in the energy storing superficial digital flexor tendon. We have also demonstrated loss of functional specialisms with ageing, leading to a hypothesised new paradigm for tendinopathy, focused on the importance of the IFM. However, to date, there have been no studies focused on structure-function specialisation or the IFM in functionally distinct human tendons. Here, we compare the positional anterior tibialis tendon and energy storing Achilles tendon, performing a detailed analysis of the composition and mechanical properties of both fascicle and IFM regions, to test the hypothesis that the IFM in the energy storing Achilles tendon has specialised composition and mechanical properties, and that these specialisations are lost with ageing. We demonstrate that the IFM is specialised in the energy storing Achilles tendon, with greater elasticity and fatigue resistance than in the positional anterior tibialis tendon. While there were few age-related alterations in mechanics, we did identify age-related alterations in the IFM proteome of the Achilles tendon specifically, which is predicted to be regulated by TGF-beta signalling and may be responsible for the trend towards decreased fatigue resistance observed in the Achilles IFM with ageing.