Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors.
ABSTRACT: The mechanical challenge of attaching elastic tendons to stiff bones is solved by the formation of a unique transitional tissue. Here, we show that murine tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, under regulation of shared regulatory elements and Krüppel-like factors (KLFs) transcription factors. High-throughput bulk and single-cell RNA sequencing of humeral attachment cells revealed expression of hundreds of chondrogenic and tenogenic genes, which was validated by in situ hybridization and single-molecule ISH. ATAC sequencing showed that attachment cells share accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis revealed enhancer signatures for most of these regions. Transgenic mouse enhancer reporter assays verified the shared activity of some of these enhancers. Finally, integrative chromatin and motif analyses and transcriptomic data implicated KLFs as regulators of attachment cells. Indeed, blocking expression of both Klf2 and Klf4 in developing limb mesenchyme impaired their differentiation.
Project description:Diabetes mellitus is associated with damage to tendons, which may result from cellular dysfunction in response to a hyperglycemic environment. Tenocytes express diminished levels of tendon-associated genes under hyperglycemic conditions. In contrast, mechanical stretch enhances tenogenic differentiation. However, whether hyperglycemia increases the non-tenogenic differentiation potential of tenocytes and whether this can be mitigated by mechanical stretch remains elusive. We explored the in vitro effects of high glucose and mechanical stretch on rat primary tenocytes. Specifically, non-tenogenic gene expression, adipogenic potential, cell migration rate, filamentous actin expression, and the activation of signaling pathways were analyzed in tenocytes treated with high glucose, followed by the presence or absence of mechanical stretch. We analyzed tenocyte phenotype in vivo by immunohistochemistry using an STZ (streptozotocin)-induced long-term diabetic mouse model. High glucose-treated tenocytes expressed higher levels of the adipogenic transcription factors <i>PPAR</i>? and C/EBPs. PPAR? was also highly expressed in diabetic tendons. In addition, increased adipogenic differentiation and decreased cell migration induced by high glucose implicated a fibroblast-to-adipocyte phenotypic change. By applying mechanical stretch to tenocytes in high-glucose conditions, adipogenic differentiation was repressed, while cell motility was enhanced, and fibroblastic morphology and gene expression profiles were strengthened. In part, these effects resulted from a stretch-induced activation of ERK (extracellular signal-regulated kinases) and a concomitant inactivation of Akt. Our results show that mechanical stretch alleviates the augmented adipogenic transdifferentiation potential of high glucose-treated tenocytes and helps maintain their fibroblastic characteristics. The alterations induced by high glucose highlight possible pathological mechanisms for diabetic tendinopathy. Furthermore, the beneficial effects of mechanical stretch on tenocytes suggest that an appropriate physical load possesses therapeutic potential for diabetic tendinopathy.
Project description:Uniparental parthenogenesis yields pluripotent stem cells without the political and ethical concerns surrounding the use of embryonic stem cells (ESCs) for biomedical applications. In the current study, we hypothesized that parthenogenetic stem cells (pSCs) could be directed to differentiate into tenocytes and applied for tissue-engineered tendon. We showed that pSCs displayed fundamental properties similar to those of ESCs, including pluripotency, clonogenicity, and self-renewal capacity. pSCs spontaneously differentiated into parthenogenetic mesenchymal stem cells (pMSCs), which were positive for mesenchymal stem cell surface markers and possessed osteogenic, chondrogenic, and adipogenic potential. Then, mechanical stretch was applied to improve the tenogenic differentiation of pMSCs, as indicated by the expression of tenogenic-specific markers and an increasing COL1A1:3A1 ratio. The pSC-derived tenocytes could proliferate and secrete extracellular matrix on the surface of poly(lactic-co-glycolic) acid scaffolds. Finally, engineered tendon-like tissue was successfully generated after in vivo heterotopic implantation of a tenocyte-scaffold composite. In conclusion, our experiment introduced an effective and practical strategy for applying pSCs for tendon regeneration. Stem Cells Translational Medicine 2017;6:196-208.
Project description:Orthopedic injuries, particularly those involving tendons and ligaments, are some of the most commonly treated musculoskeletal ailments, but are associated with high costs and poor outcomes. A significant barrier in the design of biomaterials for tendon tissue engineering is the rapid de-differentiation observed for primary tenocytes once removed from the tendon body. Herein, we evaluate the use of an anisotropic collagen-glycosaminoglycan (CG) scaffold as a tendon regeneration platform. We report the effects of structural properties of the scaffold (pore size, collagen fiber crosslinking density) on resultant tenocyte bioactivity, viability, and gene expression. In doing so we address a standing hypothesis that scaffold anisotropy and strut flexural rigidity (stiffness) co-regulate long-term maintenance of a tenocyte phenotype. We report changes in equine tenocyte specific gene expression profiles and bioactivity across a homologous series of anisotropic collagen scaffolds with defined changes in pore size and crosslinking density. Anisotropic scaffolds with higher crosslinking densities and smaller pore sizes were more able to resist cell-mediated contraction forces, promote increased tenocyte metabolic activity, and maintain and increase expression of tenogenic gene expression profiles. These results suggest that control over scaffold strut flexural rigidity via crosslinking and porosity provides an ideal framework to resolve structure-function maps relating the influence of scaffold anisotropy, stiffness, and nutrient biotransport on tenocyte-mediated scaffold remodeling and long-term phenotype maintenance.
Project description:In tendon tissue engineering, mechanical stimulus-induced differentiation is one of the most attractive techniques for stem cell-to-tenocyte differentiation in terms of cost, safety and simplicity. However, the most effective strain amplitude for differentiation using cyclic stretching remains unknown. Existing studies have not constrained cell reorientation behavior during cyclic stretching, resulting in uncertainty regarding the loads experienced by cells. In addition, strain distribution homogeneity of the culture membrane is important. Here, we improved the strain distribution uniformity of the membrane and employed a microgrooved membrane to suppress cell reorientation. Then we evaluated the most effective strain amplitude (0, 2, 4, 5, 6, or 8%) for the differentiation of mesenchymal stem cells into tenocytes by measuring mRNA expression levels. The maximum expression of all tenogenic markers was observed at a 5% strain. These results contribute to tendon tissue engineering by clarifying the most effective strain amplitude during tenogenic differentiation induction using cyclic stretching.
Project description:The incidence of tendon re-tears post-surgery is an ever present complication. It is suggested that the application of biological factors, such as bone morphogenetic protein 7 (BMP-7), can reduce complication rates by promoting tenogenic characteristics in in vitro studies. However, there remains a dearth of information in regards to the mechanisms of BMP-7 signalling in tenocytes. Using primary human tenocyte-like cells (hTLCs) from the supraspinatus tendon the BMP-7 signalling pathway was investigated: induction of the BMP associated Smad pathway and non-Smad pathways (AKT, p38, ERK1/2 and JNK); alterations in gene expression of BMP-7 associated receptors, Smad pathway components, Smad target gene (ID1) and tenogenic marker scleraxis. BMP-7 increases the expression of specific BMP associated receptors, BMPR-Ib and BMPR-II, and Smad8. Additionally, BMP-7 activates significantly Smad1/5/8 and slightly p38 pathways as indicated by an increase in phosphorylation and proven by inhibition experiments, where p-ERK1/2 and p-JNK pathways remain mainly unresponsive. Furthermore, BMP-7 increases the expression of the Smad target gene ID1, and the tendon specific transcription factor scleraxis. The study shows that tenocyte-like cells undergo primarily Smad8 and p38 signalling after BMP-7 stimulation. The up-regulation of tendon related marker genes and matrix proteins such as Smad8/9, scleraxis and collagen I might lead to positive effects of BMP-7 treatment for rotator cuff repair, without significant induction of osteogenic and chondrogenic markers.
Project description:Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting tenocyte proliferation and matrix synthesis during embryonic tendon development. However, the role of scleraxis in the growth and adaptation of adult tendons is not known. We hypothesized that scleraxis is required for tendon growth in response to mechanical loading and that scleraxis promotes the specification of progenitor cells into tenocytes. We conditionally deleted scleraxis in adult mice using a tamoxifen-inducible Cre-recombinase expressed from the Rosa26 locus (Scx?) and then induced tendon growth in Scx+ and Scx? adult mice via plantaris tendon mechanical overload. Compared with the WT Scx+ group, Scx? mice demonstrated blunted tendon growth. Transcriptional and proteomic analyses revealed significant reductions in cell proliferation, protein synthesis, and extracellular matrix genes and proteins. Our results indicate that scleraxis is required for mechanically stimulated adult tendon growth by causing the commitment of CD146+ pericytes into the tenogenic lineage and by promoting the initial expansion of newly committed tenocytes and the production of extracellular matrix proteins.
Project description:Tenomodulin (Tnmd) is a type II transmembrane glycoprotein predominantly expressed in tendons and ligaments. We found that scleraxis (Scx), a member of the Twist-family of basic helix-loop-helix transcription factors, is a transcriptional activator of Tnmd expression in tenocytes. During embryonic development, Scx expression preceded that of Tnmd. Tnmd expression was nearly absent in tendons and ligaments of Scx-deficient mice generated by transcription activator-like effector nucleases-mediated gene disruption. Tnmd mRNA levels were dramatically decreased during serial passages of rat tenocytes. Scx silencing by small interfering RNA significantly suppressed endogenous Tnmd mRNA levels in tenocytes. Mouse Tnmd contains five E-box sites in the ~1-kb 5'-flanking region. A 174-base pair genomic fragment containing a TATA box drives transcription in tenocytes. Enhancer activity was increased in the upstream region (-1030 to -295) of Tnmd in tenocytes, but not in NIH3T3 and C3H10T1/2 cells. Preferential binding of both Scx and Twist1 as a heterodimer with E12 or E47 to CAGATG or CATCTG and transactivation of the 5'-flanking region were confirmed by electrophoresis mobility shift and dual luciferase assays, respectively. Scx directly transactivates Tnmd via these E-boxes to positively regulate tenocyte differentiation and maturation.
Project description:The mechanosensitive gene tenomodulin (Tnmd) is implicated in tendon maturation and repair. However, the mechanism by which mechanical loading regulates Tnmd's expression and its role in tenocyte migration is yet to be defined. Here, we show that Tnmd and migration were upregulated in uniaxial cyclic stress-stimulated tenocytes. The knockdown of Tnmd reduced cell migration in the presence and absence of mechanical loading, suggesting that Tnmd is involved in tenocyte migration. Moreover, the treatment of stress-stimulated tenocytes with the actin inhibitor latrunculin (Lat A), histone acetyltransferase inhibitor anacardic acid (ANA), or histone demethylases inhibitor GSK-J4 suppressed Tnmd expression and tenocyte migration. These results show that actin stress fiber formation and chromatin decondensation regulates Tnmd expression, which might then regulate tenocyte migration. Thus, this study proposes the involvement of the actin and chromatin mechanotransduction pathway in the regulation of Tnmd and reveals a novel role of Tnmd in tenocyte migration. The identification of Tnmd function in tenocyte migration provides insight into the molecular mechanisms involved in Tnmd-mediated tendon repair.