Tenomodulin is necessary for tenocyte proliferation and tendon maturation.
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ABSTRACT:
SUBMITTER: Docheva D
PROVIDER: S-EPMC543433 | biostudies-other | 2005 Jan
REPOSITORIES: biostudies-other
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Project description:During embryonic development, tendons transform into a hypocellular tissue with robust tensile load-bearing capabilities. Previous work suggests that this mechanical transformation is due to increases in collagen fibril length and is dependent on mechanical stimulation via muscle activity. However, the relationship between changes in the microscale tissue structure and changes in macroscale tendon mechanics is still unclear. Additionally, the specific effect of mechanical stimulation on the multiscale structure-function relationships of developing tendons is also unknown. Therefore, the objective of this study was to measure the changes in tendon mechanics and structure at multiple length scales during embryonic development with and without skeletal muscle paralysis. Tensile testing of tendons from chick embryos was performed to determine the macroscale tensile modulus as well as the magnitude of the fibril strains and interfibrillar sliding with applied tissue strain. Embryos were also treated with either decamethonium bromide or pancuronium bromide to produce rigid or flaccid paralysis. Histology was performed to assess changes in tendon size, spacing between tendon subunits, and collagen fiber diameter. We found that the increase in the macroscale modulus observed with development is accompanied by an increase in the fibril:tissue strain ratio, which is consistent with an increase in collagen fibril length. Additionally, we found that flaccid paralysis reduced the macroscale tendon modulus and the fibril:tissue strain ratio, whereas less pronounced effects that were not statistically significant were observed with rigid paralysis. Finally, skeletal paralysis also reduced the size of collagen fibril bundles (i.e., fibers). Together, these data suggest that more of the applied tissue strain is transmitted to the collagen fibrils at later embryonic ages, which leads to an increase in the tendon macroscale tensile mechanics. Furthermore, our data suggest that mechanical stimulation during development is necessary to induce structural and mechanical changes at multiple physical length scales. This information provides valuable insight into the multiscale structure-function relationships of developing tendons and the importance of mechanical stimulation in producing a robust tensile load-bearing soft tissue.
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Project description:BACKGROUND:Tendon injury is a significant clinical problem due to poor healing and a high reinjury rate; successful treatment is limited by our poor understanding of endogenous tendon stem cells. Recent evidence suggests that adult stem cells are phenotypically diverse, even when comparing stem cells isolated from the same tissue from the same individual, and may in fact exist on a spectrum of proliferation and differentiation capacities. Additionally, the relationships between and clinical relevance of this phenotypic variation are poorly understood. In particular, tenogenic capacity has not been studied in comparison to tenogenic differentiation and cell proliferation. Toward this end, we performed a comprehensive assessment of cell proliferation and differentiation capacity toward four connective tissue lineages (tendon, cartilage, bone, and adipose) using tendon stem cell lines derived from single cells released directly from tendon tissue to (1) evaluate the differences, if any, in tenogenic potential, and (2) identify the relationships between differentiation phenotypes and proliferation capacity. METHODS:Tendon stem cells were derived from the endotenon of superficial digital flexor tendon from 3 horses. The cell suspension from each horse was separately plated simultaneously (1) at moderate density to generate a heterogenous population of cells-parent tendon cell line-and (2) at low density to separate single cells from each other to allow isolation of colonies that derive from single mother cells-clonal tendon stem cell lines. Thirty clonal tendon stem cell lines-10 from each horse-and each parent tendon cell line were assessed for tenogenesis, tri-lineage differentiation, and cell proliferation. Differentiation was confirmed by lineage-specific cell staining and quantified by the relative gene expression of lineage-specific markers. Statistical significance was determined using analysis of variance and post hoc Tukey's tests. RESULTS:Three distinct differentiation phenotypes-differentiation potency toward all 4 tissue lineages and two tri-lineage differentiation potencies-were identified in tendon clonal stem cell lines. These phenotypes were differentiation toward (1) tendon, cartilage, bone, and adipose (TCOA); (2) tendon, cartilage, and bone (TCO); and (3) tendon, cartilage, and adipose (TCA). Further, clonal cell lines that differentiated toward all four lineages had the highest expression of scleraxis and mohawk upon tenogenesis. Moreover, cell proliferation was significantly different between phenotypic groups, as evidenced by increased numbers of cumulative cell population doublings in clonal cell lines that did not differentiate toward adipose. CONCLUSIONS:Our study provides evidence of the heterogenous character of adult stem cells and identifies key differences in tendon stem cell differentiation and proliferative potentials from the same individual and from the same tendon. Isolation of tendon stem cell lines with the capacity to differentiate into all four connective tissue lineages may yield improved therapeutic benefits in clinical models of repair and promote a native, regenerative phenotype in engineered tendons. Future studies may be targeted to understanding the functional contributions of each tendon stem cell phenotype in vivo and identifying additional cell phenotypes.
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Project description:The enthesis is a mineralized fibrocartilage transition that attaches tendon to bone and is vital for musculoskeletal function. Despite recent studies demonstrating the necessity of muscle loading for enthesis formation, the mechanisms that regulate enthesis formation and mechanoresponsiveness remain unclear. Therefore, the current study investigated the role of the gap junction protein connexin 43 in these processes by deleting Gja1 (the Cx43 gene) in the tendon and enthesis. Compared with their wild-type (WT) counterparts, mice lacking Cx43 showed disrupted entheseal cell alignment, reduced mineralized fibrocartilage, and impaired biomechanical properties of the supraspinatus tendon entheses during postnatal development. Cx43-deficient mice also exhibited reduced ability to complete a treadmill running protocol but no apparent deficits in daily activity, metabolic indexes, shoulder muscle size, grip strength, and major trabecular bone properties of the adjacent humeral head. To examine enthesis mechanoresponsiveness, young adult mice were subjected to modest treadmill exercise. Gja1 deficiency in the tendon and enthesis reduced entheseal anabolic responses to treadmill exercise: WT mice had increased expression of Sox9, Ihh, and Gli1 and increased Brdu incorporation, whereas Cx43-deficient mice showed no changes or decreased levels with exercise. Collectively, the results demonstrated an essential role for Cx43 in postnatal tendon enthesis formation, function, and response to loading; results further provided evidence implicating a link between Cx43 function and the hedgehog signaling pathway. © 2020 American Society for Bone and Mineral Research.
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Project description:The process by which fibroblasts are directly reprogrammed into cardiomyocytes involves two stages; initiation and maturation. Initiation represents the initial expression of factors that induce fibroblasts to transdifferentiate into cardiomyocytes. Following initiation, the cell undergoes a period of maturation before becoming a mature cardiomyocyte. We wanted to understand the role of cardiac development transcription factors in the maturation process. We directly reprogram fibroblasts into cardiomyocytes by a combination of miRNAs (miR combo). The ability of miR combo to induce cardiomyocyte-specific genes in fibroblasts was lost following the knockdown of the cardiac transcription factors Gata4, Mef2C, Tbx5 and Hand2 (GMTH). To further clarify the role of GMTH in miR combo reprogramming we utilized a modified CRISPR-Cas9 approach to activate endogenous GMTH genes. Importantly, both miR combo and the modified CRISPR-Cas9 approach induced comparable levels of GMTH expression. While miR combo was able to reprogram fibroblasts into cardiomyocyte-like cells, the modified CRISPR-Cas9 approach could not. Indeed, we found that cardiomyocyte maturation only occurred with very high levels of GMT expression. Taken together, our data indicates that while endogenous cardiac transcription factors are insufficient to reprogram fibroblasts into mature cardiomyocytes, endogenous cardiac transcription factors are necessary for expression of maturation genes.
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