Evaluation of two distinct placental-derived membranes and their effect on tenocyte responses in vitro.
ABSTRACT: Tendon healing is a complex, multiphase process that results in increased scar tissue formation, leading to weaker tendons. The purpose of this study was to evaluate the response of tenocytes to both hypothermically stored amniotic membrane (HSAM) and dehydrated amnion/chorion membrane (dACM). Composition and growth factor release from HSAM and dACM were evaluated using proteomics microarrays. HSAM and dACM releasate was used to assess tenocyte proliferation, migration, gene expression, extracellular matrix (ECM) protein deposition, and response to inflammation. Additionally, tenocyte-ECM interactions were evaluated. HSAM and dACM contain and release growth factors relevant to tendon healing, including insulin-like growth factor I, platelet-derived growth factor, and basic fibroblast growth factor. Both dACM and HSAM promoted increased tenocyte proliferation and migration; tenocytes treated with dACM proliferated more robustly, whereas treatment with HSAM resulted in higher migration. Both dACM and HSAM resulted in altered ECM gene expression; dACM grafts alone resulted in increases in collagen deposition. Furthermore, both allografts resulted in altered tenocyte responses to inflammation with reduced transforming growth factor beta levels. Additionally, dACM treatment resulted in increased expression and production of matrix metalloprotease-1 (MMP-1), whereas HSAM treatment resulted in decreased production of MMP-1. Tenocytes migrated into and remodeled HSAM only. These results indicate that both grafts have properties that support tendon healing; however, the results presented here suggest that the responses to each type of graft may be different. Due to the complex environment during tendon repair, additional work is needed to evaluate these effects using in vivo models.
Project description:The extracellular matrix (ECM) is the primary biomechanical environment that interacts with tendon cells (tenocytes). Stresses applied via muscle contraction during skeletal movement transfer across structural hierarchies to the tenocyte nucleus in native uninjured tendons. Alterations to ECM structural and mechanical properties due to mechanical loading and tissue healing may affect this multiscale strain transfer and stress transmission through the ECM. This study explores the interface between dynamic loading and tendon healing across multiple length scales using living tendon explants. Results show that macroscale mechanical and structural properties are inferior following high magnitude dynamic loading (fatigue) in uninjured living tendon and that these effects propagate to the microscale. Although similar macroscale mechanical effects of dynamic loading are present in healing tendon compared to uninjured tendon, the microscale properties differed greatly during early healing. Regression analysis identified several variables (collagen and nuclear disorganization, cellularity, and F-actin) that directly predict nuclear deformation under loading. Finite element modeling predicted deficits in ECM stress transmission following fatigue loading and during healing. Together, this work identifies the multiscale response of tendon to dynamic loading and healing, and provides new insight into microenvironmental features that tenocytes may experience following injury and after cell delivery therapies.
Project description:The human amniotic membrane has been a subject for clinical and basic research for nearly 100 years, but weak rejection has been reported. The purpose of this research is to remove the cellular components of the amnion for eliminating its immune-inducing activity to the utmost extent. The amniotic membrane treated by acid removed the epithelial cell, fibroblast, and sponge layers and retained only the basal and dense layers. In vitro, biological effects of the new material on tenocytes were evaluated. The levels of transforming growth factor (TGF-?1), fibroblast growth factor (bFGF) proteins were measured. In vivo, the tendon injury model of chickens was constructed to observe effects on tendon adhesion and healing. The acellular amniotic membrane effectively removed the cell components of the amnion while retaining the fibrous reticular structure. Abundant collagen fibers enhanced the tensile strength of amnion, and a 3D porous structure provided enough 3D space structure for tenocyte growth. In vitro, acellular amnion resulted in the fast proliferation trend for tenocytes with relatively static properties by releasing TGF-?1 and bFGF. In vivo, the experiment revealed the mechanism of acellular amnion in promoting endogenous healing and barrier exogenous healing by evaluating tendon adhesion, biomechanical testing, and labeling fibroblasts/tendon cells and monocytes/macrophages with vimentin and CD68. The acellular amnion promotes endogenous healing and barrier exogenous healing by releasing the growth factors such as TGF-?1 and bFGF, thereby providing a new direction for the prevention and treatment of tendon adhesion.
Project description:Cell-based tendon therapies with tenocytes as a cell source need effective tenocyte in vitro expansion before application for tendinopathies and tendon injuries. Supplementation of tenocyte culture with biomolecules that can boost proliferation and matrix synthesis is one viable option for supporting cell expansion. In this in vitro study, the impacts of ascorbic acid or PDGF-BB supplementation on rabbit Achilles tenocyte culture were studied. Namely, cell proliferation, changes in gene expression of several ECM and tendon markers (collagen I, collagen III, fibronectin, aggrecan, biglycan, decorin, ki67, tenascin-C, tenomodulin, Mohawk, ?-SMA, MMP-2, MMP-9, TIMP1, and TIMP2) and ECM deposition (collagen I and fibronectin) were assessed. Ascorbic acid and PDGF-BB enhanced tenocyte proliferation, while ascorbic acid significantly accelerated the deposition of collagen I. Both biomolecules led to different changes in the gene expression profile of the cultured tenocytes, where upregulation of collagen I, Mohawk, decorin, MMP-2, and TIMP-2 was observed with ascorbic acid, while these markers were downregulated by PDGF-BB supplementation. Vice versa, there was an upregulation of fibronectin, biglycan and tenascin-C by PDGF-BB supplementation, while ascorbic acid led to a downregulation of these markers. However, both biomolecules are promising candidates for improving and accelerating the in vitro expansion of tenocytes, which is vital for various tendon tissue engineering approaches or cell-based tendon therapy.
Project description:Mechanical forces between cells and extracellular matrix (ECM) influence cell shape and function. Tendons are ECM-rich tissues connecting muscles with bones that bear extreme tensional force. Analysis of transgenic zebrafish expressing mCherry driven by the tendon determinant scleraxis reveals that tendon fibroblasts (tenocytes) extend arrays of microtubule-rich projections at the onset of muscle contraction. In the trunk, these form a dense curtain along the myotendinous junctions at somite boundaries, perpendicular to myofibers, suggesting a role as force sensors to control ECM production and tendon strength. Paralysis or destabilization of microtubules reduces projection length and surrounding ECM, both of which are rescued by muscle stimulation. Paralysis also reduces SMAD3 phosphorylation in tenocytes and chemical inhibition of TGF? signaling shortens tenocyte projections. These results suggest that TGF?, released in response to force, acts on tenocytes to alter their morphology and ECM production, revealing a feedback mechanism by which tendons adapt to tension.
Project description:Tendon injuries cause prolonged disability and never recover completely. Current mechanistic understanding of tendon regeneration is limited. Here, we use single-cell transcriptomics to identify a tubulin polymerization-promoting protein family member 3-expressing (Tppp3+) cell population as potential tendon stem cells. Through inducible lineage tracing, we demonstrate that these cells can generate new tenocytes and self-renew upon injury. A fraction of Tppp3+ cells expresses platelet-derived growth factor receptor alpha (Pdfgra). Ectopic platelet-derived growth factor-AA (PDGF-AA) protein induces new tenocyte production while inactivation of Pdgfra in Tppp3+ cells blocks tendon regeneration. These results support Tppp3+Pdgfra+ cells as tendon stem cells. Unexpectedly, Tppp3-Pdgfra+ fibro-adipogenic progenitors coexist in the tendon stem cell niche and give rise to fibrotic cells, revealing a clandestine origin of fibrotic scars in healing tendons. Our results explain why fibrosis occurs in injured tendons and present clinical challenges to enhance tendon regeneration without a concurrent increase in fibrosis by PDGF application.
Project description:Age-related tendon degeneration (tendinosis) is characterized by a phenotypic change in which tenocytes display characteristics of fibrochondrocytes and mineralized fibrochondrocytes. As tendon degeneration has been noted in vivo in areas of decreased tendon vascularity, we hypothesized that hypoxia is responsible for the development of the tendinosis phenotype, and that these effects are more pronounced in aged tenocytes. Hypoxic (1% O2 ) culture of aged, tendinotic, and young human tenocytes resulted in a mineralized fibrochondrocyte phenotype in aged tenocytes, and a fibrochondrocyte phenotype in young and tendinotic tenocytes. Investigation of the molecular mechanism responsible for this phenotype change revealed that the fibrochondrocyte phenotype in aged tenocytes occurs with decreased Rac1 activity in response to hypoxia. In young hypoxic tenocytes, however, the fibrochondrocyte phenotype occurs with concomitant decreased Rac1 activity coupled with increased RhoA activity. Using pharmacologic and adenoviral manipulation, we confirmed that these hypoxic effects on the tenocyte phenotype are linked directly to the activity of RhoA/Rac1 GTPase in in vitro human cell culture and tendon explants. These results demonstrate that hypoxia drives tenocyte phenotypic changes, and provide a molecular insight into the development of human tendinosis that occurs with aging.
Project description:OBJECTIVES:Tissue engineering is a promising approach for repair of tendon injuries. Adipose-derived mesenchymal stem cells (ADMSCs) have gained increasing research interest for their potential in improving healing and regeneration of injured tendons. The present study aimed to investigate effects of O2 tension and potential signalling pathways on AMDSC differentiation into tenocytes, in an indirect co-culture system. MATERIALS AND METHODS:Human ADMSCs were co-cultured under normoxia (20% O2 ) and also under hypoxia (2% O2 ). Tenocyte differentiation of AMDSCs and expression of hypoxia-inducible factor-1 (HIF-1?) were analysed by reverse transcription-PCR, Western blotting and immunohistochemistry. Furthermore, HIF-1? inhibitor and inducer (FG-4592) effects on differentiation of AMDSCs were studied using qPCR, immunofluorescence and Western blotting. RESULTS:Indirect co-culture with tenocytes increased differentiation of ADMSCs into tenocytes; furthermore, hypoxia further enhanced tenocyte differentiation of AMDSCs, accompanied by increased expression of HIF-1?. HIF-1? inhibitor attenuated effects of hypoxia on differentiation of ADMSCs; in contrast, FG-4592 increased differentiation of ADMSCs under both hypoxia and normoxia. CONCLUSIONS:Taken together, we found that growing ADMSCs under hypoxia, or activating expression of HIF-1? to be important in differentiation of ADMSCs, which provides a foundation for application of ADMSCs in vivo for tendon regeneration.
Project description:It was reported that substance P had beneficial effects in the healing of acute tendon injury. However, the relationship between substance P and degenerative tendinopathy development remains unclear. The purpose of this study was to determine the role of substance P in the pathogenesis of tendinopathy. Healthy and tendinopathy tendon were harvested from human and tenocytes were cultured individually. The expression levels of genes associated with tendinopathy were compared. Next, substance P was exogenously administered to the healthy tenocyte and the effect was evaluated. The results showed that tendinopathy tenocytes had higher levels of COL3A1, MMP1, COX2, SCX, ACTA2, and substance P gene expression compared to healthy tenocytes. Next, substance P treatment on the healthy tenocyte displayed similar changes to that of the tendinopathy tenocytes. These differences between the two groups were also determined by Western blot. Additionally, cells with substance P had the tendinopathy change morphologically although cellular proliferation was significantly higher compared to that of the control group. In conclusion, substance P enhanced cellular proliferation, but concomitantly increased immature collagen (type 3 collagen). Substance P plays a crucial role in tendinopathy development and could be a future therapeutic target for treatment.
Project description:Mechanical forces influence homeostasis in virtually every tissue [1, 2]. Tendon, constantly exposed to variable mechanical force, is an excellent model in which to study the conversion of mechanical stimuli into a biochemical response [3-5]. Here we show in a mouse model of acute tendon injury and in vitro that physical forces regulate the release of active transforming growth factor (TGF)-? from the extracellular matrix (ECM). The quantity of active TGF-? detected in tissue exposed to various levels of tensile loading correlates directly with the extent of physical forces. At physiological levels, mechanical forces maintain, through TGF-?/Smad2/3-mediated signaling, the expression of Scleraxis (Scx), a transcription factor specific for tenocytes and their progenitors. The gradual and temporary loss of tensile loading causes reversible loss of Scx expression, whereas sudden interruption, such as in transection tendon injury, destabilizes the structural organization of the ECM and leads to excessive release of active TGF-? and massive tenocyte death, which can be prevented by the TGF-? type I receptor inhibitor SD208. Our findings demonstrate a critical role for mechanical force in adult tendon homeostasis. Furthermore, this mechanism could translate physical force into biochemical signals in a much broader variety of tissues or systems in the body.
Project description:Mechanical loading constantly acts on tendons, and a better understanding of its effects on the tendons is essential to gain more insights into tendon patho-physiology. This study aims to investigate tendon mechanobiological responses through the use of mouse treadmill running as an in vivo model and mechanical stretching of tendon cells as an in vitro model. In the in vivo study, mice underwent moderate treadmill running (MTR) and intensive treadmill running (ITR) regimens. Treadmill running elevated the expression of mechanical growth factors (MGF) and enhanced the proliferative potential of tendon stem cells (TSCs) in both patellar and Achilles tendons. In both tendons, MTR upregulated tenocyte-related genes: collagen type I (Coll. I ?10 fold) and tenomodulin (?3-4 fold), but did not affect non-tenocyte-related genes: LPL (adipocyte), Sox9 (chondrocyte), Runx2 and Osterix (both osteocyte). However, ITR upregulated both tenocyte (Coll. I ?7-11 fold; tenomodulin ?4-5 fold) and non-tenocyte-related genes (?3-8 fold). In the in vitro study, TSCs and tenocytes were stretched to 4% and 8% using a custom made mechanical loading system. Low mechanical stretching (4%) of TSCs from both patellar and Achilles tendons increased the expression of only the tenocyte-related genes (Coll. I ?5-6 fold; tenomodulin ?6-13 fold), but high mechanical stretching (8%) increased the expression of both tenocyte (Coll. I ?28-50 fold; tenomodulin ?14-48 fold) and non-tenocyte-related genes (2-5-fold). However, in tenocytes, non-tenocyte related gene expression was not altered by the application of either low or high mechanical stretching. These findings indicate that appropriate mechanical loading could be beneficial to tendons because of their potential to induce anabolic changes in tendon cells. However, while excessive mechanical loading caused anabolic changes in tendons, it also induced differentiation of TSCs into non-tenocytes, which may lead to the development of degenerative tendinopathy frequently seen in clinical settings.