Tendon Matrix Research Articles

The Role of the Non-Collagenous Extracellular Matrix in Tendon and Ligament Mechanical Behavior: A Review.

Tendon is a connective tissue that transmits loads from muscle to bone, while ligament is a similar tissue that stabilizes joint articulation by connecting bone to bone. The 70-90% of tendon and ligament's extracellular matrix (ECM) is composed of a hierarchical collagen structure that provides resistance to deformation primarily in the fiber direction, and the remaining fraction consists of a variety of non-collagenous proteins, proteoglycans, and glycosaminoglycans (GAGs) whose mechanical roles are not well characterized. ECM constituents such as elastin, the proteoglycans decorin, biglycan, lumican, fibromodulin, lubricin, and aggrecan and their associated GAGs, and cartilage oligomeric matrix protein (COMP) have been suggested to contribute to tendon and ligament's characteristic quasi-static and viscoelastic mechanical behavior in tension, shear, and compression. The purpose of this review is to summarize existing literature regarding the contribution of the non-collagenous ECM to tendon and ligament mechanics, and to highlight key gaps in knowledge that future studies may address. Using insights from theoretical mechanics and biology, we discuss the role of the non-collagenous ECM in quasi-static and viscoelastic tensile, compressive, and shear behavior in the fiber direction and orthogonal to the fiber direction. We also address the efficacy of tools that are commonly used to assess these relationships, including enzymatic degradation, mouse knockout models, and computational models. Further work in this field will foster a better understanding of tendon and ligament damage and healing as well as inform strategies for tissue repair and regeneration.

Mesenchymal Stromal Cells Adapt to Chronic Tendon Disease Environment with an Initial Reduction in Matrix Remodeling

Tendon lesions are common sporting injuries in humans and horses alike. The healing process of acute tendon lesions frequently results in fibrosis and chronic disease. In horses, local mesenchymal stromal cell (MSC) injection is an accepted therapeutic strategy with positive influence on acute lesions. Concerning the use of MSCs in chronic tendon disease, data are scarce but suggest less therapeutic benefit. However, it has been shown that MSCs can have a positive effect on fibrotic tissue. Therefore, we aimed to elucidate the interplay of MSCs and healthy or chronically diseased tendon matrix. Equine MSCs were cultured either as cell aggregates or on scaffolds from healthy or diseased equine tendons. Higher expression of tendon-related matrix genes and tissue inhibitors of metalloproteinases (TIMPs) was found in aggregate cultures. However, the tenogenic transcription factor scleraxis was upregulated on healthy and diseased tendon scaffolds. Matrix metalloproteinase (MMPs) expression and activity were highest in healthy scaffold cultures but showed a strong transient decrease in diseased scaffold cultures. The release of glycosaminoglycan and collagen was also higher in scaffold cultures, even more so in those with tendon disease. This study points to an early suppression of MSC matrix remodeling activity by diseased tendon matrix, while tenogenic differentiation remained unaffected.

CTRP3 exacerbates tendinopathy by dysregulating tendon stem cell differentiation and altering extracellular matrix composition.

Tendinopathy, the most common disorder affecting tendons, is characterized by chronic disorganization of the tendon matrix, which leads to tendon tear and rupture. The goal was to identify a rational molecular target whose blockade can serve as a potential therapeutic intervention for tendinopathy. We identified C1q/TNF-related protein-3 (CTRP3) as a markedly up-regulated cytokine in human and rodent tendinopathy. Overexpression of CTRP3 enhanced the progression of tendinopathy by accumulating cartilaginous proteoglycans and degenerating collagenous fibers in the mouse tendon, whereas CTRP3 knockdown suppressed the tendinopathy pathogenesis. Functional blockade of CTRP3 using a neutralizing antibody ameliorated overuse-induced tendinopathy of the Achilles and rotator cuff tendons. Mechanistically, CTRP3 elicited a transcriptomic pattern that stimulates abnormal differentiation of tendon stem/progenitor cells and ectopic chondrification as an effect linked to activation of Akt signaling. Collectively, we reveal an essential role for CTRP3 in tendinopathy and propose a potential therapeutic strategy for the treatment of tendinopathy.

Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation in vivo. The transforming growth factor (TGF)-β, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-β3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-β3-induced tenogenic differentiation. The MSC were incubated with and without TGF-β3 (10 ng/ml), Y-27632 (10 μM), or both. On day 1 and day 3, the signalling pathway of TGF-β and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-β3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species (p < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-β3 and ROCK inhibition were combined (p < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-β3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-β3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.

Mesenchymal Stem Cell-Derived Extracellular Vesicles in Tendon and Ligament Repair-A Systematic Review of In Vivo Studies.

Tendon and ligament injury poses an increasingly large burden to society. This systematic review explores whether mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) can facilitate tendon/ligament repair in vivo. On 26 May 2021, a systematic search was performed on PubMed, Web of Science, Cochrane Library, Embase, to identify all studies that utilised MSC-EVs for tendon/ligament healing. Studies administering EVs isolated from human or animal-derived MSCs into in vivo models of tendon/ligament injury were included. In vitro, ex vivo, and in silico studies were excluded, and studies without a control group were excluded. Out of 383 studies identified, 11 met the inclusion criteria. Data on isolation, the characterisation of MSCs and EVs, and the in vivo findings in in vivo models were extracted. All included studies reported better tendon/ligament repair following MSC-EV treatment, but not all found improvements in every parameter measured. Biomechanics, an important index for tendon/ligament repair, was reported by only eight studies, from which evidence linking biomechanical alterations to functional improvement was weak. Nevertheless, the studies in this review showcased the safety and efficacy of MSC-EV therapy for tendon/ligament healing, by attenuating the initial inflammatory response and accelerating tendon matrix regeneration, providing a basis for potential clinical use in tendon/ligament repair.

Decellularized tendon matrix membranes prevent post-surgical tendon adhesion and promote functional repair

Adhesion often occurs after tendon injury, and results in sliding disorder and movement limitation with no ideal solution for it in clinic. In this study, an anti-adhesion membrane, i.e., decellularized tendon matrix (DTM) for tendon is successfully prepared by an optimized tendon decellularization method from homologous extracellular matrix. Microsection technology has been used to optimize the method of decellularization in order to better preserve the bioactive components in tissues and reduce the chemical reagent residues on the premise of effective decellularization with relatively shorter time and less reagents for decellularization. The physic-chemical properties and biological functions of DTM are evaluated, and high-throughput and high-precision tandem mass tags (TMT) labeling proteomics technology is used to analyze protein components of DTM, which may provide the scientific support for application of the innovative product. In vitro biosafety tests show that DTM not only is non-toxic but also promote cell proliferation. Subcutaneous implantation test confirms that DTM is completely degraded after 12 weeks and there is no obvious inflammatory reaction. The results of Achilles tendon repair in rabbits show that DTM can not only prevent tendon adhesion but also improve the quality of tendon repair, which demonstrates its tremendous application potential. Statement of SignificanceThere is no ideal solution for adhesion after tendon injury. In this study, a dense tendon anti-adhesion membrane (DTM) was successfully prepared from homologous extracellular matrix (ECM). This DTM could effectively retain bioactive ingredients, and prevent adhesion as well as improve the quality of tendon repair in vivo. An optimized decellularization method was used which could effectively decellularize tendon in a short time, better preserve bioactive components, and reduce reagent residues. For the first time, high-throughput and high-precision tandem mass tags (TMT) labeling proteomics technology was used to qualitatively and quantitatively analyze the protein composition of fresh tendon, acellular tendon and DTM, which provided not only scientific support for the application of DTM, but also comprehensive and accurate data support for related research of bovine tendons and decellularization.

In vitro and in vivo tenocyte-protective effectiveness of dehydroepiandrosterone against high glucose-induced oxidative stress

BackgroundDehydroepiandrosterone (DHEA), an adrenal steroid, has a protective role against diabetes. This study aimed to investigate the in vitro and in vivo protective effects of DHEA against high glucose-induced oxidative stress in tenocytes and tendons.MethodsTenocytes from normal Sprague-Dawley rats were cultured in low-glucose (LG) or high-glucose (HG) medium with or without DHEA. The experimental groups were: control group (LG without DHEA), LG with DHEA, HG without DHEA, and HG with DHEA. Reactive oxygen species (ROS) production, apoptosis, and messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, and interleukin-6 (IL-6) were determined. Further, diabetic rats were divided into a control group and a DHEA-injected group (DHEA group). NOX1 and NOX4 protein expression and mRNA expression of NOX1, NOX4, IL-6, matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-2, and type I and III collagens in the Achilles tendon were determined.ResultsIn rat tenocytes, DHEA decreased the expression of NOX1 and IL-6, ROS accumulation, and apoptotic cells. In the diabetic rat Achilles tendon, NOX1 protein expression and mRNA expression of NOX1, IL-6, MMP-2, TIMP-2, and type III collagen were significantly lower while type I collagen expression was significantly higher in the DHEA group than in the control group.ConclusionsDHEA showed antioxidant and anti-inflammatory effects both in vitro and in vivo. Moreover, DHEA improved tendon matrix synthesis and turnover, which are affected by hyperglycemic conditions. DHEA is a potential preventive drug for diabetic tendinopathy.

Reticulocalbin 3 is involved in postnatal tendon development by regulating collagen fibrillogenesis and cellular maturation

Tendon plays a critical role in the joint movement by transmitting force from muscle to bone. This transmission of force is facilitated by its specialized structure, which consists of highly aligned extracellular matrix consisting predominantly of type I collagen. Tenocytes, fibroblast-like tendon cells residing between the parallel collagen fibers, regulate this specialized tendon matrix. Despite the importance of collagen structure and tenocyte function, the biological mechanisms regulating fibrillogenesis and tenocyte maturation are not well understood. Here we examine the function of Reticulocalbin 3 (Rcn3) in collagen fibrillogenesis and tenocyte maturation during postnatal tendon development using a genetic mouse model. Loss of Rcn3 in tendon caused decreased tendon thickness, abnormal tendon cell maturation, and decreased mechanical properties. Interestingly, Rcn3 deficient mice exhibited a smaller collagen fibril distribution and over-hydroxylation in C-telopeptide cross-linking lysine from α1(1) chain. Additionally, the proline 3-hydroxylation sites in type I collagen were also over-hydroxylated in Rcn3 deficient mice. Our data collectively suggest that Rcn3 is a pivotal regulator of collagen fibrillogenesis and tenocyte maturation during postnatal tendon development.

The Lack of a Representative Tendinopathy Model Hampers Fundamental Mesenchymal Stem Cell Research.

Overuse tendon injuries are a major cause of musculoskeletal morbidity in both human and equine athletes, due to the cumulative degenerative damage. These injuries present significant challenges as the healing process often results in the formation of inferior scar tissue. The poor success with conventional therapy supports the need to search for novel treatments to restore functionality and regenerate tissue as close to native tendon as possible. Mesenchymal stem cell (MSC)-based strategies represent promising therapeutic tools for tendon repair in both human and veterinary medicine. The translation of tissue engineering strategies from basic research findings, however, into clinical use has been hampered by the limited understanding of the multifaceted MSC mechanisms of action. In vitro models serve as important biological tools to study cell behavior, bypassing the confounding factors associated with in vivo experiments. Controllable and reproducible in vitro conditions should be provided to study the MSC healing mechanisms in tendon injuries. Unfortunately, no physiologically representative tendinopathy models exist to date. A major shortcoming of most currently available in vitro tendon models is the lack of extracellular tendon matrix and vascular supply. These models often make use of synthetic biomaterials, which do not reflect the natural tendon composition. Alternatively, decellularized tendon has been applied, but it is challenging to obtain reproducible results due to its variable composition, less efficient cell seeding approaches and lack of cell encapsulation and vascularization. The current review will overview pros and cons associated with the use of different biomaterials and technologies enabling scaffold production. In addition, the characteristics of the ideal, state-of-the-art tendinopathy model will be discussed. Briefly, a representative in vitro tendinopathy model should be vascularized and mimic the hierarchical structure of the tendon matrix with elongated cells being organized in a parallel fashion and subjected to uniaxial stretching. Incorporation of mechanical stimulation, preferably uniaxial stretching may be a key element in order to obtain appropriate matrix alignment and create a pathophysiological model. Together, a thorough discussion on the current status and future directions for tendon models will enhance fundamental MSC research, accelerating translation of MSC therapies for tendon injuries from bench to bedside.

Preparation and biological characteristics of a bovine acellular tendon fiber material.

Acellular tendon matrix is an ideal substitute for constructing tissue engineering ligaments, but using detergents causes damage to collagen and fibrin during the process of decellularization. In this study, fresh tendons were lyophilized and separated into fresh tendon fiber (FTF) bundles, and then the cellular components in FTF were removed to prepare acellular tendon fiber (ATF) without adding chemical detergent. H&E staining and DAPI fluorescence microscopy showed no nucleus and DNA residue. Compared with FTFs, the DNA content of ATFs was significantly lower without the collagen content change before and after decellularization. The microstructure of collagen fibrils in ATFs was intact under scanning electron microscopy (SEM), and the maximum tensile load and elastic modulus between FTFs and ATFs were not statistically different. The ATF bundles were cultured with SD rat tenocytes for 72 hr and cells attachment to fiber surfaces were observed under SEM. ATF bundles were then implanted into paraspinal muscles, and histological analysis showed fibroblast-like cells within the ATFs and was similar to the control group (fresh tendon autograft) in morphology. H&E staining showed that the number of lymphocytes and plasma cells in ATF was less than that in fresh tendon autograft. ATF bundles were twisted into linear fiber materials by hand, of which the maximum breaking strength was similar to silk with same diameter. These findings demonstrated that ATFs retain their original fibril structure and mechanical properties after decellularization by trypsin and pancreatic deoxyribonuclease without detergent. Lyophilized ATFs linear fiber material provides the possibility of preparing personalized ligament and other tissue engineering scaffolds.

Inhibition of ERK 1/2 kinases prevents tendon matrix breakdown

Tendon extracellular matrix (ECM) mechanical unloading results in tissue degradation and breakdown, with niche-dependent cellular stress directing proteolytic degradation of tendon. Here, we show that the extracellular-signal regulated kinase (ERK) pathway is central in tendon degradation of load-deprived tissue explants. We show that ERK 1/2 are highly phosphorylated in mechanically unloaded tendon fascicles in a vascular niche-dependent manner. Pharmacological inhibition of ERK 1/2 abolishes the induction of ECM catabolic gene expression (MMPs) and fully prevents loss of mechanical properties. Moreover, ERK 1/2 inhibition in unloaded tendon fascicles suppresses features of pathological tissue remodeling such as collagen type 3 matrix switch and the induction of the pro-fibrotic cytokine interleukin 11. This work demonstrates ERK signaling as a central checkpoint to trigger tendon matrix degradation and remodeling using load-deprived tissue explants.

Cell-Free Book-Shaped Decellularized Tendon Matrix Graft Capable of Controlled Release of BMP-12 to Improve Tendon Healing in a Rat Model

Background: Achilles tendon (AT) defects often occur in traumatic and chronic injuries. Currently, no graft can satisfactorily regenerate parallel tendinous tissue at the defect site to completely restore AT function. Purpose: To develop a cell-free functional graft by tethering bone morphogenetic protein 12 (BMP-12) on a book-shaped decellularized tendon matrix (BDTM) and to determine whether this graft is more beneficial for AT defect healing than an autograft. Study Design: Controlled laboratory study. Methods: Canine patellar tendon was sectioned into a book shape and decellularized to fabricate a BDTM. The collagen-binding domain (CBD) was fused into the N-terminus of BMP-12 to synthesize a recombinant BMP-12 (CBD-BMP-12), which was tethered to the BDTM to prepare a cell-free functional graft (CBD-BMP-12/BDTM). After its tensile resistance, tenogenic inducibility, and BMP-12 release dynamics were evaluated, the efficacy of the graft for tendon regeneration was determined in a rat model. A total of 140 mature male Sprague-Dawley rats underwent AT tenotomy. The defect was reconstructed with reversed AT (autograft group), native BMP-12 tethered to an intact decellularized tendon matrix (IDTM; NAT-BMP-12/IDTM group), native BMP-12 tethered to a BDTM (NAT-BMP-12/BDTM group), CBD-BMP-12 tethered on an IDTM (CBD-BMP-12/IDTM group), and CBD-BMP-12 tethered on a BDTM (CBD-BMP-12/BDTM group). The rats were sacrificed 4 or 8 weeks after surgery to harvest AT specimens. Six specimens from each group at each time point were used for histological evaluation; the remaining 8 specimens were used for biomechanical testing. Results: In vitro CBD-BMP-12/BDTM was noncytotoxic, showed high biomimetics with native tendons, was suitable for cell adhesion and growth, and had superior tenogenic inducibility. In vivo the defective AT in the CBD-BMP-12/BDTM group regenerated more naturally than in the other groups, as indicated by more spindle-shaped fibroblasts embedded in a matrix of parallel fibers. The biomechanical properties of the regenerated AT in the CBD-BMP-12/BDTM group also increased more significantly than in the other groups. Conclusion: CBD-BMP-12/BDTM is more beneficial than autograft for healing AT defects in a rat model. Clinical Relevance: The findings of this study demonstrate that CBD-BMP-12/BDTM can serve as a practical graft for reconstructing AT defects.

Multi-component relaxation modelling in human Achilles tendon: Quantifying chemical shift information in ultra-short echo time imaging.

To examine multi-component relaxation modelling for quantification of on- and off-resonance relaxation signals in multi-echo ultra-short echo time (UTE) data of human Achilles tendon (AT) and compare bias and dispersion errors of model parameters to that of the bi-component model. Multi-component modelling is demonstrated for quantitative multi-echo UTE analysis of AT and supported using a novel method for determining number of MR-visible off-resonance components, UTE data from six healthy volunteers, and analysis of proton NMR measurements from ex vivo bovine AT. Cramer-Rao lower bound expressions are presented for multi- and bi-component models and parameter estimate variances are compared. Bias error in bi-component estimates is characterized numerically. Two off-resonance components were consistently detected in all six volunteers and in bovine AT data. Multi-component model exhibited superior quality of fit, with a marginal increase in estimate variance, when compared to the bi-component model. Bi-component estimates exhibited notable bias particularly in in the presence of off-resonance components. Multi-component modelling more reliably quantifies tendon matrix water components while also providing quantitation of additional non-water matrix constituents. Further work is needed to interpret the origin of the observed off-resonance signals with preliminary assignments made to chemical groups in lipids and proteoglycans.

Synergistic enhancement of tendon-to-bone healing via anti-inflammatory and pro-differentiation effects caused by sustained release of Mg2+/curcumin from injectable self-healing hydrogels.

Poor healing response after rotator cuff reconstruction is multifactorial, with the inflammatory microenvironment and deficiency of stem cell differentiation factors at the lesion site being most relevant. However, there is a lack of effective tissue engineering strategies that can simultaneously exert anti-inflammatory and pro-differentiation effects to promote rotator cuff healing.Methods: In this study, we synthesized and characterized a novel active drug delivery vector that successfully overcame the challenge of simultaneous high-efficiency loading and controlled release of Mg2+ and curcumin. The anti-inflammatory and pro-differentiation effects of the composite hydrogel were evaluated in vitro and in vivo. Moreover, healing of the rotator cuff tendon-to-bone interface was studied by histology, immunofluorescence, and biomechanical tests.Results: The composite hydrogel exhibited excellent biocompatibility and injectability, good adhesiveness, and rapid self-healing. The released curcumin showed obvious anti-inflammatory and antioxidation effects, which protected stem cells and tendon matrix. Furthermore, released Mg2+ promoted stem cell aggregation and chondrogenesis. Moreover, biomechanical tests and histological results of a rat rotator cuff tear model at 8 weeks after surgery indicated that the composite hydrogel significantly enhanced tendon-to-bone healing.Conclusions: The composite hydrogel mediated sustained in situ release of curcumin and Mg2+ to effectively promote rotator cuff tendon-to-bone healing via anti-inflammatory and pro-differentiation effects. Therefore, this composite hydrogel offers significant promise for rotator cuff repair.

Effect of Metformin on Development of Tendinopathy Due to Mechanical Overloading in an Animal Model.

Tendinopathy is a debilitating tendon disorder that affects millions of Americans and costs billions of health care dollars every year. High mobility group box 1 (HMGB1), a known tissue damage signaling molecule, has been identified as a mediator in the development of tendinopathy due to mechanical overloading of tendons in mice. Metformin (Met), a drug approved by the Food and Drug Administration used for the treatment of type 2 diabetes, specifically inhibits HMGB1. This study tested the hypothesis that Met would prevent mechanical overloading-induced tendinopathy in a mouse model of tendinopathy created by intensive treadmill running (ITR). C57BL/6J mice (female, 3 months old) were equally separated into 4 groups and treated for 24 weeks as follows: group 1 had cage control activities, group 2 received a single intraperitoneal injection of Met (50 mg/kg body weight) daily, group 3 underwent ITR to induce tendinopathy, and group 4 received daily Met injection along with ITR to inhibit HMGB1. Tendinopathic changes were assessed in Achilles tendons of all mice using histology, immunohistochemistry, and enzyme-linked immunosorbent assays. ITR induced HMGB1 release into the tendon matrix and developed characteristics of tendinopathy as evidenced by the expression of macrophage marker CD68, proinflammatory molecules (COX-2, PGE2), cell morphological changes from normal elongated cells to round cells, high levels of expression of chondrogenic markers (SOX-9, collagen type II), and accumulation of proteoglycans in tendinopathic tendons. Daily injection of Met inhibited HMGB1 release and decreased these degenerative changes in ITR tendons. Inhibition of HMGB1 by injections of Met prevented tendinopathy development due to mechanical overloading in the Achilles tendon in mice. Met may be able to be repurposed as a therapeutic option for preventing the development of tendinopathy in high-risk patients.

Regeneration of a full-thickness defect in rotator cuff tendon with umbilical cord-derived mesenchymal stem cells in a rat model.

Although rotator cuff disease is a common cause of shoulder pain, there is still no treatment method that could halt or reveres its development and progression. The purpose of this study was to investigate the efficacy of umbilical cord-derived mesenchymal stem cells (UC MSCs) on the regeneration of a full-thickness rotator cuff defect (FTD) in a rat model. We injected either UC MSCs or saline to the FTD and investigated macroscopic, histological and biomechanical results and cell trafficking. Treatment with UC MSCs improved macroscopic appearance in terms of tendon thickness at two weeks, and inflammation, defect size, swelling/redness and connection surrounding tissue and slidability at four weeks compared to the saline group. Histologically, UC MSCs induced the tendon matrix formation recovering collagen organization, nuclear aspect ratio and orientation angle of fibroblast as well as suppressing cartilage-related glycosaminoglycan compared to saline group at four weeks. The UC MSCs group also improved ultimate failure load by 25.0% and 19.0% and ultimate stress by 27.3% and 26.8% at two and four weeks compared to saline group. UC MSCs labeled with PKH26 exhibited 5.3% survival at four weeks compared to three hours after injection. This study demonstrated that UC MSCs regenerated the FTD with tendon tissue similar properties to the normal tendon in terms of macroscopic, histological and biomechanical characteristics in a rat model.

Ultrastructural evidence for telocytes in equine tendon.

The three-dimensional ultrastructure of the tendon is complex. Two main cell types are classically supported: elongated tenocytes and ovoid tenoblasts. The existence of resident stem/progenitor cells in human and equine tendons has been demonstrated, but their location and relationship to tenoblasts and tenocytes remain unclear. Hence, in this work, we carried out an ultrastructural study of the equine superficial digital flexor tendon. Although the fine structure of tendons has been previously studied using electron microscopy, the presence of telocytes, a specific type of interstitial cell, has not been described thus far. We show the presence of telocytes in the equine inter-fascicular tendon matrix near blood vessels. These telocytes have characteristic telopodes, which are composed of alternating dilated portions (podoms) and thin segments (podomers). Additionally, we demonstrate the presence of the primary cilium in telocytes and its ability to release exosomes. The location of telocytes is similar to that of tendon stem cells. The telocyte-blood vessel proximity, the presence of primary immotile cilia and the release of exosomes could have special significance for tendon homeostasis.

2020 J. Leonard Goldner Award Winner: Inhibition of HMGB1 by Metformin Prevents Mechanical Overloading-Induced Tendinopathy

Category:Basic Sciences/Biologics; SportsIntroduction/Purpose:Tendinopathy is a debilitating tendon disorder that affects millions of Americans and costs billions of healthcare dollars every year. Mechanical overloading is considered to cause the development of tendinopathy, but the underlying molecular mechanisms of tendinopathy remain unclear. High mobility group box-1 (HMGB1), an upstream potent inflammatory mediator, has been identified in high levels in early stage tendinopathy patients [1]. However, whether HMGB1 mediates tendinopathy development due to mechanical overloading is completely unknown. Metformin (Met), a hypoglycemic drug commonly used for the treatment of type II diabetes, has shown to inhibit the activity of HMGB1 via binding the acidic tail of HMGB1 [2]. In this study, we tested the hypothesis that Met prevents mechanical overloading-induced tendinopathy by inhibiting HMGB1.Methods:A total of 24 mice were divided into 4 groups and treated for 24 weeks as follows: Group 1 (Cage) with cage activities; Group 2 (Met) received daily IP injection of metformin (50 mg/kg body weight); Group 3 (ITR) ran on treadmill at 15 meters/min for 3 h/ day, 5 days a week; Group 4 (ITR+Met) ran the same protocol as that of ITR group but with daily IP injection of metformin. Six mice/group were sacrificed at 24 weeks and the Achilles and patellar tendon tissues were harvested. The tendons from the left legs were used for histochemical staining and the right for immunostaining.Results:We found that mechanical overloading induced HMGB1 release into tendon matrix (Fig. 1G, K, O). Metformin inhibited HMGB1 release (Fig. 1H, L, P). ITR induced degenerative tendinopathy as evidenced by the cell morphological changes from elongated shape in normal tendon (Fig. 2A, E, I, M) to round shape (Fig. 2C, G, K, O) and the accumulation of proteoglycans (Fig. 2K, O) in ITR tendon. Metformin injection inhibited ITR effect, which is shown by less round shaped cells and low proteoglycan levels found in metformin injected ITR tendons (Fig. 2D, H, L, P). ITR promoted the expression of chondrogenic markers (collagen II and SOX-9) in tendon (Fig. 3C, G, K, O), and metformin inhibited the expression of chondrogenic makers (Fig. 3D, H, L, P).Conclusion:Our study demonstrated that mechanical overloading induced degenerative changes in mouse tendons characterized by the presence of chondrocyte-like cells, accumulation of proteoglycans, high levels of chondrogenic marker SOX-9 and Collagen II expression. Administration of metformint reduced the degenerative responses in overloaded tendon and blocked the development of tendinopathy. These findings support the notion that mechanical overloading induces tendinopathy development by initiation of tendon inflammation via HMGB1, which leads to eventual tendon degeneration. Thus, metformin, a commonly prescribed and FDA approved drug that specifically inhibits HMGB1, can be used to prevent tendinopathy development due to mechanical overloading placed on the tendon.

Adding exogenous biglycan or decorin improves tendon formation for equine peritenon and tendon proper cells in vitro

BackgroundTendon injuries amount to one of the leading causes of career-ending injuries in horses due to the inability for tendon to completely repair and the high reinjury potential. As a result, novel therapeutics are necessary to improve repair with the goal of decreasing leg lameness and potential reinjury. Small leucine-rich repeat proteoglycans (SLRPs), a class of regulatory molecules responsible for collagen organization and maturation, may be one such therapeutic to improve tendon repair. Before SLRP supplementation can occur in vivo, proper evaluation of the effect of these molecules in vitro needs to be assessed. The objective of this study was to evaluate the effectiveness of purified bovine biglycan or decorin on tendon proper and peritenon cell populations in three-dimensional tendon constructs.MethodsEquine tendon proper or peritenon cell seeded fibrin three-dimensional constructs were supplemented with biglycan or decorin at two concentrations (5 nM or 25 nM). The functionality and ultrastructural morphology of the constructs were assessed using biomechanics, collagen content analysis, transmission electron microscopy (TEM), and gene expression by real time – quantitative polymerase chain reaction (RT-qPCR).ResultsSLRP supplementation affected both tendon proper and peritenon cells-seeded constructs. With additional SLRPs, material and tensile properties of constructs strengthened, though ultrastructural analyses indicated production of similar-sized or smaller fibrils. Overall expression of tendon markers was bolstered more in peritenon cells supplemented with either SLRP, while supplementation of SLRPs to TP cell-derived constructs demonstrated fewer changes in tendon and extracellular matrix markers. Moreover, relative to non-supplemented tendon proper cell-seeded constructs, SLRP supplementation of the peritenon cells showed increases in mechanical strength, material properties, and collagen content.ConclusionsThe SLRP-supplemented peritenon cells produced constructs with greater mechanical and material properties than tendon proper seeded constructs, as well as increased expression of matrix assembly molecules. These findings provide evidence that SLRPs should be further investigated for their potential to improve tendon formation in engineered grafts or post-injury.

Elastin levels are higher in healing tendons than in intact tendons and influence tissue compliance.

Elastic fibers containing elastin play an important role in tendon functionality, but the knowledge on presence and function of elastin during tendon healing is limited. The aim of this study was to investigate elastin content and distribution in intact and healing Achilles tendons and to understand how elastin influence the viscoelastic properties of tendons. The right Achilles tendon was completely transected in 81 Sprague-Dawley rats. Elastin content was quantified in intact and healing tendons (7, 14, and 28days post-surgery) and elastin distribution was visualized by immunohistochemistry at 14days post-surgery. Degradation of elastin by elastase incubation was used to study the role of elastin on viscoelastic properties. Mechanical testing was either performed as a cyclic test (20× 10N) or as a creep test. We found significantly higher levels of elastin in healing tendons at all time-points compared to intact tendons (4% in healing tendons 28days post-surgery vs 2% in intact tendons). The elastin was more widely distributed throughout the extracellular matrix in the healing tendons in contrast to the intact tendon where the distribution was not so pronounced. Elastase incubation reduced the elastin levels by approximately 30% and led to a 40%-50% reduction in creep. This reduction was seen in both intact and healing tendons. Our results show that healing tendons contain more elastin and is more compliable than intact tendons. The role of elastin in tendon healing and tissue compliance indicates a protective role of elastic fibers to prevent re-injuries during early tendon healing. PLAIN LANGUAGE SUMMARY: Tendons transfer high loads from muscles to bones during locomotion. They are primarily made by the protein collagen, a protein that provide strength to the tissues. Besides collagen, tendons also contain other building blocks such as, for example, elastic fibers. Elastic fibers contain elastin and elastin is important for the extensibility of the tendon. When a tendon is injured and ruptured the tissue heals through scar formation. This scar tissue is different from a normal intact tendon and it is important to understand how the tendons heal. Little is known about the presence and function of elastin during healing of tendon injuries. We have shown, in animal experiments, that healing tendons have higher amounts of elastin compared to intact tendons. The elastin is also spread throughout the tissue. When we reduced the levels of this protein, we discovered altered mechanical properties of the tendon. The healing tendon can normally extend quite a lot, but after elastin removal this extensibility was less obvious. The ability of the healing tissue to extend is probably important to protect the tendon from re-injuries during the first months after rupture. We therefore propose that the tendons heal with a large amount of elastin to prevent re-ruptures during early locomotion.