Galangin Promotes Tendon Repair Mediated by Tendon-Derived Stem Cells through Activating the TGF-ß1/Smad3 Signaling Pathway.

Tendon injury is a prevalent orthopedic disease that currently lacks effective treatment. Galangin (GLN) is a vital flavonoid found abundantly in galangal and is known for its natural activity. This study aimed to investigate the GLN-mediated molecular mechanism of tendon-derived stem cells (TDSCs) in tendon repair. The TDSCs were characterized using alkaline phosphatase staining, alizarin red S staining, oil red O staining, and flow cytometry. The effect of GLN treatment on collagen deposition was evaluated using Sirius red staining and quantitative (q)PCR, while a Western bot was used to assess protein levels and analyze pathways. Results showed that GLN treatment not only increased the collagen deposition but also elevated the mRNA expression and protein levels of multiple tendon markers like collagen type I alpha 1 (COL1A1), decorin (DCN) and tenomodulin (TNMD) in TDSCs. Moreover, GLN was also found to upregulate the protein levels of transforming growth factor ß1 (TGF-ß1) and p-Smad3 to activate the TGF-ß1/Smad3 signaling pathway, while GLN mediated collagen deposition in TDSCs was reversed by LY3200882, a TGF-ß receptor inhibitor. The study concluded that GLN-mediated TDSCs enhanced tendon repair by activating the TGF-ß1/Smad3 signaling pathway, suggesting a novel therapeutic option in treating tendon repair.

Dexamethasone treatment influences tendon healing through altered resolution and a direct effect on tendon cells.

Inflammation, corticosteroids, and loading all affect tendon healing, with an interaction between them. However, underlying mechanisms behind the effect of corticosteroids and the interaction with loading remain unclear. The aim of this study was to investigate the role of dexamethasone during tendon healing, including specific effects on tendon cells. Rats (n = 36) were randomized to heavy loading or mild loading, the Achilles tendon was transected, and animals were treated with dexamethasone or saline. Gene and protein analyses of the healing tendon were performed for extracellular matrix-, inflammation-, and tendon cell markers. We further tested specific effects of dexamethasone on tendon cells in vitro. Dexamethasone increased mRNA levels of S100A4 and decreased levels of ACTA2/α-SMA, irrespective of load level. Heavy loading + dexamethasone reduced mRNA levels of FN1 and TenC (p < 0.05), while resolution-related genes were unaltered (p > 0.05). In contrast, mild loading + dexamethasone increased mRNA levels of resolution-related genes ANXA1, MRC1, PDPN, and PTGES (p < 0.03). Altered protein levels were confirmed in tendons with mild loading. Dexamethasone treatment in vitro prevented tendon construct formation, increased mRNA levels of S100A4 and decreased levels of SCX and collagens. Dexamethasone during tendon healing appears to act through immunomodulation by promoting resolution, but also through an effect on tendon cells.

Multi-omics analysis of human tendon adhesion reveals that ACKR1-regulated macrophage migration is involved in regeneration.

Tendon adhesion is a common complication after tendon injury with the development of accumulated fibrotic tissues without effective anti-fibrotic therapies, resulting in severe disability. Macrophages are widely recognized as a fibrotic trigger during peritendinous adhesion formation. However, different clusters of macrophages have various functions and receive multiple regulation, which are both still unknown. In our current study, multi-omics analysis including single-cell RNA sequencing and proteomics was performed on both human and mouse tendon adhesion tissue at different stages after tendon injury. The transcriptomes of over 74 000 human single cells were profiled. As results, we found that SPP1+ macrophages, RGCC+ endothelial cells, ACKR1+ endothelial cells and ADAM12+ fibroblasts participated in tendon adhesion formation. Interestingly, despite specific fibrotic clusters in tendon adhesion, FOLR2+ macrophages were identified as an antifibrotic cluster by in vitro experiments using human cells. Furthermore, ACKR1 was verified to regulate FOLR2+ macrophages migration at the injured peritendinous site by transplantation of bone marrow from Lysm-Cre;R26RtdTomato mice to lethally irradiated Ackr1-/- mice (Ackr1-/- chimeras; deficient in ACKR1) and control mice (WT chimeras). Compared with WT chimeras, the decline of FOLR2+ macrophages was also observed, indicating that ACKR1 was specifically involved in FOLR2+ macrophages migration. Taken together, our study not only characterized the fibrosis microenvironment landscape of tendon adhesion by multi-omics analysis, but also uncovered a novel antifibrotic cluster of macrophages and their origin. These results provide potential therapeutic targets against human tendon adhesion.

Malvidin attenuates trauma-induced heterotopic ossification of tendon in rats by targeting Rheb for degradation via the ubiquitin-proteasome pathway.

The pathogenesis of trauma-induced heterotopic ossification (HO) in the tendon remains unclear, posing a challenging hurdle in treatment. Recognizing inflammation as the root cause of HO, anti-inflammatory agents hold promise for its management. Malvidin (MA), possessing anti-inflammatory properties, emerges as a potential agent to impede HO progression. This study aimed to investigate the effect of MA in treating trauma-induced HO and unravel its underlying mechanisms. Herein, the effectiveness of MA in preventing HO formation was assessed through local injection in a rat model. The potential mechanism underlying MA's treatment was investigated in the tendon-resident progenitor cells of tendon-derived stem cells (TDSCs), exploring its pathway in HO formation. The findings demonstrated that MA effectively hindered the osteogenic differentiation of TDSCs by inhibiting the mTORC1 signalling pathway, consequently impeding the progression of trauma-induced HO of Achilles tendon in rats. Specifically, MA facilitated the degradation of Rheb through the K48-linked ubiquitination-proteasome pathway by modulating USP4 and intercepted the interaction between Rheb and the mTORC1 complex, thus inhibiting the mTORC1 signalling pathway. Hence, MA presents itself as a promising candidate for treating trauma-induced HO in the Achilles tendon, acting by targeting Rheb for degradation through the ubiquitin-proteasome pathway.

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair.

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Novel application of in vivo microdialysis in a rat Achilles tendon acute injury model.

Tendon injury and healing involve intricate changes to tissue metabolism, biology, and inflammation. Current techniques often require animal euthanasia or tissue destruction, limiting assessment of dynamic changes in tendon, including treatment response, disease development, rupture risk, and healing progression. Microdialysis, a minimally invasive technique, offers potential for longitudinal assessment, yet it has not been applied to rat tendon models. Therefore, the objective of this study is to adapt a novel application of an in vivo assay, microdialysis, using acute injury as a model for extreme disruption of the tendon homeostasis. We hypothesize that microdialysis will be able to detect measurable differences in the healing responses of acute injury with high specificity and sensitivity. Overall results suggest that microdialysis is a promising in vivo technique for longitudinal assessment for this system with strong correlations between extracellular fluid (ECF) and dialysate concentrations and reasonable recovery rates considering the limitations of this model. Strong positive correlations were found between dialysate and extracellular fluid (ECF) concentration for each target molecule of interest including metabolites, inflammatory mediators, and collagen synthesis and degradation byproducts. These results suggest that microdialysis is capable of detecting changes in tendon healing following acute tendon injury with high specificity and sensitivity. In summary, this is the first study to apply microdialysis to a rat tendon model and assess its efficacy as a direct measurement of tendon metabolism, biology, and inflammation.NEW & NOTEWORTHY This study adapts a novel application of microdialysis to rat tendon models, offering a minimally invasive avenue for longitudinal tendon assessment. Successfully detecting changes in tendon healing after acute injury, it showcases strong correlations between extracellular fluid and dialysate concentrations. The results highlight the potential of microdialysis as a direct measure of tendon metabolism, biology, and inflammation, bypassing the need for animal euthanasia and tissue destruction.

TRIM54 alleviates inflammation and apoptosis by stabilizing YOD1 in rat tendon-derived stem cells.

Tendinopathy is a disorder of musculoskeletal system that primarily affects athletes and the elderly. Current treatment options are generally comprised of various exercise and loading programs, therapeutic modalities, and surgical interventions and are limited to pain management. This study is to understand the role of TRIM54 (tripartite motif containing 54) in tendonitis through in vitro modeling with tendon-derived stem cells (TDSCs) and in vivo using rat tendon injury model. Initially, we observed that TRIM54 overexpression in TDSCs model increased stemness and decreased apoptosis. Additionally, it rescued cells from tumor necrosis factor α-induced inflammation, migration, and tenogenic differentiation. Further, through immunoprecipitation studies, we identified that TRIM54 regulates inflammation in TDSCs by binding to and ubiquitinating YOD1. Further, overexpression of TRIM54 improved the histopathological score of tendon injury as well as the failure load, stiffness, and young modulus in vivo. These results indicated that TRIM54 played a critical role in reducing the effects of tendon injury. Consequently, these results shed light on potential therapeutic alternatives for treating tendinopathy.

Chronological Changes in the Expression and Localization of Sox9 between Achilles Tendon Injury and Functional Recovery in Mice.

Tendons help transmit forces from the skeletal muscles and bones. However, tendons have inferior regenerative ability compared to muscles. Despite studies on the regeneration of muscles and bone tissue, only a few have focused on tendinous tissue regeneration, especially tendon regeneration. Sex-determining region Y-box transcription factor 9 (Sox9) is an SRY-related transcription factor with a DNA-binding domain and is an important control factor for cartilage formation. Sox9 is critical to the early-to-middle stages of tendon development. However, how Sox9 participates in the healing process after tendon injury is unclear. We hypothesized that Sox9 is expressed in damaged tendons and is crucially involved in restoring tendon functions. We constructed a mouse model of an Achilles tendon injury by performing a 0.3 mm wide partial excision in the Achilles tendon of mice, and chronologically evaluated the function restoration and localization of the Sox9 expressed in the damaged sites. The results reveal that Sox9 was expressed simultaneously with the formation of the pre-structure of the epitenon, an essential part of the tendinous tissue, indicating that its expression is linked to the functional restoration of tendons. Lineage tracing for Sox9 expressed during tendon restoration revealed the tendon restoration involvement of cells that switched into Sox9-expressing cells after tendon injury. The stem cells involved in tendon regeneration may begin to express Sox9 after injury.

Mussel-Derived Bioadaptive Artificial Tendon Facilitates the Cell Proliferation and Tenogenesis to Promote Tendon Functional Reconstruction.

Tendon injuries range from acute-related trauma to chronic-related injuries are prevalent and bring substantial pain, functional loss, and even disability to the patients. The management of tendon injuries is tricky due to the innate limited regenerative capability of the tendon. Currently, surgical intervention of tendon injuries with artificial tendons remains the standard of care. However, most of artificial tendons are manufactured with synthetic materials, which possess relatively poor biomimetic characteristics and inadequate inherent biodegradability, hence rendering limited cell proliferation and migration for tendon healing. To address these limitations, this work develops a mussel-derived artificial tendon based on double-cross-linked chitosan modification. In this design, decellularized artificial tendon serves as a natural biomimetic scaffold to facilitate the migration and adhesion of tendon repair cells. Additionally, as the cells proliferate, the artificial tendon can be degraded to facilitate tendon regeneration. Moreover, the chitosan cross-linking further enhances the mechanical strength of artificial tendon and offers a controllable degradation. The in vitro and in vivo experimental results demonstrate that mussel-derived artificial tendon not only accelerate the tendon functional reconstruction but also enable harmless clearance at postimplantation. The finding provides a promising alternative to conventional artificial tendons and spurs a new frontier to explore nature-derived artificial tendons.

Bioactive and Elastic Emulsion Electrospun DegraPol Tubes Delivering IGF-1 for Tendon Rupture Repair.

Tendon injuries can result in two major drawbacks. Adhesions to the surrounding tissue may limit the range of motion, while fibrovascular scar formation can lead to poor biomechanical outcomes. Prosthetic devices may help to mitigate those problems. Emulsion electrospinning was used to develop a novel three-layer tube based on the polymer DegraPol (DP), with incorporated insulin-like growth factor-1 (IGF-1) in the middle layer. Scanning electron microscopy was utilized to assess the fiber diameter in IGF-1 containing pure DP meshes. Further characterization was performed with Fourier Transformed Infrared Spectroscopy, Differential Scanning Calorimetry, and water contact angle, as well as through the assessment of mechanical properties and release kinetics from ELISA, and the bioactivity of IGF-1 by qPCR of collagen I, ki67, and tenomodulin in rabbit Achilles tenocytes. The IGF-1-containing tubes exhibited a sustained release of the growth factor up to 4 days and showed bioactivity by significantly upregulated ki67 and tenomodulin gene expression. Moreover, they proved to be mechanically superior to pure DP tubes (significantly higher fracture strain, failure stress, and elastic modulus). The novel three-layer tubes intended to be applied over conventionally sutured tendons after a rupture may help accelerate the healing process. The release of IGF-1 stimulates proliferation and matrix synthesis of cells at the repair site. In addition, adhesion formation to surrounding tissue can be reduced due to the physical barrier.

Natural 3D Extra Cellular Matrix mimicking stem cells seeded decellularized scaffolds as a platform for tendon regeneration.

Achilles tendon, which connects the calf muscles to heel, is the strongest tendon in the body. Despite its strength, it is more prone to injury due to its limited blood supply. Tendon-related injuries are more common in sportspersons, people with labor-intensive work and the aged community. The currently available treatment mode is surgery which is expensive with chances of re-injury. Present study made an attempt to fabricate a tissue-engineered tendon product using decellularized tendon (DT) seeded with stem cells and bioactive components of Tinospora cordifolia extract (TCE). The bare DT tissue scaffold/substitute may also serve as a drug delivery platform for growth factors and cells with a new approach to promote tissue regeneration in clinical applications. DT construct showed good regenerative potential and easily promoted new tissue formation. Decellularization of the tendon was carried out by chemical method using tri (n-butyl) phosphate (TnBP). DT was physicochemically characterized by contact angle measurement, thermal gravimetric analysis (TGA), and mechanical testing. Rabbit adipose derived mesenchymal stem cells (RADMSCs) were isolated and phenotypically characterized by flow cytometry analysis, tri lineage differentiation, and so forth. Further, stem cell seeded DT scaffolds were prepared and found to be non-toxic by cytotoxicity, cell adhesion by scanning electron microscope (SEM) analysis, cell viability by live dead assays, and so forth. The findings of this study yield valid proof for the employability of cell-seeded DT construct as a natural scaffold in repairing injured tendons-the toughest chords of the skeleton. This is a cost effective method for the replacement of injured/damaged tendons for athletes, people in labor-intensive occupations, the elderly population, and so forth-a boon towards the repair of the tendon in damage/injury.

CCN1 expression is regulated by mechanical stimuli in tendons.

Tendon overuse injuries are common, but the processes that govern tendon response to mechanical load are not fully understood. A series of experiments of in vitro and in vivo experiments was devised to study to the relationship between mechanical stimuli and the matricellular protein Cellular Communication Network Factor 1 (CCN1) in tenocytes and tendons. First, human and murine tenocytes were subjected to cyclic uniaxial loading in order to evaluate changes in CCN1 gene expression as a response to mechanical stimuli. Then, baseline Ccn1 gene expression in different murine tendons (Achilles, patellar, forearm, and tail) was examined. Finally, changes in Ccn1 expression after in vivo unloading experiments were examined. It was found that CCN1 expression significantly increased in both human and murine tenocytes at 5 and 10% cyclical uniaxial strain, while 2.5% strain did not have any effect on CCN1 expression. At baseline, the Achilles, patellar, and forearm tendons had higher expression levels of Ccn1 as compared to tail tendons. Twenty-four hours of immobilization of the hind-limb resulted in a significant decrease in Ccn1 expression in both the Achilles and patellar tendons. In summary, CCN1 expression is up-regulated in tenocytes subjected to mechanical load and down-regulated by loss of mechanical load in tendons. These results show that CCN1 expression in tendons is at least partially regulated by mechanical stimuli.

The potential mechanism of hypoxia-induced tenogenic differentiation of mesenchymal stem cell for tendon regeneration.

Tendon injuries account up to 50% of all musculoskeletal problems and remains a challenge to treat owing to the poor intrinsic reparative ability of tendon tissues. The natural course of tendon healing is very slow and often leads to fibrosis and disorganized tissues with inferior biomechanical properties. Mesenchymal stem cells (MSC) therapy is a promising alternative strategy to augment tendon repair due to its proliferative and multilineage differentiation potential. Hypoxic conditioning of MSC have been shown to enhance their tenogenic differentiation capacity. However, the mechanistic pathway by which this is achieved is yet to be fully defined. A key factor involved in this pathway is hypoxia-inducible factor-1-alpha (HIF-1α). This review aims to discuss the principal mechanism underlying the enhancement of MSC tenogenic differentiation by hypoxic conditioning, particularly the central role of HIF-1α in mediating activation of tenogenic pathways in the MSC. We focus on the interaction between HIF-1α with fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta 1 (TGF-ß1) in regulating MSC tenogenic differentiation pathways in hypoxic conditions. Strategies to promote stabilization of HIF-1α either through direct manipulation of oxygen tension or the use of hypoxia mimicking agents are therefore beneficial in increasing the efficacy of MSC therapy for tendon repair.

Up-regulation of CREB-1 regulates tendon adhesion in the injury tendon healing through the CREB-1/TGF-ß3 signaling pathway.

AIM: To explore the mechanism of the healing of tendon tissue and anti-adhesion, and to discuss the role of the transforming growth factor-ß3 (TGF-ß3)/cAMP response element binding protein-1 (CREB-1) signaling pathway in the healing process of tendons. METHOD: All mice were divided into four groups of 1, 2, 4, and 8 weeks respectively. Each time group was divided into four treatment groups: the amplification group, the inhibition group, the negative group, and the control group. When the tendon injury model was established, the CREB-1 virus was injected into the tendon injury parts. A series of methods such as gait behaviourism, anatomy, histological examination, immunohistochemical examination and collagen staining were employed to assess the tendon healing and the protein expression of TGF-ß3, CREB-1, Smad3/7 and type I/III collagen (COL-I/III). CREB-1 virus was sent to tendon stem cells to assess the protein expression of TGF-ß1, TGF-ß3, CREB-1, COL-I/III by methods such as immunohistochemistry and Western blot. RESULTS: The amplification group showed better gait behaviourism than the inhibition group in the healing process. The amplification group also had less adhesion than the negative group. Hematoxylin-eosin (HE) staining of tendon tissue sections showed that the number of fibroblasts in the amplification group was less than the inhibition group, and the immunohistochemical results indicated that the expression of TGF-ß3, CREB-1, and Smad7 at each time point was higher than the inhibition group. The expression of COL-I/III and Smad3 in the amplification group was lower than the inhibition group at all time points. The collagen staining indicated that the ratio of type I/III collagen in the amplification group was higher than the negative group at 2,4,8 week. The CREB-1 amplification virus could promote the protein expression of TGF-ß3, CREB-1 and inhibit the protein expression of TGF-ß1 and COL-I/III in the tendon stem cells. CONCLUSION: In the process of tendon injury healing, CREB-1 could promote the secretion of TGF-ß3, so as to promote the tendon healing and have the effect of anti-adhesion in tendons. It might provide new intervention targets for anti-adhesion treatment of tendon injuries.

Exosome Cell Origin Affects In Vitro Markers of Tendon Repair in Ovine Macrophages and Tenocytes.

Tendon injuries and disease are resistant to surgical repair; thus, adjunct therapies are widely investigated, especially mesenchymal stromal cells (MSCs) and, more recently, their extracellular vesicles (MSCdEVs), for example, exosomes. Thought to act on resident and infiltrating immune cells, the role of MSCdEVs in paracrine signaling is of great interest. This study investigated how MSCdEVs differ from analogs derived from resident (tenocyte) populations (TdEV). As macrophages play a significant role in tendon maintenance and repair, macrophage signaling was compared by cytokine quantification using a multiplexed immunoassay and tenocyte migration by in vitro scratch-wound analysis. TdEV-treated macrophages decreased IL-1 and increased MIP-1 and CXCL8 expression. In addition, macrophage signaling favored collagen synthesis and tenocyte bioactivity, while reducing proangiogenic signaling when TdEVs were used in place of MSCdEVs. These in vitro data demonstrate a differential influence of exosomes on macrophage signaling, according to cell source, supporting that local cell-derived exosomes may preferentially drive healing by different means with possible different outcomes compared to MSCdEVs. Impact Statement Adipose-derived mesenchymal stromal cell (AdMSC) exosomes (EVs) can improve tendon mechanical resilience, tissue organization, and M2 macrophage phenotype predominance in response to tendon injury. This active area of investigation drives great interest in the function of these exosomes as adjunct therapies for tendon disease, particularly rotator cuff tendinopathy. However, little is known about the effects of EVs as a function of cell source, nor regarding their efficacy in preclinical translational ovine models. Herein we demonstrate a differential effect of exosomes as a function of cell source, tenocyte compared to AdMSCs, on macrophage signaling and tenocyte migration of ovine cells.

N-Acetyl-L-cysteine facilitates tendon repair and promotes the tenogenic differentiation of tendon stem/progenitor cells by enhancing the integrin α5/ß1/PI3K/AKT signaling.

BACKGROUND: Tendon injury is associated with oxidative stress, leading to reactive oxygen species (ROS) production and inflammation. N-acetyl-L-cysteine (NAC) is a potent antioxidant. However, how NAC affects the biological functions of tendon stem/progenitor cells (TSPCs) and tendon repair has not been clarified.  METHOD: The impacts of NAC on the viability, ROS production, and differentiation of TSPCs were determined with the cell counting kit-8, fluorescence staining, Western blotting, and immunofluorescence. The effect of NAC on gene transcription in TSPCs was analyzed by transcriptomes and bioinformatics and validated by Western blotting. The potential therapeutic effect of NAC on tendon repair was tested in a rat model of Achilles tendon injury. RESULTS: Compared with the untreated control, treatment with 500 µM NAC greatly promoted the proliferation of TSPCs and significantly mitigated hydrogen peroxide-induced ROS production and cytotoxicity in vitro. NAC treatment significantly increased the relative protein expression of collagen type 1 alpha 1 (COL1A1), tenascin C (TNC), scleraxis (SCX), and tenomodulin (TNMD) in TPSCs. Bioinformatics analyses revealed that NAC modulated transcriptomes, particularly in the integrin-related phosphoinositide 3-kinase (PI3K)/AKT signaling, and Western blotting revealed that NAC enhanced integrin α5ß1 expression and PI3K/AKT activation in TSPCs. Finally, NAC treatment mitigated the tendon injury, but enhanced the protein expression of SCX, TNC, TNMD, and COLIA1 in the injured tissue regions of the rats. CONCLUSION: NAC treatment promoted the survival and differentiation of TSPCs to facilitate tendon repair after tendon injury in rats. Thus, NAC may be valuable for the treatment of tendon injury.

Collagen fibrils from both positional and energy-storing tendons exhibit increased amounts of denatured collagen when stretched beyond the yield point.

Collagen molecules are the base structural unit of tendons, which become denatured during mechanical overload. We recently demonstrated that during tendon stretch, collagen denaturation occurs at the yield point of the stress-strain curve in both positional and energy-storing tendons. We were interested in investigating how this load is transferred throughout the collagen hierarchy, and sought to determine the onset of collagen denaturation when collagen fibrils are stretched. Fibrils are one level above the collagen molecule in the collagen hierarchy, allowing more direct probing of the effect of strain on collagen molecules. We isolated collagen fibrils from both positional and energy-storing tendon types and stretched them using a microelectromechanical system device to various levels of strain. We stained the fibrils with fluorescently labeled collagen hybridizing peptides that specifically bind to denatured collagen, and examined whether samples stretched beyond the yield point of the stress-strain curve exhibited increased amounts of denatured collagen. We found that collagen denaturation in collagen fibrils from both tendon types occurs at the yield point. Greater amounts of denatured collagen were found in post-yield positional fibrils than in energy-storing fibrils. This is despite a greater yield strain and yield stress in fibrils from energy-storing tendons compared to positional tendons. Interestingly, the peak modulus of collagen fibrils from both tendon types was the same. These results are likely explained by the greater crosslink density found in energy-storing tendons compared to positional tendons. The insights gained from this study could help management of tendon and other musculoskeletal injuries by targeting collagen molecular damage at the fibril level. STATEMENT OF SIGNIFICANCE: When tendons are stretched or torn, this can lead to collagen denaturation (damage). Depending on their biomechanical function, tendons are considered positional or energy-storing with different crosslink profiles. By stretching collagen fibrils instead of fascicles from both tendon types, we can more directly examine the effect of tensile stretch on the collagen molecule in tendons. We found that regardless of tendon type, collagen denaturation in fibrils occurs when they are stretched beyond the yield point of the stress-strain curve. This provides insight into how load affects different tendon sub-structures during tendon injuries and failure, which will help clinicians and researchers understand mechanisms of injuries and potentially target collagen molecular damage as a treatment strategy, leading to improved clinical outcomes following injury.

Impact of elevated serum advanced glycation end products and exercise on intact and injured murine tendons.

OVERVIEW: Delayed tendon healing is a significant clinical challenge for those with diabetes. We explored the role of advanced glycation end-products (AGEs), a protein modification present at elevated levels in serum of individuals with diabetes, on injured and intact tendons using a mouse model. Cell proliferation following tissue injury is a vital component of healing. Based on our previous work demonstrating that AGEs limit cell proliferation, we proposed that AGEs are responsible for the delayed healing process commonly observed in diabetic patients. Further, in pursuit of interventional strategies, we suggested that moderate treadmill exercise may support a healing environment in the presence of AGEs as exercise has been shown to stimulate cell proliferation in tendon tissue. MATERIALS AND METHODS: Mice began receiving daily intraperitoneal injections of bovine serum albumin (BSA)-Control or AGE-BSA injections (200µg/ml) at 16-weeks of age. A tendon injury was created in the central third of both patellar tendons. Animals assigned to an exercise group began a moderate treadmill protocol one week following injury. The intact Achilles tendon and soleus muscle were also evaluated to assess the effect of BSA and AGE-BSA on un-injured muscle and tendon. RESULTS: We demonstrate that our injection dosing and schedule lead to an increase in serum AGEs. Our findings imply that AGEs indeed modulate gene expression following a patellar tendon injury and have modest effects on gene expression in intact muscle and tendon. CONCLUSIONS: While additional biomechanical analysis is warranted, these data suggest that elevated serum AGEs in persons with diabetes may impact tendon health.

CD200 as a Potential New Player in Inflammation during Rotator Cuff Tendon Injury/Repair: An In Vitro Model.

Rotator cuff tendon (RCT) disease results from multifactorial mechanisms, in which inflammation plays a key role. Pro-inflammatory cytokines and tendon stem cell/progenitor cells (TSPCs) have been shown to participate in the inflammatory response. However, the underlying molecular mechanism is still not clear. In this study, flow cytometry analyses of different subpopulations of RCT-derived TSPCs demonstrate that after three days of administration, TNFα alone or in combination with IFNγ significantly decreases the percentage of CD146+CD49d+ and CD146+CD49f+ but not CD146+CD109+ TSPCs populations. In parallel, the same pro-inflammatory cytokines upregulate the expression of CD200 in the CD146+ TSPCs population. Additionally, the TNFα/IFNγ combination modulates the protein expression of STAT1, STAT3, and MMP9, but not fibromodulin. At the gene level, IRF1, CAAT (CAAT/EBPbeta), and DOK2 but not NF-κb, TGRF2 (TGFBR2), and RAS-GAP are modulated. In conclusion, although our study has several important limitations, the results highlight a new potential role of CD200 in regulating inflammation during tendon injuries. In addition, the genes analyzed here might be new potential players in the inflammatory response of TSPCs.

Collagen V deficiency during murine tendon healing results in distinct healing outcomes based on knockdown severity.

Tendon function is dependent on proper organization and maintenance of the collagen I tissue matrix. Collagen V is a critical regulator of collagen I fibrils, and while prior studies have shown a negative impact of collagen V deficiency on tendon healing outcomes, these studies are confounded by collagen V deficiency through tendon development. The specific role of collagen V in regulating healing tendon properties is therefore unknown. By using inducible Col5a1 knockdown models and analyzing gene expression, fibril and histological tendon morphology, and tendon mechanical properties, this study defines the isolated role of collagen V through tendon healing. Patellar tendon injury caused large changes in tendon gene expression, and Col5a1 knockdown resulted in dysregulated expression of several genes through tendon healing. Col5a1 knockdown also impacted collagen fibril size and shape without observable changes in scar tissue formation. Surprisingly, heterozygous Col5a1 knockdown resulted in improved stiffness of healing tendons that was not observed with homozygous Col5a1 knockdown. Together, these results present an unexpected and dynamic role of collagen V deficiency on tendon healing outcomes following injury. This work suggests a model of tendon healing in which quasi-static mechanics may be improved through titration of collagen fibril size and shape with modulation of collagen V expression and activity.