Tendon Fibroblasts Research Articles

An engineered tenogenic patch for the treatment of rotator cuff tear

Rotator cuff tear of shoulder is a common tendon injury. Current therapeutic strategies are unsatisfactory because of poor biomechanical properties of the repaired rotator cuff tendons. To solve this problem, a newly biofabricated tenogenic patch by uniaxial cold-drawing technology was applied for the treatment of acute rotator cuff tear and its therapeutic effects were evaluated in vitro and in vivo. The cytocompatibility results indicated that the engineered patch was capable of promoting the spreading, migration and proliferation of tendon fibroblasts in vitro. Stress–strain curves of the sutured patch indicated improved elasticity followed by the yield phenomena, nonlinear plasticity and obviously delaminated rupture. The tenogenic patch also showed significantly enhanced collagen I deposition and better tendon-to-bone healing in the rat rotator cuff tear models in vivo. Furthermore, implantation with the tenogenic patch also promoted the fibrocartilage formation in Safranin-O/Fast green-staining. The analysis of biomechanical properties showed that the tenogenic patch is capable of improving the ultimate load of the repaired tendon. Therefore, the tenogenic patch may constitute an efficient strategy to enhance tendon-to-bone healing for clinical repair applications of rotator cuff tear.

Descriptive transcriptome analysis of tendon derived fibroblasts following in-vitro exposure to advanced glycation end products.

Tendon pathologies affect a large portion of people with diabetes. This high rate of tendon pain, injury, and disease appears to manifest independent of well-controlled HbA1c and fasting blood glucose. Advanced glycation end products (AGEs) are elevated in the serum of those with diabetes. In vitro, AGEs severely impact tendon fibroblast proliferation and mitochondrial function. However, the extent that AGEs impact the tendon cell transcriptome has not been evaluated. The purpose of this study was to investigate transcriptome-wide changes that occur to tendon-derived fibroblasts following treatment with AGEs. We propose to complete a descriptive approach to pathway profiling to broaden our mechanistic understanding of cell signaling events that may contribute to the development of tendon pathology. Rat Achilles tendon fibroblasts were treated with glycolaldehyde-derived AGEs (200μg/ml) for 48 hours in normal glucose (5.5mM) conditions. In addition, total RNA was isolated, and the PolyA+ library was sequenced. We demonstrate that tendon fibroblasts treated with 200μg/ml of AGEs differentially express 2,159 gene targets compared to fibroblasts treated with an equal amount of BSA-Control. Additionally, we report in a descriptive and ranked fashion 21 implicated cell-signaling pathways. Our findings suggest that AGEs disrupt the tendon fibroblast transcriptome on a large scale and that these pathways may contribute to the development and progression of diabetic tendinopathy. Specifically, pathways related to cell cycle progression and extracellular matrix remodeling were affected in our data set and may play a contributing role in the development of diabetic tendon complications.

Effects of 6-Hydroxykaempferol: A Potential Natural Product for Amelioration of Tendon Impairment.

Tendon impairment is a common injury associated with impairment of range of motion and pain. Currently, evidence has confirmed that natural herbs contribute to orthopedics and have shown excellent results in the clinical management of tendon impairment. Shujin Huoxue tablet (SHT) and its complex prescriptions are regularly used in tendon rupture therapy with positive results. This study aimed to discover the potential molecules that promote tendon healing. The Chinese traditional medicine system pharmacological database analysis platform (TCMSP) is the primary resource. The Traditional Chinese Medicine Integrated Database and Encyclopedia of Traditional Chinese Medicine database were used as secondary databases. The GeneCards database was used to search for reported tendinopathy-related genes by keywords. Functions of the targeted genes were analyzed using Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes. Protein–protein interaction information was extracted from the STRING database. Docking study, MTT assay, quantitative real-time PCR, and migration assays were performed to obtain a better understanding of the herbs according to cell function to test the basic pharmacological action in vitro. A total of 104 disease nodes, 496 target gene nodes, 35 ingredient nodes, and one drug node were extracted. According to the TCMSP database, 6-hydroxykaempferol, which reportedly promotes the proliferation of microvascular endothelial cells, is a molecule found in SHT. We found that it promoted the proliferation and migration of tendon fibroblasts and elevated tendon repair-related gene expression. Purified 6-hydroxykaempferol promoted the proliferation and migration of tendon fibroblasts and increased their mRNA expression in tendon proliferation.

Circadian regulation of protein cargo in extracellular vesicles.

The circadian clock controls many aspects of physiology, but it remains undescribed whether extracellular vesicles (EVs), including exosomes, involved in cell-cell communications between tissues are regulated in a circadian pattern. We demonstrate a 24-hour rhythmic abundance of individual proteins in small EVs using liquid chromatography–mass spectrometry in circadian-synchronized tendon fibroblasts. Furthermore, the release of small EVs enriched in RNA binding proteins was temporally separated from those enriched in cytoskeletal and matrix proteins, which peaked during the end of the light phase. Last, we targeted the protein sorting mechanism in the exosome biogenesis pathway and established (by knockdown of circadian-regulated flotillin-1) that matrix metalloproteinase 14 abundance in tendon fibroblast small EVs is under flotillin-1 regulation. In conclusion, we have identified proteomic time signatures for small EVs released by tendon fibroblasts, which supports the view that the circadian clock regulates protein cargo in EVs involved in cell-cell cross-talk.

High glucose represses the proliferation of tendon fibroblasts by inhibiting autophagy activation in tendon injury.

Diabetic foot ulcer (DFU) is a kind of common and disabling complication of Diabetes Mellitus (DM). Emerging studies have demonstrated that tendon fibroblasts play a crucial role in remodeling phase of wound healing. However, little is known about the mechanism underlying high glucose (HG)-induced decrease in tendon fibroblasts viability. In the present study, the rat models of DFU were established, and collagen deposition, autophagy activation and cell apoptosis in tendon tissues were assessed using Hematoxylin–Eosin (HE) staining, immunohistochemistry (IHC), and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, respectively. Tendon fibroblasts were isolated from Achilles tendon of the both limbs, and the effect of HG on autophagy activation in tendon fibroblasts was assessed using Western blot analysis, Cell Counting Kit-8 (CCK-8) assay, and flow cytometry. We found that cell apoptosis was increased significantly and autophagy activation was decreased in foot tendon tissues of DFU rats compared with normal tissues. The role of HG in regulating tendon fibroblasts viability was then investigated in vitro, and data showed that HG repressed cell viability and increased cell apoptosis. Furthermore, HG treatment reduced LC3-II expression and increased p62 expression, indicating that HG repressed autophagy activation of tendon fibroblasts. The autophagy activator rapamycin reversed the effect. More importantly, rapamycin alleviated the suppressive role of HG in tendon fibroblasts viability. Taken together, our data demonstrate that HG represses tendon fibroblasts proliferation by inhibiting autophagy activation in tendon injury.

Alleviation of Diabetic Tendon Injury via Activation of Tendon Fibroblasts Autophagy under Berberine Treatment.

Berberine (BBR) is an isoquinoline alkaloid isolated from Coptis chinensis and possesses valuable pharmacological activities, including anti-inflammatory, anti-tumor, and alleviating several complications of type 2 diabetes mellitus (T2DM). However, the role of BBR in regulating diabetic tendon injury remains poorly understood. In this study, a rat model of T2DM was constructed, and cell apoptosis and autophagy were assessed in tendon tissues after BBR treatment through TdT-Mediated dUTP nick-end labeling (TUNEL) assay and immunohistochemical analysis. Tendon fibroblasts were obtained from the rat Achilles tendon, and the role of BBR in regulating cell apoptosis, the production of inflammatory cytokines, and autophagy activation were assessed using flow cytometry, quantitative real-time PCR (qRT-PCR), and western blot analysis. We demonstrated that BBR treatment significantly increased autophagy activation and decreased cell apoptosis in tendon tissues of T2DM rats. In tendon fibroblasts, BBR repressed High glucose (HG)-induced cell apoptosis and production of proinflammatory cytokines. HG treatment resulted in a decrease of autophagy activation in tendon fibroblasts, whereas BBR restored autophagy activation. More important, pharmacological inhibition of autophagy by 3-MA weakened the protective effects of BBR against HG-induced tendon fibroblasts injury. Taken together, the current results demonstrate that BBR helps relieve diabetic tendon injury by activating autophagy of tendon fibroblasts.

Electrospun Scaffold Micro-Architecture Induces an Activated Transcriptional Phenotype within Tendon Fibroblasts.

Biomaterial augmentation of surgically repaired rotator cuff tendon tears aims to improve the high failure rates (∼40%) of traditional repairs. Biomaterials that can alter cellular phenotypes through the provision of microscale topographical cues are now under development. We aimed to systematically evaluate the effect of topographic architecture on the cellular phenotype of fibroblasts from healthy and diseased tendons. Electrospun polydioxanone scaffolds with fiber diameters ranging from 300 to 4000 nm, in either a highly aligned or random configuration, were produced. Healthy tendon fibroblasts cultured for 7 days on scaffolds with highly aligned fibers demonstrated a distinctive elongated morphology, whilst those cultured on randomly configured fibers demonstrated a flattened and spread morphology. The effect of scaffold micro-architecture on the transcriptome of both healthy and diseased tendon fibroblasts was assessed with bulk RNA-seq. Both healthy (n = 3) and diseased tendon cells (n = 3) demonstrated a similar transcriptional response to architectural variants. Gene set enrichment analysis revealed that large diameter (≥2000 nm) aligned scaffolds induced an upregulation of genes involved in cellular replication and a downregulation of genes defining inflammatory responses and cell adhesion. Similarly, PDPN and CD248, markers of inflammatory or “activated” fibroblasts, were downregulated during culture of both healthy and diseased fibroblasts on aligned scaffolds with large (≥2000 nm) fiber diameters. In conclusion scaffold architectures resembling that of disordered type III collagen, typically present during the earlier phases of wound healing, resulted in tendon fibroblast activation. Conversely, scaffolds mimicking aligned diameter collagen I fibrils, present during tissue remodelling, did not activate tendon derived fibroblasts. This has implications for the design of scaffolds used during rotator cuff repair augmentation.

Exploring the Effects of 630 nm Wavelength of Light-Emitting Diode Irradiation on the Proliferation and Migration Ability of Human Biceps Tendon Fibroblast Cells.

Light-emitting diode (LED)-based photobiomodulation is used as an inducer of cell regeneration. Although numerous in vitro and in vivo orthopedic studies have been conducted, the ideal LED wavelength range for tendon healing has not yet been determined. This study, thus, focused on the effects of LED of a 630 nm wavelength on the cell viability, proliferation, and migration of human biceps tendon fibroblast cells. Human tendon fibroblast cell culture was performed using the biceps tendon of patients who had undergone biceps tenodesis. Human biceps tendon fibroblasts from two patients (male, aged 42 and 69 years) were isolated and cultured. The cell type was confirmed by a morphological analysis and using tendon and fibroblast specific markers. They were then split into three groups, with each receiving a different irradiation treatment: no LED treatment (control), 630 nm LED, and 630 nm + 880 nm LED for 20 minutes each. After the LED treatment, cell viability, proliferation, and migration assays were performed, and the results were compared between the groups. Twenty-four hours after LED treatment, cell viability and proliferation were significantly increased in the 630 nm LED and 630 nm + 880 nm LED treatment groups compared to that in the control group (p < 0.05). Under the same conditions, compared with the control group, the 630 nm LED alone treatment group showed a 3.06 ± 0.21 times higher cell migration rate (p < 0.05), and the 630 nm + 880 nm LED combination treatment group showed a 2.88 ± 0.20 times higher cell migration rate (p < 0.05) in three-dimensional migration assay. In human tendon fibroblast cells, 20 minutes of LED treatment at 630 nm and 630 nm + 880 nm exhibited significant effects on cell proliferation and migration. Our findings suggest the potential of LED therapy as an adjuvant treatment for tendon healing, and hence, further research is warranted to standardize the various parameters to further develop and establish this as a reliable treatment regimen.

The Regenerative Role of Gelatin in PLLA Electrospun Membranes for the Treatment of Chronic Massive Rotator Cuff Injuries.

Failing to regenerate native tendon tissue in chronic massive rotator cuff tears (CMRCTs) results in high retear rates after surgery. Gelatin is a hydrolyzed form of collagen which is bioactive and biocompatible. This study intends to investigate the suitability of integrating gelatin to poly (l-lactic acid) (PLLA) fibrous membranes for promoting the healing of CMRCTs. PLLA/Gelatin electrospun membranes (PGEM) are fabricated using electrospinning technology. The fourier transform infrared, static contact angles are tested sequentially. Cytocompatibility is evaluated with rat tendon fibroblasts and human umbilical endothelial cells (HUEVCs) lines. CMRCTs rat models are established and assigned into three groups (the sham group, the repaired group, and the augmentation group) to perform histomorphological and biomechanical evaluations. Gelatin is successfully integrated into PLLA fibrous membranes by the electrospinning technique. In vitro studies indicate that PGEM shows a great cytocompatibility for rat tendon fibroblasts and HUEVCs. In vivo studies find that applications of PGEM significantly promote well-aligned collagen I fibers formation and enhance biomechanical properties of the repaired tendon in CMRCTs rat models. In summary, gelatin promotes tendon fibroblasts and HUEVCs adhesion, migration, and proliferation on the PLLA fibrous membranes, and PGEM may provide a great prospect for clinical application.

Extracellular Matrix Hydrogels Promote Expression of Muscle-Tendon Junction Proteins.

Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may help understand how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue, resulting in junction-specific features. The ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue-specific pre-hydrogel digests. The ECM materials were unique in respect to protein composition and included many types of ECM proteins, not just collagens. After digestion and gelation, ECM hydrogels had similar complex viscosities that were less than type I collagen hydrogels at the same concentration. C2C12 myoblasts and tendon fibroblasts were cultured in tissue-specific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. The ECM conditioned media had only a minor effect on the upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with a varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. In addition, another junction marker, type XXII collagen, had similar expression patterns as paxillin, with smaller effect sizes. Using tissue-specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscle-tendon junctions to study the expression of myotendinous junction-specific proteins. Impact statement The muscle-tendon junction is an important feature of muscle-tendon units; however, despite crosstalk between the two tissue types, the junction is often overlooked in current research. Deconstructing the cell-matrix interactions will provide the opportunity to study significant junction-specific features and markers that should be included in tissue models of the muscle-tendon unit, while gaining a deeper understanding of the natural junction. This research aims at informing future methods to engineer a more relevant multi-tissue platform to study the muscle-tendon unit.

Association of myostatin deficiency with collagen related disease-umbilical hernia and tippy toe standing in pigs.

Herein, we investigate the high incidence of umbilical hernia and tippy-toe standing and their underlying changes in gene expression and proliferation in myostatin knockout (MSTN-/-) pigs. Thirty-six male MSTN-/- pigs were generated by somatic cell nuclear transfer (SCNT). These pigs presented a considerably high incidence of tippy-toe standing and umbilical hernia (69.4% and 61.1%, respectively). The tendon to body weight ratio was significantly lower than wild-type pigs (0.202 ± 0.017 vs 0.250 ± 0.004, respectively). The crimp length of the MSTN-/- tendon was significantly longer than that of wild-type pigs. The expression of MSTN and the activin type IIB (ACVR2B) was detected in the tendon and linea alba of MSTN-/- pigs. MSTN treatment significantly increased the phosphorylation of Smad2/3 in both tendon and linea alba fibroblasts. Type I collagen (Col1A) and Scleraxis (Scx) expression levels in the tendon and linea alba of MSTN-/- pigs were significantly lower than those in wild-type in vivo, whereas and cyclin-dependent kinase inhibitor 1 (p21) expression levels were higher. Treatment of tendon and linea alba fibroblasts with recombinant MSTN increased Col1A and Scx and decreased p21 expression in vivo. Moreover, there was a significant increase in fibroblast proliferation after treatment. The results indicated that MSTN regulates collagen expression and proliferation in tendon and linea alba fibroblasts; thus, MSTN deficiency causes collagen-related pathological features in MSTN-/- pigs. Hence, MSTN could be used as a therapeutic target for treating UH and tendon abnormalities.

Potential Mechanisms of the Impact of Hepatocyte Growth Factor Gene-Modified Tendon Stem Cells on Tendon Healing.

The therapeutic impact of stem cells is potentially largely attributable to secretion of exosomes and soluble factors. The present study evaluates the impact of hepatocyte growth factor (HGF)–expressing tendon stem cells (TSCs) on tendon healing in a rat model. Patellar tendon TSCs were isolated and underwent transfection with lentiviral vectors containing HGF or green fluorescent protein (GFP) genes. In vivo, immunohistochemistry of tendons sampled 1 week postsurgery demonstrated that all stem cell–treated groups exhibited higher numbers of CD163+ M2 monocytes and IL-10+ cells (anti-inflammatory), and lower numbers of CCR7+ M1 monocytes and IL-6+ as well as COX-2+ cells (pro-inflammatory). Effects were most pronounced in the HGF-expressing TSCs (TSCs + HGF) treated group. Histology ± immunohistochemistry of tendons sampled 4 and 8 weeks postsurgery demonstrated that all stem cell–treated groups exhibited more ordered collagen fiber arrangement and lower levels of COLIII, α-SMA, TGF-β1, and fibronectin (proteins relevant to fibroscarring). Effects were most pronounced in the TSCs + HGF–treated group. For the in vitro study, isolated tendon fibroblasts pretreated with TGF-β1 to mimic the in vivo microenvironment of tendon injury were indirectly cocultured with TSCs, TSCs + GFP, or TSCs + HGF using a transwell system. Western blotting demonstrated that all stem cell types decreased TGF-β1-induced increases in fibroblast levels of COX-2, COLIII, and α-SMA, concomitant with decreased activation of major TGF-β1 signaling pathways (p38 MAPK, ERK1/2, but not Smad2/3). This effect was most pronounced for TSCs + HGF, which also decreased the TGF-β1-induced increase in activation of the Smad2/3 signaling pathway. The presence of specific inhibitors of these pathways during fibroblast TGF-β1 stimulation also attenuated increases in levels of COX-2, COLIII, and α-SMA. In conclusion, TSCs + HGF, which exhibit HGF overexpression, may promoting tendon healing via decreasing inflammation and fibrosis, perhaps partly via inhibiting TGF-β1-induced signaling. These findings identify a novel potential therapeutic strategy for tendon injuries, warranting additional research.

Magnetic Alignment of Electrospun Fiber Segments Within a Hydrogel Composite Guides Cell Spreading and Migration Phenotype Switching.

Fibrous extracellular matrix (ECM) proteins provide mechanical structure and adhesive scaffolding to resident cells within stromal tissues. Aligned ECM fibers play an important role in directing morphogenetic processes, supporting mechanical loads, and facilitating cell migration. Various methods have been developed to align matrix fibers in purified biopolymer hydrogels, such as type I collagen, including flow-induced alignment, uniaxial tensile deformation, and magnetic particles. However, purified biopolymers have limited orthogonal tunability of biophysical cues including stiffness, fiber density, and fiber alignment. Here, we generate synthetic, cell-adhesive fiber segments of the same length-scale as stromal fibrous proteins through electrospinning. Superparamagnetic iron oxide nanoparticles (SPIONs) embedded in synthetic fiber segments enable magnetic field induced alignment of fibers within an amorphous bulk hydrogel. We find that SPION density and magnetic field strength jointly influence fiber alignment and identify conditions to control the degree of alignment. Tuning fiber length allowed the alignment of dense fibrous hydrogel composites without fiber entanglement or regional variation in the degree of alignment. Functionalization of fiber segments with cell adhesive peptides induced tendon fibroblasts to adopt a uniaxial morphology akin to within native tendon. Furthermore, we demonstrate the utility of this hydrogel composite to direct multicellular migration from MCF10A spheroids and find that fiber alignment prompts invading multicellular strands to separate into disconnected single cells and multicellular clusters. These magnetic fiber segments can be readily incorporated into other natural and synthetic hydrogels and aligned with inexpensive and easily accessible rare earth magnets, without the need for specialized equipment. 3D hydrogel composites where stiffness/crosslinking, fiber density, and fiber alignment can be orthogonally tuned may provide insights into morphogenetic and pathogenic processes that involve matrix fiber alignment and can enable systematic investigation of the individual contribution of each biophysical cue to cell behavior.

Rho-Kinase Inhibitors as a novel medication for Glaucoma Treatment – A Review of the literature

Glaucoma is a progressive optic neuropathy, characterized by loss of retinal ganglion cells with subsequent deterioration in visual field. A major clinically modifiable risk factor for glaucomatous deterioration is elevated intraocular pressure (IOP) as a sequence of an increased aqueous humor (AH) outflow resistance. Aside from the introduction of prostaglandin analogs, topical carbonic anhydrase inhibitors , and topical alpha agonists in the 1990s, a new pharmaceutical agents to lower IOP have been introduced approximately 20 years. Rho-kinase (ROCK) Inhibitors, a new pharmacological class of hypotensive glaucoma medication targeting specifically the diseased trabecular outflow pathway. Rock inhibitors function by relaxing the trabecular meshwork, which subsequently leads to improved aqueous humor outflow and lower IOP. ROCK is a downstream effector of a small-G protein Rho. In addition to improving the outflow facility of the trabecular meshwork, Rho kinase inhibitors also enhance retinal ganglion cell survival after ischemic injury and increase ocular blood flow. ROCK inhibitors were reported to block TGF-B myofibroblast differentiation of human tendon fibroblasts, suggesting that may reduce postoperative conjunctival scarring after glaucoma surgery The objectives of this review are to describe the basic science underlying the mechanism of ROCK inhibitors as a therapy to lower IOP ,their neuro-protective, and vasoactive properties .

Effects of genipin crosslinking on mechanical cell-matrix interaction in 3D engineered tendon constructs

It is well known that cells can generate endogenous forces onto the extracellular matrix, but to what extent the mechanical properties of the matrix influences these endogenous cellular forces remains unclear. We therefore sought to quantify the influence of matrix rigidity on cell-matrix interactions by inducing cross-links using increasing concentrations of genipin (0.01–1 mM) or by blocking cross-link formation using beta-aminopropionitrile (BAPN) in engineered human tendon tissue constructs. The cell-matrix mechanics of the tendon constructs were evaluated as cell-generated tissue re-tensioning and stress-relaxation responses using a novel custom-made force monitor, which can apply and detect tensional forces in real-time in addition to mechanical failure testing. Genipin treatment had no influence on the biochemical profile (hydroxyproline, glycosaminoglycan and DNA content) of the constructs and cell viability was comparable between genipin-treated and control constructs, except at the highest genipin concentration. Endogenous re-tension after unloading was significantly decreased with increasing genipin concentrations compared to controls. Mechanical failure testing of tendon constructs showed increased (56%) peak stress at the highest genipin concentration but decreased (72%) with BAPN treatment when compared to controls. Tendon construct stiffness increased with high genipin concentrations (0.1 and 1 mM) and decreased by 70% in BAPN-treated constructs, relative to the controls. These data demonstrate that human tendon fibroblasts regulate their force exertion inversely proportional to increased cross-link capacity but did so independently of matrix stiffness. Overall, these findings support the notion of an interaction between cell force generation and cross-linking, and thus a role for this interplay in mechanical homeostasis of the tissue.

Hepatocyte Growth Factor-Induced Tendon Stem Cell Conditioned Medium Promotes Healing of Injured Achilles Tendon.

Tendon repair is a medical challenge. Our present study investigated the effectiveness of acellular therapy consisting of conditioned medium (CM) of tendon stem cells (TSCs) induced with hepatocyte growth factor (HGF) in promoting the healing of injured Achilles tendon in a rat model. Proteomic analysis of soluble substances in the CM was performed using an array chip, and bioinformatic analysis was carried out to evaluate interactions among the factors. The effects of CM on viability and migratory capacity of tendon fibroblasts derived from rats with ruptured Achilles tendon were evaluated with the Cell Counting Kit 8 and wound healing assay, respectively. The expression of extracellular matrix (ECM)-related protein was assessed by western blotting. Rats with Achilles tendon injury were treated with CM by local injection for 2 weeks, and the organization of tendon fibers at the lesion site was evaluated by hematoxylin and eosin and Masson’s trichrome staining of tissue samples. The deposition and degradation of ECM proteins and the expression of inflammatory factors at the lesion site were evaluated by immunohistochemistry and immunofluorescence. Biomechanical testing was carried out on the injured tendons to assess functional recovery. There were 12 bioactive molecules in the CM, with HGF as the hub of the protein–protein interaction network. CM treatment enhanced the viability and migration of tendon fibroblasts, altered the expression of ECM proteins, promoted the organization of tendon fibers, suppressed inflammation and improved the biomechanics of the injured Achilles tendon. These results suggest that HGF stimulates the secretion of soluble secretory products by TSCs and CM promotes the repair and functional recovery of ruptured Achilles tendon. Thus, HGF-induced TSC CM has therapeutic potential for the treatment of tendinopathy.

Comparing the Efficacy of Multi-Source Serum for in vitro Culture of Equine Achilles Tendon Fibroblasts

Comparing the Efficacy of Multi-Source Serum for in vitro Culture of Equine Achilles Tendon Fibroblasts

Fibroblasts From Common Anterior Cruciate Ligament Tendon Grafts Exhibit Different Biologic Responses to Mechanical Strain

Background: Different tendons are chosen for anterior cruciate ligament (ACL) reconstruction based on perceived advantages and disadvantages, yet there is a relative paucity of information regarding biologic responsiveness of commonly used tendon grafts to mechanical strain. Purpose: To evaluate the in vitro responses of graft fibroblasts derived from tendons used for ACL reconstruction to clinically relevant strain levels. Study Design: Controlled laboratory study. Methods: Twelve quadriceps tendons (QTs), 12 patellar tendons (PTs), and 9 hamstring tendons (HTs) were harvested from skeletally mature dogs (n = 16). Tendon fibroblasts were isolated and seeded onto BioFlex plates (1 × 105 cells/well). Cells were subjected to 3 strain conditions (stress deprivation, 0%; physiologic, 4%; high, 10%) for 5 days. Media were collected for proinflammatory and metabolic assays. RNA was extracted for gene expression analysis using real-time reverse transcription polymerase chain reaction. Results: Stress deprivation elicited significantly higher metabolic activity from HT and PT cells than from QT cells (P < .001 and P = .001, respectively). There were no differences in metabolic activity among all 3 graft fibroblasts at physiologic and high strain. COL-1 expression was significantly higher in PT versus HT during physiologic strain (P = .007). No significant differences with COL-3 expression were seen. TIMP-1 (P = .01) expression was higher in PT versus HT under physiologic strain. Scleraxis expression was higher in PT versus HT (P = .007) under physiologic strain. A strain-dependent increase in PGE2 levels occurred for all grafts. At physiologic strain conditions, HT produced significantly higher levels of PGE2 versus QT (P < .001) and PT (P = .005). Conclusion: Fibroblasts from common ACL graft tissues exhibited different metabolic responses to mechanical strain. On the basis of these data, we conclude that early production of extracellular matrix and proinflammatory responses from ACL grafts are dependent on mechanical loading and graft source. Clinical Relevance: Graft-specific differences in ACL reconstruction outcomes are known to exist. Our results suggest that there are differences in the biologic responsiveness of cells from the tendon grafts used in ACL reconstruction, which are dependent on strain levels and graft source. The biologic properties of the tissue used for ACL reconstruction should be considered when selecting graft source.

Proteomics identifies differences in fibrotic potential of extracellular vesicles from human tendon and muscle fibroblasts

BackgroundFibroblasts are the powerhouses responsible for the production and assembly of extracellular matrix (ECM). Their activity needs to be tightly controlled especially within the musculoskeletal system, where changes to ECM composition affect force transmission and mechanical loading that are required for effective movement of the body. Extracellular vesicles (EVs) are a mode of cell-cell communication within and between tissues, which has been largely characterised in cancer. However, it is unclear what the role of healthy fibroblast-derived EVs is during tissue homeostasis.MethodsHere, we performed proteomic analysis of small EVs derived from primary human muscle and tendon cells to identify the potential functions of healthy fibroblast-derived EVs.ResultsMass spectrometry-based proteomics revealed comprehensive profiles for small EVs released from healthy human fibroblasts from different tissues. We found that fibroblast-derived EVs were more similar than EVs from differentiating myoblasts, but there were significant differences between tendon fibroblast and muscle fibroblast EVs. Small EVs from tendon fibroblasts contained higher levels of proteins that support ECM synthesis, including TGFβ1, and muscle fibroblast EVs contained proteins that support myofiber function and components of the skeletal muscle matrix.ConclusionsOur data demonstrates a marked heterogeneity among healthy fibroblast-derived EVs, indicating shared tasks between EVs of skeletal muscle myoblasts and fibroblasts, whereas tendon fibroblast EVs could play a fibrotic role in human tendon tissue. These findings suggest an important role for EVs in tissue homeostasis of both tendon and skeletal muscle in humans.EueS9_TM9G2gnPvs8i76-XVideo abstract

Single-cell transcriptomic analysis identifies extensive heterogeneity in the cellular composition of mouse Achilles tendons.

Tendon is a dense connective tissue that stores and transmits forces between muscles and bones. Cellular heterogeneity is increasingly recognized as an important factor in the biological basis of tissue homeostasis and disease, yet little is known about the diversity of cell types that populate tendon. To address this, we determined the heterogeneity of cell populations within mouse Achilles tendons using single-cell RNA sequencing. In assembling a transcriptomic atlas of Achilles tendons, we identified 11 distinct types of cells, including three previously undescribed populations of tendon fibroblasts. Prior studies have indicated that pericytes, which are found in the vasculature of tendons, could serve as a potential source of progenitor cells for adult tendon fibroblasts. Using trajectory inference analysis, we provide additional support for the notion that pericytes are likely to be at least one of the progenitor cell populations for the fibroblasts that compose adult tendons. We also modeled cell-cell interactions and identified previously undescribed ligand-receptor signaling interactions involved in tendon homeostasis. Our novel and interactive tendon atlas highlights previously underappreciated heterogeneity between and within tendon cell populations. The atlas also serves as a resource to further the understanding of tendon extracellular matrix assembly and maintenance and in the design of therapies for tendinopathies.