Scleraxis Research Articles

Study on anti-adhesion effect and mechanism of dynamic and static stress stimulation during early healing process of rat Achilles tendon injury

To investigate the anti-adhesive effect and underlying mechanism of dynamic and static stress stimulation on the early healing process of rat Achilles tendon injury. Achilles tendon tissues of 15 male Sprague Dawley (SD) rats aged 4-6 weeks were isolated and cultured by enzyme digestion method. Rat Achilles tendon cells were treated with tumor necrosis factor α to construct the Achilles tendon injury cell model, and dynamic stress stimulation (dynamic group) and static stress stimulation (static group) were applied respectively, while the control group was not treated. Live/dead cell double staining was used to detect cell activity, ELISA assay was used to detect the expression of α smooth muscle actin (α-SMA), and real-time fluorescence quantitative PCR was used to detect the mRNA expression of collagen type Ⅰ (COL1A1), collagen type Ⅲ (COL3A1), and Scleraxis (SCX). Thirty male SD rats aged 4-6 weeks underwent Achilles tendon suture and were randomly divided into dynamic group (treated by dynamic stress stimulation), static group (treated by static stress stimulation), and control group (untreated), with 10 rats in each group. HE staining and scoring were performed to evaluate the healing of Achilles tendon at 8 days after operation. COL1A1 and COL3A1 protein expressions were detected by immunohistochemical staining, α-SMA and SCX protein expressions were detected by Western blot, and maximum tendon breaking force and tendon stiffness were detected by biomechanical stretching test. In vitro cell experiment, when compared to the static group, the number of living cells in the dynamic group was higher, the expression of α-SMA protein was decreased, the relative expression of COL3A1 mRNA was decreased, and the relative expression of SCX mRNA was increased, and the differences were all significant ( P<0.05). In the in vivo animal experiment, when compared to the static group, the tendon healing in the dynamic group was better, the HE staining score was lower, the expression of COL1A1 protein was increased, the expression of COL3A1 protein was decreased, the relative expression of SCX protein was increased, the relative expression of α-SMA protein was decreased, and the tendon stiffness was increased, the differences were all significant ( P<0.05). Compared with static stress stimulation, the dynamic stress stimulation improves the fibrosis of the scar tissue of the rat Achilles tendon, promote the recovery of the biomechanical property of the Achilles tendon, and has obvious anti-adhesion effect.

Growth Differentiation Factor 5-Induced Mesenchymal Stromal Cells Enhance Tendon Healing.

Mesenchymal stromal cells (MSCs) have immense potential for use in musculoskeletal tissue regeneration; however, there is still a paucity of evidence on the effect of tenogenic MSCs (TMSCs) in tendon healing invivo. This study aimed to determine the effects of growth differentiation factor 5 (GDF5)-induced rabbit MSCs (rbMSCs) on infraspinatus tendon healing in a New Zealand white rabbit model. In this study, bone marrow-derived rbMSCs were isolated, and 100 ng/mL GDF5 was used to induce tenogenic differentiation in rbMSC. The effects of GDF5 on rbMSC in vitro were assessed by total collagen assay, gene expression analysis, and immunofluorescence staining of tenogenic markers; native tenocytes isolated from rabbit tendon were used as a positive control. In invivo, a window defect was created on the infraspinatus tendons bilaterally. After 3 weeks, the rabbits (n = 18) were randomly divided into six groups and repaired with various interventions: (1) surgical suture; (2) fibrin glue (FG); (3) suture and FG; (4) suture, FG, and rabbit tenocytes (rbTenocyte); (5) suture, FG, and rbMSCs, and (6) suture, FG, and TMSC. All animals were euthanized at 6 weeks postoperatively. The in vitro GDF5-induced rbMSCs (or TMSC) showed increased total collagen expression, augmented scleraxis (SCX), and type-I collagen (COL1A1) mRNA gene expression levels. Immunofluorescence showed similar expression in GDF5-induced rbMSC to that of rbTenocyte. In vivo histological analysis showed progressive tendon healing in the TMSC-treated group; cells with elongated nuclei aligned parallel to the collagen fibers, and the collagen fibers were in a more organized orientation, along with macroscopic evidence of tendon callus formation. Significant differences were observed in the cell-treated groups compared with the non-cell-treated groups. Histological scoring showed a significantly enhanced tendon healing in the TMSC- and rbMSC-treated groups compared with the rbTenocyte group. The SCX mRNA expression levels, at 6 weeks following repair, were significantly upregulated in the TMSC group. Immunofluorescence showed COL-1 bundles aligned in parallel orientation; this was further confirmed in atomic force microscopy imaging. SCX, TNC, and TNMD were detected in the TMSC group. In conclusion, GDF5 induces tenogenic differentiation in rbMSCs, and TMSC enhances tendon healing invivo compared with conventional suture repair. Impact Statement Tendon tears and degeneration are debilitating clinical conditions. To date, the suture method is the only gold standard for repairing tendons. Mesenchymal stromal cells (MSCs) have been suggested for many years for their potential in tissue regeneration, especially in tendon-degenerative conditions. Growth differentiation factor 5 (GDF5) has been reported to induce human MSC into a tenogenic lineage (or TMSC), hence a potential cell source for tendon regeneration. This study reported on the potential of rabbit MSC to differentiate into TMSC via GDF5 induction and the potential of TMSC in tendon healing in a New Zealand white rabbit infraspinatus tendon model fulfilled with the 3R principle (reduce, reuse, and replace).

Novel In Vitro Platform for Studying the Cell Response to Healthy and Diseased Tendon Matrices.

Current in vitro models poorly represent the healthy or diseased tendon microenvironment, limiting the translation of the findings to clinics. The present work aims to establish a physiologically relevant in vitro tendon platform that mimics biophysical aspects of a healthy and tendinopathic tendon matrix using a decellularized bovine tendon and to characterize tendon cells cultured using this platform. Bovine tendons were subjected to various decellularization techniques, with the efficacy of decellularization determined histologically. The biomechanical and architectural properties of the decellularized tendons were characterized using an atomic force microscope. Tendinopathy-mimicking matrices were prepared by treating the decellularized tendons with collagenase for 3 h or collagenase-chondroitinase (CC) for 1 h. The tendon tissue collected from healthy and tendinopathic patients was characterized using an atomic force microscope and compared to that of decellularized matrices. Healthy human tendon-derived cells (hTDCs) from the hamstring tendon were cultured on the decellularized matrices for 24 or 48 h, with cell morphology characterized using f-actin staining and gene expression characterized using real-time PCR. Tendon matrices prepared by freeze-thawing and 48 h nuclease treatment were fully decellularized, and the aligned structure and tendon stiffness (1.46 MPa) were maintained. Collagenase treatment prepared matrices with a disorganized architecture and reduced stiffness (0.75 MPa), mimicking chronic tendinopathy. Treatment with CC prepared matrices with a disorganized architecture without altering stiffness, mimicking early tendinopathy (1.52 MPa). hTDCs on a healthy tendon matrix were elongated, and the scleraxis (SCX) expression was maintained. On tendinopathic matrices, hTDCs had altered morphological characteristics and lower SCX expression. The expression of genes related to actin polymerization, matrix degradation and remodeling, and immune cell invasion were higher in hTDCs on tendinopathic matrices. Overall, the present study developed a physiological in vitro system to mimic healthy tendons and early and late tendinopathy, and it can be used to better understand tendon cell characteristics in healthy and diseased states.

A systematic investigation of the effects of TGF-β3 and mechanical stimulation on tenogenic differentiation of mesenchymal stromal cells in a poly(ethylene glycol)/gelatin-based hydrogel

BackgroundHigh post-surgical failure rates following tendon injury generate high medical costs and poor patient recovery. Cell-based tendon tissue engineering has the potential to produce fully functional replacement tissue and provide new strategies to restore tendon function and healing. In this endeavour, the application of mesenchymal stromal cells (MSCs) encapsulated in biomaterial scaffolds has shown great promise. However, a consensus on optimal promotion of tenogenic differentiation of MSCs has yet to be reached, although growth factors and mechanical cues are generally acknowledged as important factors. MethodsIn this study, we prepared a hydrogel cell culture system consisting of methacrylated poly(d,l-lactic acid-ethylene glycol-d,l-lactic acid) (P(LA-EG-LA)) and gelatin methacrylate (GelMA) to encapsulate human bone marrow-derived MSCs (hBMSCs). We further systematically investigated the influence of static and intermittent cyclic uniaxial strain mechanical stimulation, in combination with transforming growth factor-β3 (TGF-β3) supplementation, on tenogenic differentiation of hBMSCs. ResultsIncreased TGF-β3 concentration upregulated the tenogenic genes Scleraxis (SCX) and collagen type I (COL1A1) but showed no effects on tenascin-c (TNC) and collagen type III (COL3A1) expression. Mechanical stimulation had no observable effect on gene expression, but intermittent cyclic uniaxial strain stimulation improved matrix deposition. Together, these data provide new insights into how TGF-β3 and mechanical stimulation regulate MSC tenogenesis, with TGF-β3 promoting the expression of key tenogenic genes whilst mechanical stimulation aided matrix deposition in the engineered tissue. Furthermore, intermittent cyclic uniaxial strain at 3% elongation and 0.33 ​Hz for 1 ​h/day showed improved matrix effects compared to static strain. ConclusionTogether, the most promising result for tenogenic differentiation of hBMSCs was identified as treatment with 5 ​ng/ml TGF-β3 under intermittent cyclic uniaxial strain (3% strain; 0.33 ​Hz; 1 ​h/day). This knowledge is of importance for the development of an improved protocol for tenogenic differentiation of MSCs and thereby for tendon tissue engineering. The translational potential of this articleTissue-engineered strategies for tendon repair require a consensus on the differentiation of mesenchymal stromal cells to tenocytes, which is currently lacking. This article provides a systematic investigation of two main tenogenic differentiation conditions to further development of a tenogenic differentiation protocol.

Nonviral overexpression of Scleraxis or Mohawk drives reprogramming of degenerate human annulus fibrosus cells from a diseased to a healthy phenotype.

Intervertebral disc (IVD) degeneration is a major contributor to low back pain (LBP), yet there are no clinical therapies targeting the underlying pathology. The annulus fibrosus (AF) plays a critical role in maintaining IVD structure/function and undergoes degenerative changes such as matrix catabolism and inflammation. Thus, therapies targeting the AF are crucial to fully restore IVD function. Previously, we have shown nonviral delivery of transcription factors to push diseased nucleus pulposus cells to a healthy phenotype. As a next step in a proof-of-concept study, we report the use of Scleraxis (SCX) and Mohawk (MKX), which are critical for the development, maintenance, and regeneration of the AF and may have therapeutic potential to induce a healthy, pro-anabolic phenotype in diseased AF cells. MKX and SCX plasmids were delivered via electroporation into diseased human AF cells from autopsy specimens and patients undergoing surgery for LBP. Transfected cells were cultured over 14 days and assessed for cell morphology, viability, density, gene expression of key phenotypic, inflammatory, matrix, pain markers, and collagen accumulation. AF cells demonstrated a fibroblastic phenotype posttreatment. Moreover, transfection of SCX and MKX resulted in significant upregulation of the respective genes, as well as SOX9. Transfected autopsy cells demonstrated upregulation of core extracellular matrix markers; however, this was observed to a lesser effect in surgical cells. Matrix-degrading enzymes and inflammatory cytokines were downregulated, suggesting a push toward a pro-anabolic, anti-inflammatory phenotype. Similarly, pain markers were downregulated over time in autopsy cells. At the protein level, collagen content was increased in both MKX and SCX transfected cells compared to controls. This exploratory study demonstrates the potential of MKX or SCX to drive reprogramming in mild to moderately degenerate AF cells from autopsy and severely degenerate AF cells from surgical patients toward a healthy phenotype and may be a potential nonviral gene therapy for LBP.

Examining the Effects of In Vitro Co-Culture of Equine Adipose-Derived Mesenchymal Stem Cells With Tendon Proper and Peritenon Cells

Tendinopathies remain the leading contributor to career-ending injuries in horses because of the complexity of tendon repair. As such, cell-based therapies like injections of adipose-derived mesenchymal stem cells (ADMSCs, or MSCs) into injured tendons are becoming increasingly popular though their long-term efficacy on a molecular and wholistic level remains contentious. Thus, we co-cultured equine MSCs with intrinsic (tendon proper) and extrinsic (peritenon) tendon cell populations to examine interactions between these cells. Gene expression for common tenogenic, perivascular, and differentiation markers was quantified at 48 and 120 hours. Additionally, cellular metabolism of proliferation was examined every 24 hours for peritenon and tendon proper cells co-cultured with MSCs. MSCs co-cultured with tendon proper or peritenon cells had altered expression profiles demonstrating trend toward tenogenic phenotype with the exception of decreases in type I collagen (COL1A1). Peritenon cells co-cultured with MSCs had a trending and significant decrease in biglycan (BGN) and CSPG4 at 48 hours and 120 hours but overall significant increases in lysyl oxidase (LOX), mohawk (MKX), and scleraxis (SCX) within 48 hours. Tendon proper cells co-cultured with MSCs also exhibited increases in LOX and SCX at 48 hours. Furthermore, cell proliferation improved overall for tendon proper cells co-cultured with MSCs. The co-culture study results suggest that adipose-derived MSCs contribute beneficially to tenogenic stimulation of peritenon or tendon proper cells.

Rebalancing SMAD7/SMAD3 Signaling Reduces Adhesion Formation during Flexor Tendon Healing.

Transforming growth factor-β is a key factor in regulating adhesion formation during tendon healing. We investigated the effectiveness of SMAD family members, SMAD7 and SMAD3, in the TGF-β/Smad signaling during flexor tendon repair. Mouse flexor toe deep tendon rupture anastomosis models were made. On days 3, 7, 14, 21, and 28, the expressions of smad7 and smad3 in flexor tendon tissues were detected by RT-qPCR and western blot. Furthermore, postoperative intraperitoneal injections of SMAD7 agonists or SMAD3 antagonists were given. The degree of tendon healing was evaluated by adhesion testing and biomechanical experiments. Hematoxylin and eosin (HE) staining was used to observe the pathological changes. Immunohistochemistry was used to evaluate the expressions of collagen III, SMAD3, and SMAD7. The mRNA levels of matrix metalloproteinases, Mmp2 and Mmp9, and scleraxis (SCX) in flexor tendon tissue were detected by RT-qPCR. Smad3 expression increased and Smad7 expression decreased in flexor tendon tissue after injury. In addition, the SMAD7 agonist blocked SMAD3 phosphorylation. SMAD7 agonist and SMAD3 antagonist both improved adhesion formation during flexor tendon healing, and decreased the expressions of collagen III, Mmp9, and SCX, while increasing Mmp2 expression. This study provides a possible theoretical basis for the SMAD7-SMAD3 signal cascade during flexor tendon adhesion healing.

Synergistic effects of growth factor-based serum-free medium and tendon-like substrate topography on tenogenesis of mesenchymal stem cells

Addressing clinical challenges for tendon injuries requires a deeper understanding of the effects that biological and biophysical cues have on tenogenesis. Although prior studies have identified tenogenic growth factors (GFs) or elucidated the effects of substrate topography on tenocyte behavior, few have characterized their combined effect in the presence of a tendon-like substrate. In this study, we assessed the effect of biological (GFs) and biophysical (substrate topography) cues on tenogenic proliferation and differentiation under defined, serum-free conditions. Specifically, human bone marrow-derived mesenchymal stem cells (hMSCs) were cultured in a serum-free culture medium containing a GF cocktail comprised of fibroblast growth factor-2 (FGF-2), transforming growth factor-beta 3 (TGF-β3), and insulin-like growth factor-1 (IGF-1), either alone or in combination with tendon-like substrate topography produced by replica casting of tendon tissue sections. Our data demonstrated that the use of serum-free GF cocktail medium alone promoted hMSC proliferation, as shown via DNA staining as well as Ki67 protein levels and gene expression. In particular, gene expression of Ki67 was increased by 8.46-fold in all three donors relative to serum-free medium control. Also, serum-free GF cocktail promoted tenogenic differentiation, on the basis of expression of tendon-associated gene and protein markers, scleraxis (SCX), tenascin C (TNC), and collagen type I (COL1A1) including increased normalized collagen production by 1.4-fold in two donors relative to serum-free medium control. Interestingly, hMSCs cultured on a tendon-like substrate exhibited highly oriented cell morphology and extracellular matrix (ECM) alignment reminiscent of tendon. In particular, when this GF cocktail was combined with tendon-like topography, they showed a synergistically increased expression of tendon-related markers and anisotropic organization of ECM proteins with moderate-to-large effect sizes. Together, in addition to showing the utility of a GF cocktail for expansion and differentiation of tenocyte-like cells, our findings clearly demonstrate the synergistic relationship between GF-mediated and substrate topography-related effects on hMSC tenogenic differentiation. This information provides insights into the design of strategies that combine biological and biophysical cues for ex vivo tenocyte production and tendon tissue engineering.

N-Acetyl-L-cysteine facilitates tendon repair and promotes the tenogenic differentiation of tendon stem/progenitor cells by enhancing the integrin α5/β1/PI3K/AKT signaling

BackgroundTendon 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. MethodThe 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.ResultsCompared 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 α5β1 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.ConclusionNAC 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.

Anterior cruciate ligament remnant preservation attenuates apoptosis and enhances the regeneration of hamstring tendon graft

The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction. The ACL remnant, bone marrow, and hamstring tendons were surgically harvested from rabbits. The apoptosis rate, cell proliferation, and expression of types I and III collagen, transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and tenogenic genes (scleraxis (SCX), tenascin C (TNC), and tenomodulin (TNMD)) of the hamstring tendons were compared between the co-culture medium (ACL remnant cells (ACLRCs) and BMSCs co-culture) and control medium (BMSCs-only culture). We also evaluated the apoptosis, cell proliferation, migration, and gene expression of hamstring tenocytes with exposure to co-culture and control media. Compared to BMSCs-only culture medium, the co-culture medium showed substantially decreased early and late apoptosis rates, attenuation of intrinsic and extrinsic apoptotic pathways, and enhanced proliferation of the hamstring tendons and tenocytes. In addition, the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes in the hamstring tendons and tenocytes significantly increased in the co-culture medium compared to that in the control medium. In the presence of ACLRCs and BMSCs, the hamstring tendons and tenocytes significantly attenuated apoptosis and enhanced the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes. This in vitro study suggests that the ACLRCs mixed with BMSCs could aid regeneration of the hamstring tendon graft during ACL reconstruction.Cite this article: Bone Joint Res2023;12(1):9-21.

The Direct Effect of Nanofiber-based Vitamin D Sheet Engineered with 3D Printing for Tendon-to-bone Healing and Muscle Regeneration after Repair in a Chronic Rotator Cuff Tear Model of Rabbit (217)

Objectives:To compare the efficacy between the direct effect of nanofiber-based vitamin D sheet engineered with 3D printing (VTD sheet) and vitamin D supplementation (VTDS) as diet on tendon-to-bone healing and muscle regeneration after repair in a chronic rotator cuff tear model of rabbit.Methods:A total of 64 rabbits were randomly allocated into two groups (n=32), then each groups was allocated into four small groups (Group A, A’: VTDS only, Group B, B’: Normal diet + sheet without vitamin D, Group C, C’: Normal diet + VTD sheet, Group D, D’: VTDS + VTD sheet, n=8 each). The supraspinatus tendons which were detached and left for 6 weeks were repaired in a transosseous manner with the sheet only for groups B and B’, and VTD sheet for groups C, C’, D and D’ (Figure 1). Groups A, B, C, and D were extracted at 4weeks after repair, while groups A’, B’, C’, and D’ were extracted at 12 weeks after repair. Serum 25-OH vitamin D level was checked at the time of making tear, repair, and extraction. Regarding tendon-to-bone healing, the expression (relative ratio to control) of genes including type 1 collagen (COL1), type 3 collagen (COL3), bone morphogenic protein-2 (BMP-2), scleraxis (SCX), SOX9, and aggrecan (ACAN) was assessed at 4 weeks (Groups A, B, C, and D), and at 12 weeks (Groups A’, B’, C’, and D’) after repair. The histological and biomechanical evaluations of tendon-to-bone healing were done at 12 weeks after repair. Regarding muscle regeneration, rotator cuff muscle cross-sectional areas were measured at 4 and 12 weeks after repair. Enzyme-linked immunosorbent assay (ELISA) was done to calculate vitamin D level in muscle at 12 weeks after repair.Results:Serum vitamin D level of group D and D’ was highest among groups at the time of repair and extraction (p < 0.001). At 4 weeks after repair, mRNA expression of COL1 in group D was highest among groups (A, B, C, D; 0.86 ± 0.25, 0.90 ± 0.27, 0.93 ± 0.19, and 1.06 ± 0.25, respectively, p = 0.046). At 12 weeks after repair, group D’ showed most dense collagen density (p = 0.037) and had highest load to failure among groups (A’, B’, C’, D’; 102.3 ± 12.6 N, 99.5 ± 8.3 N, 102.3 ± 18.5 N, and 139.6 ± 25.3, respectively, p = 0.024). Regarding muscle regeneration, the cross-sectional area of muscle fiber was largest in group D and D’ at 4 and 12 weeks after repair (p < 0.05, figure 4) with highest vitamin D level of muscle by ELISA at 12 weeks after repair (p = 0.003).Conclusions:The use of nanofiber-based vitamin D sheet engineered with 3D printing may promote tendon-to-bone healing and regenerate rotator cuff muscle after repair in a chronic rabbit rotator cuff tear model.

Tenogenic Potential of Equine Fetal Mesenchymal Stem Cells Under The In Vitro Effect of Bone Morphogenetic Protein-12 (BMP-12)

Equine adult bone marrow-derived MSCs (BM-MSCs) may be induced into the tenogenic lineage after exposure with bone morphogenetic protein-12 (BMP-12). Despite fetal BM-MSCs have showed a greater differentiation potential compared to adults, the tenogenic differentiation capacity of equine fetal BM-MSC have not been reported. Thus, the aim of the present study was to evaluate the in vitro tenogenic differentiation potential of equine fetal BM-MSCs under the effect of BMP-12. Equine fetal BM-MSCs were exposed to three concentrations of BMP-12 (25, 50 and 100 ng/mL) during a 21-day culture period. Levels of mRNA of tenogenic markers decorin (DCN), tenomodulin (TNMD), scleraxis (SCX), collagen 1α1 (COL1α1) and protein expression of Col1α1 were evaluated. Plastic adherent cells exhibited specific MSC profile including expression of CD73 and lack of expression of CD34. Gene expression levels of DCN, TNMD, SCX and COL1α1 were increased in equine fetal BM-MSC exposed to three different concentrations of BMP-12 during a 21-day culture period. Equine fetal BM-MSCs displayed specific expression profiles suggesting features of MSCs and multipotent capacity. Furthermore, up-regulation of tenogenic markers DCN, TNMD, COL1α1 and SCX after exposure to different concentrations of BMP-12 suggests that equine fetal BM-MSCs have potential to activate selected genes that control tenogenic differentiation.

Synergistic effects of myogenic cells and fibroblasts on the promotion of engineered tendon regeneration with muscle derived cells

ABSTRACT Aims Tendon development requires the coordinated interaction of muscles and tendons. Muscle-derived cells (MDCs), a mixed cell population containing both myogenic and fibroblastic cell subsets, have been found to be ideal seed cells for tendon regeneration. However, the necessity of these cell types for tendon regeneration has not yet been tested. In this study, we aim to explore the possible synergistic effects of myogenic cells and fibroblasts in engineered tendon regeneration. Methods MDCs were separated into rapidly adhering cell (RAC; fibroblasts) and slowly adhering cell (SAC; myogenic cells) populations. Myogenic- and tenogenic-related molecules were analyzed by immunofluorescent staining, RT-PCR and real-time PCR. The proliferative abilities of MDCs, RACs and SACs were also evaluated. Cell-scaffold constructs were implanted into nude mice, and subsequently evaluated for their histologic, ultrastructure, gene expression, and biomechanical characteristics. Results MDCs have better proliferative activity than RAC and SAC population. RACs could express higher levels of tenogenic-related molecules tenomodulin (TNMD) and scleraxis (SCX) than SACs. Whereas SACs only expressed myogenic-related molecules MyoD. In contrast to the tendons engineered using RACs and SACs, the tendons engineered using MDCs exhibited a relatively more mature and well-organized tissue structure and ultrastructure as well as better mechanical properties. Conclusions Fibroblasts in muscle may be the primary cell population involved in tendon regeneration and that myogenic cells are an important component of the niche and control the fibroblast activity during tendon regeneration. The synergistic effects between fibroblasts and myogenic cells significantly contribute to efficient and effective regeneration of engineered tendons.

Scleraxis expressing scleral cells respond to inflammatory stimulation

The sclera is an ocular tissue rich of collagenous extracellular matrix, which is built up and maintained by relatively few, still poorly characterized fibroblast-like cells. The aims of this study are to add to the characterization of scleral fibroblasts and to examine the reaction of these fibroblasts to inflammatory stimulation in an ex vivo organotypic model. Scleras of scleraxis-GFP (SCX-GFP) mice were analyzed using immunohistochemistry and qRT-PCR for the expression of the tendon cell associated marker genes scleraxis (SCX), mohawk and tenomodulin. In organotypic tissue culture, explanted scleras of adult scleraxis GFP reporter mice were exposed to 10 ng/ml recombinant interleukin 1-ß (IL1-ß) and IL1-ß in combination with dexamethasone. The tissue was then analyzed by immunofluorescence staining of the inflammation- and fibrosis-associated proteins IL6, COX-2, iNOS, connective tissue growth factor, MMP2, MMP3, and MMP13 as well as for collagen fibre degradation using a Collagen Hybridizing Peptide (CHP) binding assay. The mouse sclera displayed a strong expression of scleraxis promoter-driven GFP, indicating a tendon cell-like phenotype, as well as expression of scleraxis, tenomodulin and mohawk mRNA. Upon IL1-ß stimulation, SCX-GFP+ cells significantly upregulated the expression of all proteins analysed. Moreover, IL1-ß stimulation resulted in significant collagen degradation. Adding the corticosteroid dexamethasone significantly reduced the response to IL1-ß stimulation. Collagen degradation was significantly enhanced in the IL1-ß group. Dexamethasone demonstrated a significant rescue effect. This work provides insights into the characteristics of scleral cells and establishes an ex vivo model of scleral inflammation.

Paediatric knee anterolateral capsule does not contain a distinct ligament: analysis of histology, immunohistochemistry and gene expression

ObjectivesThe presence of a discrete ligament within the knee anterolateral capsule (ALC) is controversial. Tendons and ligaments have typical collagens, ultrastructure, transcription factors and proteins. However, these characteristics have not been investigated in paediatric ALC. The purpose of this study was to characterise the paediatric ALC in terms of tissue ultrastructure and cellular expression of ligament markers scleraxis (SCX)—a basic helix-loop-helix transcription factor—and the downstream transmembrane glycoprotein tenomodulin (TNMD), as compared with the paediatric lateral collateral ligament (LCL) and paediatric quadriceps tendon (QT). We hypothesised that, in comparison to the LCL and QT, the ALC would possess poor collagen orientation and reduced SCX and TNMD expression.Methods15 paediatric ALCs (age 6.3±3.3 years), 5 paediatric LCLs (age 3.4±1.3 years) and 5 paediatric QTs (age 2.0±1.2 years) from fresh cadaveric knees were used in this study. Fresh-frozen samples from each region were cryosectioned and then stained with H&E to evaluate collagen alignment and cell morphology. Expression of SCX and TNMD was determined by gene expression analysis and immunohistochemistry.ResultsThe histological sections of the paediatric LCL and QT showed well-organised, dense collagenous tissue fibres with elongated fibroblasts, while the ALC showed more random collagen orientation without clear cellular directionality. The aspect ratio of cells in the ALC was significantly lower than that of the LCL and QT (p<0.0001 and p<0.0001, respectively). The normalised distribution curve of the inclination angles of the nuclei in the ALC was more broadly distributed than that of the LCL or QT, indicating random cell alignment in the ALC. SCX immunostaining was apparent in the paediatric LCL within regions of aligned fibres, while the comparatively disorganised structure of the ALC was negative for SCX. The paediatric LCL also stained positive for TNMD, while the ALC was only sparsely positive for this tendon/ligament cell-surface molecule. Relative gene expression of SCX and TNMD were higher in the LCL and QT than in the ALC.ConclusionIn this study, a distinct ligament could not be discerned in the ALC based on histology, immunohistochemistry and gene expression analysis.Level of evidenceControlled laboratory study.

Collagen and chondroitin sulfate functionalized bioinspired fibers for tendon tissue engineering application

Functional tendon tissue engineering depends on harnessing the biochemical and biophysical cues of the native tendon extracellular matrix. In this study, we fabricated highly-aligned poly(L-lactic acid) (PLLA) fibers with surfaces decorated by two of the crucial tendon ECM components, type 1 collagen (COL1) and chondroitin sulfate (CS), through a coaxial stable jet electrospinning approach. Effects of the biomimetic COL1-CS (shell)/PLLA (core) fibers on the tenogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro were investigated. Higher rates of cell spreading and proliferation are observed on the aligned COL1-CS/PLLA fibers compared to that on the plain PLLA fibers. Expression of the tendon-associated genes scleraxis (SCX) and COL1 as well as protein tenomodulin (TNMD) are significantly increased. Introduction of mechanical stimulation gives rise to synergistic effect on tenogenic differentiation of hMSCs. Higher expression of TGF-β2, TGFβR-II, and Smad3 by the cells on the COL1-CS/PLLA fiber substrates are observed, which indicates that COL1-CS/PLLA ultrafine fibers dictate the hMSC tenogenic differentiation through activating the TGF-β signaling pathway. Animal study in rat Achilles tendon repair model corroborated the promoting role of COL1-CS/PLLA in regenerating a tendon-like tissue. Thus, our highly aligned biomimicking fibers may serve as an efficient scaffolding system for functional tendon regeneration.

Induction of tenogenic differentiation of equine adipose-derived mesenchymal stem cells by platelet-derived growth factor-BB and growth differentiation factor-6.

Managingtendon healing process is complicated mainly due tothe limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and have been considered for tendon repair and regeneration. This study aimed to evaluate the capacity of equine adipose tissue-derived cells (eASCs) to differentiate into tenocytes in response to platelet-derived growth factor-BB (PDGF-BB) and growth differentiation factor-6 (GDF-6) in vitro. Frozen characterized eASCS of 3 mares were thawed and the cells were expanded in basic culture medium (DMEM supplementedwith 10% FBS). The cells at passage 5 were treated for 14 days in different conditions including: (1) control group in basic culture medium (CM), (2) induction medium as IM (CM containing L-prolin, and ascorbic acid (AA)) supplemented with PDGF-BB (20ng/ml), (3) IM supplemented with GDF-6 (20ng/ml), and (4) IM supplemented with PDGF-BB and GDF-6. At the end of culture period (14th day), tenogenic differentiation was evaluated. Sirius Red staining was used to assess collagen production, and H&E was used for assessing cell morphology. mRNA levels of collagen type 1 (colI), scleraxis (SCX), and Mohawk (MKX), as tenogenic markers, were analyzed using real-time reverse-transcription polymerase chain reaction (qPCR). H&E staining showed a stretching and spindle shape (tenocyte-like) cells in all treated groups compared to unchanged from of cells in control groups. Also, Sirius red staining data showed a significant increase in collagen production in all treated groups compared with the control group. MKX expression was significantly increased in PDGF-BB and mixed groups and COLI expression was significantly increased only in PDGF-BB group. In conclusion, our results showed that PDGF-BB and GDF-6 combinationcould induce tenogenic differentiation in eASCs. These in vitro findings could be useful for cell therapy in equine regenerative medicine.

The transcription factor scleraxis differentially regulates gene expression in tenocytes isolated at different developmental stages

The transcription factor scleraxis (SCX) is expressed throughout tendon development and plays a key role in directing tendon wound healing. However, little is known regarding its role in fetal or young postnatal tendons, stages in development that are known for their enhanced regenerative capabilities. Here we used RNA-sequencing to compare the transcriptome of adult and fetal tenocytes following SCX knockdown. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes affected in adult tenocytes, indicating that scleraxis-dependent processes may differ in these two developmental stages. Gene ontology, network and pathway analysis revealed an overrepresentation of extracellular matrix (ECM) remodelling processes within both comparisons. These included several matrix metalloproteinases, proteoglycans and collagens, some of which were also investigated in SCX knockdown tenocytes from young postnatal foals. Using chromatin immunoprecipitation, we also identified novel genes that SCX differentially interacts with in adult and fetal tenocytes. These results indicate a role for SCX in modulating ECM synthesis and breakdown and provide a useful dataset for further study into SCX gene regulation.

The Transcription Factor SCX is a Potential Serum Biomarker of Fibrotic Diseases.

Fibrosing diseases are causes of morbidity and mortality around the world, and they are characterized by excessive extracellular matrix (ECM) accumulation. The bHLH transcription factor scleraxis (SCX) regulates the synthesis of ECM proteins in heart fibrosis. SCX expression was evaluated in lung fibroblasts and tissue derived from fibrotic disease patients and healthy controls. We also measured SCX in sera from 57 healthy controls, and 56 Idiopathic Pulmonary Fibrosis (IPF), 40 Hypersensitivity Pneumonitis (HP), and 100 Systemic Sclerosis (SSc) patients. We report high SCX expression in fibroblasts and tissue from IPF patients versus controls. High SCX-serum levels were observed in IPF (0.663 ± 0.559 ng/mL, p < 0.01) and SSc (0.611 ± 0.296 ng/mL, p < 0.001), versus controls (0.351 ± 0.207 ng/mL) and HP (0.323 ± 0.323 ng/mL). Serum levels of the SCX heterodimerization partner, TCF3, did not associate with fibrotic illness. IPF patients with severely affected respiratory capacities and late-stage SSc patients presenting anti-topoisomerase I antibodies and interstitial lung disease showed the highest SCX-serum levels. SCX gain-of-function induced the expression of alpha-smooth muscle actin (α-SMA/ACTA2) in fibroblasts when co-overexpressed with TCF3. As late and severe stages of the fibrotic processes correlated with high circulating SCX, we postulate it as a candidate biomarker of fibrosis and a potential therapeutic target.

Development of a novel in vitro insulin resistance model in primary human tenocytes for diabetic tendinopathy research.

BackgroundType 2 diabetes mellitus (T2DM) had been reported to be associated with tendinopathy. However, the underlying mechanisms of diabetic tendinopathy still remain largely to be discovered. The purpose of this study was to develop insulin resistance (IR) model on primary human tenocytes (hTeno) culture with tumour necrosis factor-alpha (TNF-α) treatment to study tenocytes homeostasis as an implication for diabetic tendinopathy.MethodshTenowere isolated from human hamstring tendon. Presence of insulin receptor beta (INSR-β) on normal tendon tissues and the hTeno monolayer culture were analyzed by immunofluorescence staining. The presence of Glucose Transporter Type 1 (GLUT1) and Glucose Transporter Type 4 (GLUT4) on the hTeno monolayer culture were also analyzed by immunofluorescence staining. Primary hTeno were treated with 0.008, 0.08, 0.8 and 8.0 µM of TNF-α, with and without insulin supplement. Outcome measures include 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) assay to determine the glucose uptake activity; colourimetric total collagen assay to quantify the total collagen expression levels; COL-I ELISA assay to measure the COL-I expression levels and real-time qPCR to analyze the mRNA gene expressions levels of Scleraxis (SCX), Mohawk (MKX), type I collagen (COL1A1), type III collagen (COL3A1), matrix metalloproteinases (MMP)-9 and MMP-13 in hTeno when treated with TNF-α. Apoptosis assay for hTeno induced with TNF-α was conducted using Annexin-V FITC flow cytometry analysis.ResultsImmunofluorescence imaging showed the presence of INSR-β on the hTeno in the human Achilles tendon tissues and in the hTeno in monolayer culture. GLUT1 and GLUT4 were both positively expressed in the hTeno. TNF-α significantly reduced the insulin-mediated 2-NBDG uptake in all the tested concentrations, especially at 0.008 µM. Total collagen expression levels and COL-I expression levels in hTeno were also significantly reduced in hTeno treated with 0.008 µM of TNF-α. The SCX, MKX and COL1A1 mRNA expression levels were significantly downregulated in all TNF-α treated hTeno, whereas the COL3A1, MMP-9 and MMP-13 were significantly upregulated in the TNF–α treated cells. TNF-α progressively increased the apoptotic cells at 48 and 72 h.ConclusionAt 0.008 µM of TNF-α, an IR condition was induced in hTeno, supported with the significant reduction in glucose uptake, as well as significantly reduced total collagen, specifically COL-I expression levels, downregulation of candidate tenogenic markers genes (SCX and MKX), and upregulation of ECM catabolic genes (MMP-9 and MMP-13). Development of novel IR model in hTeno provides an insight on how tendon homeostasis could be affected and can be used as a tool for further discovering the effects on downstream molecular pathways, as the implication for diabetic tendinopathy.