Scleraxis Expression Research Articles

Nanotopographic cues and stiffness control of tendon-derived stem cells from diverse conditions.

BackgroundTendon-derived stem cells (TDSCs) are key factors associated with regeneration and healing in tendinopathy. The aim of this study was to investigate the effects of mechanical stiffness and topographic signals on the differentiation of TDSCs depending on age and pathological conditions.Materials and methodsWe compared TDSCs extracted from normal tendon tissues with TDSCs from tendinopathic Achilles tendon tissues of Sprague Dawley rats in vitro and TDSCs cultured on nanotopographic cues and substrate stiffness to determine how to control the TDSCs. The tendinopathy model was created using a chemical induction method, and the tendon injury model was created via an injury-and-overuse method. Norland Optical Adhesive 86 (NOA86) substrate with 2.48 GPa stiffness with and without 800 nm-wide nanogrooves and a polyurethane substrate with 800 nm-wide nanogrooves were used.ResultsTDSCs from 5-week-old normal tendon showed high expression of type III collagen on the flat NOA86 substrate. In the 15-week normal tendon model, expression of type III collagen was high in TDSCs cultured on the 800 nm NOA86 substrates. However, in the 15-week tendon injury model, expression of type III collagen was similar irrespective of nanotopographic cues or substrate stiffness. The expression of type I collagen was also independent of nanotopographic cues and substrate stiffness in the 15-week normal and tendon injury models. Gene expression of scleraxis was increased in TDSCs cultured on the flat NOA86 substrate in the 5-week normal tendon model (P=0.001). In the 15-week normal tendon model, scleraxis was highly expressed in TDSCs cultured on the 800 nm and flat NOA86 substrate (P=0.043). However, this gene expression was not significantly different between the substrates in the 5-week tendinopathy and 15-week tendon injury models.ConclusionDevelopment and maturation of tendon are enhanced when TDSCs from normal tendons were cultured on stiff surface, but not when the TDSCs came from pathologic models. Therapeutic applications of TDSCs need to be flexible based on tendon age and tendinopathy.

Platelet-Rich Plasma Promotes Migration, Proliferation, and the Gene Expression of Scleraxis and Vascular Endothelial Growth Factor in Paratenon-Derived Cells In Vitro.

Background:Platelet-rich plasma (PRP) is a treatment option for tendon injury because of its effective tendon-healing properties. At the early stage of tendon repair, paratenon-derived cells (PDCs) are thought to play a more important role than tendon proper–derived cells (TDCs). However, there has been no study investigating the effects of PRP on PDCs.Hypothesis:PRP promotes the migration, proliferation, and differentiation of PDCs in vitro.Study Design:Controlled laboratory study.Methods:TDCs and PDCs were isolated from the tendon proper and paratenon of rat Achilles tendons and were cultured to the third passage. PRP was prepared from the rats using the double-spin method. Third-passage TDCs and PDCs were cultured in Dulbecco’s modified Eagle medium with 2% fetal bovine serum (control group) or 2% fetal bovine serum plus 5% PRP (PRP group), and cell migration, proliferation, and differentiation were evaluated. The relative mRNA expression levels of scleraxis (Scx), tenomodulin (Tnmd), collagen type I alpha 1 (Col1a1), collagen type III alpha 1 (Col3a1), and vascular endothelial growth factor A (VEGF) were examined by quantitative real-time reverse transcription polymerase chain reaction.Results:The cell migration rate was significantly higher in the PDCs of the PRP group than in the control group (1.4-fold increase; P = 0.02). Cell proliferation was significantly higher in the PDCs of the PRP group (2.2-fold increase; P < 0.01). In the PDCs, the gene expression levels of Scx, Col1a1, and VEGF were significantly increased by PRP (Scx: 2.0-fold increase, P = 0.01; Col1a1: 5.3-fold increase, P = 0.01; VEGF: 7.8-fold increase, P = 0.01), but the gene expression level of Tnmd, a factor for tendon maturation, was significantly reduced by PRP (0.11-fold decrease; P = 0.02).Conclusion:In vitro PRP promoted migration, proliferation, and tenogenic differentiation with the upregulation of Scx in PDCs. PRP also upregulated the expression of the angiogenic marker VEGF.Clinical Relevance:Our results suggest that PRP treatment in vitro may enhance the tendon-healing properties of PDCs at the initial stage of tendon repair.

Identification of topographical architectures supporting the phenotype of rat tenocytes

Tenocytes, the main cell type of the tendon, require mechanical stimuli for their proper function. When the tenocyte environment changes due to tissue damage or by transferring tenocytes from their native environment into cell culture, the signals from the tenocyte niche are lost, leading towards a decline of phenotypic markers. It is known that micro-topographies can influence cell fate by the physical cues they provide. To identify the optimal topography-induced biomechanical niche in vitro, we seeded tenocytes on the TopoChip, a micro-topographical screening platform, and measured expression of the tendon transcription factor Scleraxis. Through machine learning algorithms, we associated elevated Scleraxis levels with topological design parameters. Fabricating micro-topographies with optimal surface characteristics on larger surfaces allowed finding an improved expression of multiple tenogenic markers. However, long-term confluent culture conditions coincided with osteogenic marker expression and the loss of morphological characteristics. In contrast, passaging tenocytes which migrated from the tendon directly on the topography resulted in prolonged elongated morphology and elevated Scleraxis levels. This research provides new insights into how micro-topographies influence tenocyte cell fate, and supports the notion that micro-topographical design can be implemented in a new generation of tissue culture platforms for supporting the phenotype of tenocytes. Statement of SignificanceThe challenge in controlling in vitro cell behavior lies in controlling the complex culture environment. Here, we present for the first time the use of micro-topographies as a biomechanical niche to support the phenotype of tenocytes. For this, we applied the TopoChip platform, a screening tool with 2176 unique micro-topographies for identifying feature characteristics associated with elevated Scleraxis expression, a tendon related marker. Large area fabrication of micro-topographies with favorable characteristics allowed us to find a beneficial influence on other tenogenic markers as well. Furthermore, passaging cells is more beneficial for Scleraxis marker expression and tenocyte morphology compared to confluent conditions. This study presents important insights for the understanding of tenocyte behavior in vitro, a necessary step towards tendon engineering.

Bone morphogenetic protein-12 inducing tenogenic differentiation of mesenchymal stem cells enhances healing of linea alba incision.

The objective of this study was to investigate the curative effects of mesenchymal stem cells' tenogenic differentiation on linea alba incision healing induced by bone morphogenetic protein-12. Mesenchymal stem cells were isolated and induced by 10 ng/ml of bone morphogenetic protein-12 for 48 h. Expression of scleraxis, collagen I and collagen III were examined at 48 h, 5 and 7 days to investigate the tenogenic differentiation. The expression of scleraxis increases continually even in the absence of bone morphogenetic protein-12 for 5 days (P<0.01). The expression of collagen I and III requires persistent inducing. Then fifty Sprague-Dawley rats were randomly divided into five groups: negative control, positive control, sham group, native mesenchymal stem cells and tenogenically differentiated mesenchymal stem cells. Tensiometric testing and modified semiquantitative histological analysis were performed to explore the curative effects. The tension levels in the positive control, sham, native mesenchymal stem cells and tenogenically differentiated mesenchymal stem cells were 44, 41.8, 51.6 and 69.7%, respectively, compared with the negative control. Tenogenically differentiated mesenchymal stem cells exhibited a greater increase in tension compared with positive control, sham and native mesenchymal stem cell groups (P<0.05). From the sections stained with Masson's Trichrome, collagen organization and amount of tenogenically differentiated mesenchymal stem cells was better than the other three groups (P<0.05). In conclusion, mesenchymal stem cells' tenogenic differentiation induced by bone morphogenetic protein-12 can enhance linea alba incision healing.

Possible association of oestrogen and Cryba4 with masticatory muscle tendon-aponeurosis hyperplasia.

Masticatory muscle tendon-aponeurosis hyperplasia, which is associated with limited mouth opening, progresses very slowly from adolescence. The prevalence rates of this disease are higher among women than among men, suggesting oestrogen involvement. As parafunctional habits are frequently observed, mechanical stress is likely involved in the pathogenesis and advancement of this disease. To elucidate the pathological condition, we examined the effect of oestrogen on tenocyte function and the relationship between mechanical stress and crystallin beta A4 (Cryba4), using murine TT-D6 tenocytes. Cell proliferation assays, RT-PCR, real-time RT-PCR, Western blot analysis and mechanical loading experiments were performed. The physiological dose of oestrogen increased the levels of scleraxis and tenomodulin in TT-D6 tenocytes. In contrast, forced expression of Cryba4 inhibited scleraxis expression in these cells. Surprisingly, oestrogen significantly promoted cell differentiation in the Cryba4-overexpressing TT-D6 tenocytes. Moreover, tensile force induced Cryba4 expression in these tendon cells. Oestrogen and Cryba4 may be associated with the progression of masticatory muscle tendon-aponeurosis hyperplasia.

Tenogenic Properties of Mesenchymal Progenitor Cells Are Compromised in an Inflammatory Environment

Transplantation of multipotent mesenchymal progenitor cells is a valuable option for treating tendon disease. Tenogenic differentiation leading to cell replacement and subsequent matrix modulation may contribute to the regenerative effects of these cells, but it is unclear whether this occurs in the inflammatory environment of acute tendon disease. Equine adipose-derived stromal cells (ASC) were cultured as monolayers or on decellularized tendon scaffolds in static or dynamic conditions, the latter represented by cyclic stretching. The impact of different inflammatory conditions, as represented by supplementation with interleukin-1β and/or tumor necrosis factor-α or by co-culture with allogeneic peripheral blood leukocytes, on ASC functional properties was investigated. High cytokine concentrations increased ASC proliferation and osteogenic differentiation, but decreased chondrogenic differentiation and ASC viability in scaffold culture, as well as tendon scaffold repopulation, and strongly influenced musculoskeletal gene expression. Effects regarding the latter differed between the monolayer and scaffold cultures. Leukocytes rather decreased ASC proliferation, but had similar effects on viability and musculoskeletal gene expression. This included decreased expression of the tenogenic transcription factor scleraxis by an inflammatory environment throughout culture conditions. The data demonstrate that ASC tenogenic properties are compromised in an inflammatory environment, with relevance to their possible mechanisms of action in acute tendon disease.

Cyclic Uniaxial Tensile Strain Enhances the Mechanical Properties of Engineered, Scaffold-Free Tendon Fibers.

The treatment of injured tendon is an ever-increasing clinical and financial burden, for which tissue-engineered replacements have shown great promise. Recently, there has been growing interest in a more regenerative approach to tissue engineering, in which the cells' abilities to self-assemble and create matrix are harnessed to create tissue constructs without the use of a scaffold. Herein, utilizing our scaffold-free technique to engineer tendon at the single fiber level, we study how applied mechanical loading, namely cyclic uniaxial strain, influences the mechanical properties and nuclear alignment of developing tendon fiber constructs. Engineered fibers were subjected to 1, 3, and 7 days of intermittent uniaxial loading (0.0-0.7% sinusoidal strain), and then characterized mechanically by constant-rate elongation to failure to obtain tensile properties and histologically to examine cytoskeletal arrangement and nuclear shape, and characterized using real-time polymerase chain reaction to measure the expression of tendon-specific makers, scleraxis and tenomodulin. Fiber peak stress, elastic modulus, toughness, and nuclear aspect ratio increased with the presence and duration of loading, while failure strain, toe-in strain, and nuclear area were unchanged. These biomechanical results suggest that cyclic strain promotes matrix deposition in a manner that increases the fiber resistance to stretch, but preserves fiber extensibility over the 7-day loading period. Over 7 days of loading, the scleraxis and tenomodulin expression increased drastically. Histologically, while there was no immediate difference in nuclear area with the addition of loading, nuclear aspect ratio significantly increased with loading duration, such that nuclei became progressively more elongated to the long axis of the fiber. Together with our biomechanical findings, such nuclear deformation suggests that cyclic strain elicits a mechanotransductive response, particularly one that modulates gene expression to promote matrix deposition during fiber development.

Nanoscaled and microscaled parallel topography promotes tenogenic differentiation of ASC and neotendon formation in vitro.

BackgroundTopography at different scales plays an important role in directing mesenchymal stem cell differentiation including adipose-derived stem cells (ASCs) and the differential effect remains to be investigated.PurposeThis study aimed to investigate the similarity and difference between micro- and nanoscaled aligned topography for inducing tenogenic differentiation of human ASCs (hASCs).MethodsParallel microgrooved PDMS membrane and a parallel aligned electrospun nanofibers of gelatin/poly-ε-caprolactone mixture were employed as the models for the study.ResultsAligned topographies of both microscales and nanoscales could induce an elongated cell shape with parallel alignment, as supported by quantitative cell morphology analysis (cell area, cell body aspect, and cell body major axis angle). qPCR analysis also demonstrated that the aligned topography at both scales could induce the gene expressions of various tenogenic markers at the 7th day of in vitro culture including tenomodulin, collagen I and collagen VI, decorin, tenascin-C and biglycan, but with upregulated expression of scleraxis and tenascin-C only in microscaled topography. Additionally, tenogenic differentiation at the 3rd day was confirmed only at microscale. Furthermore, microscaled topography was confirmed for its tenogenic induction at tissue level as neotendon tissue was formed with the evidence of mature type I collagen fibers only in parallel aligned polyglycolic acid (PGA) microfibers after in vitro culture with mouse ASCs. Instead, only fat tissue was formed in random patterned PGA microfibers.ConclusionBoth microscaled and nanoscaled aligned topographies could induce tenogenic differentiation of hASCs and micro-scaled topography seemed better able to induce elongated cell shape and stable tenogenic marker expression when compared to nanoscaled topography. The microscaled inductive effect was also confirmed at tissue level by neotendon formation in vitro.

Scleraxis regulates Twist1 and Snai1 expression in the epithelial-to-mesenchymal transition.

Numerous physiological and pathological events, from organ development to cancer and fibrosis, are characterized by an epithelial-to-mesenchymal transition (EMT), whereby adherent epithelial cells convert to migratory mesenchymal cells. During cardiac development, proepicardial organ epithelial cells undergo EMT to generate fibroblasts. Subsequent stress or damage induces further phenotype conversion of fibroblasts to myofibroblasts, causing fibrosis via synthesis of an excessive extracellular matrix. We have previously shown that the transcription factor scleraxis is both sufficient and necessary for the conversion of cardiac fibroblasts to myofibroblasts and found that scleraxis knockout reduced cardiac fibroblast numbers by 50%, possibly via EMT attenuation. Scleraxis induced expression of the EMT transcriptional regulators Twist1 and Snai1 via an unknown mechanism. Here, we report that scleraxis binds to E-box consensus sequences within the Twist1 and Snai1 promoters to transactivate these genes directly. Scleraxis upregulates expression of both genes in A549 epithelial cells and in cardiac myofibroblasts. Transforming growth factor-β induces EMT, fibrosis, and scleraxis expression, and we found that transforming growth factor-β-mediated upregulation of Twist1 and Snai1 completely depends on the presence of scleraxis. Snai1 knockdown upregulated the epithelial marker E-cadherin; however, this effect was lost after scleraxis overexpression, suggesting that scleraxis may repress E-cadherin expression. Together, these results indicate that scleraxis can regulate EMT via direct transactivation of the Twist1 and Snai1 genes. Given the role of scleraxis in also driving the myofibroblast phenotype, scleraxis appears to be a critical controller of fibroblast genesis and fate in the myocardium and thus may play key roles in wound healing and fibrosis. NEW & NOTEWORTHY The molecular mechanism by which the transcription factor scleraxis mediates Twist1 and Snai1 gene expression was determined. These results reveal a novel means of transcriptional regulation of epithelial-to-mesenchymal transition and demonstrate that transforming growth factor-β-mediated epithelial-to-mesenchymal transition is dependent on scleraxis, providing a potential target for controlling this process.

Scleraxis Regulation of Snail1 and Twist1 Gene Expression in Epithelial – Mesenchymal Transition

Epithelial‐Mesenchymal Transition (EMT) is a process by which epithelial cells lose their connectivity with each other and convert to a migratory, mesenchymal phenotype, and plays an important role in development as well as pathogenesis of diseases such as cancer and fibrosis. EMT is induced in part by the up‐regulation of the transcription factors Twist1 as well as Snai1, which directly represses transcription of E‐cadherin, an integral component of adherens junctions: tight cell‐cell connections prominent in epithelial cell sheets. Our previous data in mice demonstrated that gene knockout of the transcription factor scleraxis, which is involved in cell phenotype conversion, resulted in fewer cardiac fibroblasts, which arise developmentally from proepicardial cells undergoing EMT. We observed an increase in epithelial markers concomitant with a loss of mesenchymal markers in scleraxis null hearts, suggesting that EMT was impaired by scleraxis deletion. Our data indicated that scleraxis directly transactivates the Twist1 and Snail1 gene promoters, and that scleraxis was sufficient to induce EMT in A549 epithelial cells, but the mechanistic details of this regulation were unclear.Here we examined the Twist1 and Snail1 gene promoters for E‐boxes – CANNTG sequences to which scleraxis binds. By in silico analysis, we identified one and four putative E‐boxes in the Twist1 and Snail1 proximal promoters, respectively. Using luciferase reporter assays, we found that scleraxis directly transactivated both promoters. The single E‐box in the Twist1 promoter was required for this effect, as mutation of the E‐box abolished scleraxis‐mediated reporter induction. Conversely, three out of four E‐boxes within the Snail1 promoter were similarly required. Results for both studies were confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays, which demonstrated that scleraxis directly interacted with the E‐boxes in question. Scleraxis over‐expression resulted in up‐regulation of both Snail1 and Twist1, and increased migration of A549 epithelial cells as measured via scratch assay, indicating progression of EMT. TGFβ1, a known inducer of both EMT and scleraxis expression, increased Snail1 and Twist1 expression, but scleraxis knockdown via shRNA attenuated this induction, demonstrating that TGFβ1‐mediated EMT requires scleraxis. These findings suggest that scleraxis is an important factor in driving EMT, and may be a novel therapeutic target to arrest cell conversion in EMT‐dependent pathological processes.Support or Funding InformationDSA was supported by funding from the GETS program of the University of Manitoba. MPC was supported by an Open Operating Grant from the Canadian Institutes of Health Research (MOP136862).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interleukin-6 Promotes Proliferation but Inhibits Tenogenic Differentiation via the Janus Kinase/Signal Transducers and Activators of Transcription 3 (JAK/STAT3) Pathway in Tendon-Derived Stem Cells.

BackgroundPrevious studies demonstrated that tendon-derived stem cells (TDSCs) were vital healing cells and that mRNA expression of anti-inflammatory cytokine IL-6 was significantly upregulated in injured tendons. The aim of the present study was to investigate the effects of IL-6 on the TDSCs in vitro.Material/MethodsTDSCs isolated from the Achilles tendons in SD rats were co-cultured with various concentrations of IL-6. Cell proliferation, cell cycle analysis, quantitative real-time PCR, western blotting analysis, and statistical analysis were used in the study.ResultsThe result showed that IL-6 strongly increased proliferation capability, and induced cell cycle activation and transition into G2/M phase from G1 phase in TDSCs. However, IL-6 treatment strongly inhibited gene expression of Scleraxis, Collagen 1, Tenomodulin, Collagen 3, Early Growth Response Protein 1, Decorin, Lumican, Biglycan and Fibromodulin in TDSCs. It also strongly inhibited protein expression of tendon cell markers like scleraxis, collagen 1, collagen 3, and tenomodulin. IL-6 treatment strongly activated the JAK/Stat3 signaling pathway in TDSCs. Furthermore, WP1066, a JAK/Stat3 signaling pathway inhibitor, abrogated the effects of IL-6 on TDSCs.ConclusionsThese findings indicated that IL-6 might exert dual effects on TDSCs in vitro: strongly enhancing their proliferation but inhibiting their tenogenic differentiation via the JAK/Stat3 pathway.

Ex-vivo Tendon Repair Augmented with Bone Marrow Derived Mesenchymal Stem Cells Stimulated with Myostatin for Tenogenesis.

To investigate the effect of myostatin (GDF-8) stimulation of bone marrow derived mesenchymal stem cells (BMSCs) on tenogenesis in the setting of tendon repair. GDF-8 has demonstrated the ability to augment tenogenesis and we sought to identify if this effect could lead to the focused differentiation of pluripotential stem cells down a tenocyte lineage ex vivo. Cadaveric upper limb flexor tendons were harvested, decellularized and divided into 1 cm segments. Sutures seeded with stem cells were passed through tendon segments to simulate repair. The repaired tendons were then cultured either with or without myostatin for 3, 5, and 7 days. The experiment was also repeated with non-decellularized tendons for a total of 4 groups. The tendons were then evaluated for the expression of scleraxis and tenomodulin, two biomarkers for tendon. Myostatin stimulation led to an increase in expression of tenomodulin and scleraxis at 5 and 7 days in both the decellularized and non-decellularized tendons. Myostatin increased the differentiation of BMSCs into tenocytes and/or led to the upregulation of tenomodulin and scleraxis production by the native tenocytes present within the non-decellularized tendons. The addition of myostatin to BMSCs leads to tenocyte differentiation as evidenced by the expression of tenocyte biomarkers, scleraxis and tenomodulin. This effect is maintained in an ex vivo tendon repair model suggestive that these cells survive the passage through tendon tissue and remain metabolically active.

Comparative Analysis of Different Platelet Lysates and Platelet Rich Preparations to Stimulate Tendon Cell Biology: An In Vitro Study.

The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP) was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL) could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP); RegenLab (PRP-BCT)), platelet concentrate (apheresis, PC), and PL (freezing-thawing destruction of PC). Additionally, ten commercial allogenic PLs (AlloPL) from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP-BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1β, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration.

Extracorporeal shock waves trigger tenogenic differentiation of human adipose-derived stem cells

ABSTRACTPurposes: Incomplete tendon healing impairs the outcome of tendon ruptures and tendinopathies. Human Adipose-derived Stem Cells (hASCs) are promising for tissue engineering applications. Extracorporeal Shock Waves (ESW) are a leading choice for the treatment of several tendinopathies. In this study, we investigated the effects of ESW treatment and tenogenic medium on the differentiation of hASCs into tenoblast-like cells. Materials and Methods: hASCs were treated with ESW generated by a piezoelectric device and tenogenic medium. Quantitative real-time PCR was used to check the mRNA expression levels of tenogenic transcription factors, extracellular matrix proteins, and integrins. Western blot and immunofluorescence were used to detect collagen 1 and fibronectin. Collagen fibers were evaluated by Masson staining. Calcium deposition was assessed by Alizarin Red staining. Results: The combined treatment improved the expression of the tendon transcription factors scleraxis and eyes absent 2, and of the extracellular matrix proteins fibronectin, collagen I, and tenomodulin. Cells acquired elongated and spindle shaped fibroblastic morphology; Masson staining revealed the appearance of collagen fibers. Finally, the combined treatment induced the expression of alpha 2, alpha 6, and beta 1 integrin subunits, suggesting a possible role in mediating ESW effects. Conclusions: ESW in combination with tenogenic medium improved the differentiation of hASCs toward tenoblast-like cells, providing the basis for ESW and hASCs to be used in tendon tissue engineering.

Tenogenic differentiation of mesenchymal stem cells improves healing of linea alba incision

OBJECTIVE: The aim of this study is to investigate the curative effects of mesenchymal stem cells' (MSCs') tenogenic differentiation on Linea alba incision healing. MATERIALS AND METHODS: Autologous MSCs were isolated from rat bone marrow and cultured and induced by 10 ng/mL of bone morphogenetic protein-12 (BMP-12) for 48 h. Expression of scleraxis (SCX), collagen I, and collagen III was examined at 48 h, 5 days, and 7 days to investigate the tenogenic differentiation. Fifty Sprague-Dawley rats were randomly divided into five groups: tenogenically differentiated (group E) or native mesenchymal stem cells (group D) seeded onto collagen sponge scaffolds or only sponge scaffolds (group C) were transplanted into the linea alba incision; rats that underwent operation without implantation of anything served as the sham group (group B), and rats that did not undergo operation were used as the control group (group A). Histological analysis was performed to explore the curative effects. RESULTS: The expression of SCX increases continually even in the absence of BMP-12 for 5 days (P CONCLUSIONS: In animal experiments, MSCs' tenogenic differentiation induced by BMP-12 can dramatically enhance linea alba incision healing.

Tenogenically differentiated adipose-derived stem cells are effective in Achilles tendon repair in vivo

The purpose of this study was to characterize rat adipose-derived stem cells, induce adipose-derived stem cell tenogenesis, and analyze adipose-derived stem cell effects on tendon repair in vivo. Adipose-derived stem cells demonstrated an immunomodulatory, pro-angiogenic, and pro-proliferatory profile in vitro. Tenogenesis was induced for 1, 7, 14, and 21 days with 24 combinations of growth differentiation factor-5, 6, and 7 and platelet-derived growth factor–BB. Adipose-derived stem cells expression of scleraxis and collagen type I increased the most after 14 days of induction with growth differentiation factor-6 and platelet-derived growth factor–BB. Achilles excision defects injected with hydrogel alone (Gp2), with undifferentiated (Gp3) adipose-derived stem cells, or tenogenically differentiated (Gp4) adipose-derived stem cells exhibited improved tissue repair compared with untreated tendons (Gp1). Addition of adipose-derived stem cells improved tissue cytoarchitecture and increased expression of collagen type I and III, scleraxis, and tenomodulin. Adipose-derived stem cells significantly improved biomechanical properties (ultimate load and elastic toughness) over time more than hydrogel alone, while tenogenically differentiated adipose-derived stem cells improved the mean histological score and collagen fiber dispersion range closest to normal tendon. In addition, tendon sections treated with GFP-adipose-derived stem cells exhibited green fluorescence and positive GFP immunostaining on microscopy confirming the in vivo survival of adipose-derived stem cells that were injected into tendon defects to support the effects of adipose-derived stem cells on tissue up to 4.5 weeks post injury.

Application of Amniotic Membrane Improves Repair in a Diabetic Animal Model for Delayed Tendon Healing

Category: Basic Sciences/Biologics, Diabetes Introduction/Purpose: Tendon injuries often heal with significant scar formation and compromised biomechanical function. For diabetics, these injuries are further complicated by changes in the extracellular matrix of the tendon, leading to higher incidences of injury and slower healing. Defective or delayed healing is a result of several factors including an impaired ability to form a collagen matrix, compromised angiogenesis, and inadequate production of growth factors. Consequently, complications and re- rupture rates are higher in diabetic patients. Amnion-derived cells resulted in improved tendon healing in an otherwise healthy animal model, in part due to their ability to provide numerous regenerative cytokines to the repair site. The purpose of this study was to evaluate the effect of amniotic tissues on tendon healing in a diabetic model with impaired healing. Methods: For this study BBZDR/WOR animals, an insulin dependent Type II diabetic rodent model, were used. After appropriate anesthesia, a full thickness injury was made through the Achilles tendon; immediately following injury, the tendon was repaired using the modified Kessler method. Repaired tendons were wrapped with a 0.5 × 0.5 cm section of either a fresh hypothermically stored human amniotic membrane (HSAM), a dehydrated human amnion chorion membrane (dHACM), or left unwrapped as a control. Contralateral tendons were used as sham controls, which were exposed then immediately closed. Tendons were retrieved at 14 or 28 days and evaluated using histology, immunohistochemistry, and qPCR. At 28 days, both experimental tendons and contralateral controls from five rats per group were harvested and tested to failure and peak force, stiffness, energy uptake, and displacement at rupture were then determined. Results: At day 14, histological evaluation found significant increases in cellular recruitment at the site of injury in both dHACM and HSAM treated animals (p=0.0001). qPCR and immunofluorescence results confirmed several biomarkers implicated in tendon repair were highly expressed in the treatment groups, but not in untreated controls. In dHACM treated animals, TNF-a, TGFß-1, IL6, FLAP, Tenascin-C, and Scleraxis expression was elevated at 14 days. Control animals with only primary repair resulted in a tendon repair failure rate of 20%; whereas animals treated with HSAM or dHACM had a 6% and 0% failure rate, respectively. dHACM treated tendons also had significant improvements in biomechanical properties compared to controls including increases in the max force, stiffness and strain (47±21%, 146±90%, and 59±35% respectively). Conclusion: Augmentation of tendon repair with placental-derived membranes may improve diabetic tendon repair and reduce re-rupture rates. This study presents compelling data suggesting chorion-containing dHACM is more efficacious than the amnion- only HSAM for tendon repair. The cause for this difference is unclear, we hypothesize that cytokine content, important for tendon healing, within the chorion may be responsible. Components of placental tissue contribute different cytokines, ECM and cell populations—this study highlights that differences in components-delivered and processing techniques may impact outcomes and results may vary for different clinical applications. This study highlights a promising treatment option for a clinically challenging population.

Glutamate signaling through the NMDA receptor reduces the expression of scleraxis in plantaris tendon derived cells

BackgroundA body of evidence demonstrating changes to the glutaminergic system in tendinopathy has recently emerged. This hypothesis was further tested by studying the effects of glutamate on the tenocyte phenotype, and the impact of loading and exposure to glucocorticoids on the glutamate signaling machinery.MethodsPlantaris tendon tissue and cultured plantaris tendon derived cells were immunohisto-/cytochemically stained for glutamate, N-Methyl-D-Aspartate receptor 1 (NMDAR1) and vesicular glutamate transporter 2 (VGluT2). Primary cells were exposed to glutamate or receptor agonist NMDA. Cell death/viability was measured via LDH/MTS assays, and Western blot for cleaved caspase 3 (c-caspase 3) and cleaved poly (ADP-ribose) polymerase (c-PARP). Scleraxis mRNA (Scx)/protein(SCX) were analyzed by qPCR and Western blot, respectively. A FlexCell system was used to apply cyclic strain. The effect of glucocorticoids was studies by adding dexamethasone (Dex). The mRNA of the glutamate synthesizing enzymes Got1 and Gls, and NMDAR1 protein were measured. Levels of free glutamate were determined by a colorimetric assay.ResultsImmunoreactions for glutamate, VGluT2, and NMDAR1 were found in tenocytes and peritendinous cells in tissue sections and in cultured cells. Cell death was induced by high concentrations of glutamate but not by NMDA. Scleraxis mRNA/protein was down-regulated in response to NMDA/glutamate stimulation. Cyclic strain increased, and Dex decreased, Gls and Got1 mRNA expression. Free glutamate levels were lower after Dex exposure.ConclusionsIn conclusion, NMDA receptor stimulation leads to a reduction of scleraxis expression that may be involved in a change of phenotype in tendon cells. Glutamate synthesis is increased in tendon cells in response to strain and decreased by glucocorticoid stimulation. This implies that locally produced glutamate could be involved in the tissue changes observed in tendinopathy.

MP56-18 THE MOUSE CORPUS CAVERNOSUM GLANDUS IS BIOMECHANICALLY ANALOGOUS TO THE HUMAN CORPUS CAVERNOSUM

You have accessJournal of UrologySexual Function/Dysfunction: Peyronie’s Disease II1 Apr 2017MP56-18 THE MOUSE CORPUS CAVERNOSUM GLANDUS IS BIOMECHANICALLY ANALOGOUS TO THE HUMAN CORPUS CAVERNOSUM Matthew Hennefarth, Ling Chen, Ryan Hsi, Misun Kang, Amanda Reed-Maldonado, Guiting Lin, Marshall Stoller, Tom Lue, and Sunita Ho Matthew HennefarthMatthew Hennefarth More articles by this author , Ling ChenLing Chen More articles by this author , Ryan HsiRyan Hsi More articles by this author , Misun KangMisun Kang More articles by this author , Amanda Reed-MaldonadoAmanda Reed-Maldonado More articles by this author , Guiting LinGuiting Lin More articles by this author , Marshall StollerMarshall Stoller More articles by this author , Tom LueTom Lue More articles by this author , and Sunita HoSunita Ho More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1771AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Calcified Peyronie's plaque (CPP) is postulated to be of bone phenotype within a human penis. Rodents are used as models to investigate spatiotemporal mechanistic processes leading to CPP in humans; however, there are specific biomechanical differences between species. The comparable regions to program insults within rodents to induce the pathologically mineralized tissue similar to CPP are not well correlated. The objective of this study is to illustrate that the mouse corpus cavernosum glandus (CCG), instead of the mouse corpus cavernosum penis (CPP), is more analogous to the human corpus cavernosum (HCC). METHODS Penile tissues from 12 Scx-GFP mice with ages 4 week, 5 week, 6 week, and 1.2 years, were dissected and processed following routine histology protocols. 6μm thick sections were stained with H&E, Massons' trichrome (TRI), Verhoeff's elastin (ELA), and DAPI. All sections were imaged using bright field or fluorescence microscopy techniques. Cell orientations along collagen fibers with age were analyzed using Image J Fiji. RESULTS Figure: With age, CCG illustrated well-oriented struts at an angle of 32° to the baculum. Additionally, scleraxis and elastin expressions, and smooth muscle content increased within the struts, specifically in the 6 week-old mice. The expression of scleraxis indicated that the CCG contains mechanoactive collagenous and elastin-ridden struts that support biomechanical function of the baculum. Within the CCG of the mouse, there was more sinusoidal space in the younger age groups (4-6 weeks), and this space decreased with age, and elastin expression increased significantly (see 1 year). Compared to the CCG (A,C,E,G), the CCP (B,D,F,H) illustrated significantly lower expressions of scleraxis, elastin, and smooth muscle. CONCLUSIONS The collagen-producing fibroblasts within the CCG and CCP express scleraxis, indicative of local mechanoactive areas. Based upon orientation of cells expressing scleraxis, the biomechanical function of the CCP is to push the glans of the penis like a piston, while the CCG stiffens the penis during erection and pulls the glans of the penis back into the prepuce post-erection. The CCG is similar to the HCC with regard to compositional ratio of collagen to muscle, elastin localization, and biomechanical function and as such is a good model to study human penile physiology. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e761-e762 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Matthew Hennefarth More articles by this author Ling Chen More articles by this author Ryan Hsi More articles by this author Misun Kang More articles by this author Amanda Reed-Maldonado More articles by this author Guiting Lin More articles by this author Marshall Stoller More articles by this author Tom Lue More articles by this author Sunita Ho More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Role of Scleraxis in Intestinal Fibroblasts

Scleraxis is a transcription factor in the BHLH family originally shown to regulate the development of embryonic tissue into tendon and bone—suggesting a link to collagen production and release. Recently, scleraxis has been shown to play a role in the development cardiac fibrosis. Organ fibrosis typically follows inflammation and involves over activation of myofibroblasts (activated fibroblasts that express α‐smooth muscle actin). Intestinal fibrosis is a somewhat common complication of Inflammatory Bowel Disease (IBD), which primarily includes Crohn's Disease (CD) and Ulcerative Colitis (UC). Intestinal fibrosis is characterized by dysregulated and excessive production of collagen from fibroblasts that results in excessive contraction in the intestine, intestinal stricture and bowel obstruction. Interestingly, approximately 20–25% of IBD patients will develop fibrosis. We hypothesize that scleraxis is expressed by intestinal fibroblasts and can modify the activation of such fibroblasts. Transfection (siRNA) studies show that when scleraxis is reduced in fibroblasts there is an increase in intestinal fibroblast attachment to fibrinogen (CD11b ligand). While exposure of fibroblasts to cytokines does not appear to regulate its expression, scleraxis expression is increased in IBD intestines. Immuno‐purification studies reveal that scleraxis may have a physical link to CD11b suggesting that they may have similar signaling pathways. These results highlight scleraxis as a target molecule in the treatment of diseases involving the over‐activation of fibroblasts.Support or Funding Information5T35DK007431‐32 and and National Science Foundation RII