Expression Of Tenomodulin Research Articles

Growth differentiation factor‑5 induces tenomodulin expression via phosphorylation of p38 and promotes viability of murine mesenchymal stem cells from compact bone.

Growth differentiation factor (GDF)-5 serves a role in tissue development and tenomodulin serves an important role in the development of tendons. The effects of GDF-5 on mesenchymal stem cells (MSCs), particularly with regards to tendon bioengineering, are poorly understood. The present study aimed to investigate the effects of GDF-5 on cell viability and tenomodulin expression in MSCs from murine compact bone. MSCs were isolated from murine compact bones and confirmed by flow cytometric analysis. In addition, the adipogenic, osteoblastic and chondrocyte differentiation capabilities of the MSCs were determined. MSCs were treated with GDF-5 and the effects of GDF-5 on MSC viability were determined. The mRNA and protein expression levels of tenomodulin were detected by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. MSCs from murine compact bone were successfully isolated. GDF-5 had optimal effects on cell viability at 100 ng/ml (+36.9% of control group without GDF-5 treatment, P<0.01) and its effects peaked after 6 days of treatment (+56.6% of control group, P<0.001). Compared with the control group, treatment with 100 ng/ml GDF-5 for 4 days enhanced the mRNA expression levels of tenomodulin (3.56±0.94 vs. 1.02±0.25; P<0.05). In addition, p38 was activated by GDF-5, as determined by enhanced expression levels of phosphorylated p38 (p-p38). The GDF-5-induced protein expression levels of p-p38 and tenomodulin were markedly inhibited following treatment with SB203580, an inhibitor of p38 mitogen-activated protein kinase. These results suggested that GDF-5 treatment may increase tenomodulin protein expression via phosphorylation of p38 in MSCs from murine compact bone. These findings may aid the future development of tendon bioengineering.

Modulating microfibrillar alignment and growth factor stimulation to regulate mesenchymal stem cell differentiation.

Polymeric electrospun fibers can be tuned to match the fibrillar size and anisotropy of collagen fibers in ligaments, and can be mechanically competent. Therefore, their use is attractive when attempting to tissue engineer the bone-ligament interface. A central challenge in this field is recapitulating the cellular phenotypes observed across the bone-ligament interface. Here we demonstrated that it is possible to direct MSCs seeded onto aligned electrospun fibres towards either a ligamentogenic, chondrogenic or fibrochondrogenic phenotype upon presentation of appropriate biochemical cues. This opens the possibility of using aligned microfibrillar scaffolds that are spatially functionalized with specific growth factors to direct MSC differentiation for engineering the bone-ligament interface.

Tenogenic differentiation of tonsil-derived mesenchymal stem cells

Category: Basic Sciences/Biologics Introduction/Purpose: Human palatine tonsil-derived mesenchymal stem cells (T-MSCs) are known to be a new source of progenitor cells. Using waste tissue after tonsillectomy as a cell provider can be the biggest benefit of T-MSCs, compared with other stem cells. The purpose of this study was to investigate tenogenic differentiation of T-MSCs and to access the differential effects of TGF-ß3 on the tenogenesis of T-MSCs. Methods: Human tonsil was obtained after tonsillectomy.Using a cytometric analysis, we were able to find that the T-MSCs had typical mesenchymal stem cell markers: positive for CD73, CD90 and CD105, and negative for CD14, CD34 and CD45. Using a Transforming growth factor beta 3 (TGF-ß3), the expressions of tenocyte-specific genes and proteins, such as Collagen type I (COL1), Tenomodulin (TNMD), and Scleraxis (SCX), were measured by a quantitative polymerase chain reaction (PCR) assay, immunofluorescence staining, immunohistochemistry and Western blot analysis. Results: Quantitative PCR assay showed that TGF-ß3 significantly increased the expressions of tenocyte lineage marker genes, including Collagen type I (COL1), Tenomodulin (TNMD), and Scleraxis (SCX), at a 3-day treatment, compared with control. However, these increases were not found at long-term exposures (7 or 10 days), except that TNMD expression was maintained at 50 ng/ml at 7-day exposure to TGF-ß3 (Fig). Like genes, the protein expression levels of COL1, TNMD, and SCX were also induced in TGF-ß3-treated T-MSCsin 3-day treatments. Moreover, the protein levels were maintained for 10 days, as evidenced by immunofluorescence staining, immunohistochemistry and Western blot analyses. Conclusion: This study demonstrated that T-MSCs in tenogenic stimulation with TGF-ß3 have high tenogenic differentiation potential.

Tenogenic Differentiation of Human Embryonic Stem Cells.

Tendon healing is complex to manage because of the limited regeneration capacity of tendon tissue; stem cell-based tissue engineering approaches may provide alternative healing strategies. We sought to determine whether human embryonic stem cells (hESC) could be induced to differentiate into tendon-like cells by the addition of exogenous bone morphogenetic protein (BMP)12 (growth differentiation factor[GDF]7) and BMP13 (GDF6). hESC (SHEF-1) were maintained with or without BMP12/13 supplementation, or supplemented with BMP12/13 and the Smad signaling cascade blocking agent, dorsomorphin. Primary rat tenocytes were included as a positive control in immunocytochemistry analysis. A tenocyte-like elongated morphology was observed in hESC after 40-days continuous supplementation with BMP12/13 and ascorbic acid (AA). These cells displayed a tenomodulin expression pattern and morphology consistent with that of the primary tenocyte control. Analysis of tendon-linked gene transcription in BMP12/13 supplemented hESC demonstrated consistent expression of COL1A2, COL3A1, DCN, TNC, THBS4, and TNMD levels. Conversely, when hESCs were cultured in the presence of BMP12/13 and dorsomorphin COL3A1, DCN, and TNC gene expression and tendon matrix formation were inhibited. Taken together, we have demonstrated that hESCs are responsive to tenogenic induction via BMP12/13 in the presence of AA. The directed in vitro generation of tenocytes from pluripotent stem cells may facilitate the development of novel repair approaches for this difficult to heal tissue.

Murine Supraspinatus Tendon Detachment and Repair Model Augmented With Tendon-Derived, Activated Endothelial Cells: A New Concept in Biologic Enhancement of Tendon-to-Bone Healing

Objectives:Biologic interventions are being increasingly used to improve outcome of rotator cuff repair. Individual growth factors and stem cell-based augmentations are among the most popular approaches. In this study we investigated a novel approach in which tendon-derived, activated endothelial cells (tAECs) are used as a source of tissue-specific growth factors to stimulate tendon intrinsic stem cell niche in our established murine model of microsurgical supraspinatus tendon (SST) detachment and repair.Methods:Study protocol was approved by the IACUC. In stage 1, 20 C57BL/6 mice underwent tendon harvest from fore- and hind limbs and endothelial cells isolation and transfection with adenoviral E4 ORF1 and red fluorescent protein labelling using a proprietary protocol. In stage 2, 158 C57BL/6 mice underwent the procedure with implantation of a 10μm fibrin glue bead (FGB) at the repair site and were randomly allocated to study group (SG) and control group (CG). Study mice received 100,000 tAECs suspended in the FGB. Control mice received the FGB only. 3 mice were sacrificed on post-operative day (POD) #3 and #7 for cell viability study. 96 mice were sacrificed at 1, 2, and 4 weeks, with 10 and 6 mice in each group and time point for biomechanical and histologic evaluations, respectively. 56 mice were sacrificed on POD 5, 10, 14, and 28 for gene expression analysis using qPCR, with 7 mice in each group and time point. Biomechanical evaluation consisted of determination of failure force and site. Histologic parameters were cell count (H&E), number of vessels (factor VIII-specific antibody), number of chondrocytes and proteoglycan content (Alcian Blue), and collagen organization (picrosirius red and polarized light microscopy). 12 evaluated genes were aggrecan (ACAN), collagen1α1 (COL1), collagen3α1 (COL3), MKX, MMP-3, 13, and 14, Runx2, scleraxis (Scx), SOX9, tenomodulin (TNM) and VEGFa.Results:Fluorescent microscopy revealed viable tAECs at the repair site on POD #3 with No fluorescent activity on POD #7. Failure force in the SG was 50% higher at 2 weeks (2.50 ± 0.55 N vs 1.86 ± 0.82 N, p = 0.012) with 65% of all failures occurring at SST mid-substance, whereas 90% of failures in the CG occurred at the repair site. There was no significant difference in failure force at 4 weeks. Cell count, number of vessels, and proteoglycan content (Figure 1) were significantly higher in the SG at all time points. The number of chondrocytes was higher in the SG at 1 week (Figure 1). Quantitative analysis showed significantly superior collagen organization in the SG (Figure 2). SCX, MKX, TNM and COL3 expression were up-regulated on POD #10 and decreased on day #28 in both groups. Expression of TNM and COL3 in the SG showed significance difference at 4 weeks. ACAN, SOX9, COL1, MMP14 expression exhibited an increase starting from POD 5, followed by a significant decline towards baseline by POD 28. In the SG, SOX9 peaked at day 14. Expression of VEGFa showed an uptrend at earlier time points (Figure 3).Conclusion:tAECs increased repair strength of SST. Histologic findings were suggestive of a more vigorous cellular and vascular response, higher proteoglycan content, and ultimately, superior collagen organization. A commensurate pattern was observed in expression of relevant genes. This approach opens a new avenue for cell-based biologic augmentation of SST repair with the goal of stimulating the intrinsic stem cell niche in tendon, as opposed to incorporating exogenous cell sources.

Scleraxis is required for maturation of tissue domains for proper integration of the musculoskeletal system

Scleraxis (Scx) is a basic helix-loop-helix transcription factor that is expressed persistently in tendons/ligaments, but transiently in entheseal cartilage. In this study, we generated a novel ScxCre knock-in (KI) allele, by in-frame replacement of most of Scx exon 1 with Cre recombinase (Cre), to drive Cre expression using Scx promoter and to inactivate the endogenous Scx. Reflecting the intensity and duration of endogenous expression, Cre-mediated excision occurs in tendinous and ligamentous tissues persistently expressing Scx. Expression of tenomodulin, a marker of mature tenocytes and ligamentocytes, was almost absent in tendons and ligaments of ScxCre/Cre KI mice lacking Scx to indicate defective maturation. In homozygotes, the transiently Scx-expressing entheseal regions such as the rib cage, patella cartilage, and calcaneus were small and defective and cartilaginous tuberosity was missing. Decreased Sox9 expression and phosphorylation of Smad1/5 and Smad3 were also observed in the developing entheseal cartilage, patella, and deltoid tuberosity of ScxCre/Cre KI mice. These results highlighted the functional importance of both transient and persistent expression domains of Scx for proper integration of the musculoskeletal components.

Myostatin promotes tenogenic differentiation of C2C12 myoblast cells through Smad3

Myostatin, a member of the transforming growth factor‐β (TGF‐β) superfamily, is expressed in developing and adult skeletal muscle and negatively regulates skeletal muscle growth. Recently, myostatin has been found to be expressed in tendons and increases tendon fibroblast proliferation and the expression of tenocyte markers. C2C12 is a mouse myoblast cell line, which has the ability to transdifferentiate into osteoblast and adipocyte lineages. We hypothesized that myostatin is capable of inducing tenogenic differentiation of C2C12 cells. We found that the expression of scleraxis, a tendon progenitor cell marker, is much higher in C2C12 than in the multipotent mouse mesenchymal fibroblast cell line C3H10T1/2. In comparison with other growth factors, myostatin significantly up‐regulated the expression of the tenogenic marker in C2C12 cells under serum‐free culture conditions. Immunohistochemistry showed that myostatin inhibited myotube formation and promoted the formation of spindle‐shaped cells expressing tenomodulin. We examined signaling pathways essential for tenogenic differentiation to clarify the mechanism of myostatin‐induced differentiation of C2C12 into tenocytes. The expression of tenomodulin was significantly suppressed by treatment with the ALK inhibitor SB341542, in contrast to p38MAPK (SB203580) and MEK1 (PD98059) inhibitors. RNAi silencing of Smad3 significantly suppressed myostatin‐induced tenomodulin expression. These results indicate that myostatin has a potential role in the induction of tenogenic differentiation of C2C12 cells, which have tendon progenitor cell characteristics, through activation of Smad3‐mediated signaling.

The effects of high glucose on tendon-derived stem cells: implications of the pathogenesis of diabetic tendon disorders.

Patients with diabetes are at great risk to suffer many musculoskeletal disorders, such as tendinopathy, tendon rupture and impaired tendon healing. However, the pathogenesis of these tendon disorders still remains unclear. In this study, we aimed to investigate the effects of high glucose on cell proliferation, cell apoptosis and tendon-related markers expression of tendon-derived stem cells (TDSCs) in vitro. These findings might provide new insights into the pathogenesis of diabetic tendon disorders. The cell proliferative ability and apoptosis rate of TDSCs in different groups were evaluated by MTT assay and Annexin V-FITC/PI staining assay. The mRNA expression of tendon-related markers (Scleraxis and Collagen I alpha 1 chain) were assessed by qRT-PCR. The protein expression of tendon-related markers (Tenomodulin and Collagen I) were measured by Western blotting. The proliferative ability of TDSCs treated with high glucose (15mM and 25mM) decreased significantly at day1, day3 and day5. The cell apoptosis of TDSCs increased significantly when they were cultured with high glucose for 48h in vitro. The gene expression of Scleraxis and Collagen I alpha 1 chain in TDSCs decreased significantly when they were treated with high glucose for 24h and 48h. The protein expression of Tenomodulin and Collagen I in TDSCs decreased significantly when they were treated with high glucose for 24h and 48h. High glucose could inhibit cell proliferation, induce cell apoptosis and suppress the tendon-related markers expression of TDSCs in vitro. These findings might account for some pathological mechanisms underlying the pathogenesis of diabetic tendon disorders.

Ex vivo Tendon Repair Augmented with Bone Marrow Derived Mesenchymal Stem Cells Stimulated with Myostatin for Tenogenesis: N/A - Not a clinical study

Consulting Fee: Arthrex (Yao) We performed an ex vivo study to investigate the effect of bone marrow derived mesenchymal stem cells (BMSC) stimulated with myostatin on tenogenesis in the setting of tendon repair. We hypothesized that BMSC that were delivered to a tendon repair site after being seeded onto sutures would improve tenogenesis if they were stimulated with myostatin. Cadaveric upper limb flexor tendons were harvested, decellularized, and divided into 1cm segments. Sutures seeded with BMSC were passed through 2 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 to make a total of 4 groups. The presence of tenomodulin and scleraxis using immunofluorescence analysis was measured at 3, 5, and 7 days for all 4 groups. Viable BMSC were found at all 3 time points in all 4 groups. Myostatin stimulation lead to expression of tenomodulin and scleraxis at 5 and 7 days in both the decellularized and non-decellularized tendons. In the tendons that were not treated with myostatin, tenomodulin and scleraxis were detected only at day 7 for the decellularized tendons and at days 5 and 7 for the non-decellularized tendons. There were also more cells expressing tenomodulin and scleraxis for the groups treated with myostatin at days 5 and 7. For all 4 groups, no tenomodulin or scleraxis was detected at 3 days. •BMSC delivered to tendon repair sites with sutures can survive for at least 7 days.•There is at least a 3 day delay before the markers of tenogenesis are detectable after BMSC delivery to the tendon repair site.•Myostatin results in an earlier, more robust and sustained expression of tenomodulin and scleraxis in this tendon repair model, implying that it facilitated earlier and increased differentiation of BMSC into tenocytes and/or upregulation of tenomodulin and scleraxis production by the tenocytes present.•Treatment of BMSC with myostatin following their delivery using coated sutures to a tendon repair site may result in improved tenogenesis, implying earlier and better healing of the injured tendon.

Expression of tenocyte lineage-related factors from tonsil-derived mesenchymal stem cells.

Human palatine tonsil-derived mesenchymal stem cells (TMSCs) are known to be a new source of progenitor cells. Using waste tissue after tonsillectomy as a cell provider can be the biggest benefit of TMSCs, compared with other stem cells. The purpose of this study was to investigate tenogenic differentiation of TMSCs and to access the differential effects of transforming growth factor beta 3 (TGF-β3) on the tenogenesis of TMSCs. Human tonsil was obtained after tonsillectomy. Using a cytometric analysis, we were able to find that the TMSCs had typical mesenchymal stem cell markers: positive for CD73, CD90, and CD105, and negative for CD14, CD34, and CD45. Using TGF-β3, the expressions of tenocyte-specific genes and proteins, such as collagen type 1 (COL1), tenomodulin (TNMD), and scleraxis (SCX), were measured by a quantitative polymerase chain reaction (PCR), immunofluorescence staining, immunohistochemistry and Western blot analyses. Quantitative PCR assay showed that TGF-β3 significantly increased the expressions of tenocyte lineage marker genes, including COL1, TNMD, and SCX, at a 3-day treatment, compared with control. However, these increases were not found at long-term exposures (7 or 10 days), except that TNMD expression was maintained at 50 ng/mL at a 7-day exposure to TGF-β3. Like genes, the protein expression levels of COL1, TNMD, and SCX were also induced in TGF-β3-treated TMSCs in a 3-day treatment, which were maintained for 10 days, as evidenced by immunofluorescence staining, immunohistochemistry and Western blot analyses. This study demonstrated that TMSCs in tenogenic stimulation with TGF-β3 have a high tenogenic differentiation potential.

Tenomodulin promotes human adipocyte differentiation and beneficial visceral adipose tissue expansion

Proper regulation of energy storage in adipose tissue is crucial for maintaining insulin sensitivity and molecules contributing to this process have not been fully revealed. Here we show that type II transmembrane protein tenomodulin (TNMD) is upregulated in adipose tissue of insulin-resistant versus insulin-sensitive individuals, who were matched for body mass index (BMI). TNMD expression increases in human preadipocytes during differentiation, whereas silencing TNMD blocks adipogenesis. Upon high-fat diet feeding, transgenic mice overexpressing Tnmd develop increased epididymal white adipose tissue (eWAT) mass, and preadipocytes derived from Tnmd transgenic mice display greater proliferation, consistent with elevated adipogenesis. In Tnmd transgenic mice, lipogenic genes are upregulated in eWAT, as is Ucp1 in brown fat, while liver triglyceride accumulation is attenuated. Despite expanded eWAT, transgenic animals display improved systemic insulin sensitivity, decreased collagen deposition and inflammation in eWAT, and increased insulin stimulation of Akt phosphorylation. Our data suggest that TNMD acts as a protective factor in visceral adipose tissue to alleviate insulin resistance in obesity.

Piezoelectric nanoscaffolds: mediating tendon regeneration through activation of piezoresponsive receptors.

Event Abstract Back to Event Piezoelectric nanoscaffolds: mediating tendon regeneration through activation of piezoresponsive receptors. Marc A. Fernandez-Yague1, Dimitrios Zeugolis1, 2, Abhay Pandit1 and Manus J. Biggs1 1 National University of Ireland, Centre for Research in Medical Devices (CÚRAM), Ireland 2 National University of Ireland, Regenerative, Modular and Developmental Engineering Laboratory (REMODEL), Ireland Introduction: The design of artificial scaffolds that can recreate the unique physicomechanical features of specific cellular niches is of vital importance for tissue engineering applications. The anisotropic biomechanical properties of tendon tissues are attributed to the high degree of alignment of collagen fibrils, which provides topographical guidance for tenocytes and promote tissue organisation. However, an often overlooked physical property of collagen-rich musculoskeletal tissues is the inherent piezoelectric response to mechanical loading. The conversion of mechanical forces into electrical fields is thought to significantly modulate cell function through transmembrane voltage-gated receptors[1]. In this study, primary human tendon cells were subjected to dynamic tension on a piezoelectric fibrous scaffold developed from poly (vinylidenefluoride-co-trifluoroethylene) P(VDF-TrFE), a material capable of generating electrical charges under mechanical loading with physiological conditions. The sensitivity of the material was further modulated through the incorporation of piezoelectric Boron Nitride nanotubes (BNNTs)[2]. The nanomaterials were evaluated as regenerative scaffolds through the analysis of cellular proliferation and the expression of tendon-specific genes. Materials and Methods: P(VDF-TrFE) was dissolved in DMF/Acetone and electrospun into nanoscale fibers. To modulate electrical sensitivity, BNNTs were functionalized with a conductive polymer and incorporated into the matrix. The scaffold morphology was observed by FESEM and the electrical properties in response to cyclic loading were assessed with PFM and an in-house oscilloscope/dynamic loading system. Primary Human Tenocytes were isolated and cultured for 7, 14 and 21 days onto the scaffolds under static and mechanical loading conditions. Samples were, immunoassayed for focal adhesions (FA), Tenomodulin (TNMD), Collagen I (COL-I) and III (COL-III). Gene expression was analyzed using a Custom RT2 Profile PCR array. Results: The PDVF-TrFE/BNNT scaffolds were piezoelectric, biocompatible and mechanical stimulation promoted the maintenance of a tenospecific phenotype for up to 21 days. Preliminary results shows that cell morphology and focal adhesion distribution in cells cultured on static and dynamic conditions scaffold were modulated and piezoresponsive gene were upregulated. Despite the findings tat proliferation was lower on piezoscaffolds, tenomodulin expression (transmembrane glycoprotein expressed in Tendon) in piezoelectric nanoscaffolds and tenocyte morphology was maintained after all time points. Discussion and Conclusions: This study indicates that the piezoelectrical activity of PVDF-TrFE may be tuned by incorporation of BNNTs. Moreover, gene and protein analyses showed that piezoelectric stimulation significantly upregulated the expression of mechanosensitive and voltage gated genes. Science Foundation Ireland, Starting Investigator SIRG COFUND fellow (grant agreement no. 11/SIRG/B2135); Science Foundation Ireland Centre for Research in Medical Devices (CÚRAM) (Grant agreement no. 13/RC/2073)

Three-Dimensional Platelet-Rich Plasma Hydrogel Model to Study Early Tendon Healing

Since the experimental conditions of cell cultures may bias results, it is critical to use suitable models. This is also true in the context of tendon cell biology and the study of platelet-rich plasma (PRP) therapies and PRP-augmented cell-based therapies. We compared the culture of human tendon cells in 2 dimensions (2D) with PRP-supplemented media to culture in matching 3-dimensional (3D) PRP hydrogels. Cell proliferation, cell shape, and the pattern of gene and protein expression were examined. Our data revealed modifications in cell shape and enhanced expression of tenomodulin and scleraxis in 3D hydrogels. Additionally, protein secretion analysis using glass-based arrays specific for angiogenesis revealed differences in interleukin (IL)-6 and IL-8 protein expression between 2D cultures and 3D hydrogels, while the secretion of other angiogenic or inflammatory cytokines was unaffected. Our study suggests that 3D hydrogels are physiologically more relevant than 2D cultures in the study of tendon cells, based on cell shape, support of tenocyte proliferation, phenotype, and the pattern of gene and protein expression.

Aligned multilayered electrospun scaffolds for rotator cuff tendon tissue engineering

The rotator cuff consists of several tendons and muscles that provide stability and force transmission in the shoulder joint. Whereas most rotator cuff tears are amenable to suture repair, the overall success rate of repair is low, and massive tears are prone to re-tear. Extracellular matrix (ECM) patches are used to augment suture repair, but they have limitations. Tissue-engineered approaches provide a promising solution for massive rotator cuff tears. Previous studies have shown that, compared to nonaligned scaffolds, aligned electrospun polymer scaffolds exhibit greater anisotropy and exert a greater tenogenic effect. Nevertheless, achieving rapid cell infiltration through the full thickness of the scaffold is challenging, and scaling to a translationally relevant size may be difficult. Our goal was to evaluate whether a novel method of alignment, combining a multilayered electrospinning technique with a hybrid of several electrospinning alignment techniques, would permit cell infiltration and collagen deposition through the thickness of poly(ε-caprolactone) scaffolds following seeding with human adipose-derived stem cells. Furthermore, we evaluated whether multilayered aligned scaffolds enhanced collagen alignment, tendon-related gene expression, and mechanical properties compared to multilayered nonaligned scaffolds. Both aligned and nonaligned multilayered scaffolds demonstrated cell infiltration and ECM deposition through the full thickness of the scaffold after only 28days of culture. Aligned scaffolds displayed significantly increased expression of tenomodulin compared to nonaligned scaffolds and exhibited aligned collagen fibrils throughout the full thickness, the presence of which may account for the increased yield stress and Young’s modulus of cell-seeded aligned scaffolds along the axis of fiber alignment. Statement of SignificanceRotator cuff tears are an important clinical problem in the shoulder, with over 300,000 surgical repairs performed annually. Re-tear rates may be high, and current methods used to augment surgical repair have limited evidence to support their clinical use due to inadequate initial mechanical properties and slow cellular infiltration. Tissue engineering approaches such as electrospinning have shown similar challenges in previous studies. In this study, a novel technique to align electrospun fibers while using a multilayered approach demonstrated increased mechanical properties and development of aligned collagen through the full thickness of the scaffolds compared to nonaligned multilayered scaffolds, and both types of scaffolds demonstrated rapid cell infiltration through the full thickness of the scaffold.

Static and cyclic mechanical loading of mesenchymal stem cells on elastomeric, electrospun polyurethane meshes.

Biomaterial substrates composed of semi-aligned electrospun fibers are attractive supports for the regeneration of connective tissues because the fibers are durable under cyclic tensile loads and can guide cell adhesion, orientation, and gene expression. Previous studies on supported electrospun substrates have shown that both fiber diameter and mechanical deformation can independently influence cell morphology and gene expression. However, no studies have examined the effect of mechanical deformation and fiber diameter on unsupported meshes. Semi-aligned large (1.75 μm) and small (0.60 μm) diameter fiber meshes were prepared from degradable elastomeric poly(esterurethane urea) (PEUUR) meshes and characterized by tensile testing and scanning electron microscopy (SEM). Next, unsupported meshes were aligned between custom grips (with the stretch axis oriented parallel to axis of fiber alignment), seeded with C3H10T1/2 cells, and subjected to a static load (50 mN, adjusted daily), a cyclic load (4% strain at 0.25 Hz for 30 min, followed by a static tensile loading of 50 mN, daily), or no load. After 3 days of mechanical stimulation, confocal imaging was used to characterize cell shape, while measurements of deoxyribonucleic acid (DNA) content and messenger ribonucleic acid (mRNA) expression were used to characterize cell retention on unsupported meshes and expression of the connective tissue phenotype. Mechanical testing confirmed that these materials deform elastically to at least 10%. Cells adhered to unsupported meshes under all conditions and aligned with the direction of fiber orientation. Application of static and cyclic loads increased cell alignment. Cell density and mRNA expression of connective tissue proteins were not statistically different between experimental groups. However, on large diameter fiber meshes, static loading slightly elevated tenomodulin expression relative to the no load group, and tenascin-C and tenomodulin expression relative to the cyclic load group. These results demonstrate the feasibility of maintaining cell adhesion and alignment on semi-aligned fibrous elastomeric substrates under different mechanical conditions. The study confirms that cell morphology is sensitive to the mechanical environment and suggests that expression of select connective tissue genes may be enhanced on large diameter fiber meshes under static tensile loads.

Expression of tenomodulin in development mouse masseter muscle (731.2)

Tenomodulin (TNMD) is related to the type II transmembrane glycoprotein, which contains an angiogenesis inhibitor with an antiangiogenic cysteine‐rich C‐terminal domain. TNMD functions as an anti‐angiogenic molecule and is expressed primarily in hypovascular tissues, such as tendons and ligaments. The masseter muscle contains a tendinous skeletal element that is an important site of masticatory dysfunction. However, the relationship between the RNA transcript levels of TNMD and angiogenic factors, such as vascular endothelial growth factor (VEGF), in the connective tissue of developing mouse masseter muscle is unknown. Method: Crlj:CD1 (ICR) mice were fed and sacrificed at the age of day E12.5, E14.5 E17.5, E18.5, 0 ,5, 10,15 and 20 by an overdose of pentobarbital. Fresh samples of right masseter muscle and femoris rectus were removed for further investigation (n=3 in each group). The study followed the regulations of Nippon Dental University (i.e., Rules for the Care and Use of LaboratoryAnimals, no. 03‐34). A quantitative real‐time reverse transcription polymerase chain reaction (RT‐PCR) was used to measure the RNA expression of mouse TNMD and other endothelial markers. We also determined the protein expression of the TNMD gene and other endothelial markers using an enzyme‐linked immunosorbent assay (ELISA). Result: In the masseter muscle of developing mice aged E12.5 to 5 days, the levels of TNMD mRNAs is appeared and high from E17.5 (ANOVA, p&lt;0.001) but gradually decreased in 5‐day‐old mice (ANOVA, p&lt;0.001). In contrast, VEGF and CD31 are different expression pattern and decreased in mice aged 0 to 20 days (ANOVA,p&lt;0.001). The different expression of TNMD indicates formation of tendon in the masseter muscle at embryonic stages in contrast to that of other angiogenesis markers.

Bone Morphogenetic Protein-12 Induces Tenogenic Differentiation of Mesenchymal Stem Cells Derived from Equine Amniotic Fluid

Tendon injuries are common in race horses, and mesenchymal stem cells (MSCs) isolated from adult and foetal tissue have been used for tendon regeneration. In the present study, we evaluated equine amniotic fluid (AF) as a source of MSCs and standardised methodology and markers for their in vitro tenogenic differentiation. Plastic-adherent colonies were isolated from 12 of 20 AF samples by day 6 after seeding and 70-80% cell confluency was reached by day 17. These cells expressed mesenchymal surface markers [cluster of differentiation (CD)73, CD90 and CD105] by reverse transcription (RT)-polymerase chain reaction (PCR) and immunocytochemistry, but did not express haematopoietic markers (CD34, CD45 and CD14). In flow cytometry, the expression of CD29, CD44, CD73 and CD90 was observed in 68.83 ± 1.27, 93.66 ± 1.80, 96.96 ± 0.44 and 93.7 ± 1.89% of AF-MSCs, respectively. Osteogenic, chondrogenic and adipogenic differentiation of MSCs was confirmed by von Kossa and Alizarin red S, Alcian blue and oil red O staining, respectively. Upon supplementation of MSC growth media with 50 ng/ml bone morphogenetic protein (BMP)-12, AF-MSCs differentiated to tenocytes within 14 days. The differentiated cells were more slender, elongated and spindle shaped with thinner and longer cytoplasmic processes and showed expression of tenomodulin and decorin by RT-PCR and immunocytochemistry. In flow cytometry, 96.7 ± 1.90 and 80.9 ± 6.4% of differentiated cells expressed tenomodulin and decorin in comparison to 1.6 and 3.1% in undifferentiated control cells, respectively. Our results suggest that AF is an easily accessible and effective source of MSCs. On BMP-12 supplementation, AF-MSCs can be differentiated to tenocytes, which could be exploited for regeneration of ruptured or damaged tendon in race horses.

Regulation of Tenomodulin Expression Via Wnt/β-catenin Signaling in Equine Bone Marrow-derived Mesenchymal Stem Cells.

ABSTRACTTenomodulin has been recognized as a biomarker for tendon differentiation, and its gene expression is regulated by several transcription factors including Scleraxis and Mohawk. In this study, we found a novel regulatory mechanism of tenomodulin expression. Equine bone marrow-derived mesenchymal stem cells (BMSCs) in monolayer culture showed a low mRNA level of tenomodulin in comparison with the level in the tendon. When cultured in collagen gel containing a glycogen synthase kinase-3 (GSK-3) inhibitor (BIO), expression of tenomodulin in BMSCs increased up to the level in the tendon. Participation of GSK-3 in its gene expression was further demonstrated by a gene silencing experiment with small interference RNA corresponding to GSK-3, suggesting that Wnt/β-catenin signaling mediated expression of tenomodulin. These results were confirmed by nuclear translocation of β-catenin in BIO-treated BMSCs cultured in collagen gel. Under this culture condition, expression of tenomodulin-related transcription factors including Scleraxis and Mohawk was not affected, suggesting that Wnt/β-catenin signaling was independent from these transcription factors. Additionally, BIO strongly enhanced expression of type XIV collagen in collagen-embedded BMSCs up to the level in the tendon, and other tendon-related extracellular matrix components such as decorin and fibromodulin were also upregulated. Taken together, these results indicated that activation of Wnt/β-catenin signaling could induce differentiation of BMSCs into tenomodulin-expressing tendon cells in collagen gel.

Potential utility of cell sheets derived from the anterior cruciate ligament and synovium fabricated in temperature‐responsive culture dishes

Development of tissue-engineered materials to treat anterior cruciate ligament (ACL) injury has been limited by the lack of phenotypic markers. We investigated the feasibility of inducing ACL regeneration using cell sheet technology based on the expression of tenomodulin (TNMD) as an early phenotypic marker of ligaments. ACL remnants, the synovium surrounding cruciate ligaments (SCL), the synovium surrounding the infrapatellar fat pads (SIF), and subcutaneous fat tissue (SCF) were obtained from patients undergoing ACL reconstruction or total knee arthroplasty. ACL cell sheets and SCL-derived cell sheets were fabricated successfully A three-dimensional bioengineered ACL was generated by combining triple-layered ACL cell sheets with a bioabsorbable mesh composite. Immunohistochemical examination showed that TNMD was expressed in human ACL fibers, triple-layered ACL cell sheets, ACL remnants, SCL, and SIF, but not in SCF. Real-time PCR showed that TNMD mRNA was expressed at substantially higher levels in the ACL, SCL, and SIF than in the SCF. These results suggest that TNMD is a specific marker of the human ACL and that ACL sheets have a phenotype similar to that of the ACL. The greater expression of TNMD in the SCL- and SIF- suggests that the synovium is a potential cell source for ACL regeneration.

Effects of platelet‐rich plasma (PRP) on the healing of Achilles tendons of rats

Platelet-rich plasma (PRP) contains growth factors involved in the tissular healing process. The aim of the study was to determine if an injection of PRP could improve the healing of sectioned Achilles tendons of rats. After surgery, rats received an injection of PRP (n = 60) or a physiological solution (n = 60) in situ. After 5, 15, and 30 days, 20 rats of both groups were euthanized and 15 collected tendons were submitted to a biomechanical test using cryo-jaws before performing transcriptomic analyses. Histological and biochemical analyses were performed on the five remaining tendons in each group. Tendons in the PRP group were more resistant to rupture at 15 and 30 days. The mechanical stress was significantly increased in tendons of the PRP group at day 30. Histological analysis showed a precocious deposition of fibrillar collagen at day 5 confirmed by a biochemical measurement. The expression of tenomodulin was significantly higher at day 5. The messenger RNA levels of type III collagen, matrix metalloproteinases 2, 3, and 9, were similar in the two groups at all time points, whereas type I collagen was significantly increased at day 30 in the PRP group. In conclusion, an injection of PRP in sectioned rat Achilles tendon influences the early phase of tendon healing and results in an ultimately stronger mechanical resistance.