Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture.

Tendon self-renewal is a rare occurrence because of the poor vascularization of this tissue; therefore, reconstructive surgery using autologous tendon is often performed in severe injury cases. However, the post-surgery re-injury rate is relatively high, and the collection of autologous tendons leads to muscle weakness, resulting in prolonged rehabilitation. Here, we introduce an induced pluripotent stem cell (iPSC)-based technology to develop a therapeutic option for tendon injury. First, we derived tenocytes from human iPSCs by recapitulating the normal progression of step-wise narrowing fate decisions in vertebrate embryos. We used single-cell RNA sequencing to analyze the developmental trajectory of iPSC-derived tenocytes. We demonstrated that iPSC-tenocyte grafting contributed to motor function recovery after Achilles tendon injury in rats via engraftment and paracrine effects. The biomechanical strength of regenerated tendons was comparable to that of healthy tendons. We suggest that iPSC-tenocytes will provide a therapeutic option for tendon injury.

The glucagon-like peptide 1 receptor agonist Exendin-4 induces tenogenesis in human mesenchymal stem cells.

Tendon injuries are common and account for up to 50% of musculoskeletal injuries in the United States. The poor healing nature of the tendon is attributed to poor vascularization and cellular composition. In the absence of FDA-approved growth factors for tendon repair, engineering strategies using bioactive factors, donor cells, and delivery matrices to promote tendon repair and regeneration are being explored. Growth factor alternatives in the form of small molecules, donor cells, and progenitors offer several advantages and enhance the tendon healing response. Small drug molecules and peptides offer stability over growth factors that are known to suffer from relatively short biological half-lives. The primary focus of this study was to assess the ability of the exendin-4 (Ex-4) peptide, a glucagon-like peptide 1 (GLP-1) receptor agonist, to induce tenocyte differentiation in bone marrow-derived human mesenchymal stem cells (hMSCs). We treated hMSCs with varied doses of Ex-4 in culture media to evaluate proliferation and tendonogenic differentiation. A 20 nM Ex-4 concentration was optimal for promoting cell proliferation and tendonogenic differentiation. Tendonogenic differentiation of hMSCs was evaluated via gene expression profile, immunofluorescence, and biochemical analyses. Collectively, the levels of tendon-related transcription factors (Mkx and Scx) and extracellular matrix (Col-I, Dcn, Bgn, and Tnc) genes and proteins were elevated compared to media without Ex-4 and other controls including insulin and IGF-1 treatments. The tendonogenic factor Ex-4 in conjunction with hMSCs appear to enhance tendon regeneration.

Macrophage depletion impairs neonatal tendon regeneration.

Tendons are dense connective tissues that transmit muscle forces to the skeleton. After adult injury, healing potential is generally poor and dominated by scar formation. Although the immune response is a key feature of healing, the specific immune cells and signals that drive tendon healing have not been fully defined. In particular, the immune regulators underlying tendon regeneration are almost completely unknown due to a paucity of tendon regeneration models. Using a mouse model of neonatal tendon regeneration, we screened for immune-related markers and identified upregulation of several genes associated with inflammation, macrophage chemotaxis, and TGFß signaling after injury. Depletion of macrophages using AP20187 treatment of MaFIA mice resulted in impaired functional healing, reduced cell proliferation, reduced ScxGFP+ neo-tendon formation, and altered tendon gene expression. Collectively, these results show that inflammation is a key component of neonatal tendon regeneration and demonstrate a requirement for macrophages in effective functional healing.

Development of pluripotent stem cell-based human tenocytes.

Human pluripotent stem cells (PSCs) are used as a platform for therapeutic purposes such as cell transplantation therapy and drug discovery. Another motivation for studying PSCs is to understand human embryogenesis and development. All cell types that make up the body tissues develop through defined trajectories during embryogenesis. For example, paraxial mesoderm is considered to differentiate into several cell types including skeletal muscle cells, chondrocytes, osteocytes, dermal fibroblasts, and tenocytes. Tenocytes are fibroblast cells that constitute the tendon. The step-wise narrowing fate decisions of paraxial mesoderm in the embryo have been modeled in vitro using PSCs; however, deriving tenocytes from human-induced PSCs and their application in cell therapy have long been challenging. PSC-derived tenocytes can be used for a source of cell transplantation to treat a damaged or ruptured tendon due to injury, disorder, or aging. In this review, we discuss the latest research findings on the use of PSCs for studying the biology of tenocyte development and their application in therapeutic settings.

Moderate and intensive mechanical loading differentially modulate the phenotype of tendon stem/progenitor cells in vivo.

To examine the differential mechanobiological responses of specific resident tendon cells, we developed an in vivo model of whole-body irradiation followed by injection of either tendon stem/progenitor cells (TSCs) expressing green fluorescent protein (GFP-TSCs) or mature tenocytes expressing GFP (GFP-TNCs) into the patellar tendons of wild type C57 mice. Injected mice were subjected to short term (3 weeks) treadmill running, specifically moderate treadmill running (MTR) and intensive treadmill running (ITR). In MTR mice, both GFP-TSC and GFP-TNC injected tendons maintained normal cell morphology with elevated expression of tendon related markers collagen I and tenomodulin. In ITR mice injected with GFP-TNCs, cells also maintained an elongated shape similar to the shape found in normal/untreated control mice, as well as elevated expression of tendon related markers. However, ITR mice injected with GFP-TSCs showed abnormal changes, such as cell morphology transitioning to a round shape, elevated chondrogenic differentiation, and increased gene expression of non-tenocyte related genes LPL, Runx-2, and SOX-9. Increased gene expression data was supported by immunostaining showing elevated expression of SOX-9, Runx-2, and PPARγ. This study provides evidence that while MTR maintains tendon homeostasis by promoting the differentiation of TSCs into TNCs, ITR causes the onset of tendinopathy development by inducing non-tenocyte differentiation of TSCs, which may eventually lead to the formation of non-tendinous tissues in tendon tissue after long term mechanical overloading conditions on the tendon.

Dose-Response Tendon-Specific Markers Induction by Growth Differentiation Factor-5 in Human Bone Marrow and Umbilical Cord Mesenchymal Stem Cells.

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) are utilized in tendon tissue-engineering protocols while extra-embryonic cord-derived, including from Wharton's Jelly (hWJ-MSCs), are emerging as useful alternatives. To explore the tenogenic responsiveness of hBM-MSCs and hWJ-MSCs to human Growth Differentiation Factor 5 (hGDF-5) we supplemented each at doses of 1, 10, and 100 ng/mL of hGDF-5 and determined proliferation, morphology and time-dependent expression of tenogenic markers. We evaluated the expression of collagen types 1 (COL1A1) and 3 (COL3A1), Decorin (DCN), Scleraxis-A (SCX-A), Tenascin-C (TNC) and Tenomodulin (TNMD) noting the earliest and largest increase with 100 ng/mL. With 100 ng/mL, hBM-MSCs showed up-regulation of SCX-A (1.7-fold) at Day 1, TNC (1.3-fold) and TNMD (12-fold) at Day 8. hWJ-MSCs, at the same dose, showed up-regulation of COL1A1 (3-fold), DCN (2.7-fold), SCX-A (3.8-fold) and TNC (2.3-fold) after three days of culture. hWJ-MSCs also showed larger proliferation rate and marked aggregation into a tubular-shaped system at Day 7 (with 100 ng/mL of hGDF-5). Simultaneous to this, we explored the expression of pro-inflammatory (IL-6, TNF, IL-12A, IL-1ß) and anti-inflammatory (IL-10, TGF-ß1) cytokines across for both cell types. hBM-MSCs exhibited a better balance of pro-inflammatory and anti-inflammatory cytokines up-regulating IL-1ß (11-fold) and IL-10 (10-fold) at Day 8; hWJ-MSCs, had a slight expression of IL-12A (1.5-fold), but a greater up-regulation of IL-10 (2.5-fold). Type 1 collagen and tenomodulin proteins, detected by immunofluorescence, confirming the greater protein expression when 100 ng/mL were supplemented. In the same conditions, both cell types showed specific alignment and shape modification with a length/width ratio increase, suggesting their response in activating tenogenic commitment events, and they both potential use in 3D in vitro tissue-engineering protocols.

Different Frequency of Cyclic Tensile Strain Relates to Anabolic/Catabolic Conditions Consistent with Immunohistochemical Staining Intensity in Tenocytes.

Tenocytes are mechanosensitive cells intimately adapting their expression profile and hence, their phenotype to their respective mechanomilieu. The immunolocalization and expression intensity of tenogenic, anabolic and catabolic markers in tenocytes in response to in vitro mechanical loading have not been monitored by immunohistochemical staining (IHC). Thus, we investigated the association between IHC intensities, different stimulation frequencies, and tenogenic metabolism using a versatile mechanical stretcher. Primary tenocytes obtained from murine Achilles tendons were transferred to poly(dimethylsiloxane) (PDMS) elastomeric chamber. Chambers were cyclically stretched by 5% in uniaxial direction at a variation of tensile frequency (1 or 2 Hz) for 3 h. After stretching, cell physiology, IHC intensities of tendon-related markers, and protein level of the angiogenesis marker vascular endothelial growth factor (VEGF) were evaluated. Cell proliferation in tenocytes stimulated with 1 Hz stretch was significantly higher than with 2 Hz or without stretch, while 2 Hz stretch induced significantly reduced cell viability and proliferation with microscopically detectable apoptotic cell changes. The amount of scleraxis translocated into the nuclei and tenomodulin immunoreactivity of tenocytes treated with stretch were significantly higher than of non-stretched cells. The collagen type-1 expression level in tenocytes stretched at 1 Hz was significantly higher than in those cultivated with 2 Hz or without stretching, whereas the matrix metalloproteinase (MMP)-1 and MMP-13 immunoreactivities of cells stretched at 2 Hz were significantly higher than in those stimulated with 1 Hz or without stretching. The secreted VEGF-protein level of tenocytes stretched at 2 Hz was significantly higher than without stretching. Our IHC findings consistent with cell physiology suggest that appropriate stretching can reproduce in vitro short-term tenogenic anabolic/catabolic conditions and allow us to identify an anabolic stretching profile.

Tenocyte-imprinted substrate: a topography-based inducer for tenogenic differentiation in adipose tissue-derived mesenchymal stem cells.

Tendon tissue engineering based on stem cell differentiation has attracted a great deal of attention in recent years. Previous studies have examined the effect of cell-imprinted polydimethylsiloxane (PDMS) substrate on induction differentiation in stem cells. In this study, we used tenocyte morphology as a positive mold to create a tenocyte-imprinted substrate on PDMS. The morphology and topography of this tenocyte replica on PDMS was evaluated with scanning electron microscopy (SEM) and atomic force microscopy. The tenogenic differentiation induction capacity of the tenocyte replica in adipose tissue-derived mesenchymal stem cells (ADSCs) was then investigated and compared with other groups, including tissue replica (which was produced similarly to the tenocyte replica and was evaluated by SEM), decellularized tendon, and bone morphogenic protein (BMP)-12, as other potential inducers. This comparison gives us an estimate of the ability of tenocyte-imprinted PDMS (called cell replica in the present study) to induce differentiation compared to other inducers. For this reason, ADSCs were divided into five groups, including control, cell replica, tissue replica, decellularized tendon and BMP-12. ADSCs were seeded on each group separately and investigated by the real-time reverse transcription polymerase chain reaction (RT-PCR) technique after seven and 14 days. Our results showed that in spite of the higher effect of the growth factor on tenogenic differentiation, the cell replica can also induce tenocyte marker expression (scleraxis and tenomodulin) in ADSCs. Moreover, the tenogenic differentiation induction capacity of the cell replica was greater than tissue replica. Immunocytochemistry analysis revealed that ADSCs seeding on the cell replica for 14 days led to scleraxis and tenomodulin expression at the protein level. In addition, immunohistochemistry indicated that contrary to the promising results in vitro, there was little difference between ADSCs cultured on tenocyte-imprinted PDMS and untreated ADSCs. The results of such studies could lead to the production of inexpensive cell culture plates or biomaterials that can induce differentiation in stem cells without growth factors or other supplements.

Matrix regeneration proteins in the hypoxia-triggered exosomes of shoulder tenocytes and adipose-derived mesenchymal stem cells.

Regenerative functions of exosomes rely on their contents which are influenced by pathological stimuli, including hypoxia, in rotator cuff tendon injuries (RCTI). The hypoxic environment triggers tenocytes and adjacent adipose-derived mesenchymal stem cells (ADMSCs) to release regenerative mediators to the ECM via the exosomes which elicit autocrine/paracrine responses to protect the tendon matrix from injury. We investigated the exosomal protein contents from tenocytes and subcutaneous ADMSCs from the shoulder of Yucatan microswine cultured under hypoxic conditions (2% O2). The exosomal proteins were detected using high-resolution mass spectrometry nano-LC-MS/MS Tribrid system and were compiled using 'Scaffold' software. Hypoxic exosomes from tenocytes and ADMSCs carried 199 and 65 proteins, respectively. The key proteins identified by mass spectrometry and associated with ECM homeostasis from hypoxic ADMSCs included MMP2, COL6A, CTSD and TN-C and those from hypoxic tenocytes were THSB1, NSEP1, ITIH4 and TN-C. These findings were confirmed at the mRNA and protein level in the hypoxic ADMSCs and tenocytes. These proteins are involved in multiple signaling pathways of ECM repair/regeneration. This warrants further investigations for their translational significance in the management of RCTI.

Rotator Cuff Repair With Autologous Tenocytes and Biodegradable Collagen Scaffold: A Histological and Biomechanical Study in Sheep.

BACKGROUND: Large rotator cuff tears still represent a challenging problem in orthopaedics. The use of tenocytes on biomaterials/scaffolds for the repair of large rotator cuff defects might be a promising approach in the field of tendon regeneration. HYPOTHESIS: Cultivated autologous tenocytes seeded on a collagen scaffold lead to enhanced histological and biomechanical results after rotator cuff repair in a sheep model as compared with unseeded scaffolds in an acute setting. STUDY DESIGN: Controlled laboratory study. METHODS: At the tendon-bone junction of the infraspinatus tendon of the right foreleg of 24 sheep, a 3.5 × 1.5-cm tendon defect was created. Sheep were randomly allocated to group 1, a defect; group 2, where an unseeded collagen scaffold was implanted; or group 3, which received the implantation of a collagen scaffold seeded with autologous tenocytes. Twelve weeks postoperatively, tendon regeneration was examined histologically and biomechanically. RESULTS: The histology of the neotendons of group 3 showed better fiber patterns, a higher production of proteoglycans, and an increased genesis of collagen III in contrast to groups 1 and 2. Immunostaining revealed less tissue dedifferentiation, a more structured cartilage layer, and homogeneous cartilage-bone transition in group 3 in comparison with groups 1 and 2. Biomechanically, the tensile strength of the reconstructed tendons in group 3 (mean load to failure, 2516 N; SD, 407.5 N) was approximately 84% that of the native tendons (mean load to failure, 2995 N; SD, 223.1 N) without statistical significance. A significant difference (P = .0095) was registered between group 1 (66.9% with a mean load to failure of 2004 N; SD, 273.8 N) and the native tendons, as well as between group 2 (69.7% with a mean load to failure of 2088 N; SD, 675.4 N) and the native tendons for mean ultimate tensile strength. In breaking stress, a significant difference (P = .0095) was seen between group 1 (mean breaking stress, 1335 N/mm2; SD, 182.7 N/mm2) and the native tendons, as well as between group 2 (breaking stress, 1392 N/mm2; SD, 450.2 N/mm2) and the native tendons (mean breaking stress, 1996 N/mm2; SD, 148.7 N/mm2). Again, there was no significant difference between group 3 (mean breaking stress, 1677 N/mm2; SD, 271.7 N/mm2) and the native tendons. CONCLUSION: Autologous tenocytes seeded on collagen scaffolds yield enhanced biomechanical results after tendon-bone reconstruction as compared with unseeded scaffolds in an acute setting. Biomechanical results and histological outcomes were promising, showing that the use of autologous tenocytes with specific carrier matrices could be a novel approach for repairing rotator cuff tears. CLINICAL RELEVANCE: This study supports the use of tenocytes and scaffolds for improving the quality of tendon-bone regeneration.

Inhibition of CD44 induces apoptosis, inflammation, and matrix metalloproteinase expression in tendinopathy.

Apoptosis has emerged as a primary cause of tendinopathy. CD44 signaling pathways exert anti-apoptotic and -inflammatory effects on tumor cells, chondrocytes, and fibroblast-like synoviocytes. The aim of this study was to examine the association among CD44, apoptosis, and inflammation in tendinopathy. Expression of CD44 and apoptotic cell numbers in tendon tissue from patients with long head of biceps (LHB) tendinopathy were determined according to the histological grades of tendinopathy. Primary tenocytes from Achilles tendon of Sprague-Dawley rats 1 week after collagenase injection were cultured with an antagonizing antibody against CD44. Treatment responses were determined by evaluating cell viability and expression of tendon-related proliferation markers, inflammatory mediators, and apoptosis. The expression of CD44 and apoptosis were positively correlated with the severity of tendinopathy in the human LHB tendinopathy. Furthermore, CD44 expression and apoptotic cells were co-stained in tendinopathic tendon. Blocking the CD44 signaling pathways in rat primary tenocytes by OX-50 induced cell apoptosis and the elevated levels of cleaved caspase-3. Furthermore, they had decreased cell viability and expression of collagen type I, type III, tenomodulin, and phosphorylated AKT. In contrast, there were elevated levels of inflammatory mediators, including interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, cyclooxygenase-2, and phosphorylated NF-κB, as well as matrix metalloproteinase (MMP) family members including MMP-1, -3, -9, and -13 in tenocytes upon OX-50 treatment. This study is the first to demonstrate the association of CD44 and apoptosis in tendinopathy. Our data imply that CD44 may play a role in tendinopathy via regulating apoptosis, inflammation, and extracellular matrix homeostasis.

Regulation of the tenogenic gene expression in equine tenocyte-derived induced pluripotent stem cells by mechanical loading and Mohawk.

Cell-based therapeutic strategies afford major potential advantages in the repair of injured tendons. Generation of induced pluripotent stem cells (iPSCs) expands cell sources for "regenerative" therapy. However, its application in tendon repair is still limited and the effects remain unclear. In this study, equine tenocyte-derived iPSCs (teno-iPSCs) were generated by expressing four Yamanaka factors. Compared to parental tenocytes and bone marrow derived mesenchymal stem cells (BMSCs), the transcriptional activities of lineage-specific genes, including Mkx, Col1A2, Col14, DCN, ELN, FMOD, and TNC, were highly repressed in the resulting teno-iPSCs. Exposure to cyclic uniaxial mechanical loading increased the expression of Scx, Egr1, Col1A2, DCN, and TNC in teno-iPSCs and the expression of Scx, Egr1, DCN, and TNC in BMSCs. Reintroduction of tenogenic transcription factor Mohawk (Mkx) upregulated the expression of DCN in teno-iPSCs and the expression of Scx, Col14, and FMOD in BMSCs. Mechanical loading combined with ectopic expression of equine Mkx further enhanced the expression of Egr1, Col1A2, DCN, and TNC in teno-iPSCs and the expression of Scx, Egr1, and TNC in BMSCs. These data suggest that the repressed lineage-specific genes in the teno-iPSCs can be re-activated by mechanical loading and ectopic expression of Mkx. Our findings offer new insights into the application of iPSCs for basic and clinic research in tendon repair.

Decellularised porcine peritoneum as a tendon protector sheet.

Tissue grafts achieve high levels of compositional and mechanical integrity biomimicry and are often considered as the gold standard in clinical practice. Herein, we assessed the potential of decellularised porcine peritoneum (XenoMEM) as a tendon protector sheet and correlated its properties to a commercially available product (TenoGlide®). XenoMEM presented lower cross-linking ratio (p < 0.05), higher mechanical properties (p < 0.01), lower coefficient of friction (p < 0.01) and higher (p < 0.05) cytocompatibility with human tenocytes than TenoGlide®. In addition, XenoMEM exhibited lower (p < 0.05) immune response than TenoGlide® with macrophages. Collectively, these data support the use of XenoMEM in tendon tissue engineering.

Characterization of Tendon-Specific Markers in Various Human Tissues, Tenocytes and Mesenchymal Stem Cells.

Background: Unlike bone, cartilage, or muscle, tendon-specific markers are not well established. The purpose of the study was to investigate expression pattern and level of 6 well-known tendon-specific markers, in various human musculoskeletal tissues, tenocytes, and mesenchymal stem cells (MSCs). Methods: Musculoskeletal tissue samples of tendon, bone, cartilage, nerve, muscle, and fat were obtained from patients undergoing orthopedic surgery. Tenocytes, MSCs from bone marrow, adipose tissue, and umbilical cord were isolated from each tissue and cultured. Six tendon-specific markers, scleraxis (Scx), tenomodulin (TNMD), thrombospondin-4 (TSP-4), tenascin-C (TNC), type I collagen (Col I), and type III collagen (Col III) were investigated in tendon tissue, tenocytes, and MSCs. Results: mRNA levels of 6 tendon-specific markers were significantly higher in tendon tissue that in other connective tissues levels of Scx, TNMD, TSP-4, and Col III immediately decreased after plating tenocytes in culture dishes whereas those of TNC and Col I did not. In comparison with tendon tissue, mRNA levels pattern of Scx, TNMD, and TSP-4 in tenocytes were significantly higher than that in MSCs, but lower than in tendon tissue whereas expression pattern of TNC, Col I and III showed different pattern with each other. Conclusion: This study demonstrated that 6 commonly used tendon-specific markers were mainly expressed in tendon tissue, but that expression level and pattern of the tendon-specific markers with respect to kinds of tissues, culture duration of tenocytes and sources of MSCs.

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 SIGNIFICANCE: The 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.

Vascular Endothelial Growth Factor Enhances Proliferation of Human Tenocytes and Promotes Tenogenic Gene Expression.

BACKGROUND: In obtaining human tenocytes for tendon tissue engineering, a low proliferation rate and phenotype loss during passaging is a problem. It was the authors' aim to evaluate the influence of vascular endothelial growth factor (VEGF) on human tenocyte growth and gene expression. METHODS: Human tenocytes were exposed to human VEGF in various concentrations (5, 10, and 20 ng/ml) for 5 days. Cell proliferation was counted and expression of tendon-related genes was analyzed. RESULTS: Tenocyte count was 1.4 × 10(5)/ml, 2.7 × 10(5)/ml, 2.3 × 10(5)/ml, and 3.7 × 10(5)/ml for 0, 5, 10, and 20 ng/ml VEGF, respectively. Expression of Col1 was up-regulated 6.4 ± 4.2-fold, 60.1 ± 21.6-fold, and 15.8 ± 10.2-fold for 5, 10, and 20 ng/ml VEGF; all differences were significant with p < 0.05. Col3 was down-regulated to 0.2 ± 0.1-fold, 0.3 ± 0.1-fold, and 0.1 ± 0.03-fold for 5, 10, and 20 ng/ml VEGF; all differences were significant. Eln was up-regulated 2.3 ± 1.7-fold, 25.5 ± 10.9-fold, and 16.6 ± 9.0-fold for 5, 10, and 20 ng/ml VEGF; differences were significant for 10 and 20 ng/ml VEGF. TSC was down-regulated to 0.3 ± 0.1-fold and 0.3 ± 0.1-fold for 5 and 20 ng/ml VEGF; differences were significant for 5 and 20 ng/ml. SCX was up-regulated to 31.3 ± 8.5-fold, 49.1 ± 23.4-fold, and 20.9 ± 9.5-fold for 5, 10, and 20 ng/ml VEGF; all changes were significant. CONCLUSIONS: VEGF enhances proliferation and expression of tendon-related genes in human tenocytes. It could therefore be a useful addition for tenocyte cultivation.

Making Them Commit: Strategies to Influence Phenotypic Differentiation in Mesenchymal Stem Cells.

Tendon injuries, bone defects, and cartilage defects are complex clinical conditions leading to pain and dysfunctions. Tendon, bone, and cartilage are highly specialized and organized tissues, and the self-healing may be limited by their histologic features, or impaired by the local conditions. Furthermore, the resultant tissue often shows inferior properties compared with native tissue, leading to high rates of reruptures and revision surgeries. A growing field of research has explored tendon, bone, and cartilage regeneration using mesenchymal stem cells (MSCs), because of their multipotency, and because they are relatively easy to harvest. Great expectations arose from the use of MSCs in regenerative medicine in the last decade, although both the potential and the drawbacks of this method remain under reflection. This is a narrative review of the literature about different strategies to differentiate MSCs into tenocytes, osteoblasts, and chondrocytes. Challenges and limitations on the use of MSCs in vivo and in clinical practice are also discussed.

Optimization of differentiation time of mesenchymal-stem-cell to tenocyte under a cyclic stretching with a microgrooved culture membrane and selected measurement cells.

PURPOSE: There is a need for efficient stem cell-to-tenocyte differentiation techniques for tendon tissue engineering. More than 1 week is required for tenogenic differentiation with chemical stimuli, including co-culturing. Research has begun to examine the utility of mechanical stimuli, which reduces the differentiation time to several days. However, the precise length of time required to differentiate human bone marrow-derived mesenchymal stem cells (hBMSCs) into tenocytes has not been clarified. Understanding the precise time required is important for future tissue engineering projects. Therefore, in this study, a method was developed to more precisely determine the length of time required to differentiate hBMSCs into tenocytes with cyclic stretching stimulus. METHODS: First, it had to be determined how stretching stimulation affected the cells. Microgrooved culture membranes were used to suppress cell orientation behavior. Then, only cells oriented parallel to the microgrooves were selected and evaluated for protein synthesis levels for differentiation. RESULTS: The results revealed that growing cells on the microgrooved membrane and selecting optimally-oriented cells for measurement improved the accuracy of the differentiation evaluation, and that hBMSCs differentiated into tenocytes in approximately 10 h. CONCLUSIONS: The differentiation time corresponded to the time required for cellular cytoskeleton reorganization and cellular morphology alterations. This suggests that cells, when subjected to mechanical stimulus, secrete mRNAs and proteins for both cytoskeleton reorganization and differentiation.

Effects of cell adhesion motif, fiber stiffness, and cyclic strain on tenocyte gene expression in a tendon mimetic fiber composite hydrogel.

We recently developed a fiber composite consisting of tenocytes seeded onto discontinuous fibers embedded within a hydrogel, designed to mimic physiological tendon micromechanics of tension and shear. This study examined if cell adhesion peptide (DGEA or YRGDS), fiber modulus (50 or 1300 kPa) and/or cyclic strain (5% strain, 1 Hz) influenced bovine tenocyte gene expression. Ten genes were analyzed and none were sensitive to peptide or fiber modulus in the absence of cyclic tensile strain. Genes associated with tendon (SCX and TNMD), collagens (COL1A1, COL3A1, COL11A1), and matrix remodelling (MMP1, MMP2, and TIMP3) were insensitive to cyclic strain. Contrarily, cyclic strain up-regulated IL6 by 30-fold and MMP3 by 10-fold in soft YRGDS fibers. IL6 expression in soft YRGDS fibers was 5.7 and 3.3-fold greater than in soft DGEA fibers and stiff RGD fibers, respectively, under cyclic strain. Our findings suggest that changes in the surrounding matrix can influence catabolic genes in tenocytes when cultured in a complex strain environment mimicking that of tendon, while having minimal effects on tendon and homeostatic genes.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.