NSUN2 facilitates tenogenic differentiation of rat tendon-derived stem cells via m5C methylation of KLF2.

Introduction: Tendon-derived stem cells (TDSCs) play a critical role in tendon repair. N5-methylcytosine (m5C) is a key regulator of cellular processes such as differentiation. This study aimed to investigate the impact of m5C on TDSC differentiation and the underlying mechanism. Methods: TDSCs were isolated from rats and identified, and a tendon injury rat model was generated. Tenogenic differentiation in vitro was evaluated using Sirius red staining and quantitative real-time polymerase chain reaction, while that in vivo was assessed using immunohistochemistry and hematoxylin‒eosin staining. m5C methylation was analyzed using methylated RNA immunoprecipitation, dual-luciferase reporter assay, and RNA stability assay. Results: The results showed that m5C levels and NSUN2 expression were increased in TDSCs after tenogenic differentiation. Knockdown of NSUN2 inhibited m5C methylation of KLF2 and decreased its stability, which was recognized by YBX1. Moreover, interfering with KLF2 suppressed tenogenic differentiation of TDSCs, which could be abrogated by KLF2 overexpression. Additionally, TDSCs after NSUN2 overexpression contributed to ameliorating tendon injury in vivo. In conclusion, NSUN2 promotes tenogenic differentiation of TDSCs via m5C methylation of KLF2 and accelerates tendon repair. Conclusions: The findings suggest that overexpression of NSUN2 can stimulate the differentiation ability of TDSCs, which can be used in the treatment of tendinopathy.

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

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

Augmentation of Tendon and Ligament Repair with Fiber-Reinforced Hydrogel Composites.

This review highlights the promise of fiber-reinforced hydrogel composites (FRHCs) for augmenting tendon and ligament repair and regeneration. Composed of reinforcing fibers embedded in a hydrogel, these scaffolds provide both mechanical strength and a conducive microenvironment for biological processes required for connective tissue regeneration. Typical properties of FRHCs are discussed, highlighting their ability to simultaneously fulfill essential mechanical and biological design criteria for a regenerative scaffold. Furthermore, features of FRHCs are described that improve specific biological aspects of tendon healing including mesenchymal progenitor cell recruitment, early polarization to a pro-regenerative immune response, tenogenic differentiation of recruited progenitor cells, and subsequent production of a mature, aligned collagenous matrix. Finally, the review offers a perspective on clinical translation of tendon FRHCs and outlines key directions for future work.

Mechanobiological Strategies to Enhance Ovine (Ovis aries) Adipose-Derived Stem Cells Tendon Plasticity for Regenerative Medicine and Tissue Engineering Applications.

Adipose-derived stem cells (ADSCs) hold promise for tendon repair, even if their tenogenic plasticity and underlying mechanisms remain only partially understood, particularly in cells derived from the ovine animal model. This study aimed to characterize oADSCs during in vitro expansion to validate their phenotypic properties pre-transplantation. Moreover, their tenogenic potential was assessed using two in vitro-validated approaches: (1) teno-inductive conditioned media (CM) derived from a co-culture between ovine amniotic stem cells and fetal tendon explants, and (2) short- (48 h) and long-term (14 days) seeding on highly aligned PLGA (ha-PLGA) electrospun scaffold. Our findings indicate that oADSCs can be expanded without senescence and can maintain the expression of stemness (Sox2, Oct4, Nanog) and mesenchymal (CD29, CD166, CD44, CD90) markers while remaining negative for hematopoietic (CD31, CD45) and MHC-II antigens. Of note, oADSCs' tendon differentiation potential greatly depended on the in vitro strategy. oADSCs exposed to CM significantly upregulated tendon-related genes (COL1, TNMD, THBS4) but failed to accumulate TNMD protein at 14 days of culture. Conversely, oADSCs seeded on ha-PLGA fleeces quickly upregulated the tendon-related genes (48 h) and in 14 days accumulated high levels of the TNMD protein into the cytoplasm of ADSCs, displaying a tenocyte-like morphology. This mechano-sensing cellular response involved a complete SOX9 downregulation accompanied by YAP activation, highlighting the efficacy of biophysical stimuli in promoting tenogenic differentiation. These findings underscore oADSCs' long-term self-renewal and tendon differentiative potential, thus opening their use in a preclinical setting to develop innovative stem cell-based and tissue engineering protocols for tendon regeneration, applied to the veterinary field.

The Application of Photobiomodulation on Mesenchymal Stem Cells and its Potential Use for Tenocyte Differentiation.

Tendinopathy is a prevalent and debilitating musculoskeletal disorder. Uncertainty remains regarding its pathophysiology, but it is believed to be a combination of inflammation, damage, degenerative changes, and unsuccessful repair mechanisms. Cell-based therapy is an emerging regenerative medicine modality that uses mesenchymal stem cells (MSCs), their progeny or exosomes to promote tendon healing and regeneration. It is based on the fact that MSCs can be differentiated into tenocytes, the major cell type within tendons, and facilitate tendon repair. Photobiomodulation (PBM) is a non-invasive and potentially promising therapeutic technique that utilizes low-level light to alter intracellular processes and promote tissue healing and regeneration. Recent studies have examined the potential for PBM to improve MSC therapy use in tendinopathy by promoting viability, proliferation, and differentiation. As well as enhance tendon regeneration. This review focuses on Photobiomodulation and MSC therapy applications in regenerative medicine and their potential for tendon tissue engineering.

Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-ß3 signaling in tenogenic differentiation.

The transforming growth factor (TGF)-ß3 is a well-known inducer for tenogenic differentiation, signaling via the Smad2/3 pathway. Furthermore, other factors like extracellular matrix or mechanical force can induce tenogenic differentiation and possibly alter the response to TGF-ß3 by signaling via the Rho/ROCK pathway. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-ß3/Smad signaling in tenogenic differentiation, with the Smad2/3 molecule hypothesized as a possible interface. Cultured as monolayers or on collagen I matrices, mesenchymal stromal cells (MSC) were treated with the ROCK inhibitor Y-27632 (10 µM), TGF-ß3 (10 ng/ml) or both combined. Control cells were cultured accordingly, without Y-27632 and/or without TGF-ß3. At different time points, MSC were analyzed by real-time RT-PCR, immunofluorescence, and Western blot. Cultivation of MSC on collagen matrices and ROCK inhibition supported tenogenic differentiation and fostered the effect of TGF-ß3. The phosphorylation of the linker region of Smad2 was reduced by cultivation on collagen matrices, but not by ROCK inhibition. The latter, however, led to increased phosphorylation of the linker region of Smad3. In conclusion, collagen matrices and the Rho/ROCK signaling pathway influence the TGF-ß3/Smad2/3 pathway by regulating different phosphorylation sites of the Smad linker region.

Assessing the functional potential of conditioned media derived from amniotic epithelial stem cells engineered on 3D biomimetic scaffolds: An in vitro model for tendon regeneration.

Tendon diseases pose a significant challenge in regenerative medicine due to the limited healing capacity of this tissue. Successful tendon regeneration requires a combination of angiogenesis, immune response, and tenogenesis processes. An effective tendon engineering (TE) strategy must finely tune this systems' interplay toward homeostasis. This study explores in vitro the paracrine influence of amniotic epithelial stem cells (AECs) engineered on a validated 3D electrospun PLGA scaffolds on HUVECs (angiogenesis), PBMCs/Jurkat (immune response), and AECs (tenogenic stem cell activation). The results revealed the role of scaffold's topology and topography in significantly modulating the paracrine profile of the cells. In detail, AECs basal release of bioactive molecules was boosted in the cells engineered on 3D scaffolds, in particular VEGF-D, b-FGF, RANTES, and PDGF-BB (p < 0.0001 vs. CMCTR). Moreover, biological tests demonstrated 3D scaffolds' proactive role in potentiating AECs' paracrine inhibition on PBMCs proliferation (CM3Dvs. CTR, p < 0.001) and LPS-mediated Jurkat activation with respect to controls (CM3D and CM2Dvs. CTR, p < 0.01 and p < 0.05, respectively), without exerting any in vitro pro-angiogenic role in promoting HUVECs proliferation and tubule formation. Teno-inductive paracrine ability of AECs engineered on 3D scaffolds was assessed on co-cultured ones, which formed tendon-like structures. These latter demonstrated an upregulation of tendon-related genes (SCX, THBS4, COL1, and TNMD) and the expression TNMD and COL1 proteins. Overall, this research underscores the pivotal role of the 3D topology and topography of PLGA tendon mimetic scaffolds in orchestrating effective tendon regeneration through modulating cell behavior and crosstalk between engineered stem cells and different subpopulations in the damaged tendon.

A systematic investigation of the effects of TGF-ß3 and mechanical stimulation on tenogenic differentiation of mesenchymal stromal cells in a poly(ethylene glycol)/gelatin-based hydrogel.

Background: High post-surgical failure rates following tendon injury generate high medical costs and poor patient recovery. Cell-based tendon tissue engineering has the potential to produce fully functional replacement tissue and provide new strategies to restore tendon function and healing. In this endeavour, the application of mesenchymal stromal cells (MSCs) encapsulated in biomaterial scaffolds has shown great promise. However, a consensus on optimal promotion of tenogenic differentiation of MSCs has yet to be reached, although growth factors and mechanical cues are generally acknowledged as important factors. Methods: In this study, we prepared a hydrogel cell culture system consisting of methacrylated poly(d,l-lactic acid-ethylene glycol-d,l-lactic acid) (P(LA-EG-LA)) and gelatin methacrylate (GelMA) to encapsulate human bone marrow-derived MSCs (hBMSCs). We further systematically investigated the influence of static and intermittent cyclic uniaxial strain mechanical stimulation, in combination with transforming growth factor-ß3 (TGF-ß3) supplementation, on tenogenic differentiation of hBMSCs. Results: Increased TGF-ß3 concentration upregulated the tenogenic genes Scleraxis (SCX) and collagen type I (COL1A1) but showed no effects on tenascin-c (TNC) and collagen type III (COL3A1) expression. Mechanical stimulation had no observable effect on gene expression, but intermittent cyclic uniaxial strain stimulation improved matrix deposition. Together, these data provide new insights into how TGF-ß3 and mechanical stimulation regulate MSC tenogenesis, with TGF-ß3 promoting the expression of key tenogenic genes whilst mechanical stimulation aided matrix deposition in the engineered tissue. Furthermore, intermittent cyclic uniaxial strain at 3% elongation and 0.33 â€‹Hz for 1 â€‹h/day showed improved matrix effects compared to static strain. Conclusion: Together, the most promising result for tenogenic differentiation of hBMSCs was identified as treatment with 5 â€‹ng/ml TGF-ß3 under intermittent cyclic uniaxial strain (3% strain; 0.33 â€‹Hz; 1 â€‹h/day). This knowledge is of importance for the development of an improved protocol for tenogenic differentiation of MSCs and thereby for tendon tissue engineering. The translational potential of this article: Tissue-engineered strategies for tendon repair require a consensus on the differentiation of mesenchymal stromal cells to tenocytes, which is currently lacking. This article provides a systematic investigation of two main tenogenic differentiation conditions to further development of a tenogenic differentiation protocol.

Circ_0005736 promotes tenogenic differentiation of tendon-derived stem cells through the miR-636/MAPK1 axis.

BACKGROUND: Tendon-derived stem cells (TDSCs) are one of stem cells characterized by greater clonogenicity, tenogenesis, and proliferation capacity. Circ_0005736 has been shown to be decreased in Rotator cuff tendinopathy. Here, we investigated the function and relationship of circ_0005736 in TDSC tenogenic differentiation. METHODS: Transforming growth factor ß1 (TGF-ß1) was used to induce the tenogenic differentiation in TDSC. Cell proliferation, invasion and migration were evaluated by Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine, transwell, and wound healing assays, respectively. The detection of the levels of genes and proteins was performed by qRT-PCR and Western blot. The binding between miR-636 and circ_0005736 or MAPK1 (Mitogen-Activated Protein Kinase 1) was verified using dual-luciferase reporter assay and RIP assays. RESULTS: TGF-ß1 induced tenogenic differentiation by enhancing the production of tendon-specific markers and TDSC proliferation, invasion and migration. TGF-ß1 treatment promoted circ_0005736 expression, knockdown of circ_0005736 abolished TGF-ß1-induced tenogenic differentiation in TDSCs. Mechanistically, circ_0005736 acted as a sponge for miR-636 to up-regulate the expression of MAPK1, which was confirmed to be a target of miR-636 in TDSCs. Further rescue assays showed that inhibition of miR-636 could rescue circ_0005736 knockdown-induced suppression on TGF-ß1-caused tenogenic differentiation in TDSCs. Moreover, forced expression of miR-636 abolished TGF-ß1-caused tenogenic differentiation in TDSCs, which was rescued by MAPK1 up-regulation. CONCLUSION: Circ_0005736 enhanced TGF-ß1-induced tenogenic differentiation in TDSCs via increasing the production of tendon-specific markers and TDSC proliferation, invasion and migration through miR-636/MAPK1 axis.

Tendon Repair and Regeneration Using Bioinspired Fibrillation Engineering That Mimicked the Structure and Mechanics of Natural Tissue.

Replicating the controlled nanofibrillar architecture of collagenous tissue represents a promising approach in the design of tendon replacements that have tissue-mimicking biomechanics─outstanding mechanical strength and toughness, defect tolerance, and fatigue and fracture resistance. Guided by this principle, a fibrous artificial tendon (FAT) was constructed in the present study using an engineering strategy inspired by the fibrillation of a naturally spun silk protein. This bioinspired FAT featured a highly ordered molecular and nanofibrillar architecture similar to that of soft collagenous tissue, which exhibited the mechanical and fracture characteristics of tendons. Such similarities provided the motivation to investigate FAT for applications in Achilles tendon defect repair. In vitro cellular morphology and expression of tendon-related genes in cell culture and in vivo modeling of tendon injury clearly revealed that the highly oriented nanofibrils in the FAT substantially promoted the expression of tendon-related genes combined with the Achilles tendon structure and function. These results provide confidence about the potential clinical applications of the FAT.

Nanoscale vibration could promote tenogenic differentiation of umbilical cord mesenchymal stem cells.

Regulation of mesenchymal stem cell (MSC) fate for targeted cell therapy applications has been a subject of interest, particularly for tissues such as tendons that possess a marginal regenerative capacity. Control of MSCs' fate into the tendon-specific lineage has mainly been achieved by implementation of chemical growth factors. Mechanical stimuli or 3-dimensional (D) scaffolds have been used as an additional tool for the differentiation of MSCs into tenocytes, but oftentimes, they require a sophisticated bioreactor or a complex scaffold fabrication technique which reduces the feasibility of the proposed method to be used in practice. Here, we used nanovibration to induce the differentiation of MSCs toward the tenogenic fate solely by the use of nanovibration and without the need for growth factors or complex scaffolds. MSCs were cultured on 2D cell culture dishes that were connected to piezo ceramic arrays to apply nanovibration (30-80 nm and 1 kHz frequency) over 7 and 14 d. We observed that nanovibration resulted in significant overexpression of tendon-related markers in both gene expression and protein expression levels, while there was no significant differentiation into adipose and cartilage lineages. These findings could be of assistance in the mechanoregulation of MSCs for stem cell engineering and regenerative medicine applications.

Tenogenic differentiation of human tendon-derived stem cells induced by long non-coding RNA LINCMD1 via miR-342-3p/EGR1 axis.

BACKGROUND: Tendon-derived stem cells (TDSCs) are proposed as a potential cell-seed for the treatment of tendon injury due to their tenogenic differentiation potential. In this work, we defined the action of long non-coding RNA (lncRNA) muscle differentiation 1 (LINCMD1) in tenogenic differentiation of human TDSCs (hTDSCs). METHODS: Quantitative real-time PCR (qRT-PCR) was used to assess the levels of LINCMD1, microRNA (miR)-342-3p, and early growth response-1 (EGR1) mRNA. Cell proliferation was detected by the XTT colorimetric assay. Protein expression was quantified by western blot. hTDSCs were grown in an osteogenic medium to induce osteogenic differentiation, and the extent of osteogenic differentiation was assessed by Alizarin Red Staining (ARS). The activity of alkaline phosphatase (ALP) was measured by the ALP Activity Assay Kit. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to evaluate the direct relationship between miR-342-3p and LINCMD1 or EGR1. RESULTS: Our results showed that enforced expression of LINCMD1 or suppression of miR-342-3p accelerated the proliferation and tenogenic differentiation and reduced osteogenic differentiation of hTDSCs. LINCMD1 regulated miR-342-3p expression by binding to miR-342-3p. EGR1 was identified as a direct and functional target of miR-342-3p, and knockdown of EGR1 reversed the effects of miR-342-3p suppression on cell proliferation and tenogenic and osteogenic differentiation. Furthermore, the miR-342-3p/EGR1 axis mediated the regulation of LINCMD1 on hTDSC proliferation and tenogenic and osteogenic differentiation. CONCLUSION: Our study suggests the induction of LINCMD1 in tenogenic differentiation of hTDSCs through miR-342-3p/EGR1 axis.

Material Stiffness in Cooperation with Macrophage Paracrine Signals Determines the Tenogenic Differentiation of Mesenchymal Stem Cells.

Stiffness is an important physical property of biomaterials that determines stem cell fate. Guiding stem cell differentiation via stiffness modulation has been considered in tissue engineering. However, the mechanism by which material stiffness regulates stem cell differentiation into the tendon lineage remains controversial. Increasing evidence demonstrates that immune cells interact with implanted biomaterials and regulate stem cell behaviors via paracrine signaling; however, the role of this mechanism in tendon differentiation is not clear. In this study, polydimethylsiloxane (PDMS) substrates with different stiffnesses are developed, and the tenogenic differentiation of mesenchymal stem cells (MSCs) exposed to different stiffnesses and macrophage paracrine signals is investigated. The results reveal that lower stiffnesses facilitates tenogenic differentiation of MSCs, while macrophage paracrine signals at these stiffnesses suppress the differentiation. When exposed to these two stimuli, MSCs still exhibit enhanced tendon differentiation, which is further elucidated by global proteomic analysis. Following subcutaneous implantation in rats for 2 weeks, soft biomaterial induces only low inflammation and promotes tendon-like tissue formation. In conclusion, the study demonstrates that soft, rather than stiff, material has a greater potential to guide tenogenic differentiation of stem cells, which provides comprehensive evidence for optimized bioactive scaffold design in tendon tissue engineering.

The potential mechanism of hypoxia-induced tenogenic differentiation of mesenchymal stem cell for tendon regeneration.

Tendon injuries account up to 50% of all musculoskeletal problems and remains a challenge to treat owing to the poor intrinsic reparative ability of tendon tissues. The natural course of tendon healing is very slow and often leads to fibrosis and disorganized tissues with inferior biomechanical properties. Mesenchymal stem cells (MSC) therapy is a promising alternative strategy to augment tendon repair due to its proliferative and multilineage differentiation potential. Hypoxic conditioning of MSC have been shown to enhance their tenogenic differentiation capacity. However, the mechanistic pathway by which this is achieved is yet to be fully defined. A key factor involved in this pathway is hypoxia-inducible factor-1-alpha (HIF-1α). This review aims to discuss the principal mechanism underlying the enhancement of MSC tenogenic differentiation by hypoxic conditioning, particularly the central role of HIF-1α in mediating activation of tenogenic pathways in the MSC. We focus on the interaction between HIF-1α with fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta 1 (TGF-ß1) in regulating MSC tenogenic differentiation pathways in hypoxic conditions. Strategies to promote stabilization of HIF-1α either through direct manipulation of oxygen tension or the use of hypoxia mimicking agents are therefore beneficial in increasing the efficacy of MSC therapy for tendon repair.

Dexamethasone Is Not Sufficient to Facilitate Tenogenic Differentiation of Dermal Fibroblasts in a 3D Organoid Model.

Self-assembling three-dimensional organoids that do not rely on an exogenous scaffold but maintain their native cell-to-cell and cell-to-matrix interactions represent a promising model in the field of tendon tissue engineering. We have identified dermal fibroblasts (DFs) as a potential cell type for generating functional tendon-like tissue. The glucocorticoid dexamethasone (DEX) has been shown to regulate cell proliferation and facilitate differentiation towards other mesenchymal lineages. Therefore, we hypothesized that the administration of DEX could reduce excessive DF proliferation and thus, facilitate the tenogenic differentiation of DFs using a previously established 3D organoid model combined with dose-dependent application of DEX. Interestingly, the results demonstrated that DEX, in all tested concentrations, was not sufficient to notably induce the tenogenic differentiation of human DFs and DEX-treated organoids did not have clear advantages over untreated control organoids. Moreover, high concentrations of DEX exerted a negative impact on the organoid phenotype. Nevertheless, the expression profile of tendon-related genes of untreated and 10 nM DEX-treated DF organoids was largely comparable to organoids formed by tendon-derived cells, which is encouraging for further investigations on utilizing DFs for tendon tissue engineering.

Platelet-Derived Extracellular Vesicles Promote Tenogenic Differentiation of Stem Cells on Bioengineered Living Fibers.

Tendon mimetic scaffolds that recreate the tendon hierarchical structure and niche have increasing potential to fully restore tendon functionality. However, most scaffolds lack biofunctionality to boost the tenogenic differentiation of stem cells. In this study, we assessed the role of platelet-derived extracellular vesicles (EVs) in stem cells' tenogenic commitment using a 3D bioengineered in vitro tendon model. First, we relied on fibrous scaffolds coated with collagen hydrogels encapsulating human adipose-derived stem cells (hASCs) to bioengineer our composite living fibers. We found that the hASCs in our fibers showed high elongation and cytoskeleton anisotropic organization, typical of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted the hASCs' tenogenic commitment, prevented phenotypic drift, enhanced the deposition of the tendon-like extracellular matrix, and induced lower collagen matrix contraction. In conclusion, our living fibers provided an in vitro system for tendon tissue engineering, allowing us to study not only the tendon microenvironment but also the influence of biochemical cues on stem cell behavior. More importantly, we showed that platelet-derived EVs are a promising biochemical tool for tissue engineering and regenerative medicine applications that are worthy of further exploration, as paracrine signaling might potentiate tendon repair and regeneration.

Human Muscle-Derived Cells Are Capable of Tenogenic Differentiation and Contribution to Tendon Repair.

BACKGROUND: It has been reported that the harvested hamstring tendon for autograft could be regenerated with well-oriented fibers and uniformly distributed spindle-shaped cells after removal. However, which cell type might participate in the repair process remains unknown. PURPOSE: To investigate the tenogenic differentiation potential of human muscle-derived cells (MDCs) both in vitro and in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: Primary human MDCs and tenocytes were isolated from discarded materials during a peroneus longus tendon-harvesting procedure. Expression of tenogenic genes were evaluated and compared among MDCs, MDCs with tenogenic induction, and tenocytes. RNA sequencing was performed to evaluate the expression profile of differentiated MDCs. Human MDCs were implanted in a tendon injury model to investigate the in vivo tenogenic differentiation potential. Histologic and functional analyses were performed to evaluate the function of MDCs for tendon repair. RESULTS: The relative expression levels (in fold change) of tenogenic genes Col I, MKX, SCX, THBS4, and TNC in MDCs were significantly upregulated 11.5 ± 1.3, 957.1 ± 63.7, 19.1 ± 2.8, 61.9 ± 4.8, and 10.2 ± 2.8 after tenogenic induction, respectively. The expression profile of tenogenically differentiated MDCs was much closer to primary tenocytes. Activation of TGF-ß/Smad3 signaling significantly promoted the tenogenic differentiation ability of MDCs. Transplanted human MDCs were identified in regenerated tendon and expressed tenogenic genes. As for biomechanical properties, the failure loads in the Matrigel, transplantation, and uninjured groups were 7.2 ± 0.5, 11.6 ± 0.3, and 13.9 ± 0.7 N, while the stiffness values were 4.4 ± 1.3 × 103, 7.6 ± 0.8 × 103, and 10.9 ± 1.1 × 103 N/m. Plantarflexion force, histologic morphology, and motor function were also significantly improved after MDC transplantation in a tendon injury model. CONCLUSION: There exist cells with tenogenic differentiation potential in human skeletal muscles. Activation of TGF-ß/Smad3 signaling plays an important role in tenogenic differentiation for human MDCs. Human MDCs contribute to structural and functional repair for the injured tendon. MDCs are a potential cell source to participate in the repair process after tendon injury. CLINICAL RELEVANCE: The MDCs could be a promising cell source to repair tendon injury.

Degenerative tendon matrix induces tenogenic differentiation of mesenchymal stem cells.

PURPOSE: Mesenchymal stem cells (MSCs) react dynamically with the surrounding microenvironment to promote tissue-specific differentiation and hence increase targeted regenerative capacity. Extracellular matrix (ECM) would be the first microenvironment to interact with MSCs injected into the tissue lesion. However, degenerative tissues would have different characteristics of ECM in comparison with healthy tissues. Therefore, the influence of degenerative ECM on tissue-specific differentiation of MSCs and the formation of matrix composition need to be considered for the sophisticated therapeutic application of stem cells for tissue regeneration. METHODS: Human degenerative tendon tissues were obtained from patients undergoing rotator cuff repair and finely minced into 2 ~ 3 mm fragments. Different amounts of tendon matrix (0.005 g, 0.01 g, 0.025 g, 0.05 g, 0.1 g, 0.25 g, 0.5 g, 1 g, and 2 g) were co-cultured with bone marrow MSCs (BM MSCs) for 7 days. Six tendon-related markers, scleraxis, tenomodulin, collagen type I and III, decorin, and tenascin-C, osteogenic marker, alkaline phosphatase (ALP), and chondrogenic marker, aggrecan (ACAN), were analyzed by qRT-PCR. Cell viability and senescence-associated beta-galactosidase assays were performed. The connective tissue growth factor was used as a positive control. RESULTS: The expressions of six tendon-related markers were significantly upregulated until the amount of tendon matrix exceeded 0.5 g, the point where the mRNA expressions of all six genes analyzed started to decrease. The tendon matrix exerted an inhibitory effect on ACAN expression but had a negligible effect on ALP expression. Cell viability did not change significantly over the culture period. The amount of tendon matrix exceeding 0.01 g significantly increased the SA-ßgal activity of BM MSCs. CONCLUSION: This study successfully demonstrated tendon ECM-stimulated tenogenesis of BM MSCs through an indirect co-culture system without the use of exogenous growth factors and the alteration of cellular viability. In contrast to the initial hypothesis, the tenogenesis of BM MSCs induced with the degenerative tendon matrix accompanied cellular senescence.

Leucine rich repeat containing 32 accelerates tenogenic differentiation of tendon-derived stem cells and promotes Achilles tendon repair in rats.

Although many surgical or non-operative therapies have been developed to treat Achilles tendon injuries, the prognosis of which is often unsatisfactory. Recently, biologic approaches using multipotent stem cells like tendon-derived stem cells (TDSCs) pose a possible treatment option. To evaluate whether the Leucine rich repeat containing 32 (Lrrc32) affects the tenogenic differentiation of TDSCs and thus promotes Achilles tendon healing. TDSCs were infected with the recombinant Lrrc32-overexpressing lentivirus (LV-Lrrc32) and then locally injected into the injured site of rat. Four weeks after surgery, the Achilles tendon tissue (~0.5 cm) around the injured area was harvested for analysis. Pathological results showed that Lrrc32-overexpressing TDSCs significantly improved the morphological changes of the injured tendons. Specifically, the increased collagen-I expression and hydroxyproline content in extracellular matrix, and more orderly arrangement of the regenerated collagen fibers were observed in the Lrrc32 overexpression group. Moreover, 4 weeks after injection of Lrrc32-overexpressing TDSCs, the expression of tenocyte-related genes such as tenomodulin (Tnmd), scleraxis (Scx) and decorin (Dcn) were upregulated in the area of the healing tendon. These findings indicated that Lrrc32 promoted the tenogenic differentiation of TDSCs in vivo. Additionally, Lrrc32 overexpression also increased the expression of TGF-ß1 and p-SMAD2/3, suggesting that the beneficial effects of Lrrc32 on tendon repair might be associated with the expression of TGF-ß1 and p-SMAD2/3. Our findings collectively revealed that Lrrc32-overexpressed TDSCs promoted tendon healing more effectively than TDSCs alone.

Electrospun aligned poly(ε-caprolactone) nanofiber yarns guiding 3D organization of tendon stem/progenitor cells in tenogenic differentiation and tendon repair.

Hierarchical anisotropy structure directing 3D cellular orientation plays a crucial role in designing tendon tissue engineering scaffolds. Despite recent development of fabrication technologies for controlling cellular organization and design of scaffolds that mimic the anisotropic structure of native tendon tissue, improvement of tenogenic differentiation remains challenging. Herein, we present 3D aligned poly (ε-caprolactone) nanofiber yarns (NFYs) of varying diameter, fabricated using a dry-wet electrospinning approach, that integrate with nano- and micro-scale structure to mimic the hierarchical structure of collagen fascicles and fibers in native tendon tissue. These aligned NFYs exhibited good in vitro biocompatibility, and their ability to induce 3D cellular alignment and elongation of tendon stem/progenitor cells was demonstrated. Significantly, the aligned NFYs with a diameter of 50 µm were able to promote the tenogenic differentiation of tendon stem/progenitor cells due to the integration of aligned nanofibrous structure and suitable yarn diameter. Rat tendon repair results further showed that bundled NFYs encouraged tendon repair in vivo by inducing neo-collagen organization and orientation. These data suggest that electrospun bundled NFYs formed by aligned nanofibers can mimic the aligned hierarchical structure of native tendon tissue, highlighting their potential as a biomimetic multi-scale scaffold for tendon tissue regeneration.