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Failure to regenerate the gradient tendon–bone interface of the enthesis results in poor clinical outcomes for surgical repair. The goal of this study was to evaluate the potential of composite cell sheets for engineering of the tendon–bone interface to improve regeneration of the functionally graded tissue. We hypothesize that stacking cell sheets at early stages of differentiation into tenogenic and osteogenic progenitors will create a composite structure with integrated layers. Cell sheets were fabricated on methyl cellulose and poly(N-isopropylacrylamide) thermally reversible polymers with human adipose-derived stem cells and differentiated into progenitors of tendon and bone with chemical induction media. Tenogenic and osteogenic cell sheets were stacked, and the engineered tendon–bone interface (TM-OM) was characterized in vitro in comparison to stacked cell sheet controls cultured in basal growth medium (GM-GM), osteogenic medium (OM-OM), and tenogenic medium (TM-TM). Samples were characterized by histology, quantitative real-time polymerase chain reaction, and immunofluorescent staining for markers of tendon, fibrocartilage, and bone including mineralization, scleraxis, tenomodulin, COL2, COLX, RUNX2, osteonectin, and osterix. After 1 week co-culture in basal growth medium, TM-OM cell sheets formed a tissue construct with integrated layers expressing markers of tendon, mineralized fibrocartilage, and bone with a spatial gradient in RUNX2 expression. Tenogenic cell sheets had increased expression of scleraxis and tenomodulin. Osteogenic cell sheets exhibited mineralization 1 week after stacking and upregulation of osterix and osteonectin. Additionally, in the engineered interface, there was significantly increased gene expression of IHH and COLX, indicative of endochondral ossification. These results highlight the potential for composite cell sheets fabricated with adipose-derived stem cells for engineering of the tendon–bone interface. IMPACT STATEMENT: This study presents a method for fabrication of the tendon–bone interface using stacked cell sheets of tenogenic and osteogenic progenitors differentiated from human adipose-derived mesenchymal stem cells, resulting in a composite structure expressing markers of tendon, mineralized fibrocartilage, and bone. This work is an important step toward regeneration of the biological gradient of the enthesis and demonstrates the potential for engineering complex tissue interfaces from a single autologous cell source to facilitate clinical translation.

Summary Understanding cell recruitment in damaged tendons is critical for improvements in regenerative therapy. We recently reported that targeted disruption of transforming growth factor beta (TGFβ) type II receptor in the tendon cell lineage (Tgfbr2ScxCre) resulted in resident tenocyte dedifferentiation and tendon deterioration in postnatal stages. Here we extend the analysis and identify direct recruitment of stem/progenitor cells into the degenerative mutant tendons. Cre-mediated lineage tracing indicates that these cells are not derived from tendon-ensheathing tissues or from a Scleraxis-expressing lineage, and they turned on tendon markers only upon entering the mutant tendons. Through immunohistochemistry and inducible gene deletion, we further find that the recruited cells originated from a Sox9-expressing lineage and their recruitment was dependent on cell autonomous TGFβ signaling. The cells identified in this study thus differ from previous reports of cell recruitment into injured tendons and suggest a critical role for TGFβ signaling in cell recruitment, providing insights that may support improvements in tendon repair., Graphical abstract, Highlights • Targeted deletion of TGFβ signaling led to degenerative changes in mouse tendons • Stem/progenitor cells were recruited into the degenerative mutant tendons • The recruited cells are different from the ones so far reported in tendon injury • Recruitment was dependent on cell autonomous TGFβ signaling in the recruited cells, In this article, Schweitzer and colleagues identify the recruitment of a new population of stem/progenitors into degenerative mutant mouse tendons and demonstrate that their recruitment was dependent on cell autonomous TGFβ signaling. This provides an opportunity to directly examine the origin of tendon stem/progenitor cells and the mechanisms of their activation, which is critical for improvement in tendon repair.

The use of mesenchymal stem cells can add local improvements potential to enthesis tissue regeneration based on tropical activity through secretions of growth factors, cytokines, and vesicles (e.g. exosomes), collectively known as secretomes. This study aims to analyze secretomes characterization from adipose-derived mesenchymal stem cells seeded with enthesis tissue scaffold in hypoxic conditions and to analyze the influence of hypoxic environment to the characterization of secretomes. This is an in-vitro study using a Randomized Control Group Post-Test Only design. This study using Adipose Stem Cells (ASCs) were cultured in hypoxia (Oxygen 5%) and Normoxia (21%) condition. The scaffolds are fresh-frozen enthesis tissue and was seeded in the treatment group and compared to control. The evaluation of Scleraxis (Scx) and SRY-box (Sox9) was measured using ELISA on the 2nd, 4th, and 6th days. Comparison of Scx levels between each evaluation time showed a positive trend in a group with scaffold in hypoxia condition although it has no significant differences (p=0.085), with the highest level on day 6, that is 13,568 ng/ml. Conversely, the comparison of Sox9 showed significant differences (p=0.02) in a group with scaffold in hypoxia condition, with the highest level on day 4, that is 28,250 ng/ml. The use of enthesis scaffold seeded in adipose-derived mesenchymal stem cells in hypoxic conditions shows a positive trend as regenerative effort of injured enthesis tissue through Scleraxis and Sox9 secretomes induction.

The tenocyte niche contains biochemical and biophysical signals that are needed for tendon homeostasis. The tenocyte phenotype is correlated with cell shape in vivo and in vitro, and shape-modifying cues are needed for tenocyte phenotypical maintenance. Indeed, cell shape changes from elongated to spread when cultured on a flat surface, and rat tenocytes lose the expression of phenotypical markers throughout five passages. We hypothesized that tendon gene expression can be preserved by culturing cells in the native tendon shape. To this end, we reproduced the tendon topographical landscape into tissue culture polystyrene, using imprinting technology. We confirmed that the imprints forced the cells into a more elongated shape, which correlated with the level of Scleraxis expression. When we cultured the tenocytes for 7 days on flat surfaces and tendon imprints, we observed a decline in tenogenic marker expression on flat but not on imprints. This research demonstrates that native tendon topography is an important factor contributing to the tenocyte phenotype. Tendon imprints therefore provide a powerful platform to explore the effect of instructive cues originating from native tendon topography on guiding cell shape, phenotype, and function of tendon-related cells.

Herein, we investigate the high incidence of umbilical hernia and tippy-toe standing and their underlying changes in gene expression and proliferation in myostatin knockout (MSTN−/−) pigs. Thirty-six male MSTN−/− pigs were generated by somatic cell nuclear transfer (SCNT). These pigs presented a considerably high incidence of tippy-toe standing and umbilical hernia (69.4% and 61.1%, respectively). The tendon to body weight ratio was significantly lower than wild-type pigs (0.202 ± 0.017 vs 0.250 ± 0.004, respectively). The crimp length of the MSTN−/− tendon was significantly longer than that of wild-type pigs. The expression of MSTN and the activin type IIB (ACVR2B) was detected in the tendon and linea alba of MSTN−/− pigs. MSTN treatment significantly increased the phosphorylation of Smad2/3 in both tendon and linea alba fibroblasts. Type I collagen (Col1A) and Scleraxis (Scx) expression levels in the tendon and linea alba of MSTN−/− pigs were significantly lower than those in wild-type in vivo, whereas and cyclin-dependent kinase inhibitor 1 (p21) expression levels were higher. Treatment of tendon and linea alba fibroblasts with recombinant MSTN increased Col1A and Scx and decreased p21 expression in vivo. Moreover, there was a significant increase in fibroblast proliferation after treatment. The results indicated that MSTN regulates collagen expression and proliferation in tendon and linea alba fibroblasts; thus, MSTN deficiency causes collagen-related pathological features in MSTN−/− pigs. Hence, MSTN could be used as a therapeutic target for treating UH and tendon abnormalities.

Mesenchymal Stem Cells (MSCs) are important in regenerative medicine and tissue engineering and will be a very sensible choice for repair and regeneration of tendon. New biological practices, such as cellular therapy using stem cells, are promising for facilitating or expediting tendon therapy. Before using these cells clinically, it is best to check and confirm the optimal conditions for differentiation of these cells in the laboratory. Hence, in the present study, the impacts of PDGF-BB and GDF-6 supplementation on adipose-derived MSCs (ASCs) culture were studied. The frozen ASC were recovered and expanded in basic culture medium (DMEM with 10%FBS). The cells after passage five (P5) were treated with basic medium containing L-Prolin, Ascorbic Acid and only PDGF-BB or GDF-6 (20 ng/ml) or both of them (mix) as 3 groups for 14 days to investigate efficiency of ASCs differentiation towards tenocytes. The cells culturing in basic medium were used as control group. To validate tenogenic differentiation, H&E and Sirius Red staining were used to assess cell morphology and collagen production, respectively. In addition, mRNA levels of collagen I and III, Scleraxis and Tenomodulin as tenogenic markers were analyzed using qPCR. In all test groups, cells appeared slenderer, elongated cytoplasmic attributes compared to the control cells. The intensity of Sirius Red staining was significantly higher in GDF-6, PDGF-BB alone, than in group without supplements. The optical density was higher in the GDF-6 than PDGF-BB and mix-group. QPCR results showed that Col I and III gene expression was increased in all groups compared to the control. SCX expression was significantly increased only in the PDGF-BB group. TNMD mRNA expression was not significant among groups. In this study, we have corroborated that human ASCs are reactionary to tenogenic induction by GDF-6 and PDGF-BB alone or in combination. These outcomes will help greater insight into GDF-6 and PDGF-BB driven tenogenesis of ASCs and new directions of discovery in the design of ASC-based treatments for tendon healing.

Intermuscular bones (IBs) are ossified from tendons and only occur in lower teleosts. Positive association between the regulation of scleraxis gene (scx) and tendon development gave us reasons to speculate that the scx gene may play a potential role in regulating the development of IBs. A phylogenetic analysis conducted for this study revealed potential functional differentiation between two scx orthologues, scxa and scxb. The scxa−/− and scxb−/− zebrafish were generated through CRISPR-Cas9 technology to study the role of scx in the IB and rib development. The results showed a significant reduction of the number of IBs in adult scxa-1-/- zebrafish, with almost 70% reduction (15–25 IBs) compared to the wild type scxa-1+/+ zebrafish (76–80 IBs). In the scxa-1+/+ adults, IBs were observed in both dorsal and tail segments; however, in scxa-1-/- fish IBs were observed only in the tail segment (none in the dorsal segment). Although scxa-1−/− zebrafish had rib defects, the mutants were viable and fertile as adult fish. The scxb−/− zebrafish had the same number of IBs and same skeletal phenotype as the wild-type fish. This suggests that only scxa has a crucial role in the IB development, and confirms functional differentiation of scx orthologues. To further clarify the molecular mechanism by which scxa affects the IB development, we conducted comparative transcriptome analysis of dorsal tissue samples of scxa-1+/+ (with IBs) and scxa-1−/− (without IBs), and further verified the expression of key genes via qPCR. This is the first study to identify a gene that controls the amount of IBs in fish, and it provides a new sight into the effects of scxa on the molecular mechanism of IB development in fish.

Aging is hypothesized to be associated with changes in tendon matrix composition which may lead to alteration of tendon material properties and hence propensity to injury. Altered gene expression may offer insights into disease pathophysiology and thus open new perspectives toward designing pathophysiology‐driven therapeutics. Therefore, the current study aimed at identifying naturally occurring differences in tendon micro‐morphology and gene expression of newborn, young and old horses. Age‐related differences in the distribution pattern of tendon fibril thickness and in the expression of the tendon relevant genes collagen type 1 (Col1), Col3, Col5, tenascin‐C, decorin, tenomodulin, versican, scleraxis and cartilage oligomeric matrix protein were investigated. A qualitative and quantitative gene expression and collagen fibril diameter analysis was performed for the most frequently injured equine tendon, the superficial digital flexor tendon, in comparison with the deep digital flexor tendon. Most analyzed genes (Col1, Col3, Col5, tenascin‐C, tenomodulin, scleraxis) were expressed at a higher level in foals (age ≤ 6 months) than in horses of 2.75 years (age at which flexor tendons become mature in structure) and older, decorin expression increased with age. Decorin was previously reported to inhibit the lateral fusion of collagen fibrils, causing a thinner fibril diameter with increased decorin concentration. The results of this study suggested that reduction of tendon fibril diameters commonly seen in equine tendons with increasing age might be a natural age‐related phenomenon leading to greater fibril surface areas with increased fibrillar interaction and reduced sliding at the fascicular/fibrillar interface and hence a stiffer interfascicular/interfibrillar matrix. This may be a potential reason for the higher propensity to tendinopathies with increasing age., TEM pictures and histograms showing collagen fibril distribution changes for the SDFT and DDFT with age. Whereas a Gaussian‐like distribution of fibril diameters was observed in tendons of foals up to 6 months of age, a marked shift towards much thinner fibrils was seen in tendons of horses 2.75 years of age or older.

Background and objectives: Controlling nutrition and exercise can be two important strategies in controlling tendon health. It has been reported that resistance training and palm pollen separately can improve Scleraxis (Scx) in tendon tissue; so present study aimed to investigate the interactive effects of resistance training with ethanolic extract of palm pollen on Scx protein and gene expression levels in the tendon tissue of male adult rats. Methods: In this experimental study 30 male adult rats divided into 6 groups of 6 rats including: 1) sham, 2) training, 3) palm pollen, 4) testosterone, 5) training + palm pollen, and 6) training + testosterone. During 4 weeks, groups 2, 5, and 6 performed resistance trainings for five sessions per week; groups 3 and 5 received 100 mg/kg palm pollen for five days per week via gavage and groups 4 and 6 received 2 mg/kg testosterone propionate peritoneally. Scx protein and gene expression levels were measured in tendon tissue by Western blot and real-time PCR methods respectively. Shapiro- Wilk, one way ANOVA with Tukey’s post- hoc tests were used to analyze the findings (P≤0.05). Results: Training significantly increased Scx protein levels (P=0.005); palm pollen significantly increased Scx gene expression levels (P=0.001); training + palm pollen significantly increased Scx protein and gene expression levels (P=0.001) also training + palm pollen had more favorable effect on increase of Scx protein and gene expression levels compared to training and palm pollen alone (P=0.001). Conclusion: It seems that resistance training simultaneously with palm pollen administration can have a more favorable effect than each one alone on improving Scx protein and gene expression levels in tendon tissue of male adult rats.

Identification of a suitable cell source and bioactive agents guiding cell differentiationtowards tenogenic phenotype represents a prerequisite for advancement of cellâ based therapies for tendon repair. Human adiposeâ derived stem cells (hASCs) are apromising, yet intrinsically heterogenous population with diversified differentiationcapacities. In this work, we investigated antigenicallyâ defined subsets of hASCsexpressing markers related to tendon phenotype or associated with pluripotency thatmight be more prone to tenogenic differentiation, when compared to unsortedhASCs. Subpopulations positive for tenomodulin (TNMD+ hASCs) and stage specificearly antigen 4 (SSEAâ 4+ hASCs), as well as unsorted ASCs were cultured up to 21days in basic medium or media supplemented with TGFâ β3 (10 ng/ml), or GDFâ 5(50 ng/ml). Cell response was evaluated by analysis of expression of tendonâ relatedmarkers at gene level and protein level by real time RTâ PCR, western blot, and immu-nocytochemistry. A significant upregulation of scleraxis was observed for both sub-populations and unsorted hASCs in the presence of TGFâ β3. More prominentalterations in gene expression profile in response to TGFâ β3 were observed forTNMD+ hASCs. Subpopulations evidenced an increased collagen III and TNC deposi-tion in basal medium conditions in comparison with unsorted hASCs. In the particularcase of TNMD+ hASCs, GDFâ 5 seems to influence more the deposition of TNC.Within hASCs populations, discrete subsets could be distinguished offering variedsensitivity to specific biochemical stimulation leading to differential expression oftenogenic components suggesting that cell subsets may have distinctive roles in thecomplex biological responses leading to tenogenic commitment to be further exploredin cell based strategies for tendon tissues., The authors acknowledge the financial support from the European Union Framework Programme for Research and Innovation HORIZON 2020, under the Tendon Therapy Train Marie Skłodowska‐Curie Innovative Training Network grant agreement No. 676338, the TEAMING Grant agreement No 739572 ‐ The Discoveries CTR, and the Achilles Twinning Project No. 810850. Authors acknowledge also the European Research Council CoG MagTendon No. 772817, the FCT (Fundação para a Ciência e a Tecnologia) Project MagTT PTDC/CTM‐ CTM/29930/2017 (POCI‐01‐0145‐FEDER‐29930), and the Project NORTE‐01‐0145‐FEDER‐000021: “Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechnologies, marine‐derived biomaterials and stem cells”, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). ADE and JdB acknowledge the financial contribution of the Dutch province of Limburg in the LINK (FCL67723; “Limburg INvesteert in haar Kenniseconomie”) knowledge economy project.

Intervertebral disc (IVD) injuries are a cause of degenerative changes in adults which can lead to back pain, a leading cause of disability. We developed a model of neonatal IVD regeneration with full functional restoration and investigate the cellular dynamics underlying this unique healing response. We employed genetic lineage tracing in mice using Scleraxis (Scx) and Sonic hedgehog (Shh) to fate-map annulus fibrosus (AF) and nucleus pulposus (NP) cells, respectively. Results indicate functional AF regeneration after severe herniation injury occurs in neonates and not adults. AF regeneration is mediated by Scx-lineage cells that lose ScxGFP expression and adopt a stem/progenitor phenotype (Sca-1, days 3–14), proliferate, and then redifferentiate towards type I collagen producing, ScxGFP+ annulocytes at day 56. Non Scx-lineage cells were also transiently observed during neonatal repair, including Shh-lineage cells, macrophages, and myofibroblasts; however, these populations were no longer detected by day 56 when annulocytes redifferentiate. Overall, repair did not occur in adults. These results identify an exciting cellular mechanism of neonatal AF regeneration that is predominantly driven by Scx-lineage annulocytes.

Tendons and ligaments are structural tissues that attach to bone and are essential for joint mobility and stability in vertebrates. Tendon and ligament attachments (i.e., entheses) are often found at bony protrusions (i.e., eminences), and the shape and size of these protrusions depends on both mechanical forces and cellular cues during growth and development. The formation of tendon eminences also contributes to mechanical leverage for skeletal muscle. Fibroblast growth factor receptor (FGFR) signaling plays a critical role in bone development, and Fgfr1 and Fgfr2 are highly expressed in the perichondrium and periosteum of bone where tendon and ligament attachments can be found. However, the role of FGFR signaling in attachment development and maintenance in the limb remains unknown. In this study, we used transgenic mouse models for combinatorial knockout of Fgfr1 and/or Fgfr2 in tendon/ligament and attachment progenitors using ScxCre and measured eminence size and bone shape in the appendicular skeleton. Conditional deletion of both, but not individual, Fgfr1 and Fgfr2 in Scx progenitors led to enlarged eminences in the postnatal appendicular skeleton and smaller secondary ossification centers in long bones. In addition, Fgfr1 Fgfr2 double conditional knockout mice had more variation in the size of collagen fibrils in tendon, narrowed synovial joint spacing, and increased cell death at sites of ligament attachments, as well as decreased plasticity of mature bone compared to age-matched wildtype littermates. These findings identify a role for FGFR signaling in regulating growth and maintenance of tendon/ligament attachments and the size and shape of bony eminences.

Embryonic tendon cells have been studied in vitro to better understand mechanisms of tendon development. Outcomes of in vitro cell culture studies are easily affected by phenotype instability of embryonic tendon cells during expansion in vitro to achieve desired cell numbers, yet this has not been characterized. In the present study, we characterized phenotype stability, expansion potential, and onset of senescence in chick embryo tendon cells from low to high cell doublings. We focused on cells of Hamburger-Hamilton stages (HH) HH40 and HH42, where HH40 is the earliest stage associated with substantial increases in extracellular matrix and mechanical properties during embryonic tendon development 1 . HH40 and HH42 cells both downregulated expression levels of tendon phenotype markers, scleraxis and tenomodulin, and exhibited onset of senescence, based on p16 and p21 expression levels, cell surface area, and percentage of β-galactosidase positive cells, before significant decreases in proliferation rates were detected. These findings showed that embryonic tendon cells destabilize phenotype and become senescent earlier than they begin to decline in proliferation rates in vitro. Additionally, embryonic stage of isolation appears to have no effect on proliferation rates, whereas later stage HH42 cells downregulate phenotype and become susceptible to senescence sooner than earlier stage HH40 cells. Based on our data, we recommend chick embryo tendon cells be used before a maximum cumulative doubling level of 12 (passage 4 in this study) to avoid phenotype destabilization and onset of senescence. This article is protected by copyright. All rights reserved.

The tendon healing process is regulated by the coordinated interaction of multiple cell types and molecular processes. However, these processes are not well-defined leading to a paucity of therapeutic approaches to enhance tendon healing. Scleraxis-lineage (ScxLin) cells are the major cellular component of adult tendon and make time-dependent contributions to the healing process. Prior work from our lab and others suggests heterogeneity within the broader ScxLin population over the course of tendon healing; therefore delineating the temporal and spatial contributions of these cells is critical to understanding and improving the healing process. In the present study we utilize lineage tracing of the adult aScxLin population to determine whether these cells undergo cellular activation and subsequent myofibroblast differentiation, which is associated with both proper healing and fibrotic progression in many tissues. We show that adult aScxLin cells undergo transient activation in the organized cellular bridge at the tendon repair site, contribute to the formation of an organized neotendon, and contribute to a persistent myofibroblast population in the native tendon stubs. The mechanisms dictating this highly specialized spatial response are unknown. We therefore utilized spatial transcriptomics to better define the spatio-molecular program of tendon healing. Integrated transcriptomic analyses across the healing time-course identifies five distinct molecular regions, including key interactions between the inflammatory bridging tissue and highly reactive tendon tissue at the repair site, with adult ScxLin cells being a central player in the transition from native tendon to reactive, remodeling tendon. Collectively, these data provide important insights into both the role of adult ScxLin cells during healing as well as the molecular mechanisms that underpin and coordinate the temporal and spatial healing phenotype, which can be leveraged to enhance the healing process.

Background Extracellular vesicles from bone marrow-derived multipotent mesenchymal stromal cells (BMSC-EVs) can play important roles in the repair of injured tissues. However, no reports have investigated the role and underlying mechanisms of BMSCs-EVs in the tendon repair process. We hypothesized that BMSC-EVs may play a role in modulating inflammation during tendon healing and improving tendon repair in a rat model of patellar tendon injury. Methods First, we created window defects in the patellar tendons of Sprague–Dawley rats. Rats (n = 16) were then randomly assigned to three groups: BMSC-EVs group, Fibrin group, and control group. Rats in the BMSC-EVs group were treated with BMSC-EVs and fibrin glue (25 µg in 10 µL). Rats in the fibrin group were treated with fibrin only, and those in the control group received no treatment. Histopathology, immunohistochemistry, and gene expression analyses were performed at 2 and 4 weeks after surgery. Results At 4 weeks, tendons treated with BMSC-EVs showed regularly aligned and compact collagen fibers as compared with the disrupted scar-like healing in rats in the fibrin and control groups. The expression of genes related to tendon matrix formation and tenogenic differentiation: collagen (COL)-1a1, scleraxis (SCX), and tenomodulin (TNMD) was significantly higher in the BMSC-EVs group than in the other two groups. With histopathology, we observed significantly higher numbers of CD146+ tendon stem cells and fewer numbers of apoptotic cells and C–C chemokine receptor type 7 (CCR7)-positive proinflammatory macrophages in the BMSC-EVs group. BMSC-EVs treatment also led to an increase in the expression of anti-inflammatory mediators (IL-10 and IL-4) at 2 weeks after surgery. Conclusions Overall, our findings show that the local administration of BMSC-EVs promotes tendon healing by suppressing inflammation and apoptotic cell accumulation and increasing the proportion of tendon-resident stem/progenitor cells. These findings provide a basis for the potential clinical use of BMSC-EVs in tendon repair.

Tendon tissue engineering strategies that recreate the biophysical and biochemical native microenvironment have a greater potential to achieve regeneration. Here, we developed tendon biomimetic scaffolds using mechanically competent yarns of poly-ε-caprolactone, chitosan and cellulose nanocrystals to recreate the inherent tendon hierarchy from the nano to macro scale. These were then coated with tropoelastin (TROPO) through polydopamine linking (PDA), to mimic the native extracellular matrix (ECM) composition and elasticity. Both PDA and TROPO coatings decreased surface stiffness without masking the underlying substrate. We found that human adipose-derived stem cells (hASCs) seeded onto these TROPO biomimetic scaffolds more rapidly acquired their spindle-shape morphology and high aspect ratio characteristic of tenocytes. Immunocytochemistry shows that the PDA and TROPO-coated surfaces boosted differentiation of hASCs towards the tenogenic lineage, with sustained expression of the tendon-related markers scleraxis and tenomodulin up to 21 days of culture. Furthermore, these surfaces enabled the deposition of a tendon-like ECM, supported by the expression of collagens type I and III, tenascin and decorin. Gene expression analysis revealed a downregulation of osteogenic and fibrosis markers in the presence of TROPO when compared with the control groups, suggesting proper ECM deposition. Remarkably, differentiated cells exposed to TROPO acquired an elastogenic profile due to the evident elastin synthesis and deposition, contributing to the formation of a more mimetic matrix in comparison with the PDA-coated and uncoated conditions. In summary, our biomimetic substrates combining biophysical and biological cues modulate stem cell behavior potentiating their long-term tenogenic commitment and the production of an elastin-rich ECM., European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 706996, Teaming grant agreement No 739572 – The Discoveries CTR, European Research Council grant agreement No 726178, and ERA Chair grant agreement No 668983 - FORECAST; Biomedical Engineering Australian Mobility (BEAM) Program – Master Joint Mobility Project between EU Commission Australian Government; Fundação para a Ciência e a Tecnologia (FCT) for Post-Doc grant SFRH/BPD/112459/2015 and project SmarTendon (PTDC/NAN-MAT/30595/2017); Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund for NORTE-01-0145-FEDER-000021

Background. Cellular therapy is proposed for tendinopathy treatment. Bone marrow- (BM-MSC) and adipose tissue- (ASC) derived mesenchymal stromal cells are candidate populations for such a therapy. The first aim of the study was to compare human BM-MSCs and ASCs for their basal expression of factors associated with tenogenesis as well as chemotaxis. The additional aim was to evaluate if the donor age influences these features. Methods. Cells were isolated from 24 human donors, 8 for each group: hASC, hBM-MSC Y (age≤45), and hBM-MSC A (age>45). The microarray analysis was performed on RNA isolated from hASC and hBM-MSC A cells. Based on microarray results, 8 factors were chosen for further evaluation. Two genes were additionally included in the analysis: SCLERAXIS and PPARγ. All these 10 factors were tested for gene expression by the qRT-PCR method, and all except of RUNX2 were additionally evaluated for protein expression or secretion. Results. Microarray analysis showed over 1,400 genes with a significantly different expression between hASC and hBM-MSC groups. Eight of these genes were selected for further analysis: CXCL6, CXCL12, CXCL16, TGF-β2, SMAD3, COLLAGEN 14A1, MOHAWK, and RUNX2. In the subsequent qRT-PCR analysis, hBM-MSCs showed a significantly higher expression than did hASCs in following genes: CXCL12, CXCL16, TGF-β2, SMAD3, COLLAGEN 14A1, and SCLERAXIS (p<0.05, regardless of BM donor age). In the case of CXCL12, the difference between hASC and hBM-MSC was significant only for younger BM donors, whereas for COLLAGEN 14A1—only for elder BM donors. PPARγ displayed a higher expression in hASCs compared to hBM-MSCs. In regard to CXCL6, MOHAWK, and RUNX2 gene expression, no statistically significant differences between groups were observed. Conclusions. In the context of cell-based therapy for tendinopathies, bone marrow appears to be a more attractive source of MSCs than does adipose tissue. The age of cell donors seems to be less important than cell source, although cells from elder donors show slightly higher basal tenogenic potential than do cells from younger donors.

Tendinopathy is a common and disabling condition that is difficult to treat. The pathomolecular events behind tendinopathy remain uncertain. Micro-RNAs (miRNAs, miRs) are short non-coding RNAs that regulate gene expression and may play a role in tendinopathy development. Tenocytes were obtained from human patellar tendons in patients undergoing anterior cruciate ligament (ACL) reconstruction. Micro-RNA mimics and antagomirs for miR-30d, 26a, and 29a were separately transfected into tenocyte culture. Gene expression for scleraxis, collagen 1 alpha 1 (COL1A1), collagen 3 alpha 1 (COL3A1), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), bone morphogenic protein 2 (BMP2), bone morphogenic protein 12 (BMP12), and osteocalcin was determined for each miRNA mimic and antagomir transfection using real-time quantitative PCR (qPCR). The results showed that exogenous miR-29a downregulated BMP2 and BMP12, while miR-26a and miR-30d did not have a significant effect on tenocyte gene expression. These findings suggest miR-29a contributes to tendon homeostasis and can serve as a potential therapeutic target in treating tendinopathy.

Interleukin 1β (IL-1β) is upregulated following tendon injury. Here we demonstrate that in adult and fetal tenocytes IL-1β increases the expression of matrix metalloproteinases, tenascin-C and Sox9 and decreases the expression of scleraxis and cartilage oligomeric matrix protein. When cultured in 3-dimensional collagen gels adult and fetal tenocytes exposed to IL-1β have reduced contraction ability and generate tendon-like constructs with a lower storage modulus. In contrast, equine embryonic stem cell (ESC) derived tenocytes exposed to IL-1β exhibit no changes in gene expression and generate identical tendon-like constructs. We propose that ESC-derived tenocytes do not respond to IL-1β due to their low expression of interleukin 1 (IL-1) receptor 1 and high expression of the decoy receptor IL-1 receptor 2 and IL-1 receptor antagonist protein (IL1Ra). This may make ESC-derived tenocytes an advantageous source of cells for tissue regeneration and allow the development of novel pharmaceutical interventions to protect endogenous cells from inflammation.

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.

Tenogenic differentiation of stem cells is needed for tendon tissue engineering approaches. A current challenge is the limited information on the cellular-level changes during tenogenic induction. Tendon cells in embryonic and adult tendons possess an array of cell-cell junction proteins that include cadherins and connexins, but how these proteins are impacted by tenogenic differentiation is unknown. Our objective was to explore how tenogenic induction of mesenchymal stem cells (MSCs) using the transforming growth factor (TGF)β2 impacted protein markers of tendon differentiation and protein levels of N-cadherin, cadherin-11 and connexin-43. MSCs were treated with TGFβ2 for 21 days. At 3 days, TGFβ2-treated MSCs developed a fibroblastic morphology and significantly decreased levels of N-cadherin protein, which were maintained through 21 days. Similar decreases in protein levels were found for cadherin-11. Connexin-43 protein levels significantly increased at 3 days, but then decreased below control levels, though not significantly. Protein levels of scleraxis and tenomodulin were significantly increased at day 14 and 21, respectively. Taken together, our results indicate that TGFβ2 is an inducer of tendon marker proteins (scleraxis and tenomodulin) in MSCs and that tenogenesis alters the protein levels of N-cadherin, cadherin-11 and connexin-43. These findings suggest a role for connexin-43 early in tenogenesis, and show that early-onset and sustained decreases in N-cadherin and cadherin-11 may be novel markers of tenogenesis in MSCs.

Proper development of tendons is crucial for the integration and function of the musculoskeletal system. Currently little is known about the molecular mechanisms controlling tendon development and tendon cell differentiation. The transcription factor Scleraxis (Scx) is expressed throughout tendon development and plays essential roles in both embryonic tendon development and adult tendon healing, but few direct target genes of Scx in tendon development have been reported and genome-wide identification of Scx direct target genes in vivo has been lacking. In this study, we have generated a ScxFlag knockin mouse strain, which produces fully functional endogenous Scx proteins containing a 2xFLAG epitope tag at the carboxy terminus. We mapped the genome-wide Scx binding sites in the developing limb tendon tissues, identifying 12,097 high quality Scx regulatory cis-elements in-around 7,520 genes. Comparative analysis with previously reported embryonic tendon cell RNA-seq data identified 490 candidate Scx direct target genes in early tendon development. Furthermore, we characterized a new Scx gene-knockout mouse line and performed whole transcriptome RNA sequencing analysis of E15.5 forelimb tendon cells from Scx–/– embryos and control littermates, identifying 68 genes whose expression in the developing tendon tissues significantly depended on Scx function. Combined analysis of the ChIP-seq and RNA-seq data yielded 32 direct target genes that required Scx for activation and an additional 17 target genes whose expression was suppressed by Scx during early tendon development. We further analyzed and validated Scx-dependent tendon-specific expression patterns of a subset of the target genes, including Fmod, Kera, Htra3, Ssc5d, Tnmd, and Zfp185, by in situ hybridization and real-time quantitative polymerase chain reaction assays. These results provide novel insights into the molecular mechanisms mediating Scx function in tendon development and homeostasis. The ChIP-seq and RNA-seq data provide a rich resource for aiding design of further studies of the mechanisms regulating tendon cell differentiation and tendon tissue regeneration. The ScxFlag mice provide a valuable new tool for unraveling the molecular mechanisms involving Scx in the protein interaction and gene-regulatory networks underlying many developmental and disease processes.

Despite the requirement forScleraxis-lineage (ScxLin) cells during tendon development, the function of ScxLincells during adult tendon repair, post-natal growth, and adult homeostasis have not been defined. Therefore, we inducibly depleted ScxLincells (ScxLinDTR) prior to tendon injury and repair surgery and hypothesized that ScxLinDTRmice would exhibit functionally deficient healing compared to wild-type littermates. Surprisingly, depletion of ScxLincells resulted in increased biomechanical properties without impairments in gliding function at 28 days post-repair, indicative of regeneration. RNA sequencing of day 28 post-repair tendons highlighted differences in matrix-related genes, cell motility, cytoskeletal organization, and metabolism. We also utilized ScxLinDTRmice to define the effects on post-natal tendon growth and adult tendon homeostasis and discovered that adult ScxLincell depletion resulted in altered tendon collagen fibril diameter, density, and dispersion. Collectively, these findings enhance our fundamental understanding of tendon cell localization, function, and fate during healing, growth, and homeostasis.

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

Meniscus tears common injuries of the knee joint and a major osteoarthritis (OA) risk factor. Knowledge gaps that limit development of therapeutic approaches for meniscus injury and degeneration concern transcription factors that control the meniscus cell phenotype. Analysis of RNA-sequencing data from 37 human tissues in the Genotype-Tissue Expression (GTEx) database and RNA-sequencing data from meniscus and articular cartilage showed that transcription factor Mohawk (MKX) is highly enriched in meniscus. In human meniscus cells, MKX regulates expression of meniscus marker genes, OA-related genes and other transcription factors, including Scleraxis (SCX), SRY Box 5 (SOX5), and Runt domain-related transcription factor 2 (RUNX2). In mesenchymal stem cells, the combination of adenoviral MKX (Ad-MKX) and Transforming growth factor-β3 (TGF-β3) induced a meniscus cell phenotype. When Ad-MKX-transduced mesenchymal stem cells (MSCs) were seeded on TGF-β3-conjugated decellularized meniscus scaffold (DMS) and inserted into experimental tears in meniscus explants, they increased glycosaminoglycan content, extracellular matrix interconnectivity, cell infiltration into the DMS and improved biomechanical properties. Ad-MKX injection into mouse knee joints with experimental OA induced by surgical destabilization of the meniscus suppressed meniscus and cartilage damage, reducing OA severity. Ad-MKX injection into human OA meniscus tissue explants corrected pathogenic gene expression. These results identify MKX as a previously unidentified key transcription factor that regulates the meniscus cell phenotype. The combination of Ad-MKX with TGF-β3 is effective for differentiation of MSCs to a meniscus cell phenotype and useful for meniscus repair. MKX is a promising therapeutic target for meniscus tissue engineering, repair and prevention of OA.

Although being of utmost importance for human health and mobility, stem cell identity and hierarchical organization of musculoskeletal progenitors remain largely unexplored. Here, cells from E10.5, E12.5, and E15.5 murine limbs are analyzed by high throughput single‐cell RNA sequencing to illustrate the cellular architecture during limb development. Single‐cell transcriptional profiling demonstrates the identity and differentiation architecture of musculoskeletal stem cells (MSSC), soft and hard tissue progenitors through expression pattern of musculoskeletal markers (scleraxis [Scx], Hoxd13, Sox9, and Col1a1). This is confirmed by genetic in vivo lineage tracing. Moreover, single‐cell analyses of Scx knockout mice tissues illustrates that Scx regulates MSSC self‐renewal and proliferation potential. A high‐throughput and low‐cost multi‐tissues RNA sequencing strategy further provides evidence that musculoskeletal system tissues, including muscle, bone, meniscus, and cartilage, are all abnormally developed in Scx knockout mice. These results establish the presence of an indispensable limb Scx+Hoxd13+ MSSC population and their differentiation into soft tissue progenitors (Scx+Col1a1+) and hard tissue progenitors (Scx+Sox9+). Collectively, this study paves the way for systematically decoding the complex molecular mechanisms and cellular programs of musculoskeletal tissues morphogenesis in limb development and regeneration., This study presents a comprehensive genomics resource that reveals the transcriptome dynamics of the developing limb. Single‐cell transcriptional profiling with lineage tracing data demonstrates the identity and differentiation trajectory of musculoskeletal stem cells into soft and hard tissues. These results emphasize an indispensable role of limb Scx+ musculoskeletal stem cell population and provide additional perspective on musculoskeletal biology.

Angiotensin II (Ang II) is a primary mediator of profibrotic signaling in the heart and more specifically, the cardiac fibroblast. Ang II-mediated cardiomyocyte hypertrophy in combination with cardiac fibroblast proliferation, activation, and extracellular matrix production compromise cardiac function and increase mortality in humans. Profibrotic actions of Ang II are mediated by increasing production of fibrogenic mediators (e.g. transforming growth factor beta, scleraxis, osteopontin, and periostin), recruitment of immune cells, and via increased reactive oxygen species generation. As a result, drugs that inhibit Ang II production or action, collectively referred to as renin angiotensin system (RAS) inhibitors, are first line therapeutics for heart failure. Moreover, transient RAS inhibition has been found to persistently alter hypertensive cardiac fibroblast responses to injury providing a useful tool to identify novel therapeutic targets. This review summarizes the profibrotic actions of Ang II and the known impact of RAS inhibition on cardiac fibroblast phenotype and cardiac remodeling.

The tenocyte niche contains biochemical and biophysical signals that are needed for tendon homeostasis. The tenocyte phenotype is correlated with cell shapein vivoandin vitro, and shape-modifying cues are needed for tenocyte phenotypical maintenance. Indeed, cell shape changes from elongated to spread when cultured on a flat surface, and rat tenocytes lose the expression of phenotypical markers throughout five passages. We hypothesized that tendon gene expression can be preserved by culturing cells in the native tendon shape. To this end, we reproduced the tendon topographical landscape into tissue culture polystyrene, using imprinting technology. We confirmed that the imprints forced the cells into a more elongated shape, which correlated with the level of Scleraxis expression. When we cultured the tenocytes for seven days on flat surfaces and tendon imprints, we observed a decline in tenogenic marker expression on flat but not on imprints. This research demonstrates that native tendon topography is an important factor contributing to the tenocyte phenotype. Tendon imprints therefore provide a powerful platform to explore the effect of instructive cues originating from native tendon topography on guiding cell shape, phenotype and function of tendon-related cells.

Growth of the musculoskeletal system requires precise coordination between bone, muscle, and tendon during development. Insufficient elongation of the muscle-tendon unit relative to bone growth results in joint contracture, a condition characterized by reduction or complete loss of joint range of motion. Here we establish a novel murine model of joint contracture by targeting Smad4 for deletion in the tendon cell lineage using Scleraxis-Cre (ScxCre). Smad4ScxCre mutants develop a joint contracture shortly after birth. The contracture is stochastic in direction and increases in severity with age. Smad4ScxCre mutant tendons exhibited a stable reduction in cellularity and a progressive reduction in extracellular matrix volume. Collagen fibril diameters were reduced in the Smad4ScxCre mutants, suggesting a role for Smad4 signaling in the regulation of matrix accumulation. Although ScxCre also has sporadic activity in both cartilage and muscle, we demonstrate an essential role for Smad4 loss in tendons for the development of joint contractures. Disrupting the canonical TGFβ-pathway in Smad2;3ScxCre mutants did not result in joint contractures. Conversely, disrupting the BMP pathway by targeting BMP receptors (Alk3ScxCre/Alk6null) recapitulated many features of the Smad4ScxCre contracture phenotype, suggesting that joint contracture in Smad4ScxCre mutants is caused by disruption of BMP signaling. Overall, these results establish a model of murine postnatal joint contracture and a role for BMP signaling in tendon elongation and extracellular matrix accumulation.

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues., Highlighted Article: Analysis of conditional and temporal knockout mice reveals roles of Ebf3 in the differentiation of lateral plate mesenchymal cells towards pre-osteoblasts during sternum ossification at the boundary of the lateral and somitic mesoderm.

Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting tenocyte proliferation and matrix synthesis during embryonic tendon development. However, the role of scleraxis in the growth and adaptation of adult tendons is not known. We hypothesized that scleraxis is required for tendon growth in response to mechanical loading, and that scleraxis promotes the specification of progenitor cells into tenocytes. We conditionally deleted scleraxis in adult mice using a tamoxifen-inducible Cre-recombinase expressed from the Rosa26 locus (ScxΔ), and then induced tendon growth in Scx+ and ScxΔ adult mice via plantaris tendon mechanical overload. Compared to the wild type Scx+ group, ScxΔ mice demonstrated blunted tendon growth. Transcriptional and proteomic analyses revealed significant reductions in cell proliferation, protein synthesis, and extracellular matrix genes and proteins. Our results indicate that scleraxis is required for mechanically-stimulated adult tendon growth by causing the commitment of CD146+ pericytes into the tenogenic lineage, and by promoting the initial expansion of newly committed tenocytes and the production of extracellular matrix proteins.

Background In order to optimize the tenogenic differentiation of mesenchymal stem cells (MSCs), researchers should consider various factors. However, this requires testing numerous experimental settings, which is costly and time-consuming. We aimed to assess the differential effects of transforming growth factor beta-3 (TGF-β3) on the tenogenesis of tonsil-derived MSCs (T-MSCs) and bone marrow-derived MSCs (BM-MSCs) using response surface methodology (RSM). Methods Bone marrow and tonsillar tissue were collected from four patients; mononuclear cells were separated and treated with 5 or 10 ng/mL of TGF-β3. A full factorial experimental design with a categorical factor of 0 was employed to study the effect of tension based on T-MSCs. Eighty-four trials were fitted with RSM and then used to obtain mathematical prediction models. Results Exposure of T-MSCs and BM-MSCs to TGF-β3 increased the expression of scleraxis (SCX), tenomodulin (TNMD), decorin, collagen I, and tenascin C. Expression of most of these factors reached a maximum after 2–3 days of treatment. The model predicted that the values of the tenocyte lineage-related factors assessed would be significantly increased at 2.5 days of culture with 2.7 ng/mL of TGF-β3 for T-MSCs and at 2.3 days of culture regardless of TGF-β3 concentration for BM-MSCs. Conclusions This study demonstrated that the RSM prediction of the culture time necessary for the tenogenic differentiation of T-MSCs and BM-MSCs under TGF-β3 stimulation was similar to the experimentally determined time of peak expression of tenocyte-related mRNAs, suggesting the potential of using the RSM approach for optimization of the culture protocol for tenogenesis of MSCs.

Integrity of the musculoskeletal system is essential for the transfer of muscular contraction force to the associated bones. Tendons and skeletal muscles intertwine, but on a cellular level, the myotendinous junctions (MTJs) display a sharp transition zone with a highly specific molecular adaption. The function of MTJs could go beyond a mere structural role and might include homeostasis of this musculoskeletal tissue compound, thus also being involved in skeletal muscle regeneration. Repair processes recapitulate several developmental mechanisms, and as myotendinous interaction does occur already during development, MTJs could likewise contribute to muscle regeneration. Recent studies identified tendon-related, scleraxis-expressing cells that reside in close proximity to the MTJs and the muscle belly. As the muscle-specific function of these scleraxis positive cells is unknown, we compared the influence of two immortalized mesenchymal stem cell (MSC) lines&mdash, differing only by the overexpression of scleraxis&mdash, on myoblasts morphology, metabolism, migration, fusion, and alignment. Our results revealed a significant increase in myoblast fusion and metabolic activity when exposed to the secretome derived from scleraxis-overexpressing MSCs. However, we found no significant changes in myoblast migration and myofiber alignment. Further analysis of differentially expressed genes between native MSCs and scleraxis-overexpressing MSCs by RNA sequencing unraveled potential candidate genes, i.e., extracellular matrix (ECM) proteins, transmembrane receptors, or proteases that might enhance myoblast fusion. Our results suggest that musculotendinous interaction is essential for the development and healing of skeletal muscles.

Tendons are dense fibrous structures that attach muscles to bones. Healing of tendon injuries is a clinical challenge owing to poor regenerative potential and scarring. Here, we created reporter mice that express EGFP, driven by the promoter of the tendon-specific Scleraxis (Scx) transcription-factor gene; we then generated induced pluripotent stem cells (iPSCs) from these mice. Utilising these fluorescently labelled iPSCs, we developed a tenogenic differentiation protocol. The iPSC-derived EGFP-positive cells exhibited elevated expression of tendon-specific genes, including Scx, Mohawk, Tenomodulin, and Fibromodulin, indicating that they have tenocyte-like properties. Finally, we demonstrated that these cells promoted tendon regeneration in mice after transplantation into injured tendons reducing scar formation via paracrine effect. Our data demonstrate that the tenogenic differentiation protocol successfully provided functional cells from iPSCs. We propose that pluripotent stem cell-based therapy using this protocol will provide an effective therapeutic approach for tendon injuries.

Spatiotemporal mechanobiology resulting in penile pathologies continues to be investigated using small scale animals models such as mice. However, species-dependent functional biomechanics of a mouse penis, is not known. In this study, spatial mapping of a mechanosensitive transcription factor, scleraxis (Scx), at ages 4, 5, 6 weeks, and 1 year were generated to identify mechanoactive regions within penile tissues. Reconstructed volumes of baculum collected using micro X-ray computed tomography illustrated significantly increased baculum length with decreased porosity, and increased mineral density (p

Despite the requirement forScleraxis-lineage (ScxLin) cells during tendon development, the function of ScxLincells during adult tendon repair, post-natal growth, and adult homeostasis have not been defined. Therefore, we inducibly depleted ScxLincells (ScxLinDTR) prior to tendon injury and repair surgery and hypothesized that ScxLinDTRmice would exhibit functionally deficient healing compared to wildtype littermates. Surprisingly, depletion of ScxLincells resulted in increased biomechanical properties without impairments in gliding function at 28 days post-repair, indicative of regeneration. RNA sequencing of day 28 post-repair tendons highlighted differences in matrix-related genes, cell motility, cytoskeletal organization, and metabolism. We also utilized ScxLinDTRmice to define the effects on post-natal tendon growth and adult tendon homeostasis and discovered that adult ScxLincell depletion resulted in altered tendon collagen fibril diameter, density, and dispersion. Collectively, these findings enhance our fundamental understanding of tendon cell localization, function, and fate during healing, growth, and homeostasis.

It has been suggested that mechanical strain may elicit cell differentiation in adult somatic cells through activation of epithelial sodium channels (ENaC). However, such phenomenon has not been previously demonstrated in mesenchymal stromal cells (MSCs). The present study was thus conducted to investigate the role of ENaC in human bone marrow-derived MSCs (hMSCs) tenogenic differentiation during uniaxial tensile loading. Passaged-2 hMSCs were seeded onto silicone chambers coated with collagen I and subjected to stretching at 1 Hz frequency and 8% strain for 6, 24, 48, and 72 hours. Analyses at these time points included cell morphology and alignment observation, immunocytochemistry and immunofluorescence staining (collagen I, collagen III, fibronectin, and N-cadherin), and gene expression (ENaC subunits, and tenogenic markers). Unstrained cells at similar time points served as the control group. To demonstrate the involvement of ENaC in the differentiation process, an ENaC blocker (benzamil) was used and the results were compared to the noninhibited hMSCs. ENaC subunits’ (α,β,γ, andδ) expression was observed in hMSCs, although onlyαsubunit was significantly increased during stretching. An increase in tenogenic genes’ (collagen1,collagen3,decorin,tenascin-c,scleraxis, andtenomodulin) and proteins’ (collagen I, collagen III, fibronectin, and N-cadherin) expression suggests that hMSCs underwent tenogenic differentiation when subjected to uniaxial loading. Inhibition of ENaC function resulted in decreased expression of these markers, thereby suggesting that ENaC plays a vital role in tenogenic differentiation of hMSCs during mechanical loading.

One of the main challenges relating to tendons is to understand the regulators of the tendon differentiation program. The optimum culture conditions that favor tendon cell differentiation have not been identified. Mesenchymal stem cells present the ability to differentiate into multiple lineages in cultures under different cues ranging from chemical treatment to physical constraints. We analyzed the tendon differentiation potential of C3H10T1/2 cells, a murine cell line of mesenchymal stem cells, upon different 2D- and 3D-culture conditions. We observed that C3H10T1/2 cells cultured in 2D conditions on silicone substrate were more prone to tendon differentiation, assessed with the expression of the tendon markers Scx, Col1a1 and Tnmd as compared to cells cultured on plastic substrate. The 3D-fibrin environment was more favorable for Scx and Col1a1 expression compared to 2D cultures. We also identified TGFβ2 as a negative regulator of Tnmd expression in C3H10T1/2 cells in 2D and 3D cultures. Altogether, our results provide us with a better understanding of the culture conditions that promote tendon gene expression and identify mechanical and molecular parameters upon which we could act to define the optimum culture conditions that favor tenogenic differentiation in mesenchymal stem cells., Summary: Our results provide a better understanding of the culture conditions upon which we could act to trigger tendon cell differentiation from undifferentiated stem cells.

Digits shape is sculpted by interdigital programmed cell death during limb development. Here, we show that DNA breakage in the periphery of 5-methylcytosine nuclei foci of interdigital precursors precedes cell death. These cells showed higher genome instability than the digit-forming precursors when exposed to X-ray irradiation or local bone morphogenetic protein (BMP) treatments. Regional but not global DNA methylation differences were found between both progenitors. DNA-Methyl-Transferases (DNMTs) including DNMT1, DNMT3B and, to a lesser extent, DNMT3A, exhibited well-defined expression patterns in regions destined to degenerate, as the interdigital tissue and the prospective joint regions. Dnmt3b functional experiments revealed an inverse regulation of cell death and cartilage differentiation, by transcriptional regulation of key genes including Sox9, Scleraxis, p21 and Bak1, via differential methylation of CpG islands across their promoters. Our findings point to a regulation of cell death versus chondrogenesis of limb skeletal precursors based on epigenetic mechanisms., Sanchez-Fernandez, Lorda-Diez et al. study a possible link between DNA methylation and interdigital cell death. They use irradiation experiments, methylation, and mutagenic analyses to demonstrate that Dnmt3b inversely regulates cell death and cartilage differentiation. Their work provides insights on the regulation of apoptosis in embryonic limbs.

Age-related degenerative changes in tendon tissue represent a common cause for acute tendon pathologies. Although the regenerative potential of multipotent mesenchymal stromal cells (MSC) was reported to restore functionality in injured tendon tissue, cellular mechanisms of action remain partly unclear. Potential tenogenic differentiation of applied MSC is affected by various intrinsic and extrinsic factors. The current study presents an in vitro model to evaluate the combined extrinsic effects of decellularized equine tendon matrix, transforming growth factor beta 3 (TGFβ3) and bone morphogenetic protein 12 (BMP12) on the tenogenic fate of equine adipose tissue-derived MSC. Monolayer MSC cultures supplemented with TGFβ3 and BMP12 as well as MSC cultured on tendon matrix scaffolds preloaded with the growth factors were incubated for 3 and 5 days. Histological evaluation and real time reverse transcription polymerase chain reaction (RT-PCR) revealed that growth factor-mediated tenogenic induction of MSC was modified by the conditions of the surrounding microenvironment. While the gene expression pattern in monolayer cultures supplemented with TGFβ3 or TGFβ3 and BMP12 revealed an upregulation for collagen 1A2, collagen 3A1, tenascin c, scleraxis and mohawk ( p < 0.05 ), the presence of tendon matrix led to an upregulation of decorin and osteopontin as well as to a downregulation of smad8 ( p < 0.05). Preloading of scaffolds with either TGFβ3, or with TGFβ3 and BMP12 promoted a tenocyte-like phenotype and improved cell alignment. Furthermore, gene expression in scaffold culture was modulated by TGFβ3 and/or BMP12, with downregulation of collagen 1A2, collagen 3A1, decorin, scleraxis, smad8 and osteopontin, whereas gene expression of tenascin c was increased. This study shows that growth factor-induced tenogenic differentiation of equine MSC is markedly altered by topographical constraints of decellularized tendon tissue in vitro. While TGFβ3 represents an effective mediator for tenogenic induction, the role of BMP12 in tenogenesis may be of modulatory character and needs further evaluation.

Tendons and ligaments are connective tissues that have been comparatively less studied than muscle and cartilage/bone, even though they are crucial for proper function of the musculoskeletal system. In tendon biology, considerable progress has been made in identifying tendon-specific genes (Scleraxis, Mohawk, and Tenomodulin) in the past decade. However, besides tendon function and the knowledge of a small number of important players in tendon biology, neither the ontogeny of the tenogenic lineage nor signaling cascades have been fully understood. This results in major drawbacks in treatment and repair options following tendon degeneration. In this review, we have systematically evaluated publications describing tendon-related genes, which were studied in depth and characterized by using knockout technologies and the subsequently generated transgenic mouse models (Tg) (knockout mice, KO). We report in a tabular manner, that from a total of 24 tendon-related genes, in 22 of the respective knockout mouse models, phenotypic changes were detected. Additionally, in some of the models it was described at which developmental stages these changes appeared and progressed. To summarize, only loss of Scleraxis and TGFβ signaling led to severe tendon developmental phenotypes, while mice deficient for various proteoglycans, Mohawk, EGR1 and 2, and Tenomodulin presented mild phenotypes. These data suggest that the tendon developmental system is well organized, orchestrated, and backed up; this is even more evident among the members of the proteoglycan family, where the compensatory effects are much clearer. In future, it will be of great importance to discover additional master tendon transcription factors and the genes that play crucial roles in tendon development. This would improve our understanding of the genetic makeup of tendons, and will increase the chances of generating tendon-specific drugs to advance overall treatment strategies.

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

One of the main challenges in tendon field relies in the understanding of regulators of the tendon differentiation program. The optimum culture conditions that favor tendon cell differentiation are not identified. Mesenchymal stem cells present the ability to differentiate into multiple lineages in cultures under different cues ranging from chemical treatment to physical constraints. We analyzed the tendon differentiation potential of C3H10T1/2 cells, a murine cell line of mesenchymal stem cells, upon different 2D- and 3D-culture conditions. We observed that C3H10T1/2 cells cultured in 2D conditions on silicone substrate were more prone to tendon differentiation assessed with the expression of the tendon markersScx, Col1a1andTnmdas compared to cells cultured on plastic substrate. 3D fibrin environment was more favorable forScxandCol1a1expression compared to 2D-cultures. We also identified TGFβ2 as a negative regulator ofTnmdexpression in C3H10T1/2 cells in 2D- and 3D-cultures. Altogether, our results provide us with a better understanding of the culture conditions that promote tendon gene expression and identify mechanical and molecular parameters on which we could play to define the optimum culture conditions that favor tenogenic differentiation in mesenchymal stem cells.

Remodeling of the cardiac extracellular matrix is responsible for a number of the detrimental effects on heart function that arise secondary to hypertension, diabetes and myocardial infarction. This remodeling consists both of an increase in new matrix protein synthesis, and an increase in the expression of matrix metalloproteinases (MMPs) that degrade existing matrix structures. Previous studies utilizing knockout mice have demonstrated clearly that MMP2 plays a pathogenic role during matrix remodeling, thus it is important to understand the mechanisms that regulate MMP2 gene expression. We have shown that the transcription factor scleraxis is an important inducer of extracellular matrix gene expression in the heart that may also control MMP2 expression. In the present study, we demonstrate that scleraxis directly transactivates the proximal MMP2 gene promoter, resulting in increased histone acetylation, and identify a specific E-box sequence in the promoter to which scleraxis binds. Cardiac myo-fibroblasts isolated from scleraxis knockout mice exhibited dramatically decreased MMP2 expression; however, scleraxis over-expression in knockout cells could rescue this loss. We further show that regulation of MMP2 gene expression by the pro-fibrotic cytokine TGFβ occurs via a scleraxis-dependent mechanism: TGFβ induces recruitment of scleraxis to the MMP2 promoter, and TGFβ was unable to up-regulate MMP2 expression in cells lacking scleraxis due to either gene knockdown or knockout. These results reveal that scleraxis can exert control over both extracellular matrix synthesis and breakdown, and thus may contribute to matrix remodeling in wound healing and disease.

Bone marrow mesenchymal stem cells (BMSCs) are pluripotent cells, which have the capacity to differentiate into various types of mesenchymal cell phenotypes, including osteoblasts, chondroblasts, myoblasts and tendon fibroblasts (TFs). The molecular mechanism for tenogenic differentiation of BMSCs is still unknown. The present study investigated the effects of bone morphogenetic protein (BMP) 14 on BMSC differentiation in vitro. It was revealed that BMP14 significantly increased the expression of tendon markers (scleraxis and tenomodulin) at the mRNA and protein level, which led to the upregulation of sirtuin 1 (Sirt1) expression. The gain or loss of Sirt1 function may promote or inhibit tenogenic differentiation by deacetylating the peroxisome proliferator-activated receptor (PPAR)-γ. BMP14 also triggered the phosphorylation of c-Jun N-terminal kinase (JNK) and Smad1; overexpression of Sirt1 significantly increased the phosphorylation and knockdown of Sirt1 significantly decreased the phosphorylation. The inhibition of JNK and Smad significantly increased the acetylation of PPARγ and inhibited the expression of tenogenic differentiation markers. These results suggest that BMP14 may induce the tenogenic differentiation of BMSCs via the Sirt1-JNK/Smad1-PPARγ signaling pathway. The present study provided a cellular and molecular basis for the development of novel therapeutic strategies for tendon healing.

Tendon is a dense connective tissue that transmits high mechanical forces from skeletal muscle to bone. The transcription factor scleraxis (Scx) is a highly specific marker of both precursor and mature tendon cells (tenocytes). Mice lacking scx exhibit a specific and virtually complete loss of tendons during development. However, the functional contribution of Scx to wound healing in adult tendon has not yet been fully characterized. Here, using ScxGFP-tracking and loss-of-function systems, we show in an adult mouse model of Achilles tendon injury that paratenon cells, representing a stem cell antigen-1 (Sca-1)–positive and Scx-negative progenitor subpopulation, display Scx induction, migrate to the wound site, and produce extracellular matrix (ECM) to bridge the defect, whereas resident tenocytes exhibit a delayed response. Scx induction in the progenitors is initiated by transforming growth factor β (TGF-β) signaling. scx-deficient mice had migration of Sca-1–positive progenitor cell to the lesion site but impaired ECM assembly to bridge the defect. Mechanistically, scx-null progenitors displayed higher chondrogenic potential with up-regulation of SRY-box 9 (Sox9) coactivator PPAR-γ coactivator-1α (PGC-1α) in vitro, and knock-in analysis revealed that forced expression of full-length scx significantly inhibited Sox9 expression. Accordingly, scx-null wounds formed cartilage-like tissues that developed ectopic ossification. Our findings indicate a critical role of Scx in a progenitor-cell lineage in wound healing of adult mouse tendon. These progenitor cells could represent targets in strategies to facilitate tendon repair. We propose that this lineage-regulatory mechanism in tissue progenitors could apply to a broader set of tissues or biological systems in the body.

Changes in cell phenotype underlie many of the body's responses to altered environmental conditions, stress, or damage, resulting in both physiological and pathophysiological alterations to tissue function. Transcriptional regulators play a critical role in reprogramming cell identity and behavior. Emerging evidence has implicated the transcription factor scleraxis as a novel and powerful determiner of cell phenotype in cells that produce large quantities of extracellular matrix, including tenocytes and cardiac fibroblasts. In this review, we examine the role of scleraxis in epithelial-to-mesenchymal transition and in altering cell phenotype, and discuss the implications of this role in tissue repair and pathology. This specialized role for scleraxis makes it a potential target for therapies aimed at improving wound healing or attenuating tissue fibrosis.

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal–regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active β1-integrin–containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp–mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5β1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.