Tenogenic Lineage Research Articles

Single Cell Transcriptomics-Informed Induced Pluripotent Stem Cells Differentiation to Tenogenic Lineage.

During vertebrate embryogenesis, axial tendons develop from the paraxial mesoderm and differentiate through specific developmental stages to reach the syndetome stage. While the main roles of signaling pathways in the earlier stages of the differentiation have been well established, pathway nuances in syndetome specification from the sclerotome stage have yet to be explored. Here, we show stepwise differentiation of human iPSCs to the syndetome stage using chemically defined media and small molecules that were modified based on single cell RNA-sequencing and pathway analysis. We identified a significant population of branching off-target cells differentiating towards a neural phenotype overexpressing Wnt. Further transcriptomics post-addition of a WNT inhibitor at the somite stage and onwards revealed not only total removal of the neural off-target cells, but also increased syndetome induction efficiency. Fine-tuning tendon differentiation in vitro is essential to address the current challenges in developing a successful cell-based tendon therapy.

Writing 3D In Vitro Models of Human Tendon within a Biomimetic Fibrillar Support Platform.

Tendinopathies are poorly understood diseases for which treatment remains challenging. Relevant in vitro models to study human tendon physiology and pathophysiology are therefore highly needed. Here we propose the automated 3D writing of tendon microphysiological systems (MPSs) embedded in a biomimetic fibrillar support platform based on cellulose nanocrystals (CNCs) self-assembly. Tendon decellularized extracellular matrix (dECM) was used to formulate bioinks that closely recapitulate the biochemical signature of tendon niche. A monoculture system recreating the cellular patterns and phenotype of the tendon core was first developed and characterized. This system was then incorporated with a vascular compartment to study the crosstalk between the two cell populations. The combined biophysical and biochemical cues of the printed pattern and dECM hydrogel were revealed to be effective in inducing human-adipose-derived stem cells (hASCs) differentiation toward the tenogenic lineage. In the multicellular system, chemotactic effects promoted endothelial cells migration toward the direction of the tendon core compartment, while the established cellular crosstalk boosted hASCs tenogenesis, emulating the tendon development stages. Overall, the proposed concept is a promising strategy for the automated fabrication of humanized organotypic tendon-on-chip models that will be a valuable new tool for the study of tendon physiology and pathogenesis mechanisms and for testing new tendinopathy treatments.

Mesenchymal Stem Cell Use in Acute Tendon Injury: In Vitro Tenogenic Potential vs. In Vivo Dose Response.

Stem cell therapy for the treatment of tendon injury is an emerging clinical practice in the fields of human and veterinary sports medicine; however, the therapeutic benefit of intralesional transplantation of mesenchymal stem cells in tendonitis cases is not well designed. Questions persist regarding the overall tenogenic potential and efficacy of this treatment alone. In this study, we aimed to isolate a rat mesenchymal stem cell lineage for in vitro and in vivo use, to assess the effects of growth factor exposure in vitro on cell morphology, behavior, and tendon-associated glycoprotein production, and to assess the therapeutic potential of intralesional stem cells, as a function of dose, in vivo. First, rat adipose-derived (rAdMSC) and bone marrow-derived (rBMSC) stem cell lineages were isolated, characterized with flow cytometric analysis, and compared in terms of proliferation (MTS assay) and cellular viability (calcein AM staining). Rat AdMSCs displayed superior proliferation and more homogenous CD 73, CD 44H, and CD 90 expression as compared to rBMSC. Next, the tenogenic differentiation potential of the rAdMSC lineage was tested in vitro through isolated and combined stimulation with reported tenogenic growth factors, transforming growth factor (TGF)-β3 and connective tissue growth factor (CTGF). We found that the most effective tenogenic factor in terms of cellular morphologic change, cell alignment/orientation, sustained cellular viability, and tendon-associated glycoprotein upregulation was TGFβ3, and we confirmed that rAdMSC could be induced toward a tenogenic lineage in vitro. Finally, the therapeutic potential of rAdMSCs as a function of dose was assessed using a rat acute Achilles tendon injury model. Amounts of 5 × 105 (low dose) and 4 × 106 (high dose) were used. Subjectively, on the gross morphology, the rAdMSC-treated tendons exhibited fewer adhesions and less scar tissue than the control tendons; however, regardless of the rAdMSC dose, no significant differences in histological grade or tissue collagen I deposition were noted between the rAdMSC-treated and control tendons. Collectively, rAdMSCs exhibited appropriate stem cell markers and tenogenic potential in vitro, but the clinical efficacy of intralesional implantation of undifferentiated cells in acute tendonitis cases could not be proven. Further investigation into complementary therapeutics or specialized culture conditions prior to implantation are warranted.

Tenogenic Potential of Equine Fetal Mesenchymal Stem Cells Under The In Vitro Effect of Bone Morphogenetic Protein-12 (BMP-12)

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.

Fabrication and Plasma Surface Activation of Aligned Electrospun PLGA Fiber Fleeces with Improved Adhesion and Infiltration of Amniotic Epithelial Stem Cells Maintaining their Teno-inductive Potential.

Electrospun PLGA microfibers with adequate intrinsic physical features (fiber alignment and diameter) have been shown to boost teno-differentiation and may represent a promising solution for tendon tissue engineering. However, the hydrophobic properties of PLGA may be adjusted through specific treatments to improve cell biodisponibility. In this study, electrospun PLGA with highly aligned microfibers were cold atmospheric plasma (CAP)-treated by varying the treatment exposure time (30, 60, and 90 s) and the working distance (1.3 and 1.7 cm) and characterized by their physicochemical, mechanical and bioactive properties on ovine amniotic epithelial cells (oAECs). CAP improved the hydrophilic properties of the treated materials due to the incorporation of new oxygen polar functionalities on the microfibers’ surface especially when increasing treatment exposure time and lowering working distance. The mechanical properties, though, were affected by the treatment exposure time where the optimum performance was obtained after 60 s. Furthermore, CAP treatment did not alter oAECs’ biocompatibility and improved cell adhesion and infiltration onto the microfibers especially those treated from a distance of 1.3 cm. Moreover, teno-inductive potential of highly aligned PLGA electrospun microfibers was maintained. Indeed, cells cultured onto the untreated and CAP treated microfibers differentiated towards the tenogenic lineage expressing tenomodulin, a mature tendon marker, in their cytoplasm. In conclusion, CAP treatment on PLGA microfibers conducted at 1.3 cm working distance represent the optimum conditions to activate PLGA surface by improving their hydrophilicity and cell bio-responsiveness. Since for tendon tissue engineering purposes, both high cell adhesion and mechanical parameters are crucial, PLGA treated for 60 s at 1.3 cm was identified as the optimal construct.

Scleraxis is required for the growth of adult tendons in response to mechanical loading.

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 with the WT 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.

Electrospun PLGA Fiber Diameter and Alignment of Tendon Biomimetic Fleece Potentiate Tenogenic Differentiation and Immunomodulatory Function of Amniotic Epithelial Stem Cells.

Injured tendons are challenging in their regeneration; thus, tissue engineering represents a promising solution. This research tests the hypothesis that the response of amniotic epithelial stem cells (AECs) can be modulated by fiber diameter size of tendon biomimetic fleeces. Particularly, the effect of electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned microfibers possessing two different diameter sizes (1.27 and 2.5 µm: ha1- and ha2-PLGA, respectively) was tested on the ability of AECs to differentiate towards the tenogenic lineage by analyzing tendon related markers (Collagen type I: COL1 protein and mRNA Scleraxis: SCX, Tenomodulin: TNMD and COL1 gene expressions) and to modulate their immunomodulatory properties by investigating the pro- (IL-6 and IL-12) and anti- (IL-4 and IL-10) inflammatory cytokines. It was observed that fiber alignment and not fiber size influenced cell morphology determining the morphological change of AECs from cuboidal to fusiform tenocyte-like shape. Instead, fleece mechanical properties, cell proliferation, tenogenic differentiation, and immunomodulation were regulated by changing the ha-PLGA microfiber diameter size. Specifically, higher DNA quantity and better penetration within the fleece were found on ha2-PLGA, while ha1-PLGA fleeces with small fiber diameter size had better mechanical features and were more effective on AECs trans-differentiation towards the tenogenic lineage by significantly translating more efficiently SCX into the downstream effector TNMD. Moreover, the fiber diameter of 1.27 µm induced higher expression of pro-regenerative, anti-inflammatory interleukins mRNA expression (IL-4 and IL-10) with favorable IL-12/IL-10 ratio with respect to the fiber diameter of 2.5 µm. The obtained results demonstrate that fiber diameter is a key factor to be considered when designing tendon biomimetic fleece for tissue repair and provide new insights into the importance of controlling matrix parameters in enhancing cell differentiation and immunomodulation either for the cells functionalized within or for the transplanted host tissue.

Tendon Biomimetic Electrospun PLGA Fleeces Induce an Early Epithelial-Mesenchymal Transition and Tenogenic Differentiation on Amniotic Epithelial Stem Cells.

Background. The design of tendon biomimetic electrospun fleece with Amniotic Epithelial Stem Cells (AECs) that have shown a high tenogenic attitude may represent an alternative strategy to overcome the unsatisfactory results of conventional treatments in tendon regeneration. Methods. In this study, we evaluated AEC-engineered electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned fibers (ha-PLGA) that mimic tendon extracellular matrix, their biocompatibility, and differentiation towards the tenogenic lineage. PLGA fleeces with randomly distributed fibers (rd-PLGA) were generated as control. Results. Optimal cell infiltration and biocompatibility with both PLGA fleeces were shown. However, only ha-PLGA fleeces committed AECs towards an Epithelial-Mesenchymal Transition (EMT) after 48 h culture, inducing their cellular elongation along the fibers’ axis and the upregulation of mesenchymal markers. AECs further differentiated towards tenogenic lineage as confirmed by the up-regulation of tendon-related genes and Collagen Type 1 (COL1) protein expression that, after 28 days culture, appeared extracellularly distributed along the direction of ha-PLGA fibers. Moreover, long-term co-cultures of AEC-ha-PLGA bio-hybrids with fetal tendon explants significantly accelerated of half time AEC tenogenic differentiation compared to ha-PLGA fleeces cultured only with AECs. Conclusions. The fabricated tendon biomimetic ha-PLGA fleeces induce AEC tenogenesis through an early EMT, providing a potential tendon substitute for tendon engineering research.

Engineering the Tenogenic Niche In Vitro with Microenvironmental Tools

AbstractTendon injuries affect millions of people worldwide annually. Considering that traditional approaches (e.g., surgery, exercise, steroid injections) have failed to reinstate tendon function following injury, recent efforts have been directed toward the development of cell‐based tendon repair and regeneration therapies. However, tenocytes readily lose their phenotype in culture and implantation of naïve stem cells is often associated with ectopic bone formation. To this end, there has been a surge in efforts to engineer the optimal tenogenic niche in vitro that will either maintain the phenotype of tenocytes or direct other cell types toward tenogenic lineage. Mono‐domain biochemical (e.g., media supplements, substrate coatings, oxygen tension), biological (e.g., growth factors, co‐culture systems), or biophysical (topography, rigidity, mechanical stimulation, macromolecular crowding) cues that replicate the native tendon milieu have shown some level of success, but they have failed to provide a functional therapy. Considering the complexity of the native tissue microenvironment together with recent advances in chemistry, biology, and engineering, it is anticipated that in the years to come multifactorial approaches are likely to yield a functional cell‐based therapy for tendon repair and regeneration.

Tendon ECM modified bioactive electrospun fibers promote MSC tenogenic differentiation and tendon regeneration

Proper niche environment is the key factor for mesenchymal stem cells (MSCs) involved tendon-lineage differentiation and regeneration. In the present study, soluble tendon derived extracellular matrix (sTECM) was used to modify aligned electrospun fibers with acid neutralizing effect in order to create an optimal niche environment for tendon regeneration, and its efficacy was evaluated using mouse MSC tenogenic differentiation in vitro and rat Achilles tendon regeneration in vivo. The results showed that sTECM modified ultrafine fibers were more cytocompatible and capable of inducing a predominant tenogenic phenotype of mouse MSCs with the enhanced expression of tendon markers including scleraxis, tenomodulin, collagen III, mohawk homeobox, decorin, fibromodulin and biglycan. By contrast, pure sTECM components led to enhanced multi-lineage differentiation of mouse MSCs including tenogenic lineage. This inductive effect was further enhanced when unilateral mechanical loading was applied in vivo. Moreover, the implantation of sTECM modified scaffold created an ideal niche environment including bioactive sTECM components, topological and mechanical signals, upon in vivo implantation for in situ tendon regeneration. As the results, sTECM modified scaffold in comparison with the non-modified one regenerated higher quality tendon tissue with the features of more mature tissue structure, better tissue organization, cell density and alignment as well as better collagen ultrastructure. Quantitative analysis demonstrated significantly bigger collagen fibril diameter, stronger mechanical property and better tissue grading score and higher expression levels of tenogenic markers when compared to those of control scaffold (p < 0.05). This study demonstrated that sTECM modified pH-neutral ultrafine fibers may represent a novel bioactive scaffold for in situ tendon regeneration, which deserves further investigation in large animals.

The Subacromial Bursa is a Viable Source of Autologous Mesenchymal Stem Cells for Rotator Cuff Repair

Objectives: Chronic rotator cuff tears still represent a significant source of morbidity and functional decline in the general population. The purpose of this study was to establish protocols for isolation and expansion of bursa-derived mesenchymal stem cells (BDSCs) and to evaluate their differentiation capacity, including tenogenesis. We hypothesized that BDSCs would be capable of multilineage differentiation (including tenogenesis) and represent an important source for autologous stem cells for patients undergoing rotator cuff repair. Methods: After IRB approval, 10 patients (ages 43-65 years) scheduled to undergo arthroscopic repair for chronic rotator cuff tears were enrolled. During diagnostic arthroscopy, subacromial bursa tissue was harvested using an arthroscopic shaver and collected by attaching the outflow tubing to a specialized specimen cup. Tissue specimens were transported to our laboratory for analysis. BDSCs were isolated via adherent culture and plated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). Chondrogenic, adipogenic, and osteogenic induction media were used to induce differentiation. Tenogenic induction was performed using DMEM supplemented with varying concentrations of BMP-12, ascorbic acid, and human tenocyte-conditioned media. Alcian Blue staining was used to evaluate chondrogenesis, Oil Red O staining for adipogenesis, and Alkaline Phosphatase staining for osteogenesis. Gene expression markers for adipogenesis (ADIPOQ, FABP4, PPARγ), chondrogenesis (COL2A1 and SOX5), and osteogenesis (osteocalcin, osterix), along with primary antibodies to tenogenic markers (scleraxis, tenomodulin), were used to verify each cell lineage. Results: BDSCs isolated by adherent culture without collagen exhibited a spindle-shaped morphology characteristic of mesenchymal stem cells (MSCs), formed colonies, and demonstrated great expandability for six to eight passages without morphology changes (Figure 1A). After 3 weeks of culture, 95% (p&lt;0.0001) of the BDSCs expressed the MSC surface marker CD90 and were negative for non-MSC cell markers (CD45, CD146, CD31, and CD34) (Figure 1B and C). These BDSCs demonstrated a capacity for adipogenesis (positive Oil Red O staining, ADIPOQ, FABP4, PPARγ expression), osteogenesis (positive alkaline phosphatase staining, positive osteocalcin and scleraxis expression), chondrogenesis (positive Alcian Blue staining, positive COL2A1 and SOX5 expression), and tenogenesis (scleraxis and tenomodulin expression) (Figure 2). The results indicate that BDSCs are multipotent as evidenced by their differentiation into fat, bone, cartilage, and tendon cells. For tenogenesis, we found that 7-day incubation in DMEM supplemented with 100 ng/mL of BMP-12 and 50 μg/mL of ascorbic acid produced superior tenogenic induction. These BDSCs adapted an elongated morphology combined with the expression of both scleraxis and tenomodulin - a unique characteristic of native tenocytes. Conclusion: Our results demonstrate that subacromial bursa represents a viable source of mesenchymal stem cells. We developed a reliable protocol for isolation of BDSCs from patient bursa samples. We show that BDSCs in the presence of BMP-12 and ascorbic acid can differentiate toward a tenogenic lineage. Our work provides strong evidence that BDSCs may be a potent tool for cellular therapy and may benefit future patients who undergo surgical repair of chronic rotator cuff tears. [Figure: see text][Figure: see text]

Tropoelastin-Coated Tendon Biomimetic Scaffolds Promote Stem Cell Tenogenic Commitment and Deposition of Elastin-Rich Matrix.

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 a nano-to-macro scale. These were then coated with tropoelastin (TROPO) through polydopamine (PDA) linking, 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 toward 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.

Multifactorial bottom-up bioengineering approaches for the development of living tissue substitutes.

Bottom-up bioengineering utilizes the inherent capacity of cells to build highly sophisticated structures with high levels of biomimicry. Despite the significant advancements in the field, monodomain approaches require prolonged culture time to develop an implantable device, usually associated with cell phenotypic drift in culture. Herein, we assessed the simultaneous effect of macromolecular crowding (MMC) and mechanical loading in enhancing extracellular matrix (ECM) deposition while maintaining tenocyte (TC) phenotype and differentiating bone marrow stem cells (BMSCs) or transdifferentiating neonatal and adult dermal fibroblasts toward tenogenic lineage. At d 7, all cell types presented cytoskeleton alignment perpendicular to the applied load independently of the use of MMC. MMC enhanced ECM deposition in all cell types. Gene expression analysis indicated that MMC and mechanical loading maintained TC phenotype, whereas tenogenic differentiation of BMSCs or transdifferentiation of dermal fibroblasts was not achieved. Our data suggest that multifactorial bottom-up bioengineering approaches significantly accelerate the development of biomimetic tissue equivalents.-Gaspar, D., Ryan, C. N. M., Zeugolis, D. I. Multifactorial bottom-up bioengineering approaches for the development of living tissue substitutes.

Understanding Tendons: Lessons from Transgenic Mouse Models.

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.

Housekeeping Gene Stability in Human Mesenchymal Stem and Tendon Cells Exposed to Tenogenic Factors.

The use of biochemical inducers of mesenchymal stem cell (MSC) differentiation into tenogenic lineage represents an investigated aspect of tendon disorder treatment. Bone morphogenetic protein 12 (BMP-12) is a widely studied factor, representing along with ascorbic acid (AA) and basic fibroblast growth factor (bFGF) one of the most promising stimulus in this context so far. Quantitative gene expression of specific tenogenic marker is commonly used to assess the efficacy of these supplements. Nevertheless, the reliability of these data is strongly associated with the choice of stable housekeeping genes. To date, no published studies have evaluated the stability of housekeeping genes in MSCs during tenogenic induction. Three candidate housekeeping genes (YWHAZ, RPL13A, and GAPDH) in human MSCs from bone marrow (BMSCs), adipose tissue (ASCs), and tendon cells (TCs) supplemented with BMP-12 or AA and bFGF in comparison with control untreated cells for 3 and 10 days were evaluated. GeNorm, NormFinder, and BestKeeper tools and the comparative ΔCt method were used to evaluate housekeeping gene stability and the overall ranking was determined by using by the RefFinder algorithm. In all culture conditions, YWHAZ was the most stable gene and RPL13A was the second choice. YWHAZ and RPL13A were the two most stable genes also for ASCs and BMSCs, regardless of the time point analyzed, and for TCs at 10 days of tenogenic induction. Only for TCs at 3 days of tenogenic induction were GAPDH and YWHAZ the best performers. In conclusion, our findings will be useful for the proper selection of housekeeping genes in studies involving MSCs cultured in the presence of tenogenic factors, to obtain accurate and high-quality data from quantitative gene expression analysis.

Mysteries Behind the Cellular Content of Tendon Tissues.

A trademark feature of the mature tendon is its low cellular content, of which 90% to 95% is composed of tendon-specific cells (ie, tenoblasts and tenocytes); tenocytes are terminally differentiated.1 Differentiation of tenoblasts to tenocytes occurs in response to various growth factors and biomechanical stimuli during tendon development and growth. The remaining 5% to 10% of cellular content in tendon is composed of fibroblastic, vascular, neural, and fat cells located within the tendon sheaths (epitenon and endotenon) and chondrocytes at the bone insertion (enthesis) and compressive loading sites. Tendon cells synthesize all components of the tendon extracellular matrix, with a peak in activity during growth and a gradual decline during aging.1 Early tendon cell commitment and differentiation are not fully understood, and the topics are controversial. However, recent studies have begun to elucidate the mechanisms involved. In early development at E10.5, embryonic mesenchymal progenitors in the somites, body wall, and limb buds enter the tendon lineage and display elevated mRNA levels of the tendon-specific transcription factor scleraxis (Scx).2 Scx expression is activated by distinct fibroblast growth factors as well as transforming growth factor-β. Transcriptome comparison of Scx green fluorescent protein (ScxGFP)-positive tendon cells derived from different embryonic developmental stages resulted in a clearer understanding of the molecular events that occur during embryonic tendon cell differentiation.3 A recent study by Sugimoto et al4 strongly suggests that, at the beginning of tendon development, double-positive progenitors exist for Scx and the cartilage-specific transcription factor SRY-box containing gene 9 (Sox9). The balance in the expression levels between Scx and Sox9 determines whether cells later enter either the cartilaginous or the tenogenic lineage. Bi et al5 identified a novel cell population of resident tendon stem/progenitor cells (TSPCs) within adult tendons. Whether these cells represent a residual population of the embryonal tendon progenitors or infiltrate the tendon from exogenous sources remains unclear. TSPCs exhibit classical adult mesenchymal stem cell criteria (ie, presence of typical surface antigens, self-renewal, clonogenicity, and trilineage differentiation), in addition to expression of tendon-related genes such as Scx and tenomodulin (Tnmd).5,6 Moreover, TSPCs have the capacity to form tendon and enthesis-like tissues when implanted in vivo.5,7 The existence of TSPCs has been further confirmed in subsequent studies of human, equine, rabbit, rat, and mouse tendons.1 Tendon-derived stem cells (TDSCs) have been isolated, expanded, and suggested for use in regenerative medicine.8-10 However, creating a pure TDSC population is still somewhat difficult because of the lack of specific surface receptors. A study published in 2009 shows that cells that simultaneously express tendon and pericyte-associated marker genes are localized in the perivascular space of tendon tissues, indicating that the perivascular niche might be a source of another type of local stem/progenitor cell.11 Mienaltowski et al12 proposed that there is a regional distribution of different stem/progenitor cells within tendon, namely, in the peritenon and within the tendon proper. Comparison between these subpopulations revealed that the peritenon-derived cells have increased vascular and pericyte markers, whereas the cells derived from the tendon proper cells are more proliferative and exhibit higher levels of Scx and Tnmd. The exact steps involved in tendon differentiation during postnatal homeostasis and tendon healing remain elusive. The few articles with information on this topic suggest that the process might be mediated by adult TSPCs, perivascular stem cells, tendon intrafascicular matrix stem cells, or even a mixture of all these cell types, depending on the circumstances.1,5,11-14 Additional studies are necessary to carefully reassemble the regional cell composition of tendons and the interconnections between different cell types. The use of novel tools (eg, gene reporter, constitutive and conditional knockout, and overexpressing mouse models or lineage-tracing techniques) is urgently needed to resolve the mysteries behind the origins of these cell populations and their contribution during tendon development, growth, and maintenance. Currently, the direct comparison of TSPCs with embryonic tendon progenitors, tenoblasts, and tenocytes is impeded because of the lack of molecular markers allowing their discrimination. As a result, the isolation and expansion of pure cell populations along the tendon differentiation cascade remains difficult. Furthermore, clear terminology is needed on the different tendon cell subsets and standardized protocols for their enrichment; appropriate stem cell, progenitor, and differentiated cell segregation methods; and an efficient technique for recapitulating the differentiation process in vitro. Improving our knowledge on these topics can provide novel and fundamental understanding not only of tendon development, but also of tendon sustainability and repair.

Human adipose tissue-derived tenomodulin positive subpopulation of stem cells: A promising source of tendon progenitor cells.

Cell-based therapies are of particular interest for tendon and ligament regeneration given the low regenerative potential of these tissues. Adipose tissue is an abundant source of stem cells, which may be employed for the healing of tendon lesions. However, human adult multipotent adipose-derived stem cells (hASCs) isolated from the stromal vascular fraction of adipose tissue originate highly heterogeneous cell populations that hinder their use in specific tissue-oriented applications. In this study, distinct subpopulations of hASCs were immunomagnetic separated and their tenogenic differentiation capacity evaluated in the presence of several growth factors (GFs), namely endothelial GF, basic-fibroblast GF, transforming GF-β1 and platelet-derived GF-BB, which are well-known regulators of tendon development, growth and healing. Among the screened hASCs subpopulations, tenomodulin-positive cells were shown to be more promising for tenogenic applications and therefore this subpopulation was further studied, assessing tendon-related markers (scleraxis, tenomodulin, tenascin C and decorin) both at gene and protein level. Additionally, the ability for depositing collagen type I and III forming extracellular matrix structures were weekly assessed up to 28days. The results obtained indicated that tenomodulin-positive cells exhibit phenotypical features of tendon progenitor cells and can be biochemically induced towards tenogenic lineage, demonstrating that this subset of hASCs can provide a reliable source of progenitor cells for therapies targeting tendon regeneration.

Tissue-engineered magnetic cell sheet patches for advanced strategies in tendon regeneration.

The concept of magnetic force-based tissue engineering may assist the development of innovative solutions to treat tendon (or other tissues) disorders upon remote control of biological processes as cell migration or differentiation. Herein, we originally fabricated magnetic responsive cell sheets (magCSs) with a Tenomodulin positive subpopulation of adipose tissue derived stem cells identified to commit to the tenogenic lineage. To the best of authors knowledge, this is the first time a tendon oriented strategy resorting on magCSsis reported. Moreover, the promising role of tenogenic living constructs fabricated as magnetically responsive ECM-rich patches is highlighted, envisioning the stimulation of endogenous regenerative mechanisms. Altogether, these findings contribute to future stem cell studies and their translation toward tendon therapies.

TGF-β1 Improves Biomechanical Strength by Extracellular Matrix Accumulation Without Increasing the Number of Tenogenic Lineage Cells in a Rat Rotator Cuff Repair Model

Background: Transforming growth factor β1 (TGF-β1) positively regulates the tenogenic marker genes scleraxis (Scx) and tenomodulin (Tnmd) in mesenchymal progenitors in vitro. However, little is known about the effect of TGF-β1 on the expression of tenogenic markers during rotator cuff (RC) healing in rats. Hypothesis: TGF-β1 improves the biomechanical properties and histological maturity of reparative tissue in a rat RC repair model by stimulating the growth of tenogenic cells. Study Design: Controlled laboratory study. Methods: Adult male Sprague-Dawley rats (N = 180) underwent unilateral supraspinatus tendon-to-bone surgical repair and were randomly treated with a gelatin hydrogel presoaked in TGF-β1 (100 ng) or phosphate-buffered saline. The effects of TGF-β1 on RC healing were investigated at 2, 4, 6, 8, and 12 weeks postoperatively by immunostaining for proliferating cell nuclear antigen, by real-time reverse transcription polymerase chain reaction and in situ hybridization or immunostaining for enthesis-related markers (SRY-box containing gene 9 [Sox9], Scx, and Tnmd), and by real-time reverse transcription polymerase chain reaction and immunostaining for type I and III collagen. At 6 and 12 weeks postoperatively, biomechanical testing, micro–computed tomography, and biochemical analysis were also performed. At 2 and 4 weeks postoperatively, mesenchymal stem cell–related markers, phospho-Smad2, and matrix metalloproteinase 9 (MMP-9) and MMP-13 were assessed by immunostaining. Results: The TGF-β1-treated group had significantly higher ultimate load to failure and tissue volume at 6 and 12 weeks postoperatively and a higher collagen content at 12 weeks compared with the saline group. Tendon-related gene expression, histological maturity, cell proliferation, and mesenchymal stem cell–related marker immunoreactivity were not affected by exogenously administrated TGF-β1 at all time points. In the TGF-β1-treated group, the percentage of phospho-Smad2-positive cells within the healing tissue increased, whereas the expression of MMP-9 and MMP-13 significantly decreased at 2 and 4 weeks postoperatively. Conclusion: TGF-β1 enhances formation of tough fibrous tissues at the healing site by inhibiting MMP-9 and MMP-13 expression to increase collagen accumulation but without the growth of tenogenic lineage cells. Clinical Relevance: These findings suggest that TGF-β1 could be used for enhancing biomechanical strength after RC surgical repair.

* An Engineered Multiphase Three-Dimensional Microenvironment to Ensure the Controlled Delivery of Cyclic Strain and Human Growth Differentiation Factor 5 for the Tenogenic Commitment of Human Bone Marrow Mesenchymal Stem Cells.

At present, injuries or rupture of tendons are treated by surgical repair or conservative approaches with unpredictable clinical outcome. Alternative strategies to repair tendon defects without the undesirable side effects associated with the current options are needed. With this in mind, a tissue engineering approach has gained considerable attention as a promising strategy. Here we investigated a synthetic three-dimensional (3D) microenvironment able to interact with stem cells and inducing, via coupled biochemical and physical signals, their early commitment toward the tenogenic lineage. This multiphase 3D construct consisted of a braided hyaluronate elastic band merged with human bone marrow mesenchymal stem cells (hBMSCs) and poly-lactic-co-glycolic acid microcarriers loaded with human growth differentiation factor 5 (hGDF-5) by means of fibrin hydrogel. The multiphase structure allowed hBMSC culture under cyclic strain within a microenvironment where a controlled amount of hGDF-5 was regularly delivered. The cooperative biochemical and physical stimuli induced significantly increased expression of tenogenic markers, such as collagen type I and III, decorin, scleraxis, and tenascin-C, within only 3 days of dynamic hBMSC culture. This approach opens exciting perspectives for future development of engineered tendon tissue substitutes.