Bone marrow stromal and anterior cruciate ligament remnant cell co-culture-derived extracellular vesicles promote cell activity in both cell types.

The significance of anterior cruciate ligament (ACL) remnants during reconstruction remains unclear. Co-culturing ACL remnant cells and bone marrow stromal cells (BMSCs) may reduce apoptosis and enhance hamstring tendon activity. This study investigated whether extracellular vesicles (EVs), which facilitate cell-cell interactions, act as the active components, improving graft maturation in this co-culture. The effects of EVs on cell viability, proliferation, migration and gene expression in the rabbit ACL remnant cells and BMSCs were assessed using control (BMSC-only culture), co-culture (ACL remnant cells and BMSCs, CM) and co-culture without EVs (CM ∆ EVs) media. EVs were isolated from control (BMSC-EV) and co-culture (CM-EV) media and characterized. CM significantly enhanced the proliferation, migration and expression of transforming growth factor (TGF-ß)-, vascular endothelial growth factor (VEGF)-, collagen synthesis- and tenogenesis-related genes. However, CM-induced effects were reversed by the CM ∆ EVs treatment. CM-EV treatment exhibited higher potential to enhance proliferation, migration and gene expression in the ACL remnant cells and BMSCs than BMSC-EV and non-EV treatments. In conclusion, EVs, secreted under the coexistence of ACL remnant cells and BMSCs, primarily increase the cell viability, proliferation, migration and gene expression of collagen synthesis-, TGF-ß-, VEGF- and tenogenesis-related genes in both cell types.

Rapamycin facilitates healing of the tendon-bone interface in an aging rat model of chronic rotator cuff injury.

BACKGROUND: Tendon-bone interface (TBI) healing in chronic rotator cuff injury (CRCI) in older individuals is a common clinical challenge due to cellular senescence, as well as decreased tissue repair and regeneration. Many studies have demonstrated the antiaging, improved tissue repair, and bone regeneration properties of rapamycin (RPM) in multiple age-related diseases. This study aimed to explore the effects of RPM on TBI healing after CRCI in an aging rat model. METHODS: A CRCI model was established in 60 Sprague-Dawley rats (24 months old). Rats were then randomly allocated into the control, 0.1 µg RPM, and 1 µg RPM groups. At 4 and 8 weeks postreconstructive surgery, the supraspinatus tendon-humerus complexes were harvested for biomechanical, microimaging, histological, and immunohistochemical evaluations. RESULTS: Biomechanical testing results demonstrated that the failure load, ultimate strength, and stiffness of the 2 RPM groups were significantly higher than those of the control group at 4 and 8 weeks postoperatively. Microradiographically, both RPM groups had significantly higher values of bone mineral density and the ratio of trabecular bone volume to total volume than controls at each time point. Moreover, the RPM groups had higher histological scores and showed better regenerated TBI, characterized by better organizational tissue, more fibrocartilage cells, and more bone formation. Immunohistochemical evaluations showed that RUNX2-, SOX9-, and SCX-positive cells were significantly more in the 2 RPM groups than in the controls at each time point. CONCLUSIONS: RPM may effectively enhance CRCI healing after reconstruction by facilitating osteogenesis, tenogenesis, and fibrocartilage reformation at the TBI, as well as improving biomechanical properties.

Fibrin Scaffolds Perfused with Transforming Growth Factor-ß1 as an In Vitro Model to Study Healthy and Tendinopathic Human Tendon Stem/Progenitor Cells.

A limited understanding of tendon cell biology in healthy and pathological conditions has impeded the development of effective treatments, necessitating in vitro biomimetic models for studying tendon events. We established a dynamic culture using fibrin scaffolds, bioengineered with tendon stem/progenitor cells (hTSPCs) from healthy or diseased human biopsies and perfused with 20 ng/mL of human transforming growth factor-ß1 for 21 days. Both cell types showed long-term viability and upregulated Scleraxis (SCX-A) and Tenomodulin (TNMD) gene expressions, indicating tenogenic activity. However, diseased hTSPCs underexpressed collagen type I and III (COL1A1 and COL3A1) genes and exhibited lower SCX-A and TNMD protein levels, but increased type I collagen production, with a type I/type III collagen ratio > 1.5 by day 14, matching healthy cells. Diseased hTSPCs also showed constant high levels of pro-inflammatory cytokines, such as IL-8 and IL-6. This biomimetic environment is a valuable tool for studying tenogenic and inflammatory events in healthy and diseased tendon cells and identifying new therapeutic targets.

TGFß2-induced tenogenesis impacts cadherin and connexin cell-cell junction proteins in mesenchymal stem cells.

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)ß2 impacted protein markers of tendon differentiation and protein levels of N-cadherin, cadherin-11 and connexin-43. MSCs were treated with TGFß2 for 21 days. At 3 days, TGFß2-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 TGFß2 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.

Activation of AKT-mTOR Signaling Directs Tenogenesis of Mesenchymal Stem Cells.

Tendon repair is a clinical challenge because of the limited understanding on tenogenesis. The synthesis of type I collagen (Collagen I) and other extracellular matrix are essential for tendon differentiation and homeostasis. Current studies on tenogenesis focused mostly on the tenogenic transcriptional factors while the signaling controlling tenogenesis on translational level remains largely unknown. Here, we showed that mechanistic target of rapamycin (mTOR) signaling was activated by protenogenic growth factor, transforming growth factors beta1, and insulin-like growth factor-I. The expression of mTOR was upregulated during tenogenesis of mesenchymal stem cells (MSCs). Moreover, mTOR was downregulated in human tendinopathy tissues and was inactivated upon statin treatment. Both inhibition and depletion of AKT or mTOR significantly reduced type I collagen production and impaired tenogenesis of MSCs. Tendon specific-ablation of mTOR resulted in tendon defect and reduction of Collagen I. However, there is no evident downregulation of tendon associated collagens at the transcription level. Our study demonstrated that AKT-mTOR axis is a key mediator of tendon differentiation and provided a novel therapeutic target for tendinopathy and tendon injuries. Stem Cells 2018;36:527-539.

Mesenchymal Stem Cells Empowering Tendon Regenerative Therapies.

Tendon tissues have limited healing capacity. The incidence of tendon injuries and the unsatisfactory functional outcomes of tendon repair are driving the search for alternative therapeutic approaches envisioning tendon regeneration. Cellular therapies aim at delivering adequate, regeneration-competent cell types to the injured tendon and toward ultimately promoting its reconstruction and recovery of functionality. Mesenchymal stem cells (MSCs) either obtained from tendons or from non-tendon sources, like bone marrow (BM-MSCs) or adipose tissue (ASCs), have been receiving increasing attention over the years toward enhancing tendon healing. Evidences from in vitro and in vivo studies suggest MSCs can contribute to accelerate and improve the quality of tendon healing. Nonetheless, the exact mechanisms underlying these repair events are yet to be fully elucidated. This review provides an overview of the main challenges in the field of cell-based regenerative therapies, discussing the role of MSCs in boosting tendon regeneration, particularly through their capacity to enhance the tenogenic properties of tendon resident cells.

Tenogenesis of Decellularized Porcine Achilles Tendon Matrix Reseeded with Human Tenocytes in the Nude Mice Xenograft Model.

Cultivation of autologous human tenocytes in a cell-free xenogenic extracellular tendon matrix (xECM) could present an approach for tendon reconstruction. The aim of this study was to achieve tendon-like tissue formation by implanting decellularized porcine Achilles tendons recellularized with human hamstring tendon-derived tenocytes into nude mice. The structure of decellularized xECM was histologically monitored before being dynamically reseeded with human tenocytes. After 6⁻12 weeks in vivo, construct quality was monitored using macroscopical and histological scoring systems, vitality assay and quantitative DNA and glycosaminoglycan (GAG) assays. For comparison to tendon xECM, a synthetic polyglycolic acid (PGA) polymer was implanted in a similar manner. Despite decellularized xECM lost some GAGs and structure, it could be recellularized in vitro with human tenocytes, but the cell distribution remained inhomogeneous, with accumulations at the margins of the constructs. In vivo, the xECM constructs revealed in contrast to the PGA no altered size, no inflammation and encapsulation and a more homogeneous cell distribution. xECM reseeded with tenocytes showed superior histological quality than cell-free implanted constructs and contained surviving human cells. Their DNA content after six and 12 weeks in vivo resembled that of native tendon and xECM recellularized in vitro. Results suggest that reseeded decellularized xECM formed a tendon-like tissue in vivo.

Informing Stem Cell-Based Tendon Tissue Engineering Approaches with Embryonic Tendon Development.

Adult tendons fail to regenerate normal tissue after injury, and instead form dysfunctional scar tissue with abnormal mechanical properties. Surgical repair with grafts is the current standard to treat injuries, but faces significant limitations including pain and high rates of re-injury. To address this, we aim to regenerate new, normal tendons to replace dysfunctional tendons. A common approach to tendon tissue engineering is to design scaffolds and bioreactors based on adult tendon properties that can direct adult stem cell tenogenesis. Despite significant progress, advances have been limited due, in part, to a need for markers and potent induction cues. Our goal is to develop novel tendon tissue engineering approaches informed by embryonic tendon development. We are characterizing structure-property relationships of embryonic tendon to identify design parameters for three-dimensional scaffolds and bioreactor mechanical loading systems to direct adult stem cell tenogenesis. We will review studies in which we quantified changes in the mechanical and biochemical properties of tendon during embryonic development and elucidated specific mechanisms of functional property elaboration. We then examined the effects of these mechanical and biochemical factors on embryonic tendon cell behavior. Using custom-designed bioreactors, we also examined the effects of dynamic mechanical loading and growth factor treatment on embryonic tendon cells. Our findings have established cues to induce tenogenesis as well as metrics to evaluate differentiation. We finish by discussing how we have evaluated the tenogenic differentiation potential of adult stem cells by comparing their responses to that of embryonic tendon cells in these culture systems.

Gene expression of Postn and FGF7 in canine chordae tendineae and their effects on flexor tenocyte biology.

Chordae tendineae, referred to as heart tendinous cords, act as tendons connecting the papillary muscles to the valves in the heart. Their role is analogous to tendons in the musculoskeletal system. Despite being exposed to millions of cyclic tensile stretches over a human's lifetime, chordae tendineae rarely suffer from overuse injuries. On the other hand, musculoskeletal tendinopathy is very common and remains challenging in clinical treatment. The objective of this study was to investigate the mechanism behind the remarkable durability and resistance to overuse injuries of chordae tendineae, as well as to explore their effects on flexor tenocyte biology. The messenger RNA expression profiles of chordae tendineae were analyzed using RNA sequencing and verified by quantitative reverse transcription polymerase chain reaction  and immunohistochemistry. Interestingly, we found that periostin (Postn) and fibroblast growth factor 7 (FGF7) were expressed at significantly higher levels in chordae tendineae, compared to flexor tendons. We further treated flexor tenocytes in vitro with periostin and FGF7 to examine their effects on the proliferation, migration, apoptosis, and tendon-related gene expression of flexor tenocytes. The results displayed enhanced cell proliferation ability at an early stage and an antiapoptotic effect on tenocytes, while treated with periostin and/or FGF7 proteins. Furthermore, there was a trend of promoted tenocyte migration capability. These findings indicated that Postn and FGF7 may represent novel cytokines to target flexor tendon healing. Clinical significance: The preliminary discovery leads to a novel idea for treating tendinopathy in the musculoskeletal system using specific molecules identified from chordae tendineae.

Development of a Cissus quadrangularis-Doped Extracellular Matrix and a Hyaluronic Acid-Incorporated Scaffold for Periodontal Regeneration: An In Vitro Study.

PURPOSE: The study aimed to analyze whether adding Cissus quadrangularis (CQ) extract and the extracellular matrix of ovine tendon (TENDON) increases the regenerative potential of mesenchymal stem cells produced in hyaluronic acid (HA) scaffolds for tenogenesis. MATERIALS AND METHODS: Fifty grams of powdered CQ was mixed with 250 mL of ethanol to prepare the extract. Two grams of hyaluronic acid powder was added to 100 mL of distilled water to make the HA solution. The ovine tendon was decellularized using a mixture of 10% phosphate-buffered saline (PBS), sodium dodecyl sulfate (SDS), and Triton-X. The hydrogel samples were prepared by mixing the extracellular matrix of tendon, HA, and CQ, after which they were divided into study groups such as HA, HA + CQ, HA + TENDON, and HA + CQ + TENDON. Scanning electron microscopy (SEM) analysis, swelling analysis, differentiation analysis, compression test, compatibility assay, and tenogenesis assay were later conducted. RESULTS: The morphology of the samples was analyzed using SEM. Low levels of swelling of the hydrogels were observed. Cells were found to be viable and showed good differentiation and tenogenesis. Optimal compression levels were observed, and the properties of the prepared hydrogels were satisfactory. CONCLUSION: The results suggest that the addition of CQ considerably increases the tenogenic potential of the extracellular matrix/HA scaffold. Hence, it can be used as a regenerative material for periodontal tissue regeneration.

Bioactive 3D Electrohydrodynamic Printed Lattice Architectures Augment Tenogenesis of Tendon Stem/Progenitor Cells.

Tendon defect repair remains a tough clinical procedure that hinders functional motion in patients. Electrohydrodynamic (EHD) three-dimensional (3D) printing, as a novel strategy, can controllably fabricate biomimetic micro/nanoscale architecture, but the hydrophobic and bioinert nature of polymers might be adverse to cell-material interplay. In this work, 3D EHD printed polycaprolactone (PCL) was immobilized on basic fibroblast growth factor (bFGF) using polydopamine (PDA), and the proliferation and tenogenic differentiation of tendon stem/progenitor cells (TSPCs) in vitro was researched. A subcutaneous model was established to evaluate the effects of tenogenesis and immunomodulation. We then investigated the in situ implantation and immunomodulation effects in an Achilles tendon defect model. After immobilization of bFGF, the scaffolds profoundly facilitated proliferation and tenogenic differentiation; however, PDA had only a proliferative effect. Intriguingly, the bFGF immobilized on EHD printed PCL indicated a synergistic effect on the highest expression of tenogenic gene and protein markers at 14 days, and the tenogenesis may be induced by activating the transforming growth factor-ß (TGF-ß) signal pathway in vitro. The subcutaneous engraftment study confirmed a tendon-like structure, similar to that of the native tendon, as well as an M2 macrophage polarization effect. Additionally, the bioactive scaffold exhibited superior efficacy in new collagen formation and repair of Achilles tendon defects. Our study revealed that the topographic cues alone were insufficient to trigger tenogenic differentiation, requiring appropriate chemical signals, and that appropriate immunomodulation was conducive to tenogenesis. The tenogenesis of TSPCs on the bioactive scaffold may be correlated with the TGF-ß signal pathway and M2 macrophage polarization.

Engineered Microenvironmental Cues from Fiber-Reinforced Hydrogel Composites Drive Tenogenesis and Aligned Collagen Deposition.

Effective tendon regeneration following injury is contingent on appropriate differentiation of recruited cells and deposition of mature, aligned, collagenous extracellular matrix that can withstand the extreme mechanical demands placed on the tissue. As such, myriad biomaterial approaches have been explored to provide biochemical and physical cues that encourage tenogenesis and template aligned matrix deposition in lieu of dysfunctional scar tissue formation. Fiber-reinforced hydrogels present an ideal biomaterial system toward this end given their transdermal injectability, tunable stiffness over a range amenable to tenogenic differentiation of progenitors, and capacity for modular inclusion of biochemical cues. Here, tunable and modular, fiber-reinforced, synthetic hydrogels are employed to elucidate salient microenvironmental determinants of tenogenesis and aligned collagen deposition by tendon progenitor cells. Transforming growth factor ß3 drives a cell fate switch toward pro-regenerative or pro-fibrotic phenotypes, which can be biased toward the former by culture in softer microenvironments or inhibition of the RhoA/ROCK activity. Furthermore, studies demonstrate that topographical anisotropy in fiber-reinforced hydrogels critically mediates the alignment of de novo collagen fibrils, reflecting native tendon architecture. These findings inform the design of cell-free, injectable, synthetic hydrogels for tendon tissue regeneration and, likely, that of a range of load-bearing connective tissues.

Tendon Transection Healing Can Be Improved With Adipose-Derived Stem Cells Cultured With Growth Differentiation Factor 5 and Platelet-Derived Growth Factor.

BACKGROUND: As hand surgeons, tendon injuries and lacerations are a particularly difficult problem to treat, as poor healing potential and adhesions hamper optimal recovery. Adipose-derived stem cells (ADSCs) have been shown to aid in rat Achilles tendon healing after a puncture defect, and this model can be used to study tendon healing in the upper extremity. We hypothesized that ADSCs cultured with growth differentiation factor 5 (GDF5) and platelet-derived growth factor (PDGF) would improve tendon healing after a transection injury. METHODS: Rat Achilles tendons were transected and then left either unrepaired or repaired. Both groups were treated with a hydrogel alone, a hydrogel with ADSCs, or a hydrogel with ADSCs that were cultured with GDF5 and PDGF prior to implantation. Tissue harvested from the tendons was evaluated for gene expression of several genes known to play an important role in successful tendon healing. Histological examination of the tendon healing was also performed. RESULTS: In both repaired and unrepaired tendons, those treated with ADSCs cultured with GDF5/PDGF prior to implantation showed the best tendon fiber organization, the smallest gaps, and the most organized blood vessels. Treatment with GDF5/PDGF increased expression of the protenogenesis gene SOX9, promoted cell-to-cell connections, improved cellular proliferation, and enhanced tissue remodeling. CONCLUSIONS: Adipose-derived stem cells cultured with GDF5/PDGF prior to implantation can promote tendon repair by improving cellular proliferation, tenogenesis, and vascular infiltration. This effect results in a greater degree of organized tendon healing.

RAB23 regulates musculoskeletal development and patterning.

RAB23 is a small GTPase which functions at the plasma membrane to regulate growth factor signaling. Mutations in RAB23 cause Carpenter syndrome, a condition that affects normal organogenesis and patterning. In this study, we investigate the role of RAB23 in musculoskeletal development and show that it is required for patella bone formation and for the maintenance of tendon progenitors. The patella is the largest sesamoid bone in mammals and plays a critical role during movement by providing structural and mechanical support to the knee. Rab23 -/- mice fail to form a patella and normal knee joint. The patella is formed from Sox9 and scleraxis (Scx) double-positive chondroprogenitor cells. We show that RAB23 is required for the specification of SOX9 and scleraxis double-positive patella chondroprogenitors during the formation of patella anlagen and the subsequent establishment of patellofemoral joint. We find that scleraxis and SOX9 expression are disrupted in Rab23 -/- mice, and as a result, development of the quadriceps tendons, cruciate ligaments, patella tendons, and entheses is either abnormal or lost. TGFß-BMP signaling is known to regulate patella initiation and patella progenitor differentiation and growth. We find that the expression of TGFßR2, BMPR1, BMP4, and pSmad are barely detectable in the future patella site and in the rudimentary tendons and ligaments around the patellofemoral joint in Rab23 -/- mice. Also, we show that GLI1, SOX9, and scleraxis, which regulate entheses establishment and maturation, are weakly expressed in Rab23 -/- mice. Further analysis of the skeletal phenotype of Rab23 -/- mice showed a close resemblance to that of Tgfß2 -/- mice, highlighting a possible role for RAB23 in regulating TGFß superfamily signaling.

The LINC complex regulates Achilles tendon elastic modulus, Achilles and tail tendon collagen crimp, and Achilles and tail tendon lateral expansion during early postnatal development.

There is limited understanding of how mechanical signals regulate tendon development. The nucleus has emerged as a major regulator of cellular mechanosensation, via the linker of nucleoskeleton and cytoskeleton (LINC) protein complex. Specific roles of LINC in tenogenesis have not been explored. In this study, we investigate how LINC regulates tendon development by disabling LINC-mediated mechanosensing via dominant negative (dn) expression of the Klarsicht, ANC-1, and Syne Homology (KASH) domain, which is necessary for LINC to function. We hypothesized that LINC regulates mechanotransduction in developing tendon, and that disabling LINC would impact tendon mechanical properties and structure in a mouse model of dnKASH. We used Achilles (AT) and tail (TT) tendons as representative energy-storing and limb-positioning tendons, respectively. Mechanical testing at postnatal day 10 showed that disabling the LINC complex via dnKASH significantly impacted tendon mechanical properties and cross-sectional area, and that effects differed between ATs and TTs. Collagen crimp distance was also impacted in dnKASH tendons, and was significantly decreased in ATs, and increased in TTs. Overall, we show that disruption to the LINC complex specifically impacts tendon mechanics and collagen crimp structure, with unique responses between an energy-storing and limb-positioning tendon. This suggests that nuclear mechanotransduction through LINC plays a role in regulating tendon formation during neonatal development.

Age-related cellular and microstructural changes in the rotator cuff enthesis.

Rotator cuff injuries increase with age. The enthesis is the most frequent site of rotator cuff injury and degeneration. Understanding age-related changes of the enthesis are essential to determine the mechanism of rotator cuff injuries, degeneration, and to guide mechanistically driven therapies. In this study, we explored age-related cellular changes of the rotator cuff enthesis in young, mature, and aged rats. Here we found that the aged enthesis is typified by an increased mineralized zone and decreased nonmineralized zone. Proliferation, migration, and colony-forming potential of rotator cuff derived cells (RCECs) was attenuated with aging. The tenogenic and chondrogenic potential were significantly reduced, while the osteogenic potential increased in aged RCECs. The adipogenic potential increased in RCECs with age. This study explores the cellular differences found between young, mature, and aged rotator cuff enthesis cells and highlights the importance of using age-appropriate models, as well as provides a basis for further delineation of mechanisms and potential therapeutics for rotator cuff injuries.

Role of Yes-associated protein (YAP) in regulation of mesenchymal stem cell tenogenic differentiation.

The Yes-associated protein (YAP) transcription co-activator is recognized as a key mediator and has been implicated in the regulation of stem cell fate; however, the role of YAP in the tenogenic differentiation of mesenchymal stem cells (MSCs) is not well understood. In the present study, we characterized tenogenic differentiation of C3H10T1/2 cells induced by BMP-12 and evaluated the function of YAP in the regulation of the BMP-12-induced tenogenesis. We found that the tendon-related proteins Scleraxis, Tenascin C, and Tenomodulin were significantly increased after induction by 10 ng/ml BMP-12 for 48 h in C3H10T1/2 cells. In addition, the expression of YAP was significantly enhanced and transferred from the cytoplasm to the nucleus during tenogenic differentiation. In contrast, downregulation of YAP suppressed the cells' tenogenic differentiation, and the expression of YAP transferred from the nucleus to the cytoplasm. These results indicate that YAP is required for the expression of tenogenic markers in the tenocytic differentiation process of C3H10T1/2 cells. Collectively, we demonstrate that YAP transcription co-activator is a novel regulator in the process of BMP-12-induced MSCs tenogenesis and has a correlation with the other tenogenic regulators and markers. These results shed new light on the function of YAP in tendon healing and regeneration.

Cell-subpopulation alteration and FGF7 activation regulate the function of tendon stem/progenitor cells in 3D microenvironment revealed by single-cell analysis.

Three dimensional (3D) microenvironments more accurately replicate native microenvironments for stem cell maintenance and function compared with two dimensional (2D) microenvironments. However, the molecular mechanisms by which 3D microenvironments regulate stem cell function remain largely unexplored at the single-cell level. Here, using a single-cell analysis and functional analysis, we found not all cell-subpopulations respond to 3D microenvironments based on a systematically 3D gelatin microcarrier culture system we developed for the expansion and function maintenance of hTSPCs. 3D microenvironments alter the cell-subpopulation distribution of human tendon stem/progenitor cells (hTSPCs) by improving the proportion of ICAM1+ITGB8+ and FGF7+CYGB+ subpopulations. We also revealed the activated FGF7 signaling in the two subpopulations is responsible for the enhanced tenogenesis of hTSPCs through cell-cell interactions. The hTSPCs cultured in 3D niche with a specific cell-subpopulation structure exhibited superior stem-cell characteristics and functions both in vitro and in vivo. Together, our study demonstrates that 3D microenvironments can regulate stem-cell function by modulating the critical cell subpopulation and identifies FGF7 as a novel regulator for tenogenic differentiation and tendon regeneration.

Mesenchymal Stem Cell Sheets for Engineering of the Tendon-Bone Interface.

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.

Tendon tissue engineering: Current progress towards an optimized tenogenic differentiation protocol for human stem cells.

Tendons are integral to our daily lives by allowing movement and locomotion but are frequently injured, leading to patient discomfort and impaired mobility. Current clinical procedures are unable to fully restore the native structure of the tendon, resulting in loss of full functionality, and the weakened tissue following repair often re-ruptures. Tendon tissue engineering, involving the combination of cells with biomaterial scaffolds to form new tendon tissue, holds promise to improve patient outcomes. A key requirement for efficacy in promoting tendon tissue formation is the optimal differentiation of the starting cell populations, most commonly adult tissue-derived mesenchymal stem/stromal cells (MSCs), into tenocytes, the predominant cellular component of tendon tissue. Currently, a lack of consensus on the protocols for effective tenogenic differentiation is hampering progress in tendon tissue engineering. In this review, we discuss the current state of knowledge regarding human stem cell differentiation towards tenocytes and tendon tissue formation. Tendon development and healing mechanisms are described, followed by a comprehensive overview of the current protocols for tenogenic differentiation, including the effects of biochemical and biophysical cues, and their combination, on tenogenesis. Lastly, a synthesis of the key features of these protocols is used to design future approaches. The holistic evaluation of current knowledge should facilitate and expedite the development of efficacious stem cell tenogenic differentiation protocols with future impact in tendon tissue engineering. STATEMENT OF SIGNIFICANCE: The lack of a widely-adopted tenogenic differentiation protocol has been a major hurdle in the tendon tissue engineering field. Building on current knowledge on tendon development and tendon healing, this review surveys peer-reviewed protocols to present a holistic evaluation and propose a pathway to facilitate and expedite the development of a consensus protocol for stem cell tenogenic differentiation and tendon tissue engineering.