Expression Of Tendon-related Genes Research Articles

Cell-Based Therapies for Rotator Cuff Injuries: An Updated Review of the Literature.

This review focuses on non-surgical treatment options for rotator cuff injuries and highlights the potential of mesenchymal stem cells (MSCs) as a potential regenerative approach. MSCs, sourced from various tissues like bone marrow and adipose tissue, exhibit promising mechanisms in vitro, influencing tendon-related gene expression and microenvironment modulation. Animal studies support this, showcasing MSCs' ability to reduce inflammation, improve tissue remodeling, and enhance repaired tendon strength. Human trials, while varied and limited, suggest that MSCs might lower retear rates and enhance post-repair outcomes, but randomized controlled trials yield mixed results, emphasizing the necessity for standardized investigations. Ultimately, while cell-based therapies demonstrate an excellent safety profile, more rigorous clinical trials are necessary to determine their efficacy in improving patient outcomes and achieving lasting structural changes in rotator cuff injuries.

Viable tendon neotissue from adult adipose-derived multipotent stromal cells.

Background: Tendon healing is frequently prolonged, unpredictable, and results in poor tissue quality. Neotissue formed by adult multipotent stromal cells has the potential to guide healthy tendon tissue formation. Objectives: The objective of this study was to characterize tendon neotissue generated by equine adult adipose-derived multipotent stromal cells (ASCs) on collagen type I (COLI) templates under 10% strain in a novel bioreactor. The tested hypothesis was that ASCs assume a tendon progenitor cell-like morphology, express tendon-related genes, and produce more organized extracellular matrix (ECM) in tenogenic versus stromal medium with perfusion and centrifugal fluid motion. Methods: Equine ASCs on COLI sponge cylinders were cultured in stromal or tenogenic medium within bioreactors during combined perfusion and centrifugal fluid motion for 7, 14, or 21days under 10% strain. Viable cell distribution and number, tendon-related gene expression, and micro- and ultra-structure were evaluated with calcein-AM/EthD-1 staining, resazurin reduction, RT-PCR, and light, transmission, and scanning electron microscopy. Fibromodulin was localized with immunohistochemistry. Cell number and gene expression were compared between culture media and among culture periods (p < 0.05). Results: Viable cells were distributed throughout constructs for up to 21days of culture, and cell numbers were higher in tenogenic medium. Individual cells had a round or rhomboid shape with scant ECM in stromal medium in contrast to clusters of parallel, elongated cells surrounded by highly organized ECM in tenogenic medium after 21days of culture. Transcription factor, extracellular matrix, and mature tendon gene expression profiles confirmed ASC differentiation to a tendon progenitor-like cell in tenogenic medium. Construct micro- and ultra-structure were consistent with tendon neotissue and fibromodulin was present in the ECM after culture in tenogenic medium. Conclusion: Long-term culture in custom bioreactors with combined perfusion and centrifugal tenogenic medium circulation supports differentiation of equine adult ASCs into tendon progenitor-like cells capable of neotissue formation.

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 RNAexpression 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.

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

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

Impact of high-intensity interval training on tendon related gene expression in rat Achilles tendon

Immobilization or aging associated with limited physical activity can lead to the functional deterioration of tendons, which has become an important public health concern. Therefore, growing research is focused on the effect of exercise training on preserving tendon function. Exercise training subjects muscles and tendons to repeated mechanical stress, and in vitro studies have revealed that repetitive mechanical loading stimulates tendon cell responses to changes in the extracellular matrix and functional properties of the tendon. However, although several types of exercise training have been shown to be effective in preserving tendon function, no studies have investigated the impact of high-intensity interval training (HIIT), which involves composing short bouts of exercise with high-power output. Here, we determined whether the HIIT program enhanced tenogenic progressions by measuring the mRNA expression in rat Achilles tendons. Sixteen rats were randomly assigned into either a sedentary control group (Con, n = 8) or an HIIT group (n = 8). Rats in the HIIT group performed the program with treadmill running; the training volume was incremental (running speed, number of sets, and inclination), and training was conducted 5 days per week for 9 weeks. The rats in the HIIT group exhibited a marked decrease in the body weight and different types of fat weights, and a marked increase in different types of muscle weights. Real-time reverse transcription polymerase chain reaction analysis revealed that mRNA expressions of tendon-related genes Tnxb, Opn, and Tgfb1 were upregulated in the HIIT group compared to that in the Con group. Cross-links in mRNA expressions of collagen-related Dcn and Fmod in the HIIT group tended to be higher than in those Con group. These results suggest that HIIT induces initiation of tenogenic progression and stimulation of cross-link formation between collagen fibrils in rat Achilles tendons.

Basic calcium phosphate crystals induce the expression of extracellular matrix remodelling enzymes in tenocytes.

Basic calcium phosphate (BCP) crystals contribute to several syndromes associated with tendon disease, including acute calcific tendinitis and Milwaukee shoulder syndrome. Interactions between BCP crystals and tenocytes (tendon cells) may contribute to these clinical syndromes. This study aimed to determine the direct effects of BCP crystals on tenocyte function and viability. In vitro assays were used to assess changes in human tenocytes cultured with BCP crystals. Real-time PCR was used to determine changes in the expression of tendon-related genes and extracellular matrix remodelling enzymes (MMPs; a disintegrin and metalloproteases, ADAMTS; and tissue inhibitor of metalloproteinases, TIMPs). ELISA was used to measure protein concentrations in tenocyte supernatants. MTT and alamarBlue™ assays were used to determine changes in cell viability. BCP crystals upregulated tenocyte gene expression of MMP-1, MMP-3, ADAMTS-4 and TIMP-1 after 24 h. Time-course experiments showed expression peaked at 8 h for TIMP-1 and 48 h for MMP-1 and ADAMTS-4. Cyclooxygenase (COX)-1 gene expression was upregulated after 48 h. Tenocytes did not alter expression of scleraxis and tendon collagens, and expression of pro-inflammatory cytokines was not induced with BCP crystals. BCP crystals increased tenocyte release of prostaglandin E2 (PGE2) and MMP-1 protein after 24 h. However, neither COX-1 inhibition nor COX-2 inhibition led to consistent change in BCP crystal-induced tenocyte gene expression of extracellular matrix remodelling enzymes. BCP crystals had no effect on tenocyte viability. BCP crystals induce extracellular matrix remodelling enzymes, but not inflammatory cytokines, in tenocytes.

Tendon-Specific Dicer Deficient Mice Exhibit Hypoplastic Tendon Through the Downregulation of Tendon-Related Genes and MicroRNAs.

Tendon is a fibrous connective tissue, that is, transmitting the forces that permit body movement. However, tendon/ligament biology is still not fully understood and especially, the role of miRNAs in tendon/ligament is sparse and uncharacterized in in vivo models. The objectives of this study were to address the function of DICER using mice with tendon/ligament-specific deletion of Dicer (Dicer conditional knockout; cKO), and to identify key miRNAs in tendon/ligament. Dicer cKO mice exhibited hypoplastic tendons through structurally abnormal collagen fibrils with downregulation of tendon-related genes. The fragility of tendon did not significantly affect the tensile strength of tendon in Dicer cKO mice, but they showed larger dorsiflexion angle in gait compared with Control mice. We identified two miRNAs, miR-135a and miR-1247, which were highly expressed in the Achilles tendon of Control mice and were downregulated in the Achilles tendon of Dicer cKO mice compared with Control mice. miR-135a mimic increased the expression of tendon-related genes in injured Achilles tendon-derived fibroblasts. In this study, Dicer cKO mice exhibited immature tendons in which collagen fibrils have small diameter with the downregulation of tendon-related genes such as transcriptional factor, extracellular matrix, and miRNAs. Thus, DICER plays an important role in tendon maturation, and miR-135a may have the potential to become key miRNA for tendon maturation and healing.

Influence of mechanical and TGF-\u03b23 stimulation on the tenogenic differentiation of tonsil-derived mesenchymal stem cells

BackgroundOrganogenesis from tonsil-derived mesenchymal cells (TMSCs) has been reported, wherein tenogenic markers are expressed depending on the chemical stimulation during tenogenesis. However, there are insufficient studies on the mechanical strain stimulation for tenogenic cell differentiation of TMSCs, although these cells possess advantages as a cell source for generating tendinous tissue. The purpose of this study was to investigate the effects of mechanical strain and transforming growth factor-beta 3 (TGF-β3) on the tenogenic differentiation of TMSCs and evaluate the expression of tendon-related genes and extracellular matrix (ECM) components, such as collagen.ResultsmRNA expression of tenogenic genes was significantly higher when the mechanical strain was applied than under static conditions. Moreover, mRNA expression of tenogenic genes was significantly higher with TGF-β3 treatment than without. mRNA expression of osteogenic and chondrogenic genes was not significantly different among different mechanical strain intensities. In cells without TGF-β3 treatment, double-stranded DNA concentration decreased, while the amount of normalized collagen increased as the intensity of mechanical strain increased.ConclusionsMechanical strain and TGF-β3 have significant effects on TMSC differentiation into tenocytes. Mechanical strain stimulates the differentiation of TMSCs, particularly into tenocytes, and cell differentiation, rather than proliferation. However, a combination of these two did not have a synergistic effect on differentiation. In other words, mechanical loading did not stimulate the differentiation of TMSCs with TGF-β3 supplementation. The effect of mechanical loading with TGF-β3 treatment on TMSC differentiation can be manipulated according to the differentiation stage of TMSCs. Moreover, TMSCs have the potential to be used for cell banking, and compared to other mesenchymal stem cells, they can be procured from patients via less invasive procedures.

Nonwoven-based gelatin/polycaprolactone membrane loaded with ERK inhibitor U0126 for treatment of tendon defects

BackgroundTendon is a major component of musculoskeletal system connecting the muscles to the bone. Tendon injuries are very common orthopedics problems leading to impeded motion. Up to now, there still lacks effective treatments for tendon diseases.MethodsTendon stem/progenitor cells (TSPCs) were isolated from the patellar tendons of SD rats. The expression levels of genes were evaluated by quantitative RT-PCR. Immunohistochemistry staining was performed to confirm the presence of tendon markers in tendon tissues. Bioinformatics analysis of data acquired by RNA-seq was used to find out the differentially expressed genes. Rat patellar tendon injury model was used to evaluate the effect of U0126 on tendon injury healing. Biomechanical testing was applied to evaluate the mechanical properties of newly formed tendon tissues.ResultsIn this study, we have shown that ERK inhibitor U0126 rather PD98059 could effectively increase the expression of tendon-related genes and promote the tenogenesis of TSPCs in vitro. To explore the underlying mechanisms, RNA sequencing was performed to identify the molecular difference between U0126-treated and control TSPCs. The result showed that GDF6 was significantly increased by U0126, which is an important factor of the TGFβ superfamily regulating tendon development and tenogenesis. In addition, NBM (nonwoven-based gelatin/polycaprolactone membrane) which mimics the native microenvironment of the tendon tissue was used as an acellular scaffold to carry U0126. The results demonstrated that when NBM was used in combination with U0126, tendon healing was significantly promoted with better histological staining outcomes and mechanical properties.ConclusionTaken together, we have found U0126 promoted tenogenesis in TSPCs through activating GDF6, and NBM loaded with U0126 significantly promoted tendon defect healing, which provides a new treatment for tendon injury.

Decellularized Human Umbilical Cord Wharton Jelly Scaffold Improves Tendon Regeneration in a Rabbit Rotator Cuff Tendon Defect Model

Background: Owing to limited self-healing capacity, failure of rotator cuff tendon healing is a common complication after surgery. Biological scaffolds have garnered attention owing to their potential to enhance healing outcomes. Purpose: To verify the effect of the decellularized umbilical cord Wharton jelly (DUCWJ) scaffold as a bridging scaffold in a rabbit model of acute rotator cuff tendon defect. Study Design: Controlled laboratory study. Methods: We fabricated a DUCWJ scaffold using a physicochemical decellularized method, evaluating changes in the umbilical cord Wharton jelly before and after decellularization. Scanning electron microscopy and biomechanical testing were performed to determine the microstructure and mechanical properties. We assessed cytocompatibility and cell regulatory behavior of the scaffold toward tendon stem/progenitor cells (TSPCs). A supraspinatus tendon defect was created in 54 New Zealand White rabbits, allocated to the DUCWJ scaffold repair group and the negative control group (without scaffold). Histology, reverse transcription polymerase chain reaction, and biomechanical tensile strength were assessed at 4, 8, and 12 weeks postoperatively. Results: Decellularization completely removed cells from the umbilical cord Wharton jelly, retained a considerable amount of glycosaminoglycan and collagen, and preserved the microstructure and tensile strength. The DUCWJ scaffold facilitated migration and proliferation of TSPCs in vitro. Tendon-related gene expression revealed that the DUCWJ scaffold could maintain the tenocyte phenotype of TSPCs. In the in vivo study, the DUCWJ scaffold improved tendon healing and enhanced the biomechanical strength of repaired tendons. Histological evaluation scores of the DUCWJ group were significantly higher than those of the negative control at 4, 8, and 12 weeks after surgery (P < .05). In repaired tendon tissues, reverse transcription polymerase chain reaction findings revealed that the DUCWJ scaffold stimulated tendon development and maturation. Furthermore, an overall increase in ultimate load and tensile modulus was noted over time; the DUCWJ group presented better results than the negative control group (P < .05). Conclusion: The DUCWJ scaffold has an excellent 3-dimensional porous structure, good biocompatibility, and fundamental biomechanical characteristics, and it promotes migration, attachment, and proliferation of TSPCs. The in vivo animal study demonstrated that the DUCWJ scaffold has potential for tendon regeneration in an acute rotator cuff tendon defect model Clinical Relevance: DUCWJ scaffolds have potential as a regenerative material to augment rotator cuff healing in the clinical setting.

Effects of hypoxia on Achilles tendon repair using adipose tissue-derived mesenchymal stem cells seeded small intestinal submucosa

BackgroundThe study was performed to evaluate the feasibility of utilizing small intestinal submucosa (SIS) scaffolds seeded with adipose-derived mesenchymal stem cells (ADMSCs) for engineered tendon repairing rat Achilles tendon defects and to compare the effects of preconditioning treatments (hypoxic vs. normoxic) on the tendon healing.MethodsFifty SD rats were randomized into five groups. Group A received sham operation (blank control). In other groups, the Achilles tendon was resected and filled with the original tendon (Group B, autograft), cell-free SIS (Group C), or SIS seeded with ADMSCs preconditioned under normoxic conditions (Group D) or hypoxic conditions (Group E). Samples were collected 4 weeks after operation and analyzed by histology, immunohistochemistry, and tensile testing.ResultsHistologically, compared with Groups C and D, Group E showed a significant improvement in extracellular matrix production and a higher compactness of collagen fibers. Group E also exhibited a significantly higher peak tensile load than Groups D and C. Additionally, Group D had a significantly higher peak load than Group C. Immunohistochemically, Group E exhibited a significantly higher percentage of MKX + cells than Group D. The proportion of ADMSCs simultaneously positive for both MKX and CM-Dil observed from Group E was also greater than that in Group D.ConclusionsIn this animal model, the engineered tendon grafts created by seeding ADMSCs on SIS were superior to cell-free SIS. The hypoxic precondition further improved the expression of tendon-related genes in the seeded cells and increased the rupture load after grafting in the Achilles tendon defects.

Human Tendon Stem/Progenitor Cell Features and Functionality Are Highly Influenced by in vitro Culture Conditions.

Our understanding of tendon biology continues to evolve, thus leading to opportunities for developing novel, evidence-based effective therapies for the treatment of tendon disorders. Implementing the knowledge of tendon stem/progenitor cells (TSPCs) and assessing their potential in enhancing tendon repair could fill an important gap in this regard. We described different molecular and phenotypic profiles of TSPCs modulated by culture density, as well as their multipotency and secretory activities. Moreover, in the same experimental setting, we evaluated for different responses to inflammatory stimuli mediated by TNFα and IFNγ. We also preliminarily investigated their immunomodulatory activity and their role in regulating degradation of substance P. Our findings indicated that TSPCs cultured at low density (LD) exhibited cobblestone morphology and a reduced propensity to differentiate. A distinctive immunophenotypic profile was also observed with high secretory and promising immunomodulatory responses when primed with TNFα and IFNγ. In contrast, TSPCs cultured at high density (HD) showed a more elongated fibroblast-like morphology, a greater adipogenic differentiation potential, and a higher expression of tendon-related genes with respect to LD. Finally, HD TSPCs showed immunomodulatory potential when primed with TNFα and IFNγ, which was slightly lower than that shown by LD. A shift from low to high culture density during TSPC expansion demonstrated intermediate features confirming the cellular adaptability of TSPCs. Taken together, these experiments allowed us to identify relevant differences in TSPCs based on culture conditions. This ability of TSPCs to acquire distinguished morphology, phenotype, gene expression profile, and functional response advances our current understanding of tendons at a cellular level and suggests responsivity to cues in their in situ microenvironment.

Remote triggering of TGF-β/Smad2/3 signaling in human adipose stem cells laden on magnetic scaffolds synergistically promotes tenogenic commitment

Injuries affecting load bearing tendon tissues are a significant clinical burden and efficient treatments are still unmet. Tackling tendon regeneration, tissue engineering strategies aim to develop functional substitutes that recreate native tendon milieu. Tendon mimetic scaffolds capable of remote magnetic responsiveness and functionalized magnetic nanoparticles (MNPs) targeting cellular mechanosensitive receptors are potential instructive tools to mediate mechanotransduction in guiding tenogenic responses. In this work, we combine magnetically responsive scaffolds and targeted Activin A type II receptor in human adipose stem cells (hASCs), under alternating magnetic field (AMF), to synergistically facilitate external control over signal transduction. The combination of remote triggering TGF-β/Smad2/3 using MNPs tagged hASCs, through magnetically actuated scaffolds, stimulates overall expression of tendon related genes and the deposition of tendon related proteins, in comparison to non-stimulated conditions. Moreover, the phosphorylation of Smad2/3 proteins and their nuclear co-localization was also more evident. Overall, biophysical stimuli resulting from magnetic scaffolds and magnetically triggered cells under AMF stimulation modulate the mechanosensing response of hASCs towards tenogenesis, holding therapeutic promise. Statement of SignificanceThe concept of magnetically-assisted tissue engineering may assist the development of innovative solutions to treat tendon disorders upon remote control of biological processes as cell migration or differentiation. Herein, we originally combine a fibrous aligned superparamagnetic scaffold, based on a biodegradable polymeric blend of starch and poly-ɛ-caprolactone incorporating magnetic nanoparticles (MNPs), and human adipose stem cells (hASCs) labelled with MNPs functionalized with anti-activin receptor type IIA (ActRIIA). Constructs were stimulated using alternating magnetic field (AMF), to activate the ActRIIA and subsequent induction of TGF-β signaling, through Smad2/3 phosphorylation cascade, enhancing the expression of tendon-related markers. Altogether, these findings contribute with powerful bio-magnetic approaches to activate key tenogenic pathways, envisioning future translation of magnetic biomaterials into regenerative platforms for tendon repair.

In vitro Neo-Genesis of Tendon/Ligament-Like Tissue by Combination of Mohawk and a Three-Dimensional Cyclic Mechanical Stretch Culture System.

Tendons and ligaments are pivotal connective tissues that tightly connect muscle and bone. In this study, we developed a novel approach to generate tendon/ligament-like tissues with a hierarchical structure, by introducing the tendon/ligament-specific transcription factor Mohawk (MKX) into the mesenchymal stem cell (MSC) line C3H10T1/2 cells, and by applying an improved three-dimensional (3D) cyclic mechanical stretch culture system. In our developed protocol, a combination of stable Mkx expression and cyclic mechanical stretch synergistically affects the structural tendon/ligament-like tissue generation and tendon related gene expression. In a histological analysis of these tendon/ligament-like tissues, an organized extracellular matrix (ECM), containing collagen type III and elastin, was observed. Moreover, we confirmed that Mkx expression and cyclic mechanical stretch, induced the alignment of structural collagen fibril bundles that were deposited in a fibripositor-like manner during the generation of our tendon/ligament-like tissues. Our findings provide new insights for the tendon/ligament biomaterial fields.

High cholesterol induces apoptosis and autophagy through the ROS-activated AKT/FOXO1 pathway in tendon-derived stem cells

BackgroundHypercholesterolemia increases the risk of tendon pain and tendon rupture. Tendon-derived stem cells (TDSCs) play a vital role in the development of tendinopathy. Our previous research found that high cholesterol inhibits tendon-related gene expression in TDSCs. Whether high cholesterol has other biological effects on TDSCs remains unknown.MethodsTDSCs isolated from female SD rats were exposed to 10 mg/dL cholesterol for 24 h. Then, cell apoptosis was assessed using flow cytometry and fluorescence microscope. RFP-GFP-LC3 adenovirus transfection was used for measuring autophagy. Signaling transduction was measured by immunofluorescence and immunoblotting. In addition, Achilles tendons from ApoE −/− mice fed with a high-fat diet were histologically assessed using HE staining and immunohistochemistry.ResultsIn this work, we verified that 10 mg/dL cholesterol suppressed cell proliferation and migration and induced G0/G1 phase arrest. Additionally, cholesterol induced apoptosis and autophagy simultaneously in TDSCs. Apoptosis induction was related to increased expression of cleaved caspase-3 and BAX and decreased expression of Bcl-xL. The occurrence of autophagic flux and accumulation of LC3-II demonstrated the induction of autophagy by cholesterol. Compared with the effects of cholesterol treatment alone, the autophagy inhibitor 3-methyladenine (3-MA) enhanced apoptosis, while the apoptosis inhibitor Z-VAD-FMK diminished cholesterol-induced autophagy. Moreover, cholesterol triggered reactive oxygen species (ROS) generation and activated the AKT/FOXO1 pathway, while the ROS scavenger NAC blocked cholesterol-induced activation of the AKT/FOXO1 pathway. NAC and the FOXO1 inhibitor AS1842856 rescued the apoptosis and autophagy induced by cholesterol. Finally, high cholesterol elevated the expression of cleaved caspase-3, Bax, LC3-II, and FOXO1 in vivo.ConclusionThe present study indicated that high cholesterol induced apoptosis and autophagy through ROS-activated AKT/FOXO1 signaling in TDSCs, providing new insights into the mechanism of hypercholesterolemia-induced tendinopathy.Graphical abstractHigh cholesterol induces apoptosis and autophagy through the ROS-activated AKT/FOXO1 pathway in tendon-derived stem cells.

Early Growth Response Genes Increases Rapidly After Mechanical Overloading and Unloading in Tendon Constructs.

Tendon cells exist in a dense extracellular matrix and mechanical loading is important for the strength development of this matrix. We therefore use a three-dimensional (3D) culture system for tendon formation in vitro. The objectives of this study were to elucidate the temporal expression of tendon-related genes during the formation of artificial tendons in vitro and to investigate if early growth response-1 (EGR1), EGR2, FOS, and cyclooxygenase-1 and -2 (PTGS1 and PTGS2) are sensitive to mechanical loading. First, we studied messenger RNA (mRNA) levels of several tendon-related genes during formation of tendon constructs. Second, we studied the mRNA levels of, for example, EGR1 and EGR2 after different degrees of loading; dynamic physiologic-range loading (2.5% strain), dynamic overloading (approximately 10% strain), or tension release. The gene expression for tendon-related genes (i.e., EGR2, MKX, TNMD, COL3A1) increased with time after seeding into this 3D model. EGR1, EGR2, FOS, PTGS1, and PTGS2 did not respond to physiologic-range loading. But overloading (and tension release) lead to elevated levels of EGR1 and EGR2 (p ≤ 0.006). FOS and PTGS2 were increased after overloading (both p < 0.007) but not after tension release (p = 0.06 and 0.08). In conclusion, the expression of tendon-related genes increases during the formation of artificial tendons in vitro, including EGR2. Furthermore, the gene expression of EGR1 and EGR2 in human tendon cells appear to be sensitive to overloading and unloading but did not respond to the single episode of physiologic-range loading. These findings could be helpful for the understanding of tendon tensional homeostasis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:173-181, 2020.

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

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

Effect of micro-RNA on tenocytes and tendon-related gene expression: A systematic review.

The purpose of the review was to synthesize the current literature regarding the effect of miRNA on biological processes known to be involved in tendon and tenocyte development and homeostasis. Using multiple databases, a systematic review was performed with a customized search term crafted to identify any study examining micro-RNA in relation to tendon and/or tenocytes. Results were classified based on the following categories: Gene expression, tenocyte development and differentiation, tendon tissue repair, and tenocyte senescence. A total of 3,112 potentially relevant studies were reviewed, and after exclusion criteria was applied, 15 investigations were included in the final analysis. There were 14 specific miRNA included in this review, with 11 studies reporting on tendon-related gene expression, five reporting on tendon development and/or tenocyte differentiation, six reporting on tendon tissue repair, and five reporting on tenocyte senescence. The miR-29 family was the most commonly reported micro-RNA in the investigation. We also report on a number of micro-RNA which are associated with both positive and negative effects on tendon homeostasis. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2823-2829, 2018.

RhPDGF-BB combined with ADSCs in the treatment of Achilles tendinitis via miR-363/PI3K/Akt pathway.

To investigate the mechanism of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) and human adipose-derived stem cells (hADSCs) in the treatment of Achilles tendinitis. Biomechanical indices of stiffness, stress, and maximum load-to-failure were detected by biomechanical test. mRNA and protein levels of miR-363, p-PI3K/AKT, tendon-related genes Collagen I, Scleraxis (Scx), and Tenascin C (TNC) were measured by qRT-PCR and western blot. The proliferation of hADSCs was accessed by MTT assay. Biomechanical indices of stiffness, stress, and maximum load-to-failure, and mRNA and protein levels of tendon-related genes could be improved by rhPDGF-BB or hADSCs alone, and could be further improved by rhPDGF-BB+hADSCs. rhPDGF-BB and hADSCs downregulated the expression of miR-363 and upregulated the levels of p-PI3K/Akt, and rhPDGF-BB+hADSCs further strengthened these effects. In addition, rhPDGF-BB promoted the proliferation of hADSCs in vitro and upregulated the expression of tendon-related genes. miR-363 mimic downregulated the levels of p-PI3K/Akt, miR-363 inhibitor upregulated the levels of p-PI3K/Akt, and miR-363 mimic and PI3K/Akt pathway inhibitor LY294002 reversed the positive effect of rhPDGF-BB on the proliferation of hADSCs, which suggested that rhPDGF-BB promoted the proliferation of hADSCs via miR-363/PI3K/Akt pathway. Biomechanical indices and tendon-related genes could be improved by rhPDGF-BB and hADSCs. Moreover, rhPDGF-BB promoted the proliferation of hADSCs via miR-363/PI3K/Akt pathway, indicating that rhPDGF-BB combined with ADSCs could treat Achilles tendinitis via miR-363/PI3K/Akt pathway.

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.