Influence of mechanical and TGF-β3 stimulation on the tenogenic differentiation of tonsil-derived mesenchymal stem cells

Background Organogenesis 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. Results mRNA 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. Conclusions Mechanical 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.

Bioinspired Bimodal Micro-Nanofibrous Scaffold Promotes Tenogenic Differentiation of Tendon Stem/Progenitor Cells for Achilles Tendon Regeneration

Poor tendon repair remains a clinical problem due to the difficulties in replicating the complex multiscale hierarchical structure of native tendon. Herein, a bioinspired fibrous scaffold with bimodal micro-nanofibers and...

Mesenchymal Stromal Cells Adapt to Chronic Tendon Disease Environment with an Initial Reduction in Matrix Remodeling

Tendon lesions are common sporting injuries in humans and horses alike. The healing process of acute tendon lesions frequently results in fibrosis and chronic disease. In horses, local mesenchymal stromal cell (MSC) injection is an accepted therapeutic strategy with positive influence on acute lesions. Concerning the use of MSCs in chronic tendon disease, data are scarce but suggest less therapeutic benefit. However, it has been shown that MSCs can have a positive effect on fibrotic tissue. Therefore, we aimed to elucidate the interplay of MSCs and healthy or chronically diseased tendon matrix. Equine MSCs were cultured either as cell aggregates or on scaffolds from healthy or diseased equine tendons. Higher expression of tendon-related matrix genes and tissue inhibitors of metalloproteinases (TIMPs) was found in aggregate cultures. However, the tenogenic transcription factor scleraxis was upregulated on healthy and diseased tendon scaffolds. Matrix metalloproteinase (MMPs) expression and activity were highest in healthy scaffold cultures but showed a strong transient decrease in diseased scaffold cultures. The release of glycosaminoglycan and collagen was also higher in scaffold cultures, even more so in those with tendon disease. This study points to an early suppression of MSC matrix remodeling activity by diseased tendon matrix, while tenogenic differentiation remained unaffected.

Mechanical activation drives tenogenic differentiation of human mesenchymal stem cells in aligned dense collagen hydrogels

Tendons are force transmitting mechanosensitive tissues predominantly comprised of highly aligned collagen type I fibres. In this study, the recently introduced gel aspiration-ejection method was used to rapidly fabricate aligned dense collagen (ADC) hydrogel scaffolds. ADCs provide a biomimetic environment compared to traditional collagen hydrogels that are mechanically unstable and comprised of randomly oriented fibrils. The ADC scaffolds were shown to be anisotropic with comparable stiffness to immature tendons. Furthermore, the application of static and cyclic uniaxial loading, short-term (48 h) and high-strain (20%), resulted in a 3-fold increase in both the ultimate tensile strength and modulus of ADCs. Similar mechanical activation of human mesenchymal stem cell (MSC) seeded ADCs in serum- and growth factor-free medium induced their tenogenic differentiation. Both static and cyclic loading profiles resulted in a greater than 12-fold increase in scleraxis gene expression and either suppressed or maintained osteogenic and chondrogenic expressions. Following the 48 h mechanoactivation period, the MSC-seeded scaffolds were matured by tethering in basal medium without further external mechanical stimulation for 19 days, altogether making up 21 days of culture. Extensive cell-induced matrix remodeling and deposition of collagens type I and III, tenascin-C and tenomodulin were observed, where initial cyclic loading induced significantly higher tenomodulin protein content. Moreover, the initial short-term mechanical stimulation elongated and polarized seeded MSCs and overall cell alignment was significantly increased in those under static loading. These findings indicate the regenerative potential of the ADC scaffolds for short-term mechanoactivated tenogenic differentiation, which were achieved even in the absence of serum and growth factors that may potentially increase clinical translatability.

The 532 Nm Laser Treatment Promotes the Proliferation of Tendon- Derived Stem Cells and Up-Regulates Nr4a1 to Stimulate Tenogenic Differentiation

Background: The combination of low-level laser therapy (LLLT) and stem cell transplantation with tendon-derived stem cells (TDSCs) as seed cells provides a new treatment strategy for tendon injury. Nevertheless, the effect of LLLT on the biological behavior of TDSCs and its internal mechanisms remain unclear. This study aimed to verify the effect of LLLT with a wavelength of 532 nm on the proliferation and differentiation of TDSCs of Sprague-Dawley (SD) rats. Methods: TDSCs were isolated from Achilles tendons of SD rats and identified by cell morphology and flow cytometric analysis. Energy density gradient experiment was performed to determine the ideal energy. Then TDSCs were treated with LLLT using a wavelength of 532 nm at a fluence of 15 J/cm2. Cell response after irradiation was observed at 6, 12 and 24 hours to ascertain cell morphology and cell proliferation. The RNA expression levels of the key genes of TDSCs differentiation, including Scx, Tnmd, Mkx and Dcn, PPARγ, Sox9 and Runx2, were detected by RT-PCR. Then gene chip microarray was used to detect the expression of differential genes after 532 nm laser intervention in TDSCs, and the target genes were screened out to verify the role of target genes in this process.Results: When the 532 nm laser energy density was 15 J/cm2, the proliferation capacity of TDSCs was improved (2.73 ± 0.24 vs. 1.81 ± 0.71, P < 0.05), and the expression of genes related to tenogenic differentiation of TDSCs was significantly increased (P < 0.01), showing the potential of tenogenic differentiation. After RNA-seq and bioinformatics analyses, we speculated that Nr4a1 was involved in the tenogenic differentiation process of TDSCs regulated by 532 nm laser treatment. Subsequent experiments confirmed that Nr4a1 regulated the expression of the tenogenic differentiation genes scleraxis (Scx) and tenomodulin (Tnmd) in TDSCs, affecting the process. Conclusion: A 532 nm laser with 15 J/cm2 regulated the process of TDSC proliferation and tenogenic differentiation by up-regulating Nr4a1, which could accelerate tendon healing.

The 532 nm Laser Treatment Promotes The Proliferation of Tendon-Derived Stem Cells and Up-Regulates Nr4a1 To Stimulate Tenogenic Differentiation

Background The combination of low-level laser therapy (LLLT) and stem cell transplantation with tendon-derived stem cells (TDSCs) as seed cells provides a new treatment strategy for tendon injury. Nevertheless, the effect of LLLT on the biological behavior of TDSCs and its internal mechanisms remain unclear. The purpose of this study was to verify the effect of LLLT with a wavelength of 532 nm on the proliferation and differentiation of TDSCs of Sprague-Dawley (SD) rats. Methods TDSCs were isolated from Achilles tendons of SD rats and identified by cell morphology and flow cytometric analysis. Energy density gradient experiment was performed to determine the ideal energy. Then TDSCs were treated with LLLT using a wavelength of 532 nm at a fluence of 15J/cm2. Cell response after irradiation was observed at 6, 12 and 24 hours to ascertain cell morphology and cell proliferation. RT-PCR was used to detect the RNA expression levels of the key genes of TDSCs differentiation including Scx, Tnmd, Mkx and Dcn, PPARγ, Sox9 and Runx2. Then gene chip microarray was used to detect the expression of differential genes after 532 nm laser intervention in TDSCs, and the target genes were screened out to verify the role of target genes in this process. Results When the 532 nm laser energy density was 15 J/cm2, the proliferation capacity of TDSCs was improved (2.73 ± 0.24 vs. 1.81 ± 0.71, P < 0.05), and the expression of genes related to tenogenic differentiation of TDSCs was significantly increased (P < 0.01), showing the potential of tenogenic differentiation. After RNA-seq and bioinformatics analyses, we speculated that Nr4a1 was involved in the tenogenic differentiation process of TDSCs regulated by 532 nm laser treatment. Subsequent experiments confirmed that Nr4a1 regulated the expression of the tenogenic differentiation genes scleraxis (Scx) and tenomodulin (Tnmd) in TDSCs, affecting the process. Conclusion A 532 nm laser with 15J/cm2 regulated the process of TDSC proliferation and tenogenic differentiation by up-regulating Nr4a, which could accelerate tendon healing.

Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation in vivo. The transforming growth factor (TGF)-β, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-β3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-β3-induced tenogenic differentiation. The MSC were incubated with and without TGF-β3 (10 ng/ml), Y-27632 (10 μM), or both. On day 1 and day 3, the signalling pathway of TGF-β and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-β3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species ( p < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-β3 and ROCK inhibition were combined ( p < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-β3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-β3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.

Long noncoding RNA H19 accelerates tenogenic differentiation by modulating miR-140-5p/VEGFA signaling

Rotator cuff tear (RCT) is a common tendon injury, but the mechanisms of tendon healing remain incompletely understood. Elucidating the molecular mechanisms of tenogenic differentiation is essential to develop novel therapeutic strategies in clinical treatment of RCT. The long noncoding RNA H19 plays a regulatory role in tenogenic differentiation and tendon healing, but its detailed mechanism of action remains unknown. To elucidate the role of H19 in tenogenic differentiation and tendon healing, tendon-derived stem cells were harvested from the Achilles tendons of Sprague Dawley rats and a rat model of cuff tear was established for the exploration of the function of H19 in promoting tenogenic differentiation. The results showed that H19 overexpression promoted, while H19 silencing suppressed, tenogenic differentiation of tendon-derived stem cells (TDSCs). Furthermore, bioinformatic analyses and a luciferase reporter gene assay showed that H19 directly targeted and inhibited miR-140-5p to promote tenogenic differentiation. Further, inhibiting miR-140-5p directly increased VEGFA expression, revealing a novel regulatory axis between H19, miR-140-5p, and VEGFA in modulating tenogenic differentiation. In rats with RTC, implantation of H19-overexpressing TDSCs at the lesion promoted tendon healing and functional recovery. In general, the data suggest that H19 promotes tenogenic differentiation and tendon-bone healing by targeting miR-140-5p and increasing VEGFA levels. Modulation of the H19/miR-140-5p/VEGFA axis in TDSCs is a new potential strategy for clinical treatment of tendon injury.

Oxo-M and 4-PPBP Delivery via Multi-Domain Peptide Hydrogel Toward Tendon Regeneration

We have recently identified novel small molecules, Oxo-M and 4-PPBP, which specifically stimulates endogenous tendon stem/progenitor cells (TSCs) leading to potential regenerative healing of fully-transected tendons. Here we investigated an injectable, multi-domain peptide (MDP) hydrogel providing a controlled delivery of the small molecules for regenerative tendon healing. We investigated the release kinetics of Oxo-M and 4-PPBP from MDP hydrogels and the effect of MDP-released small molecules on tenogenic differentiation of TSCs and in vivo tendon healing. In vitro, MDP showed a sustained release of Oxo-M and 4-PPBP and a slower degradation compared to fibrin. In addition, tenogenic gene expression was significantly increased in TSC with MDP-released Oxo-M and 4-PPBP as compared to the fibrin-released. In vivo, MDP releasing Oxo-M and 4-PPBP significantly improved tendon healing, likely associated with prolonged effects of Oxo-M and 4-PPBP on suppression of M1 macrophages and promotion of M2 macrophages. Comprehensive analyses including histomorphology, digital image processing, and modulus mapping with nanoindentation consistently suggested that Oxo-M and 4-PPBP delivered via MDP further improved tendon healing as compared to fibrin-based delivery. In conclusion, MDP delivered with Oxo-M and 4-PPBP may serve as an efficient regenerative therapeutic for in situ tendon regeneration and healing.

Altered Differentiation of Tendon-Derived Stem Cells in Diabetic Conditions Mediated by Macrophage Migration Inhibitory Factor

The purpose of our study was to evaluate the role of macrophage migration inhibitory factor (MIF) in the differentiation of tendon-derived stem cells (TdSCs) under hyperglycemic conditions. In the in vivo experiment, rats were classified into diabetic (DM) and non-DM groups depending on the intraperitoneal streptozotocin (STZ) or saline injection. Twelve-week after STZ injection, the supraspinatus tendon was harvested and prepared for histological evaluation and real-time reverse transcription polymerase chain reaction for osteochondrogenic (aggrecan, BMP-2, and Sox9) and tenogenic (Egr1, Mkx, scleraxis, type 1 collagen, and Tnmd) markers. For the in vitro experiment, TdSCs were isolated from healthy rat Achilles tendons. Cultured TdSCs were treated with methylglyoxal and recombinant MIF or MIF gene knockdown to determine the effect of hyperglycemic conditions and MIF on the differentiation function of TdSCs. These conditions were classified into four groups: hyperglycemic-control group, hyperglycemic-recombinant-MIF group, hyperglycemic-knockdown-MIF group, and normal-control group. The mRNA expression of osteochondrogenic and tenogenic markers was compared among the groups. In the in vivo experiment, the mRNA expression of all osteochondrogenic and tenogenic differentiation markers in the DM group was significantly higher and lower than that in the non-DM group, respectively. Similarly, in the in vitro experiments, the expression of all osteochondrogenic and tenogenic differentiation markers was significantly upregulated and downregulated, respectively, in the hyperglycemic-control group compared to that in the normal-control group. The hyperglycemic-knockdown-MIF group demonstrated significantly decreased expression of all osteochondrogenic differentiation markers and increased expression of only some tenogenic differentiation markers compared with the hyperglycemic-control group. In contrast, the hyperglycemic-recombinant-MIF group showed significantly increased expression of all osteochondrogenic differentiation markers, but no significant difference in any tenogenic marker level, compared to the hyperglycemic-control group. These results suggest that tendon homeostasis could be affected by hyperglycemic conditions, and MIF appears to alter the differentiation of TdSCs via enhancement of the osteochondrogenic differentiation in hyperglycemic conditions. These are preliminary findings, and must be confirmed in a further study.