Differentiation Of Tendon Stem Cells Research Articles

Regulation of Tendon Stem Cell Behavior by Designed Nanoporous Topography of Microfibers.

Nano scale topography scaffold is more bioactive and biomimetic than smooth fiber topographies. Tendon stem cells (TSCs) play important roles in the tendinogenesis of tendon tissue engineering, but the effects and mechanisms of nano topography on TSC behavior are still unclear. This study determined whether the morphology, proliferation, cytoskeleton, and differentiation of TSCs are affected by topography of scaffold in vitro. The porous PA56 scaffolds were prepared with different concentration ratios of glycerol as the molecular template by electrospinning. Its topological characteristics, hydrophilicity, and degradation properties varied with glycerol proportion and movement rate of the receiving plate. Porous fibers promoted the proliferation of TSCs and the number of TSCs varied with topography. Although there was no significant difference due to the small sample size, the number of pseudopodia and cell polarizability still showed differences among different topographies. The morphology of actin cytoskeleton of TSCs showed difference among cultured on porous fibers, smooth fibers, and in culture media with no fiber, suggesting the orientation growth of cells on porous fiber. Moreover, porous fibers promoted teno-lineage differentiation of TSCs by upregulating tendon-specific gene expression. These findings provide evidence that nano porous topography scaffold promotes TSC proliferation, cytoskeleton orientation, and tenogenic differentiation.

Verteporfin attenuates trauma-induced heterotopic ossification of Achilles tendon by inhibiting osteogenesis and angiogenesis involving YAP/β-catenin signaling.

Heterotopic ossification occurs as a pathological ossification condition characterized by ectopic bone formation within soft tissues following trauma. Vascularization has long been established to fuel skeletal ossification during tissue development and regeneration. However, the feasibility of vascularization as a target of heterotopic ossification prevention remained to be further clarified. Here, we aimed to identify whether verteporfin as a widely used FDA-approved anti-vascularization drug could effectively inhibit trauma-induced heterotopic ossification formation. In the current study, we found that verteporfin not only dose dependently inhibited the angiogenic activity of human umbilical vein endothelial cells (HUVECs) but also the osteogenic differentiation of tendon stem cells (TDSCs). Moreover, YAP/β-catenin signaling axis was downregulated by the verteporfin. Application of lithium chloride, an agonist of β-catenin, recovered TDSCs osteogenesis and HUVECs angiogenesis that was inhibited by verteporfin. In vivo, verteporfin attenuated heterotopic ossification formation by decelerating osteogenesis and the vessels densely associated with osteoprogenitors formation, which could also be readily reversed by lithium chloride, as revealed by histological analysis and Micro-CT scan in a murine burn/tenotomy model. Collectively, this study confirmed the therapeutic effect of verteporfin on angiogenesis and osteogenesis in trauma-induced heterotopic ossification. Our study sheds light on the anti-vascularization strategy with verteporfin as a candidate treatment for heterotopic ossification prevention.

A unidirectional drug‐release Janus membrane based on hydrogen bonding barrier effect for preventing postoperative adhesion and promoting tissue repair

AbstractBarrier membranes incorporated with anti‐adhesion drugs have been widely used for preventing postoperative adhesion. However, the bidirectional release of drugs may interfere with internal tissue healing. Herein, a unidirectional Janus membrane composed of two functional layers is prepared by sequential electrospinning. The outer layer is designed as random polycaprolactone fibers incorporated with tannic acid/Fe3+ particles to prevent tissue adhesion, while the inner layer is designed as oriented gelatin fibers to promote tissue repair. Hydrogen bonding was employed to act as a barrier for preventing the diffusion of the tannic acid to the inner side, and thus the release of tannic acid occurs in a unidirectional manner. As a result, asymmetric biological performances of the Janus membrane are achieved. The outer face can provide anti‐inflammatory capability to the membrane and inhibit the viability of fibroblasts, while the inner face can promote the proliferation and differentiation of tendon stem cells. Tendon injured model confirms the prominent inflammation modulation, adhesion prevention, and repair promotion capabilities of the Janus membrane in vivo. This study provides a new strategy to develop advanced functional barrier membranes for postoperative adhesion prevention and internal tissue repair promotion.

LncRNA MEG3 Promotes Osteogenic Differentiation of Tendon Stem Cells Via the miR-129-5p/TCF4/β-Catenin Axis and thus Contributes to Trauma-Induced Heterotopic Ossification.

Heterotopic ossification (HO) is one of the most intractable conditions following injury to the musculoskeletal system. In recent years, much attention has been paid to the role of lncRNA in musculoskeletal disorders, but its role in HO was still unclear. Therefore, this study attempted to determine the role of lncRNA MEG3 in the formation of post-traumatic HO and further explore the underlying mechanisms. On the basis of high-throughput sequencing and qPCR validation, elevated expression of the lncRNA MEG3 was shown during traumatic HO formation. Accordingly, in vitro experiments demonstrated that lncRNA MEG3 promoted aberrant osteogenic differentiation of tendon-derived stem cells (TDSCs). Mechanical exploration through RNA pulldown, luciferase reporter gene assay and RNA immunoprecipitation assay identified the direct binding relationship between miR-129-5p and MEG3, or miR-129-5p and TCF4. Further rescue experiments confirmed the miR-129-5p/TCF4/β-catenin axis to be downstream molecular cascade responsible for the osteogenic-motivating effects of MEG3 on the TDSCs. Finally, experiments in a mouse burn/tenotomy model corroborated the promoting effects of MEG3 on the formation of HO through the miR-129-5p/TCF4/β-catenin axis. Our study demonstrated that the lncRNA MEG3 promoted osteogenic differentiation of TDSCs and thus the formation of heterotopic ossification, which could be a potential therapeutic target.

Caveolin-1 is involved in fatty infiltration and bone-tendon healing of rotator cuff tear

BackgroundCaveolin-1 has been predicted, based on RNA transcriptome sequencing, as a key gene in rotator cuff tear (RCT) and it is related to fatty infiltration. This study aims to elucidate the upstream and downstream mechanism of Caveolin-1 in fatty infiltration and bone-tendon healing after RCT in rat models.MethodsDifferentially expressed genes related to RCT were screened, followed by functional enrichment analysis and protein-protein interaction analysis. GATA6 was overexpressed and Caveolin-1 was knocked down in tendon stem cells (TSCs) to evaluate their effects on the adipogenic differentiation of TSCs. In addition, a RCT rat model was constructed and injected with lentivirus carrying oe-GATA6, oe-Caveolin-1 alone or in combination to assess their roles in fatty infiltration and bone-tendon healing.Results and conclusionCaveolin-1 was identified as a key gene involved in the RCT process. In vitro results demonstrated that Caveolin-1 knockdown inhibited adipogenic differentiation of TSCs by activating the cAMP/PKA pathway. GATA6 inhibited the transcription of Caveolin-1 and inhibited its expression, thus suppressing the adipogenic differentiation of TSCs. In vivo data confirmed that GATA6 overexpression activated the cAMP/PKA pathway by downregulating Caveolin-1 and consequently repressed fatty infiltration, promoted bone-tendon healing, improved biomechanical properties and reduced the rupture risk of injured tendon in rats after RCT. Overall, this study provides novel insights into the mechanistic action of Caveolin-1 in the fatty infiltration and bone-tendon healing after RCT.

Scavenging of reactive oxygen species can adjust the differentiation of tendon stem cells and progenitor cells and prevent ectopic calcification in tendinopathy

Tendinopathy is a common disorder that leads to pain and impaired quality of life. Recent studies revealed that osteogenic differentiation of tendon stem/progenitor cells (TSPCs) played an important role in the pathogenesis of tendon calcification and tendinopathy. In this study, we found that the growth hormone-releasing hormone agonist (GA) can prevent matrix degradation and osteogenic differentiation in TSPCs. As oxidative stress is a key factor in the osteogenic differentiation of TSPCs, we used bovine serum albumin/heparin nanoparticles (BHNPs), which have biocompatibility and drug loading capacity, to scavenge reactive oxygen species (ROS) and achieve sustained release of GA at the site of inflammation. The newly developed BHNPs@GA had a synergetic effect on reducing ROS production in TSPCs. In addition, BHNPs@GA effectively inhibited tendon calcification and promoted collagen formation in a rat model of tendinopathy. Focusing on the ROS underlying the differentiation and dedifferentiation of TSPCs, this work demonstrated that sustained release of GA targeting ROS and ectopic ossification is a practical therapeutic strategy for treating tendinopathy. Statement of SignificanceOsteogenic differentiation of tendon stem/progenitor cells (TSPCs) plays an important role in the pathogenesis of ectopic calcification in tendinopathy. In this study, we found that growth hormone-releasing hormone agonist (GA) can reduce reactive oxygen species (ROS) production and adjust TSPCs differentiation. Bovine serum albumin/heparin nanoparticles (BHNPs) were developed to encapsulate GA and achieve sustained release of GA at the site of inflammation. The developed compound, BHNPs@GA, with a synergistic effect of inhibiting ROS and thus, can effectively adjust TSPCs differentiation, inhibit tendon calcification, and promote collagen formation in tendinopathy. This study highlighted the role of ROS underlying the differentiation and dedifferentiation of TSPCs in tendinopathy, and findings may help to identify new therapeutic targets and develop novel strategy for treating tendinopathy.

TGFβ1-transfected tendon stem cells promote tendon fibrosis

BackgroundIn aged people, tendon injuries frequently occur during sporting and daily activities. In clinical practice, typical physiotherapeutic, pharmacotherapeutic, and surgical techniques do not result in the full recovery of injured tendons, which may lead to chronic degenerative disease.MethodsWe first isolated tendon stem cells (TSCs) from rats and transfected them with the TGFβ1 gene, resulting in TGFβ1-TSCs. The proliferation of TSCs was detected using the Cell Counting Kit 8, and TSCs were identified by immunofluorescence analysis and differentiation capacity analysis. Aggrecan, COL2A1, alpha smooth muscle actin (α-SMA), and p-Smad2 expression levels were detected using western blotting and quantitative reverse transcription polymerase chain reaction. Additionally, a tendon injury model was generated to explore the effect of TGFβ1 on the repair of the tendon by TSCs.ResultsCompared with fibrinogen treatment, TSC + fibrinogen or TGFβ1-TSC + fibrinogen treatment significantly promoted the fibrosis of injured tendons, as evidenced by histological analyses, with TGFβ1-TSC + fibrinogen having a greater effect than TSC + fibrinogen. In TGFβ1-TSCs, increased expression levels of aggrecan and COL2A1 indicated that TGFβ1 signaling induced chondrogenic differentiation. Meanwhile, the increased collagen and α-SMA protein levels indicated that TGFβ1 promoted fibrogenesis. Additionally, TGFβ1 stimulated the production of phosphorylated Smad2 in TSCs, which suggested that the chondrogenic and fibrogenic differentiation of TSCs, as well as tissue regeneration, may be associated with the TGFβ1/Smad2 pathway.ConclusionTGFβ1-TSC therapy may be a candidate for effective tendon fibrosis.

Palovarotene Can Attenuate Heterotopic Ossification Induced by Tendon Stem Cells by Downregulating the Synergistic Effects of Smad and NF-κB Signaling Pathway following Stimulation of the Inflammatory Microenvironment.

Heterotopic ossification (HO) is defined as the formation of bone tissues outside the bones, such as in the muscles. Currently, the mechanism of HO is still unclear. Tendon stem cells (TSCs) play important roles in the occurrence and development of HO. The inflammatory microenvironment dominated by macrophages also plays an important role in the course of HO. The commonly used clinical treatment methods, such as nonsteroidal anti-inflammatory drugs and radiotherapy, have relatively large side effects, and more efficient treatment methods are needed in clinical practice. Under physiological conditions, retinoic acid receptor (RAR) signal transduction pathway inhibits osteogenic progenitor cell aggregation and chondrocyte differentiation. We focus on palovarotene, a retinoic acid γ-receptor activator, showing an inhibitory effect on HO mice, but the specific mechanism is still unclear. This study was aimed at exploring the specific molecular mechanism of palovarotene by blocking osteogenic differentiation and HO formation of TSCs in vitro and in vivo in an inflammatory microenvironment. We constructed a coculture model of TCSs and polarized macrophages, as well as overexpression and knockdown models of the Smad signaling pathway of TCSs. In addition, a rat model of HO, which was constructed by Achilles tendon resection, was also established. These models explored the role of inflammatory microenvironment and Smad signaling pathways in the osteogenic differentiation of TSCs which lead to HO, as well as the reversal role played by palovarotene in this process. Our results suggest that, under the stimulation of inflammatory microenvironment and trauma, the injured site was in an inflammatory state, and macrophages were highly concentrated in the injured site. The expression of osteogenic and inflammation-related proteins, as well as Smad proteins, was upregulated. Osteogenic differentiation was performed in TCSs. We also found that TCSs activated Smad and NF-κB signaling pathways, which initiated the formation of HO. Palovarotene inhibited the aggregation of osteogenic progenitor cells and macrophages and attenuated HO by blocking Smad and NF-κB signaling pathways. Therefore, palovarotene may be a novel HO inhibitor, while other drugs or antibodies targeting Smad and NF-κB signaling pathways may also prevent or treat HO. The expressions of Smad5, Id1, P65, and other proteins may predict HO formation.

Adipose-derived mesenchymal stromal cell-derived exosomes promote tendon healing by activating both SMAD1/5/9 and SMAD2/3

BackgroundThe use of adipose-derived mesenchymal stromal cell-derived exosomes (ADSC-Exos) may become a new therapeutic method in biomedicine owing to their important role in regenerative medicine. However, the role of ADSC-Exos in tendon repair has not yet been evaluated. Therefore, we aimed to clarify the healing effects of ADSC-Exos on tendon injury.MethodsThe adipose-derived mesenchymal stromal cells (ADSCs) and tendon stem cells (TSCs) were isolated from the subcutaneous fat and tendon tissues of Sprague-Dawley rats, respectively, and exosomes were isolated from ADSCs. The proliferation and migration of TSCs induced by ADSC-Exos were analyzed by EdU, cell scratch, and transwell assays. We used western blot to analyze the tenogenic differentiation of TSCs and the role of the SMAD signaling pathways. Then, we explored a new treatment method for tendon injury, combining exosome therapy with local targeting using a biohydrogel. Immunofluorescence and immunohistochemistry were used to detect the expression of inflammatory and tenogenic differentiation after tendon injury, respectively. The quality of tendon healing was evaluated by hematoxylin-eosin (H&E) staining and biomechanical testing.ResultsADSC-Exos could be absorbed by TSCs and promoted the proliferation, migration, and tenogenic differentiation of these cells. This effect may have depended on the activation of the SMAD2/3 and SMAD1/5/9 pathways. Furthermore, ADSC-Exos inhibited the early inflammatory reaction and promoted tendon healing in vivo.ConclusionsOverall, we demonstrated that ADSC-Exos contributed to tendon regeneration and provided proof of concept of a new approach for treating tendon injuries.

Aspirin promotes tenogenic differentiation of tendon stem cells and facilitates tendinopathy healing through regulating the GDF7/Smad1/5 signaling pathway.

Tendinopathy is a common musculoskeletal system disorder in sports medicine, but regeneration ability of injury tendon is limited. Tendon stem cells (TSCs) have shown the definitive treatment evidence for tendinopathy and tendon injuries due to their tenogenesis capacity. Aspirin, as the representative of nonsteroidal anti-inflammatory drugs for its anti-inflammatory and analgestic actions, has been commonly used in treating tendinopathy in clinical, but the effect of aspirin on tenogenesis of TSCs is unclear. We hypothesized that aspirin could promote injury tendon healing through inducing TSCs tenogenesis. The aim of the present study is to make clear the effect of aspirin on TSC tenogenesis and tendon healing in tendinopathy, and thus provide new treatment evidence and strategy of aspirin for clinical practice. First, TSCs were treated with aspirin under tenogenic medium for 3, 7, and 14 days. Sirius Red staining was performed to observe the TSC differentiation. Furthermore, RNA sequencing was utilized to screen out different genes between the induction group and aspirin treatment group. Then, we identified the filtrated molecules and compared their effect on tenogenesis and related signaling pathway. At last, we constructed the tendinopathy model and compared biomechanical changes after aspirin intake. From the results, we found that aspirin promoted tenogenesis of TSCs. RNA sequencing showed that growth differentiation factor 6 (GDF6), GDF7, and GDF11 were upregulated in induction medium with the aspirin group compared with the induction medium group. GDF7 increased tenogenesis and activated Smad1/5 signaling. In addition, aspirin increased the expression of TNC, TNMD, and Scx and biomechanical properties of the injured tendon. In conclusion, aspirin promoted TSC tenogenesis and tendinopathy healing through GDF7/Smad1/5 signaling, and this provided new treatment evidence of aspirin for tendinopathy and tendon injuries.

Knockdown of lncRNA KCNQ1OT1 suppresses the adipogenic and osteogenic differentiation of tendon stem cell via downregulating miR-138 target genes PPARγ and RUNX2

ABSTRACTThe study aimed to investigate the mechanism and biological roles of long noncoding RNA KCNQ1OT1 in adipogenic and osteogenic differentiation of tendon stem cell. In this study, tendon injury mice model was established to detect the expression of lncRNA KCNQ1OT1, miR-138, peroxisome proliferator-activated receptor gamma (PPARγ) and runt-related gene 2 (RUNX2) using quantitative real-time PCR (qRT-PCR) and western blot. Mechanical testing was carried out to assess tendon function. Adiponectin and Osterix were used to evaluate the adipogenic and osteogenic differentiation of tendon stem cells (TSCs). The interaction between lncRNA KCNQ1OT1 and miR-138 was detected by RNA immunoprecipitation (RIP) assay and RNA pull-down assay. We found that lncRNA KCNQ1OT1, PPARγ and RUNX2 expression were significantly upregulated, while miR-138 was suppressed in tendon tissue of injured group and the separated TSCs. lncRNA KCNQ1OT1 knockdown inhibited the adipogenic and osteogenic differentiation of TSCs. Further studies indicated that lncRNA KCNQ1OT1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-138 in TSCs. Further investigations confirmed that lncRNA KCNQ1OT1 knockdown exerted anti-adipogenic and anti-osteogenic function via miR-138/PPARγ and miR-138/RUNX2 axis. Therefore, the lncRNA KCNQ1OT1/miR-138/PPARγ or RUNX2 axis modulated adipogenic and osteogenic differentiation of tendon stem cell, which might be a promising therapeutic target for tendon injuries.

Effects of young extracellular matrix on the biological characteristics of aged tendon stem cells.

Age-related changes in the properties of tendon stem cells (TSCs) may play a role in the progressive degeneration and increased risk of injury to tendon tissue. Recent reports have demonstrated that a decellularized extracellular matrix (DECM) can provide an appropriate niche to maintain the proliferation and differentiation capacity of adult stem cells. We investigated the biological effects of DECM obtained from young TSCs on the proliferation, stemness, senescence, and differentiation of the aged TSCs. Tendon stem cells were isolated from rat patellar tendons and the DECM was collected. The proliferative capacity, β-galactosidase activity, stem cell marker expression, and tenogenic differentiation potential of TSCs were assessed. Our results showed that DECM from young TSCs enhanced the proliferation and tenogenic differentiation of aged TSCs. Moreover, the senescence-associated β-galactosidase activity of aged TSCs was decreased by young DECM. After being cultured on the young DECM, the expression of stem cell markers by aged TSCs was more extensive. The young DECM preserved stem cell properties of aged TSCs. Taken together, the impaired capacity of aged TSCs can be rejuvenated by exposure to young DECM. The positive results in our study suggest that young TSC-derived DECM may provide a novel approach for the prevention and treatment of age-dependent tendon disorders.

Osteopractic total flavone promoting rat extra-articular tendon-bone healing through mTOR pathway

To explore function and related molecular mechanism of osteopractic total flavone (OTF) on tendon healing in rats. Ten male rats aged for 8 weeks were collected and weighted from 180 to 220 g. Tendon stem cells were cultivated, the third tendon stem cells were used for experiment. OTP treated with 0, 0.1, 1, 10 ng/ml were added into tendon stem cells, and expression change of ALP, Runx2, OCN, VEGF, P-S6, P-4E/BP1 were detected after 14 days. Forty male rats aged for 8 weeks (weighted 180 to 220 g) were established extra-articular tendon-bone transplanting healing model, and divided into experimental group and control group. Experimental group were treated with OTF(100 mg·kg⁻¹·d⁻¹), while control group was treated by normal saline with the same volume. Tendon-bone healing degree were detected by biomechanical testing at 3 and 6 weeks after surgery, histological detection were applied to detect tendon-bone healing and number of new vessles. After treated by OTP, ALP staining and active index detection showed there were statistical differences among 0, 0.1, 1, 10 ng/ml group. After 14 days' cultivation, western blotting results showed mTOR downstream marker protein P-S6 protein expression were gradually increased with increase of density of OTP, expression of P-4E/BP1 was reduced, while expression of Runx2, OCN, VEGF were increased. Biological detection results showed that there was no significant difference in mechanical strength between experimental group(0.78±0.05) N/mm and control group (0.51±0.02) N/mm at 3 weeks after surgery, while mechanical strength in experimental group (1.36±0.09) N/mm was higher than control group (1.01±0.08) N/mm at 6 weeks after surgery. Histological results showed maturity of tendon-bone surface cell were higher at 3 and 6 weeks in experimental group, sharpey fiber growth more density, calcification extent of mesenchyme was high, and new bone, vessels were increased. OTF could promote osteogenic differentiation of tendon stem cells through mTOR signaling in vitro, and stimulate tendon-bone healing in bone tunnel and enhance connection quality between tendon and bone.

A Novel Approach for Meniscal Regeneration Using Kartogenin-Treated Autologous Tendon Graft

Background: The meniscus is one of the most commonly injured parts of the body, and meniscal healing is difficult. Hypothesis: Kartogenin (KGN) induces tendon stem cells (TSCs) to differentiate into cartilage cells in vitro and form meniscus-like tissue in vivo. A damaged meniscus can be replaced with a KGN-treated autologous tendon graft. Study Design: Controlled laboratory study. Methods: In the in vitro experiments, TSCs were isolated from rabbit patellar tendons and cultured with various concentrations of KGN, from 0 to 1000 µM. The effect of KGN on the chondrogenesis of TSCs in vitro was investigated by histochemical staining and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The in vivo experiments were carried out on 6 New Zealand White rabbits by removing a meniscus from the rabbit knee and implanting an autologous tendon graft treated with KGN or saline. The meniscus formation in vivo was examined by histological analysis and immune staining. Results: The proliferation of TSCs was promoted by KGN in a concentration-dependent manner. Both histochemical staining and qRT-PCR showed that the chondrogenic differentiation of TSCs was increased with KGN concentration. After 3 months of implantation, the tendon graft treated with KGN formed a meniscus-like tissue with a white and glistening appearance, while the saline-treated tendon graft retained tendon-like tissue and appeared yellowish and unhealthy. Histochemical staining showed that after 3 months of implantation, the KGN-treated tendon graft had a structure similar to that of normal meniscus. Many cartilage-like cells and fibrocartilage-like tissues were found in the KGN-treated tendon graft. However, no cartilage-like cells were found in the saline-treated tendon graft after 3 months of implantation. Furthermore, the KGN-treated tendon graft was positively stained by both anti–collagen type I and type II antibodies, but the saline-treated tendon graft was not stained by collagen type II. Conclusion: The findings indicated that KGN can induce the differentiation of TSCs into cartilage-like cells in vitro and in vivo. The results suggest that KGN-treated tendon graft may be a good substitute for meniscal repair and regeneration. Clinical Relevance: This study revealed the direct effects of KGN on the chondrogenic differentiation of TSCs in vitro and in vivo. A KGN-treated autologous tendon graft induced formation of a meniscus-like tissue in vivo. This study provides a new cartilage regenerating technology for the treatment of damaged meniscus.

Effects of different mechanical stretch conditions on differentiation of rat tendon stem cells

To investigate the effects of different mechanical stretch conditions on the differentiation of rat tendon stem cells (TSCs), to find the best uniaxial cyclic stretching for TSCs tenogenic differentiation, osteogenic differentiation, and adipogenic differentiation. TSCs were isolated from the Achilles tendons of 8-week-old male Sprague Dawley rats by enzymatic digestion method and cultured. The TSCs at passage 3 were randomly divided into 5 groups: group A (stretch strength of 4% and frequency of 1 Hz), group B (stretch strength of 4% and frequency of 2 Hz), group C (stretch strength of 8% and frequency of 1 Hz), group D (stretch strength of 8% and frequency of 2 Hz), and group E (static culture). At 12, 24, and 48 hours after mechanical stretch, the mRNA expressions of the tenogenic differentiation related genes [Scleraxis (SCX) and Tenascin C (TNC)], the osteogenic differentiation related genes [runt related transcription factor 2 (RUNX2) and distal-less homeobox 5 (DLX5)], and the adipogenic differentiation related genes [CCAAT-enhancer-binding protein-α (CEBPα) and lipoprteinlipase (LPL)] were detected by real-time fluorescent quantitative PCR and the protein expressions of TNC, CEBPα, and RUNX2 were detected by Western blot. The mRNA expressions of SCX and TNC in group B were significantly higher than those in groups A, C, D, and E at 24 hours after mechanical stretch ( P<0.05). The mRNA expressions of CEBPα and LPL in group D were significantly higher than those in groups A, B, C, and E at 48 hours after mechanical stretch ( P<0.05). The mRNA expressions of RUNX2 and DLX5 in group C were significantly higher than those in groups A, B, D, and E at 24 hours after mechanical stretch ( P<0.05). Western blot detection showed that higher protein expression of TNC in group B than group E at each time point after mechanical stretch ( P<0.05), and the protein expression of CEBPα was significantly inhibited when compared with group E at 24 hours after mechanical stretch ( P<0.05). At 24 hours after mechanical stretch, the protein expression of RUNX2 in group C was significantly higher than that in group E ( P<0.05); and the protein expression of TNC was significantly lower than that in group E at 24 and 48 hours after mechanical stretch ( P<0.05). At 48 hours after mechanical stretch, the protein expression of CEBPα was significantly increased and the protein expression of TNC was significantly decreased in group D when compared with group E ( P<0.05), but no significant difference was found in the protein expression of RUNX2 between groups D and E ( P>0.05). The mechanical strain could promote differentiation of TSCs, and different parameter of stretch will lead to different differentiation. The best stretch condition for tenogenic differentiation is 4% strength and 2 Hz frequency for 24 hours; the best stretch condition for osteogenic differentiation is 8% strength and 1 Hz frequency for 24 hours; and the best stretch condition for adipogenic differentiation is 8% strength and 2 Hz frequency for 48 hours.

Effect of cyclic stretch on expression of c-fos gene in rat Achilles-derived tendon stem cells

To investigate whether mechanical stretch stimulation affects the expression of the immediate early gene c-fos mRNA in rat Achilles-derived tendon stem cells (TSCs) in vitro. TSCs were isolated from the Achilles tendons of 8 weeks old male Sprague Dawley rats by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated by a uniaxial cyclic stretching loading system under the condition of 1 Hz, respectively with 4% or 8% stretch intensity for 0, 5, 15, 30, 60, and 120 minutes. At each time point, TSCs were collected to detect c-fos mRNA expressions and to find the best time-point T max by real-time fluorescence quantitative PCR. Then, TSCs were simulated with 2%, 4%, 6%, 8%, or 12% stretch intensity for T max to observe the relative expressions of c-fos mRNA under different stretch intensities. Next, TSCs were stretched for 0, 5, or 15 minutes respectively and followed by incubation at relax status up to T max to observe the changes of c-fos mRNA expressions after short period stimulation. Finally, TSCs were stimulated with 4% or 8% stretch intensity respectively for 0, T max, or 120 minutes to detect the expressions of the tenogenic differentiation related genes [collagen type I, tenomodulin (TNMD)], the osteogenic differentiation related genes [runt related transcription factor 2 (Runx2), distal-less homeobox 5 (Dlx5)], and the adipogenic differentiation related gene [fatty acid binding protein 4 (FABP4)]. Under 4% or 8% stretch intensity, the relative expressions of c-fos mRNA significantly increased at 15 minutes ( P<0.05), reached the maximum at 30 minutes ( P<0.05), and returned to baseline at 60 minutes ( P>0.05) when compared with expression at 0 minute. Therefore, T max was 30 minutes. The stretch intensity of 2% was enough to cause the expression of c-fos mRNA at 30 minutes, and the expression was significantly higher under the stretch intensity of 6%, 8%, and 12% than 2% and 4% ( P<0.05). Even for a short period stimulation of 5 minutes, c-fos mRNA expression could still significantly increase at 30 minutes ( P<0.05). The relative expressions of differentiation related genes at 30 and 120 minutes showed no significant difference when compared with the expression at 0 minute under 4% stretch intensity ( P>0.05); but the relative expression of Runx2 gene significantly increased at 30 minutes, and the relative expressions of collagen type I, TNMD, Dlx5, and Runx2 increased at 120 minutes under 8% stretch intensity ( P<0.05). Mechanical stretch stimulation can affect the relative expression of the immediate early gene c-fos mRNA of rat Achilles-derived tendon stem cells in vitro, and there is time- and intensity-dependence. It is suggested that the mechanical stimulation with different time or intensity may affect the differentiation of TSCs at early stage. This study is meaningful for the further study on TSCs intracellular mechanical signal transfer mechanism.

MTOR REGULATES THE PROLIFERATION AND DIFFERENTIATION OF TENDON STEM CELLS: AN IN VITRO STUDY

Objectives: The mechanistic target of rapamycin (mTOR) controls cell growth and proliferation via translation regulation in eukaryotes. The present study investigated the effects of mTOR on the proliferation and differentiation of tendon stem cells (TSCs). Methods: The proliferation and differentiation ability of TSCs was tested in response to antagonist (MHY1485), and a depressor of mTOR (Rapamycin and KU0063794). CCK test was performed to test cell proliferation; quantitative real-time PCR (RT-PCR) and Western blot test were performed to evaluate the differentiation of TSCs. Results: Blocking of mTOR1 inhibited the proliferation of TSCs and blocking of mTOR2 enhanced the proliferation of TSCs; however, the effects of mTOR1 surpassed the effects of mTOR2. Blocking of mTOR1 or activation of mTOR2 induced the expression of TNC, and blocking of mTOR2 inhibited the expression of TNC. Blocking of mTOR1 by rapamycin decreased the expression of ap2. Both blocking of mTOR1 or mTOR2 had little effects on the expression of Runx2 and Sox9; however, activation of mTOR2 induced the expression of Runx2 and Sox9. Moreover, the Western blot test showed that blocking of mTOR1 by Rapamycin or the blocking of both mTOR1 and mTOR2 by KU-0062794 enhanced the expression of TNC; in addition, blocking of mTOR1 by Rapamycin enhanced the expression of c-EBPα and Sox9. However, activation of mTOR1 and mTOR2 by MHY1485 increased the expression of Runx2. Conclusions: mTOR played important roles in the proliferation and differentiation of TSCs. Furthermore, mTOR1 and mTOR2 played different roles on the proliferation and differentiation of TSCs. Blocking mTOR1 inhibited the proliferation of TSCs and played a dominant function.Blocking of mTOR1 enhanced the expression of tenocyte related genes; however, blocking of mTOR2 inhibited the expression of TNC. Blocking of mTOR1 by rapamycin decreased the expression of ap2 and activation of mTOR2 induced the expression of Runx2.

Harmful Effects of Leukocyte-Rich Platelet-Rich Plasma on Rabbit Tendon Stem Cells In Vitro

Background: Platelet-rich plasma (PRP) is now widely used as a promising treatment for patients with tendinopathy. However, the efficacy of PRP treatment for tendinopathy is controversial mainly because of inconsistent results from human clinical trials and particularly because the concentration and effect of leukocytes in PRP remain largely unknown. Hypothesis: Leukocyte-rich PRP (L-PRP) inhibits growth factor release, decreases proliferation, and induces nontenocyte differentiation of tendon stem cells (TSCs); increases catabolic cytokine concentrations; and causes inflammation and apoptosis. Thus, L-PRP has a detrimental effect on tendon stem/progenitor cells, which impairs injured tendon healing. Study Design: Controlled laboratory study. Methods: Pure PRP (P-PRP) and L-PRP were prepared from the same individual rabbit blood, and platelet numbers in each PRP product were adjusted to reach the same level. The leukocyte level in L-PRP was 4 and 8 times higher than those in whole blood and P-PRP, respectively. The growth factors in both P-PRP and L-PRP were measured by enzyme-linked immunosorbent assay kits. The morphology, stemness, proliferation, and differentiation of TSCs grown in L-PRP and P-PRP were examined by microscopy, immunocytochemistry, population doubling time, quantitative real-time polymerase chain reaction, and histological analysis. Results: L-PRP produced lower levels of growth factors, such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming growth factor (TGF)–β1, and platelet-derived growth factor (PDGF), than did P-PRP. TSC proliferation was significantly decreased in L-PRP in a concentration-dependent manner. Furthermore, TSCs cultured in P-PRP produced more collagen and formed tendon-like tissue; however, TSCs grown in L-PRP differentiated into nontenocytes and produced more inflammatory factors such as membrane-associated prostaglandin synthase (mPGES) and interleukin (IL)–1β. Moreover, L-PRP was associated with increased apoptosis. Conclusion: L-PRP has harmful effects on TSCs. Clinical Relevance: This study revealed the direct effects of different compositions of PRP on TSCs and provided basic scientific data to help understand the cellular and molecular mechanisms of the efficacy of PRP treatment in clinical use.

Mechanism of osteogenic and adipogenic differentiation of tendon stem cells induced by sirtuin 1

The aim of the present study was to assess the expression of sirtuin (Sirt)1 in tendon stem cells (TSCs) and to elucidate its association with osteogenic and adipogenic differentiation of TSCs. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analyses were performed to detect Sirt1 mRNA and protein levels in TSCs, respectively. TSCs were positive for Sirt1 expression, which was elevated by Sirt1 activator SRT1720 in a time- and concentration- dependent manner, and decreased by Sirt1 inhibitor EX527. TSCs were treated with SRT1720 and EX527 for various time periods and resulting changes in osteogenic and adipogenic protein markers were analyzed using alizarin red and oil redO staining. According to RT-qPCR and western blot analyses, the associated factors β‑catenin, Runt-related transcription factor2 (Runx2) and bone morphogenetic protein2 were elevated following increases of Sirt1 levels, while CCAAT/enhancer binding protein (CEBP)α and peroxisome proliferator-activated receptor (PPAR)γ were decreased. These results suggested that osteogenic differentiation capacity was enhanced, while adipogenic differentiation capacity declined. Further mechanistic study revealed that phosphoinositide‑3 kinase (PI3K) and AKT were decreased following activation of Sirt1. In conclusion, the present study suggested that Sirt1 promotes the osteogenic differentiation of TSCs through upregulating β‑catenin and Runx2 and inhibits the adipogenic differentiation of TSCs through the PI3K/AKT pathway with downregulation of CEBPα and PPARγ.

Strenuous Treadmill Running Induces a Chondrocyte Phenotype in Rat Achilles Tendons.

BackgroundAlthough tendinopathy is common, its underlying pathogenesis is poorly understood. This study aimed to investigate the possible pathogenesis of tendinopathy.Material/MethodsIn this study, a total of 24 rats were randomly and evenly divided into a control (CON) group and a strenuous treadmill running (STR) group. Animals in the STR group were subjected to a 12-week treadmill running protocol. Subsequently, all Achilles tendons were harvested to perform histological observation or biochemical analyses.ResultsHistologically, hypercellularity and round cells, as well as disorganized collagen fibrils, were presented in rat Achilles tendon sections from the STR group. Furthermore, our results showed that the expression of aggrecan, collagen type II (Col II), and Sex-Determining Region Y Box 9 (Sox 9) were markedly increased in the STR group compared with that in the CON group. Additionally, the mRNA expression of bone morphogenetic protein-2 (BMP-2) and biglycan was significantly up-regulated in the STR group in contrast to that in CON group.ConclusionsThese results suggest that a 12-week strenuous treadmill running regimen can induce chondrocyte phenotype in rat Achilles tendons through chondrogenic differentiation of tendon stem cells (TSCs) by BMP-2 signaling.