Matrix stiffness-mediated tenogenesis of tendon stem/progenitor cells via integrin-αm for tendon regeneration.

Tendon injuries, commonly associated with sports activities, pose significant challenges in terms of treatment and recovery due to limited tendon regeneration and the formation of proliferative scars. Stem cell-based therapy has shown promising application, but there are still challenges. Physical and biological cues are instrumental in guiding stem cell differentiation and maturation. This study focuses on exploring the effects of matrix biomechanics on tendon stem/progenitor cells (TSPCs) differentiation. We fabricated polydimethylsiloxane (PDMS) substrates with different elastic modulus to mimic the mechanical characteristics of healthy tendons. A tissue-engineered culture system was developed for tenogenesis, and pre-differentiated tissue-engineered tendons were transplanted in vivo to assess their efficacy in regenerating patella tendon injuries. Furthermore, we demonstrated that the biomechanical stimuli activated the integrin-αm to enhance the tenogenesis capacity of TSPCs. Our findings highlight the importance of biomechanics in tendon tissue engineering and provide a novel perspective for enhancing tendon regeneration.

Discovery of Muscle-Tendon Progenitor Subpopulation in Human Myotendinous Junction at Single-Cell Resolution.

The myotendinous junction (MTJ) is a complex and special anatomical area that connects muscles and tendons, and it is also the key to repairing tendons. Nevertheless, the anatomical structure and connection structure of MTJ, the cluster and distribution of cells, and which cells are involved in repairing the tissue are still unclear. Here, we analyzed the cell subtype distribution and function of human MTJ at single-cell level. We identified four main subtypes, including stem cell, muscle, tendon, and muscle-tendon progenitor cells (MTP). The MTP subpopulation, which remains the characteristics of stem cells and also expresses muscle and tendon marker genes simultaneously, may have the potential for bidirectional differentiation. We also found the muscle-tendon progenitor cells were distributed in the shape of a transparent goblet; muscle cells first connect to the MTP and then to the tendon. And after being transplanted in the MTJ injury model, MTP exhibited strong regenerative capability. Finally, we also demonstrated the importance of mTOR signaling for MTP maintenance by in vitro addition of rapamycin and in vivo validation using mTOR-ko mice. Our research conducted a comprehensive analysis of the heterogeneity of myotendinous junction, discovered a special cluster called MTP, provided new insights into the biological significance of myotendinous junction, and laid the foundation for future research on myotendinous junction regeneration and restoration.

Inhibition of miR-490-5p Promotes Human Adipose-Derived Stem Cells Chondrogenesis and Protects Chondrocytes via the PITPNM1/PI3K/AKT Axis.

MicroRNAs (miRNAs) play a pivotal role in cartilage development and homeostasis in osteoarthritis (OA). While the fundamental roles of miRNAs in cartilage degeneration have been extensively studied, their effects on chondrogenic differentiation induced by human adipose-derived stem cells (hADSCs) and the underlying mechanisms remain largely elusive. Here, we investigated the roles and mechanisms of miRNAs in hADSC chondrogenic differentiation and chondrocyte homeostasis. Using microarray analysis, we screened miRNAs expressed in the chondrogenic differentiated hADSCs and identified miR-490-5p as the most significantly down-regulated miRNA. We analyzed its expression patterns during chondrogenesis in vivo and in vitro. Our study showed that miR-490-5p overexpression promoted the transition of hADSCs from chondrogenesis to osteogenesis. In addition, based on miRNA-mRNA prediction analysis and dual-luciferase reporter assay, we proposed and proved that miR-490-5p targeted PITPNM1 by binding to its 3'-UTR and inhibiting its translation. Moreover, loss- and gain-of-function experiments identified the involvement of the PI3K/AKT signaling pathway, and a rescue experiment determined the effect and specific mechanism of the miR-490-5p/PITPNM1/PI3K/AKT axis in hADSC chondrogenic differentiation and chondrocyte homeostasis. Inhibition of miR-490-5p alleviated cartilage injury in vivo as demonstrated using the destabilization of the medial meniscus (DMM) OA model. We identified miR-490-5p as a novel modulator of hADSC-mediated chondrogenesis and chondrocyte phenotype. This study highlighted that miR-490-5p attenuated hADSC chondrogenesis and accelerated cartilage degradation through activation of the PI3K/AKT signaling pathway by targeting PITPNM1. Inhibition of miR-490-5p facilitated hADSC chondrogenic differentiation and protected chondrocyte phenotype via the PITPNM1/PI3K/AKT axis, thus providing a novel stem cell potential therapeutic target for OA treatment.

MiR-455-3p reduces apoptosis and alleviates degeneration of chondrocyte through regulating PI3K/AKT pathway.

AIMS: This study aimed to explore the functions of miR-455-3p, PTEN, and PI3K/AKT pathway in osteoarthritis. MATERIALS AND METHODS: We used the human bone marrow stem cell (BMSC), healthy chondrocytes, osteoarthritis chondrocytes (OA), and the IL-1ß/TNF-α-treated chondrocyte model to explore the relationship between miR-455-3p and PTEN. Mimic or inhibitor was used to transfect chondrocytes to determine whether miR-455-3p can regulate PTEN and influence COL2A1 and MMP13. Apoptosis was detected by flow cytometry. A luciferase report was applied to verify the targeted binding. KO mice were applied to investigate PTEN and pAKT expression and the effect on chondrocytes in vivo. KEY FINDINGS: MiR-455-3p and PTEN were reverse in chondrogenesis and healthy cartilage versus OA cartilage. Similar trends were noted in IL-1ß model. PTEN and MMP13 decreased and COL2A1 increased after overexpressing miR-455-3p, whereas the inhibition showed opposite results. Flow cytometry showed that miR-455-3p could reduce the apoptosis of chondrocytes. The results of luciferase revealed that miR-455-3p could affect fluorescence activity of PTEN by targeting its 3'-UTR. Finally, we found a marked increased in the expression of PTEN in KO mice relative to WT mice, while pAKT levels decreased. SIGNIFICANCE: It can be supported that miR-455-3p can reduce the apoptosis of chondrocytes and alleviate OA through regulating PI3K/AKT pathway, which may be expected to be a target for the treatment of osteoarthritis.

Long Non-coding RNA HOTTIP Promotes CCL3 Expression and Induces Cartilage Degradation by Sponging miR-455-3p.

Long non-coding RNAs (lncRNAs) play pivotal roles in diseases such as osteoarthritis (OA). However, knowledge of the biological roles of lncRNAs is limited in OA. We aimed to explore the biological function and molecular mechanism of HOTTIP in chondrogenesis and cartilage degradation. We used the human mesenchymal stem cell (hMSC) model of chondrogenesis, in parallel with, tissue biopsies from normal and OA cartilage to detect HOTTIP, CCL3, and miR-455-3p expression in vitro. Biological interactions between HOTTIP and miR-455-3p were determined by RNA silencing and overexpression in vitro. We evaluated the effect of HOTTIP on chondrogenesis and degeneration, and its regulation of miR-455-3p via competing endogenous RNA (ceRNA). Our in vitro ceRNA findings were further confirmed within animal models in vivo. Mechanisms of ceRNAs were determined by bioinformatic analysis, a luciferase reporter system, RNA pull-down, and RNA immunoprecipitation (RIP) assays. We found reduced miR-455-3p expression and significantly upregulated lncRNA HOTTIP and CCL3 expression in OA cartilage tissues and chondrocytes. The expression of HOTTIP and CCL3 was increased in chondrocytes treated with interleukin-1ß (IL-1ß) in vitro. Knockdown of HOTTIP promoted cartilage-specific gene expression and suppressed CCL3. Conversely, HOTTIP overexpression reduced cartilage-specific genes and increased CCL3. Notably, HOTTIP negatively regulated miR-455-3p and increased CCL3 levels in human primary chondrocytes. Mechanistic investigations indicated that HOTTIP functioned as ceRNA for miR-455-3p enhanced CCL3 expression. Taken together, the ceRNA regulatory network of HOTTIP/miR-455-3p/CCL3 plays a critical role in OA pathogenesis and suggests HOTTIP is a potential target in OA therapy.

Exosomal miR-95-5p regulates chondrogenesis and cartilage degradation via histone deacetylase 2/8.

MicroRNAs play critical roles in the pathogenesis of osteoarthritis, the most common chronic degenerative joint disease. Exosomes derived from miR-95-5p-overexpressing primary chondrocytes (AC-miR-95-5p) may be effective in treating osteoarthritis. Increased expression of HDAC2/8 occurs in the tissues and chondrocyte-secreted exosomes of patients with osteoarthritis and mediates cartilage-specific gene expression in chondrocytes. We have been suggested that exosomes derived from AC-miR-95-5p (AC-miR-95-5p-Exos) would enhance chondrogenesis and prevent the development of osteoarthritis by directly targeting HDAC2/8. Our in vitro experiments showed that miR-95-5p expression was significantly lower in osteoarthritic chondrocyte-secreted exosomes than in normal cartilage. Treatment with AC-miR-95-5p-Exos promoted cartilage development and cartilage matrix expression in mesenchymal stem cells induced to undergo chondrogenesis and chondrocytes, respectively. In contrast, co-culture with exosomes derived from chondrocytes transfected with an antisense inhibitor of miR-95-5p (AC-anti-miR-95-5p-Exos) prevented chondrogenic differentiation and reduced cartilage matrix synthesis by enhancing the expression of HDAC2/8. MiR-95-5p suppressed the activity of reporter constructs containing the 3'-untranslated region of HDAC2/8, inhibited HDAC2/8 expression and promoted cartilage matrix expression. Our results suggest that AC-miR-95-5p-Exos regulate cartilage development and homoeostasis by directly targeting HDAC2/8. Thus, AC-miR-95-5p-Exos may act as an HDAC2/8 inhibitor and exhibit potential as a disease-modifying osteoarthritis drug.