BMP signaling maintains auricular chondrocyte identity and prevents microtia development by inhibiting protein kinase A.

Elastic cartilage constitutes a major component of the external ear, which functions to guide sound to the middle and inner ears. Defects in auricle development cause congenital microtia, which affects hearing and appearance in patients. Mutations in several genes have been implicated in microtia development, yet, the pathogenesis of this disorder remains incompletely understood. Here, we show that Prrx1 genetically marks auricular chondrocytes in adult mice. Interestingly, BMP-Smad1/5/9 signaling in chondrocytes is increasingly activated from the proximal to distal segments of the ear, which is associated with a decrease in chondrocyte regenerative activity. Ablation of Bmpr1a in auricular chondrocytes led to chondrocyte atrophy and microtia development at the distal part. Transcriptome analysis revealed that Bmpr1a deficiency caused a switch from the chondrogenic program to the osteogenic program, accompanied by enhanced protein kinase A activation, likely through increased expression of Adcy5/8. Inhibition of PKA blocked chondrocyte-to-osteoblast transformation and microtia development. Moreover, analysis of single-cell RNA-seq of human microtia samples uncovered enriched gene expression in the PKA pathway and chondrocyte-to-osteoblast transformation process. These findings suggest that auricle cartilage is actively maintained by BMP signaling, which maintains chondrocyte identity by suppressing osteogenic differentiation.

Cytokine Receptor-like Factor 1 (CRLF1) and Its Role in Osteochondral Repair.

BACKGROUND: Since cytokine receptor-like factor 1 (CRLF1) has been implicated in tissue regeneration, we hypothesized that CRLF1 released by mesenchymal stem cells can promote the repair of osteochondral defects. METHODS: The degree of a femoral osteochondral defect repair in rabbits after intra-articular injections of bone marrow-derived mesenchymal stem cells (BMSCs) that were transduced with empty adeno-associated virus (AAV) or AAV containing CRLF1 was determined by morphological, histological, and micro computer tomography (CT) analyses. The effects of CRLF1 on chondrogenic differentiation of BMSCs or catabolic events of interleukin-1beta-treated chondrocyte cell line TC28a2 were determined by alcian blue staining, gene expression levels of cartilage and catabolic marker genes using real-time PCR analysis, and immunoblot analysis of Smad2/3 and STAT3 signaling. RESULTS: Intra-articular injections of BMSCs overexpressing CRLF1 markedly improved repair of a rabbit femoral osteochondral defect. Overexpression of CRLF1 in BMSCs resulted in the release of a homodimeric CRLF1 complex that stimulated chondrogenic differentiation of BMSCs via enhancing Smad2/3 signaling, whereas the suppression of CRLF1 expression inhibited chondrogenic differentiation. In addition, CRLF1 inhibited catabolic events in TC28a2 cells cultured in an inflammatory environment, while a heterodimeric complex of CRLF1 and cardiotrophin-like Cytokine (CLC) stimulated catabolic events via STAT3 activation. CONCLUSION: A homodimeric CRLF1 complex released by BMSCs enhanced the repair of osteochondral defects via the inhibition of catabolic events in chondrocytes and the stimulation of chondrogenic differentiation of precursor cells.

Ocu-miR-10a-5p promotes the chondrogenic differentiation of rabbit BMSCs by targeting BTRC-mediated Wnt/ß-catenin signaling pathway.

MicroRNAs (miRNAs) play an important role in articular cartilage damage in osteoarthritis (OA). However, the biological role of miRNAs in the chondrogenic differentiation of bone marrow mesenchymal stem cell (BMSC) remains largely unclear. Rabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated, cultured, and identified. Afterwards, rBMSCs were induced to chondrogenic differentiation, examined by Alcian Blue staining. Differentially expressed miRNAs were identified in rBMSCs between induced and non-induced groups by miRNA sequencing analysis, part of which was validated via PCR assay. Cell viability and apoptosis were assessed by CCK-8 assay and Hoechst staining. Saffron O staining was utilized to assess chondrocyte hyperplasia. The expression of specific chondrogenic markers, including COL2A1, SOX9, Runx2, MMP-13, Aggrecan, and BMP-2, were measured at mRNA and protein levels. The association between beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) and miR-10a-5p in the miRNA family from rabbit (ocu-miR-10a-5p) was determined by luciferase reporter assay. A total of 76 differentially expressed miRNAs, including 52 downregulated and 24 upregulated miRNAs, were identified in rBMSCs from the induced group. Inhibition of ocu-miR-10a-5p suppressed rBMSC viability and chondrogenic differentiation, as well as downregulated the expression of ß-catenin, SOX9, COL2A1, MMP-13, and Runx2. BTRC was predicted and confirmed as a target of ocu-miR-10a-5p. Overexpression of BTRC rescued the promoting impacts of overexpressed ocu-miR-10a-5p on chondrogenic differentiation of rBMSCs and ß-catenin expression. Taken together, our data suggested that ocu-miR-10a-5p facilitated rabbit BMSC survival and chondrogenic differentiation by activating Wnt/ß-catenin signaling through BTRC.

A synthetic, closed-looped gene circuit for the autonomous regulation of RUNX2 activity during chondrogenesis.

The transcription factor RUNX2 is a key regulator of chondrocyte phenotype during development, making it an ideal target for prevention of undesirable chondrocyte maturation in cartilage tissue-engineering strategies. Here, we engineered an autoregulatory gene circuit (cisCXp-shRunx2) that negatively controls RUNX2 activity in chondrogenic cells via RNA interference initiated by a tunable synthetic Col10a1-like promoter (cisCXp). The cisCXp-shRunx2 gene circuit is designed based on the observation that induced RUNX2 silencing after early chondrogenesis enhances the accumulation of cartilaginous matrix in ATDC5 cells. We show that the cisCXp-shRunx2 initiates RNAi of RUNX2 in maturing chondrocytes in response to the increasing intracellular RUNX2 activity without interfering with early chondrogenesis. The induced loss of RUNX2 activity in turn negatively regulates the gene circuit itself. Moreover, the efficacy of RUNX2 suppression from cisCXp-shRunx2 can be controlled by modifying the sensitivity of cisCXp promoter. Finally, we show the efficacy of inhibiting RUNX2 in preventing matrix loss in human mesenchymal stem cell-derived (hMSC-derived) cartilage under conditions that induce chondrocyte hypertrophic differentiation, including inflammation. Overall, our results demonstrated that the negative modulation of RUNX2 activity with our autoregulatory gene circuit enhanced matrix synthesis and resisted ECM degradation by reprogrammed MSC-derived chondrocytes in response to the microenvironment of the degenerative joint.

Early insights into the role of Exoc6B associated with spondyloepimetaphyseal dysplasia with joint laxity type 3 in primary ciliogenesis and chondrogenic differentiation in vitro.

BACKGROUND: Spondyloepimetaphyseal dysplasia with joint laxity type 3 (SEMDJL3) is a rare skeletal dysplasia associated with EXOC6B, a component of the exocyst complex, involved in vesicle tethering and exocytosis at the plasma membrane. So far, EXOC6B and the pathomechanisms underlying SEMDJL3 remain obscure. METHODS AND RESULTS: Exoc6b was detected largely at the perinuclear regions and the primary cilia base in ATDC5 prechondrocytes. Its shRNA lentiviral knockdown impeded primary ciliogenesis. In Exoc6b silenced prechondrocytes, Hedgehog signaling was attenuated, including when stimulated with Smoothened agonist. Exoc6b knockdown deregulated the mRNA and protein levels of Col2a1, a marker of chondrocyte proliferation at 7- and 14-days following differentiation. It led to the upregulation of Ihh another marker of proliferative chondrocytes. The levels of Col10a1, a marker of chondrocyte hypertrophy was enhanced at 14 days of differentiation. Congruently, Axin2, a canonical Wnt pathway modulator that inhibits chondrocyte hypertrophy was repressed. The expression of Mmp13 and Adamts4 that are terminal chondrocyte hypertrophy markers involved in extracellular matrix (ECM) remodelling were downregulated at 7 and 14 days of chondrogenesis. Bglap that encodes for the most abundant non-collagenous bone matrix constituent and promotes ECM calcification was suppressed at 14 days of chondrocyte differentiation. ECM mineralization was assessed by Alizarin Red staining. Gene expression and ciliogenesis were investigated by reverse transcription quantitative real-time PCR, immunoblotting, and immunocytochemistry. CONCLUSIONS: These findings provide initial insights into the potential role of Exoc6b in primary ciliogenesis and chondrogenic differentiation, contributing towards a preliminary understanding of the molecular pathomechanisms underlying SEMDJL3.

Basement membrane extract potentiates the endochondral ossification phenotype of bone marrow-derived mesenchymal stem cell-based cartilage organoids.

Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.

Gli1 labels progenitors during chondrogenesis in postnatal mice.

Skeletal growth promoted by endochondral ossification is tightly coordinated by self-renewal and differentiation of chondrogenic progenitors. Emerging evidence has shown that multiple skeletal stem cells (SSCs) participate in cartilage formation. However, as yet, no study has reported the existence of common long-lasting chondrogenic progenitors in various types of cartilage. Here, we identify Gli1+ chondrogenic progenitors (Gli1+ CPs), which are distinct from PTHrP+ or FoxA2+ SSCs, are responsible for the lifelong generation of chondrocytes in the growth plate, vertebrae, ribs, and other cartilage. The absence of Gli1+ CPs leads to cartilage defects and dwarfishness phenotype in mice. Furthermore, we show that the BMP signal plays an important role in self-renewal and maintenance of Gli1+ CPs. Deletion of Bmpr1α triggers Gli1+ CPs quiescence exit and causes the exhaustion of Gli1+ CPs, consequently disrupting columnar cartilage. Collectively, our data demonstrate that Gli1+ CPs are common long-term chondrogenic progenitors in multiple types of cartilage and are essential to maintain cartilage homeostasis.

Mesenchymal Stem Cells Cultured in a 3D Microgel Environment Containing Platelet-Rich Plasma Significantly Modify Their Chondrogenesis-Related miRNA Expression.

The aim of this work is to study the effect of platelet factors on the differentiation of mesenchymal stem cells (MSCs) to hyaline cartilage chondrocytes in a three-dimensional environment. MSCs were cultured in a microgel environment with a chondrogenic medium. The microgel consisted of microspheres that combine gelatin and platelet-rich plasma (PRP). The gelatin/PRP microdroplets were produced by emulsion. The gelatin containing the microdroplets was enzymatically gelled, retaining PRP and, just before seeding the cells, platelets were activated by adding calcium chloride so that platelet growth factors were released into the culture media but not before. Platelet activation was analyzed before activation to rule out the possibility that the gelatin cross-linking process itself activated the platelets. The gene expression of characteristic chondrogenic markers and miRNA expression were analyzed in cells cultured in a differentiation medium and significant differences were found between gelation/PRP microgels and those containing only pure gelatin. In summary, the gelatin microspheres effectively encapsulated platelets that secreted and released factors that significantly contributed to cellular chondrogenic differentiation. At the same time, the microgel constituted a 3D medium that provided the cells with adherent surfaces and the possibility of three-dimensional cell-cell contact.

CircIRAK3 exerts negative feedback regulation on inflammation by binding to HNRNP U and destabilizing proinflammatory cytokine mRNA in osteoarthritis and chondrogenesis.

Osteoarthritis (OA) is the most prevalent age-related and degenerative joint disease with limited treatment options. Previous studies have identified the therapeutic effects of mesenchymal stem cells (MSCs) therapy. Nevertheless, chronic inflammation impedes MSCs therapeutic effect. There have been reports suggesting that circular RNAs (circRNAs) are involved in OA and chondrogenesis. The combination of MSCs and circRNAs in therapies appears to be a promising option. In this study, we identified circIRAK3 as a significant regulator in cartilage degeneration and chondrogenesis through high-throughput sequencing analyses. We observed increased circIRAK3 in OA cartilage and during MSCs chondrogenesis. Knockdown of circIRAK3 resulted in excessive apoptosis, inhibited proliferation, and degradation of chondrocytes, along with the inhibition of MSCs chondrogenesis. Mechanistically, circIRAK3 bound to HNRNP U and competitively prevented its binding to IL-1ß, TNFα, and IL6 mRNA, thereby promoting mRNA degradation. Notably, circIRAK3 expression in plasma increased with higher OARSI scores. Intra-articular injection of adeno-associated virus-circIRAK3 delayed cartilage degeneration and reduced inflammation in DMM mouse model. Our study highlights a compensatory regulation network of circIRAK3 in chondrocytes in response to inflammation. CircIRAK3 has the potential to serve as a new therapeutic target for OA. Furthermore, therapies targeting circIRAK3 combined with MSCs hold promise.

Endochondral Repair of Jawbone Defects Using Periosteal Cell Spheroids.

Recapitulation of the natural healing process is receiving increasing recognition as a strategy to induce robust tissue regeneration. Endochondral ossification has been recognized as an essential reparative approach in natural jawbone defect healing. However, such an approach has been overlooked in the recent development of cell-based therapeutics for jawbone repair. Therefore, this study aimed to explore a bioinspired stem cell-based strategy for jawbone repair by mimicking the mesenchymal condensation of progenitor cells during the early endochondral ossification process. For this purpose, passage 3 of jawbone periosteum-derived cells (jb-PDCs) was cultured in our previously reported nonadherent microwells (200 µm in diameter, 148 µm in depth, and 100 µm space in between) and self-assembled into spheroids with a diameter of 96.4 ± 5.8 µm after 48 h. Compared to monolayer culture, the jb-PDC spheroids showed a significant reduction of stemness marker expression evidenced by flow cytometry. Furthermore, a significant upregulation of chondrogenic transcription factor SOX9 in both gene and protein levels was observed in the jb-PDC spheroids after 48 h of chondrogenic induction. RNA sequencing and Western blotting analysis further suggested that the enhanced SOX9-mediated chondrogenic differentiation in jb-PDC spheroids was attributed to the activation of the p38 MAPK pathway. Impressively, inhibition of p38 kinase activity significantly attenuated chondrogenic differentiation jb-PDC spheroids, evidenced by a significant decline of SOX9 in both gene and protein levels. Strikingly, the jb-PDC spheroids implanted in 6- to 8-wk-old male C57BL/6 mice with critical-size jawbone defects (1.8 mm in diameter) showed an evident contribution to cartilaginous callus formation after 1 wk, evidenced by histological analysis. Furthermore, micro-computed tomography analysis showed that the jb-PDC spheroids significantly accelerated bone healing after 2 wk in the absence of exogenous growth factors. In sum, the presented findings represent the successful development of cell-based therapeutics to reengineer the endochondral bone repair process and illustrate the potential application to improve bone repair and regeneration in the craniofacial skeleton.

[The influence of peptides on the chondrogenic differentiation of human mesenchymal stem cells during replicative aging.]

Osteoarthritis is a widespread age-related disease, that has no effective targeted therapy. In this regard, bioengineering methods are being actively developed that can stimulate the restoration of cartilage tissue. These methods include chondrogenic differentiation of stem cells, which is stimulated by various biomolecules, including short peptides and polypeptide complexes. It was studied the effect of the cartilage polypeptide complex (CPC) and AED peptide on gene expression and protein synthesis of chondrogenic differentiation - SOX9, aggrecan, type II collagen and COMP - in human mesenchymal stem cell (MSC) during replicative aging. AED peptide at the concentration of 200 ng/ml activates gene expression and protein synthesis during aging of MSCs. CPC has the same effect in the concentration 2000 ng/ml. These data indicate the stimulating effect of studied peptides on regulation of chondrogenesis and open up prospects for further investigation of their effectiveness in osteoarthritis models.

Induced regeneration of articular cartilage - identification of a dormant regeneration program for a non-regenerative tissue.

A mouse organoid culture model was developed to regenerate articular cartilage by sequential treatment with BMP2 and BMP9 (or GDF2) that parallels induced joint regeneration at digit amputation wounds in vivo. BMP9-induced chondrogenesis was used to identify clonal cell lines for articular chondrocyte and hypertrophic chondrocyte progenitor cells from digit fibroblasts. A protocol that includes cell aggregation enhanced by BMP2 followed by BMP9-induced chondrogenesis resulted in the differentiation of organized layers of articular chondrocytes, similar to the organization of middle and deep zones of articular cartilage in situ, and retained a differentiated phenotype following transplantation. In addition, the differentiation of a non-chondrogenic connective tissue layer containing articular chondrocyte progenitor cells demonstrated that progenitor cell sequestration is coupled with articular cartilage differentiation at a clonal level. The studies identify a dormant endogenous regenerative program for a non-regenerative tissue in which fibroblast-derived progenitor cells can be induced to initiate morphogenetic and differentiative programs that include progenitor cell sequestration. The identification of dormant regenerative programs in non-regenerative tissues such as articular cartilage represents a novel strategy that integrates regeneration biology with regenerative medicine.

Decreased expression of GEM in osteoarthritis cartilage regulates chondrogenic differentiation via Wnt/ß-catenin signaling.

BACKGROUND: GEM (GTP-binding protein overexpressed in skeletal muscle) is one of the atypical small GTPase subfamily members recently identified as a regulator of cell differentiation. Abnormal chondrogenesis coupled with an imbalance in the turnover of cartilaginous matrix formation is highly relevant to the onset and progression of osteoarthritis (OA). However, how GEM regulates chondrogenic differentiation remains unexplored. METHODS: Cartilage tissues were obtained from OA patients and graded according to the ORASI and ICRS grading systems. The expression alteration of GEM was detected in the Grade 4 cartilage compared to Grade 0 and verified in OA mimic culture systems. Next, to investigate the specific function of GEM during these processes, we generated a Gem knockdown (Gem-Kd) system by transfecting siRNA targeting Gem into ATDC5 cells. Acan, Col2a1, Sox9, and Wnt target genes of Gem-Kd ATDC5 cells were detected during induction. The transcriptomic sequencing analysis was performed to investigate the mechanism of GEM regulation. Wnt signaling pathways were verified by real-time PCR and immunoblot analysis. Finally, a rescue model generated by treating Gem-KD ATDC5 cells with a Wnt signaling agonist was established to validate the mechanism identified by RNA sequencing analysis. RESULTS: A decreased expression of GEM in OA patients' cartilage tissues and OA mimic chondrocytes was observed. While during chondrogenesis differentiation and cartilage matrix formation, the expression of GEM was increased. Gem silencing suppressed chondrogenic differentiation and the expressions of Acan, Col2a1, and Sox9. RNA sequencing analysis revealed that Wnt signaling was downregulated in Gem-Kd cells. Decreased expression of Wnt signaling associated genes and the total ß-CATENIN in the nucleus and cytoplasm were observed. The exogenous Wnt activation exhibited reversed effect on Gem loss-of-function cells. CONCLUSION: These findings collectively validated that GEM functions as a novel regulator mediating chondrogenic differentiation and cartilage matrix formation through Wnt/ß-catenin signaling.

Delta like Non-Canonical Notch Ligand 2 inhibits chondrogenic differentiation and cell proliferation of bone marrow mesenchymal stem cells through the Notch1 signaling pathway.

Bone marrow mesenchymal stem cells (BMSCs) is the candidate for the treatment of cartilage defects because of their directional induction potential and natural anti-inflammatory properties. As one of the non-canonical receptors of Notch1, Delta Like Non-Canonical Notch Ligand 2 (DLK2) involves in stem cells' adipogenesis and chondrogenic differentiation. However, the specific regulatory mechanism of DLK2 in the chondrogenic differentiation of BMSCs is still unclear. In this study, we found that the expression of DLK2 was reduced and the expression of Col2a1, Col10a1, Acan, Sox9, and Notch1 was raised in the process of BMSCs chondrogenic differentiation. However, the expression of Col2a1, Col10a1, Acan, and Sox9 reduced significantly, and the signal factor Notch1 and the chondrogenic differentiation capacity of BMSCs turned down in the DLK2 overexpression group. Furthermore, the expression of Col2a1, Col10a1, Acan, and Sox9 significantly enhanced, Notch1 expression was also increased, and the chondrogenic differentiation capacity of BMSCs turned up in the DLK2 suppression group. Concurrently, the proliferation of BMSCs was weakened after overexpression of DLK2, and there was no significant change in cell migration. However, the proliferation and migration of BMSCs were significantly enhanced after the inhibition of DLK2 expression. Therefore, these results suggest that DLK2 negatively regulates chondrogenic differentiation and cell proliferation in BMSCs by inhibiting the Notch1 signaling pathway.

MicroRNAs as Prognostic Markers for Chondrogenic Differentiation Potential of Equine Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are a promising cell source for cartilage tissue regeneration in animals and humans but with large interdonor variation in their in vitro chondrogenic differentiation potential. Underlying molecular mechanisms responsible for culture-expanded MSC heterogeneity remain poorly understood. In this study, we sought to identify variations in microRNA (miRNA) signatures associated with cultured equine MSC chondrogenic differentiation potential from different donors. Neocartilage tissue generated from equine cord blood-derived MSCs was categorized as having either high or low chondrogenic potential (LCP) based on their histological appearance and quantification of glycosaminoglycan deposition. Using next-generation sequencing, we identified 30 differentially expressed miRNAs among undifferentiated MSC cultures that corresponded with their chondrogenic potential. Of note, MSCs with LCP upregulated miR-146a and miR-487b-3p, which was also observed by quantitative real-time polymerase chain reaction. Our findings suggest that miRNA profiling of equine MSC cultures may have prognostic value in selecting MSC donors with regard to their chondrogenic differentiation potential.

GuiLu-ErXian Glue extract promotes mesenchymal stem cells (MSC)-Induced chondrogenesis via exosomes release and delays aging in the MSC senescence process.

ETHNOPHARMACOLOGICAL RELEVANCE: The treatment of osteoarthritis (OA) patients is a challenging problem. Mesenchymal stem cells (MSCs) are multipotent cells and play key roles in regenerative medicine for cartilage degeneration. GuiLu-ErXian Glue (GLEXG) is an herbal remedy widely used in traditional Chinese medicine to treat joint pain and disability in elderly OA patients. However, the mechanisms of how GLEXG affects MSCs-induced chondrogensis remains to be elucidated. AIM OF THE STUDY: The aim of this study was to investigate the effects of GLEXG on MSC-derived chondrogenesis, both in vitro and in vivo and its potential mechanisms. METHODS: Using human MSC (hMSCs) as in vitro model, the effects of HPLC-profiled GLEXG water extract on chondrogenic differentiation were investigated by 3D spheroid cultures under chondrogenesis-inducing medium (CIM) condition. The chondrogenesis process was evaluated by measuring the sphere sizes, chondrogenesis-related genes expression by reverse transcription real-time PCR that targeted type II/X collagens, SOX9, aggrecan, and protein expression by immunostaining. Anti-TGF-ß1 neutralization antibody was used for mechanistic study. Mono-iodoacetate (MIA) induced OA joint was used to evaluate the effects of GLEXG on in vivo model. MSCs-derived exosomes were purified for proteomics study and senescence process was evaluated by cumulative population doublings and senescence-associated ß-Galactosidase staining. RESULTS: The results showed that GLEXG enhanced hMSCs chondrogenesis and upregulated RNA expression of type II/X collagen, SOX9 and aggrecan at 0.1 µg/mL, 0.3 µg/mL in vitro. In vivo, GLEXG at the dose of 0.3 µg intraarticular (i.a.) injection rescued the MIA-induced cartilage defect. Proteomics and ingenuity pathway analysis obtained from MSCs-released exosomes suggested that senescence pathway was less activated in GLEXG group than in vehicle group. Besides, GLEXG was able to increase cumulative population doubling and delayed hMSCs senescence process after four passages in cultures. CONCLUSION: we conclude that GLEXG promotes in vitro MSC-induced chondrogenesis possibly via exosomes release and delays aging in the MSC senescence process and that treatment with GLEXG (0.3 µg, i.a.) rescued cartilage defects in rat OA knee model.

Galectin-1 promotes angiogenesis and chondrogenesis during antler regeneration.

BACKGROUND: Deer antlers are the only known mammalian structure that undergoes full regeneration. In addition, it is peculiar because when growing, it contains vascularized cartilage. The differentiation of antler stem cells (ASCs) into chondrocytes while inducing endochondral extension of blood vessels is necessary to form antler vascularized cartilage. Therefore, antlers provide an unparalleled opportunity to investigate chondrogenesis, angiogenesis, and regenerative medicine. A study found that Galectin-1 (GAL-1), which can be used as a marker in some tumors, is highly expressed in ASCs. This intrigued us to investigate what role GAL-1 could play in antler regeneration. METHODS: We measured the expression level of GAL-1 in antler tissues and cells by immunohistochemistry, WB and QPCR. We constructed antlerogenic periosteal cells (APCs, one cell type of ASCs) with the GAL-1 gene knocked out (APCGAL-1-/-) using CRISPR-CAS9 gene editing system. The effect of GAL-1 on angiogenesis was determined by stimulating human umbilical vein endothelial cells (HUVECs) using APCGAL-1-/- conditioned medium or adding exogenous deer GAL-1 protein. The effect of APCGAL-1-/- on chondrogenic differentiation was evaluated compared with the APCs under micro-mass culture. The gene expression pattern of APCGAL-1-/- was analyzed by transcriptome sequencing. RESULTS: Immunohistochemistry revealed that GAL-1 was widely expressed in the antlerogenic periosteum (AP), pedicle periosteum (PP) and antler growth center. Western blot and qRT-PCR analysis using deer cell lines further supports this result. The proliferation, migration, and tube formation assays of human umbilical vein endothelial cells (HUVECs) showed that the proangiogenic activity of APCGAL-1-/- medium was significantly decreased (P < 0.05) compared with the APCs medium. The proangiogenic activity of deer GAL-1 protein was further confirmed by adding exogenous deer GAL-1 protein (P < 0.05). The chondrogenic differentiation ability of APCGAL-1-/- was impeded under micro-mass culture. The terms of GO and KEGG enrichment of the differentially expressed genes (DEGs) of APCGAL-1-/- showed that down-regulated expression of pathways associated with deer antler angiogenesis, osteogenesis and stem cell pluripotency, such as the PI3K-AKT signaling pathway, signaling pathways regulating pluripotency of stem cells and TGF-ß signaling pathway. CONCLUSIONS: Deer GAL-1, has strong angiogenic activity, is widely and highly expressed in deer antler. The APCs can induce angiogenesis by secreting GAL-1. The knockout of GAL-1 gene of APCs damaged its ability to induce angiogenesis and differentiate into chondrocytes. This ability is crucial to the formation of deer antler vascularized cartilage. Moreover, Deer antlers offer a unique model to explore explore how angiogenesis at high levels of GAL-1 expression can be elegantly regulated without becoming cancerous.

Peptide Regulation of Chondrogenic Stem Cell Differentiation.

The search for innovative ways to treat osteoarthritis (OA) is an urgent task for molecular medicine and biogerontology. OA leads to disability in persons of middle and older age, while safe and effective methods of treating OA have not yet been discovered. The directed differentiation of mesenchymal stem cells (MSCs) into chondrocytes is considered one of the possible methods to treat OA. This review describes the main molecules involved in the chondrogenic differentiation of MSCs. The peptides synthesized on the basis of growth factors' structures (SK2.1, BMP, B2A, and SSPEPS) and components of the extracellular matrix of cartilage tissue (LPP, CFOGER, CMP, RDG, and N-cadherin mimetic peptide) offer the greatest promise for the regulation of the chondrogenic differentiation of MSCs. These peptides regulate the WNT, ERK-p38, and Smad 1/5/8 signaling pathways, gene expression, and the synthesis of chondrogenic differentiation proteins such as COL2, SOX9, ACAN, etc.

The Effect of CaV1.2 Inhibitor Nifedipine on Chondrogenic Differentiation of Human Bone Marrow or Menstrual Blood-Derived Mesenchymal Stem Cells and Chondrocytes.

Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa2+) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa2+ was highest in chondrocytes, whereas iCa2+ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa2+, Cav1.2 and the proliferation of all cells and affected iCa2+ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs (SOX9 and COL2A1 expression); however, purposeful iCa2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs.

Modulus-dependent effects on neurogenic, myogenic, and chondrogenic differentiation of human mesenchymal stem cells in three-dimensional hydrogel cultures.

Human mesenchymal stromal cells (hMSCs) are of significant interest as a renewable source of therapeutically useful cells. In tissue engineering, hMSCs are implanted within a scaffold to provide enhanced capacity for tissue repair. The present study evaluates how mechanical properties of that scaffold can alter the phenotype and genotype of the cells, with the aim of augmenting hMSC differentiation along the myogenic, neurogenic or chondrogenic linages. The hMSCs were grown three-dimensionally (3D) in a hydrogel comprised of poly(ethylene glycol) (PEG)-conjugated to fibrinogen. The hydrogel's shear storage modulus (G'), which was controlled by increasing the amount of PEG-diacrylate cross-linker in the matrix, was varied in the range of 100-2000 Pascal (Pa). The differentiation into each lineage was initiated by a defined culture medium, and the hMSCs grown in the different modulus hydrogels were characterized using gene and protein expression. Materials having lower storage moduli (G' = 100 Pa) exhibited more hMSCs differentiating to neurogenic lineages. Myogenesis was favored in materials having intermediate modulus values (G' = 500 Pa), whereas chondrogenesis was favored in materials with a higher modulus (G' = 1000 Pa). Enhancing the differentiation pathway of hMSCs in 3D hydrogel scaffolds using simple modifications to mechanical properties represents an important achievement toward the effective application of these cells in tissue engineering.