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.

A Multi-functional Hybrid System Comprised of Polydopamine Nanobottles and Biological Effectors for Cartilage Repair.

Biological effectors play critical roles in augmenting the repair of cartilage injuries, but it remains a challenge to control their release in a programmable, stepwise fashion. Herein, a hybrid system consisting of polydopamine (PDA) nanobottles embedded in a hydrogel matrix to manage the release of biological effectors for use in cartilage repair is reported. Specifically, a homing effector is load in the hydrogel matrix, together with the encapsulation of a cartilage effector in PDA nanobottles filled with phase-change material. In action, the homing effector is quickly released from the hydrogel in the initial step to recruit stem cells from the surroundings. Owing to the antioxidation effect of PDA, the recruited cells are shielded from reactive oxygen species. The cartilage effector is then slowly released from the nanobottles to promote chondrogenic differentiation, facilitating cartilage repair. Altogether, this strategy encompassing recruitment, protection, and differentiation of stem cells offers a viable route to tissue repair or regeneration through stem cell therapy.

Suppression of MALAT1 promotes human synovial mesenchymal stem cells enhance chondrogenic differentiation and prevent osteoarthritis of the knee in a rat model via regulating miR-212-5p/MyD88 axis.

Osteoarthritis (OA) is one of the most common diseases of the skeleton. Long non-coding RNAs (lncRNAs) are emerging as key players in OA pathogenesis. This work sets out to determine the function of lncRNA MALAT1 in OA and the mechanisms by which it does so. Mesenchymal stem cells isolated from the human synovial membrane are called hSMSCs. The hSMSCs' surface markers were studied using flow cytometry. To determine whether or not hSMSC might differentiate, researchers used a number of different culture settings and labeling techniques. The expression levels of associated genes and proteins were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), and immunostaining. A dual luciferase reporter experiment and RNA immunoprecipitation (RIP) test demonstrated the direct association between miR-212-5p and MALAT1 or MyD88. MALAT1 was downregulated during the chondrogenic differentiation of hSMSCs, and underexpression of MALAT1 promotes chondrogenesis in hSMSCs. Using dual luciferase reporter and RIP assays facilitated the identification of MALAT1 as a competitive endogenous RNA (ceRNA) that sequesters miR-212-5p. Additionally, the expression of MYD88 was regulated by MALAT1 through direct binding with miR-212-5p. Significantly, the effects of MALAT1 on the chondrogenic differentiation of hSMSCs were counteracted by miR-212-5p/MYD88. Furthermore, our in vivo investigation revealed that the inhibition of MALAT1 mitigated osteoarthritis progression in rat models. In conclusion, the promotion of chondrogenic differentiation in hSMSCs and the protective effect on cartilage tissue in OA can be achieved by suppressing MALAT1, which regulates the miR-212-5p/MyD88 axis.

Primary cilia in skeletal development and disease.

Primary cilia are non-motile, microtubule-based sensory organelle present in most vertebrate cells with a fundamental role in the modulation of organismal development, morphogenesis, and repair. Here we focus on the role of primary cilia in embryonic and postnatal skeletal development. We examine evidence supporting its involvement in physiochemical and developmental signaling that regulates proliferation, patterning, differentiation and homeostasis of osteoblasts, chondrocytes, and their progenitor cells in the skeleton. We discuss how signaling effectors in mechanotransduction and bone development, such as Hedgehog, Wnt, Fibroblast growth factor and second messenger pathways operate at least in part at the primary cilium. The relevance of primary cilia in bone formation and maintenance is underscored by a growing list of rare genetic skeletal ciliopathies. We collate these findings and summarize the current understanding of molecular factors and mechanisms governing primary ciliogenesis and ciliary function in skeletal development and disease.

MiR-143-3p regulates chondrogenic differentiation of synovium derived mesenchymal stem cells under mechanical stress through the BMPR2-Smad signalling pathway by targeting BMPR2.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from the synovium, known as synovium mesenchymal stem cells (SMSCs), exhibit significant potential for articular cartilage regeneration owing to their capacity for chondrogenic differentiation. However, the microRNAs (miRNAs) governing this process and the associated mechanisms remain unclear. While mechanical stress positively influences chondrogenesis in MSCs, the miRNA-mediated response of SMSCs to mechanical stimuli is not well understood. OBJECTIVE: This study explores the miRNA-driven mechano-transduction in SMSCs chondrogenesis under mechanical stress. METHODS: The surface phenotype of SMSCs was analysed by flow cytometry. Chondrogenesis capacities of SMSCs were examined by Alcian blue staining. High throughput sequencing was used to screen mechano-sensitive miRNAs of SMSCs. The RNA expression level of COL2A1, ACAN, SOX9, BMPR2 and miR-143-3p of SMSCs were tested by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-143-3p and TLR4 was confirmed by luciferase reporter assays. The protein expression levels of related genes were assessed by western blot. RESULTS: High-throughput sequencing revealed a notable reduction in miR-143-3p levels in mechanically stressed SMSCs. Gain- or loss-of-function strategies introduced by lentivirus demonstrated that miR-143-3p overexpression hindered chondrogenic differentiation, whereas its knockdown promoted this process. Bioinformatics scrutiny and luciferase reporter assays pinpointed a potential binding site for miR-143-3p within the 3'-UTR of bone morphogenetic protein receptor type 2 (BMPR2). MiR-143-3p overexpression decreased BMPR2 expression and phosphorylated Smad1, 5 and 8 levels, while its inhibition activated BMPR2-Smad pathway. CONCLUSION: This study elucidated that miR-143-3p negatively regulates SMSCs chondrogenic differentiation through the BMPR2-Smad pathway under mechanical tensile stress. The direct targeting of BMPR2 by miR-143-3p established a novel dimension to our understanding of mechano-transduction mechanism during SMSC chondrogenesis. This understanding is crucial for advancing strategies in articular cartilage regeneration.

Cartilage-Specific Gene Expression and Extracellular Matrix Deposition in the Course of Mesenchymal Stromal Cell Chondrogenic Differentiation in 3D Spheroid Culture.

Articular cartilage damage still remains a major problem in orthopedical surgery. The development of tissue engineering techniques such as autologous chondrocyte implantation is a promising way to improve clinical outcomes. On the other hand, the clinical application of autologous chondrocytes has considerable limitations. Mesenchymal stromal cells (MSCs) from various tissues have been shown to possess chondrogenic differentiation potential, although to different degrees. In the present study, we assessed the alterations in chondrogenesis-related gene transcription rates and extracellular matrix deposition levels before and after the chondrogenic differentiation of MSCs in a 3D spheroid culture. MSCs were obtained from three different tissues: umbilical cord Wharton's jelly (WJMSC-Wharton's jelly mesenchymal stromal cells), adipose tissue (ATMSC-adipose tissue mesenchymal stromal cells), and the dental pulp of deciduous teeth (SHEDs-stem cells from human exfoliated deciduous teeth). Monolayer MSC cultures served as baseline controls. Newly formed 3D spheroids composed of MSCs previously grown in 2D cultures were precultured for 2 days in growth medium, and then, chondrogenic differentiation was induced by maintaining them in the TGF-ß1-containing medium for 21 days. Among the MSC types studied, WJMSCs showed the most similarities with primary chondrocytes in terms of the upregulation of cartilage-specific gene expression. Interestingly, such upregulation occurred to some extent in all 3D spheroids, even prior to the addition of TGF-ß1. These results confirm that the potential of Wharton's jelly is on par with adipose tissue as a valuable cell source for cartilage engineering applications as well as for the treatment of osteoarthritis. The 3D spheroid environment on its own acts as a trigger for the chondrogenic differentiation of MSCs.

Research progress on the regulation of bone marrow stem cells by noncoding RNAs in adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a common spinal deformity in young women, but its pathogenesis remains unclear. The primary pathogenic factors contributing to its development include genetics, abnormal bone metabolism, and endocrine factors. Bone marrow stem cells (BMSCs) play a crucial role in the pathogenesis of AIS by regulating its occurrence and progression. Noncoding RNAs (ncRNAs) are also involved in the pathogenesis of AIS, and their role in regulating BMSCs in patients with AIS requires further evaluation. In this review, we discuss the relevant literature regarding the osteogenic, chondrogenic, and lipogenic differentiation of BMSCs. The corresponding mechanisms of ncRNA-mediated BMSC regulation in patients with AIS, recent advancements in AIS and ncRNA research, and the importance of ncRNA translation profiling and multiomics are highlighted.

Long non-coding RNA MIR22HG suppresses the chondrogenic differentiation of human adipose-derived stem cells by interacting with CTCF to upregulate CRLF1.

Improved chondrogenic differentiation of mesenchymal stem cells (MSCs) by genetic regulation is a potential method for regenerating articular cartilage. LncRNA MIR22HG has been proven to accelerate osteogenic differentiation, but the regulation mechanism of chondrogenic differentiation is still unclear. Human adipose-derived stem cells (hADSCs) have been widely utilised for bone tissue engineering applications. The present study aimed to examine the effect of MIR22HG on the chondrogenic differentiation of hADSCs. The results confirmed that MIR22HG was downregulated in the process of chondrogenic differentiation. Subsequently, gain- and loss-of-function of MIR22HG experiments showed that the overexpression of MIR22HG suppressed the deposition of cartilage matrix proteoglycans and decreased the expression of cartilage-related markers (e.g. Sox9, ACAN and Col2A1), whereas the knockdown of MIR22HG had the opposite effect. MIR22HG could bind to CTCF (CCCTC-binding factor), and CTCF could bind to the CRLF1 (cytokine receptor-like factor 1) promoter and upregulate CRLF1 gene expression. Besides, inhibition of CRLF1 can reverse the effect of MIR22HG on cell chondrogenic differentiation of hADSCs. Taken together, our outcomes reveal that MIR22HG suppressed chondrogenic differentiation by interaction with CTCF to stabilise CRLF1.

Rps6ka2 enhances iMSC chondrogenic differentiation to attenuate knee osteoarthritis through articular cartilage regeneration in mice.

Articular cartilage (AC) is most susceptible to degeneration in knee osteoarthritis (OA); however, the existing treatments for OA do not target the core link of the pathogenesis-"decreased tissue cell function activity and extracellular matrix (ECM) metabolic disorders" for effective intervention. iMSC hold lower heterogeneity and great promise in biological research and clinical applications. Rps6ka2 may play an important role in the iMSC to treat OA. In this study, the CRISPR/Cas9 gene editing Rps6ka2-/- iMSC were obtained. Effect of Rps6ka2 on iMSC proliferation and chondrogenic differentiation was evaluated in vitro. An OA model was constructed in mice by surgical destabilization of medial meniscus (DMM). The Rps6ka2-/- iMSC and iMSC were injected into the articular cavity twice-weekly for 8 weeks. In vitro experiments showed that Rps6ka2 could promote iMSC proliferation and chondrogenic differentiation. In vivo results further confirmed that Rps6ka2 could improve iMSC viability to promote ECM production to attenuate OA in mice.

Enhancement of mesenchymal stem cells' chondrogenic potential by type II collagen-based bioscaffolds.

BACKGROUND: Osteoarthritis (OA) is a common degenerative chronic disease accounting for physical pain, tissue stiffness and mobility restriction. Current therapeutic approaches fail to prevent the progression of the disease considering the limited knowledge on OA pathobiology. During OA progression, the extracellular matrix (ECM) of the cartilage is aberrantly remodeled by chondrocytes. Chondrocytes, being the main cell population of the cartilage, participate in cartilage regeneration process. To this end, modern tissue engineering strategies involve the recruitment of mesenchymal stem cells (MSCs) due to their regenerative capacity as to promote chondrocyte self-regeneration. METHODS AND RESULTS: In the present study, we evaluated the role of type II collagen, as the main matrix macromolecule in the cartilage matrix, to promote chondrogenic differentiation in two MSC in vitro culture systems. The chondrogenic differentiation of human Wharton's jelly- and dental pulp-derived MSCs was investigated over a 24-day culture period on type II collagen coating to improve the binding affinity of MSCs. Functional assays, demonstrated that type II collagen promoted chondrogenic differentiation in both MSCs tested, which was confirmed through gene and protein analysis of major chondrogenic markers. CONCLUSIONS: Our data support that type II collagen contributes as a natural bioscaffold enhancing chondrogenesis in both MSC models, thus enhancing the commitment of MSC-based therapeutic approaches in regenerative medicine to target OA and bring therapy closer to the clinical use.

PAC1 deficiency reduces chondrogenesis in atherosclerotic lesions of hypercholesterolemic ApoE-deficient mice.

BACKGROUND: Induction of chondrogenesis is associated with progressive atherosclerosis. Deficiency of the ADCYAP1 gene encoding pituitary adenylate cyclase-activating peptide (PACAP) aggravates atherosclerosis in ApoE deficient (ApoE-/-) mice. PACAP signaling regulates chondrogenesis and osteogenesis during cartilage and bone development. Therefore, this study aimed to decipher whether PACAP signaling is related to atherogenesis-related chondrogenesis in the ApoE-/- mouse model of atherosclerosis and under the influence of a high-fat diet. METHODS: For this purpose, PACAP-/-/ApoE-/-, PAC1-/-/ApoE-/-, and ApoE-/- mice, as well as wildtype (WT) mice, were studied under standard chow (SC) or cholesterol-enriched diet (CED) for 20 weeks. The amount of cartilage matrix in atherosclerotic lesions of the brachiocephalic trunk (BT) with maximal lumen stenosis was monitored by alcian blue and collagen II staining on deparaffinized cross sections. The chondrogenic RUNX family transcription factor 2 (RUNX2), macrophages [(MΦ), Iba1+], and smooth muscle cells (SMC, sm-α-actin) were immunohistochemically analyzed and quantified. RESULTS: ApoE-/- mice fed either SC or CED revealed an increase of alcian blue-positive areas within the media compared to WT mice. PAC1-/-/ApoE-/- mice under CED showed a reduction in the alcian blue-positive plaque area in the BT compared to ApoE-/- mice. In contrast, PACAP deficiency in ApoE-/- mice did not affect the chondrogenic signature under either diet. CONCLUSIONS: Our data show that PAC1 deficiency reduces chondrogenesis in atherosclerotic plaques exclusively under conditions of CED-induced hypercholesterolemia. We conclude that CED-related chondrogenesis occurs in atherosclerotic plaques via transdifferentiation of SMCs and MΦ, partly depending on PACAP signaling through PAC1. Thus, PAC1 antagonists or PACAP agonists may offer therapeutic potential against pathological chondrogenesis in atherosclerotic lesions generated under hypercholesterolemic conditions, especially in familial hypercholesterolemia. This discovery opens therapeutic perspectives to be used in the treatment against the progression of atherosclerosis.

Pleiotrophin inhibited chondrogenic differentiation potential of dental pulp stem cells.

OBJECTIVE: Studies have shown that the levels of pleiotrophin (PTN) are greatly elevated in the synovial fluid and cartilage in osteoarthritis. Therefore, the purpose of this study was to investigate the effect and mechanism of PTN on the chondrogenic differentiation of DPSCs in inflammatory and normal microenvironments. MATERIALS AND METHODS: A lentiviral vector was used to deplete or overexpress PTN in DPSCs. The inflammatory microenvironment was simulated in vitro by the addition of IL-1ß to the culture medium. The chondrogenic differentiation potential was assessed using Alcian Blue staining and the main chondrogenic markers. A dual-luciferase reporter assay was used to explore the relationship between miR-137 and PTN. RESULTS: The results showed that 0.1 ng/mL IL-1ß treatment during chondrogenic induction greatly impaired the chondrogenic differentiation of DPSCs. Supplementation with PTN and PTN overexpression inhibited chondrogenic differentiation of DPSCs, while PTN depletion promoted chondrogenic differentiation. MiR-137 negatively regulated the expression of PTN by binding to the 3'UTR of its mRNA. Moreover, miR-137 promoted chondrogenic differentiation of DPSCs in normal and inflammatory microenvironments. CONCLUSION: Our results suggest that PTN may play an inhibitory role in the chondrogenic differentiation of DPSCs in normal and inflammatory microenvironments, which is regulated by miR-137.

CD44 mediates hyaluronan to promote the differentiation of human amniotic mesenchymal stem cells into chondrocytes.

OBJECTIVES: CD44 is the major receptor for hyaluronan (HA), but its effect on HA-induced differentiation of human amnion mesenchymal stem cells into chondrocytes is unclear. This study aimed to investigate the effects and mechanisms of CD44 in HA-induced chondrogenesis. METHODS: Immunocytochemistry and toluidine blue staining were used to assess the secretion of type II collagen and aggrecan, respectively. qRT-PCR and western blotting were performed to evaluate the expression of key genes and proteins. RESULTS: The expression of aggrecan and type II collagen was downregulated after using the anti-CD44 antibody (A3D8). The transcriptional levels of chondrocytes­associated genes SRY­box transcription factor 9, aggrecan, and collagen type II alpha 1 chain were also decreased. Thus, CD44 may mediate HA-induced differentiation of hAMSCs into chondrocytes. Further investigation indicated that expression of phosphorylated (p)­Erk1/2 and p­Smad2 decreased following CD44 inhibition. The changes in the expression of p-Erk1/2 and p-Smad2 were consistent after using the ERK1/2 inhibitor (U0126) and agonist (EGF), respectively. After administering the p-Smad2 inhibitor, the expression levels of p-ERK1/2 and p-Smad2 appeared downregulated. The results showed crosstalk between Erk1/2 and Smad2. Moreover, inhibition of p-Erk1/2 and p-Smad2 significantly reduced the accumulation of aggrecan and type II collagen. CONCLUSION: These data indicate that CD44 mediates HA-induced differentiation of hAMSCs into chondrocytes by regulating Erk1/2 and Smad2 signaling.

The Effects of Chronological Age on the Chondrogenic Potential of Mesenchymal Stromal Cells: A Systematic Review.

Tissue engineering and cell therapy for regenerative medicine have great potential to treat chronic disorders. In musculoskeletal disorders, mesenchymal stromal cells (MSCs) have been identified as a relevant cell type in cell and regenerative strategies due to their multi-lineage potential, although this is likely to be a result of their trophic and immunomodulatory effects on other cells. This PRISMA systematic review aims to assess whether the age of the patient influences the chondrogenic potential of MSCs in regenerative therapy. We identified a total of 3027 studies after performing a search of four databases, including Cochrane, Web of Science, Medline, and PubMed. After applying inclusion and exclusion criteria, a total of 14 papers were identified that were reviewed, assessed, and reported. Cell surface characterization and proliferation, as well as the osteogenic, adipogenic, and chondrogenic differentiation, were investigated as part of the analysis of these studies. Most included studies suggest a clear link between aged donor MSCs and diminished clonogenic and proliferative potential. Our study reveals a heterogeneous and conflicting range of outcomes concerning the chondrogenic, osteogenic, and adipogenic potential of MSCs in relation to age. Further investigations on the in vitro effects of chronological age on the chondrogenic potential of MSCs should follow the outcomes of this systematic review, shedding more light on this complex relationship.

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.

The Effects of Mechanical Load on Chondrogenic Responses of Bone Marrow Mesenchymal Stem Cells and Chondrocytes Encapsulated in Chondroitin Sulfate-Based Hydrogel.

Articular cartilage is vulnerable to mechanical overload and has limited ability to restore lesions, which leads to the development of chronic diseases such as osteoarthritis (OA). In this study, the chondrogenic responses of human bone marrow mesenchymal stem cells (BMMSCs) and OA cartilage-derived chondrocytes in 3D chondroitin sulfate-tyramine/gelatin (CS-Tyr)/Gel) hydrogels with or without experimental mechanical load have been investigated. Chondrocytes were smaller in size, had slower proliferation rate and higher level of intracellular calcium (iCa2+) compared to BMMSCs. Under 3D chondrogenic conditions in CS-Tyr/Gel with or without TGF-ß3, chondrocytes more intensively secreted cartilage oligomeric matrix protein (COMP) and expressed collagen type II (COL2A1) and aggrecan (ACAN) genes but were more susceptible to mechanical load compared to BMMSCs. ICa2+ was more stably controlled in CS-Tyr/Gel/BMMSCs than in CS-Tyr/Gel/chondrocytes ones, through the expression of L-type channel subunit CaV1.2 (CACNA1C) and Serca2 pump (ATP2A2) genes, and their balance was kept more stable. Due to the lower susceptibility to mechanical load, BMMSCs in CS-Tyr/Gel hydrogel may have an advantage over chondrocytes in application for cartilage regeneration purposes. The mechanical overload related cartilage damage in vivo and the vague regenerative processes of OA chondrocytes might be associated to the inefficient control of iCa2+ regulating channels.

Icariin accelerates cartilage defect repair by promoting chondrogenic differentiation of BMSCs under conditions of oxygen-glucose deprivation.

This study explored the role played by combined ICA and bone mesenchymal stem cells (BMSCs) in repairing rabbit knee cartilage defects. Firstly, rabbit BMSCs were isolated and used to construct an in vitro cellular model of oxygen-glucose deprivation/reoxygenation (OGD/R). Subsequently, ICA processing, Alcian blue staining, immunofluorescence and Western blot studies were performed to evaluate the ability of BMSCs to display signs of chondrogenic differentiation. Furthermore, a rabbit knee cartilage injury model was established in vivo. International Cartilage Repair Society (ICRS) macroscopic evaluations, H&E, Alcian blue and EdU staining, as well as immunohistochemistry, were analysed cartilage repair and pathological condition of the knee cartilage tissue. Our in vitro results showed that ICA promoted the chondrogenic differentiation of BMSCs, as well as aggrecan (AGR), bone morphogenetic protein 2 (BMP2) and COL2A1 protein expression in BMSCs. In vivo experiments showed that rabbits in the BMSCs or ICA treatment group had higher ICRS scores and displayed a better restoration of cartilage-like tissue and chondrocyte expression on the surface of their cartilage defects. In conclusion, ICA or BMSCs alone could repair rabbit knee cartilage damage, and combined treatment with ICA and BMSCs showed a better ability to repair rabbit knee cartilage damage.

Caspase-9 inhibition decreases expression of Mmp9 during chondrogenesis.

Besides cell death, caspase-9 participates in non-apoptotic events, including cell differentiation. To evaluate a possible impact on the expression of chondrogenic/osteogenic factors, a caspase-9 inhibitor was tested in vitro. For this purpose, mouse forelimb-derived micromass cultures, the most common chondrogenic in vitro model, were used. The following analyses were performed based on polymerase chain reaction (PCR) arrays and real-time PCR. The expression of several chondrogenesis-related genes was shown to be altered, some of which may impact chondrogenic differentiation (Bmp4, Bmp7, Sp7, Gli1), mineral deposition (Alp, Itgam) or the remodelling of the extracellular matrix (Col1a2, Mmp9) related to endochondral ossification. From the cluster of genes with altered expression, Mmp9 showed the most significant decrease in expression, of more than 50-fold. Additionally, we determined the possible impact of caspase-9 downregulation on the expression of other Mmp genes. A mild increase in Mmp14 was observed, but there was no change in the expression of other studied Mmp genes (-2, -3, -8, -10, -12, -13). Interestingly, inhibition of Mmp9 in micromasses led to decreased expression of some chondrogenic markers related to caspase-9. These samples also showed a decreased expression of caspase-9 itself, suggesting a bidirectional regulation of these two enzymes. These results indicate a specific impact of caspase-9 inhibition on the expression of Mmp9. The localisation of these two enzymes overlaps in resting, proliferative and pre-hypertrophic chondrocytes during in vivo development, which supports their multiple functions, either apoptotic or non-apoptotic. Notably, a coincidental expression pattern was identified in Pik3cg, a possible candidate for Mmp9 regulation.

Integrated regulation of chondrogenic differentiation in mesenchymal stem cells and differentiation of cancer cells.

Chondrogenesis is the formation of chondrocytes and cartilage tissues and starts with mesenchymal stem cell (MSC) recruitment and migration, condensation of progenitors, chondrocyte differentiation, and maturation. The chondrogenic differentiation of MSCs depends on co-regulation of many exogenous and endogenous factors including specific microenvironmental signals, non-coding RNAs, physical factors existed in culture condition, etc. Cancer stem cells (CSCs) exhibit self-renewal capacity, pluripotency and cellular plasticity, which have the potential to differentiate into post-mitotic and benign cells. Accumulating evidence has shown that CSCs can be induced to differentiate into various benign cells including adipocytes, fibrocytes, osteoblast, and so on. Retinoic acid has been widely used in the treatment of acute promyelocytic leukemia. Previous study confirmed that polyploid giant cancer cells, a type of cancer stem-like cells, could differentiate into adipocytes, osteocytes, and chondrocytes. In this review, we will summarize signaling pathways and cytokines in chondrogenic differentiation of MSCs. Understanding the molecular mechanism of chondrogenic differentiation of CSCs and cancer cells may provide new strategies for cancer treatment.