Application of a variational autoencoder for clustering and analyzing in situ articular cartilage cellular response to mechanical stimuli.

In various biological systems, analyzing how cell behaviors are coordinated over time would enable a deeper understanding of tissue-scale response to physiologic or superphysiologic stimuli. Such data is necessary for establishing both normal tissue function and the sequence of events after injury that lead to chronic disease. However, collecting and analyzing these large datasets presents a challenge-such systems are time-consuming to process, and the overwhelming scale of data makes it difficult to parse overall behaviors. This problem calls for an analysis technique that can quickly provide an overview of the groups present in the entire system and also produce meaningful categorization of cell behaviors. Here, we demonstrate the application of an unsupervised method-the Variational Autoencoder (VAE)-to learn the features of cells in cartilage tissue after impact-induced injury and identify meaningful clusters of chondrocyte behavior. This technique quickly generated new insights into the spatial distribution of specific cell behavior phenotypes and connected specific peracute calcium signaling timeseries with long term cellular outcomes, demonstrating the value of the VAE technique.

Extremely low frequency-electromagnetic fields promote chondrogenic differentiation of adipose-derived mesenchymal stem cells through a conventional genetic program.

Progressive cartilage deterioration leads to chronic inflammation and loss of joint function, causing osteoarthritis (OA) and joint disease. Although symptoms vary among individuals, the disease can cause severe pain and permanent disability, and effective therapies are urgently needed. Human Adipose-Derived Stem Cells (ADSCs) may differentiate into chondrocytes and are promising for treating OA. Moreover, recent studies indicate that electromagnetic fields (EMFs) could positively affect the chondrogenic differentiation potential of ADSCs. In this work, we investigated the impact of EMFs with frequencies of 35 Hertz and 58 Hertz, referred to as extremely low frequency-EMFs (ELF-EMFs), on the chondrogenesis of ADSCs, cultured in both monolayer and 3D cell micromasses. ADSC cultures were daily stimulated for 36 min with ELF-EMFs or left unstimulated, and the progression of the differentiation process was evaluated by morphological analysis, extracellular matrix deposition, and gene expression profiling of chondrogenic markers. In both culturing conditions, stimulation with ELF-EMFs did not compromise cell viability but accelerated chondrogenesis by enhancing the secretion and deposition of extracellular matrix components at earlier time points in comparison to unstimulated cells. This study showed that, in an appropriate chondrogenic microenvironment, ELF-EMFs enhance chondrogenic differentiation and may be an important tool for supporting and accelerating the treatment of OA through autologous adipose stem cell therapy.

Stem-Cell-Driven Chondrogenesis: Perspectives on Amnion-Derived Cells.

Regenerative medicine harnesses stem cells' capacity to restore damaged tissues and organs. In vitro methods employing specific bioactive molecules, such as growth factors, bio-inductive scaffolds, 3D cultures, co-cultures, and mechanical stimuli, steer stem cells toward the desired differentiation pathways, mimicking their natural development. Chondrogenesis presents a challenge for regenerative medicine. This intricate process involves precise modulation of chondro-related transcription factors and pathways, critical for generating cartilage. Cartilage damage disrupts this process, impeding proper tissue healing due to its unique mechanical and anatomical characteristics. Consequently, the resultant tissue often forms fibrocartilage, which lacks adequate mechanical properties, posing a significant hurdle for effective regeneration. This review comprehensively explores studies showcasing the potential of amniotic mesenchymal stem cells (AMSCs) and amniotic epithelial cells (AECs) in chondrogenic differentiation. These cells exhibit innate characteristics that position them as promising candidates for regenerative medicine. Their capacity to differentiate toward chondrocytes offers a pathway for developing effective regenerative protocols. Understanding and leveraging the innate properties of AMSCs and AECs hold promise in addressing the challenges associated with cartilage repair, potentially offering superior outcomes in tissue regeneration.

Bioengineering autologous cartilage grafts for functional posterior lamellar eyelid reconstruction: A preliminary study in rabbits.

The reconstruction of posterior lamellar eyelid defects remains a significant challenge in clinical practice due to anatomical complexity, specialized function, and aesthetic concerns. The ideal substitute for the posterior lamellar should replicate the native tarsoconjunctival tissue, providing both mechanical support for the eyelids and a smooth surface for the globe after implantation. In this study, we present an innovative approach utilizing tissue-engineered cartilage (TEC) grafts generated from rabbit auricular chondrocytes and a commercialized type I collagen sponge to reconstruct critical-sized posterior lamellar defects in rabbits. The TEC grafts demonstrated remarkable mechanical strength and maintained a stable cartilaginous phenotype both in vitro and at 6 months post-implantation in immunodeficient mice. When employed as autografts to reconstruct tarsal plate defects in rabbits' upper eyelids, these TEC grafts successfully restored normal eyelid morphology, facilitated smooth eyelid movement, and preserved the histological structure of the conjunctival epithelium. When applied in bilayered tarsoconjunctival defect reconstruction, these TEC grafts not only maintained the normal contour of the upper eyelid but also supported conjunctival epithelial cell migration and growth from the defect margin towards the centre. These findings highlight that auricular chondrocyte-based TEC grafts hold great promise as potential candidates for clinical posterior lamellar reconstruction. STATEMENT OF SIGNIFICANCE: The complex structure and function of the posterior lamellar eyelid continue to be significant challenges for clinical reconstructive surgeries. In this study, we utilized autologous auricular chondrocyte-based TEC grafts for posterior lamellar eyelid reconstruction in a preclinical rabbit model. The TEC grafts exhibited native cartilaginous histomorphology and comparable mechanical strength to those of the native human tarsal plate. In rabbit models with either tarsal plate defects alone or bilayered tarsoconjunctival defects, TEC grafts successfully restored the normal eyelid contour and movement, as well as supported preservation and growth of conjunctival epithelium. This is the first study to demonstrate autologous TEC grafts can be employed for repairing tarsal plate defects, thereby offering an alternative therapeutic approach for treating posterior lamellar defects in clinic settings.

3D printing of Ceffe-infused scaffolds for tailored nipple-like cartilage development.

The reconstruction of a stable, nipple-shaped cartilage graft that precisely matches the natural nipple in shape and size on the contralateral side is a clinical challenge. While 3D printing technology can efficiently and accurately manufacture customized complex structures, it faces limitations due to inadequate blood supply, which hampers the stability of nipple-shaped cartilage grafts produced using this technology. To address this issue, we employed a biodegradable biomaterial, Poly(lactic-co-glycolic acid) (PLGA), loaded with Cell-Free Fat Extract (Ceffe). Ceffe has demonstrated the ability to promote angiogenesis and cell proliferation, making it an ideal bio-ink for bioprinting precise nipple-shaped cartilage grafts. We utilized the Ceffe/PLGA scaffold to create a porous structure with a precise nipple shape. This scaffold exhibited favorable porosity and pore size, ensuring stable shape maintenance and satisfactory biomechanical properties. Importantly, it could release Ceffe in a sustained manner. Our in vitro results confirmed the scaffold's good biocompatibility and its ability to promote angiogenesis, as evidenced by supporting chondrocyte proliferation and endothelial cell migration and tube formation. Furthermore, after 8 weeks of in vivo culture, the Ceffe/PLGA scaffold seeded with chondrocytes regenerated into a cartilage support structure with a precise nipple shape. Compared to the pure PLGA group, the Ceffe/PLGA scaffold showed remarkable vascular formation, highlighting the beneficial effects of Ceffe. These findings suggest that our designed Ceffe/PLGA scaffold with a nipple shape represents a promising strategy for precise nipple-shaped cartilage regeneration, laying a foundation for subsequent nipple reconstruction.

Postmortem chondrocyte viability in porcine articular cartilage: Influence of time, temperature, and burial under winter conditions.

The aim of the present study was to investigate the effects of time, temperature, and burial in a natural environment on the viability of chondrocytes in porcine femoral condyles using confocal laser scanning microscopy. Hind trotters from 10 pigs were buried or left unburied. Samples were collected daily and stained with a combination of vital dyes (calcein-AM and ethidium homodimer-1). The chondrocytes showed an intense staining corresponding to their vitality. In the first 3 days, viability decreased slowly and showed no statistical difference between buried and unburied samples. After the first 3 days, it decreased rapidly, with the viability of the buried samples being 66% on day 4, decreasing to 25% on day 8 and to 16% on day 10, while in the unburied samples it decreased to 43% on day 4, 13% on day 8 and 5% on day 10. Our results indicate a time, temperature, and burial dependent decrease in chondrocyte viability and suggest the use of chondrocyte viability as a marker for estimating PMI in both the natural environment and in animals, as well as its potential use in humans.

Refining the identity of mesenchymal cell types associated with murine periosteal and endosteal bone.

Single-cell RNA-seq has led to novel designations for mesenchymal cells associated with bone as well as multiple designations for what appear to be the same cell type. The main goals of this study were to increase the amount of single-cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. We created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-Cxcl12 abundant reticular (CAR), osteo-CAR, preosteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by periostin expression. Osteo-X, osteo-CAR, and preosteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in preosteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single-cell RNA-seq datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.

Injectable microspheres adhering to the cartilage matrix promote rapid reconstruction of partial-thickness cartilage defects.

An effective treatment for the irregular partial-thickness cartilage defect in the early stages of osteoarthritis (OA) is lacking. Cartilage tissue engineering is effective for treating full-thickness cartilage defects with limited area. In this study, we designed an injectable multifunctional poly(lactic-co-glycolic acid) (PLGA) microsphere to repair partial-thickness cartilage defects. The microsphere was grafted with an E7 peptide after loading the microsphere with kartogenin (KGN) and modifying the outer layer through dopamine self-polymerization. The microsphere could adhere to the cartilage defect, recruit synovial mesenchymal stem cells (SMSCs) in situ, and stimulate their differentiation into chondrocytes after injection into the articular cavity. Through in vivo and in vitro experiments, we demonstrated the ability of multifunctional microspheres to adhere to cartilage matrix, recruit SMSCs, and promote their differentiation into cartilage. Following treatment, the cartilage surface of the model group with partial-thickness cartilage defect showed smooth recovery, and the glycosaminoglycan content remained normal; the untreated control group showed significant progression of OA. The microsphere, a framework for cartilage tissue engineering, promoted the expression of SMSCs involved in cartilage repair while adapting to cell migration and growth. Thus, for treating partial-thickness cartilage defects in OA, this innovative carrier system based on stem cell therapy can potentially improve therapeutic outcomes. STATEMENT OF SIGNIFICANCE: Mesenchymal stem cells (MSCs) therapy is effective in the repair of cartilage injury. However, because of the particularity of partial-thickness cartilage injury, it is difficult to recruit enough seed cells in situ, and there is a lack of suitable scaffolds for cell migration and growth. Here, we developed polydopamine surface-modified PLGA microspheres (PMS) containing KGN and E7 peptides. The adhesion ability of the microspheres is facilitated by the polydopamine layer wrapped in them; thus, the microspheres can adhere to the injured cartilage and recruit MSCs, thereby promoting their differentiation into chondrocytes and accomplishing cartilage repair. The multifunctional microspheres can be used as a safe and potential method to treat partial-thickness cartilage defects in OA.

Redifferentiation of genetically modified dedifferentiated chondrocytes in a microcavitary hydrogel.

OBJECTIVES: We genetically modified dedifferentiated chondrocytes (DCs) using lentiviral vectors and adenoviral vectors encoding TGF-ß3 (referred to as transgenic groups below) and encapsulated these DCs in the microcavitary hydrogel and investigated the combinational effect on redifferentiation of the genetically manipulated DCs. RESULTS: The Cell Counting Kit-8 data indicated that both transgenic groups exhibited significantly higher cell viability in the first week but inferior cell viability in the subsequent timepoints compared with those of the control group. Real-time polymerase chain reaction and western blot analysis results demonstrated that both transgenic groups had a better effect on redifferentiation to some extent, as evidenced by higher expression levels of chondrogenic genes, suggesting the validity of combination with transgenic DCs and the microcavitary hydrogel on redifferentiation. Although transgenic DCs with adenoviral vectors presented a superior extent of redifferentiation, they also expressed greater levels of the hypertrophic gene type X collagen. It is still worth further exploring how to deliver TGF-ß3 more efficiently and optimizing the appropriate parameters, including concentration and duration. CONCLUSIONS: The results demonstrated the better redifferentiation effect of DCs with the combinational use of transgenic TGF-ß3 and a microcavitary alginate hydrogel and implied that DCs would be alternative seed cells for cartilage tissue engineering due to their easily achieved sufficient cell amounts through multiple passages and great potential to redifferentiate to produce cartilaginous extracellular matrix.

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.

Gulp1 regulates chondrocyte growth arrest and differentiation via the TGF-ß/SMAD2/3 pathway.

Chondrocyte differentiation is crucial for cartilage formation. However, the complex processes and mechanisms coordinating chondrocyte proliferation and differentiation remain incompletely understood. Here, we report a novel function of the adaptor protein Gulp1 in chondrocyte differentiation. Gulp1 expression is upregulated during chondrogenic differentiation. Gulp1 knockdown in chondrogenic ATDC5 cells reduces the expression of chondrogenic and hypertrophic marker genes during differentiation. Furthermore, Gulp1 knockdown impairs cell growth arrest during chondrocyte differentiation and reduces the expression of the cyclin-dependent kinase inhibitor p21. The activation of the TGF-ß/SMAD2/3 pathway, which is associated with p21 expression in chondrocytes, is impaired in Gulp1 knockdown cells. Collectively, these results demonstrate that Gulp1 contributes to cell growth arrest and chondrocyte differentiation by modulating the TGF-ß/SMAD2/3 pathway.

3D Printing of Cobalt-Incorporated Chloroapatite Bioceramic Composite Scaffolds with Antioxidative Activity for Enhanced Osteochondral Regeneration.

Osteochondral defects are often accompanied by excessive reactive oxygen species (ROS) caused by osteoarthritis or acute surgical inflammation. An inflammatory environment containing excess ROS will not only hinder tissue regeneration but also impact the quality of newly formed tissues. Therefore, there is an urgent need to develop scaffolds with both ROS scavenging and osteochondral repair functions to promote and protect osteochondral tissue regeneration. In this work, by using 3D printing technology, a composite scaffold based on cobalt-incorporated chloroapatite (Co-ClAP) bioceramics, which possesses ROS-scavenging activity and can support cell proliferation, adhesion, and differentiation, is developed. Benefiting from the catalytic activity of Co-ClAP bioceramics, the composite scaffold can protect cells from oxidative damage under ROS-excessive conditions, support their directional differentiation, and simultaneously mediate an anti-inflammatory microenvironment. In addition, it is also confirmed by using rabbit osteochondral defect model that the Co-ClAP/poly(lactic-co-glycolic acid) scaffold can effectively promote the integrated regeneration of cartilage and subchondral bone, exhibiting an ideal repair effect in vivo. This study provides a promising strategy for the treatment of defects with excess ROS and inflammatory microenvironments.

NOTCH2 sensitizes the chondrocyte to the inflammatory response of tumor necrosis factor α.

Notch regulates the immune and inflammatory response and has been associated with the pathogenesis of osteoarthritis in humans and preclinical models of the disease. Notch2tm1.1Ecan mice harbor a NOTCH2 gain-of-function and are sensitized to osteoarthritis, but the mechanisms have not been explored. We examined the effects of tumor necrosis factor α (TNFα) in chondrocytes from Notch2tm1.1Ecan mice and found that NOTCH2 enhanced the effect of TNFα on Il6 and Il1b expression. Similar results were obtained in cells from a conditional model of NOTCH2 gain-of-function, Notch22.1Ecan mice, and following the expression of the NOTCH2 intracellular domain in vitro. Recombination signal-binding protein for immunoglobulin Kappa J region partners with the NOTCH2 intracellular domain to activate transcription; in the absence of Notch signaling it inhibits transcription, and Rbpj inactivation in chondrocytes resulted in Il6 induction. Although TNFα induced IL6 to a greater extent in the context of NOTCH2 activation, there was a concomitant inhibition of Notch target genes Hes1, Hey1, Hey2, and Heyl. Electrophoretic mobility shift assay demonstrated displacement of recombination signal-binding protein for immunoglobulin Kappa J region from DNA binding sites by TNFα explaining the increased Il6 expression and the concomitant decrease in Notch target genes. NOTCH2 enhanced the effect of TNFα on NF-κB signaling, and RNA-Seq revealed increased expression of pathways associated with inflammation and the phagosome in NOTCH2 overexpressing cells in the absence and presence of TNFα. Collectively, NOTCH2 has important interactions with TNFα resulting in the enhanced expression of Il6 and inflammatory pathways in chondrocytes.

Hes1 marks peri-condensation mesenchymal cells that generate both chondrocytes and perichondrial cells in early bone development.

Bone development starts with condensations of undifferentiated mesenchymal cells that set a framework for future bones within the primordium. In the endochondral pathway, mesenchymal cells inside the condensation differentiate into chondrocytes and perichondrial cells in a SOX9-dependent mechanism. However, the identity of mesenchymal cells outside the condensation and how they participate in developing bones remain undefined. Here we show that mesenchymal cells surrounding the condensation contribute to both cartilage and perichondrium, robustly generating chondrocytes, osteoblasts, and marrow stromal cells in developing bones. Single-cell RNA-seq analysis of Prrx1-cre-marked limb bud mesenchymal cells at E11.5 reveals that Notch effector Hes1 is expressed in a mutually exclusive manner with Sox9 that is expressed in pre-cartilaginous condensations. Analysis of a Notch signaling reporter CBF1:H2B-Venus reveals that peri-condensation mesenchymal cells are active for Notch signaling. In vivo lineage-tracing analysis using Hes1-creER identifies that Hes1+ early mesenchymal cells surrounding the SOX9+ condensation at E10.5 contribute to both cartilage and perichondrium at E13.5, subsequently becoming growth plate chondrocytes, osteoblasts of trabecular and cortical bones, and marrow stromal cells in postnatal bones. In contrast, Hes1+ cells in the perichondrium at E12.5 or E14.5 do not generate chondrocytes within cartilage, contributing to osteoblasts and marrow stromal cells only through the perichondrial route. Therefore, Hes1+ peri-condensation mesenchymal cells give rise to cells of the skeletal lineage through cartilage-dependent and independent pathways, supporting the theory that early mesenchymal cells outside the condensation also play important roles in early bone development.

Analysis of N-glycosylation protein of Kashin-Beck disease chondrocytes derived from induced pluripotent stem cells based on label-free strategies with LC-MS/MS.

We aimed to compare N-glycosylation proteins in Kashin-Beck disease (KBD) chondrocytes and normal chondrocytes derived from induced pluripotent stem cells (iPSCs). KBD and normal iPSCs were reprogrammed from human KBD and normal dermal fibroblasts, respectively. Subsequently, chondrocytes were differentiated from KBD and normal iPSCs separately. Immunofluorescence was utilized to assay the protein markers of iPSCs and chondrocytes. Differential N-glycosylation proteins were screened using label-free strategies with LC-MS/MS. Bioinformatics analyses were utilized to interpret the functions of differential N-glycosylation proteins. Immunofluorescence staining revealed that both KBD-iPSCs and normal-iPSCs strongly expressed pluripotency markers OCT4 and NANOG. Meanwhile, chondrocyte markers collagen II and SOX9 are presented in KBD-iPSC-chondrocytes and normal-iPSC-chondrocytes. We obtained 87 differential N-glycosylation sites which corresponded to 68 differential proteins, which were constructed into 1 cluster. We obtained collagen type I trimer and 9 other biological processes; polysaccharide binding and 9 other molecular functions; regulation of transcription by RNA polymerase II and 9 other cellular components from GO; the Pl3K-Akt signaling pathway and 9 other KEGG pathways; peroxisome and 7 other subcellular locations; and integrin alpha chain, C-terminal cytoplasmic region, conserved site and 9 other classifications of domain annotations, and 2 networks. FGFR3 and LRP1 are expressed at higher levels in KBD-iPSC-chondrocytes, while the expressions of COL2A1, TIMP1, UNC5B, NOG, LEPR, and ITGA1 were down-regulated in KBD-iPSC-chondrocytes. The differential expressions of these N-glycosylation proteins may lead to the abnormal function of KBD chondrocytes.

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.

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.

NFATc1 marks articular cartilage progenitors and negatively determines articular chondrocyte differentiation.

The origin and differentiation mechanism of articular chondrocytes remain poorly understood. Broadly, the difference in developmental mechanisms of articular and growth-plate cartilage is still less elucidated. Here, we identified that the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) is a crucial regulator of articular, but not growth-plate, chondrocyte differentiation during development. At the early stage of mouse knee development (embryonic day 13.5), NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With development, NFATc1-expressing cells generated almost all articular chondrocytes but not chondrocytes in limb growth-plate primordium. NFATc1-expressing cells displayed prominent capacities for colony formation and multipotent differentiation. Transcriptome analyses revealed a set of characteristic genes in NFATc1-enriched articular cartilage progenitors. Strikingly, the expression of NFATc1 was diminished with articular chondrocyte differentiation, and suppressing NFATc1 expression in articular cartilage progenitors was sufficient to induce spontaneous chondrogenesis while overexpressing NFATc1 suppresses chondrogenesis. Mechanistically, NFATc1 negatively regulated the transcriptional activity of the Col2a1 gene. Thus, our results reveal that NFATc1 characterizes articular, but not growth-plate, cartilage progenitors during development and negatively determines articular chondrocyte differentiation at least partly through regulating COL2A1 gene transcription.

Within the body are about 300 joints connecting bones together. Many factors ­ including trauma, inflammation, aging, and genetic changes ­ can affect the cushion tissue covering the end of the bones in these joints known as articular cartilage. This can lead to diseases such as osteoarthritis which cause chronic pain, and in some cases disability. To treat such conditions, it is essential to know how cells in the articular cartilage are formed during development. In the embryo, most cells come from groups of progenitor cells that are programmed to produce specific types of tissue. But which progenitor cells are responsible for producing the main cells in articular cartilage, chondrocytes, and the mechanisms that govern this transformation are poorly understood. In 2016, a group of researchers found that the gene for the protein NFATc1, which is important for building bone, is also expressed in a group of progenitor cells at the site where ligaments insert into bone in mice. Inactivation of NFATc1 in these progenitor cells has also been shown to cause abnormal cartilage to form, a condition termed osteochondromas. Building on this work, Zhang, Wang et al. ­ including some of the researchers involved in the 2016 study ­ set out to find whether NFATc1 is also involved in the normal development of articular chondrocytes. To investigate, the team used genetically modified mice in which any cells with NFATc1 also had a green fluorescent protein, and tracked these cells and their progeny over the course of joint development. This led them to discover a group of NFATc1-containing progenitor cells that gave rise to almost all articular chondrocytes in the knee joint. Further experiments revealed that when NFATc1 was removed, this made the progenitors become articular chondrocytes very quickly. In contrast, when the cells had excess amounts of the protein, the formation of articular chondrocytes was significantly reduced. This suggests that the level of NFATc1 governs when progenitors develop into articular chondrocytes. These findings have provided a way to track the progenitors of articular chondrocytes throughout development and study how articular cartilage is formed. In the future, this work could help researchers develop treatment strategies for osteoarthritis and other cartilage-based diseases. However, before this can happen, further work is needed to confirm that the effects observed in this study also relate to humans.

The Expression of αvß3 and Osteopontin in Osteoarthritic Knee Cartilage and Their Correlations With Disease Severity and Chondrocyte Senescence.

Osteoarthritis (OA) is the main joint disease associated with aging. Previous studies have confirmed that both osteopontin (OPN) and αvß3 integrin are involved in the progression of knee OA. The purpose of this study was to determine the expression of OPN and αvß3 integrin and chondrocyte senescence levels in OA. Forty-six cartilage tissues from normal and knee OA patients were divided into 4 groups of normal, minor, moderate, and severe lesions based on the Mankin score. Immunohistochemistry and western blotting were used to determine the expression of αvß3, OPN, and senescent-associated-ß-galactosidase (SAß-gal) in articular cartilage. Then, Spearman's correlation was used to analyze the correlations between the Mankin scores and αvß3, OPN and SAß-gal. Pearson correlation analysis was used to analyze the correlations among αvß3, OPN, and SAß-gal. The expression of OPN, αvß3, and SAß-gal in articular cartilage was explored. αvß3, OPN, and SAß-gal proteins were all elevated in OA cartilage, and the correlation coefficient between the Mankin score and the average optical density value of αvß3, OPN, SAß-gal were r =0.60, r =0.75, and r =0.87, respectively, all P <0.001; the correlation between the average optical density value of αvß3 and OPN was r =0.3191, P <0.05; the correlation between αvß3 and SAß-gal was r =0.4955, P <0.001; and the correlation between OPN and SAß-gal was r =0.7821, P <0.001. The correlations among αvß3, OPN, and SAß-gal expression in articular cartilage might be important in OA progression and pathogenesis. Nonetheless, more research is needed to elucidate the exact contribution of αvß3, OPN, and SAß-gal to the degenerative process of OA.

Epstein-Barr virus-induced gene 3 commits human mesenchymal stem cells to differentiate into chondrocytes via endoplasmic reticulum stress sensor.

Mesenchymal stem cells (MSC) can differentiate into chondrocytes. Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed during chondrogenic differentiation and can be produced by MSC. EBI3 is also a subunit of interleukin (IL)-27 and IL-35, and it accumulates in the endoplasmic reticulum (ER) when its partners, such as IL-27 p28 and IL-35 p35, are insufficient. ER stress induced by protein accumulation is responsible for chondrogenic differentiation. However, the role of EBI3 and its relevance to the ER stress in chondrogenic differentiation of MSC have never been addressed. Here, we demonstrate that EBI3 protein is expressed in the early stage of chondrogenic differentiation of MSC. Additionally, knockdown, overexpression, or induction of EBI3 through IL-1ß inhibits chondrogenesis. We show that EBI3 localizes and accumulates in the ER of MSC after overexpression or induction by IL-1ß and TNF-α, whereas ER stress inhibitor 4-phenylbutyric acid decreases its accumulation in MSC. Moreover, EBI3 modulates ER stress sensor inositol-requiring enzyme 1 α (IRE1α) after induced by IL-1ß, and MSC-like cells coexpress EBI3 and IRE1α in rheumatoid arthritis (RA) synovial tissue. Altogether, these data demonstrate that intracellular EBI3 commits to chondrogenic differentiation by regulating ER stress sensor IRE1α.