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.

An extra-erythrocyte role of haemoglobin body in chondrocyte hypoxia adaption.

Although haemoglobin is a known carrier of oxygen in erythrocytes that functions to transport oxygen over a long range, its physiological roles outside erythrocytes are largely elusive1,2. Here we found that chondrocytes produced massive amounts of haemoglobin to form eosin-positive bodies in their cytoplasm. The haemoglobin body (Hedy) is a membraneless condensate characterized by phase separation. Production of haemoglobin in chondrocytes is controlled by hypoxia and is dependent on KLF1 rather than the HIF1/2α pathway. Deletion of haemoglobin in chondrocytes leads to Hedy loss along with severe hypoxia, enhanced glycolysis and extensive cell death in the centre of cartilaginous tissue, which is attributed to the loss of the Hedy-controlled oxygen supply under hypoxic conditions. These results demonstrate an extra-erythrocyte role of haemoglobin in chondrocytes, and uncover a heretofore unrecognized mechanism in which chondrocytes survive a hypoxic environment through Hedy.

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.

GDF5+ chondroprogenitors derived from human pluripotent stem cells preferentially form permanent chondrocytes.

It has been established in the mouse model that during embryogenesis joint cartilage is generated from a specialized progenitor cell type, distinct from that responsible for the formation of growth plate cartilage. We recently found that mesodermal progeny of human pluripotent stem cells gave rise to two types of chondrogenic mesenchymal cells in culture: SOX9+ and GDF5+ cells. The fast-growing SOX9+ cells formed in vitro cartilage that expressed chondrocyte hypertrophy markers and readily underwent mineralization after ectopic transplantation. In contrast, the slowly growing GDF5+ cells derived from SOX9+ cells formed cartilage that tended to express low to undetectable levels of chondrocyte hypertrophy markers, but expressed PRG4, a marker of embryonic articular chondrocytes. The GDF5+-derived cartilage remained largely unmineralized in vivo. Interestingly, chondrocytes derived from the GDF5+ cells seemed to elicit these activities via non-cell-autonomous mechanisms. Genome-wide transcriptomic analyses suggested that GDF5+ cells might contain a teno/ligamento-genic potential, whereas SOX9+ cells resembled neural crest-like progeny-derived chondroprogenitors. Thus, human pluripotent stem cell-derived GDF5+ cells specified to generate permanent-like cartilage seem to emerge coincidentally with the commitment of the SOX9+ progeny to the tendon/ligament lineage.

The functionality and translatability of neocartilage constructs are improved with the combination of fluid-induced shear stress and bioactive factors.

Neocartilage tissue engineering aims to address the shortcomings of current clinical treatments for articular cartilage indications. However, advancement is required toward neocartilage functionality (mechanical and biochemical properties) and translatability (construct size, gross morphology, passage number, cell source, and cell type). Using fluid-induced shear (FIS) stress, a potent mechanical stimulus, over four phases, this work investigates FIS stress' efficacy toward creating large neocartilage derived from highly passaged minipig costal chondrocytes, a species relevant to the preclinical regulatory process. In Phase I, FIS stress application timing was investigated in bovine articular chondrocytes and found to improve the aggregate modulus of neocartilage by 151% over unstimulated controls when stimulated during the maturation stage. In Phase II, FIS stress stimulation was translated from bovine articular chondrocytes to expanded minipig costal chondrocytes, yielding a 46% improvement in aggregate modulus over nonstimulated controls. In Phase III, bioactive factors were combined with FIS stress to improve the shear modulus by 115% over bioactive factor-only controls. The translatability of neocartilage was improved in Phase IV by utilizing highly passaged cells to form constructs more than 9-times larger in the area (11 × 17 mm), yielding an improved aggregate modulus by 134% and a flat morphology compared to free-floating, bioactive factor-only controls. Overall, this study represents a significant step toward generating mechanically robust, large constructs necessary for animal studies, and eventually, human clinical studies.

Luteolin Protects Chondrocytes from H2O2-Induced Oxidative Injury and Attenuates Osteoarthritis Progression by Activating AMPK-Nrf2 Signaling.

Osteoarthritis (OA) is a chronic degenerative disease featured by cartilage erosion and inflammation. Luteolin, a member of the flavonoid family, has been shown to exert anti-inflammatory and antioxidative activities. However, the potential biological effects and underlying mechanism of luteolin on chondrocytes and OA progression remain largely elusive. In this study, the potential effect and mechanism of luteolin on OA were investigated in vitro and in vivo. Our data revealed that luteolin inhibited H2O2-induced cell death, apoptosis, oxidative stress, programmed necrosis, and inflammatory mediator production in primary murine chondrocytes. In addition, luteolin could activate the AMPK and Nrf2 pathways, and AMPK serves as a positive upstream regulator of Nrf2. In vivo results demonstrated the therapeutic effects of luteolin on OA in the DMM mouse model. Collectively, our findings showed that luteolin might serve as a novel and effective treatment for OA and provided a new research direction for clinical OA therapies.

Macrophages promote cartilage regeneration in a time- and phenotype-dependent manner.

Immune regulation of osteochondral defect regeneration has not yet been rigorously characterized. Although macrophages have been demonstrated to regulate the regeneration process in various tissues, their direct contribution to cartilage regeneration remains to be investigated, particularly the functions of polarized macrophage subpopulations. In this study, we investigated the origins and functions of macrophages during healing of osteochondral injury in the murine model. Upon osteochondral injury, joint macrophages are predominantly derived from circulating monocytes. Macrophages are essential for spontaneous cartilage regeneration in juvenile C57BL/6 mice, by modulating proliferation and apoptosis around the injury site. Exogeneous macrophages also exhibit therapeutic potential in promoting cartilage regeneration in adult mice with poor regenerative capacity, possibly via regulation of PDGFRα+  stem cells, with this process being influenced by initial phenotype and administration timing. Only M2c macrophages are able to promote regeneration of both cartilage tissues and subchondral bone. Overall, we reveal the direct link between macrophages and osteochondral regeneration and highlight the key roles of relevant immunological niches in successful regeneration.

Hydrogel composite scaffolds achieve recruitment and chondrogenesis in cartilage tissue engineering applications.

BACKGROUND: The regeneration and repair of articular cartilage remains a major challenge for clinicians and scientists due to the poor intrinsic healing of this tissue. Since cartilage injuries are often clinically irregular, tissue-engineered scaffolds that can be easily molded to fill cartilage defects of any shape that fit tightly into the host cartilage are needed. METHOD: In this study, bone marrow mesenchymal stem cell (BMSC) affinity peptide sequence PFSSTKT (PFS)-modified chondrocyte extracellular matrix (ECM) particles combined with GelMA hydrogel were constructed. RESULTS: In vitro experiments showed that the pore size and porosity of the solid-supported composite scaffolds were appropriate and that the scaffolds provided a three-dimensional microenvironment supporting cell adhesion, proliferation and chondrogenic differentiation. In vitro experiments also showed that GelMA/ECM-PFS could regulate the migration of rabbit BMSCs. Two weeks after implantation in vivo, the GelMA/ECM-PFS functional scaffold system promoted the recruitment of endogenous mesenchymal stem cells from the defect site. GelMA/ECM-PFS achieved successful hyaline cartilage repair in rabbits in vivo, while the control treatment mostly resulted in fibrous tissue repair. CONCLUSION: This combination of endogenous cell recruitment and chondrogenesis is an ideal strategy for repairing irregular cartilage defects.

Tethered TGF-ß1 in a Hyaluronic Acid-Based Bioink for Bioprinting Cartilaginous Tissues.

In 3D bioprinting for cartilage regeneration, bioinks that support chondrogenic development are of key importance. Growth factors covalently bound in non-printable hydrogels have been shown to effectively promote chondrogenesis. However, studies that investigate the functionality of tethered growth factors within 3D printable bioinks are still lacking. Therefore, in this study, we established a dual-stage crosslinked hyaluronic acid-based bioink that enabled covalent tethering of transforming growth factor-beta 1 (TGF-ß1). Bone marrow-derived mesenchymal stromal cells (MSCs) were cultured over three weeks in vitro, and chondrogenic differentiation of MSCs within bioink constructs with tethered TGF-ß1 was markedly enhanced, as compared to constructs with non-covalently incorporated TGF-ß1. This was substantiated with regard to early TGF-ß1 signaling, chondrogenic gene expression, qualitative and quantitative ECM deposition and distribution, and resulting construct stiffness. Furthermore, it was successfully demonstrated, in a comparative analysis of cast and printed bioinks, that covalently tethered TGF-ß1 maintained its functionality after 3D printing. Taken together, the presented ink composition enabled the generation of high-quality cartilaginous tissues without the need for continuous exogenous growth factor supply and, thus, bears great potential for future investigation towards cartilage regeneration. Furthermore, growth factor tethering within bioinks, potentially leading to superior tissue development, may also be explored for other biofabrication applications.

Mechanical Cues: Bidirectional Reciprocity in the Extracellular Matrix Drives Mechano-Signalling in Articular Cartilage.

The composition and organisation of the extracellular matrix (ECM), particularly the pericellular matrix (PCM), in articular cartilage is critical to its biomechanical functionality; the presence of proteoglycans such as aggrecan, entrapped within a type II collagen fibrillar network, confers mechanical resilience underweight-bearing. Furthermore, components of the PCM including type VI collagen, perlecan, small leucine-rich proteoglycans-decorin and biglycan-and fibronectin facilitate the transduction of both biomechanical and biochemical signals to the residing chondrocytes, thereby regulating the process of mechanotransduction in cartilage. In this review, we summarise the literature reporting on the bidirectional reciprocity of the ECM in chondrocyte mechano-signalling and articular cartilage homeostasis. Specifically, we discuss studies that have characterised the response of articular cartilage to mechanical perturbations in the local tissue environment and how the magnitude or type of loading applied elicits cellular behaviours to effect change. In vivo, including transgenic approaches, and in vitro studies have illustrated how physiological loading maintains a homeostatic balance of anabolic and catabolic activities, involving the direct engagement of many PCM molecules in orchestrating this slow but consistent turnover of the cartilage matrix. Furthermore, we document studies characterising how abnormal, non-physiological loading including excessive loading or joint trauma negatively impacts matrix molecule biosynthesis and/or organisation, affecting PCM mechanical properties and reducing the tissue's ability to withstand load. We present compelling evidence showing that reciprocal engagement of the cells with this altered ECM environment can thus impact tissue homeostasis and, if sustained, can result in cartilage degradation and onset of osteoarthritis pathology. Enhanced dysregulation of PCM/ECM turnover is partially driven by mechanically mediated proteolytic degradation of cartilage ECM components. This generates bioactive breakdown fragments such as fibronectin, biglycan and lumican fragments, which can subsequently activate or inhibit additional signalling pathways including those involved in inflammation. Finally, we discuss how bidirectionality within the ECM is critically important in enabling the chondrocytes to synthesise and release PCM/ECM molecules, growth factors, pro-inflammatory cytokines and proteolytic enzymes, under a specified load, to influence PCM/ECM composition and mechanical properties in cartilage health and disease.

Restoring Osteochondral Defects through the Differentiation Potential of Cartilage Stem/Progenitor Cells Cultivated on Porous Scaffolds.

Cartilage stem/progenitor cells (CSPCs) are cartilage-specific, multipotent progenitor cells residing in articular cartilage. In this study, we investigated the characteristics and potential of human CSPCs combined with poly(lactic-co-glycolic acid) (PLGA) scaffolds to induce osteochondral regeneration in rabbit knees. We isolated CSPCs from human adult articular cartilage undergoing total knee replacement (TKR) surgery. We characterized CSPCs and compared them with infrapatellar fat pad-derived stem cells (IFPs) in a colony formation assay and by multilineage differentiation analysis in vitro. We further evaluated the osteochondral regeneration of the CSPC-loaded PLGA scaffold during osteochondral defect repair in rabbits. The characteristics of CSPCs were similar to those of mesenchymal stem cells (MSCs) and exhibited chondrogenic and osteogenic phenotypes without chemical induction. For in vivo analysis, CSPC-loaded PLGA scaffolds produced a hyaline-like cartilaginous tissue, which showed good integration with the host tissue and subchondral bone. Furthermore, CSPCs migrated in response to injury to promote subchondral bone regeneration. Overall, we demonstrated that CSPCs can promote osteochondral regeneration. A monophasic approach of using diseased CSPCs combined with a PLGA scaffold may be beneficial for repairing complex tissues, such as osteochondral tissue.

Pharmaceutical therapeutics for articular regeneration and restoration: state-of-the-art technology for screening small molecular drugs.

Articular cartilage damage caused by sports injury or osteoarthritis (OA) has gained increased attention as a worldwide health burden. Pharmaceutical treatments are considered cost-effective means of promoting cartilage regeneration, but are limited by their inability to generate sufficient functional chondrocytes and modify disease progression. Small molecular chemical compounds are an abundant source of new pharmaceutical therapeutics for cartilage regeneration, as they have advantages in design, fabrication, and application, and, when used in combination, act as powerful tools for manipulating cellular fate. In this review, we present current achievements in the development of small molecular drugs for cartilage regeneration, particularly in the fields of chondrocyte generation and reversion of chondrocyte degenerative phenotypes. Several clinically or preclinically available small molecules, which have been shown to facilitate chondrogenesis, chondrocyte dedifferentiation, and cellular reprogramming, and subsequently ameliorate cartilage degeneration by targeting inflammation, matrix degradation, metabolism, and epigenetics, are summarized. Notably, this review introduces essential parameters for high-throughput screening strategies, including models of different chondrogenic cell sources, phenotype readout methodologies, and transferable advanced systems from other fields. Overall, this review provides new insights into future pharmaceutical therapies for cartilage regeneration.

In Vitro Cartilage Regeneration with a Three-Dimensional Polyglycolic Acid (PGA) Implant in a Bovine Cartilage Punch Model.

Resorbable polyglycolic acid (PGA) chondrocyte grafts are clinically established for human articular cartilage defects. Long-term implant performance was addressed in a standardized in vitro model. PGA implants (+/- bovine chondrocytes) were placed inside cartilage rings punched out of bovine femoral trochleas (outer Ø 6 mm; inner defect Ø 2 mm) and cultured for 84 days (12 weeks). Cartilage/PGA hybrids were subsequently analyzed by histology (hematoxylin/eosin; safranin O), immunohistochemistry (aggrecan, collagens 1 and 2), protein assays, quantitative real-time polymerase chain reactions, and implant push-out force measurements. Cartilage/PGA hybrids remained vital with intact matrix until 12 weeks, limited loss of proteoglycans from "host" cartilage or cartilage-PGA interface, and progressively diminishing release of proteoglycans into the supernatant. By contrast, the collagen 2 content in cartilage and cartilage-PGA interface remained approximately constant during culture (with only little collagen 1). Both implants (+/- cells) displayed implant colonization and progressively increased aggrecan and collagen 2 mRNA, but significantly decreased push-out forces over time. Cell-loaded PGA showed significantly accelerated cell colonization and significantly extended deposition of aggrecan. Augmented chondrogenic differentiation in PGA and cartilage/PGA-interface for up to 84 days suggests initial cartilage regeneration. Due to the PGA resorbability, however, the model exhibits limitations in assessing the "lateral implant bonding".

Dual Delivery of TGF-ß3 and Ghrelin in Microsphere/Hydrogel Systems for Cartilage Regeneration.

Articular cartilage (AC) damage is quite common, but due to AC's poor self-healing ability, the damage can easily develop into osteoarthritis (OA). To solve this problem, we developed a microsphere/hydrogel system that provides two growth factors that promote cartilage repair: transforming growth factor-ß3 (TGF-ß3) to enhance cartilage tissue formation and ghrelin synergy TGF-ß to significantly enhance the chondrogenic differentiation. The hydrogel and microspheres were characterized in vitro, and the biocompatibility of the system was verified. Double emulsion solvent extraction technology (w/o/w) is used to encapsulate TGF-ß3 and ghrelin into microspheres, and these microspheres are encapsulated in a hydrogel to continuously release TGF-ß3 and ghrelin. According to the chondrogenic differentiation ability of mesenchymal stem cells (MSCs) in vitro, the concentrations of the two growth factors were optimized to promote cartilage regeneration.

A Review of the Use of Microparticles for Cartilage Tissue Engineering.

Tissue and organ failure has induced immense economic and healthcare concerns across the world. Tissue engineering is an interdisciplinary biomedical approach which aims to address the issues intrinsic to organ donation by providing an alternative strategy to tissue and organ transplantation. This review is specifically focused on cartilage tissue. Cartilage defects cannot readily regenerate, and thus research into tissue engineering approaches is relevant as a potential treatment option. Cells, scaffolds, and growth factors are three components that can be utilized to regenerate new tissue, and in particular recent advances in microparticle technology have excellent potential to revolutionize cartilage tissue regeneration. First, microspheres can be used for drug delivery by injecting them into the cartilage tissue or joint space to reduce pain and stimulate regeneration. They can also be used as controlled release systems within tissue engineering constructs. Additionally, microcarriers can act as a surface for stem cells or chondrocytes to adhere to and expand, generating large amounts of cells, which are necessary for clinically relevant cell therapies. Finally, a newer application of microparticles is to form them together into granular hydrogels to act as scaffolds for tissue engineering or to use in bioprinting. Tissue engineering has the potential to revolutionize the space of cartilage regeneration, but additional research is needed to allow for clinical translation. Microparticles are a key enabling technology in this regard.

Phenotype and synthesis activity of joint chondrocytes extracted from newborn rats with prenatal ethanol exposure.

Thirteen female Wistar rats were divided into two groups: one treated with ethanol and the other of untreated. Four newborns from each mother were selected and weighed, measured, and evaluated for physical characteristics. From these neonates, chondrocytes were extracted from the articular cartilages of the femur and tibia, and cultivated in a chondrogenic medium at 37oC and 5% CO2. At 7, 14, and 21 days of cultivation, alkaline phosphatase activity tests, MTT conversion to formazan, and percentage area covered by cells per field were performed. At 21 days, the percentage of PAS+ areas in 3D cultures was performed, as well as the evaluation of gene transcript expression for aggrecan, SOX-9, collagen type II, collagen X, Runx-2, and VEGF by real-time RT-PCR. The means were compared by Student's t-test. The weight of the ethanol group neonates was significantly lower than that of the controls. Chondrocyte cultures from the ethanol group showed significantly higher AP activity, MTT conversion, and cell percentage. There was higher expression of collagen type II and lower expression of SOX-9 in the ethanol group. There was no difference in the percentage of PAS+ areas in pellets and in expression of aggrecan, collagen X, Runx-2, or VEGF between groups. In conclusion, prenatal exposure to ethanol alters the phenotype and activity of offspring chondrocytes, which may be mechanisms by which endochondral bone formation is compromised by maternal ethanol consumption.

The Use of Infrapatellar Fat Pad-Derived Mesenchymal Stem Cells in Articular Cartilage Regeneration: A Review.

Cartilage is frequently damaged with a limited capacity for repair. Current treatment strategies are insufficient as they form fibrocartilage as opposed to hyaline cartilage, and do not prevent the progression of degenerative changes. There is increasing interest in the use of autologous mesenchymal stem cells (MSC) for tissue regeneration. MSCs that are used to treat articular cartilage defects must not only present a robust cartilaginous production capacity, but they also must not cause morbidity at the harvest site. In addition, they should be easy to isolate from the tissue and expand in culture without terminal differentiation. The source of MSCs is one of the most important factors that may affect treatment. The infrapatellar fat pad (IPFP) acts as an important reservoir for MSC and is located in the anterior compartment of the knee joint in the extra-synovial area. The IPFP is a rich source of MSCs, and in this review, we discuss studies that demonstrate that these cells have shown many advantages over other tissues in terms of ease of isolation, expansion, and chondrogenic differentiation. Future studies in articular cartilage repair strategies and suitable extraction as well as cell culture methods will extend the therapeutical application of IPFP-derived MSCs into additional orthopedic fields, such as osteoarthritis. This review provides the latest research concerning the use of IPFP-derived MSCs in the treatment of articular cartilage damage, providing critical information for the field to grow.

A cell-free ROS-responsive hydrogel/oriented poly(lactide-co-glycolide) hybrid scaffold for reducing inflammation and restoring full-thickness cartilage defectsin vivo.

The modulation of inflammation in tissue microenvironment takes an important role in cartilage repair and regeneration. In this study, a novel hybrid scaffold was designed and fabricated by filling a reactive oxygen species (ROS)-scavenging hydrogel (RS Gel) into a radially oriented poly(lactide-co-glycolide) (PLGA) scaffold. The radially oriented PLGA scaffolds were fabricated through a temperature gradient-guided phase separation and freeze-drying method. The RS Gel was formed by crosslinking the mixture of ROS-responsive hyperbranched polymers and biocompatible methacrylated hyaluronic acid (HA-MA). The hybrid scaffolds exhibited a proper compressive modulus, good ROS-scavenging capability, and cell compatibility.In vivotests showed that the hybrid scaffolds significantly regulated inflammation and promoted regeneration of hyaline cartilage after they were implanted into full-thickness cartilage defects in rabbits for 12 w. In comparison with the PLGA scaffolds, the neo-cartilage in the hybrid scaffolds group possessed more deposition of glycosaminoglycans and collagen type II, and were well integrated with the surrounding tissue.

Mechanical stimulation of chondrocytes regulates HIF-1α under hypoxic conditions.

We investigated the effects of hypoxia-inducible factor (HIF)-1α on articular cartilage under mechanical stimulation and the associated mechanisms. Chondrocytes, isolated from articular cartilage from the knee, hip, and shoulder joints of Wistar rats, were subjected to 20 % tensile stress under hypoxic (5% O2) conditions for 24 h. HIF-1α and aggrecan expression was significantly enhanced with mechanical stimulation under hypoxia but not significantly altered with mechanical stimulation under normoxia. The nuclear translocation of HIF-1α was enhanced by mechanical stress under hypoxia. Under both normoxia and hypoxia, a disintegrin and metalloproteinase with thrombospondin motifs (ADAM-TS) 5 expression was significantly reduced with mechanical stimulation compared to that in the group without mechanical stimulation. However, HIF-1α knockdown mitigated changes in aggrecan and ADAM-TS5 expression mediated by mechanical stimulation under hypoxia. The effects of treadmill running on HIF-1α production in the articular cartilage of rat knee joints were also analyzed. HIF-1α production increased in the moderate running group and decreased to the same levels as those in the control group in the excessive running group. This suggests that HIF-1α regulates aggrecan and ADAM-TS5 expression in response to mechanical stimulation under hypoxia and general mechanical stimulation in articular cartilage under hypoxia, while controlling cartilage homeostasis.

14-3-3 epsilon is an intracellular component of TNFR2 receptor complex and its activation protects against osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) is the most common joint disease; however, the indeterminate nature of mechanisms by which OA develops has restrained advancement of therapeutic targets. TNF signalling has been implicated in the pathogenesis of OA. TNFR1 primarily mediates inflammation, whereas emerging evidences demonstrate that TNFR2 plays an anti-inflammatory and protective role in several diseases and conditions. This study aims to decipher TNFR2 signalling in chondrocytes and OA. METHODS: Biochemical copurification and proteomics screen were performed to isolate the intracellular cofactors of TNFR2 complex. Bulk and single cell RNA-seq were employed to determine 14-3-3 epsilon (14-3-3ε) expression in human normal and OA cartilage. Transcription factor activity screen was used to isolate the transcription factors downstream of TNFR2/14-3-3ε. Various cell-based assays and genetically modified mice with naturally occurring and surgically induced OA were performed to examine the importance of this pathway in chondrocytes and OA. RESULTS: Signalling molecule 14-3-3ε was identified as an intracellular component of TNFR2 complexes in chondrocytes in response to progranulin (PGRN), a growth factor known to protect against OA primarily through activating TNFR2. 14-3-3ε was downregulated in OA and its deficiency deteriorated OA. 14-3-3ε was required for PGRN regulation of chondrocyte metabolism. In addition, both global and chondrocyte-specific deletion of 14-3-3ε largely abolished PGRN's therapeutic effects against OA. Furthermore, PGRN/TNFR2/14-3-3ε signalled through activating extracellular signal-regulated kinase (ERK)-dependent Elk-1 while suppressing nuclear factor kappa B (NF-κB) in chondrocytes. CONCLUSIONS: This study identifies 14-3-3ε as an inducible component of TNFR2 receptor complex in response to PGRN in chondrocytes and presents a previously unrecognised TNFR2 pathway in the pathogenesis of OA.