Cerium oxide (CeO2) nanospheres have limited enzymatic activity that hinders further application in catalytic therapy, but they have an \"oxidation switch\" to enhance their catalytic activity by increasing oxygen vacancies. In this study, according to the defect-engineering strategy, we developed PtCuOX/CeO2-X nanozymes as highly efficient SOD/CAT mimics by introducing bimetallic copper (Cu) and platinum (Pt) into CeO2 nanospheres to enhance the oxygen vacancies, in an attempt to combine near-infrared (NIR) irradiation to regulate microenvironment for osteoarthritis (OA) therapy. As expected, the Cu and Pt increased the Ce3+/Ce4+ ratio of CeO2 to significantly enhance the oxygen vacancies, and simultaneously CeO2 (111) facilitated the uniform dispersion of Cu and Pt. The strong metal-carrier interaction synergy endowed the PtCuOX/CeO2-X nanozymes with highly efficient SOD/CAT-like activity by the decreased formation energy of oxygen vacancy, promoted electron transfer, the increased adsorption energy of intermediates, and the decreased reaction activation energy. Besides, the nanozymes have excellent photothermal conversion efficiency (55.41%). Further, the PtCuOX/CeO2-X antioxidant system effectively scavenged intracellular ROS and RNS, protected mitochondrial function, and inhibited the inflammatory factors, thus reducing chondrocyte apoptosis. In vivo, experiments demonstrated the biosafety of PtCuOX/CeO2-X and its potent effect on OA suppression. In particular, NIR radiation further enhanced the effects. Mechanistically, PtCuOX/CeO2-X nanozymes reduced ras-related C3 botulinum toxin substrate 1 (Rac-1) and p-p65 protein expression, as well as ROS levels to remodel the inflammatory microenvironment by inhibiting the ROS/Rac-1/nuclear factor kappa-B (NF-κB) signaling pathway. This study introduces new clinical concepts and perspectives that can be applied to inflammatory diseases.

BACKGROUND: Human adipose-derived stem cells (ADSCs) exert a strong anti-inflammatory effect, and synovium-derived stem cells (SDSCs) have high chondrogenic potential. Thus, this study aims to investigate whether a combination of human ADSCs and SDSCs will have a synergistic effect that will increase the chondrogenic potential of osteoarthritis (OA) chondrocytes in vitro and attenuate the cartilage degeneration of early and advanced OA in vitro.
METHODS: ADSCs, SDSCs, and chondrocytes were isolated from OA patients who underwent total knee arthroplasty. The ADSCs-SDSCs mixed cell ratios were 1:0 (ADSCs only), 8:2, 5:5 (5A5S), 2:8, and 0:1 (SDSCs only). The chondrogenic potential of the OA chondrocytes was evaluated in vitro with a transwell assay or pellet culture with various mixed cell groups. The mixed cell group with the highest chondrogenic potential was then selected and injected into the knee joints of nude rats of early and advanced OA stages in vivo. The animals were then evaluated 12 and 20 weeks after surgery through gait analysis, von frey test, microcomputed tomography, MRI, and immunohistochemical and histological analyses. Finally, the mechanisms underlying these findings were investigated through the RNA sequencing of tissue samples in vivo and Western blot of the OA chondrocyte autophagy pathway.
RESULTS: Among the MSCs treatment groups, 5A5S had the greatest synergistic effect that increased the chondrogenic potential of OA chondrocytes in vitro and inhibited early and advanced OA in vivo. The 5A5S group significantly reduced cartilage degeneration, synovial inflammation, pain sensation, and nerve invasion in subchondral nude rat OA, outperforming both single-cell treatments. The underlying mechanism was the activation of chondrocyte autophagy via the FoxO1 signaling pathway.
CONCLUSIONS: A combination of human ADSCs and SDSCs demonstrated higher potential than a single type of stem cell, demonstrating potential as a novel treatment for OA.

One of the major processes occurring during the healing of a fractured long bone is chondrogenesis, leading to the formation of the soft callus, which subsequently undergoes endochondral ossification and ultimately bridges the fracture site. Thus, understanding the molecular mechanisms of chondrogenesis can enhance our knowledge of the fracture repair process. One such molecular process is Ca++ signaling, which is known to play a critical role in the development and regeneration of multiple tissues, including bone, in response to external stimuli. Despite the existence of various mouse models for studying Ca++ signaling, none of them were designed to specifically examine the skeletal system or the various musculoskeletal cell types. As such, we generated a genetically engineered mouse model that is specific to cartilage (crossed with Col2a1 Cre mice) to study chondrocytes. Herein, we report on the characterization of this transgenic mouse line using conditional expression of GCaMP6f, a calcium-indicator protein. Specifically, this mouse line exhibits increased GCaMP6f fluorescence following calcium binding in chondrocytes. Using this model, we show real-time Ca++ signaling in embryos, newborn and adult mice, as well as in fracture calluses. Further, robust expression of GCaMP6f in chondrocytes can be easily detected in embryos, neonates, adults, and fracture callus tissue sections. Finally, we also report on Ca++ signaling pathway gene expression, as well as real-time Ca++ transient measurements in fracture callus chondrocytes. Taken together, these mice provide a new experimental tool to study chondrocyte-specific Ca++ signaling during skeletal development and regeneration, as well as various in vitro perturbations.

Eriodictyol, a flavonoid distributed in citrus fruits, has been known to exhibit anti-inflammatory activity. In this study, destabilized medial meniscus (DMM)-induced OA model was used to investigate the protective role of eriodictyol on OA. Meanwhile, we used an IL-1β-stimulated human osteoarthritis chondrocytes model to investigate the anti-inflammatory mechanism of eriodictyol on OA. The production of nitric oxide was detected by Griess reaction. The productions of MMP1, MMP3, and PGE2 were detected by ELISA. The expression of LXRα, ABCA1, PI3K, AKT, and NF-κB were measured by western blot analysis. The results demonstrated that eriodictyol could alleviate DMM-induced OA in mice. In vitro, eriodictyol inhibited IL-1β-induced NO, PGE2, MMP1, and MMP3 production in human osteoarthritis chondrocytes. Eriodictyol also suppressed the phosphorylation of PI3K, AKT, NF-κB p65, and IκBα induced by IL-1β. Meanwhile, eriodictyol significantly increased the expression of LXRα and ABCA1. Furthermore, eriodictyol disrupted lipid rafts formation through reducing the cholesterol content. And cholesterol replenishment experiment showed that adding water-soluble cholesterol could reverse the anti-inflammatory effect of eriodictyol. In conclusion, the results indicated eriodictyol inhibited IL-1β-induced inflammation in human osteoarthritis chondrocytes through suppressing lipid rafts formation, which subsequently inhibiting PI3K/AKT/NF-κB signaling pathway.

The pericellular matrix (PCM) surrounding chondrocytes is essential for articular cartilage tissue engineering. As the current isolation methods to obtain chondrocytes with their PCM (chondrons) result in a heterogeneous mixture of chondrocytes and chondrons, regenerating the PCM using a tissue engineering approach could prove beneficial. In this study, we aimed to discern the behavior of articular chondrocytes (ACs) in regenerating the PCM in such an approach and whether this would also be true for articular cartilage-derived progenitor cells (ACPCs), as an alternative cell source. Bovine ACs and ACPCs were encapsulated in agarose microgels using droplet-based microfluidics. ACs were stimulated with TGF-β1 and dexamethasone and ACPCs were sequentially stimulated with BMP-9 followed by TGF-β1 and dexamethasone. After 0, 3, 5, and 10 days of culture, PCM components, type-VI collagen and perlecan, and ECM component, type-II collagen, were assessed using flow cytometry and fluorescence microscopy. Both ACs and ACPCs synthesized the PCM before the ECM. It was seen for the first time that synthesis of type-VI collagen always preceded perlecan. While the PCM synthesized by ACs resembled native chondrons after only 5 days of culture, ACPCs often made less well-structured PCMs. Both cell types showed variations between individual cells and donors. On one hand, this was more prominent in ACPCs, but also a subset of ACPCs showed superior PCM and ECM regeneration, suggesting that isolating these cells may potentially improve cartilage repair strategies.

Chondrocytes respond to mechanical stimuli by increasing their intracellular calcium concentration. The response depends on the cellular environment. Previous studies have investigated chondrocytes under slow strain rates or cells embedded in hydrogels, but the response of chondrocytes in their native environment under physiologically relevant cyclic loads and dynamic hydrostatic pressure has not been studied. This study investigated the calcium signaling response of in-situ chondrocytes under physiological cyclic compressive loads and hydrostatic pressure with varying frequency and load rates. Bovine cartilage explants were stained with a fluorescent calcium indicator dye and subjected to physiologically relevant cyclic loads using a custom-built loading device secured on a confocal/multiphoton microscope. Calcium fluorescence intensities of the cells were tracked and analyzed. Loading groups were compared using one-way ANOVA followed by a post-hoc test with Tukey correction (α = 0.05). The percentage of cells signaling increased in all compressive loading conditions compared to the no-load baseline. The percentage of cells responding under 1 Hz load was significantly greater than the slow ramp and 0.1 Hz group (p < 0.05). The number of compression cycles had no effect on the calcium signaling response (p > 0.05). The width and time between consecutive peaks were not different between different loading conditions (p > 0.05). Calcium signaling of in-situ chondrocytes did not increase under dynamic hydrostatic pressure of magnitudes up to 0.2 MPa at frequencies of 0.5 Hz and 0.05 Hz (p > 0.05). In conclusion, in-situ chondrocytes respond to physiological compressive loads in a strain rate-dependent manner with an increased number of responsive cells and unaltered temporal characteristics.

OBJECTIVE: Inflammatory mediators such as interleukin 6 (IL-6) are known to activate catabolic responses in chondrocytes during osteoarthritis (OA). This study aimed to investigate the role of a downstream target gene of IL-6, the serine protease inhibitor SerpinA3N, in the development of cartilage damage in OA.
METHODS: RNA sequencing was performed in murine primary chondrocytes treated with IL-6, and identified target genes were confirmed in human and murine OA cartilage samples. Male cartilage-specific Serpina3n-deficient mice and control mice underwent meniscectomy (MNX) or sham surgery at 10 weeks of age. Intra-articular injections of SerpinA3N or sivelestat (an inhibitor of leucocyte elastase (LE), a substrate for SerpinA3N) were performed in wild-type mice after MNX. Joint damage was assessed 3-9 weeks after surgery by histology and micro-CT. The effect of sivelestat was assessed in cartilage explants exposed to macrophage-derived conditioned media.
RESULTS: RNA sequencing revealed that SerpinA3N is a major target gene of IL-6 in chondrocytes. The expression of SerpinA3N is increased in OA cartilage. Conditional loss of SerpinA3N in chondrocytes aggravated OA in mice, while intra-articular injection of SerpinA3N limited joint damage. Chondrocytes did not produce serine proteases targeted by SerpinA3N. By contrast, macrophages produced LE on IL-6 stimulation. Sivelestat limited the cartilage catabolism induced by conditioned media derived from IL-6-stimulated macrophages. Additionally, an intra-articular injection of sivelestat is protected against OA in the MNX model.
CONCLUSIONS: SerpinA3N protects cartilage against catabolic factors produced by macrophages, including LE. SerpinA3N and LE represent new therapeutic targets to dampen cartilage damage in OA.

UNASSIGNED: Fibroblast growth factor 20 (Fgf20), a member of the Fgf9 subfamily, was identified as an important regulator of bone differentiation and homeostasis processes. However, the role of Fgf20 in bone physiology has not been approached yet. Here we present a comprehensive bone phenotype analysis of mice with functional ablation of Fgf20.
UNASSIGNED: The study conducts an extensive analysis of Fgf20 knockout mice compared to controls, incorporating microCT scanning, volumetric analysis, Fgf9 subfamily expression and stimulation experiment and histological evaluation.
UNASSIGNED: The bone phenotype could be detected especially in the area of​ the lumbar and caudal part of the spine and in fingers. Regarding the spine, Fgf20-/- mice exhibited adhesions of the transverse process of the sixth lumbar vertebra to the pelvis as well as malformations in the distal part of their tails. Preaxial polydactyly and polysyndactyly in varying degrees of severity were also detected. High resolution microCT analysis of distal femurs and the fourth lumbar vertebra showed significant differences in structure and mineralization in both cortical and trabecular bone. These findings were histologically validated and may be associated with the expression of Fgf20 in chondrocytes and their progenitors. Moreover, histological sections demonstrated increased bone tissue formation, disruption of Fgf20-/- femur cartilage, and cellular-level alterations, particularly in osteoclasts. We also observed molar dysmorphology, including root taurodontism, and described variations in mineralization and dentin thickness.
UNASSIGNED: Our analysis provides evidence that Fgf20, together with other members of the Fgf9 subfamily, plays a crucial regulatory role in skeletal development and bone homeostasis.

BACKGROUND: Facet joint osteoarthritis (FJOA) is a prevalent condition contributing to low back pain, particularly in the elderly population. This study aimed to investigate the potential role of Cytokine Receptor-like Factor 1 (CRLF1) in FJOA pathogenesis and its therapeutic implications.
METHODS: Bioinformatics analysis was utilized to identify CRLF1 as the target gene, followed by quantification of CRLF1 expression levels and joint degeneration degree using immunohistochemistry (IHC). In primary chondrocytes, the inhibition of CRLF1 expression by siRNA was performed, and Western blot analysis was conducted to evaluate the involvement of the extracellular matrix and MAPK/ERK signaling pathway. Flow cytometry was employed to assess the apoptosis rate of chondrocytes, while immunofluorescence (IF) was utilized to evaluate the localization of CRLF1, cleaved-caspase3, MMP13, COL2A1, and ERK.
RESULTS: The expression of CRLF1 was found to be significantly elevated in FJOA tissues compared to normal tissues. Through the use of loss-of-function assays, it was determined that CRLF1 not only enhanced the rate of apoptosis in chondrocytes, but also facilitated the degradation of the extracellular matrix in vitro. Furthermore, CRLF1 was found to activate the ERK1/2 pathways. The pro-arthritic effects elicited by CRLF1 were mitigated by treatment with the MEK inhibitor U0126 in chondrocytes.
CONCLUSIONS: These results suggest that CRLF1 enhances chondrocytes apoptosis and extracellular matrix degration in FJOA and thus may therefore be a potential therapeutic target for FJOA.

Anterior talofibular ligament injuries and osteochondral lesions of the talus present unique challenges to orthopedic surgeons. This study aimed to investigate the relevant relationship between them by analyzing the Magnetic resonance imaging (MRI) results of clinical patients and single-cell RNA sequence (scRNA seq) results of healthy talus cartilage to discuss the risk factors. Data from 164 patients from 2018 to 2023 was retrospectively analyzed. The correlation analysis between ATFL injury grade and the Hepple stage of OLT determined by MRI was performed. Publicly available single-cell RNA datasets were collected. Single-cell RNA datasets from five volunteers of healthy talus cartilage were analyzed. ATFL injury grade was relevant with the Hepple stage of OLT (P < 0.05). The results of multivariate logistic regression analysis showed that injured area was the independent influencing factor of the incidence rate and the severity of OLT (P < 0.05). The Hepple stage of OLT was relevant with AOFAS and VAS (P < 0.05). Single-cell RNA sequence results showed that among the 9 subtypes of chondrocytes, the interaction strength between HTC-A and HTC-B is the highest. Their physical interactions are mainly achieved through the CD99 signaling pathway, and factor interactions are mainly achieved through the ANGPTL signaling pathway. Anterior talofibular ligament injury may lead to osteochondral lesions of the talus. Early medical intervention should be carried out for ligament injuries to restore joint stability and avoid cartilage damage.