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

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

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.

Hyaline cartilage differentiation of fibroblasts in regeneration and regenerative medicine.

Amputation injuries in mammals are typically non-regenerative; however, joint regeneration is stimulated by BMP9 treatment, indicating the presence of latent articular chondrocyte progenitor cells. BMP9 induces a battery of chondrogenic genes in vivo, and a similar response is observed in cultures of amputation wound cells. Extended cultures of BMP9-treated cells results in differentiation of hyaline cartilage, and single cell RNAseq analysis identified wound fibroblasts as BMP9 responsive. This culture model was used to identify a BMP9-responsive adult fibroblast cell line and a culture strategy was developed to engineer hyaline cartilage for engraftment into an acutely damaged joint. Transplanted hyaline cartilage survived engraftment and maintained a hyaline cartilage phenotype, but did not form mature articular cartilage. In addition, individual hypertrophic chondrocytes were identified in some samples, indicating that the acute joint injury site can promote osteogenic progression of engrafted hyaline cartilage. The findings identify fibroblasts as a cell source for engineering articular cartilage and establish a novel experimental strategy that bridges the gap between regeneration biology and regenerative medicine.

SHP2 ablation mitigates osteoarthritic cartilage degeneration by promoting chondrocyte anabolism through SOX9.

Articular cartilage phenotypic homeostasis is crucial for life-long joint function, but the underlying cellular and molecular mechanisms governing chondrocyte stability remain poorly understood. Here, we show that the protein tyrosine phosphatase SHP2 is differentially expressed in articular cartilage (AC) and growth plate cartilage (GPC) and that it negatively regulates cell proliferation and cartilage phenotypic program. Postnatal SHP2 deletion in Prg4+ AC chondrocytes increased articular cellularity and thickness, whereas SHP2 deletion in Acan+ pan-chondrocytes caused excessive GPC chondrocyte proliferation and led to joint malformation post-puberty. These observations were verified in mice and in cultured chondrocytes following treatment with the SHP2 PROTAC inhibitor SHP2D26. Further mechanistic studies indicated that SHP2 negatively regulates SOX9 stability and transcriptional activity by influencing SOX9 phosphorylation and promoting its proteasome degradation. In contrast to published work, SHP2 ablation in chondrocytes did not impact IL-1-evoked inflammation responses, and SHP2's negative regulation of SOX9 could be curtailed by genetic or chemical SHP2 inhibition, suggesting that manipulating SHP2 signaling has translational potential for diseases of cartilage dyshomeostasis.

Catalpol promotes articular cartilage repair by enhancing the recruitment of endogenous mesenchymal stem cells.

Articular cartilage defect is challenged by insufficient regenerative ability of cartilage. Catalpol (CA), the primary active component of Rehmanniae Radix, could exert protective effects against various diseases. However, the impact of CA on the treatment of articular cartilage injuries is still unclear. In this study, full-thickness articular cartilage defect was induced in a mouse model via surgery. The animals were intraperitoneally injected with CA for 4 or 8 weeks. According to the results of macroscopic observation, micro-computed tomography CT (µCT), histological and immunohistochemistry staining, CA treatment could promote mouse cartilage repair, resulting in cartilage regeneration, bone structure improvement and matrix anabolism. Specifically, an increase in the expression of CD90, the marker of mesenchymal stem cells (MSCs), in the cartilage was observed. In addition, we evaluated the migratory and chondrogenic effects of CA on MSCs. Different concentration of CA was added to C3H10 T1/2 cells. The results showed that CA enhanced cell migration and chondrogenesis without affecting proliferation. Collectively, our findings indicate that CA may be effective for the treatment of cartilage defects via stimulation of endogenous MSCs.

PLCγ1 deficiency in chondrocytes accelerates the age-related changes in articular cartilage and subchondral bone.

Ageing is the most prominent risk for osteoarthritis (OA) development. This study aimed to investigate the role of phosphoinositide-specific phospholipase Cγ (PLCγ) 1, previously linked to OA progression, in regulating age-related changes in articular cartilage and subchondral bone. d-galactose (d-Gal) was employed to treat chondrocytes from rats and mice or injected intraperitoneally into C57BL/6 mice. RTCA, qPCR, Western blot and immunohistochemistry assays were used to evaluate cell proliferation, matrix synthesis, senescence genes and senescence-associated secretory phenotype, along with PLCγ1 expression. Subchondral bone morphology was assessed through micro-CT. In mice with chondrocyte-specific Plcg1 deficiency (Plcg1flox/flox; Col2a1-CreERT), articular cartilage and subchondral bone were examined over different survival periods. Our results showed that d-Gal induced chondrocyte senescence, expedited articular cartilage ageing and caused subchondral bone abnormalities. In d-Gal-induced chondrocytes, diminished PLCγ1 expression was observed, and its further inhibition by U73122 exacerbated chondrocyte senescence. Plcg1flox/flox; Col2a1-CreERT mice exhibited more pronounced age-related changes in articular cartilage and subchondral bone compared to Plcg1flox/flox mice. Therefore, not only does d-Gal induce senescence in chondrocytes and age-related changes in articular cartilage and subchondral bone, as well as diminished PLCγ1 expression, but PLCγ1 deficiency in chondrocytes may also accelerate age-related changes in articular cartilage and subchondral bone. PLCγ1 may be a promising therapeutic target for mitigating age-related changes in joint tissue.

Metabolomic profiles of cartilage and bone reflect tissue type, radiography-confirmed osteoarthritis, and spatial location within the joint.

Osteoarthritis is the most common chronic joint disease, characterized by the abnormal remodeling of joint tissues including articular cartilage and subchondral bone. However, there are currently no therapeutic drug targets to slow the progression of disease because disease pathogenesis is largely unknown. Thus, the goals of this study were to identify metabolic differences between articular cartilage and subchondral bone, compare the metabolic shifts in osteoarthritic grade III and IV tissues, and spatially map metabolic shifts across regions of osteoarthritic hip joints. Articular cartilage and subchondral bone from 9 human femoral heads were obtained after total joint arthroplasty, homogenized and metabolites were extracted for liquid chromatography-mass spectrometry analysis. Metabolomic profiling revealed that distinct metabolic endotypes exist between osteoarthritic tissues, late-stage grades, and regions of the diseased joint. The pathways that contributed the most to these differences between tissues were associated with lipid and amino acid metabolism. Differences between grades were associated with nucleotide, lipid, and sugar metabolism. Specific metabolic pathways such as glycosaminoglycan degradation and amino acid metabolism, were spatially constrained to more superior regions of the femoral head. These results suggest that radiography-confirmed grades III and IV osteoarthritis are associated with distinct global metabolic and that metabolic shifts are not uniform across the joint. The results of this study enhance our understanding of osteoarthritis pathogenesis and may lead to potential drug targets to slow, halt, or reverse tissue damage in late stages of osteoarthritis.

Inhibition of Phlpp1 preserves the mechanical integrity of articular cartilage in a murine model of post-traumatic osteoarthritis.

OBJECTIVE: Phlpp1 inhibition is a potential therapeutic strategy for cartilage regeneration and prevention of post-traumatic osteoarthritis (PTOA). To understand how Phlpp1 loss affects cartilage structure, cartilage elastic modulus was measured with atomic force microscopy (AFM) in male and female mice after injury. METHODS: Osteoarthritis was induced in male and female Wildtype (WT) and Phlpp1-/- mice by destabilization of the medial meniscus (DMM). At various timepoints post-injury, activity was measured, and knee joints examined with AFM and histology. In another cohort of WT mice, the PHLPP inhibitor NSC117079 was intra-articularly injected 4 weeks after injury. RESULTS: Male WT mice showed decreased activity and histological signs of cartilage damage at 12 but not 6-weeks post-DMM. Female mice showed a less severe response to DMM by comparison, with no histological changes seen at any time point. In both sexes the elastic modulus of medial condylar cartilage was decreased in WT mice but not Phlpp1-/- mice after DMM as measured by AFM. By 6-weeks, cartilage modulus had decreased from 2 MPa to 1 MPa in WT mice. Phlpp1-/- mice showed no change in modulus at 6-weeks and only a 25% decrease at 12-weeks. The PHLPP inhibitor NSC117079 protected cartilage structure and prevented signs of OA 6-weeks post-injury. CONCLUSIONS: AFM is a sensitive method for detecting early changes in articular cartilage post-injury. Phlpp1 suppression, either through genetic deletion or pharmacological inhibition, protects cartilage degradation in a model of PTOA, validating Phlpp1 as a therapeutic target for PTOA.

Disentangling the detrimental effects of local from systemic adipose tissue dysfunction on articular cartilage in the knee.

OBJECTIVE: Obesity increases osteoarthritis (OA) risk due to adipose tissue dysfunction with associated metabolic syndrome and excess weight. Lipodystrophy syndromes exhibit systemic metabolic and inflammatory abnormalities similar to obesity without biomechanical overloading. Here, we used lipodystrophy mouse models to investigate the effects of systemic versus intra-articular adipose tissue dysfunction on the knee. METHODS: Intra-articular adipose tissue development was studied using reporter mice. Mice with selective lipodystrophy of intra-articular adipose tissue were generated by conditional knockout (cKO) of Bscl2 in Gdf5-lineage cells, and compared with whole-body Bscl2 knockout (KO) mice with generalised lipodystrophy and associated systemic metabolic dysfunction. OA was induced by surgically destabilising the medial meniscus (DMM) and obesity by high-fat diet (HFD). Gene expression was analysed by quantitative RT-PCR and tissues were analysed histologically. RESULTS: The infrapatellar fat pad (IFP), in contrast to overlying subcutaneous adipose tissue, developed from a template established from the Gdf5-expressing joint interzone during late embryogenesis, and was populated shortly after birth by adipocytes stochastically arising from Pdgfrα-expressing Gdf5-lineage progenitors. While female Bscl2 KO mice with generalised lipodystrophy developed spontaneous knee cartilage damage, Bscl2 cKO mice with intra-articular lipodystrophy did not, despite the presence of synovial hyperplasia and inflammation of the residual IFP. Furthermore, male Bscl2 cKO mice showed no worse cartilage damage after DMM. However, female Bscl2 cKO mice showed increased susceptibility to the cartilage-damaging effects of HFD-induced obesity. CONCLUSION: Our findings emphasise the prevalent role of systemic metabolic and inflammatory effects in impairing cartilage homeostasis, with a modulatory role for intra-articular adipose tissue.

Mechanical loading rescues mechanoresponsiveness in a human osteoarthritis explant model despite Wnt activation.

OBJECTIVES: Optimizing rehabilitation strategies for osteoarthritis necessitates a comprehensive understanding of chondrocytes' mechanoresponse in both health and disease, especially in the context of the interplay between loading and key pathways involved in osteoarthritis (OA) development, like canonical Wnt signaling. This study aims to elucidate the role of Wnt signaling in the mechanoresponsiveness of healthy and osteoarthritic human cartilage. METHODS: We used an ex-vivo model involving short-term physiological mechanical loading of human cartilage explants. First, the loading protocol for subsequent experiments was determined. Next, loading was applied to non-OA-explants with or without Wnt activation with CHIR99021. Molecular read-outs of anabolic, pericellular matrix and matrix remodeling markers were used to assess the effect of Wnt on cartilage mechanoresponse. Finally, the same set-up was used to study the effect of loading in cartilage from patients with established OA. RESULTS: Our results confirm that physiological loading maintains expression of anabolic genes in non-OA cartilage, and indicate a deleterious effect of Wnt activation in the chondrocyte mechanoresponsiveness. This suggests that loading-induced regulation of chondrocyte markers occurs downstream of canonical Wnt signaling. Interestingly, our study highlighted contrasting mechanoresponsiveness in the model of Wnt activation and the established OA samples, with established OA cartilage maintaining its mechanoresponsiveness, and mechanical loading rescuing the chondrogenic phenotype. CONCLUSION: This study provides insights into the mechanoresponsiveness of human cartilage in both non-OA and OA conditions. These findings hold the potential to contribute to the development of strategies that optimize the effect of dynamic compression by correcting OA pathological cell signaling.

Assessment of post-trauma microstructural alterations in the rabbit knee cartilage and subchondral bone.

Early diagnosis of post-traumatic osteoarthritis (PTOA) is critical for designing better treatments before the degradation becomes irreversible. We utilized multimodal high-resolution imaging to investigate early-stage deterioration in articular cartilage and the subchondral bone plate from a sub-critical impact to the knee joint, which initiates PTOA. The knee joints of 12 adult rabbits were mechanically impacted once on the femoral articular surface to initiate deterioration. At 2- and 14-week post-impact surgery, cartilage-bone blocks were harvested from the impact region in the animals (N = 6 each). These blocks were assessed for deterioration using polarized light microscopy (PLM), microcomputed tomography (µCT), and biochemical analysis. Statistically significant changes were noted in the impact tissues across the calcified zone (CZ) at 14 weeks post-impact: the optical retardation values in the CZ of impact cartilage had a drop of 29.0% at 14 weeks, while the calcium concentration in the CZ of impact cartilage also had a significant drop at 14 weeks. A significant reduction of 6.3% in bone mineral density (BMD) was noted in the subchondral bone plate of the impact samples at 14 weeks. At 2 weeks post-impact, only minor, non-significant changes were measured. Furthermore, the impact knees after 14 weeks had greater structural changes compared with the 2-week impact knees, indicating progressive degradation over time. The findings of this study facilitated a connection between mineralization alterations and the early deterioration of knee cartilage after a mechanical injury. In a broader context, these findings can be beneficial in improving clinical strategies to manage joint injuries.

Capsular attachment on the anterosuperior femoral head-neck junction: A hypothesis about femoroacetabular impingement.

Femoroacetabular impingement (FAI), characterized by a pathological contact between the proximal femur and acetabulum, is a common precursor of hip osteoarthritis. Cam morphology is a bony prominence that causes FAI and frequently forms on the anterosuperior femoral head-neck junction. Despite anatomical consensus regarding the femoral head-neck junction as a boundary area covered by the articular cartilage and joint capsule, it remains unclear whether the joint capsule is continuous with the anterosuperior articular cartilage. For the anatomical consideration of cam morphology formation, this study aimed to investigate the histological characteristics of the capsular attachment on the anterosuperior femoral head-neck junction, particularly focusing on the presence or absence of continuity of the joint capsule to the articular cartilage. A total of 21 anterosuperior regions (seven hips each for the 12:00, 1:30, and 3:00 positions) from seven hips (three males and four females; mean age at death, 68.7 years) were histologically analyzed in this study for quantitative evaluation of the capsular thickness using histological sections stained with Masson's trichrome, as well as qualitative evaluation of the capsular attachment. The present study showed that the joint capsule, which folded proximally to the femoral head-neck junction from the recess, exhibited a blend of the fibrous and synovial regions. Notably, it not only continued with the superficial layer of the articular cartilage, but also attached to the articular cartilage via the fibrocartilage. This continuous region was relatively fibrous with dense connective tissue running in the longitudinal direction. The capsular thickness at the recess point (mean, 1.7 ± 0.9 mm) and those at the distal end of the articular cartilage (0.35 ± 0.16 mm) were significantly greater than the control value for the most superficial layer thickness of the articular cartilage (0.019 ± 0.003 mm) (Dunnett's T3, both p-value <0.001). Based on the fibrous continuity between the joint capsule and articular cartilage and its thickness, this study suggests the anatomical possibility that some mechanical stress can be transmitted from the joint capsule to the articular cartilage at the frequent sites of cam morphology.

Unraveling the Mechanisms of Hypertrophy-Induced Matrix Mineralization and Modifications in Articular Chondrocytes.

Chondrocyte hypertrophic differentiation is a main event leading to articular cartilage degradation in osteoarthritis. It is associated with matrix remodeling and mineralization, the dynamics of which is not well characterized during chondrocyte hypertrophic differentiation in articular cartilage. Based on an in vitro model of progressive differentiation of immature murine articular chondrocytes (iMACs) into prehypertrophic (Prehyp) and hypertrophic (Hyp) chondrocytes, we performed kinetics of chondrocyte differentiation from Prehyp to Hyp to follow matrix mineralization and remodeling by immunofluorescence, biochemical, molecular, and physicochemical approaches, including atomic force microscopy, scanning electron microscopy associated with energy-dispersive X-ray spectroscopy (SEM-EDS), attenuated total reflection infrared analyses, and X-ray diffraction. Chondrocyte apoptosis was determined by TUNEL assay. The results show the formation of a mineral phase 7 days after Hyp induction, which spreads within the matrices to form poorly crystalline carbonate-substituted hydroxyapatite after 14 days, then the proportions of crystalline relative to amorphous content increases over time. Hyp differentiation also induced a matrix turnover that occurs over the first 7 days, characterized by a decrease in type II collagen and aggrecan and the concomitant appearance of type X collagen. This is accompanied by an increase in the enzymatic activity of MMP-13, the main collagenase in cartilage. The number of apoptotic chondrocytes slightly increased with Hyp differentiation and SEM-EDS analyses detected phosphorus-rich structures that could correspond to apoptotic bodies. Our findings highlight the mechanisms of matrix remodeling events leading to the mineralization of articular cartilage that may occur in osteoarthritis.

Assessment of articular cartilage degradation in response to an impact injury using µMRI.

PURPOSE: Degradation of articular cartilage (AC) due to injury to the knee joint may initiate post-traumatic osteoarthritis (PTOA). Failure to diagnose the onset of the disease at an early stage makes the cure ineffective for PTOA. This study investigated the consequences of a mechanical injury to the knee in a rabbit model using microscopic magnetic resonance imaging (µMRI) at high resolution. MATERIALS AND METHODS: A mechanical injury was induced to the knee joints of 12 rabbits. Cartilage blocks were extracted from the non-impacted and impacted knee joints after 2 and 14 weeks post-impact. The specimens were studied using µMRI T2 relaxation and inductively coupled plasma analysis to determine the early degradation of the articular cartilage. RESULTS: The data established a connection between T2 relaxation time and the early progression of knee PTOA after an impact injury. T2 values were found to be higher in the impacted cartilage at both 2 and 14 weeks, in particular, T2-55° values in the impacted samples displayed a significant rise of 6.93% after 2 weeks and 20.02% after 14 weeks. Lower glycosaminoglycan measurement and higher water content in the impacted cartilage confirmed the µMRI results. CONCLUSIONS: This µMRI T2 study was able to detect cartilage damage in the impacted knees. In addition, greater degradation in the affected knees at 14 weeks than at 2 weeks indicated the progressive nature of cartilage deterioration over time. The µMRI results were in accord with the biochemical analysis, indicating the detection of early structural damage in the cartilage.

Bone marrow edema-like signal after cartilage repair does not affect outcomes in a five-year follow-up.

OBJECTIVES: Bone marrow edema-like signal (BMELS) after cartilage repair is common, but its clinical significance remains uncertain. This study aimed to investigate the clinical and structural significance of BMELS following microfracturing (MFX) and matrix-induced autologous chondrocyte implantation (MACI). METHODS: In this multicenter study, MRI examinations were performed over a period of 5 years after cartilage repair surgery (MFX n = 17; MACI n = 28) in 45 patients. Morphological assessments, including the MOCART 2.0 (magnetic resonance observation of cartilage repair tissue), quantitative imaging biomarkers (QIB) with T2 mapping of the repair tissue, and, specifically, assessment of the presence and size of BMELS, were conducted along with patient-reported outcome measures, such as the Knee injury and Osteoarthritis Outcome Score (KOOS) and the International Knee Documentation Committee (IKDC). BMELS structural and clinical assessments were obtained after 3 months, 12 months, and 60 months. Statistical analysis included the Mann-Whitney U-test, Wilcoxon rank test, Shapiro-Wilk test, and simulation-based power analysis. RESULTS: BMELS were a common finding 60 months after cartilage repair. The size of BMELS differed significantly only between MACI and MFX patients after 3 months, with larger BMELS occurring in the MFX group. There were no significant differences in patients with or without BMELS regarding the T2 ratio of the treated area, the MOCART 2.0, or clinical scores. CONCLUSION: BMELS frequently appeared after cartilage repair procedures. We could show that the postoperative size and change in the size of BMELS after MACI and MFX did not affect clinical scores, morphological MRI results, or biochemical properties of the treated area after 60 months. KEY POINTS: Question What is the clinical significance of bone marrow edema-like signal (BMELS) appearance after matrix-induced autologous chondrocyte implantation (MACI) or microfracture (MFX)? Finding There were no significant differences in patients with or without BMELS regarding the T2 ratio of the treated area, the MOCART 2.0, or clinical scores. Clinical relevance BMELS frequently appeared after cartilage repair, the appearance or the size dynamic after MACI and MFX did not affect clinical scores, morphological MRI results, or biochemical properties after 60 months.

Gaining Insight into Updated MR Imaging for Quantitative Assessment of Cartilage Injury in Knee Osteoarthritis.

PURPOSE OF THE REVIEW: Knee Osteoarthritis (KOA) entails progressive cartilage degradation, reviewed via MRI for morphology, biochemical composition, and microtissue alterations, discussing clinical advantages, limitations, and research applicability. RECENT FINDINGS: Compositional MRI, like T2/T2* mapping, T1rho mapping, gagCEST, dGEMRIC, sodium imaging, diffusion-weighted imaging, and diffusion-tensor imaging, provide insights into cartilage injury in KOA. These methods quantitatively measure collagen, glycosaminoglycans, and water content, revealing important information about biochemical compositional and microstructural alterations. Innovative techniques like hybrid multi-dimensional MRI and diffusion-relaxation correlation spectrum imaging show potential in depicting initial cartilage changes at a sub-voxel level. Integration of automated image analysis tools addressed limitations in manual cartilage segmentation, ensuring robust and reproducible assessments of KOA cartilage. Compositional MRI techniques reveal microstructural changes in cartilage. Multi-dimensional MR imaging assesses biochemical alterations in KOA-afflicted cartilage, aiding early degeneration identification. Integrating artificial intelligence enhances cartilage analysis, optimal diagnostic accuracy for early KOA detection and monitoring.

Disuse atrophy of articular cartilage can be restored by mechanical reloading in mice.

BACKGROUND: Moderate mechanical stress generated by normal joint loading and movements helps maintain the health of articular cartilage. Despite growing interest in the pathogenesis of cartilage degeneration caused by reduced mechanical stress, its reversibility by mechanical reloading is less understood. This study aimed to investigate the response of articular cartilage exposed to mechanical reloading after unloading in vivo and in vitro. METHODS AND RESULTS: Disuse atrophy was induced in the knee joint cartilage of adult mice through hindlimb unloading by tail suspension. For in vivo experiments, mice were subjected to reloading with or without daily exercise intervention or surgical destabilization of the knee joint. Microcomputed tomography and histomorphometric analyses were performed on the harvested knee joints. Matrix loss and thinning of articular cartilage due to unloading were fully or partially restored by reloading, and exercise intervention enhanced the restoration. Subchondral bone density decreased by unloading and increased to above-normal levels by reloading. The severity of cartilage damage caused by joint instability was not different even with prior non-weight bearing. For in vitro experiments, articular chondrocytes isolated from the healthy or unloaded joints of the mice were embedded in agarose gel. After dynamic compression loading, the expression levels of anabolic (Sox9, Col2a1, and Acan) and catabolic (Mmp13 and Adamts5) factors of cartilage were analyzed. In chondrocytes isolated from the unloaded joints, similar to those from healthy joints, dynamic compression increased the expression of anabolic factors but suppressed the expression of catabolic factors. CONCLUSION: The results of this study indicate that the morphological changes in articular cartilage exposed to mechanical unloading may be restored in response to mechanical reloading by shifting extracellular matrix metabolism in chondrocytes to anabolism.

BASP1 knockdown suppresses chondrocyte apoptosis and extracellular matrix degradation in vivo and in vitro: A possible therapeutic approach for osteoarthritis.

Osteoarthritis(OA) is an age-related degenerative disease involving chondrocyte apoptosis and extracellular matrix(ECM) degradation.Brain acid soluble protein 1(BASP1) has been reported to induce apoptosis.Thus, we speculated that BASP1 might regulate OA progression by inducing apoptosis, which is also the purpose of this study.The cartilage of the knee joint was collected from OA patients who received the joint replacement.In OA cartilage tissue,we found BASP1 expression was highly expressed, which inferred that BASP1 might be involved in OA.To validate our hypothesis, destabilization of the medial meniscus (DMM) surgery-induced male C57BL/6mice and interleukin-1ß (IL-1ß)-treated human chondrocytes were used to mimic the OA environment.BASP1 knockdown in mice and chondrocytes was achieved by adenovirus carried with BASP1-specific shRNA.High expression of BASP1 was observed in OA mice, which was also verified in IL-1ß-treated chondrocytes.The potential mechanism of BASP1 in OA was further explored in vitro.BASP1 knockdown alleviated IL-1ß-induced apoptosis and ECM degradation, as reflected by the decreased number of apoptotic cells and matrix metalloproteases 13 expression,and the increased collagen II expression.Our findings indicated that BASP1 knockdown alleviated OA progression by inhibiting apoptosis and ECM degradation, suggesting that inhibiting BASP1 may be a potentially applicable method for preventing OA.

Conditioned Medium of Infrapatellar Fat Stem Cells Alleviates Degradation of Chondrocyte Extracellular Matrix and Delays Development of Osteoarthritis.

INTRODUCTION: Osteoarthritis (OA) is a prevalent clinical chronic degenerative condition characterized by the degeneration of articular cartilage. Currently, drug treatments for OA come with varying degrees of side effects, making the development of new therapeutic approaches for OA imperative. Mesenchymal stem cells (MSCs) are known to mitigate the progression of OA primarily through paracrine effects. The conditioned medium (CM) derived from MSCs encapsulates a variety of paracrine factors secreted by these cells. METHODS: In this study, we investigated the effect of the CM of infrapatellar fat pad-derived MSCs (IPFSCs) on OA in vitro and in vivo, as well as and the potential underlying mechanisms. We established three experimental groups: the normal group, the OA group, and the CM intervention group. In vitro experiments, we used methods such as qPCR, Western blot, immunofluorescence, and flow cytometry to detect the impact of CM on OA chondrocytes. In vivo experiments, we evaluated the changes in the knee joints of OA rats after intra-articular injection of CM treatment. RESULTS: The results showed that injection of CM into the knee joint inhibited OA development in a rat model induced by destabilization of the medial meniscus and anterior cruciate ligament transection. The CM increased the deposition of extracellular matrix-related components (type II collagen and Proteoglycan). The activation of PI3K/AKT/NF-κB signaling pathway was induced by IL-1ß in chondrocytes, which was finally inhibited by CM-IPFSCs treatment. CONCLUSION: In summary, IPFSCs-CM may have therapeutic potential for OA.

The Mechano-Ubiquitinome of Articular Cartilage: Differential Ubiquitination and Activation of a Group of ER-Associated DUBs and ER Stress Regulators.

Understanding how connective tissue cells respond to mechanical stimulation is important to human health and disease processes in musculoskeletal diseases. Injury to articular cartilage is a key risk factor in predisposition to tissue damage and degenerative osteoarthritis. Recently, we have discovered that mechanical injury to connective tissues including murine and porcine articular cartilage causes a significant increase in lysine-63 polyubiquitination. Here, we identified the ubiquitin signature that is unique to injured articular cartilage tissue upon mechanical injury (the "mechano-ubiquitinome"). A total of 463 ubiquitinated peptides were identified, with an enrichment of ubiquitinated peptides of proteins involved in protein processing in the endoplasmic reticulum (ER), also known as the ER-associated degradation response, including YOD1, BRCC3, ATXN3, and USP5 as well as the ER stress regulators, RAD23B, VCP/p97, and Ubiquilin 1. Enrichment of these proteins suggested an injury-induced ER stress response and, for instance, ER stress markers DDIT3/CHOP and BIP/GRP78 were upregulated following cartilage injury on the protein and gene expression levels. Similar ER stress induction was also observed in response to tail fin injury in zebrafish larvae, suggesting a generic response to tissue injury. Furthermore, a rapid increase in global DUB activity following injury and significant activity in human osteoarthritic cartilage was observed using DUB-specific activity probes. Combined, these results implicate the involvement of ubiquitination events and activation of a set of DUBs and ER stress regulators in cellular responses to cartilage tissue injury and in osteoarthritic cartilage tissues. This link through the ER-associated degradation pathway makes this protein set attractive for further investigation in in vivo models of tissue injury and for targeting in osteoarthritis and related musculoskeletal diseases.