Gut-joint axis in knee synovitis: gut fungal dysbiosis and altered fungi-bacteria correlation network identified in a community-based study.

OBJECTIVES: Knee synovitis is a highly prevalent and potentially curable condition for knee pain; however, its pathogenesis remains unclear. We sought to assess the associations of the gut fungal microbiota and the fungi-bacteria correlation network with knee synovitis. METHODS: Participants were derived from a community-based cross-sectional study. We performed an ultrasound examination of both knees. A knee was defined as having synovitis if its synovium was ≥4 mm and/or Power Doppler (PD) signal was within the knee synovium area (PD synovitis). We collected faecal specimens from each participant and assessed gut fungal and bacterial microbiota using internal transcribed spacer 2 and shotgun metagenomic sequencing. We examined the relation of α-diversity, ß-diversity, the relative abundance of taxa and the interkingdom correlations to knee synovitis. RESULTS: Among 977 participants (mean age: 63.2 years; women: 58.8%), 191 (19.5%) had knee synovitis. ß-diversity of the gut fungal microbiota, but not α-diversity, was significantly associated with prevalent knee synovitis. The fungal genus Schizophyllum was inversely correlated with the prevalence and activity (ie, control, synovitis without PD signal and PD synovitis) of knee synovitis. Compared with those without synovitis, the fungi-bacteria correlation network in patients with knee synovitis was smaller (nodes: 93 vs 153; edges: 107 vs 244), and the average number of neighbours was fewer (2.3 vs 3.2). CONCLUSION: Alterations of gut fungal microbiota and the fungi-bacteria correlation network are associated with knee synovitis. These novel findings may help understand the mechanisms of the gut-joint axis in knee synovitis and suggest potential targets for future treatment.

Association between gut microbiome-related metabolites and symptomatic hand osteoarthritis in two independent cohorts.

BACKGROUND: Since gut microbiome dysbiosis can cause inflammatory disorders by affecting host metabolism, we postulate that the gut microbiome and related metabolites could play a role in hand osteoarthritis. We characterised gut microbiome-related metabolites in people with symptomatic hand osteoarthritis (SHOA) in two independent cohorts. METHODS: Using data collected from a large-sample community-based observational study (discovery cohort), we assessed the relations of the microbial function and plasma key metabolites related to altered microbial function with SHOA. Finally, we verified the relations of plasma metabolites to SHOA in an independent observational study (validation cohort). FINDINGS: In the discovery cohort (n = 1359), compared to those without SHOA, participants with SHOA had significantly altered microbial functions related to tryptophan metabolism (Q = 0.025). Therefore we measured the plasma tryptophan metabolites and found that participants with SHOA had higher levels of 5-hydroxyindoleacetic acid (odds ratio [OR] = 1.25, 95% confidence interval [CI]: 1.09-1.42) and 5-hydroxytryptophol (OR = 1.13, 95% CI: 1.04-1.23), but lower levels of indole-3-lactic acid (ILA) (OR = 0.85, 95% CI: 0.72-1.00), skatole (OR = 0.93, 95% CI: 0.88-0.99) and 3-hydroxyanthranilic acid (OR = 0.90, 95% CI: 0.85-0.96). Findings from the validation cohort (n = 142) verified that lower levels of ILA were related to SHOA (OR = 0.70, 95% CI: 0.53-0.92). INTERPRETATION: Alterations of the microbial function of tryptophan biosynthesis and tryptophan metabolites, especially lower levels of ILA, are associated with SHOA. These findings suggest the role of the microbiome and tryptophan metabolites in developing of SHOA and may contribute to future translational opportunities. FUNDING: National Key Research and Development Plan and National Natural Science Foundation of China.

Dysbiosis of gut microbiota, a potential mediator of bile acid compositions, and prevalence of hand synovitis: a community-based study.

OBJECTIVES: Hand synovitis, a potentially modifiable pathological lesion, is common and associated with pain and hand OA; nevertheless, its pathogenesis remains uncertain. This study investigated the relationship between gut microbiota dysbiosis and hand synovitis prevalence and evaluated whether bile acids mediate the association. METHODS: Participants were derived from a community-based observational study. Synovitis in each hand joint was assessed using US. Gut microbiota was evaluated using 16S ribosomal RNA amplicon sequencing on faeces, and plasma bile acids were measured by HPLC mass spectrometry. We examined the relationship between gut microbiota dysbiosis and hand synovitis prevalence, as well as the extent to which bile acids were involved in the association. RESULTS: Among 1336 participants (mean age: 63.2 years; women: 58.8%), 18.3% had prevalent hand synovitis (unilateral in 13.6% and bilateral in 4.7%). ß-diversity, but not α-diversity, of gut microbiota was significantly associated with prevalent hand synovitis. Higher relative abundance of the genus Prevotella and lower relative abundance of the genus Blautia were significantly associated with the prevalence of hand synovitis. Similar associations were also observed for laterality and the number of joints affected by hand synovitis. The association between Prevotella and hand synovitis was partially mediated through its effect on tauroursodeoxycholic acid and glycoursodeoxycholic acid, the mediation proportions being 25.7% and 21.6%, respectively. CONCLUSION: Our findings suggest that gut microbiota dysbiosis is associated with the prevalence of hand synovitis. Such an association appears to be partially mediated by plasma bile acids.

Pain Intensity and Trajectory Following Intra-Articular Injection of Mono-Iodoacetate in Experimental Osteoarthritis: A Meta-Analysis of In Vivo Studies.

OBJECTIVE: Although most frequently used in experimental osteoarthritis (OA) pain induction, intra-articular mono-iodoacetate (MIA) injection lacks concluded references for dose selection and timing of intervention. Herein, we aimed to compare the pain intensity of rats induced by different doses of MIA and explored the trajectory of pain. DESIGN: PubMed, Embase, and Web of Science were searched up to June 2021 for literatures involving MIA experiments investigating OA pain. Pain intensity was measured based on weightbearing distribution (WBD) and paw withdrawal thresholds (PWT), and the pain trajectory was constructed by evaluating pain intensity at a series of time points after MIA injection. A conventional meta-analysis was conducted. RESULTS: A total of 140 studies were included. Compared with saline, MIA injections caused significantly higher pain intensity for WBD and PWT. Dose-response relationships between different doses of MIA and pain intensity were observed (P-for-trend<0.05). A pronounced increase in pain occurred from day 0 to day 7, but the uptrend ceased between day 7 and day 14, after which the pain intensity continued to rise and reached the maximum by day 28. CONCLUSIONS: Pain intensity after intra-articular MIA injection increased in a dose-dependent manner and the pain trajectory manifested a specific pattern consistent with the pathological mechanisms of MIA-induced pain, providing possible clues for proper dose selection and timing of specific OA pain interventions.

Exploration of metformin as novel therapy for osteoarthritis: preventing cartilage degeneration and reducing pain behavior.

BACKGROUND: Metformin could activate adenosine monophosphate-activated protein kinase (AMPK) which was postulated as a potential therapeutic target for osteoarthritis. This study aimed to examine the effects of metformin on cartilage and pain in osteoarthritis mouse model. METHODS: Eighty 10-week-old male C57BL/6 mice were randomized to 6 groups: non-operation, sham-operation, destabilization of the medial meniscus (DMM)-operation with intragastric saline/metformin, and DMM-operation with intraarticular saline/metformin. Articular cartilage degeneration was examined by scanning electron microscopy (SEM) and graded using the scoring system recommended by Osteoarthritis Research Society International (OARSI). Mechanical withdrawal threshold and hind paw weight distribution were measured to assess the pain-related behavior. Cell Counting Kit-8 assay, quantificational real-time polymerase chain reaction, and western blot analysis were conducted to examine the anabolic and anti-catabolic effect of metformin and the role of AMPK in mediating its effects on interleukin-1ß stimulated primary mice chondrocytes. RESULTS: Compared with mice receiving intragastric and intraarticular saline, mice in both intragastric and intraarticular metformin displayed attenuated articular cartilage degeneration, indicated by less cartilage damage under SEM and significantly lower OARSI scores. A higher paw withdrawal threshold and a decreased weight-bearing asymmetry were observed in the intragastric and intraarticular metformin mice compared with their corresponding saline groups in DMM model of osteoarthritis. In vitro experiments showed that metformin not only decreased the level of matrix metalloproteinase 13, but also elevated type II collagen production through activating AMPK pathway. CONCLUSIONS: Metformin attenuates osteoarthritis structural worsening and modulates pain, suggesting its potential for osteoarthritis prevention or treatment.

Acceleration of chondrogenic differentiation of human mesenchymal stem cells by sustained growth factor release in 3D graphene oxide incorporated hydrogels.

Damaged articular cartilage has limited self-healing capabilities, leading to degeneration that affects millions of people. Although cartilage tissue engineering is considered a promising approach for treatment, robust and long-term chondrogenesis within a 3-dimensional (3D) scaffold remains a major challenge for complete regeneration. Most current approaches involve incorporation of transforming growth factor-ß (TGF-ß) into the scaffold, but have limited utility owing to the short functional half-life and/or rapid clearance of TGF-ß. In this study, we have tested the incorporation of graphene oxide nanosheets (GO) within a photopolymerizable poly-D, l-lactic acid/polyethylene glycol (PDLLA) hydrogel, for its applicability in sustained release of the chondroinductive growth factor TGF-ß3. We found that with GO incorporation, the hydrogel scaffold (GO/PDLLA) exhibited enhanced initial mechanical strength, i.e., increased compressive modulus, and supported long-term, sustained release of TGF-ß3 for up to 4 weeks. In addition, human bone marrow-derived mesenchymal stem cells (hBMSCs) seeded within TGF-ß3 loaded GO/PDLLA hydrogels displayed high cell viability and improved chondrogenesis in a TGF-ß3 concentration-dependent manner. hBMSCs cultured in GO/PDLLA also demonstrated significantly higher chondrogenic gene expression, including aggrecan, collagen type II and SOX9, and cartilage matrix production when compared to cultures maintained in GO-free scaffolds containing equivalent amounts of TGF-ß3. Upon subcutaneous implantation in vivo, hBMSC-seeded TGF-ß3-GO/PDLLA hydrogel constructs displayed considerably greater cartilage matrix than their TGF-ß3/PDLLA counterparts without GO. Taken together, these findings support the potential application of GO in optimizing TGF-ß3 induced hBMSC chondrogenesis for cartilage tissue engineering. STATEMENT OF SIGNIFICANCE: In this work, we have developed a graphene oxide (GO) incorporated, photocrosslinked PDLLA hybrid hydrogel for localized delivery and sustained release of loaded TGF-ß3 to seeded cells. The incorporation of GO in PDLLA hydrogel suppressed the burst release of TGF-ß3, and significantly prolonged the retention time of the TGF-ß3 initially loaded in the hydrogel. Additionally, the GO improved the initial compressive strength of the hydrogel. Both in vitro analyses and in vivo implantation results showed that the GO/PDLLA constructs seeded with human mesenchymal stem cells (hMSCs) showed significantly higher cartilage formation, compared to GO-free scaffolds containing equivalent amount of TGF-ß3. Findings from this work suggest the potential application of the GO-TGF/PDLLA hydrogel as a functional scaffold for hMSC-based cartilage tissue engineering.

Modeling appendicular skeletal cartilage development with modified high-density micromass cultures of adult human bone marrow-derived mesenchymal progenitor cells.

BACKGROUND: Animal cell-based systems have been critical tools in understanding tissue development and physiology, but they are less successful in more practical tasks, such as predicting human toxicity to pharmacological or environmental factors, in which the congruence between in vitro and clinical outcomes lies on average between 50 and 60%. Emblematic of this problem is the high-density micromass culture of embryonic limb bud mesenchymal cells, derived from chick, mouse, or rat. While estimated predictive value of this model system in toxicological studies is relatively high, important failures prevent its use by international regulatory agencies for toxicity testing and policy development. A likely underlying reason for the poor predictive capacity of animal-based culture models is the small but significant physiological differences between species. This deficiency has inspired investigators to develop more organotypic, 3-dimensional culture system using human cells to model normal tissue development and physiology and assess pharmacological and environmental toxicity. METHODS: We have developed a modified, miniaturized micromass culture model using adult human bone marrow-derived mesenchymal progenitor cells (hBM-MPCs) that is amenable to moderate throughput and high content analysis to study chondrogenesis. The number of cells per culture was reduced, and a methacrylated gelatin (gelMA) overlay was incorporated to normalize the morphology of the cultures. RESULTS: These modified human cell-based micromass cultures demonstrated robust chondrogenesis, indicated by increased Alcian blue staining and immunodetectable production of collagen type II and aggrecan, and stage-specific chondrogenic gene expression. In addition, in cultures of hBM-MPCs transduced with a lentiviral collagen type II promoter-driven GFP reporter construct, levels of GFP reporter activity correlated well with changes in endogenous collagen type II transcript levels, indicating the feasibility of non-invasive monitoring of chondrogenesis. CONCLUSIONS: The modified hBM-MPC micromass culture system described here represents a reproducible and controlled model for analyzing mechanisms of human skeletal development that may later be applied to pharmacological and environmental toxicity studies.

Infiltration and In-Tissue Polymerization of Photocross-Linked Hydrogel for Effective Fixation of Implants into Cartilage-An In Vitro Study.

Effective and biocompatible fixation of implants into cartilage defects has yet to be successfully achieved. [Poly-d,l-lactic acid/polyethyleneglycol/poly-d,l-lactic acid] (PDLLA-PEG) is a chondrosupportive scaffold that is photocross-linked using the visible-light photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). Interestingly, LAP and its monomer DLLA-EG are able to infiltrate the cartilage and form hydrogels upon the detection of light. After the infiltration of LAP and DLLA-EG into the implant and host cartilage, an interconnected and continuous hydrogel structure is formed which fixes the implant within the host cartilage. A mechanical test shows that the infiltrated group displays a significantly higher push-out force than the group that has not been infiltrated (the traditional fibrin fixation group). Surprisingly, the in-cartilage hydrogel also reduces the release of sulfated glycosaminoglycan from cartilage explants. However, infiltration does not affect the cell viability or the expression of cartilage marker genes. This new strategy thus represents a biocompatible and efficient method to fix implants into host tissues.

Condensation-Driven Chondrogenesis of Human Mesenchymal Stem Cells within Their Own Extracellular Matrix: Formation of Cartilage with Low Hypertrophy and Physiologically Relevant Mechanical Properties.

Mesenchymal stem cells (MSCs) represent a promising cell source to regenerate injured cartilage. In this study, MSCs are cultured under confluent conditions for 10 days to optimize the deposition of the extracellular matrix (mECM), which will serve as the scaffold to support MSC chondrogenesis. Subsequently, the MSC-impregnated mECM (MSC-mECM) composite is briefly treated with trypsin, allowing the MSCs to adopt a round morphology without being detached from their own mECM. The constructs are then cultured in a chondrogenic medium. Interestingly, after trypsin removal, the treated MSCs undergo an aggregation process, mimicking mesenchymal condensation during developmental chondrogenesis, specifically indicated by peanut agglutinin staining and immunodetectable N-cadherin expression, followed by robust chondrogenic differentiation. In comparison to conventional pellet culture, chondrogenically induced MSC-mECM displays a similar level of chondrogenesis, but with significantly reduced hypertrophy. The reparative capacity of the MSC-mECM derived construct is assessed using bovine cartilage explants. Mechanical testing and histology results show that engineered cartilage from MSC-mECM forms better integration with the surrounding native cartilage tissue and displays a much lower hypertrophic differentiation than that from pellet culture. Taken together, these findings demonstrate that MSC-mECM may be an efficacious stem cell-based product for the repair of hyaline cartilage injury without the use of exogenous scaffolds.

Engineering hyaline cartilage from mesenchymal stem cells with low hypertrophy potential via modulation of culture conditions and Wnt/ß-catenin pathway.

Mesenchymal stem cells (MSCs) represent a promising cell source to regenerate articular cartilage, but current chondroinduction protocols, commonly using transforming growth factor-ß (TGFß), lead to concomitant chondrocytic hypertrophy with ossification risk. Here, we showed that a 14-day culture of MSC-laden hyaluronic acid hydrogel in the presence of TGFß, followed by 7 days culture in TGFß-free medium, with the supplement of Wnt/ß-catenin inhibitor XAV939 from day 10-21, resulted in significantly reduced hypertrophy phenotype. The stability of the hyaline phenotype of the MSC-derived cartilage, generated with a standard protocol (Control) or the optimized (Optimized) method developed in this study, was further examined through intramuscular implantation in nude mice. After 4 weeks, constructs from the Control group showed obvious mineralization; in contrast, the Optimized group displayed no signs of mineralization, and maintained cartilaginous histology. Further analysis showed that TGFß treatment time affected p38 expression, while exposure to XAV939 significantly inhibited P-Smad 1/5 level, which together resulted in decreased level of Runx2. These findings suggest a novel treatment regimen to generate hyaline cartilage from human MSCs-loaded scaffolds, which have a minimal risk of eliciting endochondral ossification.

Mesenchymal stem cell-derived extracellular matrix enhances chondrogenic phenotype of and cartilage formation by encapsulated chondrocytes in vitro and in vivo.

Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21 days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14 days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo. STATEMENT OF SIGNIFICANCE: Current cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for xenogenic scaffolds, and suboptimal cartilage formation. We present here a novel technique that utilizes adult stem cell-derived extracellular matrix, as a culture substrate and/or encapsulation scaffold for human adult chondrocytes, for the repair of cartilage defects. Chondrocytes cultured in stem cell-derived matrix showed higher proliferation, better chondrocytic phenotype, and improved redifferentiation ability upon in vitro culture expansion. Most importantly, 3-dimensional constructs formed from chondrocytes folded within stem cell matrix manifested excellent cartilage formation both in vitro and in vivo. These findings demonstrate the suitability of stem cell-derived extracellular matrix as a culture substrate for chondrocyte expansion as well as a candidate bioactive matrix for cartilage regeneration.

Association Between Tibial Plateau Slopes and Anterior Cruciate Ligament Injury: A Meta-analysis.

PURPOSE: To investigate the associations of medial tibial plateau slope (MTPS), lateral tibial plateau slope (LTPS), and coronal tibial plateau slope (CTPS) with anterior cruciate ligament (ACL) injury both in the general population and in different gender subgroups. METHODS: PubMed, Ovid, Embase, and Scopus databases were searched through from inception to August 31, 2016. Observational studies reporting associations of MTPS/LTPS/CTPS with ACL injury were retrieved for analysis. Either a fixed- or random-effects model was used to calculate the overall standardized mean difference (SMD). Reviews, meeting abstracts, cadaver or animal studies, and other studies without disclosing full text were excluded in this study. RESULTS: A total of 29 studies were included. Subjects with ACL injury exhibited a significant increase in MTPS (SMD: 0.34 [95% confidence interval (CI): 0.18, 0.49]; P < .0001) and LTPS (SMD: 0.49 [95% CI: 0.30, 0.68]; P < .00001), but not in the CTPS (SMD: 0.09 [95% CI: -0.10, 0.27]; P = .36), compared with controls. Meanwhile, significant differences in MTPS and LTPS were observed in the male subgroup (SMD: 0.41 [95% CI: 0.20, 0.62]; P = .0001 and SMD: 0.55 [95% CI: 0.26, 0.85]; P = .0002, respectively) but not in the female (SMD: 0.31 [95% CI: -0.02, 0.64]; P = .06 and SMD: 0.26 [95% CI: -0.04, 0.56]; P = .09, respectively). CONCLUSIONS: The present meta-analysis showed that the increases in MTPS and LTPS were overall risk factors of ACL injury. However, these slopes would only be considered as "at risk" for males, but not for females. In addition, it was also proved that CTPS was not a risk factor of ACL injury. LEVEL OF EVIDENCE: Level III, meta-analysis of Level II and III studies.