Dose- and stage-dependent toxic effects of prenatal prednisone exposure on fetal articular cartilage development.

Prednisone is frequently used to treat rheumatoid diseases in pregnant women because of its high degree of safety. Whether prenatal prednisone exposure (PPE) negatively impacts fetal articular cartilage development is unclear. In this study, we simulated a clinical prednisone treatment regimen to examine the effects of different timings and doses of PPE on cartilage development in female and male fetal mice. Prednisone doses (0.25, 0.5, and 1 mg/kg/d) was administered to Kunming mice at different gestational stages (0-9 gestational days, GD0-9), mid-late gestation (GD10-18), or during the entire gestation (GD0-18) by oral gavage. The amount of matrix aggrecan (ACAN) and collagen type II a1(COL2a1), and expression of transforming growth factor ß1 (TGFß1) signaling pathway also demonstrated that the chondrocyte count and ACAN and COL2a1 expression reduced in fetal mice with early and mid-late PPE, with the reduction being more significant in the mice with early PPE than that in those with PPE at other stages. Prenatal exposure to different prednisone doses prevented the reduction of TGFß signaling pathway-related genes [TGFßR1, SMAD family member 3 (Smad3), SRY-box9 (SOX9)] as well as ACAN and COL2a1 mRNA expression levels in fetal mouse cartilage, with the most significant decrease after 1 mg/kg·d PPE. In conclusion, PPE can inhibit/restrain fetal cartilage development, with the greatest effect at higher clinical dose (1 mg/kg·d) and early stage of pregnancy (GD0-9), and the mechanism may be related to TGFß signaling pathway inhibition. The result of this study provide a theoretical and experimental foundation for the rational clinical use of prednisone.

The molecular complexity of COL2A1 splicing variants and their significance in phenotype severity.

Pathogenic single nucleotide variants (SNVs) found in the COL2A1 gene are associated with a broad range of skeletal dysplasias due to their impact on the structure and function of the Col2a1 protein. However, the molecular mechanisms of some nucleotide variants detected during diagnostic testing remain unclear. The interpretation of missense and splicing variants caused by SNVs poses a significant challenge for clinicians. In this work, we analyzed 22 splicing variants in the COL2A1 gene which have been found in patients with COL2A1-associated skeletal dysplasias. Using a minigene system, we investigated the impact of these SNVs on splicing and gained insights into their molecular mechanisms and genotype-phenotype correlations for each patient. The results of our study are very useful for improving the accuracy of diagnosis and the management of patients with skeletal dysplasias caused by SNVs in the COL2A1 gene.

Effect of acupuncture on arthritic pathological injury in rats with type â ¡ collagen-induced rheumatoid arthritis. / é’ˆåˆºæ”¹å–„â ¡åž‹èƒ¶åŽŸè›‹ç™½è¯±å¯¼æ€§å ³èŠ‚ç‚Žå¤§é¼ ç‚Žæ€§ååº”çš„æœºåˆ¶ç ”ç©¶.

OBJECTIVES: To observe the effect of acupuncture stimulation of "Yanglingquan"(GB34), "Zusanli"(ST36) and "Xuanzhong" (GB39) on arthritis index (AI), joint synovial membrane pathology, serum-related immunoinflammatory factors, and expressions of tumor suppressor gene mt-p53, nuclear factor kappa B (NF-κB) and peroxisome proliferator activated receptor gamma (PPARγ) in knee joint synovial tissue of rats with type Ⅱ collagen-induced arthritis (CIA), so as to explore its possible mechanisms underlying improvement of rheumatoid arthritis (RA). METHODS: Male SD rats were used in the present study. The CIA model was established by subcutaneous injection of collagen emulsion (200 µL/rat) in the tail root region on the first day and repeat (100 µL/rat) once on the 9th day. Eighteen successful CIA rats were randomized into model, medication and acupuncture groups, with 6 rats in each group. Other 6 normal rats were used as the normal control group. For rats of the medication group, leflunomide (1.9 mg/kg) was administrated by gavage, once a day, and for rats of the acupuncture group, manual acupuncture stimulation was applied to bilateral GB34, ST36, GB39 for 30 min, once a day, for 12 weeks. The arthritis index (AI) score (0-4 points) was evaluated once every week. The contents of IL-6, IL-17 and TNF-α in the serum were determined by ELISA. Histopathological changes of the ankle joint were observed by H.E. staining. The protein and mRNA expression levels of mt-p53, NF-κB p65, and PPARγ in the knee joint synovial tissue were determined by Western blot and quantitative real time PCR, separately. RESULTS: Compared with the normal control group, the AI scores at different time-points after modeling, contents of serum TNF-α, IL-6 and IL-17, expression levels of mt-p53, NF-κB p65, PPARγ proteins and mRNAs were significantly increased in the model group (P<0.01, P<0.05). In comparison with the model group, the AI scores at the 10th week in the medication group and at the 3rd, 9th and 10th week in the acupuncture group, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of mt-p53 and NF-κB p65 proteins in both medication and acupuncture groups, as well as mt-p53 and NF-κB p65 mRNAs in the medication group were apparently decreased (P<0.01, P<0.05), while the expression levels of PPARγ protein in both medication and acupuncture group and PPARγ mRNA in the medication group were significantly up-regulated (P<0.05, P<0.01). No significant differences were found between the acupuncture and medication groups in down-regulating the AI score and serum TNF-α, IL-6 and IL-17 contents. The effect of acupuncture was weaker than that of medication in down-regulating the expression of mt-p53 and NF-κB p65 proteins and mRNAs and in up-regulating PPARγ mRNA (P<0.01). H.E. results showed ankle cartilage hyperplasia, reduced joint cavity, mild fibroproliferation and inflammatory cell infiltration in the surrounding soft tissue of the ankle joint in rats of the model group, which was milder in both medication and acupuncture groups. CONCLUSIONS: Acupuncture stimulation can improve the degree of joint inflammation and swelling in CIA rats, which may be related to its effects in inhibiting the overexpression of immunoinflammatory factors in serum and regulating expression of mt-p53, NF-κB p65, PPARγ mRNAs and proteins in the synovial tissue.

A Model of Type II Collagen-Induced Spondylitis and Arthritis in F1 Hybrid Male Mice.

In this study, we tested a new model of ankylosing spondylitis in order to determine its histological and radiological features needed to investigate peripheral arthritis, spondylitis, and formation of the new bone tissues. F1 hybrid male mice (BALB/c×DBA/1), a progeny of spondylitis-susceptible BALB/c male mice and rheumatoid arthritis-susceptible DBA/1 female mice, were immunized intraperitoneally with bovine type II collagen (CII) mixed with adjuvant dimethyldioctadecylammonium bromide. Radiological and histological studies were performed at the peak of swelling, redness, and stiffness. The incidence of peripheral arthritis and spondylitis induced by CII in F1 hybrid mice were 66 and 62%, respectively. X-ray examination revealed bone erosion and spondylitis in the peripheral joints, as well as the formation of new bone tissues in the coccygeal vertebrae and between LIII and LIV vertebrae. The histological study showed lymphocyte and plasma cell infiltration, capillary dilation, congestion, and endochondral ossification of the lumbar vertebrae. This novel model of CII-induced spondylitis in F1 hybrid mice provoked axial and peripheral arthritides inducing chronic inflammation. In this model, the formation of new bone tissue in the stiff spine is characterized by endochondral ossification. The advanced model is an additional and valuable tool for investigation of the autoimmune reactions in spondylitis.

Hypoxia Preconditioned Serum (HPS) Promotes Proliferation and Chondrogenic Phenotype of Chondrocytes In Vitro.

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects remains challenging in terms of maintaining chondrogenic phenotype during in vitro chondrocyte expansion. Growth factor supplementation has been found supportive in improving ACI outcomes by promoting chondrocyte redifferentiation. Here, we analysed the chondrogenic growth factor concentrations in the human blood-derived secretome of Hypoxia Preconditioned Serum (HPS) and assessed the effect of HPS-10% and HPS-40% on human articular chondrocytes from osteoarthritic cartilage at different time points compared to normal fresh serum (NS-10% and NS-40%) and FCS-10% culture conditions. In HPS, the concentrations of TGF-beta1, IGF-1, bFGF, PDGF-BB and G-CSF were found to be higher than in NS. Chondrocyte proliferation was promoted with higher doses of HPS (HPS-40% vs. HPS-10%) and longer stimulation (4 vs. 2 days) compared to FCS-10%. On day 4, immunostaining of the HPS-10%-treated chondrocytes showed increased levels of collagen type II compared to the other conditions. The promotion of the chondrogenic phenotype was validated with quantitative real-time PCR for the expression of collagen type II (COL2A1), collagen type I (COL1A1), SOX9 and matrix metalloproteinase 13 (MMP13). We demonstrated the highest differentiation index (COL2A1/COL1A1) in HPS-10%-treated chondrocytes on day 4. In parallel, the expression of differentiation marker SOX9 was elevated on day 4, with HPS-10% higher than NS-10/40% and FCS-10%. The expression of the cartilage remodelling marker MMP13 was comparable across all culture conditions. These findings implicate the potential of HPS-10% to improve conventional FCS-based ACI culture protocols by promoting the proliferation and chondrogenic phenotype of chondrocytes during in vitro expansion.

Characteristics of a Three-Generation Family with Stickler Syndrome Type I Carrying Two Different COL2A1 Mutations.

Stickler Syndrome is typically characterized by ophthalmic manifestations including vitreous degeneration and axial lengthening that predispose to retinal detachment. Systemic findings consist of micrognathia, cleft palate, sensorineural hearing loss, and joint abnormalities. COL2A1 mutations are the most common, however, there is a lack of genotype-phenotype correlations. Retrospective, single-center case series of a three-generation family. Clinical features, surgical requirements, systemic manifestations, and genetic evaluations were collected. Eight individuals clinically displayed Stickler Syndrome, seven of whom had genetic confirmation, and two different COL2A1 mutations (c.3641delC and c.3853G>T) were identified. Both mutations affect exon 51, but display distinct phenotypes. The c.3641delC frameshift mutation resulted in high myopia and associated vitreous and retinal findings. Individuals with the c.3853G>T missense mutation exhibited joint abnormalities, but mild ocular manifestations. One individual in the third generation was biallelic heterozygous for both COL2A1 mutations and showed ocular and joint findings in addition to autism and severe developmental delay. These COL2A1 mutations exhibited distinct eye vs. joint manifestations. The molecular basis for these phenotypic differences remains unknown and demonstrates the need for deep phenotyping in patients with Stickler syndrome to correlate COL2A1 gene function and expression with ocular and systemic findings.

Intra-articular delivery of AAV vectors encoding PD-L1 attenuates joint inflammation and tissue damage in a mouse model of rheumatoid arthritis.

Objective: Rheumatoid arthritis (RA) is the most common form of autoimmune inflammatory arthritis. Intra-articular gene delivery to block proinflammatory cytokines has been studied in pre-clinical models and human clinical trials. It has been demonstrated that the level of programmed death-ligand 1 (PD-L1) is associated with rheumatoid arthritis (RA). This study examined the therapeutic role of PD-L1 by intra-articular delivery via adeno-associated virus (AAV) vectors in the mouse collagen-induced arthritis (CIA) model. Methods: Mice were intra-articularly injected with AAV5 vectors encoding human PD-L1 on day 0 and immunized with bovine type II collagen to induce CIA simultaneously. On day 49 post AAV administration, joints were collected for histo-pathological and cytokine analysis. Additionally, the systemic impacts of intra-articular injection of AAV5/PD-L1 vectors were also studied. To study the therapeutic effect of PD-L1, AAV5/PD-L1 vectors were administered into the joints of RA mice on day 21. Results: After administration of AAV5/PD-L1 vectors, strong PD-L1 expression was detected in AAV transduced joints. Joints treated with PD-L1 at the time of arthritis induction exhibited significantly less swelling and improved histopathological scores when compared to untreated joints. Additionally, the infiltration of T cells and macrophages was decreased in joints of CIA mice that received AAV5/PD-L1 vectors (P<0.05). The levels of pro-inflammatory cytokines, including IL-1, IL-6, IL-17 and TNFα, were lower in AAV5/PD-L1 treated than untreated joints (P<0.05). Furthermore, the administration of AAV5/PD-L1 vectors into the joints of CIA mice did not impact serum cytokine levels and the antibody titers to type II collagen. Biodistribution of AAV vectors after intra-articular injection showed undetectable AAV genomes in other tissues except for a low level in the liver. Similar to the results of AAV5/PD-L1 vector administration on day 0, decreased joint swelling and lower histopathological damage were observed in joints treated with AAV5/PD-L1 vectors on day 21. Conclusion: The results from this study demonstrate that local AAV mediated PD-L1 gene delivery into the joints is able to prevent the development and block the progression of arthritis in CIA mice without impacting systemic immune responses. This study provides a novel strategy to effectively treat inflammatory joint diseases using local AAV gene therapy by interference with immune checkpoint pathways.

E3 Ubiquitin Ligase Constitutive Photomorphogenic 1 Regulates Differentiation and Inflammation via MAPK Signaling Pathway in Rabbit Articular Chondrocytes.

Constitutive photomorphogenic 1 (COP1), is an E3 ubiquitin ligase that plays a role in the regulation of various cellular processes including cell growth, differentiation, and survival in mammals. In certain conditions such as overexpression or loss of function, COP1 acts either as an oncogenic protein or as a tumor suppressor by targeting specific proteins for ubiquitination-mediated degradation. However, the precise role of COP1 has not been well studied in primary articular chondrocytes. In this study, we investigated the role of COP1 in chondrocyte differentiation. Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated that COP1 overexpression reduced type II collagen expression, promoted cyclooxygenase 2 (COX-2) expression, and reduced sulfated proteoglycan synthesis, as detected by Alcian blue staining. Upon siRNA treatment, revived type II collagen, sulfated proteoglycan production, and decreased COX-2 expression. Phosphorylation of p38 kinase and ERK-1/-2 signaling pathways was regulated by COP1 upon cDNA and siRNA transfection in chondrocytes. The inhibition of the p38 kinase and ERK-1/-2 signaling pathways with SB203580 and PD98059 ameliorated the expression of type II collagen and COX-2 in transfected chondrocytes, thus suggesting that COP1 regulates differentiation and inflammation in rabbit articular chondrocytes via the p38 kinase and ERK-1/-2 signaling pathway.

[Research of epigallocatechin gallate in delaying chondrocyte senescence].

Objective: To investigate the effect of epigallocatechin gallate (EGCG) on chondrocyte senescence and its mechanism. Methods: The chondrocytes were isolated from the articular cartilage of 4-week-old Sprague Dawley rats, and cultured with type Ⅱcollagenase and passaged. The cells were identified by toluidine blue staining, alcian blue staining, and immunocytochemical staining for type Ⅱ collagen. The second passage (P2) cells were divided into blank control group, 10 ng/mL IL-1ß group, and 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 µmol/L EGCG+10 ng/mL IL-1ß group. The chondrocyte activity was measured with cell counting kit 8 after 24 hours of corresponding culture, and the optimal drug concentration of EGCG was selected for the subsequent experiment. The P2 chondrocytes were further divided into blank control group (group A), 10 ng/mL IL-1ß group (group B), EGCG+10 ng/mL IL-1ß group (group C), and EGCG+10 ng/mL IL-1ß+5 mmol/L 3-methyladenine (3-MA) group (group D). After cultured, the degree of cell senescence was detected by ß-galactosidase staining, the autophagy by monodansylcadaverine method, and the expression levels of chondrocyte-related genes [type Ⅱ collagen, matrix metalloproteinase 3 (MMP-3), MMP-13] by real-time fluorescent quantitative PCR, the expression levels of chondrocyte-related proteins (Beclin-1, LC3, MMP-3, MMP-13, type Ⅱ collagen, P16, mTOR, AKT) by Western blot. Results: The cultured cells were identified as chondrocytes. Compared with the blank control group, the cell activity of 10 ng/mL IL-1ß group significantly decreased ( P<0.05). Compared with the 10 ng/mL IL-1ß group, the cell activity of EGCG+10 ng/mL IL-1ß groups increased, and the 50.0, 100.0, and 200.0 µmol/L EGCG significantly promoted the activity of chondrocytes ( P<0.05). The 100.0 µmol/L EGCG was selected for subsequent experiments. Compared with group A, the cells in group B showed senescence changes. Compared with group B, the senescence rate of chondrocytes in group C decreased, autophagy increased, the relative expression of type Ⅱ collagen mRNA increased, and relative expressions of MMP-3 and MMP-13 mRNAs decreased; the relative expressions of Beclin-1, LC3, and type Ⅱ collagen proteins increased, but the relative expressions of P16, MMP-3, MMP-13, mTOR, and AKT proteins decreased; the above differences were significant ( P<0.05). Compared with group C, when 3-MA was added in group D, the senescence rate of chondrocytes increased, autophagy decreased, and the relative expressions of the target proteins and mRNAs showed an opposite trend ( P<0.05). Conclusion: EGCG regulates the autophagy of chondrocytes through the PI3K/AKT/mTOR signaling pathway and exerts anti-senescence effects.

Metformin regulates chondrocyte senescence and proliferation through microRNA-34a/SIRT1 pathway in osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is the most common degenerative disease in joints among elderly patients. Senescence is deeply involved in the pathogenesis of osteoarthritis. Metformin is widely used as the first-line drug for Type 2 diabetes mellitus (T2DM), and has great potential for the treatment of other aging-related disorders, including OA. However, the role of metformin in OA is not fully elucidated. Therefore, our aim here was to investigate the effects of metformin on human chondrocytes. METHODS: After metformin treatment, expression level of microRNA-34a and SIRT1 in chondrocyte were detected with quantitative real-time PCR and immunofluorescence staining. Then, microRNA-34a mimic and small interfering RNA (siRNA) against SIRT1 (siRNA-SIRT1) were transfected into chondrocyte. Senescence-associated ß-galactosidase (SA-ß-gal) staining was performed to assess chondrocyte senescence. Chondrocyte viability was illustrated with MTT and colony formation assays. Western blot was conducted to detect the expression of P16, IL-6, matrix metalloproteinase-13 (MMP-13), Collagen type II (COL2A1) and Aggrecan (ACAN). RESULTS: We found that metformin treatment (1 mM) inhibited microRNA-34a while promoted SIRT1 expression in OA chondrocytes. Both miR-34a mimics and siRNA against SIRT1 inhibited SIRT1 expression in chondrocytes. SA-ß-gal staining assay confirmed that metformin reduced SA-ß-gal-positive rate of chondrocytes, while transfection with miR-34a mimics or siRNA-SIRT1 reversed it. MTT assay and colony formation assay showed that metformin accelerated chondrocyte proliferation, while miR-34a mimics or siRNA-SIRT1 weakened this effect. Furthermore, results from western blot demonstrated that metformin suppressed expression of senescence-associated protein P16, proinflammatory cytokine IL-6 and catabolic gene MMP-13 while elevated expression of anabolic proteins such as Collagen type II and Aggrecan, which could be attenuated by transfection with miR-34a mimics. CONCLUSION: Overall, our data suggest that metformin regulates chondrocyte senescence and proliferation through microRNA-34a/SIRT1 pathway, indicating it could be a novel strategy for OA treatment.

IPSC reprogramming of two patients with spondyloepiphyseal dysplasia congenita (SEDC).

Spondyloepiphyseal dysplasia congenita (SEDC) is a severe non-lethal type 2 collagenopathy caused by pathogenic variants in the COL2A1 gene, which encodes the alpha-1 chain of type II collagen. SEDC is clinically characterized by severe short stature, degenerative joint disease, hearing impairment, orofacial anomalies and ocular manifestations. To study and therapeutically target the underlying disease mechanisms, human iPSC-chondrocytes are considered highly suitable as they have been shown to exhibit several key features of skeletal dysplasias. Prior to creating iPSC-chondrocytes, peripheral blood mononuclear cells of two male SEDC patients, carrying the p.Gly1107Arg and p.Gly408Asp pathogenic variants, respectively, were successfully reprogrammed into iPSCs using the CytoTune™-iPS 2.0 Sendai Kit (Invitrogen).

Intervertebral disk regeneration in a rat model by allopurinol-loaded chitosan/alginate hydrogel.

Intervertebral disk degeneration remains one of the most challenging health problems. In the current study, allopurinol was loaded into the chitosan nanoparticles and then incorporated into chitosan/alginate hydrogels and then further studied for its disk regeneration potential in a rat model. In vitro studies were performed to characterize the hydrogel system, including scanning electron microscopy, cell viability assay, cytoprotection assay, cell migration assay, swelling assay, and drug release assay. In vivo study was performed in a rat model of the intervertebral disk injury. Animal studies showed that allopurinol-loaded hydrogels had significantly higher disk regeneration potential compared with other experimental groups. The gene expression studies showed that the animals treated with allopurinol-loaded hydrogel had significantly higher tissue expression levels of type I and type II collagen genes than other groups. Furthermore, the tissue expression levels of nuclear factor κB (NF-κB) and glutathione peroxidase (GPx) genes were significantly lower in this group. The relative expression levels of type I collagen, type II collagen, NF-κB, and GPx genes in the allopurinol-loaded hydrogel group were 2.77 ± 0.2%, 2.86 ± 0.25%, 0.58 ± 0.03%, and 0.45 ± 0.02%, respectively. We showed for the first time that allopurinol-loaded hydrogel promoted intervertebral disk repair, which could be due to its potential to modulate oxidative stress, reduce inflammation, and improve matrix synthesis.

CD44 mediates hyaluronan to promote the differentiation of human amniotic mesenchymal stem cells into chondrocytes.

OBJECTIVES: CD44 is the major receptor for hyaluronan (HA), but its effect on HA-induced differentiation of human amnion mesenchymal stem cells into chondrocytes is unclear. This study aimed to investigate the effects and mechanisms of CD44 in HA-induced chondrogenesis. METHODS: Immunocytochemistry and toluidine blue staining were used to assess the secretion of type II collagen and aggrecan, respectively. qRT-PCR and western blotting were performed to evaluate the expression of key genes and proteins. RESULTS: The expression of aggrecan and type II collagen was downregulated after using the anti-CD44 antibody (A3D8). The transcriptional levels of chondrocytes­associated genes SRY­box transcription factor 9, aggrecan, and collagen type II alpha 1 chain were also decreased. Thus, CD44 may mediate HA-induced differentiation of hAMSCs into chondrocytes. Further investigation indicated that expression of phosphorylated (p)­Erk1/2 and p­Smad2 decreased following CD44 inhibition. The changes in the expression of p-Erk1/2 and p-Smad2 were consistent after using the ERK1/2 inhibitor (U0126) and agonist (EGF), respectively. After administering the p-Smad2 inhibitor, the expression levels of p-ERK1/2 and p-Smad2 appeared downregulated. The results showed crosstalk between Erk1/2 and Smad2. Moreover, inhibition of p-Erk1/2 and p-Smad2 significantly reduced the accumulation of aggrecan and type II collagen. CONCLUSION: These data indicate that CD44 mediates HA-induced differentiation of hAMSCs into chondrocytes by regulating Erk1/2 and Smad2 signaling.

Using CRISPR/Cas9 to generate a heterozygous COL2A1 p.R719C iPSC line (MCRIi019-A-6) model of human precocious osteoarthritis.

The human iPSC line MCRIi019-A-6 was generated using CRISPR/Cas9-mediated gene editing to introduce a heterozygous COL2A1 exon 33 c.2155C>T (p.R719C) mutation into the control human iPSC line MCRIi019-A. Both the edited and parental lines display typical iPSC characteristics, including the expression of pluripotency markers, the ability to be differentiated into the three germ lines, and a normal karyotype. This cell line, along with the isogenic control line, can be used to study the molecular pathology of precocious osteoarthritis in a human model, more broadly understand type II collagenopathies, and explore novel therapeutic targets for this class of diseases.

Triclocarban triggers osteoarthritis via DNMT1-mediated epigenetic modification and suppression of COL2A in cartilage tissues.

Triclocarban (TCC) is a widely used environmental endocrine-disrupting chemical (EDC). Articular injury of EDCs has been reported; however, whether and how TCCs damage the joint have not yet been determined. Herein, we revealed that exposure to TCC caused osteoarthritis (OA) within the zebrafish anal fin. Mechanistically, TCC stimulates the expression of DNMT1 and initiates DNA hypermethylation of the type II collagen coding gene, which further suppresses the expression of type II collagen and other extracellular matrices. This further results in decreased cartilage tissue and narrowing of the intraarticular space, which is typical of the pathogenesis of OA. The regulation of OA occurrence by TCC is conserved between zebrafish cartilage tissue and human chondrocytes. Our findings clarified the hazard and potential mechanisms of TCC towards articular health and highlighted DNMT1 as a potential therapeutic target for OA caused by TCC.

Natural history and genetic spectrum of the Turkish metaphyseal dysplasia cohort, including rare types caused by biallelic COL10A1, COL2A1, and LBR variants.

BACKGROUND: Metaphyseal chondrodysplasias are a heterogeneous group of diseases characterized by short and bowed long bones and metaphyseal abnormality. The aim of this study is to investigate the genetic etiology and prognostic findings in patients with metaphyseal dysplasia. METHODS: Twenty-four Turkish patients were included in this study and 13 of them were followed for 2-21 years. COL10A1, RMRP sequencing and whole exome sequencing were performed. RESULTS: Results: Seven heterozygous pathogenic variants in COL10A1 were detected in 17 patients with Schmid type metaphyseal chondrodysplasia(MCDS). The phenotype was more severe in patients with heterozygous missense variants (one in signal peptide domain at the N-terminus of the protein, the other, class-1 group mutation at NC1 domain) compared to the patients with truncating variants. Short stature and coxa vara deformity appeared after 3 and 5 years of age, respectively, while large femoral head resolved after the age of 13 years in MCDS group. Interestingly, one patient with severe phenotype also had a biallelic missense variant in NC1 domain of COL10A1. Three patients with biallelic mutations in RMRP had prenatal onset short stature with short limb, and typical findings of cartilage hair hypoplasia (CHH). While immunodeficiency or recurrent infections were not observed, resistant congenital anemia was detected in one. Biallelic mutation in LBR was described in a patient with prenatal onset short stature, short and curved limb and metaphyseal abnormalities. Unlike previously reported patients, this patient had ectodermal findings, similar to CHH. A biallelic COL2A1 mutation was also found in the patient with lower limb deformities and metaphyseal involvement without vertebral and epiphyseal changes. CONCLUSION: Long-term clinical characteristics are presented in a metaphyseal dysplasia cohort, including rare types caused by biallelic COL10A1, COL2A1, and LBR variants. We also point out that the domains where mutations on COL10A1 take place are important in the genotype-phenotype relationship.

Blocking TXNIP reduced IL-1ß Induced chondrocyte cell inflammation.

Osteoarthritis (OA) is the most common joint disease in the elderly and is characterized by progressive and irreversible degeneration of articular cartilage, particularly cartilage loss and callus formation. This study would like to investigate the important role and the molecular mechanism of OA progression following interleukin 1ß (IL-1ß)-induced chondrocyte injury regulated by TXNIP. In this study, high-purity mouse chondrocytes were obtained by enzymatic two-step digestion for primary culture. Toluidine blue staining and type II collagen immunofluorescence were used to identify cells through histochemical staining after slide mounting. The relative expression of TXNIP was detected by immunohistochemical staining and qRT-PCR.Aiming at the shRNA sequence of the TXNIP gene, the shRNA expression vector was constructed and packaged with lentivirus to form the lentiviral vector shTXNIP. After inhibiting the expression of TXNIP by transfecting shTXNIP into normal mouse chondrocytes, the CCK-8 kit was used for detecting its effect on cell proliferation after transfection, and the effect on chondrocyte apoptosis was detected by flow cytometry. The staining kit was used to detect the effect of TXNIP knockout on chondrocyte aging, and the differential expression of TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen genes in chondrocytes was detected by RT-PCR and Western-bolt. Western blot was used to detect the expression of upstream-related protein P-ERK, downstream-related protein NLRP3 and Caspase1 after inflammatory injury of mouse articular chondrocytes. Results showed that the expression level of TXNIP in chondrocytes induced by different concentrations of il-1ß was proportional to the concentration. After silencing TXNIP by shRNA, cell proliferation increased, chondrocyte apoptosis was weakened, and chondrocyte aging was weakened. The differential expression of genes such as TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen and the differential expression of protein levels were relatively decreased. In addition, the expression of the upstream-related protein P-ERK did not change much when TXNIP was silenced, and the expression levels of the downstream-related proteins NLRP3 and Caspase1 were slightly reduced. In conclusion, silencing TXNIP can inhibit il-1ß-induced chondrocyte apoptosis and aging, and has a positive effect on cell proliferation. However, this study has not clarified the molecular mechanism involved in TXNIP and the process of its signaling expression pathway.

Induction of type II collagen expression in M2 macrophages derived from peripheral blood mononuclear cells.

The human type II collagen (Col II), specifically expressed in chondrocytes, is a crucial component of the adult hyaline cartilage. We examine the potential of artificial induction of Col II in human peripheral blood mononuclear cells (PBMNCs) as a novel Col II provider. Human PBMNCs were purified and were treated with high doses of macrophage-colony stimulating factor (M-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), or granulocyte-colony stimulating factor (G-CSF) and examined the Col II expression at indicated days. Quantitative Col II expression was validated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), immunocytochemistry, and flow cytometry. We demonstrate that monocytes in PBMNCs can be artificially induced to express both Col II proteins and M2 macrophage markers by the high concentration of colony-stimulating factors, especially M-CSF and GM-CSF. The Col II proteins were detected on the cell membrane and in the cytoplasm by flow cytometry and immunocytostaining. Combination with IL-4 provided a synergistic effect with M-CSF/GM-CSF to trigger Col II expression in M2 macrophages. These CD206 and Col II double-expressing cells, named modified macrophages, share M2 macrophages' anti-inflammatory potency. We demonstrated that the modified macrophages could significantly attenuate the inflammatory progress of Complete Freund's adjuvant (CFA)-induced arthritis and collagen-induced arthritis in rodents. Here, we provide the first evidence that a modified macrophage population could ectopically express Col II and control the progress of arthritis in animals.

Runx2 and Runx3 differentially regulate articular chondrocytes during surgically induced osteoarthritis development.

The Runt-related transcription factor (Runx) family plays various roles in the homeostasis of cartilage. Here, we examined the role of Runx2 and Runx3 for osteoarthritis development in vivo and in vitro. Runx3-knockout mice exhibited accelerated osteoarthritis following surgical induction, accompanied by decreased expression of lubricin and aggrecan. Meanwhile, Runx2 conditional knockout mice showed biphasic phenotypes: heterozygous knockout inhibited osteoarthritis and decreased matrix metallopeptidase 13 (Mmp13) expression, while homozygous knockout of Runx2 accelerated osteoarthritis and reduced type II collagen (Col2a1) expression. Comprehensive transcriptional analyses revealed lubricin and aggrecan as transcriptional target genes of Runx3, and indicated that Runx2 sustained Col2a1 expression through an intron 6 enhancer when Sox9 was decreased. Intra-articular administration of Runx3 adenovirus ameliorated development of surgically induced osteoarthritis. Runx3 protects adult articular cartilage through extracellular matrix protein production under normal conditions, while Runx2 exerts both catabolic and anabolic effects under the inflammatory condition.