Aggrecan Research Articles

Inhibition of CC chemokine receptor 1 ameliorates osteoarthritis in mouse by activating PPAR-γ

BackgroundOsteoarthritis (OA) is a degenerative joint disease characterized by cartilage destruction and inflammation. CC chemokine receptor 1 (CCR1), a member of the chemokine family and its receptor family, plays a role in the autoimmune response. The impact of BX471, a specific small molecule inhibitor of CCR1, on CCR1 expression in cartilage and its effects on OA remain underexplored.MethodsThis study used immunohistochemistry (IHC) to assess CCR1 expression in IL-1β-induced mouse chondrocytes and a medial meniscus mouse model of destabilization of the medial meniscus (DMM). Chondrocytes treated with varying concentrations of BX471 for 24 h were subjected to IL-1β (10 ng/ml) treatment. The levels of the aging-related genes P16INK4a and P21CIP1 were analyzed via western blotting, and senescence-associated β-galactosidase (SA-β-gal) activity was measured. The expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), aggrecan (AGG), and the transcription factor SOX9 were determined through western blotting and RT‒qPCR. Collagen II, matrix metalloproteinase 13 (MMP13), and peroxisome proliferator-activated receptor (PPAR)-γ expression was analyzed via western blot, RT‒qPCR, and immunofluorescence. The impact of BX471 on inflammatory metabolism-related proteins under PPAR-γ inhibition conditions (using GW-9662) was examined through western blotting. The expression of MAPK signaling pathway-related molecules was assessed through western blotting. In vivo, various concentrations of BX471 or an equivalent medium were injected into DMM model joints. Cartilage destruction was evaluated through Safranin O/Fast green and hematoxylin–eosin (H&E) staining.ResultsThis study revealed that inhibiting CCR1 mitigates IL-1β-induced aging, downregulates the expression of iNOS, COX-2, and MMP13, and alleviates the IL-1β-induced decrease in anabolic indices. Mechanistically, the MAPK signaling pathway and PPAR-γ may be involved in inhibiting the protective effect of CCR1 on chondrocytes. In vivo, BX471 protected cartilage in a DMM model.ConclusionThis study demonstrated the expression of CCR1 in chondrocytes. Inhibiting CCR1 reduced the inflammatory response, alleviated cartilage aging, and retarded degeneration through the MAPK signaling pathway and PPAR-γ, suggesting its potential therapeutic value for OA.Graphical

Exercise following joint distraction inhibits muscle wasting and delays the progression of post-traumatic osteoarthritis in rabbits by activating PGC-1α in skeletal muscle

ObjectiveMuscle wasting frequently occurs following joint trauma. Previous research has demonstrated that joint distraction in combination with treadmill exercise (TRE) can mitigate intra-articular inflammation and cartilage damage, consequently delaying the advancement of post-traumatic osteoarthritis (PTOA). However, the precise mechanism underlying this phenomenon remains unclear. Hence, the purpose of this study was to examine whether the mechanism by which TRE following joint distraction delays the progression of PTOA involves the activation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), as well as its impact on muscle wasting.MethodsQuadriceps samples were collected from patients with osteoarthritis (OA) and normal patients with distal femoral fractures, and the expression of PGC-1α was measured. The hinged external fixator was implanted in the rabbit PTOA model. One week after surgery, a PGC-1α agonist or inhibitor was administered for 4 weeks prior to TRE. Western blot analysis was performed to detect the expression of PGC-1α and Muscle atrophy gene 1 (Atrogin-1). We employed the enzyme-linked immunosorbent assay (ELISA) technique to examine pro-inflammatory factors. Additionally, we utilized quantitative real-time polymerase chain reaction (qRT-PCR) to analyze genes associated with cartilage regeneration. Synovial inflammation and cartilage damage were evaluated through hematoxylin-eosin staining. Furthermore, we employed Masson’s trichrome staining and Alcian blue staining to analyze cartilage damage.ResultsThe decreased expression of PGC-1α in skeletal muscle in patients with OA is correlated with the severity of OA. In the rabbit PTOA model, TRE following joint distraction inhibited the expressions of muscle wasting genes, including Atrogin-1 and muscle ring finger 1 (MuRF1), as well as inflammatory factors such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in skeletal muscle, potentially through the activation of PGC-1α. Concurrently, the production of IL-1β, IL-6, TNF-α, nitric oxide (NO), and malondialdehyde (MDA) in the synovial fluid was down-regulated, while the expression of type II collagen (Col2a1), Aggrecan (AGN), SRY-box 9 (SOX9) in the cartilage, and superoxide dismutase (SOD) in the synovial fluid was up-regulated. Additionally, histological staining results demonstrated that TRE after joint distraction reduced cartilage degeneration, leading to a significant decrease in OARSI scores.TRE following joint distraction could activate PGC-1α, inhibit Atrogin-1 expression in skeletal muscle, and reduce C-telopeptides of type II collagen (CTX-II) in the blood compared to joint distraction alone.ConclusionFollowing joint distraction, TRE might promote the activation of PGC-1α in skeletal muscle during PTOA progression to exert anti-inflammatory effects in skeletal muscle and joint cavity, thereby inhibiting muscle wasting and promoting cartilage regeneration, making it a potential therapeutic intervention for treating PTOA.

Expression of Chondrogenic Potential Markers in Cultured Chondrocytes from the Human Knee Joint.

While substantial progress has been made in engineering cartilaginous constructs for animal models, further research is needed to translate these methodologies for human applications. Evidence suggests that cultured autologous chondrocytes undergo changes in phenotype and gene expression, thereby affecting their proliferation and differentiation capacity. This study was designed to evaluate the expression of chondrogenic markers in cultured human articular chondrocytes from passages 3 (P3) and 7 (P7), beyond the current clinical recommendation of P3. Cultured autologous chondrocytes were passaged from P3 up to P7, and quantitative polymerase chain reaction (qPCR) was used to assess mRNA expression of chondrogenic markers, including collagen type I (COLI), collagen type II (COLII), aggrecan (AGG), bone morphogenetic protein 4 (BMP4), transcription factor SOX-9 (SOX9), proteoglycan 4 (PGR4), and transformation-related protein 53 (p53), between P3 and P7. Except for AGG, no significant differences were found in the expression of markers between passages, suggesting the maintenance of chondrogenic potential in cultured chondrocytes. Differential expression identified between SOX9 and PGR4, as well as between COLI and SOX9, indicates that differences in chondrogenic markers are present between age groups and sexes, respectively. Overall, expression profiles of younger and male chondrocytes exhibit conversion of mature cartilage characteristics compared to their counterparts, with signs of dedifferentiation and loss of phenotype within-group passaging. These results may have implications in guiding the use of higher passaged chondrocytes for engineering constructs and provide a foundation for clinical recommendations surrounding the repair and treatment of articular cartilage pathology in both sexes.

Osteoporosis GWAS-implicated DNM3 locus contextually regulates osteoblastic and chondrogenic fate of mesenchymal stem/progenitor cells through oscillating miR-199a-5p levels.

Genome wide association study (GWAS)-implicated bone mineral density (BMD) signals have been shown to localize in cis-regulatory regions of distal effector genes using 3D genomic methods. Detailed characterization of such genes can reveal novel causal genes for BMD determination. Here, we elected to characterize the "DNM3" locus on chr1q24, where the long non-coding RNA DNM3OS and the embedded microRNA MIR199A2 (miR-199a-5p) are implicated as effector genes contacted by the region harboring variation in linkage disequilibrium with BMD-associated sentinel single nucleotide polymorphism, rs12041600. During osteoblast differentiation of human mesenchymal stem/progenitor cells (hMSC), miR-199a-5p expression was temporally decreased and correlated with the induction of osteoblastic transcription factors RUNX2 and Osterix. Functional relevance of miR-199a-5p downregulation in osteoblastogenesis was investigated by introducing miR-199a-5p mimic into hMSC. Cells overexpressing miR-199a-5p depicted a cobblestone-like morphological change and failed to produce BMP2-dependent extracellular matrix mineralization. Mechanistically, a miR-199a-5p mimic modified hMSC propagated normal SMAD1/5/9 signaling and expressed osteoblastic transcription factors RUNX2 and Osterix but depicted pronounced upregulation of SOX9 and enhanced expression of essential chondrogenic genes ACAN, COMP, and COL10A1. Mineralization defects, morphological changes, and enhanced chondrogenic gene expression associated with miR-199a-5p mimic over-expression were restored with miR-199a-5p inhibitor suggesting specificity of miR-199a-5p in chondrogenic fate specification. The expression of both the DNM3OS and miR-199a-5p temporally increased and correlated with hMSC chondrogenic differentiation. Although miR-199a-5p overexpression failed to further enhance chondrogenesis, blocking miR-199a-5p activity significantly reduced chondrogenic pellet size, extracellular matrix deposition, and chondrogenic gene expression. Taken together, our results indicate that oscillating miR-199a-5p levels dictate hMSC osteoblast or chondrocyte terminal fate. Our study highlights a functional role of miR-199a-5p as a BMD effector gene at the DNM3 BMD GWAS locus, where patients with cis-regulatory genetic variation which increases miR-199a-5p expression could lead to reduced osteoblast activity.

High Frequencies of Genetic Variants in Patients with Atypical Femoral Fractures.

This study explores the genetic factors associated with atypical femoral fractures (AFF), rare fractures associated with prolonged anti-resorptive therapy. AFF are fragility fractures that typically appear in the subtrochanteric or diaphyseal regions of the femur. While some cases resemble fractures in rare genetic bone disorders, the exact cause remains unclear. This study investigates 457 genes related to skeletal homeostasis in 13 AFF patients by exome sequencing, comparing the results with osteoporotic patients (n = 27) and Iberian samples from the 1000 Genomes Project (n = 107). Only one AFF case carried a pathogenic variant in the gene set, specifically in the ALPL gene. The study then examined variant accumulation in the gene set, revealing significantly more variants in AFF patients than in osteoporotic patients without AFF (p = 3.7 × 10-5), particularly in ACAN, AKAP13, ARHGEF3, P4HB, PITX2, and SUCO genes, all of them related to osteogenesis. This suggests that variant accumulation in bone-related genes may contribute to AFF risk. The polygenic nature of AFF implies that a complex interplay of genetic factors determines the susceptibility to AFF, with ACAN, SUCO, AKAP13, ARHGEF3, PITX2, and P4HB as potential genetic risk factors. Larger studies are needed to confirm the utility of gene set analysis in identifying patients at high risk of AFF during anti-resorptive therapy.

Chondrogenic Potential of PMSCs Cultured on Chondroitin Sulfate/Gelatin -Modified DBM Scaffold

Introduction: Osteoarthritis is one of the most common orthopedic diseases that gradually causes wear and damage to the articular Subchondral bone due to the destruction of articular cartilage. One of the basic challenges in cartilage tissue engineering is the choice of scaffold. In the design of the cartilage scaffold, it is useful to consider parameters such as porosity, water absorption, high mechanical resistance, biocompatibility, and biodegradability. Therefore, in this study, Demineralized Bone Matrix (DBM) which inherently has these characteristics to some extent, was chosen as the basic scaffold. Methods: The Gelatin/demineralized bone matrix (G/DBM) and the Chondroitin sulfate- Gelatin/demineralized bone matrix (GCS/DBM) scaffolds were prepared, respectively by incorporating gelatin or Chondroitin sulfate/gelatin solution inside DBM pores, freeze-drying and crosslinking with EDC/NHS. The physicochemical, biological characteristics, and chondrogenic potential of scaffolds were studied. Results: According to the SEM results, the size of the DBM pores in the G/DBM and GCS/DBM scaffolds decreased (from almost, 100-1500 µm to less than 200 µm) which reduced cell escape compared to the DBM scaffold. Also, crosslinking the scaffolds has greatly increased their compressive E-modulus (more than 8 times). The cytocompatibility and non-toxicity of all scaffolds were confirmed by acridine orange/ethidium bromide (AO/EB) staining. The evaluation results of chondrogenic differentiation of placenta-derived mesenchymal stem cells (PMSCs) on modified scaffolds, using the Real-time PCR method, showed that the presence of CS in the GCS/DBM scaffold improved the expression of chondrogenesis markers such as Aggrecan (AGC) (~4 times) and collagen 2 (COL-2) (~ 2.2 times) compared to the DBM scaffold. Also, Alcian blue staining and immunohistochemical analyses of the scaffolds, respectively, showed a denser and more coherent synthesis of GAGs and COL-2 protein on the GCS/DBM scaffold than on the G/DBM and DBM. Conclusion: According to the obtained results, it seems that the GCS/DBM scaffold can be a suitable scaffold in cartilage tissue engineering.

Multi-omics analysis reveals the molecular regulatory network underlying the prevention of Lactiplantibacillus plantarum against LPS-induced salpingitis in laying hens

BackgroundSalpingitis is one of the common diseases in laying hen production, which greatly decreases the economic outcome of laying hen farming. Lactiplantibacillus plantarum was effective in preventing local or systemic inflammation, however rare studies were reported on its prevention against salpingitis. This study aimed to investigate the preventive molecular regulatory network of microencapsulated Lactiplantibacillus plantarum (MLP) against salpingitis through multi-omics analysis, including microbiome, transcriptome and metabolome analyses.ResultsThe results revealed that supplementation of MLP in diet significantly alleviated the inflammation and atrophy of uterus caused by lipopolysaccharide (LPS) in hens (P < 0.05). The concentrations of plasma IL-2 and IL-10 in hens of MLP-LPS group were higher than those in hens of LPS-stimulation group (CN-LPS group) (P < 0.05). The expression levels of TLR2, MYD88, NF-κB, COX2, and TNF-α were significantly decreased in the hens fed diet supplemented with MLP and suffered with LPS stimulation (MLP-LPS group) compared with those in the hens of CN-LPS group (P < 0.05). Differentially expressed genes (DEGs) induced by MLP were involved in inflammation, reproduction, and calcium ion transport. At the genus level, the MLP supplementation significantly increased the abundance of Phascolarctobacterium, whereas decreased the abundance of Candidatus_Saccharimonas in LPS challenged hens (P < 0.05). The metabolites altered by dietary supplementation with MLP were mainly involved in galactose, uronic acid, histidine, pyruvate and primary bile acid metabolism. Dietary supplementation with MLP inversely regulates LPS-induced differential metabolites such as LysoPA (24:0/0:0) (P < 0.05).ConclusionsIn summary, dietary supplementation with microencapsulated Lactiplantibacillus plantarum prevented salpingitis by modulating the abundances of Candidatus_Saccharimonas, Phascolarctobacterium, Ruminococcus_torques_group and Eubacterium_hallii_group while downregulating the levels of plasma metabolites, p-tolyl sulfate, o-cresol and N-acetylhistamine and upregulating S-lactoylglutathione, simultaneously increasing the expressions of CPNE4, CNTN3 and ACAN genes in the uterus, and ultimately inhibiting oviducal inflammation.

Allografts for partial meniscus repair: an in vitro and ex vivo meniscus culture study.

The purpose of this study was to evaluate the treatment potential of a human-derived demineralized scaffold, Spongioflex® (SPX), in partial meniscal lesions by employing in vitro models. In the first step, the differentiation potential of human meniscal cells (MCs) was investigated. In the next step, the ability of SPX to accommodate and support the adherence and/or growth of MCs while maintaining their fibroblastic/chondrocytic properties was studied. Control scaffolds, including bovine collagen meniscus implant (CMI) and human meniscus allograft (M-Allo), were used for comparison purposes. In addition, the migration tendency of MCs from fresh donor meniscal tissue into SPX was investigated in an ex vivo model. The results showed that MCs cultured in osteogenic medium did not differentiate into osteogenic cells or form significant calcium phosphate deposits, although AP activity was relatively increased in these cells. Culturing cells on the scaffolds revealed increased viability on SPX compared to the other scaffold materials. Collagen I synthesis, assessed by ELISA, was similar in cells cultured in 2D and on SPX. MCs on micro-porous SPX (weight >0.5g/cm3) exhibited increased osteogenic differentiation indicated by upregulated expression of ALP and RUNX2, while also showing upregulated expression of the chondrogen-specific SOX9 and ACAN genes. Ingrowth of cells on SPX was observed after 28days of cultivation. Overall, the results suggest that SPX could be a promising biocompatible scaffold for meniscal regeneration.

Hyaluronic Acid-Based Injective Medical Devices: In Vitro Characterization of Novel Formulations Containing Biofermentative Unsulfated Chondroitin or Extractive Sulfated One with Cyclodextrins.

Currently, chondroitin sulfate (CS) and hyaluronic acid (HA) pharma-grade forms are used for osteoarthritis (OA) management, CS as an oral formulations component, and HA as intra-articular injective medical devices. Recently, unsulfated chondroitin, obtained through biofermentative (BC) manufacturing, has been proposed for thermally stabilized injective preparation with HA. This study aimed to highlight the specific properties of two commercial injective medical devices, one based on HA/BC complexes and the other containing HA, extractive CS, and cyclodextrins, in order to provide valuable information for joint disease treatments. Their biophysical and biomechanical features were assayed; in addition, biological tests were performed on human pathological chondrocytes. Rheological measurements displayed similar behavior, with a slightly higher G' for HA/BC, which also proved superior stability to the hyaluronidase attack. Both samples reduced the expression of specific OA-related biomarkers such as NF-kB, interleukin 6 (IL-6), and metalloprotease-13 (MMP-13). Moreover, HA/BC better ensured chondrocyte phenotype maintenance by up-regulating collagen type 2A1 (COLII) and aggrecan (AGN). Notwithstanding, the similarity of biomolecule components, the manufacturing process, raw materials characteristics, and specific concentration resulted in affecting the biomechanical and, more interestingly, the biochemical properties, suggesting potential better performances of HA/BC in joint disease treatment.

Clinical-genetic analysis of selected genes involved in the development of the human skeleton in 128 Czech patients with suspected congenital skeletal abnormalities

BackgroundCongenital skeletal abnormalities are a heterogeneous group of diseases most commonly associated with small or disproportionate growth, cranial and facial dysmorphisms, delayed bone maturation, etc. Nonetheless, no detailed genotype-phenotype correlation in patients with specific genetic variants is readily available. Ergo, this study focuses on the analysis of patient phenotypes with candidate variants in genes involved in bone growth as detected by molecular genetic analysis. MethodsIn this study we used molecular genetic methods to analyse the ACAN, COL2A1, FGFR3, IGFALS, IGF1, IGF1R, GHR, NPR2, STAT5B and SHOX genes in 128 Czech children with suspected congenital skeletal abnormalities. Pathogenic variants and variants of unclear clinical significance were identified and we compared their frequency in this study cohort to the European non-Finnish population. Furthermore, a prediction tool was utilised to determine their possible impact on the final protein. All clinical patient data was obtained during pre-test genetic counselling. ResultsPathogenic variants were identified in the FGFR3, GHR, COL2A1 and SHOX genes in a total of six patients. Furthermore, we identified 23 variants with unclear clinical significance and high allelic frequency in this cohort of patients with skeletal abnormalities. Five of them have not yet been reported in the scientific literature. ConclusionCongenital skeletal abnormalities may lead to a number of musculoskeletal, neurological, cardiovascular problems. Knowledge of specific pathogenic variants may help us in therapeutic procedures.

Altered Transcript Levels of MMP13 and VIT Genes in the Muscle and Connective Tissue of Pigs with Umbilical Hernia.

Umbilical hernia (UH) and inguinal hernia (IH) are among the most common defects in pigs, affecting their welfare and resulting in economic losses. In this study, we aimed to verify the association of previously reported differences in transcript levels of the ACAN, COL6A5, MMP13, and VIT genes with the occurrence of UH and IH. We examined mRNA levels in muscle and connective tissue from 68 animals-34 affected by UH and 34 controls. In a second cohort, we examined inguinal channel samples from 46 pigs (in four groups). We determined DNA methylation levels in muscle tissue for the UH and control animals. The transcript level of MMP13 changed in the UH cases, being upregulated and downregulated in muscle and connective tissue, respectively, and the VIT gene also showed an increased muscular mRNA level. The transcript of the ACAN gene significantly decreased in old pigs with IH. We further observed an increased DNA methylation level for one CpG site within the MMP13 gene in UH individuals. We conclude that these alterations in gene mRNA levels in the UH animals depend on the tissue and can sometimes be a consequence of, not a cause of, the affected phenotype.

Injectable hydrogel microspheres experimental research for the treatment of osteoarthritis

To prepare a novel hyaluronic acid methacrylate (HAMA) hydrogel microspheres loaded polyhedral oligomeric silsesquioxane-diclofenac sodium (POSS-DS) patricles, then investigate its physicochemical characteristics and in vitro and in vivo biological properties. Using sulfhydryl POSS (POSS-SH) as a nano-construction platform, polyethylene glycol and DS were chemically linked through the "click chemistry" method to construct functional nanoparticle POSS-DS. The composition was analyzed by nuclear magnetic resonance spectroscopy and the morphology was characterized by transmission electron microscopy. In order to achieve drug sustained release, POSS-DS was encapsulated in HAMA, and hybrid hydrogel microspheres were prepared by microfluidic technology, namely HAMA@POSS-DS. The morphology of the hybrid hydrogel microspheres was characterized by optical microscope and scanning electron microscope. The in vitro degradation and drug release efficiency were observed. Cell counting kit 8 (CCK-8) and live/dead staining were used to detect the effect on chondrocyte proliferation. Moreover, a chondrocyte inflammation model was constructed and cultured with HAMA@POSS-DS. The relevant inflammatory indicators, including collagen type Ⅱ, aggrecan (AGG), matrix metalloproteinase 13 (MMP-13), recombinant A disintegrin and metalloproteinase with thrombospondin 5 (Adamts5), and recombinant tachykinin precursor 1 (TAC1) were detected by immunofluorescence staining and real-time fluorescence quantitative PCR, with normal cultured chondrocytes and the chondrocyte inflammation model without treatment as control group and blank group respectively to further evaluate their anti-inflammatory activity. Finally, by constructing a rat model of knee osteoarthritis, the effectiveness of HAMA@POSS-DS on osteoarthritis was evaluated by X-ray film and Micro-CT examination. The overall particle size of POSS-DS nanoparticles was uniform with a diameter of about 100 nm. HAMA@POSS-DS hydrogel microspheres were opaque spheres with a diameter of about 100 μm and a spherical porous structure. The degradation period was 9 weeks, during which the loaded POSS-DS nanoparticles were slowly released. CCK-8 and live/dead staining showed no obvious cytotoxicity at HAMA@POSS-DS, and POSS-DS released by HAMA@POSS-DS significantly promoted cell proliferation (P<0.05). In the chondrocyte anti-inflammatory experiment, the relative expression of collagen type Ⅱ mRNA in HAMA@POSS-DS group was significantly higher than that in control group and blank group (P<0.05). The relative expression level of AGG mRNA was significantly higher than that of blank group (P<0.05). The relative expressions of MMP-13, Adamts5, and TAC1 mRNA in HAMA@POSS-DS group were significantly lower than those in blank group (P<0.05). In vivo experiments showed that the joint space width decreased after operation in rats with osteoarthritis, but HAMA@POSS-DS delayed the process of joint space narrowing and significantly improved the periarticular osteophytosis (P<0.05). HAMA@POSS-DS can effectively regulate the local inflammatory microenvironment and significantly promote chondrocyte proliferation, which is conducive to promoting cartilage regeneration and repair in osteoarthritis.

Expression of OLR1, ACAN, and LRP1 Genes in the Synovial Membrane Cells in Patients with Osteoarthritis after SARS-CoV2 Infection

Expression of OLR1, ACAN, and LRP1 Genes in the Synovial Membrane Cells in Patients with Osteoarthritis after SARS-CoV2 Infection

NADPH oxidase 4 deficiency attenuates experimental osteoarthritis in mice

ObjectiveLow-grade inflammation plays a pivotal role in osteoarthritis (OA) through exposure to reactive oxygen species (ROS). In chondrocytes, NADPH oxidase 4 (NOX4) is one of the major ROS producers. In...

Induced Chondrogenic Differentiation of hESCs by hESC-Derived MSCs Conditioned Medium and Sequential 3D-2D Culture System

Background and Aims: It has been proven that human mesenchymal stem cells (MSCs) conditioned medium (hMSCs-CM) can influence human embryonic stem cells (hESCs) chondrogenic differentiation. In this study, we hypothesized that conditioned medium (CM) from hESCs-derived MSCs in a sequential 3D-2D culture system could facilitate the induction of chondrogenesis in hESCs.&#x0D; Materials and Methods: CM was collected from Yazd2 (hESCs; 46, XY) derived MSCs confluent cultures and stored at -20 °C. Yazd4 hESC line (46, XX) was induced for differentiation using EB formation as 3D culture into SD (spontaneously differentiation) and CM groups (differentiation using conditioned medium) for four days. Cell culture continued in a 2D (monolayer) culture system for both groups till day 14. Ultimately, chondrogenic differentiation was assessed by Alcian blue and masson′s trichrome staining at 4 and 14 days of differentiation, and quantitative real-time polymerase chain reaction (PCR) for NANOG, MEOX1, SOX5, SOX6, SOX9, ACAN, COL2A1 and RUNX2 genes for SD and CM groups on days 0 and 4.&#x0D; Results: The gene expression profile for chondrogenic genes in the CM group was significantly more than the SD group (p&lt; 0.05). Furthermore, chemical staining assessment illustrated a significant GAG and collagen II difference between the CM and SD groups at days 4 and 14 (p&lt; 0.05).&#x0D; Conclusions: Our findings would pave the way for creating an in vitro human chondrogenesis model for further studies in the developmental biology of articular cartilage tissue, which lend itself to cell-based therapy to cure joint diseases such as osteoarthritis.

An Injectable Enzymatically Crosslinked and Mechanically Tunable Silk Fibroin/Chondroitin Sulfate Chondro‐Inductive Hydrogel

AbstractAn injectable hybrid hydrogel is synthesized, comprising silk fibroin (SF) and chondroitin sulfate (CS) through di‐tyrosine formation bond of SF chains. CS and SF are reported with excellent biocompatibility as tissue engineering scaffolds. Nonetheless, the rapid degradation rate of pure CS scaffolds presents a challenge to effectively recreate articular cartilage. As CS is one of the cartilage extracellular matrix (ECM) components, it has the potential to enhance the biological activity of SF‐based hydrogel in terms of cartilage repair. Therefore, altering the CS concentrations (i.e., 0 wt%, 0.25 wt%, 0.5 wt%, 1 wt%, and 2 wt%), which are interpenetrated between SF β‐sheets and chains, can potentially adjust the physical, chemical, and mechanical features of these hybrid hydrogels. The formation of β‐sheets by 30 days of immersion in de‐ionized (DI) water can improve the compression strength of the SF/CS hybrid hydrogels in comparison with the same SF/CS hybrid hydrogels in the dried state. Biological investigation and observation depicts proper cell attachment, proliferation and cell viability for C28/I2 cells. Gene expression of sex‐determining region YBox 9 (SOX9), Collagen II α1, and Aggrecan (AGG) exhibits positive C3H10T1/2 growth and expression of cartilage‐specific genes in the 0.25 wt% and 0.5 wt% SF/CS hydrogels.

Protective effects of equol on the cartilage and subchondral bone in ovariectomized rats with osteoarthritis.

Objective(s):This study aimed to determine the therapeutic effect of equol (EQ) on osteoporotic osteoarthritis (OP OA). Materials and Methods:Thirty-six 12-week-old female Sprague-Dawley rats were randomly divided into sham group, OP OA group, and EQ group (n=12). OP OA was induced by anterior cruciate ligament transection (ACLT) combined with ovariectomy (OVX). EQ was orally administrated (10 μg/g/day) after the operation for 12 weeks. The efficacy was evaluated by gross pathology and histopathologic evaluation. The underlying mechanism was investigated by immunohistochemical analysis, micro-computed tomography (micro-CT) scanning, and tartrate-resistant acid phosphatase (TRAP) staining. Results:EQ effectively retarded cartilage degeneration, decreased the levels of matrix metalloproteinases-13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), nuclear factor-kappa B P65 (NF-κB P65) and caspase-3, and increased the levels of collagen type II (Col-II), Col-I, aggrecan (AGG), and inhibitor of NF-κB α (IκBα) in the cartilage. In addition, EQ increased bone mineral density, improved the microstructural parameters of the subchondral bone (SB), and decreased the number of osteoclasts. Conclusion:EQ exerted a chondroprotective effect on OP OA in rats, associated with inhibition of the NF-κB signaling pathway and chondrocyte apoptosis. Furthermore, EQ showed an osteoprotective effect on SB via inhibiting osteoclastic activities.

Exploring and expanding the phenotype and genotype diversity in seven Chinese families with spondylo-epi-metaphyseal dysplasia.

Spondylo-epi-metaphyseal dysplasia (SEMD) is a heterogeneous group of disorders with different modes of inheritance and is characterized by disproportionate or proportionate short stature. To date, more than 30 disease-causing genes have been identified, and different types of SEMD exhibit greatly overlapping clinical features, which usually complicate the diagnosis. This study was performed to expand the clinical and molecular spectrum of SEMD among Chinese subjects and to explore their potential phenotype–genotype relations. We enrolled seven families including 11 affected patients with SEMD, and their clinical, radiographic, and genetic data were carefully analyzed. All the seven probands showed different degrees of short stature, and each of them exhibited additional specific skeletal manifestations; four probands had extraosseous manifestations. X-rays of the seven probands showed common features of SEMD, including vertebral deformities, irregular shape of the epiphysis, and disorganization of the metaphysis. Seven variants were identified in TRPV4 (c.694C> T, p.Arg232Cys), COL2A1 (c.654 + 1G > C; c.3266_3268del, p.Gly1089del), CCN6 (c.396 T> G, p.Cys132Trp; c.721 T>C, p.Cys241Arg), SBDS (c.258 + 2T> C), and ACAN (c.1508C> A, p.Thr503Lys) genes, and two of them were novel. Two families with TRPV4 variants showed considerable intrafamily and interfamily heterogeneities. In addition, we reported one case of SEMD with a severe phenotype caused by ACAN gene mutation. Our study expands the phenotype and genetic spectrum of SEMD and provides evidence for the phenotype–genotype relations, aiding future molecular and clinical diagnosis as well as procreative management of SEMD.

Intra-bone marrow injection of magnesium isoglyrrhizinate inhibits inflammation and delays osteoarthritis progression through the NF-κB pathway

ObjectiveOsteoarthritis (OA) presents cartilage damage in addition to chronic inflammation. However, self-recovery of damaged cartilage in an inflammatory environment is not possible. Mesenchymal stem cells (MSCs) in the bone marrow are a source of regenerative repair of damaged cartilage. To date, whether intra-luminal administration of the bone marrow can delay the progression of OA is still unknown. This study, therefore, aimed to explore the role of intra-bone marrow injection of Magnesium isoglycyrrhizinate (MgIG) in delaying the OA progression and to investigate the underlying mechanism.MethodsRabbit OA models were established using the anterior cruciate ligament transection method while a catheter was implanted into the bone marrow cavity. 1 week after surgery, MgIG treatment was started once a week for 4 weeks. The cartilage degradation was analyzed using hematoxylin–eosin staining, Masson’s trichrome staining and Alcian blue staining. Additionally, the pro-inflammatory factors and cartilage regeneration genes involved in the cartilage degeneration and the underlying mechanisms in OA were detected using enzyme-linked immunosorbent assay, quantitative real-time PCR (qRT-PCR) and Western blotting.ResultsThe results of histological staining revealed that intra-bone marrow injection of MgIG reduced degeneration and erosion of articular cartilage, substantially reducing the Osteoarthritis Research Society International scores. Furthermore, the productions of inflammatory cytokines in the bone marrow cavity and articular cavity such as interleukin-1β(IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) were inhibited upon the treatment of MgIG. At the same time, the expression of alkaline phosphate, tartrate-resistant acid phosphatase-5b (TRAP-5b) and C-telopeptides of type II collagen (CTX-II) in the blood also decreased and was positively correlated. On the contrary, cartilage-related genes in the bone marrow cavity such as type II collagen (Col II), Aggrecan (AGN), and SRY-box 9 (SOX9) were up-regulated, while matrix metalloproteinase-3 (MMP-3) was down-regulated. Mechanistically, MgIG was found to exert an anti-inflammatory effect and impart protection to the cartilage by inhibiting the NF-κB pathway.ConclusionIntra-bone marrow injection of MgIG might inhibit the activation of the NF-κB pathway in the progression of OA to exert an anti-inflammatory effect in the bone marrow cavity and articular cavity, thereby promoting cartilage regeneration of MSCs in the bone marrow, making it a potential new therapeutic intervention for the treatment of OA.

First study on the effect of transforming growth factor beta 1 and insulin-like growth factor 1 on the chondrogenesis of elephant articular chondrocytes in a scaffold-based 3D culture model.

Background and Aim:Osteoarthritis (OA) is recognized as a degenerative joint disease that leads to chronic pain and low quality of life in animals. Captive elephants, the largest land mammals with a long lifespan, are more prone to develop OA due to restricted spaces and insufficient physical activity. This study aimed to investigate the effect of transforming growth factor-β1 (TGF-β1) and insulin-like growth factor 1 (IGF-1) on elephant chondrogenesis in a scaffold culture of articular chondrocytes.Materials and Methods:Elephant chondrocytes-seeded gelatin scaffolds were cultured in chondrogenic media with or without 10 ng/mL of TGF-β1 or IGF-1 alone or 5–10 ng/mL of their combination for up to 21 days. The mRNA expression of cartilage-specific anabolic genes, ACAN and COL2A1, was analyzed using a real-time reverse transcription-polymerase chain reaction. The amounts of sulfated glycosaminoglycans (sGAGs) in conditioned media and contents in cultured scaffolds were determined through dimethylmethylene blue assay. Cell morphology, accumulation of proteoglycans, and details of the cultured scaffolds were determined using hematoxylin-eosin staining, safranin O staining, and scanning electron microscopy (SEM), respectively.Results:TGF-β1 alone significantly upregulated ACAN gene expression but not COL2A1, while IGF-1 alone did not enhance both ACAN and COL2A1 genes. The combination significantly upregulated both mRNA expression levels of ACAN and COL2A1 gene at day 14. The sGAGs accumulation and contents in the treatment groups, except IGF-1 tended to be higher than the controls, concomitantly with the production of the extracellular matrix, showed the formation of a cartilage-like tissue through histological and SEM analyses.Conclusion:Together, our results suggest that the single treatment of TGF-β1 has a selective effect on ACAN gene, while the combined growth factors seem to be an advantage on elephant chondrogenesis. This three-dimensional culture model is probably helpful for developing cartilage regeneration in vitro and is further applied in tissue engineering for OA treatment in vivo.