Chondrocyte Cell Research Articles

Development and characterization of an in vitro fluorescently tagged 3D bone-cartilage interface model

Three-dimensional cultures are widely used to study bone and cartilage. These models often focus on the interaction between osteoblasts and osteoclasts or osteoblasts and chondrocytes. A culture of osteoblasts, osteoclasts and chondrocytes would represent the cells that interact in the joint and a model with these cells could be used to study many diseases that affect the joints. The goal of this study was to develop 3D bone-cartilage interface (3D-BCI) that included osteoblasts, osteocytes, osteoclasts, and cartilage. Fluorescently tagged cell lines were developed to assess the interactions as cells differentiate to form bone and cartilage. Mouse cell line, MC3T3, was labeled with a nuclear GFP tag and differentiated into osteoblasts and osteocytes in Matrigel. Raw264.7 cells transfected with a red cytoplasmic tag were added to the system and differentiated with the MC3T3 cells to form osteoclasts. A new method was developed to differentiate chondrocyte cell line ATDC5 in a cartilage spheroid, and the ATDC5 spheroid was added to the MC3T3 and Raw264.7 cell model. We used an Incucyte and functional analysis to assess the cells throughout the differentiation process. The 3D-BCI model was found to be positive for TRAP, ALP, Alizarin red and Alcian blue staining to confirm osteoblastogenesis, osteoclastogenesis, and cartilage formation. Gene expression confirmed differentiation of cells based on increased expression of osteoblast markers: Alpl, Bglap, Col1A2, and Runx2, cartilage markers: Acan, Col2A1, Plod2, and osteoclast markers: Acp5, Rank and Ctsk. Based on staining, protein expression and gene expression results, we conclude that we successfully developed a mouse model with a 3D bone-cartilage interface.

Integrin α2β1 deficiency enhances osteogenesis via BMP-2 signaling for accelerated fracture repair

Previous studies have shown that the absence of the collagen-binding integrin α2β1 confers protection against osteoporosis, primarily by enhancing osteoblast-mediated matrix formation, with a particular increase in collagen type I production. This study aimed to elucidate the mechanism underlying this increased matrix production. Our findings demonstrate that osteoblasts lacking integrin α2 secrete a pro-osteogenic factor that activates both TGF-β and BMP signaling pathways. Among these, BMP-2 was identified as the key signaling protein responsible for this effect, as its expression was significantly upregulated during osteoblast differentiation. Moreover, integrin α2 deficiency led to earlier and elevated BMP-2 secretion at the cell surface during osteogenesis, which promoted accelerated osteoblast differentiation. This phenomenon likely contributes to enhanced matrix production in aging animals, providing a protective effect against osteoporosis.To explore the broader implications of this phenotype, we utilized a fracture healing model. In integrin α2-deficient 12 weeks old female mice, elevated serum levels of BMP-2 were detected during the early stages of fracture repair. This upregulation of BMP signaling within the fracture callus accelerated the healing process, resulting in faster formation and mineralization of the cartilaginous callus. Additionally, the elevated BMP-2 levels facilitated earlier differentiation of chondrocytic cells, evidenced by the premature appearance of collagen type II- and type X-positive cells during endochondral ossification. Despite the accelerated healing, the overall biomechanical integrity of the repaired fractures remained uncompromised.Thus, the modulation of integrin α2β1 presents a promising therapeutic target for enhancing fracture repair by regulating BMP-2 signaling in a physiologically relevant manner.

Chemofunctionalization of knitted silk to improve interface connection in a nano/micro scaffold based on polycaprolactone-chitosan-multi-walled carbon nanotube/silk

Nano/micro hybrid scaffolds in long-term healing tissue engineering can simultaneously offer both mechanical and biological properties. In this study, a hybrid scaffold was fabricated through electrospinning of polycaprolactone (PCL)-chitosan (Cs)/ multi-walled carbon nanotubes (MWCNTs) based nanofibers onto a chemically functionalized knitted silk substrate (F-Silk) and the scaffold were evaluated with regard to morphology, chemical and crystalline structure, hydrophilicity, mechanical properties, bioactivity, biodegradability, and cellular behavior. Chemical functionalization of silk using N-hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) resulted in greater integrity in the formation of nanofibers onto the microfibers. The presence of MWCNTs significantly reduced the contact angle of the scaffolds from 79.72° ± 2.72 to 68.92° ± 5.63. Chemical functionalization of silk, the presence of nanofiber coating, and the presence of MWCNTs increased the ultimate tensile strength of the hybrid scaffolds by 18 %, 20 %, and 30 % compared to raw silk fabric, respectively. The presence of MWCNTs and chemical functionalization of knitted silk increased the bioactivity and reduced the degradation rate of hybrid scaffolds. The increase in the amount of carboxyl groups as a result of adding 0.5 wt% of MWCNTs significantly improved the adhesion, growth and proliferation of chondrocyte cells on the hybrid scaffolds as observed through cell morphology. According to the obtained results, hybrid scaffold based on PCL-Cs-MWCNTs/F-silk can be a suitable option for further research in cartilage tissue engineering.

TNFα-Related Chondrocyte Inflammation Models: A Systematic Review.

Tumor necrosis factor alpha (TNFα), as a key pro-inflammatory cytokine, plays a central role in joint diseases. In recent years, numerous models of TNFα-induced cartilage inflammation have been developed. However, due to the significant differences between these models and the lack of consensus in their construction, it becomes difficult to compare the results of different studies. Therefore, we summarized and compared these models based on important parameters for model construction, such as cell source, cytokine concentration, stimulation time, mechanical stimulation, and more. We attempted to analyze the advantages and disadvantages of each model and provide a compilation of the analytical methods used in previous studies. Currently, TNFα chondrocyte inflammation models can be categorized into four main types: monolayer-based, construct-based, explant-based TNFα chondrocyte inflammation models, and miscellaneous TNFα chondrocyte inflammation models. The most commonly used models were the monolayer-based TNFα chondrocyte inflammation models (42.86% of cases), with 10 ng/mL TNFα being the most frequently used concentration. The most frequently used chondrocyte cell passage is passage 1 (50%). Human tissues were most frequently used in experiments (51.43%). Only five articles included models with mechanical stimulations. We observed variations in design conditions between different models. This systematic review provides the essential experimental characteristics of the available chondrocyte inflammation models with TNFα, and it provides a platform for better comparison between existing and new studies in this field. It is essential to perform further experiments to standardize each model and to find the most appropriate experimental parameters.

8519 Investigating the Role of Protein Inhibitor of Activated STAT1 (Pias1) in Growth Plate Chondrocyte Maturation

Abstract Disclosure: J. Baronas: None. U. Ahmed: None. J.N. Hirschhorn: None. N.E. Renthal: None. Development of growth plate chondrocytes is a complex process that involves a wide array of regulatory elements. Recently our laboratory conducted a genome wide CRISPR knockout (KO) screen of growth plate chondrocyte maturation to discover novel genes and gene networks in the proliferation and maturation of chondrocytes. Among targets uncovered is Protein Inhibitor of Activated STAT 1 (Pias1), an E3 SUMO ligase that plays a multi-faceted role in genetic regulation, driving post-translational modifications, directly binding to transcription factors, and functioning as a DNA-binding protein. While Pias1 has been shown to be involved in the regulation of many cellular processes across multiple tissues, its function in growth plate development and chondrocyte maturation has been previously unstudied. In our genome-wide screen, our laboratory identified that loss of Pias1 led to early maturation of chondrocytes. This project seeks to investigate the role of Pias1 in chondrocyte maturation and differentiation. We first developed a Pias1-KO chondrocyte cell line, observing upregulation of hypertrophic markers Cd200 and Col10a1 in KOs relative to control via qPCR, in addition to proliferative regulators IHH and Pth1R. Through Bulk-RNAseq of the same cell line, we identified additional chondrocyte markers as upregulated in KO, as well as osteoblast marker Sp7. Members of the HoxD family, regulators of limb development, were downregulated. Cell culture data suggests that loss of Pias1 leads to increased cell proliferation and increased matrix production, indicating dysregulation of multiple chondrocyte processes. Immunohistochemistry of micromass culture sections also suggests morphological differences between KO and control, with KO cells significantly larger in diameter. Early SUMO-1 profiling yields limited evidence of decreased global SUMOylation in KO cells, one possible mechanistic explanation for the observed dysregulation. Ongoing studies are focused on identifying specific SUMOylation targets of Pias1 in chondrocytes, analyzing proteomic differences in KO and control, seeking putative DNA-binding sites of Pias1, and observing in vivo murine Pias1-KO growth plates. Our findings suggest that PIAS1 plays a crucial role in the growth plate by prolonging growth and delaying terminal hypertrophy. Our study aims to provide a better understanding of the regulatory pathways involved in growth plate development and related phenotypes such as height, and contribute to the development of novel therapies for growth-related disorders, such as achondroplasia and hypochondroplasia. Presentation: 6/1/2024

Analysis of Patients Who Undergo Index Arthroscopy With Biopsy but Not Implantation for Staged Chondrocyte Cell Transplantation.

Autologous chondrocyte implantation (ACI) and matrix-induced autologous chondrocyte implantation (MACI) are 2-stage procedures requiring an index full-thickness cartilage biopsy. Only a portion of patients ultimately undergo second-stage ACI/MACI. To identify patients with articular cartilage defects who underwent arthroscopic debridement with biopsy for ACI/MACI and compare those who did with those who did not proceed with implantation within 2 years after biopsy. Additionally, the authors sought to identify why patients did not proceed with implantation. Case-control study; Level of evidence, 3. Patients who underwent arthroscopy and autologous chondrocyte biopsy from January 1, 2015, to December 31, 2019, and who had minimum 2-year follow-up data were grouped into those who proceeded with second-stage ACI/MACI (implant group; n = 97) and those who did not (biopsy group; n = 63). Demographic factors, cartilage defect characteristics, and preoperative International Knee Documentation Committee (IKDC) scores were analyzed. Patients in both groups were evaluated postoperatively using the IKDC, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Single Assessment Numeric Evaluation (SANE), and visual analog scale (VAS) for pain, and patients who did not undergo implantation were asked for their reasoning. Body mass index (BMI) (P < .001) and Outerbridge grades at index arthroscopy (P = .047) were significantly higher for the implant group than the biopsy group. Both groups had significantly improved IKDC scores from their initial presentation to final follow-up (implant group: 46.4 ± 16.2 preoperative vs 69.6 ± 20.6 postoperative [P < .001]; biopsy group: 47.2 ± 15.9 preoperative vs 70.7 ± 19.1 postoperative [P < .001]); however, the level of improvement did not differ significantly between groups. Postoperative WOMAC, SANE, and VAS pain scores were also similar between groups. In the biopsy group, 23 patients (37%) cited symptom resolution or activity level improvement after initial arthroscopy as the reason for not proceeding with implantation. Patients who proceeded to the second stage of chondrocyte implantation via either ACI or MACI had higher-grade articular defects and higher BMI compared with those who underwent biopsy with concomitant debridement chondroplasty alone. Postoperative outcomes were similar between the groups.

IN VITRO PROLIFERATIVE EFFICACY OF AN INNOVATIVE NATURAL CALCIUM FORMULATION IN BONE AND MUSCLE CELLS

Calcium is an essential macronutrient with a critical role in structural, functional and metabolic processes, particularly bone and muscle systems. Although dietary intake typically meets calcium needs, uneven food habits, anti-nutrients and various pathophysiological conditions can lead to deficiencies. Addressing this gap, Dabur India Limited developed an Innovative Natural Calcium Formulation (INCF) featuring natural organic calcium, plant-derived vitamin D3 and lemon juice. Two dosage forms providing 250 mg and 500 mg of calcium were developed and tested for efficacy using in vitro proliferation of Chondrocyte (C20A4 - Bone) and Muscle (L6 rat skeletal) cells at noncytotoxic concentrations. Results showed chondrocyte proliferation of 9.9% - 24.4% and skeletal muscle proliferation of 10.94% - 28.46% compared to untreated control. These findings indicate the potential of INCF as an effective calcium supplement.

Mitomycin C as an Anti-Persister Strategy against Klebsiella pneumoniae: Toxicity and Synergy Studies.

The combination of several therapeutic strategies is often seen as a good way to decrease resistance rates, since bacteria can more easily overcome single-drug treatments than multi-drug ones. This strategy is especially attractive when several targets and subpopulations are affected, as it is the case of Klebsiella pneumoniae persister cells, a subpopulation of bacteria able to transiently survive antibiotic exposures. This work aims to evaluate the potential of a repurposed anticancer drug, mitomycin C, combined with the K. pneumoniae lytic phage vB_KpnM-VAC13 in vitro and its safety in an in vivo murine model against two clinical isolates of this pathogen, one of them exhibiting an imipenem-persister phenotype. At the same time, we verified the absence of toxicity of mitomycin C at the concentration using the human chondrocyte cell line T/C28a2. The viability of these human cells was checked using both cytotoxicity assays and flow cytometry.

Radiosensitizing Effect of PARP Inhibition on Chondrosarcoma and Chondrocyte Cells Is Dependent on Radiation LET.

Chondrosarcoma is a rare malignant tumor that forms in bone and cartilage. The primary treatment involves surgical removal of the tumor with a margin of healthy tissue. Especially if complete surgical removal is not possible, radiation therapy and chemotherapy are used in conjunction with surgery, but with a generally low efficiency. Ongoing researches are focused on understanding the genetic and molecular basis of chondrosarcoma following high linear energy transfer (LET) irradiation, which may lead to treatments that are more effective. The goal of this study is to evaluate the differential effects of DNA damage repair inhibitors and high LET irradiation on chondrosarcoma versus chondrocyte cells and the LET-dependency of the effects. Two chondrosarcoma cell lines with different IDH mutation status and one chondrocyte cell line were exposed to low LET (X-ray) and high LET (carbon ion) irradiation in combination with an Olaparib PARP inhibitor. Cell survival and DNA repair mechanisms were investigated. High LET irradiation drastically reduced cell survival, with a biological efficiency three times that of low LET. Olaparib significantly inhibited PARylation in all the tested cells. A significant reduction in cell survival of both chondrosarcoma and chondrocyte cells was observed following the treatment combining Olaparib and X-ray. PARP inhibition induced an increase in PARP-1 expression and a reduced effect on the cell survival of WT IDH chondrosarcoma cells. No radiosensitizing effect was observed in cells exposed to Olaparib paired with high LET irradiation. NHEJ was activated in response to high LET irradiation, neutralizing the PARP inhibition effect in both chondrosarcoma cell lines. When high LET irradiation is not available, PARP inhibition could be used in combination with low LET irradiation, with significant radiosensitizing effects on chondrosarcoma cells. Chondrocytes may be affected by the treatment combination too, showing the need to preserve normal tissues from radiation fields when this kind of treatment is suggested.

Hyaluronic acid-alginate hydrogel stimulates the differentiation of neonatal mouse testicular cells into hepatocyte-like and other cell lineages in three-dimensional culture.

Extracellular matrix (ECM)-derived hydrogels are frequently used in three-dimensional (3D) cell culture and organoid formation in several tissues. However, in the 3D cultivation of testicular cells, the hyaluronic acid (HA) hydrogel has not received as much attention. This study examined the effects of three distinct composites, including HA-alginate (HA-Alg), HA-alginate-collagen (HA-Alg-Col), and HA-alginate-decellularized ECM (HA-Alg-dECM), on mouse testicular cell culture and in vitro spermatogenesis. For the creation of composites, the concentration of biomaterials used was 0.5% HA, 1% alginate, 2.5mg/mL collagen, and 25mg/mL dECM derived from the testicles of Rams. After 3D culture of 5 days post-partum (dpp) mouse testicular cells for 14 days, HA-Alg was selected as a superior composite due to the greater number and size of the produced organoids. Then, cell culture was rerun by HA-Alg for 14 days, which was later extended for an additional 28 days. In addition, the 3D culture of 10 dpp mouse testicular cells was used to compare with 5 dpp mice on day 14. The morphology and gene expression were analyzed using appropriate techniques. On day 14, the HA-Alg hydrogel showed significantly more organoids in terms of size and number than the other two groups (p<0.05); nevertheless, none of the groups showed the expected signs of testis organoids. Remarkably, on day 14, the histology and immunostaining tests revealed features of hepatocyte-like cells (HLCs) and albumin production as a marker of HLC functionality. Furthermore, the analysis of gene expression verified the significant expression of angiogenesis markers (p<0.01). After the extended culture to 28 days, 5 dpp testicular cells once more differentiated into erythrocytes and HLCs, while a small number of organoids showed the characteristic of renal cells. Cell culture of 10 dpp mice for 14 days showed a wide range of cell lineages, including renal, glandular, chondrocyte, and hepatocyte-like cells in comparison to the 5 dpp mice. While the HA-Alg composite did not support spermatogenesis in the 3D culture of mouse testicular cells, it demonstrated an unpredicted potential for promoting the differentiation of neonate mouse testicular cells into HLC, erythrocytes, and other cell lineages.

Hyaluronic Acid-Based Microparticles with Lubrication and Anti-Inflammation for Alleviating Temporomandibular Joint Osteoarthritis.

The pathogenesis of temporomandibular joint osteoarthritis (TMJOA) is closely associated with mechanical friction, which leads to the up-regulation of inflammatory mediators and the degradation of articular cartilage. Injectable drug-loaded microparticles have attracted widespread interest in intra-articular treatment of TMJOA by providing lubrication and facilitating localized drug delivery. Herein, a hyaluronic acid-based microparticle is developed with excellent lubrication properties, drug loading capacity, antioxidant activity, and anti-inflammatory effect for the treatment of TMJOA. The microparticles are facilely prepared by the self-assembly of 3-aminophenylboronic acid-modified hyaluronic acid (HP) through hydrophobic interaction in an aqueous solution, which can further encapsulate diol-containing drugs through dynamic boronate ester bonds. The resulting microparticles demonstrate excellent injectability, lubrication properties, radical scavenging efficiency, and antibacterial activity. Additionally, the drug-loaded microparticles exhibit a favorable cytoprotective effect on chondrocyte cells invitro under an oxidative stress microenvironment. Invivo experiments validate that intra-articular injection of drug-loaded microparticles effectively alleviates osteoporosis-like damage, suppresses inflammatory response, and facilitates matrix regeneration in the treatment of TMJOA. The HP microparticles demonstrate excellent injectability and encapsulation capacity for diol-containing drugs, highlighting its potential as a versatile drug delivery vehicle in the intra-articular treatment of TMJOA.

Electrochemical and Fluorescence MnO2-Polymer Dot Electrode Sensor for Osteoarthritis-Based Peroxisomal β-Oxidation Knockout Model.

A coenzyme A (CoA-SH)-responsive dual electrochemical and fluorescence-based sensor was designed utilizing an MnO2-immobilized-polymer-dot (MnO2@D-PD)-coated electrode for the sensitive detection of osteoarthritis (OA) in a peroxisomal β-oxidation knockout model. The CoA-SH-responsive MnO2@D-PD-coated electrode interacted sensitively with CoA-SH in OA chondrocytes, triggering electroconductivity and fluorescence changes due to cleavage of the MnO2 nanosheet on the electrode. The MnO2@D-PD-coated electrode can detect CoA-SH in immature articular chondrocyte primary cells, as indicated by the significant increase in resistance in the control medium (R24h = 2.17 MΩ). This sensor also sensitively monitored the increase in resistance in chondrocyte cells in the presence of acetyl-CoA inducers, such as phytol (Phy) and sodium acetate (SA), in the medium (R24h = 2.67, 3.08 MΩ, respectively), compared to that in the control medium, demonstrating the detection efficiency of the sensor towards the increase in the CoA-SH concentration. Furthermore, fluorescence recovery was observed owing to MnO2 cleavage, particularly in the Phy- and SA-supplemented media. The transcription levels of OA-related anabolic (Acan) and catabolic factors (Adamts5) in chondrocytes also confirmed the interaction between CoA-SH and the MnO2@D-PD-coated electrode. Additionally, electrode integration with a wireless sensing system provides inline monitoring via a smartphone, which can potentially be used for rapid and sensitive OA diagnosis.

Auricular cartilage regeneration using chondroitin sulfate-based hydrogel with mesenchymal stem cells in rabbits.

Cartilage is an avascular and alymphatic tissue that lacks the intrinsic ability to undergo spontaneous repair and regeneration in the event of significant injury. The efficacy of conventional therapies for invasive cartilage injuries is limited, thereby prompting the emergence of cartilage tissue engineering as a possible alternative. In this study, we fabricated three-dimensional hydrogel films utilizing sodium alginate (SA), gelatin (Gel), and chondroitin sulfate (CS). These films were included with Wharton's jelly mesenchymal stem cells (WJ-MSCs) and intended for cartilage tissue regeneration. The hydrogel film that were prepared underwent evaluation using various techniques including scanning electron microscope (SEM), Fourier transform infrared (FTIR) spectroscopy, assessment of the degree of swelling, degradation analysis, determination of water vapor transmission rate (WVTR), measurement of water contact angle (WCA), evaluation of mechanical strength, and assessment of biocompatibility. The rabbit ear cartilage regeneration by hydrogel films with and without of WJ-MSCs was studied by histopathological investigations during 15, 30, and 60 days. The hydrogel films containing CS exhibited superior metrics compared to other nanocomposites such as better mechanical strength (12.87 MPa in SA/Gel compared to 15.56 in SA/Gel/CS), stability, hydrophilicity, WVTR (3103.33 g/m2/day in SA/Gel compared to 2646.67 in nanocomposites containing CS), and swelling ratio (6.97 to 12.11% in SA/Gel composite compared to 5.03 to 10.90% in SA/Gel/CS). Histopathological studies showed the presence of chondrocyte cells in the lacunae on the 30th day and the complete restoration of the cartilage tissue on the 60th day following the injury in the group of SA/Gel/CS hydrogel containing WJ-MSCs. We successfully fabricated a scaffold composed of alginate, gelatin, and chondroitin sulfate. This scaffold was further enhanced by the incorporation of Wharton's jelly mesenchymal stem cells. Our findings demonstrate that this composite scaffold has remarkable biocompatibility and mechanical characteristics. The present study successfully demonstrated the therapeutic potential of the SA-Gel-CS hydrogel containing WJ-MSCs for cartilage regeneration in rabbits.

The iPSC secretome is beneficial for in vitro propagation of primary osteoarthritic chondrocytes cell lines

BackgroundOne of the obstacles to autologous chondrocyte implantation (ACI) is obtaining a large quantity of chondrocytes without depletion of their properties. The conditioned medium (CM) from different subpopulations of stem cells (mesenchymal stromal cells (MSC) or induced pluripotent stem cells (iPSC)) could be a gamechanger. MSCs' potential is related to the donor's health and age, which could be omitted when, as a source, iPSCs are used. There is a lack of data regarding their use in the chondrocyte culture expansion. Thus, we wanted to verify whether iPSC-CM could be beneficial for the cell culture of primary chondrocyte cells. MethodsWe added the iPSC-CMs from GPCCi001-A and ND 41658*H cells to the culture of primary chondrocyte cell lines isolated from OA patients (n = 6) for other two passages. The composition of the CM was evaluated using Luminex technology. Then, we analysed the senescence, proliferation rate and using flow cytometry: viability, distribution of cell cycle phases, production of reactive oxygen species (ROS) and double-strand breaks. The cartilage-related markers were evaluated using Western blot and immunofluorescence. Additionally, a three-dimensional cell culture was used to determine the potential to form cartilage particles. ResultsiPSC-CM increased proliferation and diminished cell ROS production and senescence. CM influenced the cartilage-related protein expression and promoted the growth of cartilage particles. The cell exposed to CM did not lose the ECM proteins, suggesting the chondroprotective effect for prolonged culture time. ConclusionOur preliminary results suggest a beneficial effect on maintaining chondrocyte biology during in vitro expansion.

Sargassum polysaccharide attenuates osteoarthritis in rats and is associated with the up-regulation of the ITGβ1-PI3K-AKT signaling pathway

BackgroundOsteoarthritis (OA) presents a formidable challenge, characterized by as-yet-unclear mechanical intricacies within cartilage and the dysregulation of bone homeostasis. Our preliminary data revealed the encouraging potential of a Sargassum polysaccharide (SP), in promoting chondrogenesis. The aim of our study is to comprehensively assess the therapeutic effects of SP on OA models and further elucidate its potential mechanism. MethodsThe protective effects of SP were initially evaluated in an inflammation-induced human chondrocyte (C28) cell model. CCK-8 assays, Alcian blue staining, RT-qPCR and Western blotting were used to verify the chondrogenesis of SP in vitro. To assess the efficacy of SP in vivo, surgically induced medial meniscus destabilization (DMM) OA rats underwent an 8-week SP treatment. The therapeutic effects of SP in OA rats were comprehensively evaluated using X-ray imaging, micro-computed tomography (μ-CT), histopathological analysis, as well as immunohistochemical and immunofluorescent staining. Following these assessments, we delved into the potential signaling pathways of SP in inflammatory chondrocytes utilizing RNA-seq analysis. Validation of these findings was conducted through RT-qPCR and western blotting techniques. ResultsSP significantly enhance the viability of C28 chondrocytes, and increased the secretion of acidic glycoproteins. Moreover, SP stimulated the expression of chondrogenic genes (Aggrecan, Sox9, Col2a1) and facilitated the synthesis of Collagen II protein in C28 inflammatory chondrocytes. In vivo experiments revealed that SP markedly ameliorated knee joint stenosis, alleviated bone and cartilage injuries, and reduced the histopathological scores in the OA rats. μ-CT analysis confirmed that SP lessened bone impairments in the medial femoral condyle and the subchondral bone of the tibial plateau, significantly improving the microarchitectural parameters of the subchondral bone. Histopathological analyses indicated that SP notably enhanced cartilage quality on the surface of the tibial plateau, leading to increased cartilage thickness and area. Immunohistochemistry staining and immunofluorescence staining corroborated these findings by showing a significant promotion of Collagen II expression in OA joints treated with SP. RNA-seq analysis suggest that SP's effects were mediated through the regulation of the ITGβ1-PI3K-AKT signaling axis, thereby stimulating chondrogenesis. Verification through RT-qPCR and Western blot analyses confirmed that SP significantly upregulated the expression of ITGβ1, p110δ, AKT1, ACAN, and Col2a1. Notably, knock-down of ITGβ1 using siRNA in C28 chondrocytes inhibited the expression of ITGβ1, p110δ, AKT1, and ACAN. However, these inhibitory effects were not completely reversed by supplemental SP intervention. ConclusionsIn summary, our findings reveal that SP significantly enhances chondrogenesis both in vitro and in vivo, alleviating OA progression both in bone and cartilage. The observed beneficial effects are intricately linked to the activation of the ITGβ1-PI3K-AKT signaling axis. The translational potential of this articleOur research marks the first instance unveiling the advantageous effects and underlying mechanisms of SP in OA treatment. With its clinical prospects, SP presents compelling new evidence for the advancement of a next-generation polysaccharide drug for OA therapy.

Extracellular matrix coated three-dimensional-printed polycaprolactone scaffolds containing curcumin for cartilage tissue engineering applications

Extracellular matrix (ECM) is widely used for clinical purposes in tissue engineering (TE). Since ECM does not have favorable mechanical properties, its use has been limited. Therefore, it is helpful to use synthetic polymers such as polycaprolactone (PCL) to improve the mechanical properties of ECM-based scaffolds. PCL scaffolds were prepared via the three-dimensional (3D) printing method. Cartilaginous ECM was obtained from bovine femur, and then it was decellularized and solubilized. PCL scaffolds were functionalized using 1,6-hexanediamine; consequently, the scaffolds were coated with solubilized decellularized ECM (SDECM) using two types of crosslinkers; namely, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) and genipin. Genipin-crosslinked SDECM-coated (PCL/ECM-Gen) scaffolds and EDC/NHS-crosslinked SDECM-coated (PCL/ECM-EN) scaffolds were characterized by different tests. Following loading the curcumin (Cur) on the scaffolds, the Cur release rate was investigated. Finally, human chondrocyte cells were cultured on the scaffolds to explore cell viability, cell attachment, and histological studies. Following functionalization via amine groups, 5% SDECM was used to coat the scaffolds, and this increased the wettability and cell viability of the PCL. Genipin crosslinked and coated the SDECM more efficiently compared to the EDC/NHS and led to lower porosity, water absorption capacity, degradation rate, higher cell proliferation, and cell attachment. Genipin-crosslinked Cur-loaded (PCL/ECM-Gen + Cur) scaffolds showed higher cell viability but lower antibacterial activity compared to EDC/NHS-crosslinked Cur-loaded (PCL/ECM-EN + Cur) scaffolds, which may indicate EDC/NHS-induced cytotoxicity. This study elucidates the value of PCL/ECM-Gen + Cur scaffolds as highly biocompatible scaffolds that can be considered a promising tool for cartilage TE applications.

Leveraging single cell multiomic analyses to identify factors that drive human chondrocyte cell fate.

The identity and integrity of the articular cartilage lining our joints are crucial to pain-free activities of daily living. Here we identified a gene regulatory landscape of human chondrogenesis at single cell resolution, which is expected to open new avenues of research aimed at mitigating cartilage diseases that affect hundreds of millions of individuals world-wide.

Evaluation of the effects of decellularized umbilical cord Wharton's Jelly ECM on polyhydroxy butyrate electrospun scaffolds: A new strategy for cartilage tissue engineering

The field of regenerative medicine has recently witnessed the emergence of the allogeneic decellularized extracellular matrix as a scaffold with natural biological signals in tissue engineering. In the present study, to prevent immune rejection and disease transmission, Wharton's Jelly extracellular matrix (WJM) derived from the human umbilical cord was first decellularized (DWJM). Eventually, the fibrous scaffolds based on poly 3-hydroxybutyrate (PHB) and five different concentrations (0, 1, 3, 5, and 10 wt%) of DWJM were fabricated through electrospinning. The results demonstrate reducing the diameter of the fibers, improved mechanical properties, hydrophilicity, and degradation rate in the scaffold containing 3 wt % DWJM. Excellent biocompatibility and increased chondrocyte cell adherence rate on the substrate of PHB-DWJM 3 % scaffolds compared with pure PHB scaffolds can be attributed to the high amounts of Collagen, sulfated glycosaminoglycans (GAGs), and hyaluronic acid (HA) in the DWJM. In conclusion, the physicochemical properties and biological activities of PHB-DWJM scaffolds indicate that blending DWJM with PHB electrospun scaffold can provide a suitable environment for articular cartilage tissue regeneration.

Intra-articular administration of extra-virgin olive oil in degenerative osteoarthritis

BackgroundWe aimed to analyze the outcomes of intraarticular extra virgin olive oil (EVOO) injection on mechanically induced rabbit knee osteoarthritis (OA) by studying the morphological, histological, and radiological findings.MethodsThe study was conducted on 32 New Zealand White rabbits. The randomly numbered subjects were divided into two main groups. The rabbits numbered 1 to 16 were selected to be the group to receive EVOO, and the remaining were selected into a control group. Both groups were separated into two subgroups for short-term (five weeks) and long-term (10 weeks) follow-up. Anterior cruciate ligament transection was applied on the left knees of all the rabbits via medial parapatellar arthrotomy to simulate knee instability. Immediately after the surgical procedure, 0.2 cc of EVOO was injected into the knee joint of rabbits numbered 1–16, and the control group received 0.2 cc of sterile saline. On the 14th day, long-term group subjects were administered another dose of 0.2 cc EVOO intraarticularly.ResultsThe gross morphological scores of the control group subjects were significantly different from the EVOO group for both short-term (p = 0,055) and long-term (p = 0,041) scores. In parallel, the MRI results of the EVOO subjects were significantly different from the control group for both short-term and long-term follow-up assessment scores (p = 0.017, p = 0.014, respectively). The Mankin scoring results showed that there were statistically significant differences between the EVOO and control group in the comparison of both total scores (p = 0.001 for short-term and p = 0.004 for long-term) and subgroup scoring, including macroscopic appearance, chondrocyte cell number, staining, and Tidemark integrity in both short-term (p = 0.005, p = 0.028, p = 0.001, p = 0.005, respectively) and long-term assessments (p = 0.002, p = 0.014, p < 0.001, p = 0. 200, respectively).ConclusionsWe have observed promising outcomes of intra-articular application of extra virgin olive oil in the treatment of acute degenerative osteoarthritis in rabbit knees. Due to its potential cartilage restorative and regenerative effects, EVOO, when administered intra-articularly, may be a promising agent to consider for further research in the treatment of OA.

KIF22 regulates mitosis and proliferation of chondrocyte cells

Point mutations in KIF22 have been linked to spondyloepimetaphyseal dysplasia with joint laxity, type 2 (SEMDJL2). Skeletal features of SEMDJL2 include short stature and joint laxity. Mechanisms underlying these limb abnormalities are unknown. Here in this manuscript, we have investigated the function of KIF22 in chondrocytes. Quantitative PCR and immunostaining revealed that Kif22 was highly expressed in proliferating-zone growth-plate chondrocytes. Kif22 knockdown resulted in defective mitotic spindle formation and reduced cell proliferation. Forced expression of SEMDJL-associated mutant Kif22 constructs likewise induced abnormal mitotic spindle morphology and reduced proliferation. Mice expressing a KIF22 truncation mutant had shorter growth plates and shorter tibial bones compared to wild-type mice. These results suggest that KIF22 regulates mitotic spindle formation in proliferating chondrocytes thereby linking the stunted longitudinal bone growth observed in SEMDJL2 to failures of chondrocyte division.