Co-culture Of Chondrocytes Research Articles

Gene therapy for fat-1 prevents obesity-induced metabolic dysfunction, cellular senescence, and osteoarthritis

Obesity is one of the primary risk factors for osteoarthritis (OA), acting through cross talk among altered biomechanics, metabolism, adipokines, and dietary free fatty acid (FA) composition. Obesity and aging have been linked to cellular senescence in various tissues, resulting in increased local and systemic inflammation and immune dysfunction. We hypothesized that obesity and joint injury lead to cellular senescence that is typically associated with increased OA severity or with aging and that the ratio of omega-6 (ω-6) to omega-3 (ω-3) FAs regulates these pathologic effects. Mice were placed on an ω-6-rich high-fat diet or a lean control diet and underwent destabilization of the medial meniscus to induce OA. Obesity and joint injury significantly increased cellular senescence in subcutaneous and visceral fat as well as joint tissues such as synovium and cartilage. Using adeno-associated virus (AAV) gene therapy for fat-1, a fatty acid desaturase that converts ω-6 to ω-3 FAs, decreasing the serum ω-6:ω-3 FA ratio had a strong senomorphic and therapeutic effect, mitigating metabolic dysfunction, cellular senescence, and joint degeneration. In vitro coculture of bone marrow-derived macrophages and chondrocytes from control and AAV8-fat1-treated mice were used to examine the roles of various FA mediators in regulating chondrocyte senescence. Our results suggest that obesity and joint injury result in a premature "aging" of the joint as measured by senescence markers, and these changes can be ameliorated by altering FA composition using fat-1 gene therapy. These findings support the potential for fat-1 gene therapy to treat obesity- and/or injury-induced OA clinically.

The Phenotype of Mesenchymal Stromal Cell and Articular Chondrocyte Cocultures on Highly Porous Bilayer Poly-L-Lactic Acid Scaffolds Produced by Thermally Induced Phase Separation and Supplemented with Hydroxyapatite.

Bilayer scaffolds could provide a suitable topology for osteochondral defect repair mimicking cartilage and subchondral bone architecture. Hence, they could facilitate the chondro- and osteogenic lineage commitment of multipotent mesenchymal stromal cells (MSCs) with hydroxyapatite, the major inorganic component of bone, stimulating osteogenesis. Highly porous poly-L-lactic acid (PLLA) scaffolds with two layers of different pore sizes (100 and 250 µm) and hydroxyapatite (HA) supplementation were established by thermally induced phase separation (TIPS) to study growth and osteogenesis of human (h) MSCs. The topology of the scaffold prepared via TIPS was characterized using scanning electron microscopy (SEM), a microCT scan, pycnometry and gravimetric analysis. HMSCs and porcine articular chondrocytes (pACs) were seeded on the PLLA scaffolds without/with 5% HA for 1 and 7 days, and the cell attachment, survival, morphology, proliferation and gene expression of cartilage- and bone-related markers as well as sulfated glycosaminoglycan (sGAG) synthesis were monitored. All scaffold variants were cytocompatible, and hMSCs survived for the whole culture period. Cross-sections revealed living cells that also colonized inner scaffold areas, producing an extracellular matrix (ECM) containing sGAGs. The gene expression of cartilage and bone markers could be detected. HA represents a cytocompatible supplement in PLLA composite scaffolds intended for osteochondral defects.

THE ROLE OF PERICYTES IN REGULATING CARTILAGE DEGRADATION AND FIBROSIS

Pericytes are contractile, motile cells that surround the capillary. Recent studies have shown that pericytes promoted joint fibrosis and induced subchondral bone angiogenesis, indicating the role of pericytes in osteoarthritis (OA). However, whether pericytes are involved in regulating inflammatory and catabolic response, as well as fibrotic repair of cartilage is still unclear. Here we used 2D and 3D models to investigate the communication of pericytes and chondrocytes under inflammatory osteoarthritis conditions.CD34-CD146+ pericytes were isolated and sorted from human bone marrow. Human OA chondrocytes were isolated from OA joints. In 2D studies, monolayer cultured chondrocytes were treated +/- pericyte conditioned media, +/- 1ng/ml IL1β for 24h. In 3D studies, pericytes and chondrocytes were cultured within fibrin gel in 3D polyurethane scaffolds, separately or combined for 7 days, followed by treatment of +/- IL1β for another 7 days (Fig 2A). The inflammatory response, catabolic activity and expression of fibrosis markers of chondrocytes and pericytes were measured by ELISA and/or q-rtPCR.Pericytes had weak inflammatory, catabolic and fibrotic response to IL1β (data not shown). The 2D study showed that pericyte conditioned media promoted inflammation, catabolism and fibrosis markers of chondrocytes, in the absence of IL1β treatment (Figure 1). However, study in 3D showed that coculture of chondrocytes and pericytes reduced the inflammatory and catabolic response of chondrocytes to IL1β and induced fibrosis markers in chondrocytes (Figure 2).Pericytes are involved in regulating inflammatory response, catabolic response and fibrosis of chondrocytes. The opposite results from 2D and 3D experiments indicate the variety of the regulatory role of pericytes in the interaction with chondrocytes within different culture models. The underlying mechanism is under evaluation with on-going studies.AcknowledgementsThis study was funded by SINPAIN project, from European Union's Horizon Europe research and innovation programme under Grant Agreement NO. 101057778. Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them.For any figures or tables, please contact the authors directly.

IL-6 induces periostin production in human ACL remnants: a possible mechanism causing post-traumatic osteoarthritis

ObjectivePerostin (POSTN) and IL-6 consistently elevated after ACL injury, and ACL has been proposed as the major source of POSTN. However, there is a lack of evidence whether IL-6 induces ACL remnants to produce POSTN. This study aimed to investigate the effect of IL-6 on POSTN production in ACL fibroblasts, which may help us understand more about the mechanism of PTOA after ACL injury and ACL reconstruction.MethodsACL remnants were harvested from 27 patients undergoing ACL reconstruction. Quantitative real-time polymerase chain reaction (PCR) was performed to examine the POSTN gene expression of ACL fibroblasts after treatment of different concentrations of IL-6. The POSTN protein production of ACL fibroblasts was determined using western blot analysis. The blockers of possible signaling pathways, including PI3K/Akt, Ras/MAPK, and JAK/STAT pathways, were added to test whether the effect of IL-6 on ACL fibroblast could be attenuated. ACL fibroblast and chondrocyte co-culture was carried out to determine the influence of ACL and IL-6 on chondrocytes.ResultsQuantitative real-time PCR showed that IL-6 time-dependently and dose-dependently increased POSTN gene expression of ACL fibroblast. Western blot analysis also revealed that IL-6 dose-dependently induced POSTN protein production. Regarding the chronicity of ACL injury, the POSTN protein production was comparable between ACL remnants which were derived within 3 months of injury and at least 6 months after injury. PI3K/Akt blockers could attenuate the effect of IL-6 on ACL remnants, whereas Ras/MAPK and JAK/STAT did not decrease POSTN production. The coexistence of ACL and IL-6 induced more MMP-13 and ADAMTS-4 by chondrocytes.ConclusionsIL-6 induced ACL remnants to produce POSTN. This effect could be attenuated by the PI3K/Akt blocker. Coexistence of IL-6 and ACL remnants may accelerate post-traumatic arthritis.

Higher ratios of chondrocyte to mesenchymal stem cells elevate the therapeutic effects of extracellular vesicles harvested from chondrocyte/mesenchymal stem cell co-culture on osteoarthritis in a rat model.

Extracellular vesicles (EVs) may have a key therapeutic role and offer an innovative treatment for osteoarthritis (OA). Studies have shown that ratio of MSC/chondrocyte could affect their therapeutic outcomes. Here, we investigate the chondrogenic potential and therapeutic effect of EVs derived from MSCs and chondrocytes in the naïve, chondrogenically primed, and co-culture states to treat OA. EVs are isolated from naïve MSCs (M-EV), chondrogenically primed MSCs (cpM-EV), chondrocytes (C-EV), and co-cultures of chondrocytes plus MSCs at ratios of 1:1 (C/M-EV), 2:1 (2C/M-EV), and 4:1 (4C/M-EV). We characterized the isolated EVs in terms of surface markers, morphology, size, and zeta potential, and evaluated their chondrogenic potential in vitro by qRT-PCR and histological analyses. Next, these EVs were intra-articularly injected into osteoarthritic cartilage of a rat model and assessed by radiography, gait parameters, and histological and immunohistochemical analyses. EVs obtained from chondrocytes co-cultured with MSCs resulted in improved matrix production and functional differentiation. Our research showed that close proximity between the two cell types was essential for this response, and improved chondrogenesis and matrix formation were the outcomes of this interaction in vitro. Furthermore, in the in vivo rat OA model induced by a monoiodoacetate (MIA), we observed recovery from OA by increasing ratio of the C/M-derived EV group compared to the other groups. Our findings show that the increasing chondrocyte ratio to MSC leads to high chondrogenic induction and the therapeutic effect of harvested EVs for cartilage repair.

Potential of secretome of human fetal cartilage progenitor cells as disease modifying agent for osteoarthritis

AimsOsteoarthritis (OA) is caused by an imbalance in the synthesis and degradation of cartilage tissue by chondrocytes. Therefore, a therapeutic agent for OA patients that can positively affect both synthesis and degradation is needed. However, current nonsurgical treatments for OA can barely achieve satisfactory long-term outcomes in cartilage repair. Human fetal cartilage progenitor cells-secretome (ShFCPC) has shown potent anti-inflammatory and tissue-repair effects; however, its underlying mechanisms and effects on OA have rarely been systematically elucidated. This study aims to analyze and evaluate the potency of ShFCPC in modifying OA process. Main methodsHerein, secreted proteins enriched in ShFCPC have been characterized, and their biological functions both in vitro and in vivo in an OA model are compared with those of human bone marrow-derived mesenchymal stem cells-secretome (ShBMSC) and hyaluronan (HA). Key findingsSecretome analysis has shown that ShFCPC is significantly enriched with extracellular matrix molecules involved in many effects of cellular processes required for homeostasis during OA progression. Biological validation in vitro has shown that ShFCPC protects chondrocyte apoptosis by suppressing the expression of inflammatory mediators and matrix-degrading proteases and promotes the secretion of pro-chondrogenic cytokines in lipopolysaccharide-induced coculture of human chondrocytes and SW982 synovial cells compared with ShBMSC. Moreover, in a rat OA model, ShFCPC protects articular cartilage by reducing inflammatory cell infiltration and M1/M2 macrophage ratio in the synovium, which directly contributes to an increase in immunomodulatory atmosphere and enhances cartilage repair compared to ShBMSC and HA. SignificanceOur findings support clinical translations of ShFCPC as a novel agent for modifying OA process.

Cartilage 3D bioprinting for rhinoplasty using adipose-derived stem cells as seed cells: Review and recent advances.

Nasal deformities due to various causes affect the aesthetics and use of the nose, in which case rhinoplasty is necessary. However, the lack of cartilage for grafting has been a major problem and tissue engineering seems to be a promising solution. 3D bioprinting has become one of the most advanced tissue engineering methods. To construct ideal cartilage, bio-ink, seed cells, growth factors and other methods to promote chondrogenesis should be considered and weighed carefully. With continuous progress in the field, bio-ink choices are becoming increasingly abundant, from a single hydrogel to a combination of hydrogels with various characteristics, and more 3D bioprinting methods are also emerging. Adipose-derived stem cells (ADSCs) have become one of the most popular seed cells in cartilage 3D bioprinting, owing to their abundance, excellent proliferative potential, minimal morbidity during harvest and lack of ethical considerations limitations. In addition, the co-culture of ADSCs and chondrocytes is commonly used to achieve better chondrogenesis. To promote chondrogenic differentiation of ADSCs and construct ideal highly bionic tissue-engineered cartilage, researchers have used a variety of methods, including adding appropriate growth factors, applying biomechanical stimuli and reducing oxygen tension. According to the process and sequence of cartilage 3D bioprinting, this review summarizes and discusses the selection of hydrogel and seed cells (centered on ADSCs), the design of printing, and methods for inducing the chondrogenesis of ADSCs.

The Impact of Different Ratios of Chondrocytes and Mesenchymal Stem Cells on Condrogenic Potential of Extracellular Vesicles Derived from Coculture of Chondrocytes and Mesenchymal Stem Cells

هدف: عدم توانایی ترمیم خود به‫خودی بافت غضروف به‫دنبال ایجاد آسیب‌ مفصلی، نیازمند یک رویکرد درمانی موثر است. اخیرا نقش وزیکول‌های خارج سلولی (EV) در ارتباطات سلولی و ترمیم بافت از طریق تنظیم فرایندهای سلولی مورد توجه قرار گرفته است. هدف این مطالعه، مقایسه توانایی غضروف­زایی EVهای مشتق از هم‌کشتی کندروسیت/سلول بنیادی مزانشیمی با نسبت‌های 1 به 2 و 1 به 4 در شرایط برون‌تنی است. مواد و روش‌ها: در این راستا، سلول‌ها جداسازی و مشخصه­یابی شدند. محیط‌های رویی سلول‌های مورد نظر جمع­آوری و EV با استفاده از دستگاه اولتراسانتریفیوژ استخراج شد. EVها از لحاظ اندازه، مورفولوژی و بیان مارکرهای سطحی بررسی شدند. توانایی تمایز سلول‌های بنیادی مزانشیمی به کندروسیت در حضور غلظت‌های مختلف EVهای (۵۰، ۱۰۰ و۱۵۰ میکروگرم‌بر‌میلی‌لیتر) در ۲۱ روز بررسی شد و بیان مارکرهای غضروفی با استفاده از qRT-PCR و مطالعات بافتی ارزیابی شد. نتایج: CHO/MSC-EV 1/2 و CHO/MSC-EV 1/4 کروی‌شکل و به ترتیب با اندازه ۱۵/۸ ±۶۶/۵۱ و ۰۳/۶ ±۱۱/۱۹ نانومتر بوده و مارکرهای سطحی ویژه‌ی EVها شامل CD9 و CD81 در دو گروه بیان شد. افزایش بیان بالاتر مارکرهای ویژه­ی غضروفی به‫ویژه Col II وهمچنین ترشح آمینوگلیکان وپروتئوگلیکان در غلظت‌های 100 و150 میکروگرم بر میلی‌لیتر CHO/MSC-EV 1/2 نسبت به CHO/MSC-EV 1/4مشاهده شد. نتیجه‌گیری: نتایج حاصل از این مطالعه پتانسیل تمایز به غضروف EVها را به‫ویژه در وزیکول‌های مشتق از CHO/MSC-EV 1/2 نشان داد. استفاده از EVها که توانایی غضروف‌زایی بالا دارند، به‫دلیل عدم ایمنی‌زایی و خطر تومورزایی می‌تواند در ترمیم بافت غضروف، موفقیت‌آمیز باشد.

Evaluation of a co-culture of rapidly isolated chondrocytes and stem cells seeded on tri-layered collagen-based scaffolds in a caprine osteochondral defect model

Cartilage has poor regenerative capacity and thus damage to the joint surfaces presents a major clinical challenge. Recent research has focussed on the development of tissue-engineered and cell-based approaches for the treatment of cartilage and osteochondral injuries, with current clinically available cell-based approaches including autologous chondrocyte implantation and matrix-assisted autologous chondrocyte implantation. However, these approaches have significant disadvantages due to the requirement for a two-stage surgical procedure and an in vitro chondrocyte expansion phase which increases logistical challenges, hospital times and costs. In this study, we hypothesized that seeding biomimetic tri-layered scaffolds, with proven regenerative potential, with chondrocyte/infrapatellar fat pad stromal cell co-cultures would improve their regenerative capacity compared to scaffolds implanted cell-free. Rapid cell isolation techniques, without the requirement for long term in vitro culture, were utilised to achieve co-cultures of chondrocytes and stromal cells and thus overcome the limitations of existing cell-based techniques. Cell-free and cell-seeded scaffolds were implanted in osteochondral defects, created within the femoral condyle and trochlear ridge, in a translational large animal goat model. While analysis showed trends towards delayed subchondral bone healing in the cell-seeded scaffold group, by the 12 month timepoint the cell-free and cell-seeded groups yield cartilage and bone tissue with comparable quality and quantity. The results of the study reinforce the potential of the biomimetic tri-layered scaffold to repair joint defects but failed to demonstrate a clear benefit from the addition of the CC/FPMSC co-culture to this scaffold. Taking into consideration the additional cost and complexity associated with the cell-seeded scaffold approach, this study demonstrates that the treatment of osteochondral defects using cell-free tri-layered scaffolds may represent a more prudent clinical approach.

Mechanically Derived Tissue Stromal Vascular Fraction Acts Anti-inflammatory on TNF Alpha-Stimulated Chondrocytes In Vitro.

Enzymatically isolated stromal vascular fraction (SVF) has already shown to be effective as a treatment for osteoarthritis (OA). Yet, the use of enzymes for clinical purpose is highly regulated in many countries. Mechanical preparation of SVF results in a tissue-like SVF (tSVF) containing intact cell–cell connections including extracellular matrix (ECM) and is therefore less regulated. The purpose of this study was to investigate the immunomodulatory and pro-regenerative effect of tSVF on TNFα-stimulated chondrocytes in vitro. tSVF was mechanically derived using the Fractionation of Adipose Tissue (FAT) procedure. Characterization of tSVF was performed, e.g., cellular composition based on CD marker expression, colony forming unit and differentiation capacity after enzymatic dissociation (from heron referred to as tSVF-derived cells). Different co-cultures of tSVF-derived cells and TNFα-stimulated chondrocytes were analysed based on the production of sulphated glycosaminoglycans and the anti-inflammatory response of chondrocytes. Characterization of tSVF-derived cells mainly contained ASCs, endothelial cells, leukocytes and supra-adventitial cells. tSVF-derived cells were able to form colonies and differentiate into multiple cell lineages. Co-cultures with chondrocytes resulted in a shift of the ratio between tSVF cells: chondrocytes, in favor of chondrocytes alone (p < 0.05), and IL-1β and COX2 gene expression was upregulated in TNFα-treated chondrocytes. After treatment with (a conditioned medium of) tSVF-derived cells, IL-1β and COX2 gene expression was significantly reduced (p < 0.01). These results suggest mechanically derived tSVF stimulates chondrocyte proliferation while preserving the function of chondrocytes. Moreover, tSVF suppresses TNFα-stimulated chondrocyte inflammation in vitro. This pro-regenerative and anti-inflammatory effect shows the potential of tSVF as a treatment for osteoarthritis.

Autologous Oxygen-Releasing Nano-Biomimetic Scaffold with Chondrocytes Promotes Joint Repair After Trauma

Our study assesses the role of a scaffold constructed by co-culture of autologous oxygen-releasing biomimetic scaffold (AONS) and chondrocytes in joint repair after trauma. A composite scaffold structure was used and a scaffold constructed of AONS and chondrocytes was transplanted into SD rats to create models of patellar cartilage fracture and hip osteochondral fracture, respectively followed by analysis of cell proliferation by immunofluorescence method, osteogenesis-related gene expression by RT-PCR, chondrocytes apoptosis by TUNEL staining. The blank control group and AONS composite chondrocytes have significant differences in apoptosis and cell proliferation of two fracture types (P &lt;0.05). The autologous oxygen-releasing nanometers at 4 and 8 weeks showed a significant difference in the number of PCNA and TUNEL cells between biomimetic scaffold and chondrocytes in two groups (P &lt; 0.05). The AONS and chondrocytes were effective for two types of fractures at 1, 4 and 8 weeks. The expression of various markers of intrachondral osteogenesis was decreased and the markers of hip osteochondral fracture were increased significantly (P &lt; 0.05). Joint recovery was better than patellar cartilage fractures. The AONS composite chondrocyte scaffold promotes repair of patellar cartilage fractures and hip osteochondral fractures with a better effect on hip osteochondral fractures.

Infrapatellar fat pad adipose-derived stem cells co-cultured with articular chondrocytes from osteoarthritis patients exhibit increased chondrogenic gene expression

AimThe variable results in clinical trials of adipose tissue-derived stem cells (ASCs) for chondral defects may be due to the different ex vivo culture conditions of the ASCs which are implanted to treat the lesions. We sought to determine the optimal in vitro chondrocyte co-culture condition that promotes infrapatellar fat pad-derived (IFPD) ASC chondrogenic gene expression in a novel co-culture combination.MethodsIn our study, we utilized an in vitro autologous co-culture of IFPD ASCs and articular chondrocytes derived from Kellgren–Lawrence Grade III/IV osteoarthritic human knee joints at ASC-to-chondrocyte seeding log ratios of 1:1, 10:1, and 100:1. Gene expression following in vitro co-culture was quantified by RT-qPCR with a panel comprising COL1A1, COL2A1, COL10A1, L-SOX5, SOX6, SOX9, ACAN, HSPG2, and COMP for chondrogenic gene expression.ResultsThe chondrogenic gene expression profiles from co-cultures were greater than would be expected from an expression profile modeled from chondrocyte and ASC-only monocultures. Additionally, chondrogenic gene expression decreased with increasing ASC-to-chondrocyte seeding ratios.ConclusionsThese findings provide insight into the mechanisms underlying clinical ASC therapies and signifies that IFPD ASCs pre-conditioned by chondrocyte co-culture may have improved chondrogenic potential for cartilage repair. This model can help further understand IFPD ASCs in chondral and osteochondral repair and the chondrogenic pathways involved.5w6sq_raPc9bkGgSr2Nbk9Video

Microvalve Bioprinting of MSC-Chondrocyte Co-Cultures.

Recent improvements within the fields of high-throughput screening and 3D tissue culture have provided the possibility of developing in vitro micro-tissue models that can be used to study diseases and screen potential new therapies. This paper reports a proof-of-concept study on the use of microvalve-based bioprinting to create laminar MSC-chondrocyte co-cultures to investigate whether the use of MSCs in ACI procedures would stimulate enhanced ECM production by chondrocytes. Microvalve-based bioprinting uses small-scale solenoid valves (microvalves) to deposit cells suspended in media in a consistent and repeatable manner. In this case, MSCs and chondrocytes have been sequentially printed into an insert-based transwell system in order to create a laminar co-culture, with variations in the ratios of the cell types used to investigate the potential for MSCs to stimulate ECM production. Histological and indirect immunofluorescence staining revealed the formation of dense tissue structures within the chondrocyte and MSC-chondrocyte cell co-cultures, alongside the establishment of a proliferative region at the base of the tissue. No stimulatory or inhibitory effect in terms of ECM production was observed through the introduction of MSCs, although the potential for an immunomodulatory benefit remains. This study, therefore, provides a novel method to enable the scalable production of therapeutically relevant micro-tissue models that can be used for in vitro research to optimise ACI procedures.

Static Magnetic Fields Enhance the Chondrogenesis of Mandibular Bone Marrow Mesenchymal Stem Cells in Coculture Systems.

Objectives Combining the advantages of static magnetic fields (SMF) and coculture systems, we investigated the effect of moderate-intensity SMF on the chondrogenesis and proliferation of mandibular bone marrow mesenchymal stem cells (MBMSCs) in the MBMSC/mandibular condylar chondrocyte (MCC) coculture system. The main aim of the present study was to provide an experimental basis for obtaining better cartilage tissue engineering seed cells for the effective repair of condylar cartilage defects in clinical practice. Methods MBMSCs and MCCs were isolated from SD (Sprague Dawley) rats. Flow cytometry, three-lineage differentiation, colony-forming assays, immunocytochemistry, and toluidine blue staining were used for the identification of MBMSCs and MCCs. MBMSCs and MCCs were seeded into the lower and upper Transwell chambers, respectively, at a ratio of 1 : 2, and exposed to a 280 mT SMF. MBMSCs were harvested after 3, 7, or 14 days for analysis. CCK-8 was used to detect cell proliferation, Alcian blue staining was utilized to evaluate glycosaminoglycan (GAG), and western blotting and real-time quantitative polymerase chain reaction (RT-qPCR) detected protein and gene expression levels of SOX9, Col2A1 (Collagen Type II Alpha 1), and Aggrecan (ACAN). Results The proliferation of MBMSCs was significantly enhanced in the experimental group with MBMSCs cocultured with MCCs under SMF stimulation relative to controls (P < 0.05). GAG content was increased, and SOX9, Col2A1, and ACAN were also increased at the mRNA and protein levels (P < 0.05). Conclusions Moderate-intensity SMF improved the chondrogenesis and proliferation of MBMSCs in the coculture system, and it might be a promising approach to repair condylar cartilage defects in the clinical setting.

Osteoblasts Regulate the Expression of ADAMTS and MMPs in Chondrocytes through ERK Signaling Pathway.

Degradative enzymes such as matrix metalloproteinase (MMP) and disintegrin metalloproteinase with platelet thrombin-sensitive protein-like motifs (ADAMTS) play a key role in the development of osteoarthritis (OA). We aimed to investigate the effects of OA subchondral osteoblasts on the expression of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 in chondrocytes and the regulation of mitogen-activated protein kinase (MAPK) signaling pathway. A rat knee OA model was constructed by cutting the anterior cruciate ligament of the knee joints, and normal rat articular cartilage chondrocytes (N-ACC), OA rat articular cartilage chondrocytes (O-ACC), normal subchondral bone osteoblasts (N-SBO), and OA subchondral bone osteoblasts (O-SBO) were isolated and extracted. The expressions of O-ACC and O-SBO COL1 and COL2 were detected respectively. Chondrocytes were identified by immunofluorescence of COL2 and toluidine blue staining, and osteoblasts were identified by COL1 immunofluorescence, alkaline phosphatase (ALP), and Alizarin Red staining. Gene expression of COL1, COL2, and aggrecan in normal chondrocytes and OA chondrocytes, and gene expression of osteoblast ALP and osteocalcin (OCN) were detected by RT-PCR to identify the two chondrocytes and the two osteoblast phenotypes. The constructing N-ACC group, O-ACC group, N-ACC + N-SBO group, N-ACC + O-SBO group, O-ACC + N-SBO group, O-ACC + O-SBO group, I+ N-ACC + O-SBO group, and I + O-ACC + O-SBO group cell cultures, and the expression of ERK, ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes in chondrocytes cultured for 0, 24, 48, and 72 h were detected by RT-PCR. The protein expressions of pERK, ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 were detected by Western blot. · The X-ray showed that the knee joint space of the affected limb became narrow.. · The results of RT-PCR of COL2 and aggrecan gene in OA and normal chondrocytes suggest that the relative expression of COL2 in OA articular chondrocytes (0.24 ± 0.07) issignificantly lower than that in normal cartilage (0.61 ± 0.07) (p < 0.05). The relative expression of AGG (0.37 ± 0.16) in OA chondrocytes was significantly lower than that of normal chondrocytes AGG (1.30 ± 0.25) (p < 0.05). The expression of COL1 was very low, and was not statistically significant.. · The results of RT-PCR of the osteoblast ALP and OCN gene indicated that gene expression of ALP (12.30 ± 1.17) and OCN (20.47 ± 4.19)was upregulated when compared with the relative expression of ALP (4.66 ± 0.71) (p < 0.05) and OCN (12.17 ± 2.76) (p < 0.05) in normal osteoblasts, indicating that osteoblasts of OA have greater osteogenic potential than normal osteoblasts.. · The expressions of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes and proteins in OA chondrocytes or normal chondrocytes were basically unchanged when they were cocultured with normal osteoblasts. Indirect coculture of OA osteoblasts and chondrocytes could promote the expression of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes and proteins in chondrocytes. Overexpression of ADAMTS and MMP in coculture systems can be reversed by MAPK-ERK inhibitors.. · OA subchondral bone osteoblasts can promote the overexpression of ADAMTS and MMPs in chondrocytes.. · The ERK signaling pathway may be involved in the regulation of the effect of subchondral bone osteoblasts on chondrocytes..

3D printed PCL/GelMA biphasic scaffold boosts cartilage regeneration using co-culture of mesenchymal stem cells and chondrocytes: In vivo study

Cartilage injury is difficult to self-repair, which poses a challenge to scaffold design and selection of seeded cells for cartilage tissue engineering. In this study, we presented a biphasic scaffold consisting of a cells-encapsulated biodegradable gelatin methacrylate (GelMA) hydrogel on the upper layer and a macroporous poly(ε-caprolactone) (PCL) scaffold filled with cells-encapsulated GelMA hydrogel on the lower layer to support cartilage regeneration using co-culture of bone marrow mesenchyml stem cells (BMSCs) and costal chondrocytes (CChon). We determined that the synergistic effect of BMSCs and costal chondrocytes was sufficient to induce chondrogenesis, but cartilage regeneration was better under TGF-β3 supplement. Memorably, when implanted in a rat osteochondral defect model for 12 weeks, the PCL/GelMA biphasic scaffolds with cells co-culture showed excellent cartilage regenerative capability, and enabled the Young’s modulus of engineered cartilage comparable to that of native cartilage. Furthermore, the implantation of biphasic scaffold resulted in less mechanical pain than cartilage defects by gait analysis. Together, both in vitro and in vivo results demonstrated that the PCL/GelMA biphasic scaffold with co-culture of BMSCs and chondrocytes is a promising scaffold for cartilage regeneration, which pave a new way to solve the key issues of clinical cartilage repair.

RANKL expression in chondrocytes and its promotion by lymphotoxin-α in the course of cartilage destruction during rheumatoid arthritis.

We investigated the expression and localization of the receptor activator nuclear factor κB ligand (RANKL) in cartilage from patients with rheumatoid arthritis (RA) of relevance to cartilage degeneration. We also examined the role of exogenous lymphotoxin (LT)-α on RANKL expression in human chondrocytes and its effect on in vitro osteoclast differentiation. Cartilage and synovial fluid samples were obtained from 45 patients undergoing total joint replacement surgery or joint puncture, including 24 patients with osteoarthritis (OA) and 21 patients with RA. RANKL expression in articular cartilage was examined by immunohistochemistry. LT-α concentrations in synovial fluid were measured using an enzyme-linked immunosorbent assay (ELISA). Normal human chondrocytes were stimulated with LT-α, and the relative mRNA levels of RANKL, osteoprotegerin (OPG), matrix metalloproteinase-9, and vascular endothelial growth factor were examined by real-time polymerase chain reaction. Soluble RANKL protein in culture media was measured using ELISA, and membrane-bound RANKL protein in cells was examined by western blotting. Co-cultures of human chondrocytes with peripheral blood mononuclear cells (PBMCs) were stimulated with macrophage-colony stimulating factor and LT-α, and osteoclast differentiation was evaluated by staining for tartrate-resistant acid phosphatase. LT-α concentrations were higher in RA synovial fluid than in OA samples. The population of RANKL-positive chondrocytes of RA cartilage was higher than that of OA cartilage, and correlated with cartilage degeneration. Stimulation of cultured human chondrocytes by LT-α increased RANKL expression, the RANKL/OPG ratio, and angiogenic factors. Membrane-bound RANKL in chondrocytes was up-regulated after stimulation of LT-α, whereas soluble RANKL in culture medium did not increase. Co-cultures of human chondrocytes and PBMCs demonstrated that LT-α stimulated human chondrocytes to produce RANKL and induced osteoclastic differentiation of PBMCs. RANKL produced by chondrocytes may contribute to cartilage destruction during RA and LT-α could promote the expression of RANKL in human chondrocytes.

Crosstalk Between Mesenchymal Stromal Cells and Chondrocytes: The Hidden Therapeutic Potential for Cartilage Regeneration.

Cartilage injuries following trauma create a puzzling clinical scenario. The finite reparative potential of articular cartilage is well known, and injuries are associated with an increased risk of osteoarthritis. Cell-based therapies have spotlighted chondrocytes and mesenchymal stromal cells (MSCs) as the functional unit of articular cartilage and theprogenitor cells, respectively. The available clinical treatments cannot reproduce the biomechanical properties of articular cartilage and call for continuous investigations into alternative approaches. Co-cultures of chondrocytes and MSCs are an attractive in vitro system to step closer to the in vivo multicellular environment's complexity. Research on the mechanisms of interaction between both cell types will reveal essential cues to understand cartilage regeneration. This review describes the latest discoveries on these interactions, along with advantages and main challenges in vitro and in vivo. The successful clinical translation of in vitro studies requires establishing rigorous standards and clinically relevant research models and an organ-targeting therapeutic strategy.

A tough polysaccharide-based cell-laden double-network hydrogel promotes articular cartilage tissue regeneration in rabbits

Hydrogels used for cartilage tissue engineering purposes must be designed to mimic the biological and physical features of the native extracellular matrix (ECM). Here, we developed three different types of physical and chemical chondro-inductive hydrogels with the controlled drug release ability based on sulfated alginate (SAlg) and chitosan (CS). Bioconjugation of catechol to SAlg (SAlg-Cat) resulted in formation of SAlg-Cat/CS hydrogels with higher mechanical stability than physical SAlg/CS hydrogel. In the chemical double-network hydrogel (DN/SAlg-Cat), the presence of catechol and amine groups within the network resulted in dynamic and reversible bonding with energy dissipation. Our DN/SAlg-Cat hydrogel with initial compressive modulus of 34.86 kPa exhibited adequate compressive strength (421.45 kPa), toughness (89.69 kJ/m3), ability to self-heal and recoverability, which could be exceptional for utilizing in a load-bearing tissue like cartilage. Among hydrogels, the DN/SAlg-Cat hydrogel displayed less degradation rate (6.81%) and higher mechanical stability (81% retention of stiffness) in the presence of lysozyme (15 μg/mL) during 21 days. Incorporation of the sulfate and catechol groups within these hydrogels mediated the sustained release of TGF-β1 and kartogenin (KGN) chondrogenic agents, and resulted in proper chondrogenesis of the encapsulated mesenchymal stem cell (MSC) and chondrocyte (CH) co-culture. The catechol-enriched hydrogels (SAlg-Cat/CS and DN/SAlg-Cat) showed better regeneration of full-thickness articular defects in rabbits after 12 weeks due to the high affinity of the catechol group for cell adhesion, tissue integration, and immobilization of the biological factors. Our results demonstrated a role for a biomimetic microenvironment in efficient chondrogenesis.

Combination of chondrocytes and chondrons improves extracellular matrix production to promote the repairs of defective knee cartilage in rabbits.

BackgroundChondrons are composed of chondrocytes and the surrounding pericellular matrix (PCM) and function to enhance chondrocyte-mediated cartilage tissue engineering. This study aimed at investigating the potential effect of combined chondrocytes with chondrons on the production of proteoglycan and collagen-II (Col-2) and the repair of defective knee cartilage in rabbits.MethodsChondrocytes and chondrons were isolated from the knee cartilage of rabbits, and cultured alone or co-cultured for varying periods in vitro. Their morphology was characterized by histology. The levels of aggrecan (AGG), Col-2 and glycosaminoglycan (GAG) expression were quantified by qRT-PCR, Alcian blue-based precipitation and ELISA. The effect of combined chondrocytes with chondrons in alginate spheres on the repair of defective knee cartilage was examined in rabbits.ResultsThe isolated chondrocytes and chondrons displayed unique morphology and began to proliferate on day 3 and 6 post culture, respectively, accompanied by completely degenerated PCM on day 6 post culture. Evidently, chondrocytes had stronger proliferation capacity than chondrons. Longitudinal analyses indicated that culture of chondrons, but not chondrocytes, increased AGG mRNA transcripts and GAG levels with time and Col-2 mRNA transcripts only on day 3 post culture. Compared with chondrocytes or chondrons alone, co-culture of chondrocytes and chondrons significantly up-regulated AGG and Col-2 expression and GAG production, particularly at a ratio of 1:1. Implantation with chondrocytes and chondrons at 1:1 significantly promoted the repair of defective knee cartilage in rabbits, accompanied by reduced the Wakiteni scores with time.ConclusionCombined chondrons with chondrocytes promoted the production of extracellular matrix and the repair of defective knee cartilage in rabbits.The translational potential of this articleThis study explores that the combination of chondrons and chondrocytes may be new therapeutic strategy for cartilage tissue engineering and repair of defective cartilage.