Chondrocyte Migration Research Articles

Intra-articular injection of placental mesenchymal stromal cells ameliorates pain and cartilage anabolism/catabolism in knee osteoarthritis.

Background: Knee Osteoarthritis (kOA), the most common joint degenerative disorder, lacks effective therapeutics. Placenta-derived mesenchymal stromal cells (PMSCs) are effective in tissue repairing and generation, which have potential in treating kOA. This study aimed to determine the anti-kOA efficacy of PMSCs and to explore its action mode. Methods: Flow cytometry and three-line differentiation were performed for identification of PMSCs. In vivo, a rat kOA model established by anterior cruciate ligament transection (ACLT) surgery was used to evaluate the efficacy of PMSCs. Histopathological HE and SO staining with Osteoarthritis Research Society International scoring were conducted, and cartilage expressions of MMP13 and Col2 were measured by immunohistochemistry. Pain behavior parameters by mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL), were measured. In vitro, wound healing and cell immunofluorescence assays were conducted to detect the proliferation and migration ability of chondrocytes treated with PMSCs conditioned medium (PMSCs-CM). Quantitative real-time PCR (qRT-PCR) and Western blot (WB) assays were applied to explore the molecular action of PMSCs on chondrocytes. Results: The results of flow cytometry indicated that the surface markers of PMSCs (CD73 > 95%, CD90 > 95%, and CD34 < 2%) were consistent with the typical mesenchymal stromal cells. The in vivo data showed that PMSCs significantly reversed the kOA progression by protection of cartilage, regulation of anabolic (Col2) and catabolic (MMP13) expressions, and relief of pain symptoms. The in vitro data showed that PMSCs promoted chondrocyte proliferation and migration and significantly restored the IL-1β-induced abnormal gene expressions of Col2, Mmp13, Adamts4, Adamts5 and Sox9 and also restored the abnormal protein expressions of Col2, Mmp13 and Sox9 of chondrocytes. The molecular actions of PMSCs on chondrocytes in nested co-culture way or in conditioned medium way were similar, confirming a paracrine-based mode of action. Conclusion: This study demonstrated PMSCs' anti-kOA efficacy and its paracrine-based action mode, providing novel knowledge of PMSCs and suggesting it as a promising cell therapy for treatment of kOA.

Exosomes Derived from Runx2-Overexpressing BMSCs Enhance Cartilage Tissue Regeneration and Prevent Osteoarthritis of the Knee in a Rabbit Model.

Osteoarthritis is the leading disease of joints worldwide. Osteoarthritis may be treated by exosomes derived from Runx2-overexpressed bone marrow mesenchymal stem cells (R-BMSCs-Exos). R-BMSCs-Exos would promote the proliferation, migration, and phenotypic maintenance of articular chondrocytes. BMSCs were transfected with and without Runx2. Exosomes derived from BMSCs and R-BMSCs (BMSCs-Exos and R-BMSCs-Exos) were isolated and identified. Proliferation, migration, and phenotypic maintenance were determined in vitro and compared between groups. The mechanism for activation of Yes-associated protein (YAP) was investigated using small interfering RNA (siRNA). The exosomes' preventive role was determined in vivo using Masson trichrome and immunohistochemical staining. R-BMSCs-Exos enhance the proliferation, migration, and phenotypic maintenance of articular chondrocytes based on the YAP being activated. R-BMSCs-Exos prevent knee osteoarthritis as studied in vivo through a rabbit model. Findings emphasize the efficacy of R-BMSCs-Exos in preventing osteoarthritis. Potential source of exosomes is sorted out for the advantages and shortcomings. The exosomes are then modified based on the molecular mechanisms to address their limitations. Such exosomes derived from modified cells have the role in future therapeutics.

Preparation of Silk Fibroin/Chitosan/Nano-Hydroxyapatite and Its Effect on the Repair of Cartilage Damage in Rats

This study was aimed to prepare a silk fibroin (SF)/chitosan (CS)/nano-Hydroxyapatite (n-HAp) composite scaffold for repair of cartilage damage. A four-layer bionic scaffold SF/CS/n-HAp was constructed by cryogenic freezing and forming. Sodium Tripolyphosphate (STPP) was used as a cross-linking agent to post-process the scaffold to improve the mechanical strength of the scaffold. The various materials prepared were characterized. The SF/CS/n-HAp scaffold prepared in this study was cream-colored cylindrical and elastic. It can show variability after external force was applied, and can be restored to its original appearance after elimination of the external force. The diameter of the scaffold was 5 cm, and the total thickness was about 1 cm. The scanning electron microscope (SEM) results showed that the surface of the scaffold material was smooth, the pore size changed gradually, and the connectivity among the holes was good. The pore size distribution of the entire scaffold material was between 100 μm∼300 μm, and the pore size range was suitable for the adhesion, expansion, and migration of chondrocytes and osteoblasts. The mechanical performance test results showed that the composite scaffold had a compression modulus of 706 kPa, which can meet the mechanical performance requirements for repair of smaller articular cartilage damage. In addition, the porosity of each layer of the scaffold was more than 91%, which was conducive to the adhesion of cells on the surface of the material. The cell experiment results revealed that the composite scaffold material was beneficial to the growth and proliferation of cells. Under osteogenic and chondrogenic induction culture, bone marrow stromal cells (BMSCs) can differentiate in the direction of osteogenesis and cartilage. The results of animal experiments revealed that the SF/CS/n-HAp material group showed better effect than the blank control group in repairing rat cartilage defects, and the Wayne score and O’Driscoll score were also significantly higher than the blank control group. This suggested that the biomimetic scaffold SF/CS/n-HAp prepared in this study showed good physical properties and biocompatibility, and had certain prospects in the application of repairing cartilage damage.

Exosomes derived from umbilical cord mesenchymal stem cells protect cartilage and regulate the polarization of macrophages in osteoarthritis

BackgroundOsteoarthritis (OA) is one of the most common joint diseases and a major global public health concern. Mesenchymal stem cells (MSCs) have been widely used for the treatment of OA owing to their paracrine secretion of trophic factors, a phenomenon in which exosomes may play a major role. Here, we investigate the potential of exosomes from human umbilical cord-derived MSCs (hUC-MSCs-Exos) in alleviating OA.MethodsThe hUC-MSCs-Exos were harvested from hUC-MSC-conditioned medium using ultracentrifugation. Rats with surgically-induced OA were intra-articularly injected with hUC-MSCs-Exos. The effect of hUC-MSCs-Exos in repairing osteoarticular cartilage was assessed using hematoxylin and eosin (HE) staining, safranin-O and fast green staining and immunohistochemistry. The in vitro experiments were further carried out to verify the therapeutic effect. The effects of hUC-MSCs-Exos on the proliferation and migration of human chondrocytes were evaluated using the cell counting kit-8, EdU-555 cell proliferation kit, and transwell assays. Annexin V-FITC/PI staining were used to evaluate the effect of exosomes on chondrocyte apoptosis. An in vitro model of human articular chondrocytes treated with interleukin 1 beta (IL-1β) was used to evaluate the effects of exosomes, analyses involved using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting. The role of hUC-MSCs-Exos in macrophage polarization was examined in the monocyte cell line, Tohoku Hospital Pediatrics-1 (THP-1) by qRT-PCR and immunofluorescence.ResultsThe results showed that hUC-MSCs-Exos prevented severe damage to the knee articular cartilage in the rat OA model. We confirmed the high efficacy of hUC-MSCs-Exos in promoting chondrocyte proliferation and migration and inhibiting chondrocyte apoptosis. Additionally, hUC-MSCs-Exos could reverse IL-1β-induced injury of chondrocytes and regulate the polarization of macrophages in vitro.ConclusionsThere is potential for hUC-MSCs-Exos to be used as a treatment strategy for OA.

Characterization of Migratory Cells From Bioengineered Bovine Cartilage in a 3D Co-culture Model

Background: Chondrocyte migration in native cartilage is limited and has been implicated as one of the reasons for the poor integration of native implants. Through use of an in vitro integration model, it has previously been shown that cells from bioengineered cartilage can migrate into the native host cartilage during integration. Platelet-rich plasma (PRP) treatment further enhanced integration of bioengineered cartilage to native cartilage in vitro. However, it is not known how PRP treatment of the bioengineered construct promotes this. Hypothesis: PRP supports cell migration from bioengineered cartilage and these migratory cells have the ability to accumulate cartilage-like matrix. Study Design: Controlled laboratory study. Methods: Osteochondral-like constructs were generated by culturing primary bovine chondrocytes on the top surface of a porous bone substitute biomaterial composed of calcium polyphosphate. After 1 week in culture, the constructs were submerged in PRP and placed adjacent, but 2 mm distant, to a native bovine osteochondral plug in a co-culture model for 2 weeks. Cell migration was monitored using phase-contrast imaging. Cell phenotype was determined by evaluating the gene expression of matrix metalloprotease 13 (MMP-13), Ki67, and cartilage matrix molecules using quantitative polymerase chain reaction. When tissue formed, it was assessed by histology, immunohistochemistry, and quantification of matrix content. Results: PRP treatment resulted in the formation of a fiber network connecting the bioengineered cartilage and native osteochondral plug. Cells from both the bioengineered cartilage and the native osteochondral tissue migrated onto the PRP fibers and formed a tissue bridge after 2 weeks of culture. Migratory cells on the tissue bridge expressed higher levels of collagen types II and I (COL2, COL1), Ki67 and MMP-13 mRNA compared with nonmigratory cells in the bioengineered cartilage. Ki67 and MMP-13–positive cells were found on the edges of the tissue bridge. The tissue bridge accumulated COL1 and COL2 and aggrecan and contained comparable collagen and glycosaminoglycan content to the bioengineered cartilage matrix. The tissue bridge did not reliably develop in the absence of cells from the native osteochondral plug. Conclusion: Bioengineered cartilage formed by bovine chondrocytes contains cells that can migrate on PRP fibers and form cartilaginous tissue. Clinical Relevance: Migratory cells from bioengineered cartilage may promote cartilage integration. Further studies are required to determine the role of migratory cells in integration in vivo.

Extracellular vesicles derived from human umbilical cord mesenchymal stem cells alleviate osteoarthritis of the knee in mice model by interacting with METTL3 to reduce m6A of NLRP3 in macrophage

BackgroundOsteoarthritis (OA) is a prevalent degenerative joint disease that not only significantly impairs the quality of life of middle-aged and elderly individuals but also imposes a significant financial burden on patients and society. Due to their significant biological properties, extracellular vesicles (EVs) have steadily received great attention in OA treatment. This study aimed to investigate the influence of EVs on chondrocyte proliferation, migration, and apoptosis and their protective efficacy against OA in mice.MethodsThe protective impact of EVs derived from human umbilical cord mesenchymal stem cells (hucMSCs-EVs) on OA in mice was investigated by establishing a mouse OA model by surgically destabilizing the medial meniscus (DMM). Human chondrocytes were isolated from the cartilage of patients undergoing total knee arthroplasty (TKA) and cultured with THP-1 cells to mimic the in vivo inflammatory environment. Levels of inflammatory factors were then determined in different groups, and the impacts of EVs on chondrocyte proliferation, migration, apoptosis, and cartilage extracellular matrix (ECM) metabolism were explored. N6-methyladenosine (m6A) level of mRNA and methyltransferase-like 3 (METTL3) protein expression in the cells was also measured in addition to microRNA analysis to elucidate the molecular mechanism of exosomal therapy.ResultsThe results indicated that hucMSCs-EVs slowed OA progression, decreased osteophyte production, increased COL2A1 and Aggrecan expression, and inhibited ADAMTS5 and MMP13 overexpression in the knee joint of mice via decreasing pro-inflammatory factor secretion. The in vitro cell line analysis revealed that EVs enhanced chondrocyte proliferation and migration while inhibiting apoptosis. METTL3 is responsible for these protective effects. Further investigations revealed that EVs decreased the m6A level of NLRP3 mRNA following miR-1208 targeted binding to METTL3, resulting in decreased inflammatory factor release and preventing OA progression.ConclusionThis study concluded that hucMSCs-EVs inhibited the secretion of pro-inflammatory factors and the degradation of cartilage ECM after lowering the m6A level of NLRP3 mRNA with miR-1208 targeting combined with METTL3, thereby alleviating OA progression in mice and providing a novel therapy for clinical OA treatment.

Wharton's jelly mesenchymal stem cell-derived small extracellular vesicles as natural nanoparticles to attenuate cartilage injury via microRNA regulation

The main strategy of tissue repair and regeneration focuses on the application of mesenchymal stem cells and cell-based nanoparticles, but there are still multiple challenges that may have negative impacts on human safety and therapeutic efficacy. Cell-free nanotechnology can effectively overcome these obstacles and limitations. Mesenchymal stem cell (MSC)-derived natural small extracellular vesicles (sEVs) represent ideal nanotherapeutics due to their low immunogenicity and lack of tumorigenicity. Here, sEVs harvested from Wharton’s jelly mesenchymal stem cells (WJMSCs) were identified. In vitro results showed that WJMSC-sEVs efficiently entered chondrocytes in the osteoarthritis (OA) model, further promoted chondrocyte migration and proliferation and modulated immune reactivity. In vivo, WJMSC-sEVs notably promoted chondrogenesis, which was consistent with the effect of WJMSCs. RNA sequencing results revealed that sEV-microRNA-regulated biocircuits can significantly contribute to the treatment of OA, such as by promoting the activation of the calcium signaling pathway, ECM-receptor interaction pathway and NOTCH signaling pathway. In particular, let-7e-5p, which is found in WJMSC-sEVs, was shown to be a potential core molecule for promoting cartilage regeneration by regulating the levels of STAT3 and IGF1R. Our findings suggest that WJMSC-sEV-induced chondrogenesis is a promising innovative and feasible cell-free nanotherapy for OA treatment.

Fibrin glue does not assist migration and proliferation of chondrocytes in collagenic membranes: an in vitro study

BackgroundSome authors secured the membrane during matrix-induced autologous chondrocyte implantation (mACI) with fibrin glue or did not use a formal fixation. The real impact of fibrin glue addition on chondrocytes migration and proliferation has not yet been clarified. This study evaluated the impact of fibrin glue on a chondrocyte loaded collagenic membrane.MethodsA resorbable collagen I/III porcine derived membrane commonly employed in AMIC was used for all experiments. Chondrocytes from three difference donors were used. At 1-, 2-, 3-, 4-, 6-, and at 8-week the membranes were embedded in Mounting Medium with Dapi (ABCAM, Cambridge, UK). The Dapi contained in the mounting medium ties the DNA of the cell nucleus and emits a blue fluorescence. In this way, the spreading of the cells in the membrane can be easily monitored. The outcomes of interest were to evaluate (1) cell migration and (2) cell proliferation within the porous membrane layer. DAPI/nuclei signals were analysed with fluorescence microscope under a magnification of 100-fold.ResultsThe no-fibrin group demonstrated greater migration of the cells within the membrane. Although migration resulted higher in the no-fibrin group at every follow-up, this difference was significant only at week 1 (P < 0.001), 2 (P = 0.004), and 3 (P = 0.03). No difference was found at week 3, 6, and 8. The no-fibrin group demonstrated greater proliferation of the chondrocytes within the membrane. These differences were significant at week 4 (P < 0.0001), 6 (P < 0.0001), 8 (P < 0.0001).ConclusionThe use of fibrin glue over a resorbable membrane leads to lower in vitro proliferation and migration of chondrocytes.

Nanohydroxyapatite Hydrogel Can Promote the Proliferation and Migration of Chondrocytes and Better Repair Talar Articular Cartilage.

As an important load-bearing part of the body, joints are prone to articular cartilage degradation during exercise, resulting in joint pain, swelling, and deformity, which has an adverse impact on patients' life quality and social medical security. Therefore, this study aims to test an effective biopolymer scaffold in promoting the growth of chondrocytes in talus. Hydrogel (Gel)-nanohydroxyapatite (nHA) was invented as a new type of biopolymer scaffold for osteoarthritis treatment in this research. To detect the effects of Gel-nHA on guidance, cartilage matrix secretion, mineralization, proliferation, and migration of chondrocyte, we cultured chondrocytes to study the biological properties of nHA. It was found that Gel could guide chondrocytes to permeate and migrate, so it could be used as acellular matrix scaffolds for chondrocyte regeneration. In addition, nHA could stimulate chondrocytes to secrete cartilage matrix, such as type II collagen and mucopolysaccharide (GAGs). At the same time, nHA help to induce chondrocyte mineralization and stimulate the secretion of type X collagen, so as to better maintain the integrity of bone cartilage interface. In Gel-nHA, chondrocyte viability could be better maintained, and the proliferation and migration of chondrocytes could be better promoted, so as to better repair the articular cartilage of talus. Therefore, the Gel-nHA scaffold is expected to become an effective method for repairing talus cartilage in the future.

Fibroblast growth factor receptor 1-bound extracellular vesicle as novel therapy for osteoarthritis.

Osteoarthritis (OA) is a joint condition that causes significant impairment of the chondrocyte. The gradual degradation of the cartilage lining of one or more freely moving joints, as well as persistent inflammation, are the causes of osteoarthritis. Current medications focus on alleviating symptoms rather than curing the condition. The aim of this review is to evaluate the potential use of fibroblast growth factor receptor 1-bound extracellular vesicle as novel therapy for osteoarthritis. This review article was completed by searching for the keywords “Fibroblast Growth Factor Receptor 1”, “Extracellular Vesicle”, and “Osteoarthritis” in various journals in several search engines. Of the 102 scientific articles found, 95 were found suitable to be used as material in the making of this article. The upregulated amount of FGFR1 (fibroblast growth factor receptors) signalling suggesting the progression of degenerative cartilage that commonly seen in osteoarthritis (OA) patients. Several studies showed that the involvement of extracellular vesicles (EV) derived from MSCs could enhance cartilage repair and protect the cartilage from degradation. EVs have the potential to deliver effects to specific cell types through ligand-receptor interactions and several pathway mechanisms related with the FGFR1. EVs and FGFR1-bound Evs have been postulated in recent years as possible therapeutic targets in human articular cartilage. The protective benefits on both chondrocytes and synoviocytes in OA patients can be achieved by administering the MSC-EVs that may also stimulate chondrocyte proliferation and migration EVs have a promising potential to become a novel therapy for treating patients with OA. However, further researches are need to be conducted to discover further the application of this therapy.

PTH (1-34) enhances the therapeutic effect of bone marrow mesenchymal stem cell-derived exosomes by inhibiting proinflammatory cytokines expression on OA chondrocyte repair in vitro

BackgroundThe effects of bone marrow mesenchymal stem cells (BMSCs) during the treatment of cartilage damage have been proven to be attributed to paracrine mechanisms, particularly the effect of exosomes. Exosomes from different batches are inhomogeneous, and different treatment effects are observed between samples. The purpose of this research was to find more effective and homogeneous exosomes for the repair of chondrocytes in osteoarthritis (OA). We observed the potential effects and possible mechanisms of exosomes derived from parathyroid hormone (PTH) (1-34)-preconditioned BMSCs (ExoPTH) in the alleviation of OA.Materials and methodsExosomes derived from BMSCs (ExoBMSC) and ExoPTH were isolated by differential centrifugation. Primary rat chondrocytes were used to establish the OA model by interleukin 1 beta (IL-1β) in vitro. The effects of these two types of exosomes on OA chondrocyte proliferation, migration, apoptosis, and extracellular matrix formation were measured and compared. We observed changes in IL-2, TNF-α, and IL-6 levels via Western blotting (WB), and quantitative real-time PCR (qRT–PCR).ResultsWe successfully extracted ExoBMSC and ExoPTH and established an IL-1β-induced OA model in primary chondrocytes from rats. Our study showed that IL-2, TNF-α, and IL-6 levels increased significantly in OA chondrocytes; however, both ExoBMSC and ExoPTH reduced the levels of IL-2, TNF-α, and IL-6. In addition, ExoPTH exhibited stronger anti-inflammatory effects. ExoPTH had a more marked effect on proliferation, migration, and production of the extracellular matrix (Col-II) in OA chondrocytes than ExoBMSC at 24 h.ConclusionExoPTH increased the migration, proliferation, and chondral matrix formation of OA chondrocytes in vitro. In OA chondrocyte therapy, the potential mechanism of ExoPTH might involve the inhibition of production of proinflammatory cytokines. Although the two types of exosomes had some similar effects, most effects of ExoPTH were better than those of ExoBMSC, so ExoPTH may have a better ability to alleviate OA.

Non-Mulberry Silk Fiber-Based Composite Scaffolds Containing Millichannels for Auricular Cartilage Regeneration

Tissue engineering has made significant progress as a cartilage repair alternative. It is crucial to promote cell proliferation and migration within three-dimensional (3D) bulk scaffolds for tissue regeneration through either chemical gradients or physical channels. In this study, by developing optimized silk fiber-based composite scaffolds, millimeter-scaled channels were created in the corresponding scaffolds via facile physical percussive drilling and subsequently utilized for auricular cartilage regeneration. We found that by the introduction of poly-l-lactic acid porous microspheres (PLLA PMs), the channels incorporated into the Antheraea pernyi (Ap) silk fiber-based scaffolds were reinforced, and the mechanical features were well maintained. Moreover, Ap silk fiber-based scaffolds reinforced by PLLA PMs containing channels (CMAF) exhibited excellent chondrocyte proliferation, migration, and synthesis of cartilage-specific extracellular matrix (ECM) in vitro. The biological evaluation in vivo revealed that CMAF had a higher chondrogenic capability for an even deposition of the specific ECM component. This study suggested that multihierarchical CMAF may have potential application for auricular cartilage regeneration.

Extracellular Vesicles from Hypoxic Pretreated Urine-Derived Stem Cells Enhance the Proliferation and Migration of Chondrocytes by Delivering miR-26a-5p.

ObjectiveStem-cell therapy is a promising treatment for cartilage defects. The newly identified urine-derived stem cells (USCs), which have multipotency and sufficient proliferative ability, are promising candidates for several tissue engineering therapies. In this study, we investigated the role of USC extracellular vehicles (EVs) in promoting the proliferation and migration of chondrocytes.DesignUSCs were characterized by measuring induced multipotent differentiation and flow cytometry analysis of surface marker expression. The EVs were isolated from USCs under normoxic conditions (nor-EVs) and hypoxic conditions (hypo-EVs). Transmission electron microscopy and western blot analysis characterized the EVs. The chondrocytes were cultured in the USC-EVs. CCK-8 assay and EdU staining detected the proliferation of chondrocytes, and transwell assay detected their migration. miR-26a-5p expression in EVs was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The target relationship of miR-26a-5p and phosphatase and tensin homolog (PTEN) was predicted and confirmed. The roles of EVs-miR-26a-5p and PTEN on the proliferation and migration of chondrocytes were also investigated.ResultsHypo-EVs showed a superior effect in promoting the proliferation and migration of chondrocytes than nor-EVs. Mechanistically, USC-EVs delivered miR-26a-5p into chondrocytes to overexpress miR-26a-5p. PTEN was identified as an miR-26a-5p target in chondrocytes. The effects of EVs-miR-26a-5p on promoting the proliferation and migration of chondrocytes were mediated by its regulation of PTEN.ConclusionOur study suggested that hypoxic USC-EVs may represent a promising strategy for osteoarthritis by promoting the proliferation and migration of chondrocytes via miR-26a-5p transfer.

Increased migratory activity and cartilage regeneration by superficial-zone chondrocytes in enzymatically treated cartilage explants

BackgroundLimited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage.MethodsFull-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, −AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-β were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared.ResultsSuperficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-β was associated with higher migratory activity of the superficial-zone chondrocytes.ConclusionsIn enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-β combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration.

Chondroitin Sulfate Enhances Proliferation and Migration via Inducing β-Catenin and Intracellular ROS as Well as Suppressing Metalloproteinases through Akt/NF-κB Pathway Inhibition in Human Chondrocytes.

Chondroitin sulfate (CS) is found in humans' cartilage, bone, cornea, skin, and arterial wall. It consists of the foundation substance in the extracellular matrix (ECM) of connective tissue. The oral supplement form of CS is clinically used in treating osteoarthritis (OA). Cell migration was observed by the transwell assay. The EMT, Akt/IKK/IκB pathways, TIMPs, collagen and MMPs in cell lysate were determined by Western blotting. The expression of MMP activity was determined by gelatin zymography. The production of reactive oxygen species (ROS) was determined by using a fluorescence spectrophotometer. In the current report, we demonstrated that CS can increase the cell proliferation and migration of chon-001 chondrocytes. Treatment with CS induced the epithelial-mesenchymal transition and increased the expression of type II collagen and TIMP-1/TIMP2 and inhibited the expressions and activities of metalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2). The phosphorylation of Akt, IκB kinase (IKK), IκB and p65 was decreased by CS. CS treatment resulted in β-catenin production and XAV939, a β-catenin inhibitor, and inhibited the cell proliferation by CS treatment. In addition, also significantly induced intracellular ROS generation. Treatment with antioxidant propyl gallate blocked cell migration induced by CS. We demonstrated that CS induced cell proliferation and migration of chondrocytes by inducing β-catenin and enhancing ROS production. Moreover, our studies demonstrated that CS can increase the activity of chondrocytes and help patients with osteoarthritis to restore cartilage function.

Exosomes derived from hypoxia preconditioned mesenchymal stem cells laden in a silk hydrogel promote cartilage regeneration via the miR-205–5p/PTEN/AKT pathway

Tissue engineering has promising prospects for cartilage regeneration. However, there remains an urgent need to harvest high quality seed cells. Bone marrow mesenchymal cells (BMSCs), and in particular their exosomes, might promote the function of articular chondrocytes (ACs) via paracrine mechanisms. Furthermore, preconditioned BMSCs could provide an enhanced therapeutic effect. BMSCs naturally exist in a relatively hypoxic environment (1%–5% O2); however, they are usually cultured under higher oxygen concentrations (21% O2). Herein, we hypothesized that hypoxia preconditioned exosomes (H-Exos) could improve the quality of ACs and be more conducive to cartilage repair. In our study, we compared the effects of exosomes derived from BMSCs preconditioned with hypoxia and normoxia (N-Exos) on ACs, demonstrating that H-Exos significantly promoted the proliferation, migration, anabolism and anti-inflammation effects of ACs. Furthermore, we confirmed that hypoxia preconditioning upregulated the expression of miR-205–5p in H-Exos, suggesting that ACs were promoted via the miR-205–5p/PTEN/AKT pathway. Finally, an injectable silk fibroin (SF) hydrogel containing ACs and H-Exos (SF/ACs/H-Exos) was utilized to repair cartilage defects and effectively promote cartilage regeneration in vivo. The application of SF/ACs/H-Exos hydrogel in cartilage regeneration therefore has promising prospects. Statement of significanceCartilage tissue engineering (CTE) has presented a promising prospect. However, the quality of seed cells is an important factor affecting the repair efficiency. Our study demonstrates for the first time that the exosomes derived from hypoxia preconditioned BMSCs (H-Exos) effectively promote the proliferation, migration and anabolism of chondrocytes and inhibit inflammation through miR-205–5p/PTEN/AKT pathway. Furthermore, we fabricated an injectable silk fibrion (SF) hydrogel to preserve and sustained release H-Exos. A complex composed of SF hydrogel, H-Exos and chondrocytes can effectively promote the regeneration of cartilage defects. Therefore, this study demonstrates that hypoxia pretreatment could optimize the therapeutic effects of BMSCs-derived exosomes, and the combination of exosomes and SF hydrogel could be a promising therapeutic method for cartilage regeneration.

Hypoxia-Preconditioned Extracellular Vesicles from Mesenchymal Stem Cells Improve Cartilage Repair in Osteoarthritis.

In the past decade, mesenchymal stem cells (MSCs) have been widely used for the treatment of osteoarthritis (OA), and extracellular vesicles (EVs) may play a major role in the efficacy of this treatment. Hypoxia can change the cargo and biological functions of MSC-derived EVs (MSC-EVs). The aim of the present study was to determine whether the effects of hypoxia-preconditioned MSC-EVs on OA cartilage repair are superior to normoxia-preconditioned MSC-EVs. By using in vitro and in vivo OA models, we verified that hypoxia-preconditioned MSC-EVs improved chondrocyte proliferation and migration and suppressed chondrocyte apoptosis to a greater extent than normoxia-preconditioned MSC-EVs. Furthermore, we found that hypoxia altered the microRNA expression in MSC-EVs and identified four differentially expressed microRNAs: hsa-miR-181c-5p, hsa-miR-18a-3p, hsa-miR-376a-5p, and hsa-miR-337-5p. Bioinformatics analysis revealed that hypoxic pretreatment may promote cartilage repair by stimulating chondrocyte proliferation and migration and suppressing chondrocyte apoptosis through the miRNA-18-3P/JAK/STAT or miRNA-181c-5p/MAPK signaling pathway. Therefore, hypoxia-preconditioned EVs may be a novel treatment for OA.

PDGF-AA promotes gap junction intercellular communication in chondrocytes via the PI3K/Akt pathway

ABSTRACT Background Gap junction intercellular communication (GJIC) plays an important role in cell growth, development and homeostasis. Connexin 43 (Cx43) is an important half-channel protein responsible for gap junction formation. Platelet-derived growth factor AA (PDGF-AA) regulates the proliferation, migration, metabolism, apoptosis and cell cycle of chondrocytes. However, the role of PDGF-AA in gap junction intercellular communication in chondrocytes is not fully understood. In the current study, we performed experiments to explore the effect of PDGF-AA on GJIC and its underlying biomechanical mechanism. Methods qPCR was performed to determine the expression of PDGF, PDGFR and connexin family genes in chondrocytes and/or cartilage. A scrape loading/dye transfer assay was used to determine GJIC. Western blot analysis was applied to detect the expression of Cx43 and PI3K/Akt signaling pathway proteins. Immunofluorescence staining was utilized to examine protein distribution. Scanning electron microscopy was used to delineate the morphology of chondrocytes. Results Expression of PDGF-A mRNA was highest among the PDGF family in chondrocytes and cartilage tissues. PDGF-AA promoted functional GJIC formation in chondrocytes by upregulating the expression of Cx43. Enhanced functional GJIC formation in chondrocytes induced by PDGF-AA occurred through the activation of PI3K/Akt signaling and its nuclear accumulation. Conclusion For the first time, this study provides evidence demonstrating the role of PDGF-AA in cell-to-cell communication in chondrocytes through mediating Cx43 expression.

Exosomes derived from platelet-rich plasma administration in site mediate cartilage protection in subtalar osteoarthritis

Subtalar osteoarthritis (STOA) is often secondary to chronic ankle sprains, which seriously affects the quality of life of patients. Due to its etiology and pathogenesis was not studied equivocally yet, there is currently a lack of effective conservative treatments. Although they have been used for tissue repair, platelet-rich plasma-derived exosomes (PRP-Exo) have the disadvantage of low retention and short-lived therapeutic effects. This study aimed to determine whether incorporation of PRP-Exo in thermosensitive hydrogel (Gel) increased their retention in the joint and thereby playing a therapeutic role on STOA due to chronic mechanical instability established by transecting lateral ligaments (anterior talofibular ligament (ATFL)/calcaneal fibular ligament (CFL)). PRP-Exo incorporated Gel (Exo-Gel) system, composed of Poloxamer-407 and 188 mixture-based thermoresponsive hydrogel matrix in an optimal ratio, was determined by its release ability of Exo and rheology of Gel response to different temperature. The biological activity of Exo-Gel was evaluated in vitro, and the therapeutic effect of Exo-Gel on STOA was evaluated in vivo. Exo released from Exo-Gel continuously for 28 days could promote the proliferation and migration of mouse bone mesenchymal stem cells (mBMSCs) and chondrocytes, at the same time enhance the chondrogenic differentiation of mBMSCs, and inhibit inflammation-induced chondrocyte degeneration. In vivo experiments confirmed that Exo-Gel increased the local retention of Exo, inhibited the apoptosis and hypertrophy of chondrocytes, enhanced their proliferation, and potentially played the role in stem cell recruitment to delay the development of STOA. Thus, Delivery of PRP-Exo incorporated in thermosensitive Gel provides a novel approach of cell-free therapy and has therapeutic effect on STOA.Graphical

The role of fibroblast growth factor 8 in cartilage development and disease.

Fibroblast growth factor 8 (FGF‐8), also known as androgen‐induced growth factor (AIGF), is presumed to be a potent mitogenic cytokine that plays important roles in early embryonic development, brain formation and limb development. In the bone environment, FGF‐8 produced or received by chondrocyte precursor cells binds to fibroblast growth factor receptor (FGFR), causing different levels of activation of downstream signalling pathways, such as phospholipase C gamma (PLCγ)/Ca2+, RAS/mitogen‐activated protein kinase‐extracellular regulated protein kinases (RAS/MAPK‐MEK‐ERK), and Wnt‐β‐catenin‐Axin2 signalling, and ultimately controlling chondrocyte proliferation, differentiation, cell survival and migration. However, the molecular mechanism of FGF‐8 in normal or pathological cartilage remains unclear, and thus, FGF‐8 represents a novel exploratory target for studies of chondrocyte development and cartilage disease progression. In this review, studies assessing the relationship between FGF‐8 and chondrocytes that have been published in the past 5 years are systematically summarized to determine the probable mechanism and physiological effect of FGF‐8 on chondrocytes. Based on the existing research results, a therapeutic regimen targeting FGF‐8 is proposed to explore the possibility of treating chondrocyte‐related diseases.