Chondrocyte Migration Research Articles

The Fibroblast-Like Synoviocyte Derived Exosomal Long Non-coding RNA H19 Alleviates Osteoarthritis Progression Through the miR-106b-5p/TIMP2 Axis.

Osteoarthritis (OA) is a common degenerative joint disease that affects people worldwide. The interaction between fibroblast-like synoviocytes (FLSs) and chondrocytes may play a vital role in OA disease pathology. However, the underlying mechanisms by which FLSs exert regulatory effects on chondrocytes still need to be elucidated. Exosomes, small membrane vesicles secreted from living cells, are known to play a variety of roles in mediating cell-to-cell communication through the transferring of biological components such as non-coding RNAs and proteins. Here, we investigate the cellular processes of chondrocytes regulated by FLS-derived exosomes and the mechanisms of action underlying the functions of exosomes in OA pathogenesis. We observed that exosome-mediated cartilage repair was characterized by increased cell viability and migration as well as alleviated matrix degradation. Using chondrocyte cultures, the enhanced cellular proliferation and migration during exosome-mediated cartilage repair was linked to the exosomal lncRNA H19-mediated regulation of the miR-106b-5p/TIMP2 axis. Transfection of miR-106-5p mimics in chondrocytes significantly decreased cell proliferation and migration, promoted matrix degradation characterized by elevated MMP13 and ADAMTS5 expression, and reduced the expression of COL2A1 and ACAN in chondrocytes. Furthermore, we found that TIMP2 was directly regulated by miR-106-5p. Co-transfections of miR-106-5p mimics and TIMP2 resulted in higher levels of COL2A1 and ACAN, but lower levels of MMP13 and ADAMTS5. Together, these observations demonstrated that the lncRNA H19 may promote chondrocyte proliferation and migration and inhibit matrix degradation in OA possibly by targeting the miR-106b-5p/TIMP2 axis. In the future, H19 may serve as a potential therapeutic target for the treatment of OA.

Hyaluronic acid-based shape memory cryogel scaffolds for focal cartilage defect repair

Purpose: Traumatic joint injuries can result in focal cartilage defects that regenerate poorly due to a lack of blood supply and low chondrocyte density. Cell-laden hydrogel scaffolds are under study but provide little flexibility in modulating their pore size, interconnectivity, swelling and stiffness, properties required for a conducive biomimetic environment. In this study, we engineered an injectable and biomimetic shape memory hyaluronic acid (HA)-based cryogel scaffolds with a macroporous and highly interconnected network in three-dimensions seeded with chondrocytes. Due to their unique ability to withstand up to 90% strains and rapidly recover their shape back, they can be arthroscopically delivered to fill up focal chondral defects for cartilage repair. Here we studied whether chondrocytes encapsulated within cryogels remain viable and metabolically active following injection and provide a superior micro-environment than conventional (nanoporous) hydrogels for cartilage regeneration. We also investigated how RGD-functionalized cryogel affects chondrocyte adhesion, migration and proliferation. Methods: Synthesis of HA-based Cryogels and Hydrogels: HA cryogels were prepared using HA modified with glycidyl methacrylate (HAGM) and either Acryl-PEG-G4RGDS to synthesis gels functionalized with RGD (Cryo RGD+) or Acryl-PEG-methoxy to synthesize gels without RGD (Cryo RGD-) in deionized (DI) water. The mixture was cast in 6 mm diam x 1.5 mm cylindrical discs using APS/TEMED as our initiator system. The samples were frozen at -20°C and allowed to cryopolymerize overnight. Next, cryogels were brought to RT and washed/sanitized prior to cell seeding. HA hydrogels (Hydro RGD+) were fabricated via photopolymerization by mixing HAGM, RGD, and lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) photo-initiator in DI water. Chondrocyte seeding and in-vitro culture:Chondrocytes harvested from calf knee femoral condyle cartilage were seeded onto each scaffold and cultured for 15 days. Injectability test: Cell-laden cryogels were incubated overnight to enable complete cell adhesion and subsequently syringe injected through a 16-gauge needle before incubation for another 24h following which cell viability and metabolism were quantified. Biochemical analysis: At the end of 15-day culture, gel samples were digested to quantify the cell metabolism (Alamar Blue), DNA (PicoGreen) and GAG (DMMB) content. Cell viability and cytoskeleton staining: Gel samples were fixed and stained for nucleus, actin, dead cells and confocal imaged at 40x. Results: Shape memory HA-based cryogels fully recovered their shape back following syringe injection through a 16-gauge needle without impacting cell metabolism or viability (Figs 1A-C). Scanning electron microscopy (SEM) images showed an average pore size of 49.2 ± 15 μm (Fig 1D) rendering a macroporous structure with high interconnectivity, which was 7x higher than hydrogels (Fig 1E). This unique macrostructural architecture enables efficient transport of nutrients and metabolites thereby offering a superior microenvironment for chondrocytes; both cell metabolism and GAG content was 2x higher in cryogels than hydrogels after 15-day culture (Figs 1F-G). Additionally, a significant amount of GAG (∼70 μg) was released from both cryogels into media owing to their macroporous structure; no released GAG was measured in media from Hydro RGD+ (Fig 1G). The presence of RGD (cell-adhesion peptide) did not improve cell adhesion or biosynthesis rates. Chondrocytes formed larger organoid-like structures in Cryo RGD- while remaining homogenously dispersed in the presence of RGD in Cryo RGD+ (Fig 1H). Conclusions: Syringe injectable HA-based shape memory cryogels provide a conducive microenvironment for chondrocyte adhesion, proliferation and matrix biosynthesis for use in repair of cartilage defects.

KLF10 is upregulated in osteoarthritis and inhibits chondrocyte proliferation and migration by upregulating Acvr1 and suppressing inhbb expression

BackgroudOsteoarthritis (OA) is a common disease caused by chondrocyte dysfunction. KLF10 is a member of the Sp1-like transcription factor family that is involved in regulating osteoblasts, but its expression and regulatory mechanism(s) in chondrocytes are unclear. In the present study, we aimed to investigate the regulatory role of KLF10 on the pathological process of OA. MethodsKLF10 expression in the cartilaginous tissue of patients with osteoarthritis (OA) was evaluated by immunohistochemistry (IHC). Next, we generated an OA mouse model, and the histological changes in cartilage tissue were verified using H&E staining, Safranin O-Fast Green staining, and IHC assays. KLF10 expression in the articular chondrocytes of OA mice was determined by qRT-PCR and Western blotting. To investigate the role of KLF10 in regulating cell proliferation and migration, a KLF10 overexpression plasmid was constructed and transfected into primary mouse chondrocytes. Subsequently, we used RNA sequencing (RNA-seq) to screen differentially expressed genes in chondrocytes with or without KLF10 overexpression. qRT-PCR was used for verification purposes. ResultsWe found that KLF10 was upregulated in the cartilaginous tissue of patients with OA as well as in cartilaginous tissue of the OA mouse model. KLF10 overexpression inhibited the proliferation and migration of chondrocytes. Furthermore, RNA sequencing results identified increased expression of Acvr1 and decreased expression of Inhbb in cells overexpressing KLF10. Changes in mRNA expression of Acvr1 and Inhbb were confirmed by qRT-PCR. ConclusionsKLF10 inhibits chondrocyte proliferation and migration by regulating the expression of Acvr1 and Inhbb in both human and mouse OA. These data suggest that KLF10 may be a potential therapeutic target for the treatment of OA.

Intra-articular delivery of extracellular vesicles secreted by chondrogenic progenitor cells from MRL/MpJ superhealer mice enhances articular cartilage repair in a mouse injury model

BackgroundChondrogenic progenitor cells (CPCs) have high self-renewal capacity and chondrogenic potential. Intra-articular delivery of purified mesenchymal stem cells (MSCs) from MRL/MpJ “superhealer” mice increased bone volume during repair and prevents post-traumatic arthritis. Recently, although extracellular vesicles released from MSCs have been used widely for treating OA, the application of extracellular vesicles secreted by CPCs from MRL/MpJ mice in OA therapy has never been reported. In this study, we evaluated the effects of extracellular vesicles secreted by CPCs from control CBA (CBA-EVs) and MRL/MpJ mice (MRL-EVs) on proliferation and migration of murine chondrocytes. We also determined here if weekly intra-articular injections of CBA-EVs and MRL-EVs would repair and regenerate surgically induced model in mice.MethodsCPC surface markers were detected by flow cytometry. CBA-EVs and MRL-EVs were isolated using an ultrafiltration method. Nanoparticle tracking analysis, transmission electron microscopy, and western blots were used to identify extracellular vesicles. CBA-EVs and MRL-EVs were injected intra-articularly in a mouse model of surgical destabilization of the medial meniscus (DMM)-induced OA, and histological and immunohistochemistry analyses were used to assess the efficacy of exosome injections. We used miRNA-seq analysis to analyze the expression profiles of exosomal miRNAs derived from CBA-EVs as well as MRL-EVs. Cell-counting and scratch assays were used to evaluate the effects of CBA-EVs and MRL-EVs on proliferation and migration of murine chondrocytes, respectively. Meanwhile, a specific RNA inhibitor assesses the roles of the candidate miRNAs in CPC-EV-induced regulation of function of chondrocytes.ResultsBoth CBA-EVs and MRL-EVs stimulated chondrocyte proliferation and migration, but MRL-EVs exerted a stronger effect than CBA-EVs. The similar result was also observed in in vivo study, which indicated that injecting either CBA-EVs or MRL-EVs attenuated OA, but MRL-EVs showed a superior therapeutic effect in comparison with CBA-EVs. The results of bioinformatics analyses revealed that the differentially expressed exosomal miRNAs participated in multiple biological processes. We identified 80 significantly upregulated and 100 downregulated miRNAs. Moreover, we found that the top 20 differentially expressed exosomal miRNAs connected OA repair to processes such as AMPK signaling, regulation of autophagy, and insulin signaling. Notably, miRNA 221-3p were highly enriched in MRL-Exos and treatment with miR 221-3p inhibitor markedly decreased chondrocyte proliferation and migration induced by CBA-EVs or MRL-EVs in vitro.ConclusionsThis is the first study to demonstrate MRL-EVs had a greater therapeutic effect on the treatment of OA than CBA-EVs. This study will hopefully provide new insight into the pathogenesis, prevention, and treatment of OA.

Synovial fibroblast-derived exosomal microRNA-106b suppresses chondrocyte proliferation and migration in rheumatoid arthritis via down-regulation of PDK4.

Fibroblast-derived exosomes have been reported to transfer microRNAs to recipient cells, where they regulate target gene expression, which is of interest for understanding the basic biology of inflammation, tissue homeostasis, and development of therapeutic approaches. Initial microarray-based analysis carried out in this study identified the rheumatoid arthritis (RA)-related differentially expressed gene pyruvate dehydrogenase kinase 4 (PDK4). Subsequently, the upstream regulatory microRNA-106b (miR-106b) of PDK4 was predicted with bioinformatic analyses. A collagen-induced arthritis (CIA)-induced mouse model was established, and exosomes were isolated from synovial fibroblasts (SFs) and transferred into chondrocytes to identify the role of exosomes in rheumatoid arthritis (RA). We found that PDK4 was poorly expressed in RA cartilage tissues and chondrocytes, while miR-106b was highly expressed in RA SFs and SF-derived exosomes. Notably, PDK4 was confirmed as a target gene of miR-106b. Over-expression of PDK4 promoted the proliferation and migration abilities of chondrocytes and inhibited their apoptosis as well as affected the receptor activator of nuclear factor kappa B ligand (RANKL)/RANK/osteoprotegerin (OPG) system. Meanwhile, miR-106b was delivered from SFs to chondrocytes through exosomes, which suppressed chondrocyte proliferation and migration and accelerated apoptosis as well as affected the RANKL/RANK/OPG system via down-regulation of PDK4. Furthermore, in vivo results validated that miR-106b inhibition could relieve CIA-induced RA. Taken together, SF-derived exosomal miR-106b stimulates RA initiation by targeting PDK4, indicating a physiologically validated potential approach for the prevention and treatment of RA. KEY MESSAGES: PDK4 is decreased in chondrocytes of RA, while miR-106b is increased in SFBs. PDK4 promotes proliferation and migration of chondrocytes. miR-106b could target 3'UTR of PDK4 gene. SFB-exosomal miR-106b inhibits proliferation and migration of chondrocytes. Inhibition of miR-106b attenuates RA progression in a CIA mouse model.

Pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration

BackgroundThe mesenchymal stem cell (MSC) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. MSCs are “environmentally responsive” to local micro-environmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. We have previously shown that exposures of MSCs to pulsed electromagnetic fields (PEMFs) enhanced MSC chondrogenesis. Here, we investigate the influence of PEMF exposure over the paracrine activity of MSCs and its significance to cartilage regeneration.MethodsConditioned medium (CM) was generated from MSCs subjected to either 3D or 2D culturing platforms, with or without PEMF exposure. The paracrine effects of CM over chondrocytes and MSC chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and MSCs was assessed.ResultsWe show that benefits of magnetic field stimulation over MSC-derived chondrogenesis can be partly ascribed to its ability to modulate the MSC secretome. MSCs cultured on either 2D or 3D platforms displayed distinct magnetic sensitivities, whereby MSCs grown in 2D or 3D platforms responded most favorably to PEMF exposure at 2 mT and 3 mT amplitudes, respectively. Ten minutes of PEMF exposure was sufficient to substantially augment the chondrogenic potential of MSC-derived CM generated from either platform. Furthermore, PEMF-induced CM was capable of enhancing the migration of chondrocytes and MSCs as well as mitigating cellular inflammation and apoptosis.ConclusionsThe findings reported here demonstrate that PEMF stimulation is capable of modulating the paracrine function of MSCs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. The PEMF-induced modulation of the MSC-derived paracrine function for directed biological responses in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical applications.

Platelet-Rich Fibrin Facilitates One-Stage Cartilage Repair by Promoting Chondrocytes Viability, Migration, and Matrix Synthesis.

The main aim of this study is to develop a one-stage method to combine platelet-rich fibrin (PRF) and autologous cartilage autografts for porcine articular cartilage repair. The porcine chondrocytes were treated with different concentrations of PRF-conditioned media and were evaluated for their cell viability and extracellular glycosaminoglycan (GAG) synthesis during six day cultivation. The chemotactic effects of PRF on chondrocytes on undigested cartilage autografts were revealed in explant cultures. For the in vivo part, porcine chondral defects were created at the medial femoral condyles of which were (1) left untreated, (2) implanted with PRF combined with hand-diced cartilage grafts, or (3) implanted with PRF combined with device-diced cartilage grafts. After six months, gross grades, histological, and immunohistochemical analyses were compared. The results showed that PRF promotes the viability and GAG expression of the cultured chondrocytes. Additionally, the PRF-conditioned media induce significant cellular migration and outgrowth of chondrocytes from undigested cartilage grafts. In the in vivo study, gross grading and histological scores showed significantly better outcomes in the treatment groups as compared with controls. Moreover, both treatment groups showed significantly more type II collagen staining and minimal type I collagen staining as compared with controls, indicating more hyaline-like cartilage and less fibrous tissue. In conclusion, PRF enhances the viability, differentiation, and migration of chondrocytes, thus, showing an appealing capacity for cartilage repair. The data altogether provide evidences to confirm the feasibility of a one-stage, culture-free method of combining PRF and cartilage autografts for repairing articular cartilage defects. From translational standpoints, these advantages benefit clinical applications by simplifying and potentiating the efficacy of cartilage autograft transplants.

Down-regulation of MiR-138-5p Protects Chondrocytes ATDC5 and CHON-001 from IL-1 β-induced Inflammation Via Up-regulating SOX9.

Osteoarthritis (OA) pertains to a chronic disease of degenerative joints distinguished by articular cartilage destruction, subchondral bone remodeling, osteophyte formation, and inflammatory changes. Chondrocyte apoptosis is inextricably linked to cartilage degeneration. SRY-related high-mobility-group-box 9 (SOX9) is a well-acknowledged transcription factor in the chondrogenesis. Nevertheless, the detailed function of miR-138-5p/SOX9 in OA remains to be fully clarified. qRT-PCR was performed to measure the expressions of miR-138-5p and SOX9 mRNA in OA and normal cartilage tissues and cells. Human chondrocyte cell lines, CHON-001 and ATDC5, were treated with different doses of interleukin-1β (IL-1β) to simulate the inflammatory response environment of OA. miR-138-5p mimics, miR-138-5p inhibitors, and SOX9 small interfering RNA (siRNA) were constructed and transfected into CHON-001 and ATDC5 cells. CCK-8 was conducted to determine the cell viability and transwell assay was used to monitor the migration of cells. Western blot was carried out to detect the expressions of apoptosis- related factors. Enzyme-linked immunosorbent assay (ELISA) was adopted to measure the contents of inflammatory factors. TargetScan predicted SOX9 was a target gene of miR-138-5p, which was then verified by luciferase assay. miR-138-5p expression was down-regulated in OA and regulated SOX9 expression. The downregulation of miR-138-5p facilitated the proliferation and migration of CHON-001 and ATDC5 cells, while impeded their apoptosis and inflammatory response. Besides, down-regulated SOX9 can counteract the promoting effect of down-regulated miR-138-5p on the proliferation and migration of chondrocytes. miR-138-5p can arrest the proliferation and migration of CHON-001 and ATDC5 via restraining SOX9, and facilitate the apoptosis and inflammation. This study revealed the protective effect of down-regulated miR-138-5p on the inflammatory injury of chondrocytes caused by IL-1β.

MiR-23a-3p-abundant small extracellular vesicles released from Gelma/nanoclay hydrogel for cartilage regeneration

ABSTRACT Articular cartilage has limited self-regenerative capacity and the therapeutic methods for cartilage defects are still dissatisfactory in clinic. Recent studies showed that exosomes derived from mesenchymal stem cells promoted chondrogenesis by delivering bioactive substances to the recipient cells, indicating exosomes might be a novel method for repairing cartilage defect. Herein, we investigated the role and mechanism of human umbilical cord mesenchymal stem cells derived small extracellular vesicles (hUC-MSCs-sEVs) on cartilage regeneration. In vitro results showed that hUC-MSCs-sEVs promoted the migration, proliferation and differentiation of chondrocytes and human bone marrow mesenchymal stem cells (hBMSCs). MiRNA microarray showed that miR-23a-3p was the most highly expressed among the various miRNAs contained in hUC-MSCs-sEVs. Our data revealed that hUC-MSCs-sEVs promoted cartilage regeneration by transferring miR-23a-3p to suppress the level of PTEN and elevate expression of AKT. Moreover, we fabricated Gelatin methacrylate (Gelma)/nanoclay hydrogel (Gel-nano) for sustained release of sEVs, which was biocompatible and exhibited excellent mechanical property. In vivo results showed that hUC-MSCs-sEVs containing Gelma/nanoclay hydrogel (Gel-nano-sEVs) effectively promoted cartilage regeneration. These results indicated that Gel-nano-sEVs have a promising capacity to stimulate chondrogenesis and heal cartilage defects, and also provided valuable data for understanding the role and mechanism of hUC-MSCs-sEVs in cartilage regeneration.

Small extracellular vesicles from human adipose-derived stem cells attenuate cartilage degeneration

ABSTRACT Osteoarthritis (OA) is a chronic degenerative disease of articular cartilage that is the most common joint disease worldwide. Mesenchymal stem cells (MSCs) have been the most extensively explored for the treatment of OA. Recently, it has been demonstrated that MSC-derived extracellular vesicles (EVs) may contribute to the potential mechanisms of MSC-based therapies. In this study, we investigated the therapeutic potential of human adipose-derived stem cells EVs (hASC-EVs) in alleviating OA, along with the mechanism. EVs were isolated from the culture supernatants of hASCs by a multi-filtration system based on the tangential flow filtration (TFF) system. The isolated EVs were characterised using dynamic light scattering (DLS), transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and flow cytometry analysis. The hASC-EVs not only promoted the proliferation and migration of human OA chondrocytes, but also maintained the chondrocyte matrix by increasing type Ⅱ collagen synthesis and decreasing MMP-1, MMP-3, MMP-13 and ADAMTS-5 expression in the presence of IL-1β in vitro. Intra-articular injection of hASC-EVs significantly attenuated OA progression and protected cartilage from degeneration in both the monosodium iodoacetate (MIA) rat and the surgical destabilisation of the medial meniscus (DMM) mouse models. In addition, administration of hASC-EVs inhibited the infiltration of M1 macrophages into the synovium. Overall results suggest that the hASC-EVs should be considered as a potential therapeutic approach in the treatment of OA.

A decellularized scaffold derived from squid cranial cartilage for use in cartilage tissue engineering.

Decellularized cartilage scaffold (DCS) is an emerging substitute for cartilage defect application. However, it is hard to preserve an intact structure of such scaffolds derived from terrestrial animals, because of their dense and compact constitution. In contrast, squid (Dosidicus gigas) cranial cartilage, which possesses a relatively loose structure, could be easily decellularized using mild conditions and it retains the original microstructures of extracellular matrix (ECM). Herein, decellularized squid cranial cartilage scaffold (DSCS) was fabricated successfully after substantially removing the cells, which contained abundant ECM components (proteoglycans and type II collagen). Microscopic structure results showed that the DSCS possesses a relatively smooth and dense surface with a favorable interconnected inner porous structure for cell growth. DSCS exhibited excellent biomechanics and hydrophilicity. In vitro experiments indicated that the scaffold extracts were not toxic to cells, and were amenable to chondrocyte migration. Meanwhile, chondrocytes seeded in DSCS could maintain a favorable viability and present a spreading morphology. Furthermore, the in vivo experiments revealed that both cell-free scaffold and cell-laden scaffold exert promotive effects for the regeneration of cartilage in a full-thickness rabbit cartilage defect model. Taken together, these results suggested that DSCS presents a novel and promising cell-free therapeutic choice for cartilage tissue engineering.

Exosomes produced from 3D cultures of umbilical cord mesenchymal stem cells in a hollow-fiber bioreactor show improved osteochondral regeneration activity

Animal and clinical studies have shown that mesenchymal stem cells (MSCs) play an important role in cartilage repair. The therapeutic effect of mesenchymal stem cells based therapies has been increasingly demonstrated to exosome-mediated paracrine secretion. Here, we investigated the cellular processes and mechanism of exosomes produced by conventional 2D culture (2D-Exos) and exosomes produced from 3D culture (3D-Exos) of umbilical MSCs (U-MSCs) in a hollow-fiber bioreactor for the treatment of cartilage repair. We found that the yield of 3D-Exos was 7.5-fold higher than that of 2D-Exos. The in vitro experiments indicated that both 2D-Exos and 3D-Exos can stimulate chondrocyte proliferation, migration, and matrix synthesis, and inhibit apoptosis, with 3D-Exos exerting a stronger effect than 2D-Exos. This effect was partly attributed to the activation of transforming growth factor beta 1 and Smad2/3 signaling. The injection of 2D-Exos and 3D-Exos showed enhanced gross appearance and attenuated cartilage defect; however, 3D-Exos showed a superior therapeutic effect than 2D-Exos. In summary, our study provides novel insights into the chondroprotective effects of exosomes produced from 3D culture of U-MSCs in a hollow-fiber bioreactor. Because of its promising biological function and high yield, 3D-Exos may become a promising therapeutic method for the treatment of cartilage defects.

Expression of Galectins-1 in Rat Traumatic Osteoarthritis and Its Regulation Mechanism

Traumatic arthritis is a common orthopedic surgery disease that seriously affects the health of patients. The expression and role of Galectins in traumatic osteoarthritis remains unclear. SD rats were divided into control group and osteoarthritis model group. Real-time PCR and ELISA were used to analyze Galectins-1 expression. Chondrocytes were isolated and cultured and divided into control group, Galectins-1 siRNA group and Galectins-1 group followed by analysis of proliferation of chondrocytes by MTT assay, cell migration by Transewell chamber, expression of RUNX2 and ADAMTS-4/5 by Real-time PCR, and PI3K/Akt by Western blot. Galectins-1 mRNA and secretion in synovial fluid was significantly reduced in model group compared to control (P < 0.05). Transfection of Galectins-1-pcDNA3.1 plasmid into chondrocytes of osteoarthritic rats significantly increased the expression of Galectins-1, promoted chondrocyte proliferation and cell migration, and downregulated RUNX2 and ADAMTS-4/5 (P < 0.05). Up-regulation of Galectins-1 blocked the expression of PI3K/Akt signaling pathway. Transfection of Galectins-1 siRNA significantly reduced the expression of Galectins-1, inhibited chondrocyte proliferation and cell migration, and upregulated RUNX2 and ADAMTS-4/5 (P < 0.05). Down-regulation of Galectins-1 up-regulated PI3K/Akt signaling pathway. Galectins-1 expression is reduced in joint tissues in rat model of traumatic osteoarthritis. Up-regulation of Galectins-1 expression can promote chondrocyte proliferation and migration by regulating PI3K/Akt signaling pathway, which may reduce chondrocyte damage in rats with traumatic osteoarthritis.

NR4A1 promotes TNF‑α‑induced chondrocyte death and migration injury via activating theAMPK/Drp1/mitochondrial fission pathway.

Nuclear receptor subfamily 4 group A member 1 (NR4A1)-induced chondrocyte death plays a critical role in the development of osteoarthritis through poorly defined mechanisms. The present study aimed to investigate the role of NR4A1 in regulating chondrocyte death in response to tumor necrosis factor-α (TNF-α) and cycloheximide (CHX) treatment, with a focus on mitochondrial fission and the AMP-activated protein kinase (AMPK) signaling pathway. The results demonstrated that NR4A1 was significantly upregulated in TNF-α and CHX exposed chondrocytes. Increased NR4A1 triggered mitochondrial fission via the AMPK/dynamin-related protein 1 (Drp1) pathway, resulting in mitochondrial dysfunction, and mitochondrial permeability transition pore (mPTP) opening-related cell death. Furthermore, excessive mitochondrial fission impaired chondrocyte migration through imbalance of F-actin homeo-stasis. Inhibiting NR4A1 attenuated TNF-α and CHX-induced mitochondrial fission and, thus, reduced mitochondrial dysfunction in chondrocytes, mPTP opening-related cell death and migration injury. Altogether, the present data confirmed that mitochondrial fission was involved in NR4A1-mediated chondrocyte injury via regulation of mitochondrial dysfunction, mPTP opening-induced cell death and F-actin-related migratory inhibition.

Chondrocyte colonisation of a tissue-engineered cartilage substitute under a mechanical stimulus

Cell-free collagen scaffolds as cartilage substitute for small focal defects show promising results in first clinical studies. However, chondrocyte migration between scaffolds and the colonisation process of a cell-free implant is yet to be fully understood. We here focus on mechanobiological interdependencies between cell migration and mechanical stimulus in a 3D environment. We develop an in vitro model composed of a human chondrocyte-seeded collagen base and adjacent cell-free collagen type I scaffolds of varying collagen concentrations. Constructs are either cultured statically or dynamically under the influence of a physiological compression (0.5Hz, 0.5% initial strain). After 20 days we identify vital chondrocytes inside all collagen implants, proving that chondrocytes migrated from the underlying scaffold into the implants. Chondrocytes have not colonised the entire sample and are predominantly found in the bottom of the implant. In static culture conditions, a nearly equal cell number is found inside of all collagen scaffolds. In dynamic culture, the total amount of cells is increased by 30% to 320%, with the highest population in a commercial implant. Differences in cell population between the materials in dynamic culturing can be referred to differences in mechanical properties of the scaffolds, such as strain-rate insensitivity fostering the colonisation process.

Autologous Micro-Fragmented Adipose Tissue as Stem Cell-Based Natural Scaffold for Cartilage Defect Repair

Osteoarthritis (OA) poses a tough challenge worldwide. Adipose-derived stem cells (ASCs) have been proved to play a promising role in cartilage repair. However, enzymatic digestion, ex vivo culture and expansion, with significant senescence and decline in multipotency, limit their application. The present study was designed to obtain micro-fragmented adipose tissue (MFAT) through gentle mechanical force and determine the effect of this stem cell-based natural scaffold on repair of full-thickness cartilage defects. In this study, ASCs sprouted from MFAT were characterized by multi-differentiation induction and flow cytometry. Scratch and transwell migration assays were operated to determine whether MFAT could promote migration of chondrocytes in vitro. In a rat model, cartilage defects were created on the femoral groove and treated with intra-articular injection of MFAT or PBS for 6 weeks and 12 weeks (n = 12). At the time points, the degree of cartilage repair was evaluated by histological staining, immunohistochemistry and scoring, respectively. Two unoperated age-matched animals served as native controls. ASCs derived from MFAT possessed properties to differentiate into adipocytes, osteocytes and chondrocytes, with expression of mesenchymal stem cell markers (CD29, 44, 90) and no expression of hematopoietic markers (CD31, 34, 45). In addition, MFAT could significantly promote migration of chondrocytes. MFAT-treated defects showed improved macroscopic appearance and histological evaluation compared with PBS-treated defects at both time points. After 12 weeks of treatment, MFAT-treated defects displayed regular surface, high amount of hyaline cartilage, intact subchondral bone reconstruction and corresponding formation of type I, II, and VI collagen, which resembled the normal cartilage. This study demonstrates the efficacy of MFAT on cartilage repair in an animal model for the first time, and the utility of MFAT as a ready-to-use therapeutic alternative to traditional stem cell therapy.

Medicina rigenerativa in ortopedia e traumatologia: la stimolazione biofisica

Cartilage lesions are considered to be the first cause of disability in the active population. Treating cartilage injuries is often a challenge for orthopaedic surgeons, owing to the poor regenerative potential of chondrocytes. Pulsed electromagnetic fields (PEMFs) improve cellular proliferation and migration, and ECM induction. PEMFs during or after surgery for chondral lesion have been demonstrated to be useful in inducing better repair and migration of chondrocytes into the bioscaffolds, and to lead to improvement in patient symptoms after surgery. In this review of the literature, we collected a significative number of studies, which support using biophysical stimulation, in particular PEMFs, as first-line treatment during and after surgery to treat chondral lesion.

Chondrocytes From Device-Minced Articular Cartilage Show Potent Outgrowth Into Fibrin and Collagen Hydrogels.

Background:Transplantation of autologous minced cartilage is an established procedure to repair chondral lesions. It relies on the migration of chondrocytes out of cartilage particles into a biomaterial. So far, there is no efficient way to finely mince cartilage. No consensus exists on the nature of the biomaterial to be used to promote chondrocyte migration.Purpose/Hypothesis:This study aimed to investigate the potential clinical use of a custom-made mincing device as well as a possible alternative biomaterial to fibrin glue. The device was tested for its effect on chondrocyte viability and on subsequent chondrocyte migration into either a fibrin or a collagen gel. We hypothesized that device mincing would allow finer cutting and consequently more cell migration and that the gelation mechanism of the collagen biomaterial, which uses the clotting of platelet-rich plasma, would enhance matrix production by outgrown chondrocytes.Study Design:Controlled laboratory study.Methods:Cartilage from 12 patients undergoing knee arthroplasty was taken from the femoral condyles and subsequently either hand minced or device minced. The viability and the degree of outgrowth were quantified with live/dead assay on the generated cartilage particles and on the gels in which these particles were embedded, respectively. Matrix deposition in the biomaterials by the outgrown cells was investigated with histology.Results:The device allowed rapid mincing of the cartilage and produced significantly smaller pieces than hand mincing. The initial chondrocyte viability in cartilage particles dropped by 25% with device mincing as compared with no mincing. However, the viability in hand-minced, device-minced, and unminced samples was no longer different after 7 and 28 days in culture. Outgrowth scores were similar among the 3 groups. Fibrin and collagen biomaterials equally supported chondrocyte outgrowth and survival, but neither promoted matrix deposition after in vitro culture.Conclusion:The outgrowth potential, the viability after 28 days in culture, and the matrix deposition were not different between the mincing techniques and the tested biomaterials, yet device mincing is faster and results in significantly smaller cartilage particles.Clinical Relevance:Device mincing could become the standard method to mince cartilage for second-generation cartilage repair techniques.

Decellularized cartilage matrix scaffolds with laser-machined micropores for cartilage regeneration and articular cartilage repair

Decellularized allogeneic and xenogeneic articular cartilage matrix scaffolds (CMS) are considered ideal scaffolds for cartilage regeneration owing to their heterogeneous architecture, and biochemical and biomechanical properties of native articular cartilage. However, the dense structure of the articular cartilage extracellular matrix, particularly the arrangement of collagen fibers, limits cellular infiltration, leading to poor cartilage regeneration. In addition, the incomplete removal of xenograft cells is associated with immunogenic reaction in the host. To facilitate the migration of chondrocytes into scaffolds and the rate of decellularization processing, we applied a carbon dioxide laser technique to modify the surface of porcine CMS while retaining major properties of the scaffold. By optimizing the laser parameters, we introduced orderly, lattice-arranged conical micropores of suitable depth and diameter onto the cartilage scaffold surface without affecting the cartilage shape or mechanical properties. We found that laser-modified CMS (LM-CMS) could enhance the degree of decellularization and were conducive to cell adhesion, as compared with the intact CMS. Decellularized scaffolds were seeded with rabbit-derived chondrocytes and cultured for 8 weeks in vitro. We found that cell-scaffold constructs formed cartilage-like tissue within the micropores and on the scaffold surface. In vivo, we found that cell-scaffold constructs subcutaneously implanted into the flanks of nude mice formed ivory-white neocartilage with high contents of DNA and cartilage matrix components, as well as good mechanical strength as compared with native CMS. Furthermore, scaffolds combined with autogenous chondrocytes induced neocartilage and better structural restoration at 8 weeks after transplantation into rabbit knee articular cartilage defects. In conclusion, decellularized xenogeneic CMS with laser-machined micropores offers an ideal scaffold with high fidelity for the functional reconstruction of articular cartilage.

Synovial Fibroblasts-Derived Exosomes Overexpressing microRNA-106b Inhibits Chondrocyte Proliferation and Migration via Down-Regulation of PDK4 in Rheumatoid Arthritis

Background: Rheumatoid arthritis (RA), an autoimmune disorder, affects people of all demographics, and exosome-derived microRNAs (miRs) have also been documented to play an important role in inflammatory disease. In view of this, our study is performed to investigate the roles of synovial fibroblasts-derived exosomes expressing miR-106b in RA via regulating PDK4. Methods: The differentially expressed genes were screened by analyzing the microarray chips related to RA. The expression of PDK4 and miR-106b were determined, and their regulatory effects on RA were evaluated. Meanwhile, exosomes derived from synovial fibroblasts (SFBs) were isolated and transferred into chondrocytes to identify the role of exosomes in RA. Furthermore, the effects of SFBs-derived exosomes overexpressing miR-106b on chondrocyte proliferation and migration were assessed using lentiviral packaged antagomir miR-106b in vivo. Results: It was found that PDK4 was down-regulated while miR-106b was up-regulated in RA. PDK4 was a target gene of miR-106b. When PDK4 was over-expressed in chondrocytes, the abilities of proliferation and migration of chondrocytes were enhanced and the RANKL/RANK/OPG system was inhibited. However, when the miR-106b was transferred into the chondrocytes by exosomes derived from SFBs, the opposite growth characteristics were identified in chondrocytes. Moreover, the investigation in vivo revealed that inhibition of miR-106b in SFBs improved the symptoms of RA in mice. Conclusion: Collectively, our study demonstrates that the SFBs-derived exosomes overexpressing miR-106b aggravates RA and affected RANKL/RANK/OPG system via down-regulation of PDK4. Funding Statement: This study was supported by grants from National Natural Science Foundation of China (no.81302576), Six Talent Peak Research Project in Jiangsu Province (no. 2015-WSN-105), the 333 Project of Jiangsu Province, Jiangsu Province Youth Medical Talent Project, Yangzhou City's Key Medical Talents Program, as well as by Jiangsu Provincial Natural Science Foundation of China (no. BK20131234). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: This study was conducted with the approval of the Ethics Committee of Affiliated Hospital of Yangzhou University. All participating patients signed written informed consent documentation prior to enrollment into the study.