Aggrecan Gene Expression Research Articles

Magneto-mechanical Stimulation of Bone Marrow Mesenchymal Stromal Cells for Chondrogenic Differentiation Studies

Mechanical interaction of cells and their surroundings are prominent in mechanically active tissues such as cartilage. Chondrocytes regulate their growth, matrix synthesis, metabolism, and differentiation in response to mechanical loadings. Cells sense and respond to applied physical forces through mechanosensors such as integrin receptors. Herein, we examine the role of mechanical stimulation of integrins in regards to their mechanotransduction ability to promote chondrogenesis. For this purpose, magnetic nanoparticles were chemically bonded to cell membrane mechanoreceptors and stimulated. Histological results showed the endocytosis of nanoparticles over the experimental period, pointing out the inefficient mechanical stimulation of the mechanoreceptors. Moreover, gene expression analysis only showed significant upregulation for SOX9, whereas type II collagen and aggrecan gene expression were not significantly different from the control group. Our results suggest that magneto-mechanical stimulation studies using magnetic nanoparticles should not only focus on the mechanical aspects, but also the interaction of magnetic nanoparticles with intracellular machinery should be investigated as well.

Microtia patients: Auricular chondrocyte ECM is promoted by CGF through IGF-1 activation of the IGF-1R/PI3K/AKT pathway.

To explore the effectiveness of the insulin-like growth factor 1receptor (IGF-1R)/PI3K/AKT pathway in promoting the synthesis of the auricular chondrocyte extracellular matrix (ECM) using concentrated growth factor (CGF). Chondrocytes isolated from the remnant auricular cartilage of microtia patients were randomly divided into different experimental and control groups, then stimulated with a reagent.IGF-1 released by CGF was quantified by enzyme-linked immunosorbent assay. Glycosaminoglycan (GAG), proteoglycan, and type II collagen (COLAII) were examined by histological and immunohistological analysis. Expression levels of IGF-1R, pIGF-1R, PI3K, pPI3K, AKT, pAKT, COLAII, and Aggrecan were detected by western blot analysis technique andgene expression was tested by real-time polymerase chain reaction. CGF significantly stimulated the synthesis of COLAII and Aggrecan and increased the phosphorylation levels of IGF-1R, PI3K, and AKT. Small interfering RNA IGF-1 blocked ECM synthesis, COLAII and Aggrecan gene expression, and IGF-1R/PI3K/AKT activation. Inhibitor AG1024 and LY294002 significantly inhibited ECM synthesis and the phosphorylation of IGF-1R, PI3K, and AKT. CGF-released IGF-1 stimulates the synthesis of the auricular chondrocyte ECM via the IGF-1R/PI3K/AKT signaling pathway.

Intra-Articular Injection of Fructus Ligustri Lucidi Extract Attenuates Pain Behavior and Cartilage Degeneration in Mono-Iodoacetate Induced Osteoarthritic Rats.

Fructus Ligustri Lucidi (FLL) has been widely used as a traditional Chinese medicine (TCM) for treating soreness and weakness of waist and knees. It has potential for treating OA owing to its kidney-tonifying activity with bone-strengthening effects, but there is so far no report of its anti-OA effect. This study established a rat OA model by intra-articular (IA) injection of mono-iodoacetate (1.5 mg) and weekly treated by IA administration of FLL at 100 μg/mL for 4 weeks. Thermal withdrawal latency, mechanical withdrawal threshold, and spontaneous activity were tested for evaluation of pain behavior, and histopathological (HE, SO, and ABH staining) and immunohistochemical (Col2, Col10, and MMP13) analyses were conducted for observation of cartilage degradation. In vitro effect of FLL on chondrocytes was evaluated by MTT assay and qPCR analysis. Moreover, HPLC analysis was performed to determine its chemoprofile. The pain behavioral data showed that FLL attenuated joint pain hypersensitivity by increasing thresholds of mechanical allodynia and thermal hyperalgesia as well as spontaneous activity. The histopathological result showed that FLL reversed OA cartilage degradation by protecting chondrocytes and extracellular matrix in cartilage, and the immunohistochemical analysis revealed its molecular actions on protein expressions of MMP13, Col2, and Col10 in cartilage. The MTT assay showed its proliferative effects on chondrocytes, and qPCR assay clarified its mechanism associated with gene expressions of Mmp13, Col2, Col10, Adamts5, Aggrecan, and Runx2 in TNF-α treated chondrocytes. Our results revealed an anti-OA effect of FLL on pain behavior and cartilage degradation in OA rats and clarified a molecular mechanism in association with the suppression of chondrocyte hypertrophy and catabolism. IA FLL can be regarded as novel and promising option for OA therapy.

Possible reparative effect of low-intensity pulsed ultrasound (LIPUS) on injured meniscus.

Menisci are a pair of crescent-shaped fibrocartilages, particularly of which their inner region of meniscus is an avascular tissue. It has characteristics similar to those of articular cartilage, and hence is inferior in healing. We previously reported that low-intensity pulsed ultrasound (LIPUS) treatment stimulates the production of CCN2/CTGF, a protein involved in repairing articular cartilage, and the gene expression of major cartilage matrices such as type II collagen and aggrecan in cultured chondrocytes. Therefore, in this present study, we investigated whether LIPUS has also favorable effect on meniscus cells and tissues. LIPUS applied with a 60mW/cm2 intensity for 20min stimulated the gene expression and protein production of CCN2 via ERK and p38 signaling pathways, as well as gene expression of SOX9, aggrecan, and collagen type II in human inner meniscus cells in culture, and slightly stimulated the gene expression of CCN2 and promoted the migration in human outer meniscus cells in culture. LIPUS also induced the expression of Ccn2, Sox9, Col2a1, and Vegf in rat intact meniscus. Furthermore, histological evaluations showed that LIPUS treatment for 1 to 4weeks promoted healing of rat injured lateral meniscus, as evidenced by better and earlier angiogenesis and extracellular matrix synthesis. The data presented indicate that LIPUS treatment might prevent meniscus from degenerative change and exert a reparative effect on injured meniscus via up-regulation of repairing factors such as CCN2 and that it might thus be useful for treatment of an injured meniscus as a non-invasive therapy.

Interleukin-1β exacerbates the catabolic effects of human nucleus pulposus cells through activation of the Nuclear Factor kappa B signaling pathway under hypoxic conditions.

To determine the regulatory role of interleukin 1 beta (IL-1β) in the Nuclear Factor kappa B (NF-κB) -mediated catabolic effects of the nucleus pulposus cells in human intervertebral disc degeneration under hypoxic conditions. Human nucleus pulposus cells were cultured and exposed to IL-1β under hypoxic or normoxic environments, with or without NF-κB inhibition. The cell growth was determined using cell counting kit-8; gene and protein expressions were analyzed by Real-time polymerase chain reaction and Western blotting, respectively. Co-treatment with IL-1β and hypoxia decreased cell viability in human nucleus pulposus cells. There was a positive effect of IL-1β on human nucleus pulposus cells under hypoxia, which was through the up-regulation of matrix metalloproteinase-3 (MMP-3), MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5. IL-1β-induced expressions of MMP-3, MMP-9, ADAMTS-4, and ADAMTS-5 under hypoxia were accompanied by increased activation of NF-κB. Inhibition of NF-κBp65 by small interfering RNA or specific inhibitor BAY11-7082 blocked IL-1β-dependent gene upregulation of MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 in a hypoxic environment. The gene expression of aggrecan was decreased by IL-1β under hypoxic conditions, which was reversed by either BAY11-7082 or NF-κBp65 knockdown. IL-1β and hypoxia synergetically contributed to the catabolic effects of the nucleus pulposus cells by upregulating the expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 through the activation of NF-κB signaling pathway, indicating that the NF-κB signaling pathway is a key mediator of intervertebral disc degeneration.

Extraculuar material promoted C3H10T1/2 chondrogenic differentiation than Pellet cultured in vitro

Objective In order to understand the chondrogenesis differentiation of mesenchymal stem cells in either hydrogel or pellet culture, we applied the two methods and reveal the possible mechanism and for further investigation. Methods In C3H10T1/2 chondrogenic differentiation, we apply extracellular matrix hydrogel mixed the cell suspensions of freshly prepared (including scaling chondroitin sulfate, sodium hyaluronate synthesis and cross-linking agent) co-culture system and high cell density pellet formed by centrifugation. Chondrogenic differentiation of C3H10T1/2 was induced by treatment with TGF-β3 (10 ng/ml), dexamethasone (100 nmol/L), ascorbic acid (50 ug/ml), 1∶100 dilution ITS+Premix and high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum. And high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum is for control group. Histochemistry staining was utilized to identify extracellular proteoglycan and real-time PCR was performed to assess gene expression of SOX9, collagen IIa1/Xa1 and aggrecan for the 1st, 2nd and 3rd week respectively. Results In the hydrogel model for 3 weeks chondrogenic differentiation, the expression of master transcription factor SOX9 was upregulated in both culture models. While the marker genes of collagen IIa1 and collagen Xa1 were all promoted in hydrogel culture, the aggrecan gene expression was peaked in pellet culture. In addition, immunocytochemistry analysis of the hydrogel and pellet for 3 week illustrated the expression of extracellular matrix and more obviously in the hydrogel model. Conclusion In compared with pellet culture, the MSCs in the hydrogel were more likely promoted chondrogenesis leading to the eventual expression of marker genes. And the hydrogel would be applied in regeneration of cartilage injury. Key words: Mesenchymal stem cells; Cellular microenvironment; Chondrocytes

Modulation of matrix metabolism by ATP-citrate lyase in articular chondrocytes

Certain dysregulated chondrocyte metabolic adaptive responses such as decreased activity of the master regulator of energy metabolism AMP-activated protein kinase (AMPK) promote osteoarthritis (OA). Metabolism intersects with epigenetic and transcriptional responses. Hence, we studied chondrocyte ATP-citrate lyase (ACLY), which generates acetyl-CoA from mitochondrial-derived citrate, and modulates acetylation of histones and transcription factors. We assessed ACLY in normal and OA human knee chondrocytes and cartilages by Western blotting and immunohistochemistry, and quantified acetyl-CoA fluorometrically. We examined histone and transcription factor lysine acetylation by Western blotting, and assessed histone H3K9 and H3K27 occupancy of iNOS, MMP3, and MMP13 promoters by chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR). We analyzed iNOS, MMP3, MMP13, aggrecan (ACAN), and Col2a1 gene expression by RT-qPCR. Glucose availability regulated ACLY expression and function, nucleocytosolic acetyl-CoA, and histone acetylation. Human knee OA chondrocytes exhibited increased ACLY activation (assessed by Ser-455 phosphorylation), associated with increased H3K9 and H3K27 acetylation. Inhibition of ACLY attenuated IL-1β-induced transcription of iNOS, MMP3, and MMP13 by suppressing acetylation of p65 NF-κB, H3K9, and H3K27, blunted release of NO, MMP3, and MMP13, and also reduced SOX9 acetylation that promoted SOX9 nuclear translocation, leading to increased aggrecan and Col2a1 mRNA expression. ACLY is a novel player involved in regulation of cartilage matrix metabolism. Increased ACLY activity in OA chondrocytes increased nucleocytosolic acetyl-CoA, leading to increased matrix catabolism via dysregulated histone and transcription factor acetylation. Pharmacologic ACLY inhibition in OA chondrocytes globally reverses these changes and stimulates matrix gene expression and AMPK activation, supporting translational investigation in OA.

Pooled Platelet-Rich Plasma Lysate Therapy Increases Synoviocyte Proliferation and Hyaluronic Acid Production While Protecting Chondrocytes From Synoviocyte-Derived Inflammatory Mediators

Platelet-rich plasma (PRP) preparations are being used with moderate success to treat osteoarthritis (OA) in humans and in veterinary species. Such preparations are hindered, however, by being autologous in nature and subject to tremendous patient and processing variability. For this reason, there has been increasing interest in the use of platelet lysate preparations instead of traditional PRP. Platelet lysate preparations are acellular, thereby reducing concerns over immunogenicity, and contain high concentrations of growth factors and cytokines. In addition, platelet lysate preparations can be stored frozen for readily available use. The purpose of this study was to evaluate the effects of a pooled allogeneic platelet-rich plasma lysate (PRP-L) preparation on equine synoviocytes and chondrocytes challenged with inflammatory mediators in-vitro to mimic the OA joint environment. Our hypothesis was that PRP-L treatment of inflamed synoviocytes would protect chondrocytes challenged with synoviocyte conditioned media by reducing synoviocyte pro-inflammatory cytokine production while increasing synoviocyte anti-inflammatory cytokine production. Synoviocytes were stimulated with either interleukin-1β (IL-1β) or lipopolysaccharide (LPS) for 24 h followed by no treatment or treatment with platelet-poor plasma lysate (PPP-L) or PRP-L for 48 h. Synoviocyte growth was evaluated at the end of the treatment period and synoviocyte conditioned media was assessed for concentrations of hyaluronic acid (HA), IL-1β, tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). Chondrocytes were then challenged for 48 h with synoviocyte conditioned media from each stimulation and treatment group and examined for gene expression of collagen types I (COL1A1), II (COL2A1), and III (COL3A1), aggrecan (ACAN), lubricin (PRG4), and matrix metallopeptidase 3 (MMP-3) and 13 (MMP-13). Treatment of inflamed synoviocytes with PRP-L resulted in increased synoviocyte growth and increased synoviocyte HA and IL-6 production. Challenge of chondrocytes with conditioned media from PRP-L treated synoviocytes resulted in increased collagen type II and aggrecan gene expression as well as decreased MMP-13 gene expression. The results of this study support continued investigation into the use of pooled PRP-L for the treatment of osteoarthritis and warrant further in-vitro studies to discern the mechanisms of action of PRP-L.

Cytokine-induced interleukin-1 receptor antagonist protein expression in genetically engineered equine mesenchymal stem cells for osteoarthritis treatment.

BackgroundA combination of tissue engineering methods employing mesenchymal stem cells (MSCs) together with gene transfer takes advantage of innovative strategies and highlights a new approach for targeting osteoarthritis (OA) and other cartilage defects. Furthermore, the development of systems allowing tunable transgene expression as regulated by natural disease‐induced substances is highly desirable.MethodsBone marrow‐derived equine MSCs were transduced with a lentiviral vector expressing interleukin‐1 receptor antagonist (IL‐1Ra) gene under the control of an inducible nuclear factor‐kappa B‐responsive promoter and IL‐1Ra production upon pro‐inflammatory cytokine stimulation [tumor necrosis factor (TNF)α, interleukin (IL)‐1β] was analysed. To assess the biological activity of the IL‐1Ra protein that was produced and the therapeutic effect of IL‐1Ra‐expressing MSCs (MSC/IL‐1Ra), cytokine‐based two‐ and three‐dimensional in vitro models of osteoarthritis using equine chondrocytes were established and quantitative real‐time polymerase chain reaction (PCR) analysis was used to measure the gene expression of aggrecan, collagen IIA1, interleukin‐1β, interleukin‐6, interleukin‐8, matrix metalloproteinase‐1 and matrix metalloproteinase‐13.ResultsA dose‐dependent increase in IL‐1Ra expression was found in MSC/IL‐1Ra cells upon TNFα administration, whereas stimulation using IL‐1β did not lead to IL‐1Ra production above the basal level observed in nonstimulated cells as a result of the existing feedback loop. Repeated cycles of induction allowed on/off modulation of transgene expression. In vitro analyses revealed that IL‐1Ra protein present in the conditioned medium from MSC/IL‐1Ra cells blocks OA onset in cytokine‐treated equine chondrocytes and co‐cultivation of MSC/IL‐1Ra cells with osteoarthritic spheroids alleviates the severity of the osteoarthritic changes.ConclusionsThus, pro‐inflammatory cytokine induced IL‐1Ra protein expression from genetically modified MSCs might represent a promising strategy for osteoarthritis treatment.

Effects of low concentrated BMP-7 administered by co-cultivation or plasmid transfection on human osteoarthritic chondrocytes.

While BMP-7 has proven to be one of the most potent growth factors in cartilage tissue engineering, protein concentration and route of administration remain a matter of debate. Here we investigated the effects of a low concentration of BMP-7 on human osteoarthritic chondrocytes administered by protein co-cultivation and plasmid transfection. Freshly released (P0) or in vitro propagated chondrocytes (P2) were cultivated in a collagen type-I gel for 3 weeks in vitro or in nude mice. Seeded chondrocytes were treated with 50 ng/mL BMP-7 directly added to the medium or were subject to transient BMP-7 plasmid transfection prior to gel cultivation. Untreated specimens served as a control. After recovery, samples were investigated by histological and immunohistochemical staining and real-time PCR. In vitro, collagen type-II protein production was enhanced, and it was stored mainly pericellularly. Collagen type-II and aggrecan gene expression were enhanced in both treatment groups. After nude mouse cultivation, col-II protein production was further enhanced, but specimens of the BMP-7 transfection group revealed a clustering of col-II positive cells. Gene expression was strongly upregulated, chondrocyte number was increased and the differentiated phenotype prevailed. In general, freshly released chondrocytes (P0) proved to be superior to chondrocytes pre-amplified in vitro (P2). Both BMP-7 co-cultivation and plasmid transfection of human osteoarthritic chondrocytes led to improved cartilage repair tissue. Nevertheless, the col-II distribution following BMP-7 co-cultivation was homogeneous, while samples produced by transient transfection revealed a col-II clustering.

Delta-like 2 negatively regulates chondrogenic differentiation.

Delta-like 2 (Dlk2), a glycoprotein highly homologous to Dlk1, belongs to the Notch/Delta/Serrata family. Dlk2 has been shown to be an important regulator of adipogenesis; however, its role in other cellular differentiation processes is still unknown. Therefore, in this study, we aimed to determine the role of Dlk2 in chondrogenic differentiation. We found that Dlk2 overexpression promoted the growth of ATDC5 cells but inhibited insulin-induced ATDC5 chondrogenic differentiation, as supported by the reduction in cartilage matrix formation and gene expression of aggrecan (acan), collagentype II (col2a1) and X (col10a1). In contrast, Dlk2 silencing inhibited the proliferation of ATDC5 cells but enhanced their chondrogenic differentiation. We then evaluated the roles of mitogen-activated protein kinases (MAPKs), which are activated by insulin during the chondrogenesis of ATDC5 cells. Overexpression of Dlk2 protein strongly promoted the activation of p38, but not extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK). Moreover, as expected, Dlk2 silencing inhibited the activation of p38, but had no effect on the ERK1/2 and JNK pathways. Finally, we also detected the expression of Dlk2 in mouse epiphyseal cartilage during embryo development. The expression of the Dlk2 protein in the limb bud could be detected at embryonic day 11.5; additionally, it was found to decrease in the superficial zones, but remained unchanged in the deep/hypertrophic zones. In conclusion, our results suggested that Dlk2 acted as an important regulator of chondrogenesis through the p38 pathway. These findings may lead to strategies for the treatment of cartilage-related diseases such as osteoarthritis.

Human Wharton's Jelly Cells Activate Degenerative Nucleus Pulposus Cells In Vitro.

To investigate the interaction between human Wharton's jelly cells (WJCs) and degenerative nucleus pulposus cells (NPCs), human WJCs were cocultured with degenerative NPCs with or without direct cell-cell contact. WJCs were isolated from the human umbilical cord and degenerative NPCs were isolated from the surgically obtained degenerative intervertebral disc tissue. The isolated WJCs positively expressed CD73, CD105, CD90, CD29, CD166, and human leukocyte antigen (HLA)-ABC, but negatively expressed CD34, CD45, and HLA-DR. After coculturing with three different WJCs:NPCs ratios for 7 days, the real-time polymerase chain reaction showed that the relative gene expression of nucleus pulposus (NP)-marker genes [aggrecan, type II collagen, and SRY-type HMG box-9 (SOX-9)] was significantly upgraded in all coculture groups (all p < 0.05 compared with control groups). Coculture either with or without cell-cell contact significantly activated the expression of NP-maker genes than controls, but coculture with cell-cell contact yielded a higher gene expression than coculture without cell-cell contact. In coculturing with cell-cell contact and WJCs:NPCs of 25:75, the relative gene expression of aggrecan, type II collagen, SOX-9 for WJCs yielded the highest increase by 721-, 1507-, and 1463-folds, respectively (all p < 0.05 compared with WJCs control). In contrast, the highest relative gene expression of aggrecan, type II collagen, SOX-9 for NPCs was 112-, 84-, and 109-folds, respectively, in coculture with cell-cell contact and in WJCs:NPCs of 75:25 (all p < 0.05 compared with NPCs control). In conclusion, the data indicated that coculturing human WJCs with degenerative NPCs induced the NP-like cell differentiation of WJCs and restored the biological status of degenerative NPCs and coculture WJCs and NPCs with direct cell-cell contact yielded more favorable gene expressions.

Early Signs of Bone and Cartilage Changes Induced by Treadmill Exercise in Rats.

This study aims to investigate the earliest alterations of bone and cartilage tissues as a result of different exercise protocols in the knee joint of Wistar rats. We hypothesize that pretraining to a continuous intense running protocol would protect the animals from cartilage degeneration. Three groups of animals were used: (i) an adaptive (pretraining) running group that ran for 8 weeks with gradually increasing velocity and time of running followed by a constant running program (6 weeks of 1.12 km/hour running per day); (ii) a non-adaptive running (constant running) group that initially rested for 8 weeks followed by 6 weeks of constant running; and (iii) a non-running (control) group. At weeks 8, 14, and 20 bone and cartilage were analyzed. Both running groups developed mild symptoms of cartilage irregularities, such as chondrocyte hypertrophy and cell clustering in different cartilage zones, in particular after the adaptive running protocol. As a result of physical training in the adaptive running exercise a dynamic response of bone was detected at week 8, where bone growth was enhanced. Conversely, the thickness of epiphyseal trabecular and subchondral bone (at week 14) was reduced due to the constant running in the period between 8 and 14 weeks. Finally, the intermediate differences between the two running groups disappeared after both groups had a resting period (from 14 to 20 weeks). The adaptive running group showed an increase in aggrecan gene expression and reduction of MMP2 expression after the initial 8 weeks running. Thus, the running exercise models in this study showed mild bone and cartilage/chondrocyte alterations that can be considered as early-stage osteoarthritis. The pretraining adaptive protocol before constant intense running did not protect from mild cartilage degeneration. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Course-, dose-, and stage-dependent toxic effects of prenatal dexamethasone exposure on fetal articular cartilage development

Dexamethasone, a synthetic long-acting glucocorticoid, is routinely used for treating mothers at risk for preterm delivery. However, intrauterine overexposure to glucocorticoids induces low birth weight and cartilage dysplasia in offspring. Also, the “critical window” and safe dose of this treatment are largely unknown. This study investigated the course-, dose-, and stage-dependent toxic effects and the possible mechanisms of prenatal dexamethasone exposure (PDE) on fetal development and articular cartilage development. Pregnant mice (C57BL/6) received subcutaneous injection of dexamethasone (0.8 mg/kg d) once on gestational day (GD) 15 or once a day from GD 15 to 17, or received various doses of dexamethasone (0, 0.2, 0.8, and 1.2 mg/kg d) on GD 15–17, or received dexamethasone (0.8 mg/kg d) at early stage (GD 12–14) or late stage of pregnancy (GD 15–17). Offspring’s knee joints were harvested at birth for morphological analyses and detection of gene expression. Repeated PDE significantly suppressed fetal and articular cartilage development, which were characterized by decreased body weight and body length, coarse articular cartilage surfaces, and reduced gene and protein expression of Col2a1 and aggrecan. For those newborns treated with repeated PDE at different doses, the toxic effects on fetal and articular cartilage development were observed at doses of 0.8 and 1.2 mg/kg d, whereas no obvious toxic effects were observed at the dose of 0.2 mg/kg d. Moreover, PDE at 0.8 mg/kg d during the early embryonic stage induced stronger toxic effects on fetal and articular cartilage development, compared with PDE during the late embryonic stage. Detection of gene expression showed that the TGFβ signaling pathway in the articular cartilage was down-regulated after PDE. Taken together, PDE induces fetal developmental toxicity and articular cartilage developmental toxicity in a course-, dose-, and stage-dependent manner.

Independent chondrogenic potential of canine bone marrow-derived mesenchymal stem cells in monolayer expansion cultures decreases in a passage-dependent pattern.

Although chondroinductive growth factors are considered necessary for chondrogenesis of bone marrow-derived mesenchymal stem cells (BMSC), independent and spontaneous chondrogenesis has been previously demonstrated in adult horses, bovine calves and adult human BMSC. Surprisingly, adult canine BMSC under similar culture conditions previously failed to demonstrate chondrogenesis. The present study evaluated independent chondrogenic potential of BMSC sourced from three young dogs in the absence of known chondroinductive factors. BMSC were culture expanded in 10% DMEM up to third passage (P3). At each passage, the phenotype of BMSC was evaluated by RT-PCR gel electrophoresis and qPCR. BMSC exhibited a chondrogenic phenotype in the absence of dexamethasone and TGF-β1 as verified by the expression of Sox-9, type II collagen and aggrecan. Sox-9 was significantly downregulated (P<0.05) from P1−P3 compared to P0 while type II and X collagen, and aggrecan were significantly downregulated at P3 compared to P0. There was a significant (P<0.01) negative correlation between passaging and Sox-9, type II collagen and aggrecan gene expression. These results indicate that independent chondrogenic potential and phenotype retention of BMSC decreases in a passage-dependent pattern. Therefore, caution should be exercised for future experiments evaluating the chondrogenic potential of BMSC after extensive expansion cultures in 10% DMEM.

Pentosan Polysulfate Sodium Restores the Phenotype of Dedifferentiated Monolayer Canine Articular Chondrocytes Cultured in Alginate Beads

Autologous chondrocyte transplantation is a promising option for the repair of isolated osteoarthritic cartilage lesions that requires isolation and expansion of chondrocytes from a small cartilage biopsy prior to implantation. However, when cultured in vitro, chondrocytes lose their stable phenotype and dedifferentiate to fibroblastic-like cells. The study investigated the potential of pentosan polysulfate (PPS) sodium to restore the phenotype of dedifferentiated monolayer articular chondrocytes. Canine articular chondrocytes isolated from four cartilage samples were culture expanded to establish primary culture. First passage chondrocytes were cultured as alginate beads for 18 days under normoxia in PPS concentrations of 0, 1, 5, 15 and 40 μg/mL in 20% DMEM. Effect of PPS on type I, II and X collagen, aggrecan and Runx2 gene expression were evaluated by real-time PCR. Runx2, HIF-1α and HIF-2α protein expression were evaluated by Western blot and proteoglycan deposition was determined by Alcian blue stain. Dedifferentiated chondrocytes fully retained their phenotype as evidenced by increased synthesis of cartilage-specific genes, type II collagen and aggrecan mRNA with complete suppression of type I and X collagen at PPS concentrations of 15 and 40 μg/mL. Compared to the control, type II collagen and aggrecan mRNA were significantly upregulated (P<0.05) at 5, 15 and 40 μg/mL and 5 and 15 μg/mL PPS, respectively. PPS significantly enhanced proteoglycan with peak deposition at 5 μg/mL compared to control. HIF-1α and HIF-2α proteins were detectable at protein level for the first time under normoxia condition in alginate culture. The study demonstrates for the first time the restoration of dedifferentiated canine articular chondrocytes phenotype by combining alginate encapsulation with culture in PPS without the addition of known chondrocytic growth factors. The study confirms PPS as novel chondroinductive factor with potential to offer a solution to the major challenges that exist in cartilage tissue engineering.

Effects of microRNA-140 gene transfection with nucleus localization signal linked nucleic kinase substrate short peptide conjugated chitosan on rabbit articular chondrocytes

To investigate the effects of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) conjugated chitosan (CS) ( NNSCS) mediated the transfection of microRNA-140 (miR-140) in rabbit articular chondrocytes in vitro. Recombinant plasmid GV268-miR-140 and empty plasmid GV268 were combined with NNSCS to form NNSCS/pDNA complexes, respectively. Chondrocytes were isolated and cultured through trypsin and collagenase digestion from articular cartilage of newborn New Zealand white rabbits. The second generation chondrocytes were divided into 3 intervention groups: normal cell control group (group A), NNSCS/GV268 empty plasmid transfection group (group B), and NNSCS/GV268-miR-140 transfection group (group C). NNSCS/GV268 and NNSCS/GV268-miR- 140 complexes were transiently transfected into cells of groups B and C. After transfection, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expressions of exogenous miR-140; Annexin Ⅴ-FITC/PI double staining and MTT assay were used to detect the effect of exogenous miR-140 on apoptosis and proliferation of transfected chondrocytes; the expressions of Sox9, Aggrecan, and histone deacetylase 4 (Hdac4) were detected by RT-qPCR. RT-qPCR showed that the expression of miR-140 in group C was significantly higher than that in groups A and B ( P<0.05). Compared with groups A and B, the apoptosis rate in group C was decreased and the proliferation activity was improved, Sox9 and Aggrecan gene expressions were significantly up-regulated, and Hdac4 gene expression was significantly down-regulated ( P<0.05). There was no significant difference in above indexes between groups A and B ( P>0.05). Exogenous gene can be carried into the chondrocytes by NNSCS and expressed efficiently, the high expression of miR-140 can improve the biological activity of chondrocytes cultured in vitro, which provides important experimental basis for the treatment of cartilage damage diseases.

Spatiotemporal variations in gene expression, histology and biomechanics in an ovine model of tendinopathy.

Flexor tendinopathy is a common problem affecting humans and animals. Tendon healing is poorly understood and the outcomes of conservative and surgical management are often suboptimal. While often considered a localized injury, recent evidence indicates that in the short term, tendinopathic changes are distributed widely throughout the tendon, remote from the lesion itself. Whether these changes persist throughout healing is unknown. The aim of this study was to document gene expression, histopathological and biomechanical changes that occur throughout the superficial digital flexor tendon (SDFT) up to 16 weeks post-injury, using an ovine surgical model of tendinopathy. Partial tendon transection was associated with decreased gene expression for aggrecan, decorin, fibromodulin, tissue inhibitors of metalloproteinases (TIMPS 1, 2 and 3), collagen I and collagen II. Gene expression for collagen III, lumican and matrix metalloproteinase 13 (MMP13) increased locally around the lesion site. Expression of collagen III and MMP13 decreased with time, but compared to controls, collagen III, MMP13 and lumican expression remained regionally high throughout the study. An increase in TIMP3 was observed over time. Histologically, operated tendons had higher pathology scores than controls, especially around the injured region. A chondroid phenotype was observed with increased cellular rounding and marked proteoglycan accumulation which only partially improved with time. Biomechanically, partial tendon transection resulted in a localized decrease in elastic modulus (in compression) but only at 8 weeks postoperatively. This study improves our understanding of tendon healing, demonstrating an early ‘peak’ in pathology characterized by altered gene expression and notable histopathological changes. Many of these pathological changes become more localized to the region of injury during healing. Collagen III and MMP13 expression levels remained high close to the lesion throughout the study and may reflect the production of tendon tissue with suboptimal biomechanical properties. Further studies evaluating the long-term response of tendon to injury (6–12 months) are warranted to provide additional information on tendon healing and provide further understanding of the mechanisms underlying the pathology observed in this study.

Modulating microfibrillar alignment and growth factor stimulation to regulate mesenchymal stem cell differentiation.

Polymeric electrospun fibers can be tuned to match the fibrillar size and anisotropy of collagen fibers in ligaments, and can be mechanically competent. Therefore, their use is attractive when attempting to tissue engineer the bone-ligament interface. A central challenge in this field is recapitulating the cellular phenotypes observed across the bone-ligament interface. Here we demonstrated that it is possible to direct MSCs seeded onto aligned electrospun fibres towards either a ligamentogenic, chondrogenic or fibrochondrogenic phenotype upon presentation of appropriate biochemical cues. This opens the possibility of using aligned microfibrillar scaffolds that are spatially functionalized with specific growth factors to direct MSC differentiation for engineering the bone-ligament interface.

Wnt5a induces catabolic signaling and matrix metalloproteinase production in human articular chondrocytes.

Aberrant Wnt signaling may contribute to osteoarthritis (OA) but the Wnt family members involved have not been fully identified. The purpose of this study was to investigate the role of Wnt5a as a potential mediator of cartilage destruction in OA. Immunohistochemistry to detect Wnt5a was performed using normal and OA human articular cartilage. Cultured normal human chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus or recombinant Wnt5a protein with or without pretreatment using a panel of signaling inhibitors. Expression of Wnt5a, anabolic genes and catabolic genes were determined by quantitative real-time PCR. Production of Wnt5a protein and matrix metalloproteinases (MMPs) as well as activation of signaling proteins were analyzed by immunoblotting. Wnt5a was present in human articular cartilage with OA changes and its expression and secretion were increased in FN-f stimulated chondrocytes. FN-f stimulated Wnt5a production through the c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) pathways. Wnt5a reduced aggrecan gene expression after 48h of treatment. Wnt5a seemed to promote MMP1, -3, and -13 expression as well as MMP1 and MMP13 protein production in normal human chondrocytes. Wnt5a inhibitor peptides did not affect FN-f induced MMP production. Wnt5a activated β-catenin independent signaling including calmodulin-dependent protein kinase II (CaMKII), JNK, p38, ERK1/2, p65 and Akt. Inhibition of JNK, p38, ERK, PI-3 kinase and CaMKII by specific signaling inhibitors suppressed Wnt5a mediated MMP1 and MMP13 production. Wnt5a is present in human OA cartilage and can promote chondrocyte catabolic activity through non-canonical Wnt signaling, which suggests a potential role in OA.