Vorinostat ameliorates IL-1α-induced reduction of type II collagen by inhibiting the expression of ELF3 in chondrocytes.

Osteoarthritis (OA) is a common joint disease that ultimately causes physical disability and imposes an economic burden on society. Cartilage destruction plays a key role in the development of OA. Vorinostat is an oral histone deacetylase (HDAC) inhibitor and has been used for the treatment of T-cell lymphoma. Previous studies have reported the anti-inflammatory effect of HDAC inhibitors in both in vivo and in vitro models. However, it is unknown whether vorinostat exerts a protective effect in OA. In this study, our results demonstrate that treatment with vorinostat prevents interleukin 1α (IL-1α)-induced reduction of type II collagen at both gene and protein levels. Treatment with vorinostat reduced the IL-1α-induced production of mitochondrial reactive oxygen species (ROS) in T/C-28a2 cells. Additionally, vorinostat rescued the IL-1α-induced decrease in the expression of the collagen type II a1 (Col2a1) gene and the expression of Sry-related HMG box 9 (SOX-9). Importantly, we found that vorinostat inhibited the expression of matrix metalloproteinase-13 (MMP-13), which is responsible for the degradation of type II collagen. Furthermore, vorinostat suppressed the expression of E74-like factor 3 (ELF3), which is a key transcription factor that plays a pivotal role in the IL-1α-induced reduction of type II collagen. Also, the overexpression of ELF3 abolished the protective effects of vorinostat against IL-1α-induced loss of type 2 collagen by inhibiting the expression of SOX-9 whilst increasing the expression of MMP-13. In conclusion, our findings suggest that vorinostat might prevent cartilage destruction by rescuing the reduction of type II collagen, mediated by the suppression of ELF3.

Effect of Whole Tissue Culture and Basic Fibroblast Growth Factor on Maintenance of Tie2 Molecule Expression in Human Nucleus Pulposus Cells.

Previous work showed a link between Tie2+ nucleus pulposus progenitor cells (NPPC) and disc degeneration. However, NPPC remain difficult to maintain in culture. Here, we report whole tissue culture (WTC) combined with fibroblast growth factor 2 (FGF2) and chimeric FGF (cFGF) supplementation to support and enhance NPPC and Tie2 expression. We also examined the role of PI3K/Akt and MEK/ERK pathways in FGF2 and cFGF-induced Tie2 expression. Young herniating nucleus pulposus tissue was used. We compared WTC and standard primary cell culture, with or without 10 ng/mL FGF2. PI3K/Akt and MEK/ERK signaling pathways were examined through western blotting. Using WTC and primary cell culture, Tie2 positivity rates were 7.0 ± 2.6% and 1.9 ± 0.3% (p = 0.004), respectively. Addition of FGF2 in WTC increased Tie2 positivity rates to 14.2 ± 5.4% (p = 0.01). FGF2-stimulated expression of Tie2 was reduced 3-fold with the addition of the MEK inhibitor PD98059 (p = 0.01). However, the addition of 1 µM Akt inhibitor, 124015-1MGCN, only reduced small Tie2 expression (p = 0.42). cFGF similarly increased the Tie2 expression, but did not result in significant phosphorylation in both the MEK/ERK and PI3K/Akt pathways. WTC with FGF2 addition significantly increased Tie2 maintenance of human NPPC. Moreover, FGF2 supports Tie2 expression via MEK/ERK and PI3K/Akt signals. These findings offer promising tools and insights for the development of NPPC-based therapeutics.

FSH directly regulates chondrocyte dedifferentiation and cartilage development.

Previous studies suggest that postmenopausal osteoarthritis is linked to a decrease in estrogen levels. However, whether follicle-stimulating hormone (FSH), the upstream hormone of estrogen, affects cartilage destruction and thus contributes to the onset of osteoarthritis has never been explored. To evaluate the potential involvement of FSH in joint degeneration and to identify the molecular mechanisms through which FSH influences chondrocytes, mouse cartilage chondrocytes and the ATDC5 chondrocyte cell line were treated with FSH and inhibitors of intracellular signaling pathways. We observed that FSH induces chondrocyte dedifferentiation by decreasing type II collagen (Coll-II) synthesis. Chondrocyte cytoskeleton reorganization was also observed after FSH treatment. The FSH-induced decrease in Coll-II was rescued by ERK-1/2 inhibition but aggravated by p38 inhibition. In addition, knocking down the FSH receptor (Fshr) by using Fshr siRNA abolished chondrocyte dedifferentiation, as indicated by the increased expression of Coll-II. Inhibition of the protein Gαi by pertussis toxin (PTX) also restored FSH-inhibited Coll-II, suggesting that Gαi is downstream of FSHR in chondrocyte dedifferentiation. FSHß antibody blockade prevented cartilage destruction and cell loss in mice. Moreover, decreased Coll-II staining due to the progression of aging could be rescued by blocking FSH. Thus, we suggest that high circulating FSH, independent of estrogen, is an important regulator in chondrocyte dedifferentiation and cartilage destruction.

lncRNA SNHG16 promotes the occurrence of osteoarthritis by sponging miR­373­3p.

Osteoarthritis (OA) is a common age­related joint disorder, for which no effective disease­modifying drugs are currently available. Long non­coding RNAs (lncRNAs) are involved in the occurrence of OA. lncRNA small nucleolar RNA host gene 16 (SNHG16) has been reported to regulate inflammation; however, the exact biological function of SNHG16 in OA and its underlying mechanism of action remain unclear. In this study, gene and protein expression levels were detected using reverse transcription­quantitative PCR and western blotting, respectively. Cell apoptosis was analyzed using flow cytometry and ELISA was performed to detect TNF­α levels. The interactions between lncRNA SNHG16 and microRNA (miR)­373­3p were examined using the dual­luciferase reporter assay. lncRNA SNHG16 was upregulated in OA tissue compared with normal joint tissue. The expression levels of collagen II were significantly reduced in OA tissue compared with normal tissue. Similarly, aggrecan expression levels were significantly reduced in IL­1ß­treated CHON­001 cells compared with the controls. In addition, the protein expression levels of MMP13 were significantly increased in OA tissues and IL­1ß­treated CHON­001 cells compared with the controls. SNHG16 knockdown significantly increased the expression levels of aggrecan, and decreased the expression levels of MMP13, cleaved caspase­3 and p21 in IL­1ß­treated CHON­001 cells. In addition, IL­1ß induced CHON­001 cell apoptosis, while SNHG16 knockdown decreased IL­1ß­induced apoptosis. Furthermore, the luciferase activity assay suggested that SNHG16 negatively regulated miR­373­3p in OA. Finally, the results suggested that the proinflammatory effect of IL­1ß on CHON­001 cells was significantly reduced by SNHG16 knockdown. In conclusion, lncRNA SNHG16 knockdown significantly limited the progression of OA by sponging miR­373­3p in vitro, which suggested that SNHG16 may serve as a potential therapeutic target for OA.

Comparative intra-articular gene transfer of seven adeno-associated virus serotypes reveals that AAV2 mediates the most efficient transduction to mouse arthritic chondrocytes.

Osteoarthritis (OA) is the most common arthropathy, characterized by progressive degeneration of the articular cartilage. Currently, there are no disease-modifying approaches for OA treatment. Adeno-associated virus (AAV)-mediated gene therapy has recently become a potential treatment for OA due to its exceptional characteristics; however, the tropism and transduction efficiency of different AAV serotypes to articular joints and the safety profile of AAV applications are still unknown. The present study aims to screen an ideal AAV serotype to efficiently transfer genes to arthritic cartilage. AAV vectors of different serotypes expressing eGFP protein were injected into the knee joint cavities of mice, with all joint tissues collected 30 days after AAV injection. The transduction efficiency of AAVs was quantified by assessing the fluorescent intensities of eGFP in the cartilage of knee joints. Structural and morphological changes were analyzed by toluidine blue staining. Changes to ECM metabolism and pyroptosis of chondrocytes were determined by immunohistochemical staining. Fluorescence analysis of eGFP showed that eGFP was expressed in the cartilage of knee joints injected with each AAV vector. Quantification of eGFP intensity indicated that AAV2, 7 and 8 had the highest transduction efficiencies. Both toluidine blue staining and Mankin score showed that AAV6 aggravated cartilage degeneration. The analysis of key molecules in ECM metabolism suggested that AAV5 and 7 significantly reduced collagen type II, while AAV9 increased ADAMTS-4 but decreased MMP-19. In addition, transduction with AAV2, 5, 7 and 8 had no obvious effect on pyroptosis of chondrocytes. Comprehensive score analysis also showed that AAV2 had the highest score in intra-articular gene transfer. Collectively, our findings point to AAV2 as the best AAV serotype candidate for gene transfer on arthritic cartilage, resulting in minimal impact to ECM metabolism and pyroptosis of chondrocytes.

The role of microRNA-3085 in chondrocyte function.

MicroRNAs have been shown to play a role in cartilage development, homeostasis and breakdown during osteoarthritis. We previously identified miR-3085 in humans as a chondrocyte-selective microRNA, however it could not be detected by Northern blot. The aim of the current study was to prove that miR-3085 is a microRNA and to investigate the function of miR-3085 in signaling pathways relevant to cartilage homeostasis and osteoarthritis. Here, we confirm that miR-3085 is a microRNA and not another class of small RNA using (1) a pre-miR hairpin maturation assay, (2) expression levels in a Dicer null cell line, and (3) Ago2 pulldown. MicroRNA-3085-3p is expressed more highly in micromass than monolayer cultured chondrocytes. Transfection of miR-3085-3p into chondrocytes decreases expression of COL2A1 and ACAN, both of which are validated as direct targets of miR-3085-3p. Interleukin-1 induces the expression of miR-3085-3p, at least in part via NFκB. In a feed-forward mechanism, miR-3085-3p then potentiates NFκB signaling. However, at early time points after transfection, its action appears to be inhibitory. MyD88 has been shown to be a direct target of miR-3085-3p and may be responsible for the early inhibition of NFκB signaling. However, at later time points, MyD88 knockdown remains inhibitory and so other functions of miR-3085-3p are clearly dominant. TGFß1 also induces the expression of miR-3085-3p, but in this instance, it exerts a feedback inhibition on signaling with SMAD3 and SMAD4 shown to be direct targets. This in vitro analysis shows that miR-3085-3p functions in chondrocytes to induce IL-1-signaling, reduce TGFß1 signaling, and inhibit expression of matrix genes. These data suggest that miR-3085-3p has a role in chondrocyte function and could contribute to the process of osteoarthritis.

CDMP1 promotes type II collagen and aggrecan synthesis of nucleus pulposus cell via the mediation of ALK6.

OBJECTIVE: Destruction of extracellular matrix (ECM), especially collagen II and aggrecan, is an essential feature of intervertebral disc degeneration (IDD). This project planned to elucidate the role of cartilage-derived morphogenetic protein-1 (CDMP-1) in the collagen II and aggrecan synthesis of nucleus pulposus (NP) cells under the IL-1ß induced degeneration. PATIENTS AND METHODS: We cultured human primary NP cells in the different concentrations of IL-1ß medium and analyzed the CDMP-1 level. Recombinant human CDMP-1 protein was used to co-culture with IL-1ß to investigate its effects on collagen II and aggrecan synthesis of NP cells. Additionally, the bone morphogenetic protein type IB receptor (ALK6) gene silenced and upregulated NP cells were used to evaluate the function of ALK6 in the CDMP-1 treated NP cells. Collagen II, aggrecan, MMP9, MMP13, and TIMP4 expression level were analyzed to assess the ECM stability of NP cells. RESULTS: CDMP-1 gene expression decreased in the IL-1ß treated NP cells with a dose-dependent. Appropriate CDMP-1 protein supplement contributed to the collagen II and aggrecan production, the suppression of MMP9 and MMP13, and the upregulation of TIMP4. However, the silencing of ALK6 rejected the positive function of CDMP-1 on the collagen II and aggrecan; on the contrary, ALK6 upregulation magnified the CDMP-1 induced collagen II and aggrecan production. CONCLUSIONS: CDMP-1 is efficient in promoting the collagen II and aggrecan synthesis of NP cells, which is probably based on the mediation of ALK6.

Extracellular Nucleotides Selectively Induce Migration of Chondrocytes and Expression of Type II Collagen.

The migration of chondrocytes from healthy to injured tissues is one of the most important challenges during cartilage repair. Additionally, maintenance of the chondrogenic phenotype remains another limitation, especially during monolayer culture in vitro. Using both the differentiated and undifferentiated chondrogenic ATDC5 cell line, we showed that extracellular nucleotides are able to increase the migration rate of chondrocytes without affecting their chondrogenic phenotype. We checked the potency of natural nucleotides (ATP, ADP, UTP, and UDP) as well as their stable phosphorothioate analogs, containing a sulfur atom in the place of one nonbridging oxygen atom in a phosphate group. We also detected P2y1, P2y2, P2y4, P2y6, P2y12, P2y13, and P2y14 mRNA transcripts for nucleotide receptors, demonstrating that P2y1 and P2y13 are highly upregulated in differentiated ATDC5 cells. We showed that ADPßS, UDPßS, and ADP are the best stimulators of migration of differentiated chondrocytes. Additionally, ADP and ADPßS positively affected the expression of type II collagen, a structural component of the cartilage matrix.

Temporal and spatial expression of Sox9, Pax1, TGF-ß1 and type I and II collagen in human intervertebral disc development.

PURPOSE: An accurate understanding of cellular biochemical changes in human intervertebral disc (IVD)s and the corresponding mechanisms during the developmental process still remain unknown and important for investigating the function of critical factors in normal IVD development as well as ascertaining the therapeutic targets for the IVD degeneration. METHODS: Under ethical conditions, human fetal cervical IVDs at 4, 5, and 6 months of pregnancy were collected at abortion surgery. Normal adult human C3-C7 cervical IVDs were taken from cadaveric donors. Sox9, Pax1, TGF-ß1 and type I/II collagen protein and RNA were detected. The number of positive cells was counted to calculate the optical density value for each factor. RESULTS: Sox9, Pax1, and TGF-ß1 expression in the IVD was remarkably reduced with the developmental stage. The location of high expression of Sox9, Pax1, and TGF-ß1 changed with the developmental stage, and migrated from the nucleus pulposus to the annulus fibrosus and endplate. Higher Sox9, Pax1, and TGF-ß1 expression was finally observed around the sclerotome of the vertebral body. The anabolism of type I/II collagens is significantly increased in the IVD in the mid-trimester fetus. CONCLUSIONS: Sox9, Pax1 and TGF-ß1 participate in the developmental process of the human IVD and vertebral body. However, these factors show a separate expression of mRNA and protein, suggesting that they are expressed in the strict time and spatial order.

Anti-Inflammatory Effect of Carprofen Is Enhanced by Avocado/Soybean Unsaponifiables, Glucosamine and Chondroitin Sulfate Combination in Chondrocyte Microcarrier Spinner Culture.

OBJECTIVE: Osteoarthritis is a painful, chronic joint disease affecting man and animals with no known curative therapies. Palliative nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used but they cause adverse side effects prompting the search for safer alternatives. To address this need, we evaluated the anti-inflammatory activity of avocado/soybean unsaponifiables (ASU), glucosamine (GLU), and chondroitin sulfate (CS) with or without the NSAID carprofen. DESIGN: Canine chondrocytes were propagated in microcarrier spinner culture and incubated with (1) control medium, (2) ASU (8.3 µg/mL) + GLU (11 µg/mL) + CS (20 µg/mL) combination for 24 hours; and/or carprofen (40 ng/mL). Cultures were next incubated with control medium alone or IL-1ß (10 ng/mL) for another 24 hours. Production of PGE2, IL-6, IL-8, and MCP-1 (also known as CCL-2) were measured by ELISA. RESULTS: Chondrocytes proliferated in microcarrier spinner culture and produced type II collagen and aggrecan. Stimulation with IL-1ß induced significant increases in PGE2, IL-6, IL-8, and MCP-1 production. The increases in production were suppressed by carprofen as well as [ASU+GLU+CS]. The combination of carprofen and [ASU+GLU+CS] reduced PGE2 production significantly more than either preparation alone. The inhibitory effect of carprofen on IL-6, IL-8, and MCP-1 production was significantly less than that of [ASU+GLU+CS], whereas the combination did not reduce the production of these molecules significantly more than [ASU+GLU+CS] alone. CONCLUSIONS: The potentiating effect of [ASU+GLU+CS] on low-dose carprofen was identified in chondrocyte microcarrier spinner cultures. Our results suggest that the combination of low-dose NSAIDs like carprofen with [ASU+GLU+CS] could offer a safe, effective management for joint pain.

SIRT3 retards intervertebral disc degeneration by anti-oxidative stress by activating the SIRT3/FOXO3/SOD2 signaling pathway.

OBJECTIVE: The objective of this paper is to determine whether SIRT3 could retard intervertebral disc degeneration and study the mechanism. MATERIALS AND METHODS: We chose the 3-month mice to establish intervertebral disc degeneration model and study the effect of SIRT3 on the intervertebral disc by Western blotting, quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), immunohistochemistry. Mouse nucleus pulposus cells were cultured to study the exact mechanism. RESULTS: The expression of SIRT3 was decreased in degenerated human nucleus pulposus. Intervertebral discs of mice treated with theacrine expressed more collagen II and less collagen X. In addition, nucleus pulposus cells stimulated with interleukin-1ß (IL-1ß) expressed less SIRT3 than that in the control group and nucleus pulposus cells with SIRT3 overexpress vectors expressed more collagen II FOXO3a and superoxide dismutase 2 (SOD2), indicating that SIRT3 could improve the intervertebral disc degeneration by anti-oxidative stress. CONCLUSIONS: SIRT3 is a protective factor for intervertebral discs and can reduce oxidative stress in the intervertebral disc.

Dexmedetomidine exerts dual effects on human annulus fibrosus chondrocytes depending on the oxidative stress status.

Dexmedetomidine (Dex) is an anesthetic widely used in lumbar discectomy, but its effect on chondrocytes remains unclear. Dex is speculated to promote cartilage degeneration by activating α-2 adrenergic receptor. However, the antioxidative and anti-inflammatory effects of Dex implied the potential chondrocyte protective effect under stress conditions. The present study aimed to determine the effect of Dex on chondrocytes under non-stress and stress conditions. Chondrocytes were isolated from human annulus fibrosus (AF) tissues and oxidative stress was induced by treatment with 1 mM hydrogen peroxide (H2O2). Chondrocytes were treated with Dex alone or in combination with H2O2 Treatment with Dex alone decreased mRNA expression of COL2A1 and increased that of MMP-3 and MMP-13, thus contributing to cartilage degeneration. However, Dex prevented H2O2-induced death and degeneration of chondrocytes partly by enhancing antioxidant capacity. Mechanistically, Dex attenuated H2O2-mediated activation of NF-κB and NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), both of which play key roles in inflammation and inflammatory damage. Dex inactivated NLRP3 through the suppression of NF-κB and JNK signals. Co-treatment with Dex and H2O2 increased protein level of XIAP (X-linked inhibitor-of-apoptosis, an anti-apoptosis protein), compared with H2O2 treatment alone. H2O2 treatment increased the expression of neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) that is a ubiquitin ligase targeting XIAP. However, Dex decreased the amount of NEDD4 adhering to XIAP, thus protecting XIAP protein from NEDD4-mediated ubiquitination and degradation. Given that surgery inevitably causes oxidative stress and inflammation, the protective effect of Dex on chondrocytes during oxidative stress is noteworthy and warrants further study.

Local intra-articular injection of rapamycin inhibits NLRP3 activity and prevents osteoarthritis in mouse DMM models.

This study investigated the influence of autophagy on the expression of Collagen type II and light chain 3 (LC-3) in the articular cartilage of osteoarthritis (OA) models. The expression of OA associated biomarkers namely Matrix metalloproteinase (MMP-13), NOD-, LRR- and pyrin domain-containing 3 (NLRP3) induced by destabilizing the medial meniscus operation (DMM) were also investigated. A total of 60 C57BL/6 mice were divided into (1) control; (2) DMM2; (3) DMM8; (4) rapamycin 2 weeks; and (5) rapamycin 8 weeks groups. Saffranin O-Fast green staining, histomorphometry and immunohistochemical methods were used for analysis. In the DMM group, the expression of the OA biomarkers MMP-13, NLRP3 significantly increased, whilst Collagen II and LC-3B levels were significantly lower than other experimental groups. We hypothesized that NLRP3 inhibits autophagy activation and delays disease progression.

Type II collagen peptide Coll2-1 is an actor of synovitis.

OBJECTIVE: We evaluated the ability of Coll2-1, a type II collagen peptide, to activate pro-inflammatory pathways in synovial cells and to induce arthritis in Lewis rats. METHOD: Human synoviocytes and chondrocytes from knee OA patients were cultured for 24 h with/without Coll2-1 and/or purified immunoglobulin G (AS0619) binding specifically this peptide, and/or CLI-095, a TLR-4 signaling inhibitor and/or apocynin and diphenyleneiodonium, Reactive oxygen species (ROS) production inhibitors. The Interleukin (IL)-8 and Vascular Endothelium Growth Factor (VEGF) expression, the IL-8 production, the IκB-α and p65 phosphorylation and ROS were evaluated. Coll2-1 peptide, bovine type II collagen (CIA), streptococcal cell wall (SCW) or saline solution were injected into Lewis rats. The Coll2-1 peptide was injected subcutaneously (SC; 20-200µg/100µl/animal) or intra-articularly (IA; 0.5-5µg/50µl/animal) and compared to CIA injected in SC (200µg/100µl/animal) and SCW in IA (5µg/50µl/animal). The animals were injected on day 0 and monitored for 28 days. Histological lesions assessment was performed using an arthritis score. RESULTS: Coll2-1 peptide significantly increased IL-8 gene expression and production by synoviocytes. AS0619 and CLI-095 significantly decreased IL-8 expression. Coll2-1 induced p65 and IκBα phosphorylation and oxidative stress inhibitors decreased it. In human chondrocytes culture, Coll2-1 significantly increased MMP-3 and VEGF gene expression. In Lewis rats, CIA, SCW or Coll2-1 injection triggered arthritis. Like CIA or SCW, Coll2-1 induced synovitis, loss of cartilage proteoglycans, cartilage structure lesion and subchondral bone remodeling. CONCLUSIONS: Coll2-1 activates synoviocytes to produce IL-8 and induces arthritis in rat. These findings suggest that neutralizing Coll2-1 could be a therapeutic approach of arthritis.

The influence of probiotic diet and chondroitin sulfate administration on Ptgs2, Tgfb1 and Col2a1 expression in rat knee cartilage during monoiodoacetate-induced osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a common worldwide disease induced by a wide range of biochemical processes, mainly inflammation and degradation of collagen. The aim of this study, was to describe the effect of a multistrain probiotic (PB) and chondroitin sulfate (CS), administered separately or in combination, on the expression of Ptgs2, Tgfb1 and Col2a1 during monoiodoacetate-induced OA in male rats. METHODS: OA was induced in male rats by injecting monoiodoacetate in right hind knee. Therapeutic groups received 3 mg/kg of CS for 28 days and/or 1.4 g/kg of multistrain PB for 14 days. Knee cartilage were taken 30 days after monoiodoacetate injection. RNA was extracted and the expression of Ptgs2, Tgfb1 and Col2a1 were analyzed using SYBR Green 1-step real-time quantitative polymerase chain reaction. RESULTS: Induction of OA caused an upregulation in Ptgs2, Tgfb1 expression, and downregulation of Col2a1. Separate administration of PB and CS reduced Ptgs2 and Tgfb1 expressions. Their combined administration significantly decreased the expression of these pro-inflammatory cytokines, comparable to controls. Expression of Col2a1 showed similar behavior, with upregulation in therapeutic group with separate administration and the cumulative effects in case of co-administration. CONCLUSIONS: The multistrain PB diet may offer a perspective to improve the standard treatment of OA and, necessitates further investigation with clinical trials.

Synovium extra cellular matrices seeded with transduced mesenchymal stem cells stimulate chondrocyte maturation in vitro and cartilage healing in clinically-induced rat-knee lesions in vivo.

Osteoarthritis (OA) is a progressive disease associated with cartilage injury and its inherently limited repair capability. Synovium-based cellular constructs (sConstructs) are proposed as possible treatments. Equine sConstructs were produced from decellularized synovium-based extracellular matrix scaffolds (sECM) seeded with synovium-derived mesenchymal stem cells (sMSC), and engineered to express green fluorescent protein (GFP), or bone morphogenetic protein-2 (BMP-2). Survival, distribution, and chondrogenic potential of the sConstructs in vitro and in vivo were assessed. sConstructs in co-culture with chondrocytes increased chondrocyte proliferation, viability, and Col II production, greatest in BMP-2-sConstructs. Chondrocyte presence increased the production of hyaluronic acid (HA), proteoglycan (PG), and BMP-2 by the sConstructs in a positive feedback loop. sECM alone, or GFP- or BMP-2-sConstructs were implanted in synovium adjacent to clinically created full-thickness rat-knee cartilage lesions. At 5 weeks, the lesion area and implants were resected. Gross anatomy, adjacent articulate cartilage growth and subchondral bone repair were scored; and peripheral, central and cartilage lesion measurements taken. For all scores and measurements, sConstruct implants were significantly greater than controls, greatest with the BMP-2-sConstructs. Immunohistochemistry demonstrated migration of endogenous cells into the sECM, with greater cellularity in the constructs with intense positive GFP staining confirming engraftment of implanted sMSC and continued gene expression. In summary, exposing cartilage to sConstructs was chondrogenic in vitro and in vivo, and resulted in substantially increased growth in vivo. This effect was mediated, in part, by soluble ECM and cell factors and upregulation of anabolic growth proteins, such as BMP-2. This work is "proof of concept" that sConstructs surgically implanted adjacent to cartilage damage can significantly improve cartilage and subchondral bone repair, and potentially prevent the progression of OA.

MicroRNA-23c inhibits articular cartilage damage recovery by regulating MSCs differentiation to chondrocytes via reducing FGF2.

OBJECTIVE: The aim of the study was to explore the role of microRNA-23c in the differentiation of marrow stromal cells (MSCs) to chondrocytes and its potential mechanism. MATERIALS AND METHODS: MSCs were first isolated from rat bone marrow for cell culture. Surface antigens of MSCs (CD29 and CD34) were identified by flow cytometry. MSCs were induced for chondrogenic differentiation in MCDM (Mesenchymal Stem Cell Chondrogenic Differentiation Medium) for 0, 3, and 7 days, respectively, followed by detection of RUNX2, microRNA-23c and FGF2 expressions by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Alcian blue staining was performed to access proteoglycan deposition in MSCs transfected with microRNA-23c mimics or inhibitor. Western blot was conducted to detect the protein expressions of ACAN and COL2A1 in MSCs. The binding condition between microRNA-23c and FGF2 was verified by dual-luciferase reporter gene assay. Finally, MSCs were co-transfected with microRNA-23c mimics and FGF2 overexpression plasmid for rescue experiments. RESULTS: On the fourth day of MSCs isolation, MSCs were in an elongated shape. Flow cytometry results showed positive expression of CD29 and negative expression of CD34, which were consistent with MSCs phenotype. QRT-PCR data elucidated that the mRNA levels of RUNX2 and FGF2 gradually increased, whereas microRNA-23c expression decreased with the prolongation of chondrogenic differentiation. Transfection of microRNA-23c mimics in MSCs remarkably elevated microRNA-23c expression. Alcian blue staining showed that microRNA-23c overexpression results in less proteoglycan deposition in MSCs than that of controls. Both mRNA and protein expressions of ACAN and COL2A1 decreased after microRNA-23c overexpression. Dual-luciferase reporter gene assay confirmed that FGF2 binds to microRNA-23c. Further Western blot results demonstrated that FGF2 expression is negatively regulated by microRNA-23c. FGF2 overexpression reversed the inhibitory effects of microRNA-23c on proteoglycan deposition, as well as expressions of ACAN and COL2A1. CONCLUSIONS: MicroRNA-23c expression decreases during chondrogenic differentiation of MSCs, which inhibits MSCs differentiation to chondrocytes by inhibiting FGF2.

Chondrocytes Promote Vascularization in Fracture Healing Through a FOXO1-Dependent Mechanism.

Chondrocytes play an essential role in fracture healing by producing cartilage, which forms an anlage for endochondral ossification that stabilizes the healing fracture callus. More recently it has been appreciated that chondrocytes have the capacity to produce factors that may affect the healing process. We examined the role of chondrocytes in angiogenesis during fracture healing and the role of the transcription factor forkhead box-O 1 (FOXO1), which upregulates wound healing in soft tissue. Closed fractures were induced in experimental mice with lineage-specific FOXO1 deletion by Cre recombinase under the control of a collagen-2α1 promoter element (Col2α1Cre+ FOXO1L/L ) and Cre recombinase negative control littermates containing flanking loxP sites (Col2α1Cre- FOXO1L/L ). Experimental mice had significantly reduced CD31+ new vessel formation. Deletion of FOXO1 in chondrocytes in vivo suppressed the expression of vascular endothelial growth factor-A (VEGFA) at both the protein and mRNA levels. Overexpression of FOXO1 in chondrocytes in vitro increased VEGFA mRNA levels and VEGFA transcriptional activity whereas silencing FOXO1 reduced it. Moreover, FOXO1 interacted directly with the VEGFA promoter and a deacetylated FOXO1 mutant enhanced VEGFA expression whereas an acetylated FOXO1 mutant did not. Lastly, FOXO1 knockdown by siRNA significantly reduced the capacity of chondrocytes to stimulate microvascular endothelial cell tube formation in vitro. The results indicate that chondrocytes play a key role in angiogenesis which is FOXO1 dependent and that FOXO1 in chondrocytes regulates a potent angiogenic factor, VEGFA. These studies provide new insight into fracture healing given the important role of vessel formation in the fracture repair process. © 2018 American Society for Bone and Mineral Research.

Macromolecular crowding as a means to assess the effectiveness of chondrogenic media.

Chondrocyte-based tissue engineering requires in vitro cell expansion, which is associated with phenotypic losses, decrease in Collagen Type II synthesis and increase in Collagen Type I synthesis. Another major obstacle in clinical translation of chondrocyte-based therapies is the lack of extracellular matrix (ECM) in the engineered cartilage substitutes. Various research and commercially available media claim that they can maintain chondrogenic phenotype, whereas macromolecular crowding (MMC) has been shown to increase tissue-specific ECM deposition and maintain cell phenotype in vitro. Herein, we hypothesised that the combination of chondrogenic media with MMC will enable chondrogenic phenotype maintenance during in vitro expansion and increase cartilage-specific ECM deposition, enabling that way the development of a tissue-engineered cartilage substitute. Immunocytochemistry analysis of Passage 3 human chondrocytes in normal media in monolayer revealed that MMC significantly increased Collagen Type I deposition, whereas no statistical difference was observed in Collagen Type II deposition. When Passage 3 human chondrocytes were cultured in normal media and alginate beads, immunocytochemistry analysis revealed that MMC increased, albeit not significantly, both Collagen Type I and Collagen Type II deposition. Subsequently, human chondrocytes were expanded up to Passage 6 in either fetal bovine serum or human serum and redifferentiated using commercially available chondrogenic media in either monolayer or alginate beads. Immunocytochemistry analysis revealed that MMC, independently of the serum used, significantly increased Collagen Type I deposition in human-redifferentiated monolayer and alginate bead chondrocyte cultures, whereas almost no Collagen Type II was detected. These data clearly illustrate that an optimal chondrogenic medium is still elusive.

Effect of Hyperbaric Oxygen on Proliferation and Gene Expression of Human Chondrocytes: An In Vitro Study.

PURPOSE: The present study investigated the effects of hyperbaric oxygen (HBO) on human chondrocyte proliferation and gene expression patterns. METHODS: Chondrocyte cultures were transferred to a HBO chamber and exposed to 100% oxygen for 7 consecutive days. Within groups, pressure was varied between 1 and 2 atm and duration of HBO administration was varied among 60, 90, and 120 minutes. Cell counts were performed using the WST-1 assay at 1, 3, 5, and 7 days after initiation of HBO treatment to obtain data to plot a growth curve. Gene expression of apoptosis markers PARP and caspase 3, as well as cartilage specific proteins collagen II and COMP, were detected by reverse transcription polymerase chain reaction. RESULTS: The experiments showed that in vitro administration of HBO inhibit chondrocyte growth. When applied compression was increased up to 2 atm, chondrocyte cell count was reduced by half at days 3 and 7 in association with an upregulation of the apoptosis markers PARP and caspase 3 as well as the cartilage specific proteins collagen II and COMP. No significant differences were monitored from varied duration of daily treatment. CONCLUSION: Chondrocyte growth was inhibited in vitro by treatment of HBO. This inhibitory effect was even increased by elevating the applied pressure, while molecular testing showed reduced chondrocyte growth. Higher levels of HBO inhibited cell growth even more, but up-regulation of apoptosis specific markers and cartilage specific proteins were seen during administration of high oxygen levels. Thus, it has to be evaluated that there is a critical level of hypo-/hyperoxia required to stimulate or at least maintain chondrocyte cell proliferation.