Chondrocytes In Monolayer Culture Research Articles

Effects of mechanical vibrations on chondrocytes in monolayer culture for regenerative cartilage

Effects of mechanical vibrations on chondrocytes in monolayer culture for regenerative cartilage

In vitro chondrotoxicity of bupivacaine, levobupivacaine and ropivacaine and their effects on caspase activity in cultured canine articular chondrocytes.

Bupivacaine, levobupivacaine and ropivacaine are potent, long acting, amide-type local anesthetics that have several clinical applications including intra-articular administration. The objectives of this study were to evaluate their in vitro effects on cell viability and caspase activity to elucidate whether they activate the extrinsic or intrinsic pathways of apoptosis in canine articular chondrocytes. Chondrocytes in monolayer culture were treated with culture medium as the control, or with 0.062% (0.62 mg/mL) bupivacaine, 0.062% levobupivacaine, and 0.062% ropivacaine for 24 hr. Cell viability was evaluated using the live/dead, 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), and Cell Counting Kit-8 (CCK-8) assays. Evaluation of caspase-3, caspase-8, and caspase-9 activity was performed using colorimetric assays. The MTT and CCK-8 assays were used to evaluate the effect of caspase inhibitors on local anesthetic chondrotoxicity. All three local anesthetics decreased chondrocyte viability after 24 hr (P<0.001). Apoptosis was induced through both the extrinsic and intrinsic pathways. Bupivacaine increased caspase-3, caspase-8, and caspase-9 activity (P<0.001). Levobupivacaine increased caspase-3 (P=0.03) while ropivacaine did not significantly upregulate activity for all three caspases. Caspase inhibition did not suppress bupivacaine chondrotoxicity whereas inhibition of caspase-8 and caspase-9 decreased ropivacaine chondrotoxicity and mildly attenuated levobupivacaine chondrotoxicity. In summary, the level of chondrotoxicity, the type of caspase activated, the level of caspase activation, and the response to caspase inhibitors was dependent on the type of local anesthetic. Therefore, ropivacaine may be a safer choice for intra-articular administration compared to levobupivacaine and bupivacaine.

Investigation of MicroRNA Biomarkers in Equine Distal Interphalangeal Joint Osteoarthritis.

Osteoarthritis of the equine distal interphalangeal joint is a common cause of lameness. MicroRNAs from biofluids are promising biomarkers and therapeutic candidates. Synovial fluid samples from horses with mild and severe equine distal interphalangeal joint osteoarthritis were submitted for small RNA sequencing. The results demonstrated that miR-92a was downregulated in equine synovial fluid from horses with severe osteoarthritis and there was a significant increase in COMP, COL1A2, RUNX2 and SOX9 following miR-92a mimic treatment of equine chondrocytes in monolayer culture. This is the first equine study to evaluate the role of miR-92a in osteoarthritic chondrocytes in vitro.

Sustained release of locally delivered celecoxib provides pain relief for osteoarthritis: a proof of concept in dog patients

Drug delivery platforms that allow for gradual drug release after intra-articular administration have become of much interest as a treatment strategy for osteoarthritis (OA). The aim of this study was to investigate the safety and efficacy of an intra-articular sustained release formulation containing celecoxib (CXB), a cyclooxygenase-2 (COX-2) selective inhibitor. Amino acid-based polyesteramide microspheres (PEAMs), a biodegradable and non-toxic platform, were loaded with CXB and employed in two in vivo models of arthritis: an acute inflammatory arthritis model in rats (n=12), and a randomized controlled study in chronic OA dog patients (n=30). In parallel, the bioactivity of sustained release of CXB was evaluated in monolayer cultures of primary dog chondrocytes under inflammatory conditions. Sustained release of CXB did not alleviate acute arthritis signs in the rat arthritis model, based on pain measurements and synovitis severity. However, in OA dog patients, sustained release of CXB improved limb function as objective parameter of pain and quality of life based on gait analysis and owner questionnaires. It also decreased pain medication dependency over a 2-month period and caused no adverse effects. Prostaglandin E2 levels, a marker for inflammation, were lower in the synovial fluid of CXB-treated dog OA patients and in CXB-treated cultured dog chondrocytes. These results show that local sustained release of CXB is less suitable to treat acute inflammation in arthritic joints, while safe and effective in treating pain in chronic OA in dogs.

Clodronate disodium is neither cytotoxic nor cytoprotective to normal and recombinant equine interleukin-1β-treated joint tissues in vitro.

To determine the effects of clodronate disodium (CLO) on control and recombinant equine interleukin-1β (IL-1β)-treated equine joint tissues. In vitro experimental study. Cartilage explants, chondrocytes, and synoviocytes (n=3 horses). Monolayer cultures of chondrocytes and synoviocytes from three horses were subjected to: control media (CON), 5ng/ml CLO (C/low), 50 ng/ml CLO (C/med), 100 ng/ml CLO (C/high), with and without IL-1β, and 10ng/ml IL-1β (IL) alone for 72 hours. Cartilage explants from three horses were subjected to CON, IL, C/low, and C/med with and without IL-1β for 72 hours. Culture media was analyzed for prostaglandin-E2 (PGE2 ), interleukin-6 (IL-6), and nitric oxide (NO). Explant media was analyzed for glycosaminoglycan (GAG) content and NO. At 72 hours, explant and monolayer culture viability were assessed, and explant GAG content was measured. IL-1β treatment resulted in higher media concentrations of GAG, NO, PGE2 , and IL-6 compared to the CON treatment (p < .05), demonstrating a catabolic effect of IL-1β on explants and monolayer cultures. CLO treatments did not increase media concentrations of GAG, NO, PGE2 , or IL-6 compared to CON, indicating no cytotoxic effect. Nevertheless, CLO treatments administered to IL-1β-treated monolayer cultures and explants did not significantly reduce the inflammatory response regardless of concentration. CLO did not demonstrate cytotoxic nor cytoprotective effects in normal and IL-1β-stimulated chondrocytes, synoviocytes or explants in culture. This study does not support the use of CLO as an anti-inflammatory treatment. Further research is necessary to confirm any anti-inflammatory effects of CLO on joint tissues.

Norepinephrine modulates IL-1\u03b2-induced catabolic response of human chondrocytes

BackgroundThe influence of the sympathetic nervous system (SNS) on metabolism of bone and cartilage expressing β-adrenergic receptors (AR) was suggested. Here, we investigated whether the SNS functions as a modulator of cartilage metabolism induced by interleukin-1beta (IL-1β).MethodsHuman articular chondrocytes and articular cartilage were collected from patients with osteoarthritis (OA). Chondrocyte monolayer and cartilage explant culture were stimulated with IL-1β. The activity of β-ARs was modulated by an agonist, norepinephrine (NE), and antagonists, including propranolol, atenolol, nebivolol, and nadolol.ResultsThe levels of β1-, β2-, and β3-AR in OA cartilage and IL-1β-treated chondrocytes were lower than normal cartilage and untreated cells. Treatment of chondrocytes with IL-1β and β-blockers, including propranolol, atenolol, nebivolol, and nadolol, for 6 h significantly upregulated IL-1β-induced expression of MMP-1, -3, and − 13, compared to chondrocytes treated with IL-1β alone, indicating that antagonism of β-AR confers catabolic signals. On the other hand, NE antagonized IL-1β-induced catabolic response. In addition, NE significantly inhibited IL-1β-induced release of glycosaminoglycan (GAG) from cartilage explant culture. In addition, β-AR activity significantly affected IL-1β-stimulated phosphorylation of JNK and ERK. These results indicate that β-AR signal is associated with cartilage metabolism.ConclusionsOur findings showed that β-ARs is a regulator of cartilage catabolism induced with IL-1β.

The anabolic effect of inorganic polyphosphate on chondrocytes is mediated by calcium signalling.

Inorganic polyphosphates (polyP) are polymers composed of phosphate residues linked by energy-rich phosphoanhydride bonds. As polyP can bind calcium, the hypothesis of this study is that polyP enters chondrocytes and exerts its anabolic effect by calcium influx through calcium channels. PolyP treatment of cartilage tissue formed in 3D culture by bovine chondrocytes showed an increase in proteoglycan accumulation but only when calcium was also present at a concentration of 1.5 mM. This anabolic effect could be prevented by treatment with either ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid or the calcium channel inhibitors gadolinium and nifedipine. Calcium and polyP cotreatment of chondrocytes in monolayer culture resulted in calcium oscillations that were polyP chain length specific and were inhibited by gadolinium and nifedipine. The calcium influx resulted in increased gene expression of sox9, collagen type II, and aggrecan which was prevented by treatment with either calphostin, an inhibitor of protein kinase C, and W7, an inhibitor of calmodulin; suggesting activation of the protein kinase C-calmodulin pathway. Tracing studies using 4',6-diamidino-2-phenylindole, Mitotracker Red, and/or Fura-AM staining showed that polyP was detected in the nucleus, mitochondria, and intracellular vacuoles suggesting that polyP may also enter the cell. PolyP colocalizes with calcium in mitochondria. This study demonstrates that polyP requires the influx of calcium to regulate chondrocyte matrix production, likely via activating calcium signaling. These findings identify the mechanism regulating the anabolic effect of polyP in chondrocytes which will help in its clinical translation into a therapeutic agent for cartilage repair.

In Vitro Effects of Bupivacaine on the Viability and Mechanics of Native and Engineered Cartilage Grafts

Background: Although the toxic effects of bupivacaine on chondrocyte monolayer culture have been well described, its cellular and mechanical effects on native and engineered articular cartilage remain unclear. For the repair of articular cartilage defects, fresh autologous and allogenic cartilage grafts are commonly used, and engineered cell-based therapies are emerging. The outcome of grafting therapies aimed at repairing damaged cartilage relies largely on maintaining proper viability and mechanical suitability of the donor tissues. Purpose: To investigate the in vitro effects of single bupivacaine exposure on the viability and mechanics of 2 cartilage graft types: native articular cartilage and engineered neocartilage. Study Design: Controlled laboratory study. Methods: Articular cartilage explants were harvested from the bovine stifle femoral condyles, and neocartilage constructs were engineered from bovine stifle chondrocytes using the self-assembling process, a scaffold-free approach to engineer cartilage tissue. Both explants and neocartilage were exposed to chondrogenic medium containing a clinically applicable bolus of 0.5%, 0.25%, or 0% (control) bupivacaine for 1 hour, followed by fresh medium wash and exchange. Cell viability and matrix content (collagen and glycosaminoglycan) were assessed at t = 24 hours after treatment, and compressive mechanical properties were assessed with creep indentation testing at t = 5 to 6 days after treatment. Results: Single bupivacaine exposure was chondrotoxic in both explants and neocartilage, with 0.5% bupivacaine causing a significant decrease in chondrocyte viability compared with the control condition (55.0% ± 13.4% vs 71.9% ± 13.5%; P < .001). Bupivacaine had no significant effect on matrix content for either tissue type. There was significant weakening of the mechanical properties in the neocartilage when treated with 0.5% bupivacaine compared with control, with decreased aggregate modulus (415.8 ± 155.1 vs 660.3 ± 145.8 kPa; P = .003), decreased shear modulus (143.2 ± 14.0 vs 266.5 ± 89.2 kPa; P = .002), and increased permeability (14.7 ± 8.1 vs 6.6 ± 1.7 × 10−15 m4/Ns; P = .009). Bupivacaine exposure did not have a significant effect on the mechanical properties of native cartilage explants. Conclusion: Single bupivacaine exposure resulted in significant chondrotoxicity in native explants and neocartilage and significant weakening of mechanical properties of neocartilage. The presence of abundant extracellular matrix does not appear to confer any additional resistance to the toxic effects of bupivacaine. Clinical Relevance: Clinicians should be judicious regarding the use of intra-articular bupivacaine in the setting of articular cartilage repair.

Effect of cadmium on the viability on monolayer cultures of synoviocytes, chondrocytes, and Hoffa: A preliminary study.

Osteoarthritis (OA) is the gradual loss of articular cartilage and involves several tissues, such as the synovial membrane, meniscus, ligaments, and adipose tissue known as Hoffa fat pad. There are largely unexplored factors that lead to OA development, such as the impact of exposure to heavy metals like cadmium (Cd) on the viability of cells in the knee joint tissue. The objective of this report was to identify the cell type with the highest susceptibility to Cd toxicity with respect to cell viability and death. Our findings showed that a concentration as low as 3 μM cadmium chloride for 12 h affects the viability of synovial cells, and a concentration of 10 μM affects Hoffa cells. Our results suggest that Cd can affect the viability of synovial and chondral cells primarily. In contrast, Hoffa cells were less susceptible, likely because Cd favors the production of pro-inflammatory cytokines before triggering their death as part of its damage mechanism at the articular level.

Alterations in characteristics of canine articular chondrocytes in non-passaged long-term monolayer culture: Matter of differentiation, dedifferentiation and redifferentiation.

This study investigated the effects of culture time on phenotype stability of canine articular chondrocytes (CACs) in non-passaged long-term monolayer culture. Third passage (P3) CACs isolated from four cartilage samples were seeded at three different initial seeding densities (0.2 × 104, 1.0 × 104 and 5.0 × 104 cells/cm2) and maintained in monolayer condition up to 8 weeks without undergoing subculture after confluence. The characteristic changes of chondrocytes during the culture period were evaluated based on the cell morphology, cell proliferation, glycosaminoglycans (GAGs) content, DNA quantification, mRNA expression and ultrastructure of chondrocytes. Chondrocytes maintained under post-confluence condition exhibited a capability to grow and proliferate up to 4 weeks. Alcian blue staining and Dimethylmethylene blue (DMMB) assay revealed that the extracellular matrix (ECM) synthesis was increased in a time-dependent manner from 2 to 8 weeks. The chondrocyte mRNA expression profile was dramatically affected by prolonged culture time, with a significant downregulation of collagen type I, whereas the expression of collagen type II, aggrecan, Sox9 and matrix metalloproteinase 13 (MMP-13) were significantly upregulated. In addition, transmission electron microscopy (TEM) result indicated dilation of rough endoplasmic reticulum (RER) in these long-term monolayer cultured chondrocytes. These findings demonstrate that the chondrocytes phenotype could be partially redifferentiated through the spontaneous redifferentiation process in long-term cultures using standard culture medium without the addition of chondrogenic supplements or tissue-culture scaffolds.

Adseverin, an actin binding protein, regulates articular chondrocyte phenotype.

Chondrocytes dedifferentiate as a result of monolayer culture for cell number expansion. This is associated with the development of an elongated shape, increased actin polymerization, development of stress fibres, and expression of contractile molecules. Given the changes in actin status with dedifferentiation, the hypothesis of this study was that adseverin, an actin severing and capping protein, plays a role in regulating chondrocyte phenotype and function. This study reports that serial passaging of articular chondrocytes in monolayer culture resulted in loss of adseverin protein expression as early as Day 14 of culture and remained repressed in Passage 2 (P2) cells. Knockdown of adseverin by siRNA in primary chondrocytes promoted an increase in cell size and an elongated shape, actin stress fibres, decreased G-/F-actin ratio, and increased number of actin-free barbed ends. The cells also showed increased expression of the contractile genes and proteins, vinculin and α-smooth muscle actin, and increased ability to contract collagen gels. These are all features of dedifferentiation. These effects were due to adseverin as adseverin overexpression following transfection of the green fluorescent protein-adseverin plasmid partially reversed all of these changes in P2 chondrocytes. Furthermore, sox9 and aggrecan chondrogenic gene expression was upregulated, and collagen type I genes expression was downregulated with adseverin overexpression. The change in aggrecan mRNA expression had functional consequence as these cells exhibited increased total proteoglycan synthesis. These findings demonstrate that adseverin regulates features indicative of redifferentiation in passaged articular chondrocytes through modulation of the actin cytoskeleton status and potentially may regulate the maintenance of phenotype in primary chondrocytes.

Microencapsulation of human osteoarthritis chondrocytes in collagen microsphere - an in vitro model for osteoarthritis studies

Purpose: Understanding the biology of human osteoarthritis chondrocytes (hOACs) and their interactions with extrinsic factors is critical to future development of innovative therapeutic treatment for osteoarthritis (OA). Monolayer culture of chondrocytes represents a simple in vitro model to use. However, native chondrocyte phenotypes usually lost their phenotype rapidly upon isolation and expansion in vitro, via a phenomenon called de-differentiation. 3D culture models usually better maintain the phenotype of cells as that in native tissues. As a result, in vitro 3D cultures of hOACs are important models for pathophysiology studies and drug screening. Here, we aim to investigate a 3D collagen microencapsulation-based in vitro model for hOACs by hypothesizing that hOACs will resume their chondrogenic phenotype and maintain responsive to external factors. Methods: hOACs were isolated from OA cartilage samples harvested from multiple patients during total knee reconstruction (TKR) surgery according to a protocol approved by the Institutional Review Board with informed consent. The isolated cells were expanded before microencapsulating into tiny collagen microspheres for subsequent 3D cultures. The change in the chondrocyte phenotype (Sox9 and collagen II expression as markers) and the OA phenotype (Runx2, collagen X and MMP13 as markers) was evaluated over time. 2D monolayer culture and traditional 3D pellet culture were used as controls. Results: Both 3D models (pellet culture and collagen microsphere) resumed the lost chondrocyte phenotype after expansion in 2D monolayer cultures. The 3D collagen microsphere model, as compared with the pellet model, better retained the OA phenotype, with lower sox9, and higher Runx2 and MMP13 gene expression, although the protein expression of these markers did not vary significantly. We further validated the collagen microsphere model by demonstrating the responsiveness of the microencapsulated hOACs towards a number of external factors including serum-free medium (ITS), hypoxia and TGF beta, all are known to stimulate a more chondrogenic phenotype. Conclusions: This work showed better phenotype maintenance of human OACs in two different 3D culture models, which are collagen microsphere platform developed by our group and the traditional pellet culture, as compared to the traditional monolayer cultures. It also highlights the significance and versatility of the collagen microsphere platform in maintaining primary hOACs. The ability of hOACs in developing responsiveness to well-known external factors suggests the possibility to use the 3D collagen microsphere as a model for screening of potential disease-modifying modalities for regenerative medicine.

Fibrin-hyaluronic acid hydrogel-based delivery of antisense oligonucleotides for ADAMTS5 inhibition in co-delivered and resident joint cells in osteoarthritis

To date no disease-modifying drugs for osteoarthritis (OA) are available, with treatment limited to the use of pain killers and prosthetic replacement. The ADAMTS (A Disintegrin and Metallo Proteinase with Thrombospondin Motifs) enzyme family is thought to be instrumental in the loss of proteoglycans during cartilage degeneration in OA, and their inhibition was shown to reverse osteoarthritic cartilage degeneration. Locked Nucleic Acid (LNA)-modified antisense oligonucleotides (gapmers) released from biomaterial scaffolds for specific and prolonged ADAMTS inhibition in co-delivered and resident chondrocytes, is an attractive therapeutic strategy. Here, a gapmer sequence identified from a gapmer screen showed 90% ADAMTS5 silencing in a monolayer culture of human OA chondrocytes. Incorporation of the gapmer in a fibrin-hyaluronic acid hydrogel exhibited a sustained release profile up to 14 days. Gapmers loaded in hydrogels were able to transfect both co-embedded chondrocytes and chondrocytes in a neighboring gapmer-free hydrogel, as demonstrated by flow cytometry and confocal microscopy. Efficient knockdown of ADAMTS5 was shown up to 14 days in both cell populations, i.e. the gapmer-loaded and gapmer-free hydrogel. This work demonstrates the use applicability of a hydrogel as a platform for combined local delivery of chondrocytes and an ADAMTS-targeting gapmer for catabolic gene modulation in OA.

A Human Chondrocyte-Derived In Vitro Model of Alcohol-Induced and Steroid-Induced Femoral Head Necrosis.

BackgroundWorldwide, femoral head necrosis (FHN), which is also known as avascular necrosis of the femoral head or osteonecrosis of the femoral head, affects millions of people. Excess alcohol intake and steroid use are two common associations with FHN, but their pathogenesis remains unknown. The aim of this study was to develop an in vitro model using human chondrocytes to study alcohol-induced and steroid-induced FHN.Material/MethodsIn this study, the in vitro model used a monolayer culture of articular chondrocytes derived from patients with non-traumatic FHN (Ficat and Arlet, Stage III). Normal chondrocytes were obtained from patients with femoral neck fracture resulting from road traffic accident (Garden, Stage IV). Alcohol-stimulated and steroid-stimulated articular chondrocytes were evaluated by a cell proliferation assay, measurement of calcium levels (alizarin red), measurement of alkaline phosphatase (ALP) levels, detection of glycosaminoglycan (GAG) secretion using safranin O histochemical staining, and analysis of cartilage-specific genes, ACAN, SOX9, OPG, TGF-β, RANKL, and RUNX2, using quantitative real-time polymerase chain reaction (qRT-PCR).ResultsBoth alcohol and steroids, but especially steroids, accelerated the degradation of cartilage by suppression of chondrogenesis while promoting chondrocyte hypertrophy and activating osteogenic differentiation, as assessed by cell proliferation assay, detection of glycosaminoglycan (GAG) secretion, and analysis of cartilage-specific genes.ConclusionsA human chondrocyte-derived in vitro model of alcohol-induced and steroid-induced FHN demonstrated chondrocyte hypertrophy and activated osteogenic differentiation.

Nano-hydroxyapatite/collagen film as a favorable substrate to maintain the phenotype and promote the growth of chondrocytes cultured invitro.

Autologous chondrocyte implantation (ACI) has emerged as a novel approach to cartilage repair through the use of harvested chondrocytes. However, the expansion of the chondrocytes from the donor tissue in vitro is restricted by the limited cell numbers and the dedifferentiation of the chondrocytes. The present study investigated the effect of collagen-based films, including collagen, hydroxyapatite (HA)/collagen (HC) and in situ synthesis of nano-HC (nHC), on monolayer cultures of chondrocytes. As a substrate for the chondrocytes monolayer culture in vitro, nHC was able to restrain the dedifferentiation of chondrocytes and facilitate cell expansion, which was detected by methyl thiazolyl tetrazolium assay, scanning electron microscopy, calcein-acetoxymethyl/propidium iodide staining, hematoxylin and eosin staining, Safranin O staining, immunohistochemical staining and reverse transcription-quantitative polymerase chain reaction. Furthermore, the nHC films significantly facilitated cell growth and enhanced the expression of cartilage-specific extracellular matrix (ECM) components, including aggrecan and type II collagen. In addition, nHC films markedly downregulated the expression of collagen type I, an indicator of dedifferentiation. The results indicated that nHC, a collagen-based substrate optimized by nanoparticles, was able to better support cell growth and preserve cell phenotype compared with collagen alone or HC. The nHC film, which favors cell growth and prevents the dedifferentiation of chondrocytes, may therefore serve as a useful cartilage-like ECM for chondrocytes. In conclusion, nHC film is a promising substrate for the culture of chondrocytes in cell-based therapy.

Mesenchymal Stromal/stem Cell-derived Extracellular Vesicles Promote Human Cartilage Regeneration In Vitro.

Osteoarthritis (OA) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. Recently, allogeneic human mesenchymal stromal/stem cells (MSC) entered clinical trials as a novel therapy for OA. Increasing evidence suggests that therapeutic efficacy of MSC depends on paracrine signalling. Here we investigated the role of extracellular vesicles (EVs) secreted by human bone marrow derived MSC (BMMSC) in human OA cartilage repair.Methods: To test the effect of BMMSC-EVs on OA cartilage inflammation, TNF-alpha-stimulated OA chondrocyte monolayer cultures were treated with BMMSC-EVs and pro-inflammatory gene expression was measured by qRT-PCR after 48 h. To assess the impact of BMMSC-EVs on cartilage regeneration, BMMSC-EVs were added to the regeneration cultures of human OA chondrocytes, which were analyzed after 4 weeks for glycosaminoglycan content by 1,9-dimethylmethylene blue (DMMB) assay. Furthermore, paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-O) and type II collagen (immunostaining).Results: We show that BMMSC-EVs inhibit the adverse effects of inflammatory mediators on cartilage homeostasis. When co-cultured with OA chondrocytes, BMMSC-EVs abrogated the TNF-alpha-mediated upregulation of COX2 and pro-inflammatory interleukins and inhibited TNF-alpha-induced collagenase activity. BMMSC-EVs also promoted cartilage regeneration in vitro. Addition of BMMSC-EVs to cultures of chondrocytes isolated from OA patients stimulated production of proteoglycans and type II collagen by these cells.Conclusion: Our data demonstrate that BMMSC-EVs can be important mediators of cartilage repair and hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis.

A-674563 increases chondrocyte marker expression in cultured chondrocytes by inhibiting Sox9 degradation

The implantation of autologous chondrocytes is a therapeutic treatment for articular cartilage damage. However, the benefits are limited due to the expansion of chondrocytes in monolayer culture, which causes loss of chondrocytic characters. Therefore, culture conditions that enhance chondrocytic characters are needed. We screened 5822 compounds and found that A-674563 enhanced the transcription of several chondrocyte marker genes, including Col2a1, Acan and Col11a2, in mouse primary chondrocytes. Experiments using cycloheximide, MG132 and bafilomycin A1 have revealed that Sox9 is degraded through the ubiquitin-proteasome pathway and that A-674563 inhibits this degradation, resulting in larger amount of Sox9 protein. RNA sequencing transcriptome analysis showed that A-674563 increases the expression of the gene that encodes ubiquitin-specific peptidase 29, which is known to induce the deubiquitination of proteins. Although the precise mechanism remains to be determined, our findings indicated that A-674563 could contribute to culture conditions that expand chondrocytes without losing chondrocytic characters.

Role of Fibulin 3 in Aging-Related Joint Changes and Osteoarthritis Pathogenesis in Human and Mouse Knee Cartilage.

The EFEMP1 gene encoding fibulin 3 is specifically expressed in the superficial zone (SZ) of articular cartilage. The aims of this study were to examine the expression patterns of fibulin 3 in the knee joints during aging and during osteoarthritis (OA) and to determine the role of fibulin 3 in the pathogenesis of OA. Immunohistochemical analysis was performed on normal and OA knee cartilage samples from humans and mice. Experimental OA was induced in wild-type and fibulin 3-/- mice, and the severity of OA was evaluated by histologic scoring. To examine fibulin 3 function, human chondrocyte monolayer cultures were transfected with small interfering RNA (siRNA), followed by quantitative polymerase chain reaction and Western blot analyses. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transduced with an EFEMP1 lentivirus and analyzed for markers of chondrogenesis. Fibulin 3 was specifically expressed in the SZ of normal knee joint cartilage from humans and mice, and the expression levels declined with aging. Both aging-related OA and experimental OA were significantly more severe in fibulin 3-/- mice compared with wild-type mice. Fibulin 3 expression was high in undifferentiated human BM-MSCs and decreased during chondrogenesis. Suppression of fibulin 3 by siRNA significantly increased the expression of SOX9, type II collagen, and aggrecan in human articular chondrocytes, while overexpression of fibulin 3 inhibited chondrogenesis in BM-MSCs. Fibulin 3 is specifically expressed in the SZ of articular cartilage and its expression is reduced in aging and OA. Fibulin 3 regulates differentiation of adult progenitor cells, and its aging-related decline is an early event in the pathogenesis of OA. Preventing aging-associated loss of fibulin 3 or restoring it to normal levels in SZ chondrocytes has the potential to delay or prevent the onset of OA.

DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro

DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full-scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome-wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5-azacytidine (5-AzaC). This research revealed that the general methylation level of CpG was increased while the COL-1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5-AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL-1A1 promoter was increased after 5-AzaC treatment. The COL-1A1 expression level was increased while that of SOX-9 was decreased during the chondrocyte dedifferentiation. 5-AzaC treatment up-regulated the SOX-9 expression while down-regulated the COL-1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage-specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. J. Cell. Physiol. 232: 1708-1716, 2017. © 2016 Wiley Periodicals, Inc.

Cell compaction influences the regenerative potential of passaged bovine articular chondrocytes in an ex vivo cartilage defect model

The loss and degradation of articular cartilage tissue matrix play central roles in the process of osteoarthritis (OA). New models for evaluating cartilage repair/regeneration are thus of great value for transferring various culture systems into clinically relevant situations. The repair process can be better monitored in exvivo systems than in invitro cell cultures. I have therefore established an exvivo defect model prepared from bovine femoral condyles for evaluating cartilage repair by the implantation of cells cultured in various ways, e.g., monolayer-cultured cells or suspension or pellet cultures of articular bovine chondrocytes representing different cell compactions with variable densities of chondrocytes. I report that the integrin subunit α10 was significantly upregulated in suspension-cultured bovine chondrocytes at passage P2 compared with monolayer-cultured cells at P1 (p=0.0083) and P2 (p<0.05). Suspension-cultured cells did not promote cartilage repair when compared with implanted monolayer-cultured chondrocytes and pellets: 24.0±0.66% for suspension cells, 46.4±2.9% for monolayer cells, and 127.64±0.90% for pellets (p<0.0001) of the original defect volume (percentage of defect). Additional cultivation with chondrogenesis-promoting growth factors TGF-β1 and BMP-2 revealed an enhancing effect on cartilage repair in all settings. The advantage and innovation of this system over invitro differentiation (e.g., micromass, pellet) assays is the possibility of examining and evaluating cartilage regeneration in an environment in which implanted cells are embedded within native surrounding tissue at the defect site. Such exvivo explants might serve as a better model system to mimic clinical situations.