Bovine Articular Chondrocytes Research Articles

Expressions of local renin-angiotensin system components in chondrocytes.

In 2013, we reported that local reninangiotensin system (local RAS) components express during the hypertrophic differentiation of chondrocytes and can modulate it, using ATDC5 cell line that involves differentiation from mesenchymal stem cells to calcified hypertrophic chondrocytes. However, the expressions of local RAS components in normal chondrocytes have not been revealed yet. The purpose of this study is to examine the expression of the local RAS components in chondrocytes in vivo and the conditions allowing the expression. We stained five major regions of 8-week-old C57BL/6 adult mice in which chondrocytes exist, including epiphyseal plates and hyaline cartilages, with antibodies to local RAS components. We also examined the expression of local RAS components in the cultured bovine’s articular cartilage chondrocytes using quantitative reverse transcription polymerase chain reaction and western blot analysis. In result, hypertrophic chondrocytes of epiphyseal plates included in the tibia and the lamina terminals expressed local RAS components. However, hyaline chondrocytes, including the knee articular cartilages, the parenchyma of nasal septums and of the tracheal walls, did not express local RAS components. Cultured bovine’s articular cartilage chondrocytes also did not express local RAS components. However, inducing hypertrophy by administering interleukin-1β or tumor necrosis factor-α, the cultured articular chondrocytes also expressed angiotensin II type 1 receptor and angiotensin II type 2 receptor. In conclusion, local RAS components express particularly in chondrocytes which occur hypertrophy and do not in hyaline chondrocytes. The results are in accord with our previous in vitro study. We think this novel knowledge is important to investigate cartilage hypertrophy and diseases induced by hypertrophic changes like osteoarthritis.

Effect of Transforming Growth Factor‐β (TGF‐β) on Sox9 function and chondrocyte phenotype (LB40)

TGF‐β enhances Sox9 protein expression as well as downstream targets that regulate extracellular matrix production in bovine articular chondrocytes (BACs). The extracellular matrix protein 3‐Prime‐Phoshoadenosine 5‐Prime‐Phosphosulfate Synthase 2 (Papss2) has been associated with spondyloepimetaphyseal dysplasias in humans and is required for proper sulfation of proteoglycans in cartilage. Our work and that of others has shown that TGF‐β can regulate Papss2 expression in articular cartilage. We aim to identify whether Sox9 is involved downstream of TGF‐β in the regulation of Papss2 mRNA expression.Using primary BACs in micromass cultures, we analyzed the effect of TGF‐β (5ng/ml) on chondrocyte phenotype, Sox9 protein expression, stability, cellular localization and activity. We also assessed the effect of TGF‐β and/or Sox9 overexpression on the mRNA expression of Papss2, Prg4 and GAPDH via quantitative real‐time PCR.We found that treatment with TGF‐β enhanced proteoglycan content and delayed chondrocyte hypertrophic differentiation in BACs. We also observed an increase in Sox9 protein levels and stability, without changes in mRNA. The increase in Sox9 protein correlated with enhanced Col2a‐reporter activity. Importantly, we found Papss2 to be upregulated via Sox9 when wild‐type (WT)‐Sox9 was overexpressed via adenoviral transduction (50 MOI/cell). Interestingly, treatment +/‐ TGF‐β when Sox9 was overexpressed did not further increase Papss2 mRNA, supporting the hypothesis that Sox9 works downstream of TGF‐ β. However, TGF‐β, but not Sox9, increased Prg4 mRNA expression, suggesting specificity of signaling. A GAPDH control was examined and remained unchanged in response to either treatment. The regulation of Papss2 by Sox9 represents a novel paradigm involving TGF‐β‐dependent signaling in articular chondrocytes.Grant Funding Source: Supported by NIAMS‐ NIH

Variation of mechanical property of single-walled carbon nanotubes-treated cells explored by atomic force microscopy.

With a range of biological properties, single-walled carbon nanotubes (SWCNTs) are a promising material for nanobiotechnology. Concerns about their potential effect on human health have led to the interest in understanding the interaction between SWCNTs and cells. There are many reports showing the potential cellular effects of SWCNTs but this issue is quite controversially discussed in the literature. In this study, we used conventional biological evaluation methods and atomic force microscopy (AFM) to compare the effects of SWCNTs on three different cell types: bovine articular chondrocytes, human bone marrow-derived mesenchymal stem cells and HeLa cells. No obvious effects of SWCNTs on cell morphology and viability were observed during 3 days in vitro culture. However, SWCNTs significantly increased the Young's modulus of all the three types of cells. The effect of SWCNTs on Young's modulus was in an increasing order of Hela cells < chondrocytes < mesenchymal stem cells. AFM was shown to be a useful tool for investigation of the effect of nanomaterials on mechanical property of cells.

Sprifermin (rhfgf18) has an anabolic effect on human osteoarthritic chondrocytes involving fgfr3 and erk1/2 but not p38α and JNKS

Purpose: Restoring the articular surface of degraded or largely injured cartilage necessitates cartilage re-cellularization and the stimulation of cartilage matrix molecule production. Sprifermin (currently in a phase II clinical trial for knee osteoarthritis) is a growth factor that is involved in chondrogenesis and exerts an anabolic effect on cartilage. Using a 3D cell culture system, we previously demonstrated the ability of Sprifermin to stimulate cartilage matrix molecule expression and to promote the chondrocyte phenotype in bovine and porcine articular chondrocytes. In the present work, we aimed to confirm this effect in osteoarthritic chondrocytes of human origin (hOA). In parallel, the pathways involved in Sprifermin signaling were investigated in porcine chondrocytes. Methods: Primary osteoarthritic chondrocytes were isolated from the cartilage of patients who underwent total knee replacement. Cells were cultivated for a few days in monolayer first and then for one week in scaffold-free 3D culture before starting the treatment. The latter consisted in the incubation with Sprifermin [100 ng/mL] permanently or one day/week for a total period of four weeks. Results were compared to a control culture without Sprifermin. Biochemical assays, quantitative PCR (qPCR) and histology were used to characterize the 3D constructs. FGFR expression by qPCR and activation of various kinases (with the human phospho-MAPK array kit from R&D Systems) upon Sprifermin stimulation were assessed on freshly isolated hOA chondrocytes. The effects of a panel of inhibitors (MAPK inhibitors and one pan-FGFR inhibitor) were tested in porcine cells. For this, freshly isolated chondrocytes from a pig hip were cultivated for 7 days in monolayer with 100 ng/mL of Sprifermin in presence of three concentrations of the inhibitors (0.1, 0.3 and 1 μM or 1, 3 and 10 μM depending on their potency). Sprifermin has been previously shown to strongly decrease the collagen I expression (monitored by qPCR) and to promote a round cell morphology (evaluated by actin staining) of porcine chondrocytes in monolayer. These parameters were used to evaluate the impact of the various inhibitors. Results: A 3D scaffold-free culture was used to test the effect of Sprifermin on hOA chondrocytes. In this setting Sprifermin [1day/week] has been found to have a positive effect on the cell content and to greatly increase the size and matrix content of the 3D constructs. In accordance with the literature, FGFR1 and 3 exhibited the highest expression among FGFRs in hOA chondrocytes. In addition, Sprifermin effects were suppressed by FGFR inhibition. In porcine chondrocytes cultivated in monolayer, Sprifermin strongly inhibited collagen I expression and promoted a round cell shape. Both effects were completely inhibited in presence of the pan-FGFR inhibitor PD173074 in a dose-dependent manner. Because FGF18 does not bind FGFR1, in chondrocytes Sprifermin signaling involves mainly FGFR3. Upon incubation with Sprifermin [100 ng/mL], ERK1/2 was activated as well as p38γ, but not p38α or the JNKs. The ERK inhibitor PD325901 completely inhibited the effect of Sprifermin on cell morphology at 100 nM. The JNK inhibitor (SR3306) and p38 inhibitors (BIRB-796 and SB203580) did not have any effect. The effect of these inhibitors on collagen I expression is still under investigation. Taken together these results indicate that ERK1/2 is involved in Sprifermin signaling whereas p38α and JNKs are not. Conclusions: As observed in previous studies with bovine and porcine chondrocytes, Sprifermin was found to exert anabolic effects in hOA chondrocytes. These effects seem to be mediated by FGFR3 and ERK1/2 activation. Others demonstrated that ERK1/2 is also involved in FGF2-FGFR1 signaling but in this case led to a catabolic response. This difference can be possibly explained by the fact that JNK and p38 are also activated by FGF2-FGFR1 signaling but not by FGF18-FGFR3.

Cellular uptake of single-walled carbon nanotubes in 3D extracellular matrix-mimetic composite collagen hydrogels.

Carbon nanotubes (CNTs) exhibit intrinsic unique physical and chemical properties that make them attractive candidates for biological and biomedicine applications. An efficient cellular uptake of CNTs is vital for many of these applications. However, most of the cellular uptake studies have been performed with a two-dimensional cell culture system. In this study, cellular uptake of single-walled carbon nanotubes (SWCNTs) was investigated by using a three-dimensional cell culture system. Bovine articular chondrocytes cultured in SWCNTs/collagen composite hydrogels maintained their proliferation capacity when compared to the culture in collagen hydrogels. Ultraviolet-visible-near-infrared spectroscopy analysis revealed a high amount of SWCNTs were internalized by cells. Confocal Raman imaging showed that most of the internalized SWCNTs were distributed in the perinuclear region. The results indicated that SWCNTs could be internalized by chondrocytes when SWCNTs were incorporated in the three-dimensional biomimetic collagen hydrogels.

Primary cilia disassembly down-regulates mechanosensitive hedgehog signalling: a feedback mechanism controlling ADAMTS-5 expression in chondrocytes

SummaryObjectiveHedgehog signalling is mediated by the primary cilium and promotes cartilage degeneration in osteoarthritis. Primary cilia are influenced by pathological stimuli and cilia length and prevalence are increased in osteoarthritic cartilage. This study aims to investigate the relationship between mechanical loading, hedgehog signalling and cilia disassembly in articular chondrocytes.MethodsPrimary bovine articular chondrocytes were subjected to cyclic tensile strain (CTS; 0.33 Hz, 10% or 20% strain). Hedgehog pathway activation (Ptch1, Gli1) and A Disintegrin And Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS-5) expression were assessed by real-time PCR. A chondrocyte cell line generated from the Tg737ORPK mouse was used to investigate the role of the cilium in this response. Cilia length and prevalence were quantified by immunocytochemistry and confocal microscopy.ResultsMechanical strain upregulates Indian hedgehog expression and activates hedgehog signalling. Ptch1, Gli1 and ADAMTS-5 expression were increased following 10% CTS, but not 20% CTS. Pathway activation requires a functioning primary cilium and is not observed in Tg737ORPK cells lacking cilia. Mechanical loading significantly reduced cilium length such that cilia became progressively shorter with increasing strain magnitude. Inhibition of histone deacetylase 6 (HDAC6), a tubulin deacetylase, prevented cilia disassembly and restored mechanosensitive hedgehog signalling and ADAMTS-5 expression at 20% CTS.ConclusionsThis study demonstrates for the first time that mechanical loading activates primary cilia-mediated hedgehog signalling and ADAMTS-5 expression in adult articular chondrocytes, but that this response is lost at high strains due to HDAC6-mediated cilia disassembly. The study provides new mechanistic insight into the role of primary cilia and mechanical loading in articular cartilage.

Articular Chondrocyte Redifferentiation in 3D Co-cultures with Mesenchymal Stem Cells

In this work, we evaluated the ability of 3D co-cultures with mesenchymal stem cells (MSCs) to redifferentiate monolayer expanded articular chondrocytes (ACs) and produce cartilaginous extracellular matrix at varying stages of the dedifferentiation process and further examined the dependency of this effect on the culture medium composition. Primary bovine ACs were expanded in monolayers for up to nine population doublings to obtain seven cell stocks with gradually increasing levels of dedifferentiation. Culture expanded ACs were then seeded as monocultures and co-cultures with rabbit bone marrow-derived MSCs (30:70 ratio of ACs-to-MSCs) on porous scaffolds. Parallel cultures were established for each cell population in serum-containing growth medium and serum-free induction medium supplemented with dexamethasone and TGF-β3. After 3 weeks, all groups were analyzed for DNA content, glycosaminoglycan (GAG) and hydroxyproline (HYP) production, and chondrogenic gene expression. Significant enhancements in cellularity, GAG content and GAG/HYP ratio, and chondrogenic phenotype were observed in the induction medium compared to growth medium at all levels of AC expansion. Furthermore, primary co-cultures showed similarly enhanced chondrogenesis compared to monocultures in both culture media, whereas passaged ACs benefitted from co-culturing only in the induction medium. We conclude that co-cultures of ACs and MSCs can produce superior in vitro engineered cartilage in comparison to pure AC cultures, due to both heterotypic cellular interactions and decreased need for monolayer expansion of biopsied chondrocytes. While the initial level of AC dedifferentiation affected the quality of the engineered constructs, co-culture benefits were realized at all stages of AC expansion when suitable chondroinductive culture medium was used.

Collagen Scaffolds with Controlled Insulin Release and Controlled Pore Structure for Cartilage Tissue Engineering

Controlled and local release of growth factors and nutrients from porous scaffolds is important for maintenance of cell survival, proliferation, and promotion of tissue regeneration. The purpose of the present research was to design a controlled release porous collagen-microbead hybrid scaffold with controlled pore structure capable of releasing insulin for application to cartilage tissue regeneration. Collagen-microbead hybrid scaffold was prepared by hybridization of insulin loaded PLGA microbeads with collagen using a freeze-drying technique. The pore structure of the hybrid scaffold was controlled by using preprepared ice particulates having a diameter range of 150–250 μm. Hybrid scaffold had a controlled pore structure with pore size equivalent to ice particulates and good interconnection. The microbeads showed an even spatial distribution throughout the pore walls. In vitro insulin release profile from the hybrid scaffold exhibited a zero order release kinetics up to a period of 4 weeks without initial burst release. Culture of bovine articular chondrocytes in the hybrid scaffold demonstrated high bioactivity of the released insulin. The hybrid scaffold facilitated cell seeding and spatial cell distribution and promoted cell proliferation.

Co-administration of hyaluronic acid with local anaesthetics shows lower cytotoxicity than local anaesthetic treatment alone in bovine articular chondrocytes

ObjectiveTo study the effect of hyaluronic acid (HA) on local anaesthetic chondrotoxicity in vitro.MethodsChondrocytes were harvested from bovine femoral condyle cartilage and isolated using collagenase-containing media. At 24 hours after seeding 15 000 cells per well onto a 96-well plate, chondrocytes were treated with media (DMEM/F12 + ITS), PBS, 1:1 lidocaine (2%):PBS, 1:1 bupivacaine (0.5%):PBS, 1:1 lidocaine (2%):HA, 1:1 bupivacaine (0. 5%):HA, or 1:1 HA:PBS for one hour. Following treatment, groups had conditions removed and 24-hour incubation. Cell viability was assessed using PrestoBlue and confirmed visually using fluorescence microscopy.ResultsMedia-treated groups had a mean of 1.55×104 cells/well (sem 783). All treated cells showed statistically significant reduced viability when compared with media alone (all p < 0.003). Cells treated with bupivacaine + HA (6.70×103 cells/well (sem 1.10×103)) survived significantly more than bupivacaine (2.44×103 cells/well (sem 830)) (p < 0.001). Lidocaine + HA (1.45×103 cells/well (sem 596)) was not significantly more cytotoxic than lidocaine (2.24×103 cells/well (sem 341)) (p = 0.999). There was no statistical difference between the chondrotoxicities of PBS (8.49×103 cells/well (sem 730) cells/well) and HA (4.75×103 cells/well (sem 886)) (p = 0.294).ConclusionsHA co-administration reduced anaesthetic cytotoxicity with bupivacaine but not lidocaine, suggesting different mechanisms of injury between the two. Co-administered intra-articular injections of HA with bupivacaine, but not lidocaine, may protect articular chondrocytes from local anaesthetic-associated death.Cite this article: Bone Joint Res 2013;2:270–5.

Endogenous versus exogenous growth factor regulation of articular chondrocytes

Anabolic growth factors that regulate the function of articular chondrocytes are candidates for articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating articular chondrocyte reparative functions. We treated adult bovine articular chondrocytes with exogenous recombinant insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-β1), or with the genes encoding these growth factors for endogenous production. Treatment effects were measured as change in chondrocyte DNA content, glycosaminoglycan production, and aggrecan gene expression. We found that IGF-I stimulated chondrocyte biosynthesis similarly when delivered by either exogenous or endogenous means. In contrast, exogenous TGF-β1 stimulated these reparative functions, while endogenous TGF-β1 had little effect. Endogenous TGF-β1 became more bioactive following activation of the transgene protein product. These data indicate that effective mechanisms of growth factor delivery for articular cartilage repair may differ for different growth factors. In the case of IGF-I, gene therapy or protein therapy appear to be viable options. In contrast, TGF-β1 gene therapy may be constrained by a limited ability of chondrocytes to convert latent complexes to an active form.

BEHAVIOR OF ARTICULAR CHONDROCYTES ON NANOENGINEERED SURFACES

In tissue engineering, surface modification has becomes one of the leading methods to enhance initial cell attachment and subsequent cellular growth, differentiation and tissue formation. This work studied growth and behavior of primary bovine articular chondrocytes on self-assembled multilayer nanofilms composed of: polyelectrolytes [poly(styrene sulfonate) (PSS), poly-L-lysine (PLL), poly-D-lysine (PDL), chondroitin sulfate (CS), poly(ethyleneimine) (PEI), poly(dimethyldiallylammonium chloride) (PDDA), poly(ethylene glycol) amine (PEG - NH2)] and proteins [bovine serum albumin (BSA), collagen, fibronectin, laminin]. These biomaterials were used to build mono-, bi-, and tri-layer nanofilm architectures. Potential cytotoxic effects were assessed using Live/Dead assay and cell proliferation was quantified using MTT assay. Bright field and fluorescence microscopy were used to analyze chondrocyte morphology. ImageJ software was used to analyze the number, mean area, circularity and Feret's diameter of viable cells. Cumulative results demonstrated that chondrocyte growth; proliferation and functionality were dependent on initial cell density, nanofilm thickness and material composition of nanofilms.

Trophic stimulation of articular chondrocytes by late‐passage mesenchymal stem cells in coculture

Coculture of mesenchymal stem cells (MSCs) with articular chondrocytes (ACs) increases glycosaminoglycan (GAG) accumulation compared to monoculture. MSCs might (1) differentiate into ACs (progenitor role) and/or (2) stimulate AC matrix metabolism (trophic role). MSCs lose the ability to undergo chondrogenesis after extended passaging. We hypothesized that MSCs act predominantly as progenitors, and that late-passage MSCs without chondrogenic potential would be unable to increase GAG in coculture. Early- and late-passage human MSCs (hMSCs) were grown in pellet monoculture under chondrogenic conditions and in pellet coculture with bovine ACs. Chondrogenesis was assessed with GAG quantification, safranin-O staining, and quantitative PCR (qPCR). Contributions of human and bovine cells were assessed with species-specific qPCR and human-specific immunostaining. Late-passage hMSCs did not undergo chondrogenesis in monoculture with chondrogenic stimuli or in coculture with ACs. Early-passage hMSCs underwent chondrogenesis only in response to chondrogenic stimuli. Coculture pellets in both cases accumulated as much GAG/DNA as monoculture AC pellets. Aggrecan transcription was not increased in coculture. Late-passage hMSCs that do not undergo chondrogenesis are capable of stimulating GAG accumulation in coculture with ACs. This supports a trophic effect of hMSCs on ACs. hMSCs may have therapeutic utility even after prolonged passaging.

Preparation of collagen scaffolds with controlled pore structures and improved mechanical property for cartilage tissue engineering

Appropriate pore structures and mechanical properties are required for scaffolds that are used for tissue engineering and regenerative medicine. In this study, pre-prepared ice particulates were used as a porogen material to prepare collagen porous scaffolds with well-controlled pore structures and improved mechanical properties. Porogen ice particulates initiated the formation of interconnected large spherical pores surrounded by small pores. The large spherical pores were well compacted and increased the elastic modulus of the scaffolds. The unique pore structures facilitated cell penetration, resulting in a homogeneous cell distribution throughout the scaffolds. The excellent mechanical properties protected the scaffolds from deformation during cell culturing and implantation. The collagen porous scaffolds facilitated cartilage regeneration when bovine articular chondrocytes were cultured in these scaffolds. The use of pre-prepared ice particulates as a porogen material proved to be a useful method to control the pore structure and improve the mechanical properties of collagen-based porous scaffolds.

Chondrotoxic effect of intraarticular bupivacaine administration

We read the article ‘‘The comparison of intraarticular morphine-bupivacaine and tramadol-bupivacaine in postoperative analgesia after arthroscopic anterior cruciate ligament reconstruction’’ by Hosseini et al. [5] recently published in this journal with great interest. The article describes a randomised, double blind, controlled trial study of ASA I–II patients undergoing arthroscopic ACL reconstruction performed under general anaesthesia who were given a combination of drugs intraarticularly for postoperative analgesia and pain control. Study was approved by the research ethics committees in Shahid Sadoughi University of Medical Sciences, and written informed consents were obtained from 60 male patients. Patients were randomly allocated into three groups. The MB group (n = 20) received 10 mg morphine and 0.5 % bupivacaine, the TB group (n = 20) received 100 mg tramadol and 0.5 % bupivacaine and the control group (n = 20) received 20 ml of isotonic saline intraarticularly at the end of the operation. The authors concluded that intraarticular morphine–bupivacaine combination provides more effective pain relief, longer analgesic duration, less supplemental analgesic postoperative requirements, shorter unassisted ambulation and discharge time when compared to intraarticular tramadol–bupivacaine injection and isotonic saline after ACL reconstruction arthroscopy. In side effects assesment, none of the patients had respiratory depression, and there was no significant difference between the groups in terms of nausea and vomiting. It is interesting that there was no mention or even concern of chondrotoxic effect of bupivacaine in this article. Chu et al. [4] have shown in 2006 that 0.5 % bupivacaine solution is cytotoxic to bovine articular chondrocytes and articular cartilage in vitro after only 15–30 min exposure. The authors suggested caution in the intraarticular use of 0.5 % bupivacaine. Toxicity of bupivacaine was demonstrated on human articular chondrocytes too [6]. Piper et al. compared the in vitro viability after exposure of full-thickness knee cartilage explants and cultured chondrocytes to 0.9 % normal saline solution, 0.5 % ropivacaine and 0.5 % bupivacaine, respectively, after 30 min. Chondrocyte viability in cartilage explants and the viability of cultured chondrocytes were significantly lower after treatment with bupivacaine as compared to ropivacaine. A few years later, in vivo animal study was conducted to determine whether a single intraarticular injection of 0.5 % bupivacaine results in chondrocyte morbidity and rapid chondrolysis [3]. Reduced chondrocyte density without cartilage tissue loss 6 months after a single intraarticular injection of 0.5 % bupivacaine suggested bupivacaine toxicity. The effects of bupivacaine were milder than those of an injection of 0.6 % monoiodoacetate (positive control), which resulted in chondrolysis over the same time period. As the risk of chondrotoxic effect of bupivacaine clearly exists, we would like to know why the adverse effect of bupivacaine was completely ignored in the article. Unfortunately, period of time when the study has been conducted was not specified in the Hosseini et al. [5] article. We are curious if the authors were aware of the adverse chondrotoxic effect of bupivacaine and if it was mentioned in the N. V. Filipcic (&) Department of Anaesthesiology and Intensive Care, University Hospital Merkur, Zagreb University, Zajceva 19, 10000 Zagreb, Croatia e-mail: natashav7@gmail.com

Preparation of collagen porous scaffolds with a gradient pore size structure using ice particulates

Direct comparison using scaffolds with a gradient pore size structure is important to investigate the effect of pore size on tissue regeneration. Collagen porous scaffolds with a pore size gradient were prepared by using pre-prepared ice particulates as a porogen material. The ice particulates had diameters of 150–250, 250–355, 355–425 and 425–500μm. The gradient collagen scaffolds had well-interconnected pore structures with compactly packed spherical pores. The gradient pore scaffolds were used for culture of bovine articular chondrocytes, directly examining the effect of pore size on cartilage regeneration. Chondrocytes adhered and showed a homogenous distribution throughout the scaffolds. In vivo implantation results indicated that the micropores in the scaffolds prepared with ice particulates in the range of 150–250μm showed the best effect on cartilage regeneration.

SAT0318 Could Adrenomedullin be Considered as a Treatment for Osteoarthritis?

BackgroundOsteoarthritis (OA) is a frequent articular disease which generally affects elderly people. Major hallmarks of OA are chondrolysis, chondrocyte apoptosis and local inflammation. Of note, there is no treatment for...

Role of insulin in Sox9 mediated COL2A1 expression in passaged articular chondrocytes

Purpose: One of the factors preventing clinical application of regenerative medicine to degenerative cartilage diseases is a suitable source of cells. Cartilage tissue is sparsely cellular and chondrocytes are the only cell type but under in vitro culture conditions they lose their phenotype along with the ability to generate hyaline cartilaginous tissue. Here we describe a reliable serum and growth factor free culture system to reverse this change in phenotype. Methods: Bovine articular chondrocytes (BAC) were passaged twice to allow for cell number expansion (P2) and cultured at high density on 3D collagen type II-coated membranes in media supplemented with ITS+ with 4.5g/l glucose, proline and 10-8 M dexamethasone (SF3D). Cultures were grown for up to 4 weeks. Inhibition studies were initiated on day 2 with 100uM HNMPA-(AM3). For FACS cells were harvested with Trypsin, stained with antibodies reactive with CD105-PE or CD44-PE (eBioscience, US) and analyzed by EPICS XL FACS and Kaluza analysis software (Beckman Coulter, US). ChIP-qPCR was carried out after in-situ cross-linking with 0.75% formaldehyde. Cells were sonicated in 1% Triton-100 and 1% SDS lysis buffer (Vibracell 30sec ON and 60sec OFF, 20x) and 250-900 bp DNA fragmentation was confirmed by agarose gel electrophoresis. Purified DNA was analyzed by qPCR using primers within the enhancer region of COL2A1. For immunoprecipitation cells were extracted in Ripa buffer and incubated with Sox9 antibody, the immune complex was harvested with Protein A/G beads. Extracts were immunoblotted with antibodies reactive with Sox6 and Med12. Results: 99% of P2 cells in monolayer culture were CD44+ and 40% were CD105+. They showed differential gene expression with high type 1 collagen and low type II collagen. Gene expression of other cartilage associated genes was lower in P2 cells when compared with the primary P0 chondrocytes. When placed in SF3D the P2 cells expressed chondrogenic genes and accumulated extracellular matrix (ECM) rich in proteoglycans (PGs) and type II collagen. Decreasing insulin receptor (IR) with HNMPA-(AM3) inhibited collagen and PGs synthesis and reduced expression of col II, col XI, IR and IGF1R genes as well as Sox6 and 9 proteins. Co-IP and ChIP analysis on inhibited cells showed binding of co-activators Sox6 and Med12 with Sox9 but reduced Sox9-Col2A1 binding. Figure 1. Photomicrographs of the tissue formed by P2 when cultured in SC3D culture show no matrix accumulation (far left panel) SF3D show accumulation of matrix rich in PGs evident by Toluidine Blue staining (second panel). SF3D tissue stained positively for collagen type II (red) and were predominantly negative for collagen type I (green) (third panel) similar to native cartilage tissue stained with the same antibodies (fourth panel). Conclusions: Here we describe a novel serum/ xenogeneic compound/ growth factor- free culture system which promotes hyaline-like cartilage tissue formation by insulin mediated regulation of Sox9-Col2A1 binding. This is a critical step towards the ultimate goal of developing autologous patient specific tissue engineered hyaline-like cartilage tissue suitable to use for repair/replacement of damaged articular cartilage. The suitability of this system for use in regenerative medicine approaches needs to be examined in vivo. Acknowledgements: Supported by DoD.

Agrin expression is downregulated in OA and is required for chondrocyte differentiation

Purpose: Background: Agrin is a large basement membrane heparan sulphate proteoglycan best known for its function at the neuromuscular junction. It is responsible for synapse formation via binding to the MuSK and LRP4 receptor complex, leading to Acetylcholine receptor aggregation. Mice deficient of Agrin die perinatally as a result of respiratory failure. Agrin is also expressed in other cell types including chondrocytes of the developing growth plate, and agrin-deficient embryos in which agrin expression had been rescued at the neuromuscular junction develop skeletal abnormalities 1. Therefore it is possible that Agrin may play a role in cartilage homeostasis. Aims: The aim of this project is to determine if agrin plays a functional role in the homeostasis of the articular cartilage and in osteoarthritis. Methods: Human adult articular cartilage explants were obtained from preserved or damaged cartilage from individuals undergoing joint replacement for osteoarthritis. Experimental osteoarthritis was induced in 10 week old mice by destabilization of the medial meniscus2. Sham-operated controlateral knees were used as controls. Knees were collected 8weeks after surgery, decalcified and embedded in paraffin. Bovine primary articular chondrocytes (BPAC) were isolated by pronase/collagenase digestion from the articular cartilage of the metatarso-phalangeal joints of 18 month old calves from a local slaughterhouse. The BPAC and the human chondrogenic cell line C28/I2 were cultured in micromass to promote differentiation and extracellular matrix production. For gain- and loss-of-function experiments, chondrocytes were transfected with a mammalian expression vector encoding for full-length Agrin (FL-Agrin) or with Agrin siRNA's respectively. Accumulation of cartilage-specific extracellular matrix production, rich in highly sulphated glycosaminoglycans (GAGs), was quantified by alcian blue staining at pH 0.2 and alizarin red staining. Chondrogenic potential was measured using pellet analysis as previously described3, BPAC pellets were cultured for 14 days, weighed, sectioned and stained with alizarin red and safranin o. Gene expression analysis of Agrin, Col2A1, Aggrecan, Sox9 was performed by real time PCR. Immunofluorescence was used to determine the expression levels of Agrin at protein level in chondrocytes cultured in monoloayer and in paraffin-embedded tissue sections. Results: Agrin was expressed in healthy adult human and bovine articular cartilage. Agrin was signifcantly downregulated in human osteoarthritic cartilage. This downregulation was also replicated in experimental murine osteoarthritis, indicating that it is a consequence rather than a cause of osteoarthritis. Agrin expression was partially retained in the articular cartilage of sham operated knees, indicating that inflammation alone (present both in DMM and in sham) is not sufficient to induce Agrin downregulation, but cartilage damage is necessary. Endogenous expression of Agrin was confirmed in C28/I2 and in BPAC. Overexpression of FL-Agrin resulted in enhanced differentiation as demonstrated by upregulated the cartilage key transcription factor Sox9. . Knock-down of Agrin by siRNA resulted in reduced GAG production in C28/I2 and chondrocyte de-differentiation as documented by decreased expression Sox9, Col2A1 and Aggrecan mRNA. Conclusions: Agrin expressed in healthy adult articular cartilage and is downregulated in osteoarthritis; - Agrin is anabolic in cultured articular chondrocytes; - Agrin is required for chondrocyte differentiation in vitro.

Genetic epidemiology of Scheuermann's disease

Genetic epidemiology of Scheuermann's disease

Computational modeling of nutrient utilization in engineered cartilage

This study presents a mathematical model for simulating cartilaginous culture of chondrocytes seeded in scaffolds and for investigating the effects of glucose and oxygen concentration and pH value on cell metabolic rates. The model can clearly interpret the unexplained experimental observation (Sengers BG, Heywood HK, Lee DA, Oomens CWJ, Bader DL. Nutrient utilization by bovine articular chondrocytes: A combined experimental and theoretical approach. J Biomech Eng. 2005;127:758-766.), which showed that the oxygen concentration within the scaffold may increase instead of continuously decreasing in static cartilaginous culture of chondrocytes. Results from simulation demonstrate that when cells metabolize glucose and form lactate under high glucose concentration conditions, the acidity in the culture environment increases, inhibiting cell metabolic rates in the process. Consequently, the rate of oxygen consumption decreases in later stages of cell culture. As oxygen can be replenished through the free surface of the culture medium, oxygen concentration within the scaffold increases rather than decreases over time in the acidic environment. Different initial glucose concentration yields different results. In low glucose concentration conditions, oxygen concentration basically keeps decreasing with culture time. This is because the pH in the environment does not significantly change because of slower glycolysis rate in low glucose concentration cases, forming less lactic acid. From the simulation results, additional information regarding in vitro culture of chondrocytes is obtained. The correlations between nutrient consumption, lactate secretion, and pH changes during cell culture are also understood and may serve as a reference for in vitro cell culture research of tissue engineering.