Bovine Articular Chondrocytes Research Articles

Cartilage-derived cells display heterogeneous pericellular matrix synthesis in agarose microgels

The pericellular matrix (PCM) surrounding chondrocytes is essential for articular cartilage tissue engineering. As the current isolation methods to obtain chondrocytes with their PCM (chondrons) result in a heterogeneous mixture of chondrocytes and chondrons, regenerating the PCM using a tissue engineering approach could prove beneficial. In this study, we aimed to discern the behavior of articular chondrocytes (ACs) in regenerating the PCM in such an approach and whether this would also be true for articular cartilage-derived progenitor cells (ACPCs), as an alternative cell source. Bovine ACs and ACPCs were encapsulated in agarose microgels using droplet-based microfluidics. ACs were stimulated with TGF-β1 and dexamethasone and ACPCs were sequentially stimulated with BMP-9 followed by TGF-β1 and dexamethasone. After 0, 3, 5, and 10 days of culture, PCM components, type-VI collagen and perlecan, and ECM component, type-II collagen, were assessed using flow cytometry and fluorescence microscopy. Both ACs and ACPCs synthesized the PCM before the ECM. It was seen for the first time that synthesis of type-VI collagen always preceded perlecan. While the PCM synthesized by ACs resembled native chondrons after only 5 days of culture, ACPCs often made less well-structured PCMs. Both cell types showed variations between individual cells and donors. On one hand, this was more prominent in ACPCs, but also a subset of ACPCs showed superior PCM and ECM regeneration, suggesting that isolating these cells may potentially improve cartilage repair strategies.

Immunomodulatory potential of mesenchymal stromal cell-derived extracellular vesicles in chondrocyte inflammation.

Osteoarthritis (OA) affects a large percentage of the population worldwide. Current surgical and nonsurgical concepts for treating OA only result in symptom-modifying effects. However, there is no disease-modifying therapy available. Extracellular vesicles released by mesenchymal stem/stromal cells (MSC-EV) are promising agents to positively influence joint homeostasis in the osteoarthritic surroundings. This pilot study aimed to investigate the effect of characterized MSC-EVs on chondrogenesis in a 3D chondrocyte inflammation model with the pro-inflammatory cytokine TNFα. Bovine articular chondrocytes were expanded and transferred into pellet culture at passage 3. TNFα, human MSC-EV preparations (MSC-EV batches 41.5-EVi1 and 84-EVi), EVs from human platelet lysate (hPL4-EV), or the combination of TNFα and EVs were supplemented. To assess the effect of MSC-EVs in the chondrocyte inflammation model after 14 days, DNA, glycosaminoglycan (GAG), total collagen, IL-6, and NO release were quantified, and gene expression of anabolic (COL-II, aggrecan, COMP, and PRG-4), catabolic (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5), dedifferentiation (COL-I), hypertrophy (COL-X, VEGF), and inflammatory (IL-8) markers were analyzed; histological evaluation was performed using safranin O/Fast Green staining and immunohistochemistry of COL I and II. For statistical evaluation, nonparametric tests were chosen with a significance level of p < 0.05. TNFα supplementation resulted in catabolic stimulation with increased levels of NO and IL-6, upregulation of catabolic gene expression, and downregulation of anabolic markers. These findings were supported by a decrease in matrix differentiation (COL-II). Supplementation of EVs resulted in an upregulation of the chondrogenic marker PRG-4. All MSC-EV preparations significantly increased GAG retention per pellet. In contrast, catabolic markers and IL-8 expression were upregulated by 41.5-EVi1. Regarding protein levels, IL-6 and NO release were increased by 41.5-EVi1. Histologic and immunohistochemical evaluations indicated a higher differentiation potential of chondrocytes treated with 84-EVi. MSC-EVs can positively influence chondrocyte matrix production in pro-inflammatory surroundings, but can also stimulate inflammation. In this study MSC-EV 41.5-EVi1 supplementation increased chondrocyte inflammation, whereas MSC-84-EVi supplementation resulted a higher chondrogenic potential of chondrocytes in 3D pellet culture. In summary, the selected MSC-EVs exhibited promising chondrogenic effects indicating their significant potential for the treatment of OA; however, the functional heterogeneity in MSC-EV preparations has to be solved.

Hydroquinone, an Environmental Pollutant, Affects Cartilage Homeostasis through the Activation of the Aryl Hydrocarbon Receptor Pathway.

Exposure to environmental pollutants has a proven detrimental impact on different aspects of human health. Increasing evidence has linked pollution to the degeneration of tissues in the joints, although through vastly uncharacterised mechanisms. We have previously shown that exposure to hydroquinone (HQ), a benzene metabolite that can be found in motor fuels and cigarette smoke, exacerbates synovial hypertrophy and oxidative stress in the synovium. To further understand the impact of the pollutant on joint health, here we investigated the effect of HQ on the articular cartilage. HQ exposure aggravated cartilage damage in rats in which inflammatory arthritis was induced by injection of Collagen type II. Cell viability, cell phenotypic changes and oxidative stress were quantified in primary bovine articular chondrocytes exposed to HQ in the presence or absence of IL-1β. HQ stimulation downregulated phenotypic markers genes SOX-9 and Col2a1, whereas it upregulated the expression of the catabolic enzymes MMP-3 and ADAMTS5 at the mRNA level. HQ also reduced proteoglycan content and promoted oxidative stress alone and in synergy with IL-1β. Finally, we showed that HQ-degenerative effects were mediated by the activation of the Aryl Hydrocarbon Receptor. Together, our findings describe the harmful effects of HQ on articular cartilage health, providing novel evidence surrounding the toxic mechanisms of environmental pollutants underlying the onset of articular diseases.

3D culture of bovine articular chondrocytes in viscous medium encapsulated in agarose hydrogels for investigation of viscosity influence on cell functions.

The mechanical properties of an extracellular microenvironment can affect cell functions. The effects of elasticity and viscoelasticity on cell functions have been extensively studied with hydrogels of tunable mechanical properties. However, investigation of the viscosity effect on cell functions is still very limited and it can be tricky to explore how viscosity affects cells in three-dimensional (3D) culture due to the lack of appropriate tools. In this study, agarose hydrogel containers were prepared and used to encapsulate viscous media for 3D cell culture to investigate the viscosity effect on the functions of bovine articular chondrocytes (BACs). Polyethylene glycol of different molecular weights was used to adjust culture medium viscosity in a large range (72.8-679.2 mPa s). The viscosity affected gene expression and secretion of cartilagenious matrices, while it did not affect BAC proliferation. The BACs cultured in the lower viscosity medium (72.8 mPa s) showed a higher level of cartilaginous gene expression and matrix secretion.

The functionality and translatability of neocartilage constructs are improved with the combination of fluid-induced shear stress and bioactive factors.

Neocartilage tissue engineering aims to address the shortcomings of current clinical treatments for articular cartilage indications. However, advancement is required toward neocartilage functionality (mechanical and biochemical properties) and translatability (construct size, gross morphology, passage number, cell source, and cell type). Using fluid-induced shear (FIS) stress, a potent mechanical stimulus, over four phases, this work investigates FIS stress' efficacy toward creating large neocartilage derived from highly passaged minipig costal chondrocytes, a species relevant to the preclinical regulatory process. In Phase I, FIS stress application timing was investigated in bovine articular chondrocytes and found to improve the aggregate modulus of neocartilage by 151% over unstimulated controls when stimulated during the maturation stage. In Phase II, FIS stress stimulation was translated from bovine articular chondrocytes to expanded minipig costal chondrocytes, yielding a 46% improvement in aggregate modulus over nonstimulated controls. In Phase III, bioactive factors were combined with FIS stress to improve the shear modulus by 115% over bioactive factor-only controls. The translatability of neocartilage was improved in Phase IV by utilizing highly passaged cells to form constructs more than 9-times larger in the area (11×17mm), yielding an improved aggregate modulus by 134% and a flat morphology compared to free-floating, bioactive factor-only controls. Overall, this study represents a significant step toward generating mechanically robust, large constructs necessary for animal studies, and eventually, human clinical studies.

From Chondrocytes to Chondrons, Maintenance of Phenotype and Matrix Production in a Composite 3D Hydrogel Scaffold.

Osteoarthritis (OA) is a degenerative disease characterized by articular cartilage (AC) degradation that affects more than 30 million people in the USA. OA is managed with symptom-alleviating medications. Matrix-assisted autologous chondrocyte transplantation (MACT) is a tissue-engineered option, but current products are expensive and lack mechanical tunability or processability to match defect mechanical properties and anatomical shapes. Here, we explore the efficacy of a biocompatible hydrogel-based scaffold composed of sodium alginate, gelatin, and gum Arabic—referred to by SA–GEL–GA—to support bovine articular chondrocyte (bAChs) proliferation, pericellular matrix (PCM), and extracellular matrix (ECM) production. bAChs were grown for 45 days in SA–GEL–GA. Their viability, their live/dead status, histological staining, biochemical assays for glycosaminoglycans (GAGs) and collagen, atomic force microscopy (AFM) imaging, and immunofluorescence staining of collagen I, collagen II, aggrecan, and CD44 were assessed. We found that SA–GEL–GA was not cytotoxic, induced cellular proliferation by 6.1-fold while maintaining a round morphology, and supported ECM deposition by producing 3.9-fold more GAG compared to day 0. bAChs transformed into chondrons and produced a PCM enriched with collagen II (3.4-fold), aggrecan (1.7-fold), and CD44 (1.3-fold) compared to day 0. In summary, SA–GEL–GA supported the proliferation, ECM production, and PCM production of bAChs in vitro.

Activation of transient receptor potential vanilloid 4 protects articular cartilage against inflammatory responses via CaMKK/AMPK/NF-\u03baB signaling pathway

Transient receptor potential vanilloid 4 (TRPV4) plays an important role in chondrocytes via Ca2+ signaling. However, its role in the progression of osteoarthritis is unclear. This study aimed to evaluate the effects of TRPV4 activation on articular cartilage and chondrocytes stimulated with interleukin (IL)-1β. Bovine and human articular chondrocytes were stimulated with various agents, including IL-1β, GSK1016790A (GSK101; a TRPV4 agonist), Compound C (an AMP-activated protein kinase (AMPK) inhibitor), and STO-609 (a calmodulin-dependent protein kinase kinase (CaMKK) inhibitor), and were processed for Western blot analysis and real-time PCR. The dimethylmethylene blue (DMMB) assay and Safranin O staining were also performed. GSK101 reversed the IL-1β-induced increase in expression of matrix metalloproteinase (MMP)-13 and decrease in expression of aggrecan. GSK101 also decreased proteoglycan release in the DMMB assay and retained Safranin O staining of articular cartilage tissue. Furthermore, GSK101 increased AMPK phosphorylation and decreased IL-1β-induced nuclear factor kappa B (NF-κB) phosphorylation. Compound C and STO-609 reversed the suppressive effects of GSK101 on NF-κB activation and MMP-13 expression. In conclusion, TRPV4 activation had chondroprotective effects on articular cartilage stimulated with IL-1β by activating CaMKK/AMPK and suppressing the NF-κB pathway. TRPV4 activators may offer a promising therapeutic option for preventing the progression of osteoarthritis.

Adrenomedullin and truncated peptide adrenomedullin(22-52) affect chondrocyte response to apoptotis in vitro: downregulation of FAS protects chondrocyte from cell death

Chondrocyte apoptosis may have a pivotal role in the development of osteoarthritis. Interest has increased in the use of anti-apoptotic compounds to protect against osteoarthritis development. In this work, we investigated the effect of adrenomedullin (AM), a 52 amino-acid hormone peptide, and a 31 amino-acid truncated form, AM(22-52), on chondrocyte apoptosis. Bovine articular chondrocytes (BACs) were cultured under hypoxic conditions to mimic cartilage environment and then treated with Fas ligand (Fas-L) to induce apoptosis. The expression of AM and its calcitonin receptor-like receptor (CLR)/receptor activity-modifying protein (RAMP) (receptor/co-receptor) was assessed by immunostaining. We evaluated the effect of AM and AM(22-52) on Fas-L-induced chondrocyte apoptosis. FAS expression was appreciated by RT-qPCR and immunostainings. The expression of hypoxia-inducible factor 1α (HIF-1α), CLR and one co-receptor (RAMP2) was evidenced. With BACs under hypoxia, cyclic adenosine monophosphate production increased dose-dependently with AM stimulation. AM significantly decreased caspase-3 activity (mean 35% decrease; p = 0.03) as a marker of Fas-L-induced apoptosis. Articular chondrocytes treated with AM showed significantly reduced cell death, along with downregulated Fas expression and production, as compared with AM(22-52). AM decreased articular chondrocyte apoptosis by downregulating a Fas receptor. These findings may pave the way for novel therapeutic approaches in osteoarthritis.

Preparation of PLGA-collagen hybrid scaffolds with controlled pore structures for cartilage tissue engineering

Scaffolds used for cartilage tissue engineering should have high mechanical strength and well-controlled pore structure to provide suitable microenvironments for functional tissue regeneration. In this study, hybrid scaffolds which had open and interconnected pore structure and high mechanical property were prepared by hybridization of PLGA mesh and collagen using ice particulates as porogen templates. Embossing ice particulates template was used to form open pore structures on the scaffold surfaces. Free ice particulates were used to generate interconnected bulk pores in the scaffolds. Hybridization with PLGA mesh provided the scaffolds with high mechanical property. Bovine articular chondrocytes were cultured in the hybrid scaffolds. The unique pore structures facilitated the homogeneous distribution of chondrocytes and cartilaginous matrices throughout the scaffolds. Subcutaneous implantation demonstrated cartilage-like tissue regeneration. The hybrid scaffolds should have a high potential for cartilage tissue engineering.

Polyphenols suppress inducible oxidative stress in human osteoarthritic and bovine chondrocytes

Reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in chondrocyte senescence and cartilage aging, pathogenesis of osteoarthritis (OA), and rheumatoid arthritis. Naturally occurring polyphenolic compounds (PPCs), such as curcumin (turmeric), resveratrol (grape), and epigallocatechin-3-gallate (EGCG) (green tea), have been known for their anti-inflammatory and chondroprotective effects. However, the potential protective effects of these PPCs against oxidative stress in chondrocytes are unclear. To investigate this, bovine articular chondrocytes and human osteoarthritic chondrocytes were pre-treated with PPCs at varying concentrations, and then exposed to hydrogen peroxide (H2O2) as an ROS inducer or S-nitroso-N-acetylpenicillamine (SNAP) as a NO donor. Alternatively, chondrocytes were co-treated with polyphenols and H2O2. Intracellular ROS/NO were measured using a fluorescent dye technique (H2DCF-DA for ROS; DAF-FM for NO). Our findings showed that PPC pre-/co-treatment inhibited both H2O2-induced ROS and SNAP-induced NO at different concentrations in both bovine chondrocytes and human osteoarthritic chondrocytes. Curcumin also increased glutathione peroxidase activity in the presence of H2O2 in bovine chondrocytes. Taken together, these findings indicate that PPCs are capable of suppressing oxidative stress- induced responses in chondrocytes, which may have potential therapeutic value for OA clinical application.

Combination of bone morphogenetic protein 9 and transforming growth factor BETA-1 improves cartilaginous matrix formation by equine chondrocytes in three-dimensional culture

Purpose: Osteoarthritis is not only common in humans, but also highly prevalent in horses. In vitro, chondrocytes are used to study disease processes under controlled conditions and the effects of (new) drugs at the cellular level. Bone morphogenic protein 9 (BMP9) has been shown to be a potent modulator of cartilage development by bovine articular chondrocytes. The hypertrophic effect of BMP9 could be antagonized by a low dose of TGFβ-1. We compared the effects of BMP9 and TGFβ-1 on cartilaginous matrix deposition by equine chondrocytes in pellet culture and hypothesized that BMP9 would synergize with a low dose of TGFβ-1 to preserve the healthy chondrocyte phenotype. Methods: Pellets from healthy equine chondrocytes (P1; n = 2 Shetland Pony donors) were cultured for 28 days with chondrogenic medium alone or supplemented with the following: TGFβ-1-low (0.1 ng/ml), TGFβ-1-standard (10 ng/ml), BMP9 (50ng/ml), combination-low (50 ng/ml BMP9 + 0.1 ng/ml TGFβ-1), combination-standard: (50 ng/ml BMP9 + 10 ng/ml TGFβ-1), or combination-low for one week, followed by TGFβ-1-standard (i.e. Switch group). Bern scoring and collagen type-2 (COL2) staining of histology sections and glycosaminoglycan (GAG) and DNA analsyis were performed at days 7, 21, and 28. Gene expression of chondrogenic markers (aggrecan, collagen type-2, TGFβ-1) and hypertrophic or fibrosis markers (ADAMTS5, collagen type-1, collagen type-3, MMP3, MMP13) were measured at day 7 and 28. Statistical analysis was conducted between TGβ-1-standard and the other groups. Results: Analysis of Safranin-O/Fast-green stained sections demonstrated higher Bern scores for the BMP9-treated groups at all time points (fig.1). At day 7, COL2 staining was stronger for groups that received BMP9. GAG content normalized for DNA was significantly higher for the BMP9 and combination-low group at days 21 and 28. At both time points, aggrecan expression was significantly higher than standard in all groups that received BMP9 during the entire culture period. However, COL2 expression was significantly higher for combination groups only, at day 7. MMP3 was significantly upregulated in the BMP9 and combination-low groups at day 28, whereas MMP13 expression was significantly downregulated in these two groups at the same time point. Conclusions: BMP9 improved matrix deposition by equine chondrocytes based on pellet size, GAG/DNA, and intensity of Safranin-O and COL2 staining. Although the combination of BMP9 with low doses of TGFβ-1 upregulated MMP3 expression, it downregulated the expression of the late hypertrophy marker MMP13 while upregulating aggrecan and COL2 expression. Further analysis of hypertrophy markers and underlying signalling pathways should be considered for a deeper understanding of the observed synergistic effect. Altogether, a combination of BMP9 with a low dose of TGFβ-1 significantly improved chondrogenesis of equine chondrocytes.

Enhanced chondrogenic phenotype of primary bovine articular chondrocytes in Fibrin-Hyaluronan hydrogel by multi-axial mechanical loading and FGF18

Current treatments for cartilage lesions are often associated with fibrocartilage formation and donor site morbidity. Mechanical and biochemical stimuli play an important role in hyaline cartilage formation. Biocompatible scaffolds capable of transducing mechanical loads and delivering bioactive instructive factors may better support cartilage regeneration.In this study we aimed to test the interplay between mechanical and FGF-18 mediated biochemical signals on the proliferation and differentiation of primary bovine articular chondrocytes embedded in a chondro-conductive Fibrin-Hyaluronan (FB/HA) based hydrogel.Chondrocytes seeded in a Fibrin-HA hydrogel, with or without a chondro-inductive, FGFR3 selective FGF18 variant (FGF-18v) were loaded into a joint-mimicking bioreactor applying controlled, multi-axial movements, simulating the natural movements of articular joints. Samples were evaluated for DNA content, sulphated glycosaminoglycan (sGAG) accumulation, key chondrogenic gene expression markers and histology.Under moderate loading, samples produced particularly significant amounts of sGAG/DNA compared to unloaded controls. Interestingly there was no significant effect of FGF-18v on cartilage gene expression at rest. Following moderate multi-axial loading, FGF-18v upregulated the expression of Aggrecan (ACAN), Cartilage Oligomeric Matrix Protein (COMP), type II collagen (COL2) and Lubricin (PRG4). Moreover, the combination of load and FGF-18v, significantly downregulated Matrix Metalloproteinase-9 (MMP-9) and Matrix Metaloproteinase-13 (MMP-13), two of the most important factors contributing to joint destruction in OA. Biomimetic mechanical signals and FGF-18 may work in concert to support hyaline cartilage regeneration and repair. Statement of significanceArticular cartilage has very limited repair potential and focal cartilage lesions constitute a challenge for current standard clinical procedures. The aim of the present research was to explore novel procedures and constructs, based on biomaterials and biomechanical algorithms that can better mimic joints mechanical and biochemical stimulation to promote regeneration of damaged cartilage.Using a hydrogel-based platform for chondrocyte 3D culture revealed a synergy between mechanical forces and growth factors. Exploring the mechanisms underlying this mechano-biochemical interplay may enhance our understanding of cartilage remodeling and the development of new strategies for cartilage repair and regeneration.

Effects of Sprifermin, IGF1, IGF2, BMP7, or CNP on Bovine Chondrocytes in Monolayer and 3D Culture.

ABSTRACTOne possible approach to treat osteoarthritis (OA) is to counteract cartilage degeneration with anabolic compounds that stimulate chondrocyte proliferation and/or extracellular matrix (ECM) production. Several molecules including sprifermin (recombinant human fibroblast growth factor [FGF18]), insulin‐like growth factor‐1 [IGF1] and ‐2 [IGF2], C‐type natriuretic peptide [CNP], and bone metamorphic protein 7 [BMP7] have been shown to have these characteristics both in vitro and in vivo. However, it is not known how these molecules compare each other regarding their effect on phenotype and stimulation of ECM production in primary chondrocytes. The effects of sprifermin, IGF1, IGF2, CNP, and BMP7 were evaluated on bovine articular chondrocytes, first in monolayer to determine their effective concentrations, and then in three‐dimensional (3D) culture at concentrations of 100 ng/ml for sprifermin; 300 ng/ml for IGF1, IGF2, and BMP7; and 10 nM for CNP. In 3D culture, the effects of a permanent exposure or a cyclic exposure consisting of 24 h incubation per week with the compounds were evaluated. All growth factors increased ECM production and cell proliferation to a similar extent but CNP had almost no effect on bovine chondrocytes. Sprifermin was more effective with cyclic exposure, IGF1, and IGF2 with permanent exposure, and BMP7 showed similar results with both exposures. Regarding the cell phenotype, sprifermin appeared to be the only compound favoring the chondrocyte phenotype; it decreased type I collagen expression and had no hypertrophic effect. Together, these results confirmed that sprifermin is a promising disease‐modifying OA drug. © 2019 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:653–662, 2020

Inhibition of CD44 intracellular domain production suppresses bovine articular chondrocyte de-differentiation induced by excessive mechanical stress loading

CD44 fragmentation is enhanced in chondrocytes of osteoarthritis (OA) patients. We hypothesized that mechanical stress-induced enhancement of CD44-intracellular domain (CD44-ICD) production plays an important role in the de-differentiation of chondrocytes and OA. This study aimed to assess the relationship between CD44-ICD and chondrocyte gene expression. Monolayer cultured primary bovine articular chondrocytes (BACs) were subjected to cyclic tensile strain (CTS) loading. ADAM10 inhibitor (GI254023X) and γ-secretase inhibitor (DAPT) were used to inhibit CD44 cleavage. In overexpression experiments, BACs were electroporated with a plasmid encoding CD44-ICD. CTS loading increased the expression of ADAM10 and subsequent CD44 cleavage, while decreasing the expression of SOX9, aggrecan, and type 2 collagen (COL2). Overexpression of CD44-ICD also resulted in decreased expression of these chondrocyte genes. Both GI254023X and DAPT reduced the production of CD44-ICD upon CTS loading, and significantly rescued the reduction of SOX9 expression by CTS loading. Chemical inhibition of CD44-ICD production also rescued aggrecan and COL2 expression following CTS loading. Our findings suggest that CD44-ICD is closely associated with the de-differentiation of chondrocytes. Excessive mechanical stress loading promoted the de-differentiation of BACs by enhancing CD44 cleavage and CD44-ICD production. Suppression of CD44 cleavage has potential as a novel treatment strategy for OA.

Scaffoldless tissue-engineered cartilage for studying transforming growth factor beta-mediated cartilage formation.

Reduced transforming growth factor beta (TGF-β) signaling is associated with osteoarthritis (OA). TGF-β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF-β's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-β signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-β that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-β type I receptor inhibitor, SB431542, was used to attenuate TGF-β signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-β receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-β signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-β signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.

Dynamic formation of cellular aggregates of chondrocytes and mesenchymal stem cells in spinner flask.

ObjectivesCellular aggregates are readily applicable in cell‐based therapy. The effects of agitation and inoculation density on the aggregation of cells in spinner flask and the molecular mechanism of aggregation were investigated.Materials and methodsThe aggregation kinetics of cells in spinner flask was evaluated with bovine articular chondrocytes (bACs), rabbit bone marrow‐derived mesenchymal stem cells (rMSCs) and their mixture. The morphology of cellular aggregates was studied with scanning electron microscopy and gene expression of cell adhesion‐related molecules was analysed.ResultsIt was shown that suspension culture in spinner flask induced the aggregation of bACs and rMSCs. Both cells exhibited increased aggregation rate and aggregate size with decreasing agitation rate and increasing cell inoculation density. Additionally, aggregate size increased with extended culture time. By analysing gene expression of integrin β1 and cadherin, it was indicated that these molecules were potentially involved in the aggregation process of bACs and rMSCs, respectively. Aggregates composed of both bACs and rMSCs were also prepared, showing rMSCs in the core and bACs in the periphery.ConclusionsCellular aggregates were prepared in dynamic suspension culture using spinner flask, the key parameters to the aggregation process were identified, and the molecular mechanism of aggregation was revealed. This would lay a solid foundation for the large‐scale production of cellular aggregates for cell‐based therapy, such as cartilage regeneration.

Regulation of articular chondrocyte catabolic genes by growth factor interaction.

Osteoarthritis is characterized by a loss of articular cartilage homeostasis in which degradation exceeds formation. Several growth factors have been shown to promote cartilage formation by augmenting articular chondrocyte anabolic activity. This study tests the hypothesis that such growth factors also play an anticatabolic role. We transferred individual or combinations of the genes encoding insulin-like growth factor-I, bone morphogenetic protein-2, bone morphogenetic protein-7, transforming growth factor-β1, and fibroblast growth factor-2, into adult bovine articular chondrocytes and measured the expression of catabolic marker genes encoding A disintegrin and metalloproteinase with thrombospondin motifs-4 and -5, matrix metalloproteinases-3 and -13, and interleukin-6. When delivered individually, or in combination, these growth factor transgenes differentially regulated the direction, magnitude, and time course of expression of the catabolic marker genes. In concert, the growth factor transgenes regulated the marker genes in an interactive fashion that ranged from synergistic inhibition to synergistic stimulation. Synergistic stimulation prevailed over synergistic inhibition, reaching maxima of 15.2- and 2.7-fold, respectively. Neither the magnitude nor the time course of the effect of the transgene combinations could be predicted on the basis of the individual transgene effects. With few exceptions, the data contradict our hypothesis. The results demonstrate that growth factors that are traditionally viewed as chondrogenic tend also to promote catabolic gene expression. The competing actions of these potential therapeutic agents add an additional level of complexity to the selection of regulatory factors for restoring articular cartilage homeostasis or promoting repair.

Simvastatin promotes restoration of chondrocyte morphology and phenotype

In this study we examined whether the action of simvastatin affects re-differentiation of passaged chondrocytes and if so, whether this was mediated via changes in cholesterol or cholesterol intermediates. Bovine articular chondrocytes, of varying passage number, human knee chondrocytes and rat chondrosarcoma chondrocytes were treated with simvastatin and examined for changes in mRNA and protein expression of markers of the chondrocyte phenotype as well as changes in cell shape, proliferation and proteoglycan production. In all three models, while still in monolayer culture, simvastatin treatment alone promoted changes in phenotype and morphology indicative of re-differentiation most prominent being an increase in SOX9 mRNA and protein expression. In passaged bovine chondrocytes, simvastatin stimulated the expression of SOX9, ACAN, BMP2 and inhibited the expression of COL1 and α-smooth muscle actin. Co-treatment of chondrocytes with simvastatin plus exogenous cholesterol—conditions that had previously reversed the inhibition on CD44 shedding, did not alter the effects of simvastatin on re-differentiation. However, the co-treatment of chondrocytes with simvastatin together with other pathway intermediates, mevalonate, geranylgeranylpyrophosphate and to a lesser extent, farnesylpyrophosphate, blocked the pro-differentiation effects of simvastatin. Treatment with simvastatin stimulated expression of SOX9 and COL2a and enhanced SOX9 protein in human OA chondrocytes. The co-treatment of OA chondrocytes with mevalonate or geranylgeranylpyrophosphate, but not cholesterol, blocked the simvastatin effects. These results lead us to conclude that the blocking of critical protein prenylation events is required for the positive effects of simvastatin on the re-differentiation of chondrocytes.

Activation of the renin-angiotensin system in mice aggravates mechanical loading-induced knee osteoarthritis.

Epidemiological studies have shown an association between hypertension and knee osteoarthritis (OA). The purpose of this study was to investigate whether activation of the renin–angiotensin system (RAS) can aggravate mechanical loading-induced knee OA in mice. Eight-week-old male Tsukuba hypertensive mice (THM) and C57BL/6 mice were divided into four groups: i) running THM group, ii) running C57BL/6 mice group, iii) non-running THM group, and iv) non-running C57BL/6 mice group. Mice in the running group were forced to run (25 m/min, 30 min/day, 5 days/week) on a treadmill. All mice in the four groups (n=10 in each group) were euthanized after 0, 2, 4, 6, or 8 weeks of running or natural breeding. Cartilage degeneration in the left knees was histologically evaluated using the modified Mankin score. Expression of Col X, MMP-13, angiotensin type 1 receptor (AT1R), and AT2R was examined immunohistochemically. To study the effects of stimulation of the AT1R in chondrocytes by mechanical loading and/or Angiotensin II (AngII) on transduction of intracellular signals, phosphorylation levels of JNK and Src were measured in bovine articular chondrocytes cultured in three-dimensional agarose scaffolds. After 4 weeks, the mean Mankin score for the lateral femoral condylar cartilage was significantly higher in the THM running group than in the C57BL/6 running group and non-running groups. AT1R and AT2R expression was not detected at 0 weeks in any group but was noted after 4 weeks in the THM running group. AT1R expression was also noted at 8 weeks in the C57BL/6 running group. The expression levels of AT1R, COL X, and MMP-13 in chondrocytes were significantly higher in the THM running group than in the control groups. Positive significant correlations were noted between the Mankin score and the rate of AT1R-immunopositive cells, between the rates of AT1R- and Col X-positive cells, and between the rates of AT1Rand AT2R-positive cells. The phosphorylation level of JNK was increased by cyclic compression loading or addition of AngII to the cultured chondrocytes and was reversed by pretreatment with an AT1R blocker. A synergistic effect on JNK phosphorylation was observed between compression loading and AngII addition. Transgene activation of renin and angiotensinogen aggravated mechanical load-induced knee OA in mice. These findings suggest that AT1R expression in chondrocytes is associated with early knee OA and plays a role in the progression of cartilage degeneration. The RAS may be a common molecular mechanism involved in the pathogenesis of hypertension and knee OA.

High glycine concentration increases collagen synthesis by articular chondrocytes in vitro: acute glycine deficiency could be an important cause of osteoarthritis

Collagen synthesis is severely diminished in osteoarthritis; thus, enhancing it may help the regeneration of cartilage. This requires large amounts of glycine, proline and lysine. Previous works of our group have shown that glycine is an essential amino acid, which must be present in the diet in large amounts to satisfy the demands for collagen synthesis. Other authors have shown that proline is conditionally essential. In this work we studied the effect of these amino acids on type II collagen synthesis. Bovine articular chondrocytes were cultured under a wide range of different concentrations of glycine, proline and lysine. Chondrocytes were characterized by type II collagen immunocytochemistry of confluence monolayer cultures. Cell growth and viability were assayed by trypan blue dye exclusion method. Type II collagen was measured in the monolayer, every 48 h for 15 days by ELISA. Increase in concentrations of proline and lysine in the culture medium enhances the synthesis of type II collagen at low concentrations, but these effects decay before 1.0 mM. Increase of glycine as of 1.0 mM exceeds these effects and this increase continues more persistently by 60–75%. Since the large effects produced by proline and lysine are within the physiological range, while the effect of glycine corresponds to a much higher range, these results demonstrated a severe glycine deficiency for collagen synthesis. Thus, increasing glycine in the diet may well be a strategy for helping cartilage regeneration by enhancing collagen synthesis, which could contribute to the treatment and prevention of osteoarthritis.