Equine Chondrocytes Research Articles

RNA Interference and BMP-2 Stimulation Allows Equine Chondrocytes Redifferentiation in 3D-Hypoxia Cell Culture Model: Application for Matrix-Induced Autologous Chondrocyte Implantation.

As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine.

Analysis of clusterin expression changes as a biomarker of osteoarthritis

Purpose: The discovery and validation of arthritis-related biomarkers and establishment of methodology for proteomic studies in osteoarthritis (OA) are needed. Proteomics strategies have identified many proteins that may relate to pathological mechanisms of OA, however targeted approaches are required to validate the roles of these proteins. This study aimed to use mass spectrometry and western blotting to identify peptides from several proteins in the secretome of chondrocytes, cartilage explants and osteochondral biopsies treated with inflammatory cytokines over a 2-week period, to evaluate their potential as biomarkers of OA progression. Methods: Healthy cartilage was obtained from fetlock joints of skeletally mature horses, euthanized for unrelated veterinary reasons. Cartilage explants were isolated using a 6 mm biopsy, with discs placed into wells (3 discs per 1 ml DMEM + 1% Pen/Strep) before incubation for 24 hours (37 °C, 5% CO2). After this equilibration period, the media was removed and replaced with either fresh DMEM + 1% Pen/Strep or DMEM supplemented with 1% Pen/Strep containing TNFα and IL-1β both at 10ng/ml. Explants were culture for 7–14 days with the cytokines replaced every 4th day. For cell based assays chondrocytes were isolated from tissue using 70U pronase for 1hr at 37 °C and overnight digestion at 37°C using a 0.2% collagenase II solution. The cell suspension was filtered and washed before being seeded into culture flasks and cultured until confluence was reached (37°C, 5% CO2). Once cultures were established cells were split into two groups: healthy control (DMEM supplemented with 1% Pen/Strep and 10% foetal calf serum) or stimulated cells (DMEM as above plus TNFα and IL-1β both at 10ng/ml). Chondroyctes were cytokine-stimulated for up to one week. Cells were used in experiments up to the 2nd passage. Results: Mass spectrometry data showed that peptides representative of clusterin were found to decrease following 7 days of inflammatory stimulation. Western blotting of secreted proteins in media of cartilage explants or chondrocyte showed that clusterin expression was reduced following 7 days of cytokine treatment. Catabolic matrix metalloproteinase enzymes MMP1, MMP3 and MMP13, as well the matrix component cartilage oligomeric protein (COMP) were all found to have an increased abundance in the media of the cytokine treated samples. This data was supported by qPCR for clusterin gene expression which showed initially mRNA levels increased 3 day after inflammatory stimulation but expression was lost after 7 days. Western blotting of media from the osteochondral biopsies showed an increase in clusterin expression after 7 days of inflammatory stimulation however clusterin protein expression could not be detected after 14 days of treatment, indicating a delayed response compared to cartilage tissue alone. Conclusions: The equine chondrocytes, cartilage explant and osteochondral biopsy models exhibited highest clusterin secretion in untreated cultures. IL-1β and TNFα treatment caused a reduction in clusterin secretion. Clusterin acts as a chaperone to aid protein refolding in situations of stress and is constitutively secreted by mammalian cells. IL-1β and TNFα appear to interrupt clusterin secretion and therefore the protection it may offer healthy functioning cells. Previous studies have reported variable data, with some studies indicating a decrease in clusterin in OA, while others indicate an increase in clusterin expression. Our results suggest the clusterin increases immediately after inflammatory stimulation but is lost after prolonged exposure. Therefore, levels of secreted clusterin may be a candidate biomarker for OA progression.

Effects of xylazine and dexmedetomidine on equine articular chondrocytes in vitro

ObjectiveTo assess the effects of xylazine and dexmedetomidine on equine chondrocytes, in vitro. Study designProspective, experimental study. Study materialEquine articular chondrocytes from five male horses. MethodsChondrocytes were isolated from healthy equine articular cartilage of the metacarpo/metatarsophalangeal joints. Cell viability was assessed using the WST-8 assay by exposing chondrocytes to xylazine (0.5, 1, 2, 4, 8, 16.6, 25, 50 mg mL−1) or dexmedetomidine (0.001, 0.005, 0.01, 0.05, 0.175, 0.25 mg mL−1) for 15, 30 and 60 minutes. Based on the results of these tests, cells were treated with xylazine (1, 4, 25 mg mL−1) or dexmedetomidine (0.05, 0.175, 0.25 mg mL−1) for 15 minutes to further evaluate: cell viability by neutral red uptake; cell membrane integrity by lactate dehydrogenase release and by fluorescence microscopy with Hoechst 33342 and propidium iodide (PI), and apoptosis by flow cytometry using double staining with annexin V-fluorescein isothiocyanate/PI and by cell morphology. ResultsBoth drugs reduced cell viability in a dose-dependent manner. Specifically, all xylazine concentrations, except 0.5 mg mL−1 and 1 mg mL−1, significantly reduced cell viability, whereas the effects of dexmedetomidine were evident only at 0.175 mg mL−1 and 0.25 mg mL−1. The highest concentrations of xylazine (25 mg mL−1) and dexmedetomidine (0.25 mg mL−1) caused loss of membrane integrity. Cell morphology and flow cytometry analyses demonstrated signs of late apoptosis in xylazine-treated cells, and signs of late apoptosis and necrosis in dexmedetomidine-treated cells. Conclusions and clinical relevanceThis study offers new insights into the potential chondrotoxicity induced by dexmedetomidine and xylazine. Therefore, the intra-articular administration of α2-agonists should be conducted with care, especially for doses of ≥ 4 mg mL−1 of xylazine and 0.175 mg mL−1 and 0.25 mg mL−1 of dexmedetomidine.

Generation of Immortalized Equine Chondrocytes With Inducible Sox9 Expression Allows Control of Hypertrophic Differentiation.

Immortalization of chondrocytes enables long term in vitro culture; however, the chondrogenic capacity of transformed cells varies, thus highlighting the need to develop a proliferative and tuneable chondrocyte cell line where hypertrophic differentiation can be controlled. In this study the SV40 large T antigen and human telomerase reverse transcriptase were employed to immortalize pooled equine chondrocytes through lentiviral vector mediated transduction either singly or on combination. Transformed chondrocytes proliferated stably over multiple passages, but resulted in significantly lower expression of chondrocyte specific collagen II mRNA (P < 0.0001) and up regulation of the hypertrophic marker collagen X (P < 0.0001) in three dimensional cultures. A Col2a1 promoter driven GFP reporter was constructed for real time monitoring of chondrogenic differentiation and a significant increase in promoter activation was observed in cultures treated with the growth factor TGFβ-3 (P < 0.05). To recapitulate the native articular chondrocyte phenotype we further transduced large T antigen immortalized chondrocytes with lentiviral vectors allowing either constitutive or doxycycline inducible expression of Sox9. In 3D cultures, the Sox9 over-expressing chondrocytes secreted significantly higher levels of extracellular matrix polysaccharide glycosaminoglycan (P < 0.05), while up-regulating collagen II and Aggrecan mRNA (P < 0.05) in both expression systems with a similar patterns observed with imunohistochemical staining. High levels of collagen X mRNA and protein were maintained with constitutive sox9 reflecting hypetrophic differentiation but significantly lower expression could be achieved with inducible Sox9. In conclusion, immortalization of equine chondrocytes results in stable proliferation but a reduction of chondrogenic potential whilst modulation of sox9 expression enabled control of hypertrophic characteristics. J. Cell. Biochem. 118: 1201-1215, 2017. © 2016 Wiley Periodicals, Inc.

Giant crystals inside mitochondria of equine chondrocytes

The present study reports for the first time the presence of giant crystals in mitochondria of equine chondrocytes. These structures show dark contrast in TEM images as well as a granular substructure of regularly aligned 1–2 nm small units. Different zone axes of the crystalline structure were analysed by means of Fourier transformation of lattice-resolution TEM images proving the crystalline nature of the structure. Elemental analysis reveals a high content of nitrogen referring to protein. The outer shape of the crystals is geometrical with an up to hexagonal profile in cross sections. It is elongated, spanning a length of several micrometres through the whole cell. In some chondrocytes, several crystals were found, sometimes combined in a single mitochondrion. Crystals were preferentially aligned along the long axis of the cells, thus appearing in the same orientation as the chondrocytes in the tissue. Although no similar structures have been found in the cartilage of any other species investigated, they have been found in cartilage repair tissue formed within a mechanically stimulated equine chondrocyte construct. Crystals were mainly located in superficial regions of cartilage, especially in joint regions of well-developed superficial layers, more often in yearlings than in adult horses. These results indicate that intramitochondrial crystals are related to the high mechanical stress in the horse joint and potentially also to the increased metabolic activity of immature individuals.

Hydrogel-based reinforcement of 3D bioprinted constructs

Progress within the field of biofabrication is hindered by a lack of suitable hydrogel formulations. Here, we present a novel approach based on a hybrid printing technique to create cellularized 3D printed constructs. The hybrid bioprinting strategy combines a reinforcing gel for mechanical support with a bioink to provide a cytocompatible environment. In comparison with thermoplastics such as -polycaprolactone, the hydrogel-based reinforcing gel platform enables printing at cell-friendly temperatures, targets the bioprinting of softer tissues and allows for improved control over degradation kinetics. We prepared amphiphilic macromonomers based on poloxamer that form hydrolysable, covalently cross-linked polymer networks. Dissolved at a concentration of 28.6%w/w in water, it functions as reinforcing gel, while a 5%w/w gelatin-methacryloyl based gel is utilized as bioink. This strategy allows for the creation of complex structures, where the bioink provides a cytocompatible environment for encapsulated cells. Cell viability of equine chondrocytes encapsulated within printed constructs remained largely unaffected by the printing process. The versatility of the system is further demonstrated by the ability to tune the stiffness of printed constructs between 138 and 263 kPa, as well as to tailor the degradation kinetics of the reinforcing gel from several weeks up to more than a year.

Modified amphiphilic copolymer based hydrogels as reinforcement for 3D biofabrication

Modified amphiphilic copolymer based hydrogels as reinforcement for 3D biofabrication

A dual crosslinking strategy for reinforcing gelatine-methacrylamide hydrogels for tissue repair

Event Abstract Back to Event A dual crosslinking strategy for reinforcing gelatine-methacrylamide hydrogels for tissue repair Ferry Melchels1, 2, Kaj Blokland2, Mylène D. Ruijter2 and Jos Malda2, 3 1 Heriot-Watt University, Institute of Biological Chemistry, Biophysics and Bioengineering, United Kingdom 2 University Medical Center Utrecht, Department of Orthopaedics, Netherlands 3 Utrecht University, Faculty of Veterinary Medicine, Netherlands Introduction: Hydrogels are a suitable class of materials for cell-based cartilage repair, as they provide a 3D environment that can stimulate encapsulated cells to proliferate, differentiate, and form new tissue [1]. Their mechanical properties however are generally poor, and most measures taken to improve the mechanical properties make it a less permissive environment for cellular processes. There is a need for strategies that reinforce hydrogels without compromising chondrocyte performance. Here, we studied the reinforcement of cell-laden gelatine-methacrylamide (gelMA) hydrogels by thermal (physical) gelation prior to UV-initiated (chemical) crosslinking. Experimental Methods: Gelatine was methacrylamide-functionalised as reported previously [2]. Solutions of 10% or 20% gelMA + 0.1% Irgacure 2959 photo-initiator were crosslinked by exposure to 15 min UV-A, either in the sol state (purely chemical gel = C) or after thermal ageing at 5 °C (physical-chemical dual-crosslinked gel = PC). Compressive moduli of C and PC gels were measured after 24 h free swelling at 37 °C, whilst stiffness development of purely physical gels (P) was measured by oscillatory shear rheometry at 5 °C. All gels were subjected to proteolytic degradation in 0.05% trypsin + 0.006% EDTA. Chondrocytes were isolated from equine articular cartilage and passaged once before encapsulation in gels at 5.106 cells/mL under the same conditions as described above. After 24 h, live/dead and Alamar blue assays were performed. Cell-laden constructs (10% C, 10% PC and 20% C) were cultured in chondrogenic medium for 6 weeks, then assayed for DNA and glycosaminoglycan (GAG) content using Picogreen and DMMB, respectively. Results and Discussion: After photo-crosslinking, 10% gelMA gels crosslinked directly from the sol state (Figure 1, C) had a stiffness of 24 kPa. On the contrary, when allowed to gel thermally prior to photo-crosslinking, the stiffness increased up to 165 kPa for 10% dual-crosslinked gels (PC), reaching similar values as 20% gelMA gels crosslinked from the sol state (not shown). By comparison, the sum of stiffness of a purely physical gel and a purely chemical gel is only 51 kPa (P+C). Figure 1: Stiffness development of dual-crosslinked 10% gelMA gels, as a function of thermal gelation time prior to photo-initiated crosslinking. P = purely physical gel (thermal gelation) C = purely chemical gel (crosslinked from solution) P + C = sum of physical and chemical gel (for reference) PC = dual-crosslinked gel Whilst the stiffness of 10% PC gels was similar to that of 20% C gels, proteolytic degradation was much faster, and more akin to 10% C gels (Figure 2). Faster enzymatic degradation indicates the 10% dual-crosslinked gels being more permissive to matrix remodelling by encapsulated cells than 20% chemical gels. This was not yet corroborated in terms of cartilage tissue formation by encapsulated equine chondrocytes; after 6 weeks of culture, GAG/DNA was similar for all conditions evaluated. Nevertheless, the cold treatment did not reduce cell viability, nor chondrogenesis. Experiments are ongoing to reveal differential behaviour within the different gel types by other cell types. Figure 2: Proteolytic degradation of gelMA gels. Mass loss of dual-crosslinked (PC) 10 % gelMA gels compared to 10% and 20% only chemically crosslinked (C) gels. Discussion: Hydrogels that allow cells to develop new tissue are generally mechanically weak. Here, we present a straightforward method for the reinforcement of cell-laden gelMA gels, which showed seven-fold increase in stiffness whilst having no negative influence on cell viability, and only slightly decreased enzymatic degradation rates. Conclusion: Thermal ageing of gelatine-methacrylamide gels prior to UV-initiated crosslinking leads to a large increase in compressive stiffness, whilst still providing a permissive environment for enzymatic degradation, and for survival and tissue formation by encapsulated cells. Kim van Dorenmalen for technical assistance; European Union’s for funding under grant no. 309962 (HydroZONES) and no. 272286 (PrintCART); The Dutch Arthritis Foundation for funding

Effects of Ghrelin on metabolic activity of equine cartilage affected with inflammatory degeneration: preliminary isolation, characterization of equine cultured chondrocytes and assessment of LPS-induced inflammatory stress.

Ghrelin, the natural ligand for GH secretagogue receptors, influences the activity of both human and rat joint chondrocytes, modulating the synthesis of eicosanoids in the cartilage 1 . This project aims to assess the possible protective effects of Ghrelin on equine cultured chondrocytes exposed to lipopolysaccharide (LPS), as a model of osteoarthritic joint. In the preliminary step of our study, we have characterized equine chondrocyte primary cell cultures and standardized the detrimental effects LPS on chondrocytes health. Equine articular cartilage was collected with aseptic procedure and equine chondrocytes were isolated by overnight incubation. Cells were cultured in monolayer culture to obtain third-passage (P3) chondrocytes and specific cartilaginous genes were identified by qualitative PCR. P3 chondrocytes were treated with increasing LPS concentrations (1-100 ng/ml) and the effects of LPS were assessed with the apoptosis-induction test and fluorescence microscopy after 1, 3, 6, 12, 24, 48 h of treatment. PCR evidenced type II collagenases and aggrecan expression in isolated cells confirming the isolation of chondrocytes. Healthy chondrocytes (HC) showed no fluorescence, necrotic chondrocytes (NC) red fluorescence, early apoptotic chondrocytes (EAC) green fluorescence and advanced apoptotic chondrocytes (AAC) mixed green-red fluorescence. In LPS-stimulated chondrocytes, there was a trend throughout an increased percentage of AAC that became equal to or overwhelmed the percentage of HC after 3 hours for all LPS treatments and lasted for 6 hours after the highest LPS concentration used. On the basis of these results, the concentration of LPS 100 ng/ml for 6 h will be used for subsequent experiments.

Crosslinkable hydrogels derived from cartilage, meniscus, and tendon tissue.

Decellularized tissues have proven to be versatile matrices for the engineering of tissues and organs. These matrices usually consist of collagens, matrix-specific proteins, and a set of largely undefined growth factors and signaling molecules. Although several decellularized tissues have found their way to clinical applications, their use in the engineering of cartilage tissue has only been explored to a limited extent. We set out to generate hydrogels from several tissue-derived matrices, as hydrogels are the current preferred cell carriers for cartilage repair. Equine cartilage, meniscus, and tendon tissue was harvested, decellularized, enzymatically digested, and functionalized with methacrylamide groups. After photo-cross-linking, these tissue digests were mechanically characterized. Next, gelatin methacrylamide (GelMA) hydrogel was functionalized with these methacrylated tissue digests. Equine chondrocytes and mesenchymal stromal cells (MSCs) (both from three donors) were encapsulated and cultured in vitro up to 6 weeks. Gene expression (COL1A1, COL2A1, ACAN, MMP-3, MMP-13, and MMP-14), cartilage-specific matrix formation, and hydrogel stiffness were analyzed after culture. The cartilage, meniscus, and tendon digests were successfully photo-cross-linked into hydrogels. The addition of the tissue-derived matrices to GelMA affected chondrogenic differentiation of MSCs, although no consequent improvement was demonstrated. For chondrocytes, the tissue-derived matrix gels performed worse compared to GelMA alone. This work demonstrates for the first time that native tissues can be processed into crosslinkable hydrogels for the engineering of tissues. Moreover, the differentiation of encapsulated cells can be influenced in these stable, decellularized matrix hydrogels.

Influence of 1α, 25-dihydroxyvitamin D3 [1, 25(OH)2D3] on the expression of Sox 9 and the transient receptor potential vanilloid 5/6 ion channels in equine articular chondrocytes.

BackgroundSox 9 is a major marker of chondrocyte differentiation. When chondrocytes are cultured in vitro they progressively de-differentiate and this is associated with a decline in Sox 9 expression. The active form of vitamin D, 1, 25 (OH)2D3 has been shown to be protective of cartilage in both humans and animals. In this study equine articular chondrocytes were grown in culture and the effects of 1, 25 (OH)2D3 upon Sox 9 expression examined. The expression of the transient receptor potential vanilloid (TRPV) ion channels 5 and 6 in equine chondrocytes in vitro, we have previously shown, is inversely correlated with de-differentiation. The expression of these channels in response to 1, 25 (OH)2D3 administration was therefore also examined.ResultsThe active form of vitamin D (1, 25 (OH)2D3) when administered to cultured equine chondrocytes at two different concentrations significantly increased the expression of Sox 9 at both. In contrast 1, 25 (OH)2D3 had no significant effect upon the expression of either TRPV 5 or 6 at either the protein or the mRNA level.ConclusionsThe increased expression of Sox 9, in equine articular chondrocytes in vitro, in response to the active form of vitamin D suggests that this compound could be utilized to inhibit the progressive de-differentiation that is normally observed in these cells. It is also supportive of previous studies indicating that 1α, 25-dihydroxyvitamin D3 can have a protective effect upon cartilage in animals in vivo. The previously observed correlation between the degree of differentiation and the expression levels of TRPV 5/6 had suggested that these ion channels may have a direct involvement in, or be modulated by, the differentiation process in vitro. The data in the present study do not support this.

Optimization of a nonviral transfection system to evaluate Cox-2 controlled interleukin-4 expression for osteoarthritis gene therapy in vitro.

Gene therapy appears to have the potential for achieving a long-term remedy for osteoarthritis (OA). However, there is a risk of adverse reactions, especially when using cytomegalovirus-controlled expression. To provide a safe application, we focused on the expression of therapeutic cytokines [e.g. interleukin (IL)-4] in a disease-responsive manner by use of the previously cloned Cox-2 promoter as 'genetic switch'. In the present study, we report the functionality of a controlled gene therapeutic system in an equine osteoarthritic cell model. Different nonviral transfection reagents were tested for their efficiency on equine chondrocytes stimulated with equine IL-1β or lipopolysaccharide to create an inflammatory environment. To optimize the transfection, we successfully redesigned the vector by excluding the internal ribosomal entry site (IRES). The functionality of our Cox-2 promoter construct with respect to expressing IL-4 was proven at the mRNA and protein levels and the anti-inflammatory potential of IL-4 was confirmed by analyzing the expression of IL-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-3 and tumor necrosis factor (TNF)-α using a quantitative polymerase chain reaction. Nonviral transfection reagents yielded transfection rates from 21% to 44% with control vectors with and without IRES, respectively. Stimulation of equine chondrocytes resulted in a 20-fold increase of mRNA expression of IL-1β. Such exogenous stimulation of chondrocytes transfected with pNCox2-IL4 led to an increase of IL-4 mRNA expression, whereas expression of inflammatory mediators decreased. The timely link between these events confirms the anti-inflammatory potential of synthesized IL-4. We consider that this approach has significant potential for translation into a useful anti-inflammation therapy. Molecular tools such as the described therapeutic plasmid pave the way for a local-controlled, self-limiting gene therapy.

Andrographolide Exerts Chondroprotective Activity in Equine Cartilage Explant and Suppresses Interleukin-1 β -Induced MMP-2 Expression in Equine Chondrocyte Culture.

Cartilage erosion in degenerative joint diseases leads to lameness in affected horses. It has been reported that andrographolide from Andrographis paniculata inhibited cartilage matrix-degrading enzymes. This study aimed to explore whether this compound protects equine cartilage degradation in the explant culture model and to determine its effect on matrix metalloproteinase-2 (MMP-2) expression, a matrix-degrading enzyme, in equine chondrocyte culture. Equine articular cartilage explant culture was induced by 25 ng/mL interleukin-1β, a key inducer of cartilage degeneration, in cultures with or without andrographolide ranging from 10 to 50 μM. After 3–21 days, they were analyzed for the markers of cartilage degradation. It was found that interleukin-1β increased the release of sulfated glycosaminoglycans and hyaluronan from the explants into the culture media consistently with the decrease in uronic acid and collagen content in the cartilage explants. These catabolic effects were inhibited when cotreated with interleukin-1β and andrographolide. In primary equine chondrocytes, andrographolide suppressed interleukin-1β-induced MMP-2 mRNA expression and MMP-2 activity in the culture medium. These results confirmed the in vitro potent chondroprotective activities of this compound which were performed in cartilage explants and on a cellular level. These may indicate the application of andrographolide for therapeutic use in equine degenerative joint diseases.

AB0092 Controlled Expression of Endogenous Interleukin-4 for Gene Therapeutic Approaches towards Rheumatic Disorders

BackgroundRheumatic diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA) rank among the most common musculoskeletal disorders around the globe accounting for marked loss of life quality, considerable disabilities and...

Effect of osmotic stress on the expression of TRPV4 and BKCa channels and possible interaction with ERK1/2 and p38 in cultured equine chondrocytes

The metabolic activity of articular chondrocytes is influenced by osmotic alterations that occur in articular cartilage secondary to mechanical load. The mechanisms that sense and transduce mechanical signals from cell swelling and initiate volume regulation are poorly understood. The purpose of this study was to investigate how the expression of two putative osmolyte channels [transient receptor potential vanilloid 4 (TRPV4) and large-conductance Ca(2+)-activated K(+) (BKCa)] in chondrocytes is modulated in different osmotic conditions and to examine a potential role for MAPKs in this process. Isolated equine articular chondrocytes were subjected to anisosmotic conditions, and TRPV4 and BKCa channel expression and ERK1/2 and p38 MAPK protein phosphorylation were investigated using Western blotting. Results indicate that the TRPV4 channel contributes to the early stages of hypo-osmotic stress, while the BKCa channel is involved in responding to elevated intracellular Ca(2+) and mediating regulatory volume decrease. ERK1/2 is phosphorylated by hypo-osmotic stress (P < 0.001), and p38 MAPK is phosphorylated by hyperosmotic stress (P < 0.001). In addition, this study demonstrates the importance of endogenous ERK1/2 phosphorylation in TRPV4 channel expression, where blocking ERK1/2 by a specific inhibitor (PD98059) prevented increased levels of the TRPV4 channel in cells exposed to hypo-osmotic stress and decreased TRPV4 channel expression to below control levels in iso-osmotic conditions (P < 0.001).

Effects of cyclic equibiaxial mechanical stretch on α-BK and TRPV4 expression in equine chondrocytes

BackgroundChondrocytes are regularly exposed to load-induced stimuli and have the capability to sense and respond to applied mechanical stress. However, the mechanisms involved in chondrocyte mechanotransduction are not clearly understood. The purpose of this study was to explore the effects of cyclic equibiaxial mechanical stretch on the expression of α-BK and TRPV4 channels.FindingsFreshly isolated equine articular chondrocytes were subjected to mechanical stress (8% elongation at frequency of 0.5 Hz for 8 h). Western blotting was used to investigate the expression of BKCa and TRPV4 channel proteins. Mechanical stretch increased the expression of BKCa channels by 1.8 fold but TRPV4 expression was not affected.ConclusionsUpregulation of BKCa channel may be the result of direct membrane stretch or elevated intracellular Ca2+.

Effects of interleukin-6 and interleukin-1β on expression of growth differentiation factor-5 and Wnt signaling pathway genes in equine chondrocytes

To determine the effects of interleukin (IL)-6 and IL-1β stimulation on expression of growth differentiation factor (GDF)-5 and Wnt signaling pathway genes in equine chondrocytes. Macroscopically normal articular cartilage samples from 6 horses and osteochondral fragments (OCFs) from 3 horses. Chondrocyte pellets were prepared and cultured without stimulation or following stimulation with IL-6 or IL-1β for 1, 2, 12, and 48 hours; expression of GDF-5 was determined with a quantitative real-time PCR assay. Expression of genes in various signaling pathways was determined with microarrays for pellets stimulated for 1 and 2 hours. Immunohistochemical analysis was used to detect GDF-5, glycogen synthase kinase 3β (GSK-3β), and β-catenin proteins in macroscopically normal cartilage samples and OCFs. Chondrocytes stimulated with IL-6 had significantly higher GDF-5 expression within 2 hours versus unstimulated chondrocytes. Microarray analysis of Wnt signaling pathway genes indicated expression of GSK-3β and coiled-coil domain containing 88C increased after 1 hour and expression of β-catenin decreased after 2 hours of IL-6 stimulation. Results of immunohistochemical detection of proteins were similar to microarray analysis results. Chondrocytes in macroscopically normal articular cartilage and OCFs had immunostaining for GDF-5. Results indicated IL-6 stimulation decreased chondrocyte expression of the canonical Wnt signaling pathway transactivator β-catenin, induced expression of inhibitors of the Wnt pathway, and increased expression of GDF-5. This suggested IL-6 may inhibit the Wnt signaling pathway with subsequent upregulation of GDF-5 expression. Anabolic extracellular matrix metabolism in OCFs may be attributable to GDF-5 expression. This information could be useful for development of cartilage repair methods.

Three-dimensional assembly of tissue-engineered cartilage constructs results in cartilaginous tissue formation without retainment of zonal characteristics

Articular cartilage has limited regenerative capabilities. Chondrocytes from different layers of cartilage have specific properties, and regenerative approaches using zonal chondrocytes may yield better replication of the architecture of native cartilage than when using a single cell population. To obtain high seeding efficiency while still mimicking zonal architecture, cell pellets of expanded deep zone and superficial zone equine chondrocytes were seeded and cultured in two layers on poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT-PBT) scaffolds. Scaffolds seeded with cell pellets consisting of a 1:1 mixture of both cell sources served as controls. Parallel to this, pellets of superficial or deep zone chondrocytes, and combinations of the two cell populations, were cultured without the scaffold. Pellet cultures of zonal chondrocytes in scaffolds resulted in a high seeding efficiency and abundant cartilaginous tissue formation, containing collagen type II and glycosaminoglycans (GAGs) in all groups, irrespective of the donor (n = 3), zonal population or stratified scaffold-seeding approach used. However, whereas total GAG production was similar, the constructs retained significantly more GAG compared to pellet cultures, in which a high percentage of the produced GAGs were secreted into the culture medium. Immunohistochemistry for zonal markers did not show any differences between the conditions. We conclude that spatially defined pellet culture in 3D scaffolds is associated with high seeding efficiency and supports cartilaginous tissue formation, but did not result in the maintenance or restoration of the original zonal phenotype. The use of pellet-assembled constructs leads to a better retainment of newly produced GAGs than the use of pellet cultures alone.

Influence of Corticosteroids on Interleukin‐1β‐Stimulated Equine Chondrocyte Gene Expression

To compare the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on expression of selected chondrocyte genes in recombinant equine interleukin-1β (reIL-1β) stimulated articular cartilage explants. In vitro experiment. Horses (n = 6). Articular cartilage explants from 2- to 3- year-old horses were exposed to reIL-1β in the presence and absence of TA and MPA at 10(-7) and 10(-6) M. Resting levels of mRNA of anabolic and catabolic genes of chondrocyte origin were quantified using qPCR after 6- and 12-hour incubations. Genes of interest included aggrecan interglobular domain, aggrecan, and collagen II, matrix metalloproteinases 3 and 13 (MMP3, MMP 13), aggrecanase 1, tissue inhibitor of matrix metalloproteinases 1 and 2 (TIMP 1, TIMP 2), BCL 2, vascular endothelial growth factor, and cyclooxygenase 2 (COX 2). IL-1β significantly influenced the expression of most transcripts. MPA and TA inhibited the induction of MMP 13 at 6 and 12 hours; an effect that was significant at 6 hours with MPA at 10(-7) M and TA at 10(-6) M. Similarly, COX 2 was induced by reIL-1β and MPA and TA significantly inhibited its upregulation. TIMP 2 expression was reduced by reIL-1β, an effect that was significantly abrogated by MPA and TA. There were no significant differences observed between glucocorticoids for any gene studied. No differential effects of MPA or TA on chondrocytic gene expression were identified suggesting that any divergent influences of these glucocorticoids on chondrocyte metabolism are posttranslational.

Equine Articular Chondrocytes on MACT Scaffolds for Cartilage Defect Treatment

Treatment of cartilage defects poses challenging problems in human and veterinary medicine, especially in horses. This study examines the suitability of applying scaffold materials similar to those used for human cartilage regeneration on equine chondrocytes. Chondrocytes gained from biopsies of the talocrural joint of three horses were propagated in 2D culture and grown on two different scaffold materials, hyaluronan (HYAFF®) and collagen (BioGide®), and evaluated by light and electron microscopy. The equine chondrocytes developed well in both types of materials. They were vital and physiologically highly active. On the surface of the scaffolds, they formed cell multilayers. Inside the hyaluronan web, the chondrocytes were regularly distributed and spanned the large scaffold fibre distances by producing their own matrix sheath. Half-circle-like depressions occasionally found in the cell membrane were probably related to movement on the flexible matrix sheath. Inside the dense collagen scaffold, only single cells were found. They passed through the scaffold strands by cell shape adaptation. This study showed that the examined scaffold materials can be used for equine chondrocyte cultivation. Chondrocytes tend to form multilayers on the surface of both, very dense and very porous scaffolds, and have strategies to span between and move in large gaps.