Equine Fibroblast-like Synoviocytes Research Articles

Interaction with macrophages attenuates equine fibroblast-like synoviocyte ADAMTS5 (aggrecanase-2) gene expression following inflammatory stimulation.

ABSTRACTThe joint synovium consists of a heterogeneous cell population, chiefly comprised of macrophages, and fibroblast‐like synoviocytes (FLS). An inter‐species co‐culture model was developed to examine interactions between these cells. Equine FLS and the canine macrophage line DH82 were differentially labeled using fluorescent markers and results from direct co‐culture compared with those from both indirect co‐culture, and conditioned media experiments. The transcript expression of IL‐1β, IL‐6, ADAMTS4, and ADAMTS5 in each cell type were determined using species‐specific qPCR assays. Lipopolysaccharide stimulation of EFLS rapidly increased IL‐1β, IL‐6, ADAMTS4, and ADAMTS5 mRNAs. The induction of ADAMTS5 was significantly reduced when equine FLS were cultured with DH82 cells directly or indirectly. Exposure of equine FLS to denatured conditioned media also significantly reduced ADAMTS5 induction. DH82 cells increased interleukin‐1β expression substantially following LPS stimulation. However, knockdown of interleukin‐1β in DH82 cells, or inhibition of NF‐κB in equine FLS prior to co‐culture did not change the inhibitory effect on equine FLS ADAMTS5 gene expression. This work indicates that macrophages can influence FLS gene expression through a soluble mediator, and modulate the expression of an enzyme critical in osteoarthritis pathology during inflammatory stimulation. © 2018 The Authors. Journal of Orthopaedic Research® Published by WileyPeriodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 36:2178–2185, 2018.

In vitro evaluation of equine fibroblast-like synoviocytes viability treated with doxycycline

Objective- The purpose of present study was to investigate the viability of equine fibroblast-like synoviocytes (FLSs) treated with doxycycline. Design- Experimental study. Sample population- FLSs from metacarpophalangeal joints of six skeletally mature horses. Methods- FLSs were established from synovial fluids of healthy joints. The cells were treated with different concentrations (1, 5, 10, 50, 100, 150, 300, 400 µg/ml) or without doxycycline for 48-hour. Viability of FLSs was determined by MTT assay and the Trypan blue dye exclusion method. Results- No significant differences were observed between viability of FLSs cultures treated with doxycycline until 150 µg/ml and control group (P>0.05). Doxycycline at 300 and 400 µg/ml significantly decreased FLSs viability (P<0.05). FLSs viability were 74.28% and 59.07% in 300 and 400 µg/ml, respectively, when measured by the MTT assay. Also FLSs viability at 300 and 400 µg/ml of doxycycline were 68.10% and 43.26%, respectively with Trypan blue exclusion method. Conclusion and Clinical Relevance- These findings demonstrate that doxycycline was not toxic for equine FLSs at concentration equal or less than 150 µg/ml in vitro. Further studies are needed to investigate the safety, efficacy and detrimental effects of doxycycline in equine joints.

Identification of Reliable Reference Genes for Quantitative Real-Time PCR in Equine Fibroblast-Like Synoviocytes Treated by Doxycycline

Real-time quantitative polymerase chain reaction is a powerful and accurate technique to evaluate gene transcription patterns in different cells under biological and pathological conditions. One of the critical steps in the quantitation of transcription profiles is accurate normalization. In this critical step, use of appropriate stable reference gene(s) would be important. The purpose of this study was to identify the most stable housekeeping genes in equine fibroblast-like synoviocytes (FLS) treated with doxycycline from a panel of candidate genes. The gene transcription levels of six commonly used reference genes (GAPDH, ACTB, PPIA, HPRT1, IPO8, and MRPL19) were determined in normal equine FLS or treated by doxycycline and the combination of both. After applying the BestKeeper, geNorm, and NormFinder programs, to this set of genes, there were differences in the ranked order in the three defined groups. But, overall resultants of all three analysis programs determined that the candidate reference genes could be ranked as HPRT1, ACTB, GAPDH, PPIA, MRPL19, and IPO8. To more accurate normalization of gene quantitative data, using of HPRT1, ACTB, and GAPDH combination as reference gene panels is recommended for equine FLS during normal condition or treated by doxycycline.

MRNA expression of genes involved in inflammation and haemostasis in equine fibroblast-like synoviocytes following exposure to lipopolysaccharide, fibrinogen and thrombin

BackgroundStudies in humans have shown that haemostatic and inflammatory pathways both play important roles in the pathogenesis of joint disease. The aim of this study was to assess mRNA expression of haemostatic and inflammatory factors in cultured equine fibroblast-like synoviocytes exposed to lipopolysaccharide (LPS), fibrinogen and thrombin. Synovial membranes were collected from metacarpo-phalangeal joints of 6 skeletally mature horses euthanized for non-orthopaedic reasons. Passage 4 fibroblast-like synoviocytes were left non-treated or treated with either 0.1 μg/ml LPS, 5 mg/ml fibrinogen or 5 U/ml thrombin and harvested at time points 0, 6, 24 and 48 h. mRNA expression of serum amyloid A (SAA), interleukin-6 (IL-6), monocyte chemotactic protein 1 (MCP-1), tissue factor (TF), plasminogen activator inhibitor 1 (PAI-1), urokinase plasminogen activator (uPA), vascular endothelial growth factor (VEGF) and protease activator receptor 1 (PAR-1) was assessed using quantitative real time reverse transcriptase PCR.ResultsLPS caused a significant increase in mRNA expression of SAA, IL-6, MCP-1 and uPA, and a decrease in TF, PAI-1 and PAR-1 when compared to non-treated cells. Treatment with thrombin resulted in increased mRNA expression of SAA, IL-6, MCP-1 and PAI-1, and a decreased PAR-1 expression compared to non-treated cells. The fibrinogen-treated synoviocytes showed significantly increased mRNA expression of IL-6, MCP-1, TF and PAI-1, and decreased PAR-1 expression compared to non-treated cells.ConclusionLPS, fibrinogen and thrombin induced an increased gene expression of inflammatory markers in isolated equine fibroblast-like synoviocytes. LPS caused changes in gene expression promoting increased fibrinolysis, while fibrinogen and thrombin changed the gene expression resulting potentially in reduced fibrinolysis. Overall, it appeared that both inflammatory and haemostatic stimuli affected expression of genes involved in inflammatory and haemostatic pathways, supporting their importance in equine joint diseases.

Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study.

Introduction. Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions.Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson’s Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via dimethylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay.Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity was inversely proportional to the concentration of PLLA used. PLLA coating did not prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions produced non-uniform cellular distribution.Discussion/Conclusion. FLS-seeding of PGA scaffolds cultured in a rotating bioreactor resulted in the most optimal cell and matrix characteristics seen in this study. Cells grew only in the pores of the OPLA sponge, and could not adhere to the PLLA coating of PGA scaffold, due to the hydrophobic property of PLA. While PGA culture in a bioreactor produced measureable GAG, no culture technique produced visible collagen. For this reason, and due to the dissolution of PGA scaffolds, the culture conditions and scaffolds described here are not recommended for inducing fibrochondrogenesis in equine FLS for meniscal tissue engineering.

Effects of growth factors on equine synovial fibroblasts seeded on synthetic scaffolds for avascular meniscal tissue engineering

Across species, the avascular portion of the knee meniscus cannot heal spontaneously if severely injured. The most common treatment is meniscectomy which results in osteoarthritis. The objective of this study was to assess the fibrochondrogenic potential of equine fibroblast-like synoviocytes (FLS) seeded on scaffolds under the influence of growth factors in vitro to determine the potential of developing a novel cell-based repair strategy. Cultured FLS were seeded onto synthetic scaffolds in a rotating bioreactor under the influence of three growth factor regimens: none, basic fibroblast growth factor (bFGF) alone, and bFGF plus transforming growth factor (TGF-β 1) and insulin-like growth factor (IGF-1). Constructs were analyzed for mRNA expression and production of fibrochondroid extracellular matrix constituents. Type II collagen and aggrecan mRNA were significantly higher in growth factor-treated groups ( p < 0.05). Despite sub-optimal extracellular matrix production, FLS can exhibit fibrochondral characteristics and may have potential for cell-based tissue engineering for avascular meniscal regeneration.