Rabbit Synovial Cells Research Articles

Synergistic Effects of Icariin and Extracellular Vesicles Derived from Rabbit Synovial Membrane-Derived Mesenchymal Stem Cells on Osteochondral Repair via the Wnt/β-Catenin Pathway.

Osteochondral defects (OCDs) are localized areas of damaged cartilage and underlying subchondral bone that can produce pain and seriously impair joint function. Literature reports indicated that icariin (ICA) has the effect of promoting cartilage repair. However, its mechanism remains unclear. Here, we explored the effects of icariin and extracellular vesicles (EVs) from rabbit synovial-derived mesenchymal stem cells (rSMSCs) on repairing of OCDs. Rabbit primary genicular chondrocytes (rPGCs), knee skeletal muscle cells (rSMCKs), and rSMSCs, and extracellular vesicles derived from the latter two cells (rSMCK-EVs and rSMSC-EVs) were isolated and identified. The rPGCs were stimulated with ICA, rSMSC-EVs either separately or in combination. The rSMCK-EVs were used as a control. After stimulation, chondrogenic-related markers were analyzed by quantitative RT-PCR and western blotting. Cell proliferation was determined by the CCK-8 assay. The preventative effects of ICA and SMSC-EVs in vivo were determined by H&E and toluidine blue staining. Immunohistochemical analyses were performed to evaluate the levels of COL2A1 and β-catenin in vivo. Results. In vitro, the proliferation of rPGCs was markedly increased by ICA treatment in a dose-dependent manner. When compared with ICA or rSMSC-EVs treatment alone, combined treatment with ICA and SMSC-EVs produced stronger stimulative effects on cell proliferation. Moreover, combined treatment with ICA and rSMSC-EVs promoted the expression of chondrogenic-related gene, including COL2A1, SOX-9, and RUNX2, which may be via the activation of the Wnt/β-catenin pathway. In vivo, combined treatment with rSMSC-EVs and ICA promoted cartilage repair in joint bone defects. Results also showed that ICA or rSMSC-EVs both promoted the COL2A1 and β-catenin protein accumulation in articular cartilage, and that was further enhanced by combined treatment with rSMSC-EVs and ICA. Our findings highlight the promising potential of using combined treatment with ICA and rSMSC-EVs for promoting osteochondral repair.

Melatonin alleviates d-galactose-decreased hyaluronic acid production in synovial membrane cells via Sirt1 signalling.

Hyaluronic acid (HA) exerts a critical role in the lubricating and buffering properties of synovial fluid in joints. The production of HA is regulated by growth factors, hormones, inflammatory cytokines and mechanical load. The reduction of HA contributes to the progression of osteoarthritis. Herein, we found that d-galactose (d-gal) induced the senescence of rabbit synovial membrane cells, accompanied by decreased HA production. The mRNA level of HA synthase 2 (HAS2) was downregulated by d-gal, as analysed by real-time polymerase chain reaction. Melatonin, an endocrine hormone, can regulate the homeostasis of bone and cartilage. We found that melatonin treatment attenuated d-gal-induced cell senescence and decreased the expression of p21, p16 and pp65 proteins. Melatonin could reverse HA production and maintain HAS2 expression. Furthermore, we revealed that Sirt1 signalling was required for melatonin effects. Sirt1 inhibitor could counteract melatonin-mediated HA production and HAS2 expression. Additionally, Sirt1 overexpression directly antagonized d-gal-induced cell aging and HA downregulation. Taken together, our results suggest that melatonin-Sirt1 signal has a protective effect on synovial membrane cells, enhancing HA synthesis and interrupting cell senescence.

Repair of osteochondral defects using injectable chitosan-based hydrogel encapsulated synovial fluid-derived mesenchymal stem cells in a rabbit model

The regeneration of hyaline articular cartilage remains a major challenge due to the limited potential for cartilage to self-repair. Mesenchymal stem cell and hydrogel scaffold-based cartilage tissue engineering is a promising technique for articular cartilage therapy. The purpose of this study was to investigate the use of rabbit synovial fluid mesenchymal stem cells (rbSF-MSCs) encapsulated in an injectable chitosan-based hydrogel to repair full-thickness cartilage defects in femoral patellar grooves in rabbits. The rbSF-MSCs were obtained from rabbit synovial fluid and the surface markers of rbSF-MSCs were coincidental to the identification criteria of MSCs according to flow cytometry. The rbSF-MSCs were able to differentiate into osteogenic, adipogenic and chondrogenic lineages. In the present study, rbSF-MSCs encapsulated in glycol chitosan (GC) and benzaldehyde capped poly (ethylene oxide) (OHC-PEO-CHO) hydrogel were introduced into rabbits to repair articular cartilage defects. The modulus of the hydrogel could be regulated by the concentrations of GC and OHC-PEO-CHO and the hydrogel has a good biocompatibility to rbSF-MSCs. Assessment of in vivo repair indicates using hydrogel/rbSF-MSCs was superior to using the hydrogel scaffold only and the untreated control based on gross appearance and histological grading and evaluation. These preliminary findings suggest using the injectable chitosan-based hydrogel as a scaffold and rbSF-MSCs as seed cells is an alternative for tissue engineering of in vivo treatments for cartilage defects and these rbSF-MSCs allografts may be promising for use in clinical applications.

Repair of articular cartilage defects with intra-articular injection of autologous rabbit synovial fluid-derived mesenchymal stem cells

BackgroundThe role of rabbit synovial fluid-derived mesenchymal stem cells (rbSF-MSCs) in cartilage defect repair remains undefined. This work evaluates the in vivo effects of rbSF-MSCs to repair knee articular cartilage defects in a rabbit model.MethodsCartilage defects were made in the patellar grooves of New Zealand white rabbits. The rbSF-MSCs were generated from the knee cavity by arthrocentesis. Passage 5 rbSF-MSCs were assayed by flow cytometry. The multipotency of rbSF-MSCs was confirmed after 3 weeks induction in vitro and the autologous rbSF-MSCs and predifferentiated rbSF-MSCs were injected into the synovial cavity. The intra-articular injection was performed once a week for 4 weeks. The animals were euthanized and the articular surfaces were subjected to macroscopic and histological evaluations at 8 and 12 weeks after the first intra-articular injection.ResultsHyaline-like cartilage was detected in the defects treated with rbSF-MSCs, while fibrocartilage tissue formed in the defects treated with chondrocytes induced from rbSF-MSCs.ConclusionsOur results suggest that autologous undifferentiated rbSF-MSCs are favorable to articular cartilage regeneration in treating cartilage defects.

The study of leucocyte and platelet-rich plasma combined with rabbit synovial stem cells for the repair of knee joint cartilage injury

Objective To investigate the effect of platelet-rich plasma (L-PRP) combined with rabbit synovium-derived mesenchymal stem cells (SMSCs) on cartilage repair in knee-joint injured. Methods 60 healthy New Zealand white rabbits were used to establish models of knee-joint cartilage defects, SMSCs were separated, purified and identified, and then L-PRP was prepared using two step centrifugation. The levels of platelet derived growth factor-BB (PDGF-BB), transforming growth factor-β1 (TGF-β1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) in whole blood and L-PRP were determined using enzyme linked immunosorbent assay (ELISA). After 12 weeks of modeling, the injured cartilage was grossly observed, and hematoxylin-eosin (HE) staining was used to observe cartilage repair under light microscope; histological examination was performed according to the O’Driscoll histologic standard. Results The levels of platelet in L-PRP increased to (135.77±50.56)×104/μl were more 6 times than whole blood [(22.38±3.20)×104/μl, P=0.001], and the levels of PDGF-BB, TGF-β1, bFGF, VEGF in L-PRP were increased as (32.50±5.10) ng/ml, (124.26±32.30) ng/ml, (87.14±12.50) pg/ml, (174.50±31.22) pg/ml significantly higher than whole blood as [(6.28±1.11) ng/ml, (21.72±3.70) ng/ml, (25.85±4.52) pg/ml, (37.87±5.33) pg/ml], the differences were statistically significant (P=0.005, 0.003, 0.008, 0.002). Post-operation 12 weeks, the results from grossly observation, HE staining and histological examination indicated that the repair effect in L-PRP group, SMSCs group, L-PRP combined with SMSCs group was significantly better than in normal saline group, L-PRP and SMSCs group, respectively. Conclusion Both L-PRP and SMSCs be able to promote cartilage repair of knee-joint injured, and L-PRP combined with SMSCs group was be superior to L-PRP and SMSCs group respectively. Key words: Knee-joint; Cartilage injury; Platelet-rich plasma; Rabbit synovium-derived mesenchymal stem cells; Stem cells

Methylglyoxal activates NF-κB nuclear translocation and induces COX-2 expression via a p38-dependent pathway in synovial cells

AimsThere is growing evidence of an increased prevalence of osteoarthritis (OA) among people with diabetes. Synovial inflammation and increased expression of cyclooxygenase-2 (COX-2) are two key features of patients with OA. Methylglyoxal (MGO) is a common intermediate in the formation of advanced glycation end-products, and its concentration is also typically higher in diabetes. In this study, we investigated the effects of the treatment of different MGO concentrations to rabbit HIG-82 synovial cells on COX-2 expression. Main methodsThe MGO induced COX-2 mRNA expression was detected by quantitative polymerase chain reaction. The MGO induced COX-2 protein production and its signaling pathways were detected by western blotting. The nuclear factor-kappa B (NF-κB) nuclear translocation by MGO was examined by immunofluorescence. Key findingsIn the present study, we find that MGO has no toxic effects on rabbit synovial cells under the experimental conditions. Our analysis demonstrates that MGO induced COX-2 mRNA and protein production. Moreover, MGO induces p38-dependent COX-2 protein expression as well as the phosphorylations of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and Akt/mammalian target of rapamycin (mTOR)/p70S6K; however, inhibition of JNK and Akt/mTOR/p70S6K phosphorylations further activates COX-2 protein expression. Furthermore, MGO is shown to activate of nuclear factor-kappa B (NF-κB) nuclear translocation. SignificanceOur results suggest that MGO can induce COX-2 expression via a p38-dependent pathway and activate NF-κB nuclear translocation in synovial cells. These results provide insight into the pathogenesis of the synovial inflammation under the diabetic condition associated with higher MGO levels.

Boiogito, a Kampo medicine, improves hydrarthrosis in a rat model of knee osteoarthritis.

BackgroundHydrarthrosis, which is associated with knee pain and limited range of motion, decreases the quality of life (QOL) of patients with osteoarthritis (OA). The Kampo medicine boiogito is prescribed for the treatment of knee OA with hydrarthrosis; however, its precise mechanisms of action remain unknown. The purposes of this study were to assess the pharmacological effects of boiogito and its mechanisms of action on joint effusion in rats with surgically induced OA.MethodsA rat OA model was produced by transecting the anterior (cranial) cruciate ligament, medial collateral ligament, and medial meniscus in the right knee joints of 7-week-old female Wistar rats. The rats were given chow containing boiogito (1 or 2 %) or indomethacin (0.002 %) for 4 weeks after surgical transection. Levels of interleukin-1β (IL-1β) and hyaluronic acid (HA) were measured by enzyme-linked immunosorbent assay. Knee joint pain was assessed using an incapacitance tester. Osmotic water permeability in cultured rabbit synovial cells was assessed using stopped-flow analysis.ResultsIncreased synovial fluid volume and knee joint pain were observed in rats with surgically induced OA. In rats with OA, levels of IL-1β and HA in the articular cavity were higher but concentration of HA in synovial fluid was lower than in sham-operated rats, suggesting excessive synovial fluid secretion. Administration of boiogito improved hydrarthrosis, IL-1β, and HA concentrations and alleviated knee joint pain in rats with OA. Indomethacin reduced IL-1β and knee joint pain but failed to improve hydrarthrosis or HA concentration in rats with OA. Osmotic water permeability in synovial cells, which is related to the function of the water channel aquaporin, was decreased by treatment with boiogito.ConclusionBoiogito ameliorates the increased knee joint effusion in rats with OA by suppressing pro-inflammatory cytokine IL-1β production in the articular cavity and regulating function of water transport in the synovium. The improvement of hydrarthrosis by boiogito results in the increased HA concentration in synovial fluid, thus reducing joint pain. Boiogito may be a clinically useful treatment of QOL in patients with OA with hydrarthrosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0979-7) contains supplementary material, which is available to authorized users.

In vitro anti-inflammatory activity of fractionated Euphorbia hirta aqueous extract on rabbit synovial fibroblasts.

Euphorbia hirta has been reported to possess anti-inflammatory activity. This study was carried out to determine the prostaglandin E 2 (PGE 2 ) inhibition activity of the fractions of the E. hirta aqueous extract on rabbit synovial fibroblast cells (HIG-82). E. hirta aqueous extract was fractionated into five fractions (fractions A, B, C, D, and E) by reversed phase flash chromatography. Rabbit synovial fibroblast cells (HIG-82) were activated with phorbol myristate acetate and treated with the fractions. The amount of PGE 2 released into the medium was measured by enzyme-linked immunosorbent assay. Fraction A (0.1, 1, and 10 μg/ml) had the greatest PGE 2 inhibitory effect among the five fractions, and showed a greater extent of PGE 2 inhibition compared to the aqueous extract. In contrast, Fraction E had the greatest stimulatory effect on PGE 2 release. Fraction A of the aqueous extract inhibited the production of PGE 2 from activated HIG-82 cells to a greater extent than the crude aqueous extract. Bioactive compounds with anti-inflammatory activity are likely to be concentrated in Fraction A of E. hirta aqueous extract.

15-Deoxy-Δ12,14-prostaglandin J2 induces PPARγ- and p53-independent apoptosis in rabbit synovial cells

A ligand of peroxisome proliferator-activated receptor γ (PPARγ), 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) induces apoptosis in various cells. However, the mechanism appears to be complex and cell-type specific. We investigated the mechanism of 15d-PGJ2-induced apoptosis of rabbit synovial cells. Exposure to 15d-PGJ2 resulted in DNA fragmentation accompanied by caspase-3 and -9 activations in the cells, suggesting occurrence of mitochondria-mediated apoptosis. Although the exposure also induced remarkable increase in p53 protein, its transcriptional activity was rather reduced, suggesting non-necessity of p53 in 15d-PGJ2-induced apoptosis. Covalent binding of 15d-PGJ2 to cellular proteins including p53 resulted in their insolubilization. N-acetylcysteine inhibited not only the 15d-PGJ2-induced apoptotic events but also the protein insolubilizations via its interaction with 15d-PGJ2. The studies using a PPARγ-agonist and -antagonist showed noninvolvement of PPARγ in 15d-PGJ2-induced apoptosis. The pre-exposure to pro-inflammatory cytokines did not affect the cytotoxicity of 15d-PGJ2 in synovial cells.Taken together, these results show that 15d-PGJ2 induces a mitochondria-mediated apoptotic pathway in p53- and PPARγ-independent manners.

Synthesis of hyaluronan and superficial zone protein in synovial membrane cells modulated by fluid flow

Hyaluronan (HA) and superficial zone protein (SZP) distribute in joint structures and play a crucial role in joint lubrication. The aim of this study was to examine the effect of fluid flow on the synthesis of both HA and SZP in synovial membrane cells. Shear stress was applied by fluid flow to the rabbit synovial membrane cell line, HIG-82. The mRNA levels of HA synthase 2 (HAS2) , HA synthase 3 (HAS3), and SZP were examined by real-time PCR. The levels of HA and SZP protein were determined by sandwich ELISA and western blotting, respectively. The expression of SZP protein was increased by the application of low-magnitude shear stress, whereas high-magnitude shear stress decreased expression of SZP protein. Meanwhile, the level of HA protein in culture was decreased when high-magnitude shear stress was applied. The levels of both HAS2 and HAS3 mRNAs were down-regulated by high-magnitude shear stress, resulting in a significant decrease in HA concentration. In conclusion, it is shown that the application of shear stress to synovial membrane cells substantially affects the synthesis of both HA and SZP, which are inhibited if excessive stress is applied.

Treatment of Partial Growth Arrest Using an In Vitro-generated Scaffold-free Tissue-engineered Construct Derived From Rabbit Synovial Mesenchymal Stem Cells

Injuries to the epiphyseal plate sometimes result in partial growth arrest, which can lead to the development of angular deformities and limb length discrepancies in growing children. The aim of this study was to develop a new treatment for partial growth arrest of the physis. For this purpose, we investigated the feasibility of an in vitro-generated scaffold-free tissue-engineered construct (TEC) derived from synovial mesenchymal stem cells (MSCs) in a rabbit growth arrest model. An experimental model for growth arrest was created by excising the medial half of the proximal growth plate of tibias from 6-week-old New Zealand White rabbits. Three experimental groups were set to evaluate TEC implantation: group 1, no implantation as controls; group 2, implantation of bone wax as additional controls; and group 3, implantation of TEC in the lesion. In group 1, all damaged growth plates were arrested and angular deformities appeared 4 weeks later. In groups 2 and 3, angular deformities were less than in the control group. Histologic images showed bone bridges developed at the damaged growth plate in group 1. Regeneration of growth plates was recognized in groups 2 and 3. Histologic examination showed greater regeneration of the growth plate in group 3 than in group 2. In addition, MSCs in the TEC differentiated into proliferative and prehypertrophic chondrocyte-like cells. A scaffold-free 3D TEC made using cultured synovium-derived MSCs differentiated into proliferative and prehypertrophic chondrocyte-like cells. The results of this experimental study suggest that scaffold-free 3D TEC made using cultured synovium-derived MSCs can be a new approach for the repair of epiphyseal injury. Clinical effectiveness of a scaffold-free 3D TEC for growth arrest remains to be determined.

Formulation and Evaluation of Quercetin Polycaprolactone Microspheres for the Treatment of Rheumatoid Arthritis

Quercetin had been shown to be effective in the management of arthritis. However, bioavailability of quercetin is a concern for such treatment. This work aims at the development of intra-articular drug delivery system by controlled release of quercetin (loaded in microspheres) for the management of rheumatoid arthritis. Polycaprolactone has been used for the preparation of microspheres (with quercetin) using the solvent evaporation method. The physio-chemical characterisation of polycaprolactone-loaded quercetin microspheres was carried out to obtain information about particle size distribution, drug loading efficiency, morphology, thermal properties, polymorphism and release trends in phosphate-buffered saline at pH 7.4 and 37°C. Quercetin-loaded polycaprolactone microspheres were found to be biocompatible as evidenced from in vitro and in vivo studies using a rabbit synovial cells and Wistar rats, respectively. Quercetin release from microspheres of selected formulations showed biphasic nature due to initial burst effect followed by a controlled release. These results suggest that optimised quercetin-loaded polycaprolactone microspheres may be the viable strategy for controlled release of quercetin in the joint cavity for more than 30 days by intra-articular injection to treat rheumatoid arthritis.

Chondrogenesis of synovium-derived mesenchymal stem cells in gene-transferred co-culture system

A co-culture strategy has been developed in this study wherein rabbit synovial mesenchymal stem cells (SMSCs) are co-cultured with growth factor (GF) transfected articular chondrocytes. Toward this end, both SMSCs and early passage rabbit articular chondrocytes that had been adenovirally transduced with transforming growth factor-beta 3 (TGF-β3) gene were separately encapsulated in alginate beads and co-cultured in the same pool of chondrogenic medium. The chondrocytes act as transfected companion cells (TCCs) providing GF supply to induce chondrogenic differentiation of SMSCs that play the role of therapeutic progenitor cells (TPCs). Against the same TCC based TGF-β3 release profile, the co-culture was started at different time points (Day 0, Day 10 and Day 20) but made to last for identical periods of exposure (30 days) so that the exposure conditions could be optimized in terms of initiation and duration. Transfection of TCCs prevents the stem cell based TPCs from undergoing the invasive procedure. It also prevents unpredictable complications in the TPCs caused by long-term constitutive over-expression of a GF. The adenovirally transfected TCCs exhibit a transient GF expression which results in a timely termination of GF supply to the TPCs. The TCC-sourced transgenic TGF-β3 successfully induced chondrogenesis in the TPCs. Real-time PCR results show enhanced expression of cartilage markers and immuno/histochemical staining for Glycosaminoglycans (GAG) and Collagen II also shows abundant extracellular matrix (ECM) production and chondrogenic morphogenesis in the co-cultured TPCs. These results confirm the efficacy of directing stem cell differentiation towards chondrogenesis and cartilage tissue formation by co-culturing them with GF transfected chondrocytes.

Modulation of Hyaluronan Fragmentation by Interleukin-1 Beta in Synovial Membrane Cells

Hyaluronan (HA) plays a crucial role in the lubricating and buffering properties of synovial fluid. The purpose of this study was to examine the effects of interleukin (IL)-1beta on HA degradation in cultured synovial membrane cells. The rabbit synovial membrane cell line HIG-82 was cultured with and without IL-1beta. The amounts of HA of varying molecular weights in the medium were analyzed using high-performance liquid chromatography, the mRNA levels of HA synthase (HAS) and hyaluronidase (HYAL) were analyzed by means of real-time PCR, and HYAL activity was analyzed by HA zymography. The amounts of HA with a molecular weight lower than 300 kDa, and between 300 and 1900 kDa, in the culture medium of HIG-82 cells were significantly higher in the presence of IL-1beta. However, the amount of HA with a molecular weight greater than 1900 kDa was significantly lower in the presence of IL-1beta. Both HAS2 and HAS3 mRNA levels were upregulated by treatment with IL-1beta. So, too, were the levels of HYAL1 and HYAL2 mRNA, which resulted in enhanced HYAL activity. However, HYAL activity was inhibited by transfection of HYAL2-siRNA. Our results suggest that IL-1beta is a crucial factor in the fragmentation of HA in inflammatory joints.

Mechanical stretch enhances NF- B-dependent gene expression and poly(ADP-ribose) synthesis in synovial cells

Temporomandibular joint disorders (TMD) show complex symptoms associated with inflammation, pain and degeneration of the peripheral tissues including synovium. Although it is believed that excessive mechanical stress on synovium causes development of TMD, the molecular mechanism by which mechanical stress triggers TMD has still remained unclear. In order to examine the effect of mechanical stress on synoviocytes, rabbit synovial cells were cyclically stretched in vitro. The stretch efficiently increased the gene expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and NF-kappaB responsive reporter gene constructs. The interruption of NF-kappaB activating pathway by inhibitors resulted in the abrogation of those expressions, indicating the pivotal role of NF-kappaB in the mechanical stretch-mediated COX-2 and iNOS expressions. In parallel, the stretch remarkably increased NO production and poly(ADP-ribose) (PAR) synthesis, suggesting that excessive amounts of NO causes DNA injury and in turn activates PAR synthesis by poly(ADP-ribose) polymerase (PARP). The inhibition of PAR synthesis by a PARP inhibitor or a radical scavenger enhanced the mechanical stretch-induced gene expressions in a NF-kappaB-independent manner, implying an involvement of PARP in the gene expression. Taken together, these results demonstrate that mechanical stress on synovial cells not only induces gene expressions of COX-2 and iNOS but also affects PAR synthesis.

Interleukin‐1 β increases gap junctional communication among synovial fibroblasts via the extracellular‐signal‐regulated kinase pathway

The gap junction protein, Cx43 (connexin 43), has been implicated in the aetiology of osteoarthritis. Studies have revealed that the size and number of gap junctions increase in synovial biopsies from patients with osteoarthritis. Furthermore, pharmacological inhibition of Cx43 function has been shown to reduce IL-1beta (interleukin-1beta)-induced metalloproteinase production by synovial fibroblasts in vitro. In the present study, we examined the link between IL-1beta and Cx43 function. We demonstrated that treatment of a rabbit synovial fibroblast cell line with IL-1beta markedly increased the level of the Cx43 protein in a concentration- and time-dependent manner. The impact on Cx43 protein levels appeared to occur post-transcriptionally, as mRNA levels are unaffected by IL-1beta administration. Additionally, we showed by fluorescence microscopy that IL-1beta alters the cellular distribution of Cx43 to cell-cell junctions and is concomitant with a striking increase in gap junction communication. Furthermore, we demonstrated that the increase in Cx43 protein, and the associated change in protein localization and gap junction communication following IL-1beta treatment, are dependent upon activation of the ERK (extracellular-signal-regulated kinase) signalling cascade. These data show that IL-1beta acts through the ERK signalling cascade to alter the expression and function of Cx43 in synovial fibroblasts.

Effects of mechanical load on the expression and activity of hyaluronidase in cultured synovial membrane cells

Hyaluronan (HA) has lubricating and buffering functions in joints. Mechanical load is a regulatory factor of HA metabolism in joints, and HA synthesis by synovial membrane cells is modulated by mechanical load. However, the effects of mechanical load on HA catabolism by hyaluronidase (HYAL) in synovial membrane cells remain unclear. The purpose of this study was to evaluate the effects of cyclic tensile load on the expression and activity of HYAL in synovial membrane cells. A cyclic tensile load of 22.8% cell elongation was applied to cultured rabbit synovial membrane cells for 3 to 48 h with or without cycloheximide. HYAL1 and HYAL2 mRNA levels were evaluated by means of real-time polymerase chain reaction (PCR) analysis. HYAL activity in the cell culture was analyzed by means of HA zymography with or without HYAL2-small interfering (si) RNA. Levels of both HYAL1 and HYAL2 mRNA were up-regulated significantly (p < 0.01) by the cyclic tensile load with or without cycloheximide. HYAL activity was detected in the loaded cell cultures and was suppressed substantially by HYAL2-siRNA. HYAL activity was undetectable in unloaded cell cultures. These results show that a cyclic tensile load induces the expression and activity of HYAL in synovial membrane cells, suggesting that increased HYAL by mechanical load affects HA catabolism in synovial fluid.

IL-1 beta and IL-6 stimulate the production of platelet-activating factor (PAF) by cultured rabbit synovial cells.

The aim of this study was to determine whether synovial cells are capable of producing PAF in the presence of cytokines such as IL-1 beta and IL-6 and other stimuli. Synovial cells were obtained from joints of healthy rabbits. PAF production was assayed by measurement of serotonin release in rabbit platelets and the incorporation of 3H-acetate into PAF. Synovial cells produced PAF after 5 min of incubation with ionophore A23187, reaching the maximal amount at 15 min (4.3 +/- 0.7 x 10(-3) pmol of PAF/mg protein, P < 0.005, n = 4), and declining afterwards. The treatment of synoviocytes with IL-1 beta and IL-6 induced synthesis of PAF after 5 min of stimulation, reaching the greatest production at 15 min with IL-6 and 30 min with IL-1 beta (3.6 +/- 1.1 x 10(-3) and 3.3 +/- 1.2 pmol of PAF/mg protein, respectively, P < 0.05, n = 4). The incubation of the cells with PMSF, an inhibitor of acetylhydrolase, before the addition of the stimuli, increased the incorporation rate of 3H-acetate, suggesting a rapid degradation of PAF by synoviocytes. These results demonstrate that synovial cells can produce PAF after stimulation with agonists, such as ionophore, and cytokines. Thus, PAF, acting alone or with other mediators, could amplify the inflammatory joint reaction.

CLONING OF HUMAN INTERLEUKIN-10 GENE AND TRANSGENE EXPRESSION IN RABBIT SYNOVIAL CELLSIN VITRO

In this work, the full-length open reading frame of the human interleukin-10 (hIL-10) gene was amplified through reverse transcription–polymerase chain reaction (RT-PCR), and then PCR products were inserted into pcDNA4/HisMax to construct an eukaryotic expression vector. After optimization by green fluorescent protein (GFP), recombinant hIL-10 genes were transfected and expressed in rabbit synovial cells compounded with liposome in vitro. In cell culture supernatant, rhIL-10 was detected using enzyme-linked immunosorbent assay (ELISA) at time intervals of 12 hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days. After 12 hours of transfection, ELISA showed that transgene expression of hIL-10 in rabbit synovial cells was elevated; at 72 hours, hIL-10 expression reached its peak value; and then it declined gradually until 7 days, compared with the control. After 14 days, transgene expression ceased. Gene cloning of hIL-10 and its transgene expression in synovial cells therefore gives a basis for the gene therapy of rheumatoid arthritis.

Effect of local viral transfer of interleukin 10 gene on a rabbit arthritis model induced by interleukin 1β

Interleukin 1beta (IL-1beta) is the principal mediator in the pathogenesis of rheumatoid arthritis. Continuous injection of interleukin 1beta (IL-1beta) into the knee articular cavities of animals can induce models that resemble rheumatoid arthritis. The objective of this study was to evaluate the feasibility of local recombinant retrovirus viral interleukin 10 (rRV-vIL-10) gene transfer treatment of a rabbit model of arthritis induced by IL-1beta. An hIL-1beta-induced rabbit rheumatoid arthritis model was established using the MFG-hIL-1beta-neo-HIG-82 cell line, which is capable of continuous secretion of hIL-1beta. After transfecting the rabbit synovial fibroblast cell line (MFG-hIL-1beta-neo-HIG-82) with rRV-vIL-10, G418 was then added to identify the positive clone. The rRV-vIL-10 positive clone was injected into the established rabbit rheumatoid arthritis model through intra-articular injection. Successful gene transfer was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The levels of IL-1beta before and after treatment were determined by enzyme- linked immunosorbent assay. Retrovirus vector was an effective vector both to synoviocytes in vitro and synovium tissue in vivo as confirmed by RT-PCR and immunohistochemistry. The rabbit arthritis model treated with rRV-vIL-10 showed a dramatic remission of arthritis and a decline in the level of cytokines such as IL-1beta. Retrovirus-mediated transfection of vIL-10 successfully transferred the gene into rabbit synovium ex vivo and was able to suppress intra-articular inflammation response to IL-1beta.