Intradiscal injection of monosodium iodoacetate induces intervertebral disc degeneration in an experimental rabbit model

Background Establishing an optimal animal model for intervertebral disc (IVD) degeneration is essential for developing new IVD therapies. The intra-articular injection of monosodium iodoacetate (MIA), which is commonly used in animal models of osteoarthritis, induces cartilage degeneration and progressive arthritis in a dose- and time-dependent manner. The purpose of this study was to determine the effect of MIA injections into rabbit IVDs on the progression of IVD degeneration evaluated by radiographic, micro-computerized tomography (micro-CT), magnetic resonance imaging (MRI), and histological analyses. Methods In total, 24 New Zealand White (NZW) rabbits were used in this study. Under general anesthesia, lumbar discs from L1–L2 to L4–L5 had a posterolateral percutaneous injection of MIA in contrast agent (CA) (L1–L2: CA only; L2–L3: MIA 0.01 mg; L3–L4: 0.1 mg; L4–L5: 1.0 mg; L5–L6: non-injection (NI) control). Disc height was radiographically monitored biweekly until 12 weeks after injection. Six rabbits were sacrificed at 2, 4, 8, and 12 weeks post-injection and processed for micro-CT, MRI (T2-mapping), and histological analyses. Three-dimensional (3D) disc height in five anatomical zones was evaluated by 3D reconstruction of micro-CT data. Results Disc height of MIA-injected discs (L2–L3 to L4–L5) gradually decreased time-dependently (P < 0.0001). The disc height of MIA 0.01 mg-injected discs was significantly higher than those of MIA 0.1 and 1.0 mg-injected discs (P < 0.01, respectively). 3D micro-CT analysis showed the dose- and time-dependent decrease of 3D disc height of MIA-injected discs predominantly in the posterior annulus fibrosus (AF) zone. MRI T2 values of MIA 0.1 and 1.0 mg-injected discs were significantly decreased compared to those of CA and/or NI controls (P < 0.05). Histological analyses showed progressive time- and dose-degenerative changes in the discs injected with MIA (P < 0.01). MIA induced cell death in the rabbit nucleus pulposus with a high percentage, while the percentage of cell clones was low. Conclusions The results of this study showed, for the first time, that the intradiscal injection of MIA induced degenerative changes of rabbit IVDs in a time- and dose-dependent manner. This study suggests that MIA injection into rabbit IVDs could be used as an animal model of IVD degeneration for developing future treatments.

Inhibition of SYK and cSrc kinases can protect bone and cartilage in preclinical models of osteoarthritis and rheumatoid arthritis

The pathophysiology of osteoarthritis (OA) includes the destruction of subchondral bone tissue and inflammation of the synovium. Thus, an effective disease-modifying treatment should act on both of these pathogenetic components. It is known that cSrc kinase is involved in bone and cartilage remodeling, and SYK kinase is associated with the inflammatory component. Thus the aim of this study was to characterize the mechanism of action and efficacy of a small molecule multikinase inhibitor MT-SYK-03 targeting SYK and cSrc kinases among others in different in vitro and in vivo arthritis models. The selectivity of MT-SYK-03 kinase inhibition was assayed on a panel of 341 kinases. The compound was evaluated in a set of in vitro models of OA and in vivo OA and RA models: surgically-induced arthritis (SIA), monosodium iodoacetate-induced arthritis (MIA), collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA). MT-SYK-03 inhibited cSrc and SYK with IC50 of 14.2 and 23 nM respectively. Only five kinases were inhibited > 90% at 500 nM of MT-SYK-03. In in vitro OA models MT-SYK-03 reduced hypertrophic changes of chondrocytes, bone resorption, and inhibited SYK-mediated inflammatory signaling. MT-SYK-03 showed preferential distribution to joint and bone tissue (in rats) and revealed disease-modifying activity in vivo by halving the depth of cartilage erosion in rat SIA model, and increasing the pain threshold in rat MIA model. Chondroprotective and antiresorptive effects were shown in a monotherapy regime and in combination with methotrexate (MTX) in murine and rat CIA models; an immune-mediated inflammation in rat AIA model was decreased. The obtained preclinical data support inhibition of cSrc and SYK as a viable strategy for disease-modifying treatment of OA. A Phase 2 clinical study of MT-SYK-03 is to be started.

Protective Effects of Annatto Tocotrienol and Palm Tocotrienol-Rich Fraction on Chondrocytes Exposed to Monosodium Iodoacetate

Background: This study aimed to compare the chondroprotective efficacy and mechanism of annatto tocotrienol (AnTT) and palm tocotrienol-rich fraction (PT3) using SW1353 chondrocytes treated with monosodium iodoacetate (MIA). Methods: The chondrocytes were incubated with AnTT or PT3 in advance or concurrently with MIA for 24 h. The viability of the cells was tested with an MTT assay. The 8-isoprostane F2-α, extracellular matrix proteins, metalloproteinase and sex-determining region Y box protein 9 (SOX9) levels were determined using immunoassays. Results: AnTT and PT3 reversed an MIA-induced decrease in chondrocyte viability when incubated together with MIA (p < 0.05). Prior incubation with both mixtures did not produce the same effects. AnTT and PT3 cotreatment could suppress 8-isoprostane F2-α level in chondrocytes exposed to MIA (p < 0.01). Co-exposure to tocotrienols and MIA increased the type II collagen/type I collagen ratio in chondrocytes (p < 0.01). In addition, the co-exposure of AnTT and MIA for 24 h significantly upregulated SOX9, type II collagen and aggrecan levels (p < 0.05), which was not observed with co-exposure of PT3 and MIA, AnTT or PT3 exposure alone. Conclusion: AnTT and PT3 could prevent a reduction in chondrocyte viability following MIA exposure by reducing oxidative stress. In addition, AnTT might induce self-repair and anabolic activities in chondrocytes challenged with MIA.

The Xiaogu San Attenuates Pain and Cartilage Damage in Rats with Monosodium Iodoacetate Induced Osteoarthritis

Background: Osteoarthritis (OA) is a degenerative joint disease with an increasing incidence associated with increased life expectancy. The application of traditional Chinese medicine in the treatment of OA has become a research hotspot. Objective: This study investigated the effects of XGS externally applied to osteoarthritic joints and analyze its effect on pain in in monosodium iodoacetate (MIA)-induced OA rats. This study also evaluates potential mechanisms behind the anti-osteoarthritic effects of XGS. Methods: A total of 24 Sprague Dawley rats were evenly and randomly divided into three separate groups including the normal control (NC), OA and XGS groups. MIA (50 μL, 10 mg/mL) was injected into the left knee joints of the rats to induce OA. After 7 days, The rats of XGS group were given XGS (0.45 g) that was externally applied to the left knee joint, were fixed with gauze and continuously administered XGS for 28 days. Morphological changes in tissues and organs were examined using H&E staining. Biochemical indicators were measured using an automatic biochemical analyzer. Inflammatory cytokines were detected using ELISA kits and immunohistochemistry. RNA-based high-throughput sequencing (RNA-seq) was performed to detect differential expression of mRNAs in normal and MIA–induced OA rats. Results: Stride of the left leg was extended in rats, matrix increased on cartilage tissue surfaces and inflammatory cytokines were reduced when treated with XGS. RNA-seq results revealed that the PI3K-Akt signaling pathway is activated in the OA model. The qRT-PCR showed that The expression levels of Tnn, Col6a6, Igf1 and Lamb1 were up-regulated by XGS. Conclusions: Altogether, this work demonstrated potential therapeutic effects of XGS in rats with OA induced by MIA. The XGS may be considered an alternative therapy to manage OA.

Effects of Dipsacus asperoides and Phlomis umbrosa Extracts in a Rat Model of Osteoarthritis

The implementation of the Nagoya Protocol highlighted the importance of identifying alternative herbal products that are as effective as traditional medicine. Dipsacus asperoides and Phlomis umbrosa, two species used in the Korean medicine ‘Sok-dan’, are used for the treatment of bone- and arthritis-related diseases, and they are often mixed or misused. To identify herbal resources with similar efficacy, we compared the effects of D. asperoides extract (DAE) and P. umbrosa extract (PUE) on osteoarthritis (OA) in a monosodium iodoacetate (MIA)-induced OA rat model. Weight-bearing distribution, serum cytokines, histopathological features, and the expression of matrix metalloproteinases (MMPs) of knee joint tissues were examined in the OA rats treated with DAE and PUE (200 mg/kg) for 21 days. DAE and PUE restored weight-bearing distribution, inhibited the production of serum cytokines, and alleviated the histopathological features of the OA knee tissue. DAE or PUE treatment decreased OA-induced overexpression of MMP-2, MMP-9, and MMP-13 in the knee joint tissue. This study demonstrated the efficacy of both DAE and PUE in an MIA-induced OA model, providing a basis for the clinical use of these products in traditional Korean medicine.

Soluble CCR2 Gene Therapy Controls Joint Inflammation, Cartilage Damage, and the Progression of Osteoarthritis by Targeting MCP-1

Background: Osteoarthritis (OA) is the most common type of degenerative arthritis and affects the entire joint, causing pain, joint inflammation, and cartilage damage. Various risk factors are implicated in causing OA, and in recent years, a lot of research and interest have been directed toward chronic low-grade inflammation in OA. Monocyte chemoattractant protein-1 (MCP-1; also called CCL2) acts through C-C chemokine receptor type 2 (CCR2) in monocytes and is a chemotactic factor of monocytes that plays an important role in the initiation of inflammation. The targeting of CCL2–CCR2 is being studied as part of various topics including the treatment of OA.Methods: In this study, we evaluated the potential therapeutic effects the sCCR2 E3 gene may exert on OA. The effects of sCCR2 E3 were investigated in animal experiments consisting of intra-articular injection of sCCR2 E3 in a monosodium iodoacetate (MIA)-induced OA rat model. The effects after intra-articular injection of sCCR2 E3 (fusion protein encoding 20 amino acids of the E3 domain of the CCL2 receptor) in a monosodium iodoacetate-induced OA rat model were compared to those in rats treated with empty vector (mock treatment) and full-length sCCR2.Results: Pain improved with expression of the sCCR2 gene. Improved bone resorption upon sCCR2 E3 gene activation was confirmed via bone analyses using micro-computed tomography. Histologic analyses showed that the sCCR2 E3 gene exerted protective effects against cartilage damage and anti-inflammatory effects on joints and the intestine.Conclusions: These results show that sCCR2 E3 therapy is effective in reducing pain severity, inhibiting cartilage destruction, and suppressing intestinal damage and inflammation. Thus, sCCR2 E3 may be a potential therapy for OA.

Evaluation of Different Intraarticular Injection Therapies with Gait Analysis in a Rat Osteoarthritis Model

Objective: Osteoarthritis (OA) is a degenerative disease that causes serious damage to joints, especially in elderly patients. The aim of study was to demonstrate the effectiveness of intraarticular therapies that are currently used or recently popularized in the treatment of OA. Design: The baseline values were determined by walking the rats on the CatWalk system. Afterwards, a monosodium iodoacetate (MIA)-induced knee OA model was created with intraarticular MIA, and the rats were walked again on the CatWalk system and post-OA values were recorded. At this stage, the rats were divided into 4 groups, and intraarticular astaxanthin, intraarticular corticosteroid, intraarticular hyaluronic acid, and intraarticular astaxanthin + hyaluronic acid were applied to the groups, respectively. The rats were walked once more and posttreatment values were obtained. Nine different dynamic gait parameters were used in the comparison. Results: Significant changes were measured in 6 of the 9 dynamic gait parameters after the MIA-induced knee OA model. While the best improvement was observed in run duration ( P = 0.0022), stride length ( P < 0.0001), and swing speed ( P = 0.0355) in the astaxanthin group, the results closest to basal values in paw print length ( P < 0.0001), paw print width ( P = 0.0101), and paw print area ( P = 0.0277) were seen in the astaxanthin + hyaluronic acid group. Conclusion: Astaxanthin gave better outcomes than corticosteroid and hyaluronic acid in both dynamic gait parameters and histological examinations. Intraarticular astaxanthin therapy can be a good alternative to corticosteroid and hyaluronic acid currently used in intraarticular therapy to treat OA.

PTX-3 Secreted by Intra-Articular-Injected SMUP-Cells Reduces Pain in an Osteoarthritis Rat Model

Mesenchymal stem cells (MSCs) are accessible, abundantly available, and capable of regenerating; they have the potential to be developed as therapeutic agents for diseases. However, concerns remain in their further application. In this study, we developed a SMall cell+Ultra Potent+Scale UP cell (SMUP-Cell) platform to improve whole-cell processing, including manufacturing bioreactors and xeno-free solutions for commercialization. To confirm the superiority of SMUP-Cell improvements, we demonstrated that a molecule secreted by SMUP-Cells is capable of polarizing inflammatory macrophages (M1) into their anti-inflammatory phenotype (M2) at the site of injury in a pain-associated osteoarthritis (OA) model. Lipopolysaccharide-stimulated macrophages co-cultured with SMUP-Cells expressed low levels of M1-phenotype markers (CD11b, tumor necrosis factor-α, interleukin-1α, and interleukin-6), but high levels of M2 markers (CD163 and arginase-1). To identify the paracrine action underlying the anti-inflammatory effect of SMUP-Cells, we employed a cytokine array and detected increased levels of pentraxin-related protein-3 (PTX-3). Additionally, PTX-3 mRNA silencing was applied to confirm PTX-3 function. PTX-3 silencing in SMUP-Cells significantly decreased their therapeutic effects against monosodium iodoacetate (MIA)-induced OA. Thus, PTX-3 expression in injected SMUP-Cells, applied as a therapeutic strategy, reduced pain in an OA model.

A novel mice model of acute flares in osteoarthritis elicited by intra-articular injection of cultured mast cells

Purpose Mast cells are multifunctional in osteoarthritis (OA), and infiltration of activated mast cells likely contributes to disease severity and progression. However, the detailed mechanisms of action are unclear. The purpose of this study was to elucidate the role of mast cell infiltration in OA at histological level using a new mice model and to investigate pharmacological inhibitory effects of existing mast cell stabilizers in this model. Methods Mice were injected intra-articularly with monosodium iodoacetate (MIA 0.5 mg) or PBS on day 0, and PBS, with or without mast cells (MC: 1 × 106 cells) on day 14. They were divided into four groups: OA flare (MIA + MC), OA (MIA + PBS), MC non-OA (PBS + MC), and PBS non-OA (PBS + PBS). In OA flare, the MC stabilizer drug (tranilast: 400 mg/kg/day) or PBS was administered intraperitoneally from days 15 to 21. Results Histologically, modified Mankin score of the OA flare was significantly higher than that of OA (7.0 [1.8] vs. 3.3 [1.3], P < 0.05), and a larger number of mast cells was observed in OA flare than in OA (34.5 [6.3]/mm2 vs. 27.2 [2.3]/mm2, P < 0.05) on day 22. OA flare also showed acute exacerbation of pain and increased gene expression of pro-inflammatory cytokines and aggrecanase compared with OA. Administration of tranilast to OA flare-up provoked significant improvements in term of histological changes, pain, and gene expression at day 22. Conclusion Our novel model possibly mimics OA flare conditions, which may open a new strategy of disease-modifying treatment for OA, focused on controlling the multiple functions of mast cells.