Cross-linked hyaluronic acid hydrogel (cHA gel) and dexamethasone (Dex) have been used to treat knee osteoarthritis (OA) in clinical practice owing to their chondroprotective and anti-inflammatory effects, respectively. The aim of the present study was to compare the treatment effects of the cHA gel pre-mixed with/without Dex in a surgery-induced osteoarthritis model in rats. Anterior cruciate ligament transection (ACLT) surgery was performed on the right knee of rats to induce OA. Male 2-month-old Sprague-Dawley rats were randomly divided into five groups (n = 10/per group): (1) ACLT + saline; (2) ACLT + cHA gel; (3) ACLT + cHA-Dex (0.2 mg/mL) gel; (4) ACLT + cHA-Dex (0.5 mg/mL) gel; (5) Sham + saline. Intra-joint injections were performed four weeks after ACLT in the right knee. All animals were euthanized at 12 weeks post-surgery. Cartilage damage and changes in the synovial membrane were assessed by micro X-ray, Indian ink articular surface staining, Safranin-O/Fast Green staining, immunohistochemistry, hematoxylin and eosin staining of the synovial membrane, and quantitative reverse transcription-polymerase chain reaction for changes in gene expression. Micro X-ray revealed that the knee joint treated with the cHA-Dex gel was wider than those treated with cHA gel alone or saline. The cHA-Dex gel group had less Indian ink staining (indicator of cartilage fibrillation) than the cHA gel or saline injection groups. Safranin-O/Fast Green staining indicated that increased proteoglycan staining and less cartilage damage were found in the cHA-Dex gel group compared with the cHA gel or saline injection groups. Quantification of histology findings from saline, cHA gel, cHA-Dex (0.2 mg/mL) gel, cHA-Dex (0.5 mg/mL) gel, and sham groups were 5.84 ± 0.29, 4.50 ± 0.87, 3.00 ± 1.00, 2.00 ± 0.48, and 0.30 ± 0.58 (p < 0.05), respectively. A strong staining of type II collagen was found in both the cHA-Dex gel groups compared with saline group or cHA alone group. Similar result was found for the mRNA level of aggrecan and opposite result for type X collagen. Hematoxylin and eosin staining in the synovial membrane showed less synovial lining cell layers and reduced inflammatory cell infiltration in cHA-Dex gel-treated animals compared with saline or cHA only groups. Altogether, cHA-Dex gel has better chondroprotective and anti-inflammatory effects in rat surgery-induced osteoarthritis than cHA alone.

To determine if ipriflavone, a novel and safe inhibitor of Indian hedgehog (Ihh) signaling, can attenuate cartilage degeneration by blocking the Ihh pathway. Human chondrocytes were used to evaluate Ihh signaling, cell proliferation, apoptosis, gene, and protein expression of chondrocytes by cell proliferation and apoptosis assays, real-time qPCR, and Western blotting at 48 h after ipriflavone treatment. Human cartilage explants were further used to validate the cell culture results. The effects of ipriflavone on cartilage degeneration in vivo were assessed using the rat ACLT OA model. Two-month-old male SD rats were randomized into 3 groups (n = 75): (1) sham, (2) ACLT alone, and (3) ACLT+ ipriflavone. Ipriflavone was administered intragastrically at 24 h after ACLT for 6 weeks. The extent of OA progression was evaluated by the OARSI score and immunohistochemistry at 12 weeks after surgery. The Ihh signaling pathway and OA-related genes were quantified by real-time PCR. Cell proliferation in the cells treated with ipriflavone was increased to 36.40% ± 1.32% (5 μM) and 28.54% ± 0.74% (10 μM) from 11.99% ± 0.35% (DMSO) (P < 0.001), and apoptosis was decreased to 12.64% ± 3.7% (5 μM) and 15.18% ± 3.13% (10 μM) from 25.76% ± 5.1% (DMSO) (P < 0.05). Ipriflavone blocked Runx-2 mainly through the Smo-Gli2 pathway. A similar result was found in the cartilage explant culture. Ihh signaling in vivo was inhibited in animals treated with ipriflavone. Safranin-O staining revealed a less cartilage damage with lower OARSI scores (P < 0.05) in the ipriflavone-treated animals compared with untreated animals. The gene expression of Smo and Gli2 was inhibited significantly by ipriflavone (P < 0.05). The OA-related gene and protein type X, MMP-13, and type II collagen-C fragment were reduced, while type II collagen and Agg were increased in the ipriflavone-treated animals (P < 0.05). Catabolic genes were disrupted by blocking the Ihh pathway. This finding suggests that disruption of Ihh signaling with ipriflavone provides chondral protection in rat posttraumatic OA.

Background: Gouty arthritis (GA) is a type of inflammatory joint disease in which the deposition of monosodium urate (MSU) crystals inside the joints leads to severe inflammatory reactions, such as synovitis. Cross-linked hyaluronic acid hydrogel (cHA gel) has been shown to have a chondroprotective effect, while dexamethasone (Dex) is an anti-inflammatory drug; both have been used in clinical practice to a certain extent. This study was designed to compare the therapeutic effects of cHA gel pre-mixed with Dex (cHA-Dex gel) or without Dex (cHA gel alone) in a rat model of GA. Methods: In this study, 2-month-old male Sprague-Dawley (SD) rats were used. The GA model was established by intra-articular injection of a solution containing 40 mg/mL MSU crystals and 0.1 mg/mL lipopolysaccharide (LPS) dissolved in saline, and this procedure was repeated every 2 weeks. The rats were subsequently randomly allocated into four groups ( n = 8 per group): the MSU/LPS + saline group, the MSU/LPS + cHA gel group, the MSU/LPS + cHA gel containing 0.5 mg/mL Dex (cHA-Dex gel) group, and a negative control group. One day after MSU/LPS injection, the designated treatments were administered via intra-articular injection only once. Various examinations were performed 1 day after the fifth MSU/LPS induction. Behavioral indicators included the degree of knee joint swelling, the capacity of the limb to bear weight, grip strength, mechanical pain threshold, acetone test results, and the gait scoring. Imaging and histological assessments included micro X-ray, Indian ink staining, safranin O-fast green staining, immunohistochemistry, and synovial hematoxylin-eosin (HE) staining. Biochemical and molecular biological markers were evaluated by serum enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (RT-qPCR). The detected targets included interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor α (TNF-α), matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-9 (MMP-9) expression levels. Eight rats per group (n = 8) were used for all assays, with cartilage from two rats pooled to yield four biological replicates per group (n = 4) for RT-qPCR. Results: The findings showed that the deposition of intra-articular MSU/LPS led to pathological changes, including inflammation and cartilage damage. Micro X-ray scans showed that the knee joint structure treated with cHA-Dex gel had a much better level of integrity than the MSU/LPS + saline and MSU/LPS + cHA gel groups. In the MSU/LPS + cHA-Dex gel group, Indian ink staining was less, and safranin O-fast green staining showed an increase in proteoglycan (PG) content and a decrease in the extent of cartilage damage. Histological quantitative analysis showed statistically significant differences in the relevant scores ( p < 0.05). Immunohistochemical staining indicated strong positive staining for type II collagen (COL II), while expression levels of MMP-3 and MMP-9 were downregulated. HE staining of synovial tissue showed that the synovial lining cell layer became thinner, with fewer infiltrating inflammatory cells. Analysis of serum and cartilage tissues showed that cHA-Dex gel significantly lowered the level of inflammatory factors. Conclusion: Altogether, the cHA gel combined with Dex demonstrated superior therapeutic outcomes in comparison to cHA gel alone in a rat model of GA. The presented work provides preclinical evidence for a localized cHA-Dex gel delivery strategy that may complement existing clinical treatments for GA, with further validation required for translational application.