Local delivery of triamcinolone acetonide in an aclt model of osteoarthritis impairs joint regeneration, possibly by inhibition of wound healing

Abstract

INTRODUCTION: Corticosteroids such as triamcinolone acetonide (TAA) are used to alleviate pain symptoms of osteoarthritic patients. Although usually pain relief is achieved, the effect wears off with time. The use of biomaterial-based local delivery systems would overcome this problem. However, little is known on the the appropriate dosing of corticosteroids in the joint. In a previous study on collagenase-induced mild OA in the rat, a limited effect on cartilage degeneration was found of TAA, either as bolus or in a polyesteramide microsphere (PEAMs) controlled release platform system. In the current study, safety, effectivity and possible side effects of TAA locally delivered by the microsphere platform at different dosages was studied in an anterior cruciate ligament transection (ACLT) and partial meniscectomy (DMM) model of rat OA, which is a more severe OA model. As damage of ligamentous structures leading to instability is a contraindication for corticosteroid therapy, despite lack of clear evidence with this respect, this was further examined. METHODS: The Utrecht University Ethical Committee for Animal Care and Use approved the study protocol (# 2014.III.10.086). OA was induced by transection of the anterior cruciate ligament and partial meniscectomy in 24 rats. Four weeks after surgery, the experimental joint was injected intra-articularly with PEAMs loaded with 0.70 mg, 1.0 mg or 1.6 mg TAA. Empty PEAMs were included as control, in addition to the contralateral healthy and untreated joints. PEAMs were administered by two injections on two consecutive days. Serum TAA levels were determined the first two weeks post-injection using HPLC analysis. Weekly pressure plate measurements were performed to monitor static weight bearing and thereby indirectly pain during a 16 week follow up. Post-mortem, histology (Mankin score) and microCT scans of all knee joints showed disease progression and possible effects on bone tissue. From healthy animals, patella, collateral and cruciate ligament explants were cultured in expansion medium consisting of DMEM (high glucose, GlutaMAX(TM), pyruvate) supplemented with 10% fetal bovine serum, 1% ascorbic acid 2-phosphate and 1% penicillin/streptomycin for 10 days with or without TAA (100 μM) added to the medium. After culture, the presence of fibroblasts migrating out of the tissue and adhering to the bottom of the culture dish was assessed. Differences in calcium deposition by microCT were evaluated by one-way analysis of variance (ANOVA). In vitro data on ligament cell outgrowth was categorized into “migration/adherence” and “no migration/adherence” and a Fisher's Exact test was performed to evaluate significant differences in the migration/adherence of ligament fibroblasts in the presence or absence of TAA. P<0.05 was taken to indicate statistical significance. RESULTS: Load bearing did not differ between the OA hind limb compared to the untreated limb in any of the treatments (n=6 per group), although degeneration was moderate (Mankin score 6). In the PEAM+TAA treated rats, serum TAA was undetectable 8 days after injection. MicroCT analysis revealed extensive dystrophic calcification in all TAA-treated knee joints (figure 1 and 2). Histopathological analysis of knee joints treated with TAA showed a significant increase in cartilage degeneration, reflected in a loss of tidemark, cartilage structure degeneration, and chondrocyte cell damage, with basal degeneration. No effects were found of empty microspheres. Cell outgrowth from ligaments was almost completely inhibited by TAA, independent of the ligament type studied (table 1). DISCUSSION: Although moderate degeneration was induced, pain could not be detected.in the ALCT OA model. Empty microspheres were found to be safe, but dystrophic calcification and destruction of the knee joint was observed with the TAA-loaded microspheres, independent of the dose. Ligament fibroblasts did not migrate out of cultured explants and adhere to the culture dish in the presence of TAA, indicating that TAA might impair wound healing processes in damaged joints thereby extending the instability of the joint. Controversy remains on the treatment of OA with corticosteroids, since both beneficial and adverse effects have been reported: dystrophic calcifications have also been reported in human patients. We hypothesize that in the present OA model, where joint instability is induced by transection of the cruciate ligaments and partial meniscectomy, the continuous presence of TAA impairs joint stabilization and tissue healing, thereby providing support for the notion that damage-induced instability of a joint is a contra-indication for corticosteroid treatment. Calcification may suggest inhibition of clearance of cell debris by immune cells, although corticosteroids have been suggested to directly induce calcification without alterations in osteogenic markers in MSCs (de Mos et al., 2007). Future studies may show the effect of slow release of TAA at lower dosages and/or in different joint disease models. Effects on joint pain of local TAA delivery may be more appropriately studied by using models such as the peptidoglycan polysaccharide-induced pain model. (Figure Presented).