Controlled biomaterial-based corticosteroid release might circumvent multiple injections and the accompanying risks, such as hormone imbalance and muscle weakness, in osteoarthritic (OA) patients. For this purpose, microspheres were prepared from an amino acid-based polyester amide (PEA) platform and loaded with triamcinolone acetonide (TAA). TAA loaded microspheres were shown to release TAA for over 60days in PBS. Furthermore, the bioactivity lasted at least 28days, demonstrated by a 80–95% inhibition of PGE2 production using TNFα-stimulated chondrocyte culture, indicating inhibition of inflammation. Microspheres loaded with the near infrared marker NIR780-iodide injected in healthy rat joints or joints with mild collagenase-induced OA showed retention of the microspheres up till 70days after injection. After intra-articular injection of TAA-loaded microspheres, TAA was detectable in the serum until day seven. Synovial inflammation was significantly lower in OA joints injected with TAA-loaded microspheres based on histological Krenn scores. Injection of TAA-loaded nor empty microspheres had no effect on cartilage integrity as determined by Mankin scoring. In conclusion, the PEA platform shows safety and efficacy upon intra-articular injection, and its extended degradation and release profiles compared to the currently used PLGA platforms may render it a good alternative. Even though further in vivo studies may need to address dosing and readout parameters such as pain, no effect on cartilage pathology was found and inflammation was effectively lowered in OA joints.

Purpose: Triamcinolone acetonide (TAA) is a glucocorticoid which is amongst others used to inhibit joint inflammation and pain via intra-articular injection. The development of controlled release systems would obviate the need for multiple injections. However, it is not quite clear what is the effect of continuous vs short exposure of TAA on articular cartilage metabolism. Furthermore, the direct mechanism of action of TAA on chondrocytes is not known. TAA can block the NF-kβ signaling pathway in vitro and as NF-kβ binds to the mouse COX2 promoter, we hypothesized that TAA inhibits joint inflammation by inhibiting COX2 activity. This in vitro study focused on the effect of TAA and the timing of its administration on articular chondrocyte extracellular matrix synthesis and COX2 expression. Methods: Canine articular chondrocytes (ACs) of three different non- chondrodystrophic dogs were cultured in pellets at 200,000 cells per pellet in chondrogenic medium consisting of DMEM (high glucose, GlutaMAX(TM), pyruvate) supplemented with 2% insulin-transferrin-selenium-X, 2% ascorbic acid 2-phosphate, 2% bovine serum albumin and 1% penicillin/streptomycin. Pellets were exposed to 0.1 μM TAA during different time-points of the 14-day culture. Conditions were continuous exposure (d1 t/m d14), first and last three days of culture (d1-3 and d11-13) and twice 1 day exposure (d1 and d8). Negative controls consisted of pellets cultured without TAA and positive controls consisted of pellets cultured in the presence of TGF-β1. The dimethylmethylene blue (DMMB) assay was used to determine GAG content, release and production. Quantitative DNA content of all pellets was evaluated using the PicoGreen assay. Production of COX-2 and collagen II content were evaluated by immunohistochemistry. Statistical analysis of differences between conditions was done by ANOVA with Tukey posthoc testing. Results: Continuous exposure to TAA slightly reduced GAG content of tissue generated by canine chondrocytes compared to control pellets, whereas short exposure greatly reduced this. Release was not clearly affected and adding up GAG release to GAG tissue content showed that GAG production was strongly inhibited (Figure 1). No clear differences in collagen II distribution were noted in the various conditions. COX2 was present in control pellets and pellets continuously exposed to TAA, but completely absent in pellets exposed to TAA for short periods (Figure 2). Conclusions: Timing of exposure to TAA greatly affects the anabolic response of chondrocytes, and intra-articular administration of TAA through a controlled-release platform appears to be safer for cartilage matrix integrity than short exposure, as occurs with single bolus injections. However, COX2 expression in pellets exposed continuously to TAA may indicate that also the anti-inflammatory action is limited. One explanation for the limited effects of continuous exposure may lie in corticosteroid receptor downregulation. Follow up studies will be extended towards human chondrocytes and address corticosteroid regulation of regeneration through inflammatory pathways.Figure 2. COX-2 staining in pellets of chondrocytes cultured for 14 days in the continuous presence of TAA or twice a 1 day exposure to 0.1 uM TAA. No COX-2 staining was observed upon two days of exposure, wheras ample COX-2 was found in tissue regenerated in the continuous presence of TAA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)