In vitro release of corticosteroids in a new model of slow release platform in the treatment of osteoarthritis.

Purpose: Corticosteroids (CS) are used for several treatments including intra-articular treatment of osteoarthritis. However, these can have severe side-effects, especially if patients have to be treated several times in the same joint. Applying CS in a controlled release manner (CS applied in microspheres (MS)), can possibly reduce the number of injections with CS needed and also prevent side-effects. The aim of this in vitro study is to show the efficacy of MS loaded with CS on human TNF-alphastimulated osteoarthritic (OA) chondrocytes in an in vitro model. Methods: TNF-alpha stimulated OA chondrocytes from three human donors were incubated in the presence of MS loaded with 5 wt% of triamcinolone acetonide (TAA). MS were added to transwells at amounts estaimted to release in total 1x10-7 M TAA/3 days. Every three days the cells and medium were collected and the transwells were transferred to wells with freshly plated and stimulated OA chondrocytes. The total duration of the tests was 28 days. As controls we used non-stimulated chondrocytes and TNF-alpha-stimulated OA chondrocytes cultured in the presence of a bolus of 1x10-7 mol TAA and empty (without TAA) MS. The bioactivity of the TA released from the loaded MS was tested using PGE2 production as an outcome parameter of inflammation (using PGE2 ELISA). Results: Because of the large fluctuation of the levels of PGE2 at the different time points in the TNF-alpha-stimulated cells, all levels of PGE2 determined in the treatment and control groups were divided by the level of PGE2 in the TNF-alpha-stimulated cells (table 1). The PGE2- level in the cells treated with a bolus of 1x10-7 mol TAA reached a maximum of 4.6%. The highest percentage of PGE2 in the MS loaded with 1x10-7 mol TAA/3 days group was 5.2% at time-point 8 (28 days follow-up) (table 2). Although the levels of PGE2 in the empty MS and non-TNF-alpha-stimulated cells compared to the negative control and the levels of PGE2 in the empty MS and TNF-alpha-stimulated cells compared to TNF-alpha-stimulated cells, seem to be higher at the first time point(s), this difference is never significant. The levels of PGE2 in the empty MS and non-TNF-alpha-stimulated cells compared to the negative control and the levels of PGE2 in the empty MS and TNF-alphastimulated cells compared to TNF-alpha-stimulated cells showed no significant differences indicating that the MS are not causing any additional inflammation (table 3 and 4). Conclusions: This study showed that MS loaded with TAA significantly reduce inflammation in TNF-alpha induced OA chondrocytes over a period of at least 28 days. The slow-release platform using MS loaded with TAA in this study showed in vitro promising results for long term inhibition of inflammation. In vivo studies (still in testing phase) showed presence of intra-articular MS (using optical imaging) at least 8 weeks after intra-articular injection in a knee of a rat. (Table Presented).