Abstract
Introduction
Human Mesenchymal stem cells (hMSCs) are a promising source for articular cartilage repair. Unfortunately, under in vitro conditions, chondrogenically differentiated hMSCs have the tendency to undergo hypertrophy similar to growth plate chondrocytes. Retinoic acid (RA) signalling plays a key role in growth plate hypertrophy. Whilst RA agonists block chondrogenesis and foster hypertrophy during later stages, RAR inverse agonists (IA) enhance chondrogenesis when applied early in culture. Therefore, we hypothesized that treatment with RAR IA will attenuate hypertrophy in chondrogenically differentiated hMSCs. To test this hypothesis, we analysed early (initial chondrogenic differentiation) and late treatment (hypertrophy stage) of hMSCs with an RAR IA.
Methods
Pellets of passage 2 hMSCs were formed in V-bottom well plates by centrifugation and pre-differentiated in a chemically defined medium containing 10ng/mL TGFß (CM+) for 14 days. Thereafter, pellets were cultured for an additional 14 days under 6 conditions: CM+, CM- (w/out TGFß), and hypertrophic medium (CM- with 25 ng/ml BMP 4, w/out dexamethasone). Each of these first three conditions was additionally supplemented with the RA receptor (RAR) inverse agonist BMS493 (BMS) at 2μM after 14 days of chondrogenic pre-differentiation. One additional BMP4 group was supplemented with BMS from the beginning of chondrogenic differentiation until day 14. The pellets were assessed for gene expression (Col 2, Col 10, Col 1 and MMP13) and histologically using dimethyl methylene blue (DMMB), alkaline phosphatase staining (ALP) and collagen II and X immunohistochemistry.
Results
Hypertrophy was reduced by addition of BMS at day 14 and further reduced by addition from the beginning. BMS treatment resulted in smaller cells under hypertrophic conditions, higher collagen II content in chondrogenic groups and reduction in collagen X production and ALP activity in every condition. Gene expression data for hypertrophic markers, collagen X and MMP13, were upregulated under the influence of BMP4 but a distinct downregulation in MMP13 expression was shown upon addition of BMS during the late stage differentiation and further reduced upon addition during early stage chondrogenesis. Furthermore, Collagen X expression was reduced by early BMS treatment.
Discussion
The treatment with the RAR IA, BMS, attenuated hypertrophic changes in chondrogenically differentiated hMSCs as demonstrated by histology, immunohistochemistry and PCR. These findings suggest an additional approach to attenuate hypertrophy in chondrogenically differentiated hMSCs. Current studies are exploring the timing and dose of BMS to most efficaciously prevent hypertrophy.
