Study On The Role Of Fibroblast Growth Factor Receptor-3 (FGFR3) In Osteogenesis Of Mice

Objective FGFR3, which is mainly expressed in resting , proliferating and prehypertrophic chondrocytes, plays a negative role that inhibiting the proliferation and differentiation of chondrocytes during chondrogenesis. The activating mutation of FGFR3 can cause Achondroplasia (ACH). FGFR3 may also influence osteogenesis. In this study, we plan to explore the effect and mechanism of FGFR3 on osteogenesis using cell co-culture and E16.5 tibia culture models.Material and Methods1. cell co-culture experiments:(1) To set up the co-culture of wild type osteoblasts with primary chondrocytes from either wild type mouse (WT) or achondroplasia mice (ACH) using TranswellTM.(2) Detection of the expression of cbfa-1,OP and OC of osteoblasts which were co-cultured with either ACH or WT chondrocytes or without chondrocytes.The mRNA levels of BMP4 in chondrocytes from ACH and WT mice were also examined by real-time PCR.(3) As BMP proteins can substitute for each other in experimental systems, we have used BMP2 protein to mimic the role of BMP4. After adding exogenous rh-BMP2 in the coculture group of osteoblast with the chondrocytes from ACH mouse, the expressions of ossification marker genes cbfa-1,OP,OC were detected quantitatively.2. Culture of E16.5 tibia(1)The in vitro mouse embryonic tibia culture model was established in vitro(2)After adding exogenous rh-BMP2 to the cultured tibia from E16.5 mouse, the bone formation was evaluated by gross measurement, safranine[0]O -fast green staining and quantitative detection of cbfa-1, OP, OC expressions. As control, the contralateral tibia were cultured without rh-BMP2 treatment. Results1. cell experimentThe expression of cbfa-1, OP, OC was higher than the osteoblasts were cultured alone when the osteoblasts were co-cultured with chondrocyte, regardless the ACH/WT. Compared with the group coculture with WT chondrocytes, the expressions of cbfa-1,OP,OC in the osteoblast co-cultured with chondrocytes of ACH mouse decreased, suggesting the function of ossification was restrained. Tthe expression of BMP4 in the chondrocytes of ACH mouse also decreased,compared with wildtype chondrocytes. After adding exogenous rh-BMP2, the expressions of cbfa-1, OP, OC in the osteoblasts cocultured with chondrocytes of ACH mouse increased,which suggested the osteogenesis was recovered.2. Embryonic tibia culture experimentCompared with untreated group, the total length and the length of calcification region of the cultured embryonic tibias from ACH mouse were increased after adding exogenous rhBMP2. Histological analysis also showed that the area of calcification region is enlarged. Besides, the expressions of cbfa-1 of cultured embryonic tibias derived from ACH mouse were up-regulated obviously after adding exogenous rh-BMP2, compared to the control, which suggested that the osteogenesis was enhanced.ConclusionFGFR3 could indirectly inhibit osteogenesis through downregulating BMP4 expression of chondrocytes.