Study On The Role Of Fibroblast Growth Factor Receptor-2 (FGFR2) During Tibia Fracture Healing In Adult Mice

Objective FGFR2 is one of the multiple important signaling molecules involved in skeleton development.More than 30 gain-of-function mutations in FGFR2 are responsible for several clinically distinct craniosynostosis syndromes in humans.Studies have suggested the participation of FGFR2 in fracture healing, but its underlying molecular mechanism is still largely unknown. Using mouse tibia fracture model,this study not only studied the spatial and temporal expression pattens of FGFR2 during fracture healing, but also explored the effect of the decreased FGFR2 expression on fracture healing process and expression of some molecules involved in skeleton development and fracture healing.Material and Methods 8-9 weeks old littermates generated from the intercross between heterozygous FGFR2^{neo250/+ mice} (genotype is FGFR2-Neo-250 MT/WT; designated as MT) and Wild type mice (C57BL; designated as WT) were used. The expression of FGFR2 in FGFR2^neo250/+ mice is down-regulated by the insertion of neomysin(neo) into intron 8 of FGFR2, which interferes with the transcription of FGFR2. Mouse genotype was identified by PCR using primers specific for the sequences of neo in FGFR^neo250/+ mice. The instablized tibia fracture model was established using mice of the above mentioned two groups. X-ray pictures of the fractured right tibiae of WT and FGFR2^neo250/+ mice were taken to observe the general healing process of the fracture; hematoxylin-eosin (HE) staining was used to detect the histological changes of the fracture callus. In situ hybridization was used to detect the spatial and temporal expression patterns of FGFR2, Colâ…¡ , Col â…©, Ihh, OC in vivo;Brdu incorporation assay was used to examine the proliferation activities of different cells in fracture callus;TdT-mediated dUTP nick end-labeling(TUNEL) was used to detect the apoptotic cells in the fracture callus at different stages of healing.Results Radiographs showed that on day 21 after fracture the calcified callus atfracture site was clear and the gap at fracture site disappeared completely in both groups, indicating that there was no obvious difference in the general healing process of fracture.Histological changes were observed by HE staining: Cartilagious callus between WT and MT in both groups began to appear at day 5 post fracture and apparently increased between day 7-10 post fracture. At postfracture day 14 the hypertrophic chondrocytes in callus began to be replaced by woven bone, there was no difference between two groups. At day 21, chondrocytes in callus were completely replaced by woven bone in WT mice and there are much more woven bone formed in WT mice, but there was still some remnant chondrocytes within the callus of MT mice.From the above results, it's suggested that the fracture healing of MT mice was histologically retarded than that of WT mice.The proliferation activities of cells were checked by Brdu incoporation assay, the spatial and temporal expressions of FGFR2 > Col IK Col X, Ihlu OC were examined by in situ hybridization.At day 3 after fracture the FGFR2 were expressed by cells underlying periosteum at fracture site, these cells simultaneously expressed OC,a marker for mature osteoblasts. The ratio of FGFR2-positve cells and expression level of FGFR2 were higher in WT mice than that in MT mice, and proliferation activity of these cells were stronger in WT mice than that in MT mice, indicating FGFR2 may promote proliferation of osteoblasts during fracture healing.At day 5 after fracture the FGFR2 were not found in proliferative chondrocytes at fracture site, while these cells expressed Col II ,the marker of chondrocyte.FGFR2 were, however, expressed by cells underlying periosteum at fracture site, these cells simultaneously expressed OC. The ratio of FGFR2-positve cells and expression level of FGFR2 were higher in WT mice than that in MT mice, and proliferation of these cells were more obvious in WT mice than that in MT mice.At day 7 after fracture the FGFR2 ^ Ihh and Col X were expressed by prehypertrophic chondrocytes at fracture site, the location and expression level of FGFR2 were comparable to that of Ihh. There are more proliferative chondrocytes in WT mice than in MT mice, indicating FGFR2 may promote proliferation of chondocytes, and inhibit differentiation of prehypertrophic chondrocytes by regulating expession of Ihh.At day 10 post fracture, the FGFR2 and Col X were still expressed by hypertrophic chondrocytes at fracture site,the ratio of FGFR2-positve cells and expression level ofFGFR2 were higher in WT mice than that in MT mice.At day 14 after fracture woven bone began to replace the apoptotic hypertrophic chondrocytes at fracture site. The FGFR2 and Col X were expressed by hypertrophic chondrocytes at fracture site,the ratio of FGFR2-positve cells and expression level of FGFR2 were higher in WT mice than in MT. There are more proliferative osteoblasts and apoptotic hypertrophic chondrocytes in WT mice than in MT mice, indicating the FGFR2 may promote apoptosis of hypertrophic chondrocytes.At day 21 after fracture,there were no chondrocytes and expression of FGFR2 and COL X at fracture site of WT mice.But there were still some remnant hypertrophic chondrocytes in MT mice,these cells expressed FGFR2 and COL X, some of which underwent apoptosis, further indicating FGFR2 is involved in the apoptosis of hypertrophic chondrocytes.Conclusion1. Down-regulation of FGFR2 does not affect the general healing process of fractured tibiae in mice.2. Down-regulation of FGFR2 may cause histologically retarded fracture healing.3. FGFR2 mRNA is expressed mainly by osteoblasts underlying periosteum and by prehypertrophic chondrocytes and hypertrophic chondrocytes in fracture callus.4. FGFR2 may promote proliferation of osteoblasts at fracture callus.5. FGFR2 may promote proliferation of chondocytes and inhibit differentiation of prehypertrophic chondrocytes by regulating expession of Ihh; FGFR2 also may promote apopotosis of hypertrophic chondrocytes.