The Experimental Research On The Role Of Fibroblast Growth Factor Receptor-3 (FGFR3) During Tibia Fracture Healing In Adult Mice

Objective The present study was conducted to observe the histomorphological changes of callus during fracture healing in adult mouse tibias. We also detected the spatial and temporal expression patterns of FGFR3 gene and the effects of apoptosis in callus during fracture healing. We then analyzed the potential roles of FGFR3 during fracture healing.Material and methods 50 wild type male mice, 6~8 weeks old, weighted from 22 to 28 grams, were used in the study. After being fractured, they were randomly divided into five groups as follows (ten mice each group): d3 (3 days after fracture), d5, d7, d14 and d28. All the mice were given an intraperitoneal injection (0.015~0.017ml/g body weight) of 2.5% Avertin, to provide approximately 15~20 min of deep anesthesia. The left tibias of the mice were fractured transversally after the intramedullary insertion of steel pin. Radiographs were obtained on d7, d14, d21 and d28 under anesthesia. Mice were sacrificed on day 3, 5, 7, 14 and 28 by cervical dislocation and the inserted pins were removed. The fractured tibias and contralateral controls were harvested and fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 0℃ overnight immediately. Then tissues were decalcified in 15% buffered EDTA (PH7.6) and dehydrated in a graded ethanol series and embedded in paraffin as described. 6μm sections were prepared and placed on clean or 10% polylysine-treated slides. In situ hybridization was used to detect the spatial and temporal expression patterns of FGFR3 gene while TdT-mediated dUTP nick end-labeling (TUNEL) was used to detect the effects of apoptosis in callus at the different stages of healing. TRAP stain was used to illustrate the function of osteoclasts in callus. Histological changes were obtained by HE staining.Results Radiographs showed that the fracture site was apparent on d7 while the calcified callus was clear on d14. The callus linked the fracture gap was enlarged and condensed on d21. The gap disappeared till d28, and the cortical bone remodeling was apparent at this moment. Histological changes were showed by HE staining: A number of fusiform cells were infiltrated at the fracture site and the organization of hematoma was apparent on d3. Cells of germinal layer in endosteum and periosteum responded to injury and began to proliferate while some condensed mesenchymal cells near the fracture site began to differentiate directly into osteoblasts and others in the granulation tissues differentiated into chondrocytes on d5. Callus was composed of premature and mature chondrocytes. Some chondrocytes near the cortical bone differentiated into hypertrophic chondrocytes and newly formed trabecula bone surrounded by osteoblasts could be observed on d7. Chondrocytes in callus were all replaced by osteoblasts on d14 and much more wave bone and mature bone tissue was formed. Bony bridge was seen till d28. The function of osteoclasts was showed by TRAP staining: There were no TRAP-positive cells seen in granulation tissue on d3 and d5. A small number of TRAP-positive cells could be seen from d7 after fracture and reached a peak on d14, suggesting that osteoclasts were active. There remained many TRAP-positive osteoclasts on d28. TRAP-positive osteoclasts were occasionally detected in normal tibias.The spetial and temporal expression of FGFR3 mRNA was showed by in situ hybridization. The ratio of FGFR3-positive cells was negative in normal tibia and callus on d28. It was very low on d3 (1.1±0.4%) while it raised significantly on d5 (38.5±3.2%) and reached a peak on d7 (93.6±5.8%) and then decreased obviously on d14 (5.2±1.7%). Under the microscope, we found that FGFR3 mRNA was hardly detected in granulation tissue on d3 after fracture. FGFR3 positive-signal was clear in the cytoplasm of prehypertrophic chondrocytes in soft callus on d 5. FGFR3 mRNA was strongly expressed in prehypertrophic chondrocytes and hypertrophic chondrocytes in soft callus on d7 while it disappeared on d14 and d28 because chondrocytes were replaced by osteoblasts. FGF...