The Role Of FGFR3 In Mouse Bone Remodeling And Fracture Healing

There are two major models of bone formation, intramembranous ossification and endochondral ossification. Both models of ossification involve condensation of mesenchyme, differentiation of mesenchymal cells into osteoblast, secretion and mineralization of extracellular matrix. Bone remodeling is important for the maintenance of normal bone structure and function in adults.Fibroblast growth factor receptors 3 (FGFR3) is one of a family of 4 membrane bound receptor tyrosine kinases (FGFR1-4) that mediate signals of at least 23 fibroblast growth factors (FGF1-23), which plays an important role in skeleton development and diseases. FGFR3 is a negative regulator of bone development. The gain-of-function mutation of FGFR3 can lead to three chondrodysplasia syndromes including achondroplasia (ACH), hypochondroplasia (HCH) and thanatophoric dysplasia (TD). There have been many studies on the role of FGFR3 in chondrogenesis, however, the role of FGFR3 in bone formation is still elusive.Some gain-of-function mutations of FGFR3 have been found in human to result in early fontanel closure, which suggests that FGFR3 may regulate osteogenesis directly. There were osteopenia and defective bone mineralization in adult FGFR3 knockout mice, which further suggests that FGFR3 may regulate osteogenesis and bone remodeling, but the mechanism is unclear. Moreover, the expressions of osteogenic markers such as Cbfa1, OP and OC were enhanced in the growth plates of achondroplasia mice (ACH) on postnatal day 15. How about the bone remodeling of adult ACH mice?Fracture healing is a complicated regeneration process which to some extent recapitulates bone development. Multiple molecules involved in skeleton development also play important roles during fracture healing, and are potential good candidates to be modified to improve fracture repair. FGF signaling is well known for its role in bone regeneration, and FGFR3 plays an important role in skeleton development, what is its role in bone fracture healing?Thus, we used mouse model mimicking human achondroplasia resulting from gain-of-function mutation of FGFR3 (FGFR3G369C/+ mice) to explore the role of FGFR3 in bone remodeling and fracture healing.MethodsPart I The role of FGFR3 in bone remodeling1. Dual beam X-Ray absorptiometry, Micro CT and three-point bending test were used to analyze the bone mass and biomechanical properties of femurs of adult mice.2. Histomorphometric measurement and real-time quantitative RT-PCR (qRT-PCR) were used to evaluate osteoblastogenesis and osteoclastogenesis in vivo.3. Cell culture in vitro was used to examine the differentiation of osteoblasts and the formation of osteoclasts. The level of Erk1/2 phosphorylation was detected by Western Blot.Part II The role of FGFR3 in bone fracture healing1. The expression of FGFR3 in callus was detected by in situ hybridization.2. X-ray analysis, histological methods and three-point bending test were used to evaluate bone fracture healing and the biomechanical properties of fractured tibiae.3. The differentiation of chondrocytes and osteoclast formation in callus were assessed by histological methods and qRT-PCR.ResultsPart I Osteopenia and defective mineralization in adult FGFR3G369C/+ mice1. The adult FGFR3G369C/+ mice showed decreased bone mineral density and trabecular number. The biomechanical properties of cortical bone were changed.2. The proliferation of bone marrow stromal cells was decreased. The differentiation of osteoblasts in FGFR3G369C/+ mice was accelerated, however the mineralization was decreased. The expressions of bone markers such as Cbfa1, OP and OC were increased, while the activity of Erk1/2 pathway was enhanced.3. The formation and absorptive function of osteoclasts in FGFR3G369C/+ mice were enhanced, which was accompanied with high mRNA level of TRAP and MMP9. Enhanced Erk1/2 phosphorylation was observed in osteoclasts of FGFR3G369C/+ mice.Part II FGFR3G369C/+ mice showed delayed fracture healing1. FGFR3 is expressed in proliferating, prehypertrophic and hypertrophic chondrocytes at fracture site on day 7, 10 and 14.2. The fractured tibiae of FGFR3G369C/+ mice showed delayed union that was evidenced by the residual cartilage in the callus tissues of mutants on day 21. These tibiae also showed decreased maximum load to failure at the same time.3. The differentiation of chondrocytes in callus of FGFR3G369C/+ mice was impaired. The expressions of cartilage marker and related genes such as COL2A1, COL10A1 and SOX9 in callus were delayed or down-regulated on day 3 and 7.4. Osteoclast formation in callus of FGFR3G369C/+ mice was decreased on day10 and 14, but increased on day 21.Conclusions:1. Osteopenia and defective mineralization were found in adult FGFR3G369C/+ mice, which may be related to the impaired osteogenesis and increased activation of osteclasts.2. Gain-of-function mutation of FGFR3 inhibits proliferation of bone marrow stromal cells, and promotes the differentiation of osteoblasts but depresses their mineralization by up-regulating Cbfa1 partially through Erk1/2 pathway.3. Gain-of-function mutation of FGFR3 increases the formation and absorptive function of osteoclasts, which may be related to Erk1/2 pathway.4. Gain-of-function mutation of FGFR3 results in delayed fracture healing by inhibiting chondrocyte differentiation and resorption.