| Chapter One Establishment and Evaluation of Refined Bone Fracture Model in Mice and Analyses on Fracture Healing CharacteristicsObjective:To study the establishment of a refined bone fracture model in mice and evaluate the effect of the model. To analyze and compare the fracture healing characteristics of two chosen refined fracture models in mice.Methods:A unilateral open tibial transverse fracture with intramedullary needle fixation model and a unilateral closed femoral transverse fracture with intramedullary needle fixation model were established on C57BL/6wild-type mice. The characteristics of bone fracture healing of these mice were serially analyzed and compared by X-ray, micro-CT and histomorphometry. The observations of average body weights, mobility and gait changes were also included. Time points were set by day4,7,10,14,21,28and35postoperatively.Results:1. The one batch successful rate of model establishment was94.2%on tibiae and was80.0%on femurs. There were significant differences of fracture line clearance in two groups by day14and28postoperatively (p<0.05).2. Callus mineralized volume fraction and connectivity density of calluses on tibiae were significantly higher in comparison to calluses on femurs by day10,14,21and28postoperatively (p<0.05).3. A larger cartilage area was observed by day7and a larger bone area was observed by10,14,21and28postoperatively in calluses on tibiae when compared to calluses on femurs (p<0.05).4. The cartilaginous callus fraction reached peak level in tibial tissues by day7and in femoral tissues by day10postoperatively.5. The recover period from fracture in terms of average body weights, mobility and gait changes was shorter in mice operated by tibial fracture.Conclusion:The refined unilateral open tibial transverse fracture with intramedullary needle fixation model is ideal for bone fracture healing studies. Chapter Two Study of the Physiological Characteristics of Bone in Cell-specifc Bmp2Conditional Knockout MiceObjective:To determine the stability and feasibility of using osteoblast-or chondrocyte-specific Bmp2conditional knockout (cKO) mice as a platform in researches related to bone fracture healing.Methods:To generate osteoblast-or chondrocyte-specific Bmp2cKO mice, Bmp2fx/fx mice were bred with either Collal-Cre or Col2al-Cre transgenic mice. The characteristics of bone tissue harvested from these mice and Cre-negative control mice were serially analyzed and compared by body weight measurement, X-ray, micro-CT, histomorphometry and quantitative gene expression analysis with time points of2weeks,1month and2months after birth.Results:1. The average body weight of chondrocyte-specific Bmp2cKO mice was significantly lower in comparison to the rest two mice groups by2weeks after birth.2. There was a short and curve deformity of tail in osteoblast-specific Bmp2cKO mice in all time points observed and the body shape of chondrocyte-specific Bmp2cKO mice was smaller than Cre-negative control mice by2weeks after birth.3. Bone volume and bone mineral density of chondrocyte-specific Bmp2cKO mice were significantly decreased in comparison to Cre-negative control mice by2weeks and1month after birth (p<0.05).4. There was no obvious difference in all three groups in histomorphometry analysis.5. Expression of Sox9was significantly higher than Cre-negative control mice by2weeks after birth while the levels of Col2al and colX were significantly lower (p<0.05).Conclusion:The physiological characteristics of bone in mice from all three groups were very similar by2months after birth and it was of good stability and feasibility to perform fracture healing study on such mice. Chapter Three Determination of the Biological Role of Endogenous BMP2in Bone Fracture Healing of MiceObjective:To determine the specific role of endogenous Bmp2in cartilage and bone formation phases of fracture healing and the specific cell population where endogenous Bmp2is expressed and required.Methods:A unilateral open tibial transverse fracture with intramedullary needle fixation model was established on osteoblast-or chondrocyte-specific Bmp2cKO mice and Cre-negative control mice. The characteristics of bone fracture healing of these mice were serially analyzed and compared by X-ray, micro-CT, histomorphometry, biomechanical torsion testing and quantitative gene expression analysis with time points of day7,10,14,21and28postoperatively.Results:1. Callus mineralized volume fraction and connectivity density of chondrocyte-specific Bmp2cKO mice at the fracture sites were significantly decreased in comparison to Cre-negative control mice by day7,10,14and21postoperatively (p<0.05).2. A less trabecular bone area but larger cartilage area in samples of chondrocyte-specific Bmp2cKO mice was observed by day10,14and21postoperatively (p<0.05).3. Maximum torque, stiffness and toughness were significantly decreased in samples of day10,14and21postoperatively of chondrocyte-specific Bmp2cKO mice compared with Cre-negative controls (p<0.05).4. Expression of Sox9boosted up to a significantly high level by day10and14, and descended to similar level with control ones by day21and28. The levels of Col2al and colX remained lower by day7and10and were higher than Cre-negative control mice by day14postoperatively (p<0.05).Conclusion:Endogenous Bmp2in chondrocytes plays a pivotal role in cartilaginous callus maturation during the early stage of bone fracture healing.
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