Effects Of Osteoclast Conditional Knockout PDK1 On Bone Remodeling And Bone Injury Healing In Mice

Research Background&ObjectiveOsteoporosis（OP）is a group of bone diseases caused by multiple causes.It was characterized by decreased bone mineral density per unit volume and degeneration of bone microstructure,leading to increased bone fragility,decreased bone strength and systemic metabolic bone disease prone to fracture.Osteolysis and bone defect are the serious complications of common clinical orthopedic diseases and osteoporosis diseases,and also one of the orthopedic treatment problems.At present,the most important treatment for osteolysis and bone defect is surgery,but bone nonunion caused by various reasons is still an important factor affecting the healing and repair of bone injuries.Therefore,aiming at the above scientific problems,this study constructed and analyzed the differentiation and functional changes of osteoclasts in the process of bone reconstruction in 3-phosphoinositid-dependent kinase 1（PDK1）gene conditional knockout mice,as well as the process of bone regeneration,healing and repair after bone injury.It aims to clarify the mechanism of PDK1 regulating the function of osteoclasts on bone remodeling and healing of bone injury.Research Methods⑴Construction of Ovariectomy（OVX）osteoporosis model,Polymethyl Methacrylate（PMMA）skull osteolysis model and skull defect model respectively.⑵Hologic Discovery Dual-Energy X-ray bone densitometer was performed on PDK1 ^f/f;Ctsk-Cre(PDK1^-/-,KO)and C57BL/6 mice at the age of12 weeks and 3 months after OVX surgery to observe and analyze the bone mineral density of the mice.⑶10 days after osteolysis of the skull and 4 weeks after the drilling of the skull bone defect,specimens were taken for Micro-CT scanning respectively to observe and analyze the changes of bone resorption and dissolution and the changes of bone defect healing and repair.Hematoxylin-eosin（H&E）staining and Masson trichromatic staining were used to observe the repair and healing of resorption and dissolution sites and bone defect sites at the histological level.Tartrate Resistant Acid Phosphatase（TRAP）staining and PDK1 immunohistochemical detection of skull bone tissue sections were used to detect the number and shape change of osteoclasts and PDK1brown positive osteoclasts in the bone resorption lacuna and adjacent bone tissue area during in bone resorptive dissolution sites and bone injury repair.Results⑴Hologic Discovery Dual-Energy X-ray absorptiometry showed that compared with C57BL/6 mice,the whole-body BMD and bone mass of KO group were significantly increased.⑵Micro-CT scan and reconstruction results of mouse skull showed that 10 days after the mouse skull osteolysis was implanted with PMMA particles,the area of skull destruction and dissolution in the PMMA+C57BL/6 group was significantly increased compared with the Sham group,and the area of skull destruction and dissolution in the PMMA+KO group was significantly decreased compared with the PMMA+C57BL/6 group.At 4 weeks after the operation of bone defect,the defect of mice in the C57BL/6group and the KO group was obviously healed.There were a lot of new bone trabeculae and new bone connected to the surrounding normal bone cortex in the skull defect area.Compared with the C57BL/6 group,the skull defect healing was not significant in the KO group（P>0.05）.⑶Hematoxylin-eosin（H&E）staining showed that 10 days after PMMA particles were implanted,the skull bone resorption was significantly increased in PMMA+C57BL/6 group,and a large number of inflammatory granulation tissue invasion and scar tissue formation were observed on the skull surface,as well as a large number of inflammatory cells（such as macrophages,multinucleated giant cells,fibroblasts,etc.）and osteoclasts were also observed.Compared with Sham group,the area of skull bone destruction and dissolution in PMMA+C57BL/6 group was significantly increased（P<0.01）.The dissolving area of the skull in PMMA+KO group was not significant compared with the Sham group（P>0.05）,but was significantly reduced compared with PMMA+C57BL/6 group（P<0.01）.At 4weeks after the operation of bone defect,the bone formation at the posterior edge of the bilateral parietal bone defect was observed by histological section.It was shown that there were no obvious lymphocytes,macrophages or plasma cells around the bone defect,and no obvious chronic inflammation.There was obvious new bone tissue between the edge of the defect and the dura mater of the skull.However,there was no obvious bone tissue formation in the center of the defect,which was filled with a large amount of connective tissue.The new bone tissue is braided bone,and the bone cells are not fully differentiated and mature,and the bone cells are large and numerous,and the collagen fibers are thick and arranged in a braided pattern.However,the original skull tissue is lamellar bone,and the bone cells are relatively mature,and the number and volume of the bone cells are small,and the collagen fibers are arranged in a lamellar pattern.Therefore,H&E staining can better distinguish the new bone tissue from the original skull tissue.Compared with the C57BL/6 group,the number of new bone in the skull defect area was not significantly increased in KO group（P<0.01）.⑷Masson staining showed that 10 days after PMMA particles were implanted,compared with the Sham group,PMMA+C57BL/6group showed loose and significantly increased collagenous fibrous tissues in the sagittal suture of PMMA particles,all of which were blue-green.Compared with the PMMA+C57BL/6 group,the collagen fiber tissue in the PMMA+KO group was significantly reduced,and there was no significant difference between the PMMA+KO group and the Sham group.At 4 weeks after the operation of bone defect,compared with the C57BL/6 group,the collagenous fibrous tissues in the skull defect area in KO group were loosely arranged and the color was obviously light and bluish-green.However,the collagen fibers of C57BL/6group were dense red-blue and the difference was significant.⑸TRAP staining revealed that 10 days after PMMA particles were implanted,the number of osteoclasts in each group was significantly increased,with bright red cytoplasmic positive osteoclasts visible in many resorbed lacunae and adjacent bone tissue areas under light microscopy.Compared with Sham group,the number of osteoclasts in PMMA+C57BL/6 group was significantly increased（P<0.01）.The number of osteoclasts in PMMA+KO group was significantly decreased compared with PMMA+C57BL/6 group（P<0.01）,and there was no significant difference between PMMA+KO group and Sham group（P>0.05）.At4 weeks after the operation of bone defect,TRAP staining showed that typical osteoclasts were formed in both groups.Osteoclasts are mainly concentrated on the surface of new bone trabeculae,and the number of cells increased significantly.Under light microscope,many bone resorption lacunas and adjacent bone tissue areas were observed.Compared with C57BL/6 group,the number of osteoclasts in KO group was significantly increased（P<0.01）.⑹Immunohistochemical staining results showed that 10 days after PMMA particles were implanted,there were many PDK1-positive brown cells were visible in bone resorptive lacunae and adjacent bone tissue areas in each group under light microscope.Compared with Sham group,the number of PDK1brown positive cells in PMMA+C57BL/6 group was significantly increased（P<0.01）.The number of PDK1 brown positive cells in PMMA+KO group was significantly decreased compared with PMMA+C57BL/6 group（P<0.01）,and there was no significant difference between PMMA+KO group and Sham group（P>0.05）.At 4 weeks after the operation of bone defect,there were many PDK1positive brown cells in resorbed bone lacunae and adjacent bone tissue areas under light microscopy in both groups.Compared with C57BL/6 group,the number of PDK1 positive brown cells in KO group was significantly decreased（P<0.01）.Conclusion⑴PDK1 gene knockout in osteoclasts can increase the bone mineral density per unit volume and significantly increase the whole-body bone density and bone mass,which can prevent osteoporosis;⑵PDK1 gene knockout in osteoclasts can inhibit PMMA granule-induced osteolysis of mouse skull,and reduce bone resorption function;⑶In the early and middle stages of bone injury healing and repair,PDK1 gene knockout in osteoclasts may delay the formation of bone callus in the defect edge cartilage by inhibiting the excessive generation of osteoclasts in bone callus.