Effect Of CKIP-1 On Different Structures Of Mouse Femur

[Background] Osteoporosis is a common chronic,aging-related skeletal disease.In China,osteoporosis affects nearly 100 million people in the health and quality of life.It involves a wide range of people,high incidence,but the treatment effect is not satisfactory.At the same time,osteoporosis is closely linked to oral and maxillofacial surgery,oral implantation,oral repair and periodontal disease and other disciplines.Regardless of the treatment of osteoporosis,fracture healing,oral implant and other diseases,there are some requirements for bone mass,How to better improve bone mass for osteoporosis and other bone disease treatment has great significance.In recent years,the rise of gene therapy for the rehabilitation of osteoporosis has brought hope.At the same time,more and more related genes are also taken seriously,including CKIP-1 and other genes.CKIP-1(Casein Kinase-2 Interaction Protein-1)has been found to be a negative regulatory gene for bone formation in recent years.In 2008,Nature Cell Biology reported that CKIP-1 knockout mice were normally born with a significant increase in bone formation rate,bone mass and bone mineral density.But how CKIP-1 affects bone different structures(cancellous bone,cortical bone,cartilage)have not been reported.[Purpose] In this study,we compare the femur come from the CKIP-1 gene knockout mice and wild type mice which come from the same nest and sex mice,Through Micro-CT scanning,histological staining and immunohistochemical methods.To compare the differences between cancellous bone,cortical bone and cartilage in CKIP-1 knockout mice and wild-type mice.The aim of this study was to further understand the role of the gene in bone formation and osteoporosis.[Methods] 1,the wild type(WT)and CKIP-1 knockout(KO)mice were propagated.At the 12 th week,8 pairs of male KO and WT were used to perform micro-CT scan and H & E staining to compare the bone mass of cancellous bone and cortical bone.2,the difference of total collagen content between cancellous bone and cortical bone of WT and KO mice was compared by tricuspid staining.The difference of the proportion of type I collagen in total collagen content in WT and KO mice was compared by Sirius red staining.3,the differences in the femoral cartilage of WT and KO mice were compared by tricuspid staining.The differences in the bone mass of femoral cartilage between WT and KO mice were compared by safranine O staining.4,the difference of newborn bone and the number of osteoblasts between cancellous bone and cortical bone of WT and KO mice were compared by toluidine blue staining.The difference of mineralization deposition rate(MAR)between cancellous bone and cortical bone of WT and KO mice was compared by tetracycline-calcein double fluorescent labeling.5,the difference of the osteoclasts number in the femur growth plate(GP),cancellous bone(TB)and cortical bone(CB)were compared by TRAP staining in WT and KO mice.6,The differences of CKIP-1,WNT signaling pathway activator Axin2 and osteogenic differentiation marker protein COL I were compared by immunohistochemical staining in WT and KO mice.[Results] 1,Analysis of Micro-CT results of cancellous bone,In group KO,bone volume/total volume(BV/TV),trabecular number(Tb.N)and trabecular thickness(Tb.Th)than that in WT group increased by 3.9%,0.7(1/mm),5.3×10-3(mm);bone surface/ bone volume(BS/BV),trabecular separation(Tb.Sp)respectively decreased by 24.4(1 /mm),0.04(mm).Cortical bone Micro-CT results analysis showed that the KO group and WT group of cortical bone area(Ct.Ar),cortical bone width of the perimeter(Ct.Id.Pm),cortical bone width of the outer perimeter(Ct.Od.Pm),cortical bone thickness(Ct.Th),cortical bone volume(Ct.BV)were no significant difference.HE staining results analysis showed that trabecular area(Tb.Ar),trabecular number(Tb.N)and trabecular width(Tb.Wi)in the KO group were 4.35%,0.7(1 / mm),5 × 10-3(mm)higher than those in the WT group;trabecular separation(Tb.Sp)decreased by 0.06(mm)in the WT group;bone thickness(Ct.Th)c were no significant difference.The results of H&E staining were consistent with the results of Micro-CT analysis.2,Cancellous bone Ponceau trichrome staining results analysis showed that the total amount of collagen in KO group(Tb.Col.Ar)was 3.7% higher than that of the WT group;cortical bone Ponceau trichrome staining results showed that the total collagen group(Ct.Col.Ar)had no significant difference.Sirius red staining results showed that KO group cancellous bone the proportion of type I collagen in total collagen content(Tb.Col I),cortical bone the proportion of type I collagen in total collagen content(Ct.Col I)compared with the WT group showed no significant difference.3,Cartilage Ponceau trichrome stain results showed that cartilage bone in KO group compared with WT group decreased.The results of safranine O staining showed that the cartilage bone mass(Cl.Ar)of the KO group was 0.04(mm2)lower than that of the WT group.4,Cancellous bone with toluidine blue staining results showed that the new bone in KO group increased;trabecular osteoblast number(Tb.Ob.N)KO group compared with WT group has nearly doubled.The cortical bone of toluidine blue staining results showed that KO group cortical bone osteoblast number(Ct.Ob.N)compared with the WT group no significant difference.Cancellous bone tetracycline calcein double fluorescence labeling results showed that KO group trabecular mineral apposition rate(Tb.MAR)than the WT group increased 1.7(m/ day).The cortical bone tetracycline calcein double fluorescence labeling results showed that KO group cortical bone mineral apposition rate(Ct.MAR)compared with the WT group no significant difference.5,the results of TRAP staining showed that there was no significant difference between the two groups in the number of osteoclasts in growth plate,cancellous bone and cortical bone.6,Immunohistochemical staining showed that the expression of CKIP-1 protein in group KO was negative,and the expression of WT was positive.The average optical density value of Axin2 protein(Axin2.Tb.MOD)in KO group was 0.013 higher than that in WT group,and the expression of cortical bone in group was negative in the two groups.The average optical density of COL I protein(COL I.Tb.MOD)in group KO was 0.01 higher than that in group WT,and the average optical density of COL I protein in cortical bone(COL I.Ct.MOD)no significant difference in two group.[Conclusion] CKIP-1 negatively regulates bone mass,may be due to the decreased expression of CKIP-1,promote osteoblast collagen secretion and accelerating the total rate of ossification of cartilage,thereby promoting cancellous bone formation rate,while the gene does not affect cortical bone and osteoclast related bone resorption,as the application of the OP gene therapy clinical indications provide a theoretical basis for.Specific conclusions are as follows:1,CKIP-1 gene can negatively regulate the changes of bone mass in cancellous bone,while the change of cortical bone mass has almost no effect.2,CKIP-1 gene can negatively regulate cancellous bone collagen amount,but the change of the proportion of type I collagen in total collagen content has little effect.The changes of cortical bone collagen content and the proportion of type I collagen in total collagen content had almost no effect.3,CKIP-1 gene can regulate cartilage bone mass.Guess: CKIP-1 knockout accelerated the rate of cartilage ossification.4,CKIP-1 gene can negatively regulate the new bone formation and bone formation rate in cancellous bone.But had little effect on the change of bone formation and bone formation rate in cortical bone.5,CKIP-1 almost no effect on osteoclastic bone resorption.6,CKIP-1 gene can negatively regulate the differentiation of osteoblasts and is related to the classical WNT signaling pathway.