Systematic Administration Of Eldecalcitol Accelerated Bone Defects Healing In Rats

Background:Bone defect,generally caused by trauma,infection and tumor,is one of the problems that need to be solved in clinic.Meanwhile,the bone defect healing is a complex process that involved in temporal and spatial regulation implemented by more than one kind of cell and signal.The treatments of bone defect consist of bone grafting,bone graft substitute materials,and Ilizarov and so on.Nevertheless,what we should mention was that surgery complications and the complicacy of operation were not easy to handle.In terms of bone graft substitute materials,that inadequate capacity of osteogenesis and absorption were the problems that we could not neglect.Therefore,a defect repairing technology that less prone to infection and companied with complications might be a promising choice for bone tissue engineering.Vitamin D plays a very important role in regulating the homeostasis of serum calcium in vivo.Eldecalcitol,a novel active vitamin D analog,has lower affinity(one eighth)to the VDR,whereas higher binding affinity to the vitamin D binding protein(DBP),compared with 1?,25(OH)2D3,which equipped eldecalcitol in lasting function and high efficiency.1?,25(OH)2D3 could significantly inhibit the postmenopausal osteoporosis.With further research,1?,25(OH)2D3 can significantly lower the risk of fracture in patients with osteoporosis.In recent years,a large number of active vitamin D analogues have been optimized on the basis of the original drugs.Eldecalcitol was approved as a therapeutic drug for the treatment of osteoporosis for its strong effects on the reduction of bone resorption and improvement of bone mineral density(BMD).In addition,it can form bone minimodeling whereas independent of the bone resorption.Osteoporosis is characterized by a reduction in bone mass,which is resulted from bone resorption and bone microstructure.It has been shown that eldecalcitol has the characteristics of inhibiting bone resorption and increasing bone mass,and thus it has been favored in the treatment of osteoporosis than bone defect.Bone defect healing is a complex biological process involving multiple cellular interactions and precise regulation of multiple signals in time and space.Osteoblasts,osteoclasts and osteocytes are the three major functional cells in bone defect healing,and play very important roles in bone regeneration.Therefore,we systemic administrated eldecalcitol to repair bone defect,so as to explore its influence on the three major functional cells as well as the healing of bone defect,with the hope that the results would be a theoretic cornerstone of bone defect repair.Purpose:The aim of this experiment was to elucidate the effects of systemic administration of eldecalcitol on factors secreted from osteoblast and osteoclast at the early stage of the restoration of bone defect healing,and on mineralization factors at the late stage of the restoration of bone defect healing in rats.Materials and Methods:8-week-old Wistar rats(48)randomly divided into two groups:control group(CON group)and eldecalcitol group(ELD group).The femurs were used to generate bone defect models(3mm×5mm×1mm,widthxlengthxdepth),and the defect was covered with a collagen membrane.ELD group was administrated with eldecalcitol(50ng/kg body weight)intragastrically once every other day.Femora were harvested at 1,2,4 and 8 weeks post-surgery.Decalcified tissue slices were prepared for immunohistochemical examination to detect the expression of ALP,CK,OCN,DMP1and FGF23.Bone biomarkers of RANKL,OPG and OCN were detected by western blot and RT-PCR.Results:1.Application of ELD to increase bone mass The hematoxylin and eosin(HE)staining showed that,at the early stage of bone defect healing(1,2 weeks),the whole extent of the defect was abounded with newly formed fibrous connective tissue and woven bone both in CON and ELD groups,but,the volume of newly formed woven bone in ELD groups was extremely larger than CON group.At the late stage of bone defect healing(4,8 weeks),the amounts of the new bone were more in the ELD group than in CON group.At 8 weeks,the bope volume/tissue volume ratio(BV/TV)was no significantly difference between two groups.2.Effect of ELD on bone formation and bone resorptionThe double staining of ALP&TRAP showed that,both at early and late stage of the restoration of bone defect healing,the number of TRAP-positive osteoclast in the CON group was significantly more,while the ALP expression was lower than that in the ELD group.The immunohistochemical analysis of cathepsin-K(CK)showed that,at the early stage of bone defect healing(1,2 weeks),the CK positive osteoclasts were located on the surface of the new bone,and the number of CK positive osteoclasts in CON group was relatively more compared with ELD group.At the late stage of bone defect healing(4,8 weeks),the number of cathepsin-K positive osteoclasts in the ELD group was significantly decreased compared with CON group.The western blot analysis showed that,both at early and late stage of bone defect(1,2,4,8 weeks),the level of RANKL expression was markedly downregulated,meanwhile the level of OPG expression was upregulated by ELD.And,ratio of RANKL/OPG was significantly downregulated by ELD.The RT-PCR analysis revealed that,both at early and late stage of bone defect(1,2,4,8 weeks),the level of RANKL mRNA expression was markedly downregulated,meanwhile the level of OPG mRNA expression was upregulated by ELD.As well as the ratio of RANKL/OPG was significantly downregulated by ELD,which tended to be an inhibitory effect on bone resorption.3.Effect of ELD on the maturity of new boneMasson staining revealed that,at the early stage of bone defect healing(1,2 weeks),the volume of unmineralized bone tissue was no significantly difference in both two groups,at 1 week.At 2 weeks,the amount of mineralized bone tissue in the ELD group was much higher than in the CON group.At the late stage of bone defect healing(4,8 weeks),cortical layer was smoother with fewer cracks inside,featured as fine compact bone,in the ELD group compared with CON group.Compared with CON group,the collagen/bone volume ratio(COL/BV)in ELD group was significantly reduced.4.Effect of ELD on osteoblast mineralization factorsThe immunohistochemical analysis of OCN showed that,at the early stage of bone defect healing(1,2 weeks),the expression of OCN was no significantly difference in both two groups,at 1 week.At 2 weeks,the expression of OCN in ELD group,located in osteoblast cytoplasm,was evidently higher than that in CON group.At the late stage of bone defect healing(4,8 weeks),the expression of OCN,transferred to bone matrix,was extremely accumulated in ELD group,compared with CON group.The western blot analysis showed that,excepted 1 week,the expression of OCN protein level was markedly upregulated by ELD,compared with control group at early and late stage of the restoration of bone defect(2,4,8 weeks).The RT-PCR analysis revealed that the expression of OCNmRNA was significantly upregulated by ELD,both at early and late stage of the restoration of bone defect.5.the effect of ELD on the osteocyte mineralization factorsThe immunohistochemical analysis of DMP1 showed that,at the late stage of bone defect healing,the number of DMP1 positive osteocytes in ELD group was evidently increased,compared with CON group.The immunohistochemical analysis of FGF23 showed that,at the late stage of bone defect healing,the number of FGF23 positive osteocytes in ELD group was evidently increased,compared with CON group.Conclusion:Systemic administration of eldecalcitol accelerated bone formation via inhibiting RANK/RANKL/OPG signaling pathway in the early stage of bone defect healing and promoted bone mineralization via regulating the expression of mineralization factors in the late stage of bone defect healing in rats.