Experimental Study On Synthesis Of Bone-targeted Osthole And Its Effect On Osteoporisis By Regulating OPG/RANKL System In Vitro

Objective Using water soluble chitosan derivative(NSC) as a bone-targeted carrier, bone targeted osthole(NSC-OST) were prepared;To observe the effect of bone-targeted osthole on cell proliferation,osteoporogeterin(OPG) and receptor activator of NF-κB ligand(RANKL) expression of rat osteoblast(OB) cultured in vitro;To observe the effect and explore the quantity and bone resorption function of osteoclasts in vitro.To analyze the prevention and treatment mechanism of osteoporosis.Methods①NSC-OST was prepared by the physical entrapping and comprehensive dialysis.②The osteoblasts were separated and cultured from the skull of new-born rats,and the osteoblasts were identified by alkaline phosphatase staining in7days, the osteoblasts were identified by alizarin red staining in14days;The osteoblasts were randomly divided into five groups:the control group,10-7mmol/L group,10-6mmol/L group,10-5mmol/L group and10-4mmol/L group. Rat osteoblast cultured in vitro was treated by a series of different, concentration of NSC-OST for different time respectively.MTT method was used to test the proliferation of osteoblasts.Rat osteoblast cultured in vitro was treated by a series of different concentration of NSC-OST for7days,ELISA method was used to test the OPG and RANKL expression of osteoblasts.③The osteoclasts were separated and cultured from the femur of new-born rats.Ditto for the groups and the method of intervention.The osteoclasts were identified through tartrate-resistant acid phosphatese (TRAP) staining in7days,at the same time,TRAP(+) cells were examined;The lacuna induced by osteoclast bone resorption were identified by toluidine blue staining,Areas of lacuna induced by osteoclast bone resorption on the bone slices was measured by computer image processing.Results①The research has successfully synthesis N-octyl-O-sulfonylurea acyl chitosan,which has the structure of water soluble chitosan derivative.②The cells cultured in vitro showed representative morphological and biological characteristics of osteoblasts.Alkaline phosphatese and Alizarin red staining presented positive results;After24h and72h of treatment, the NSC-OST with final concentration of10-5mmol/L can promote the proliferation of osteoblast(P <0.05).The NSC-OST with final concentration of10-6mmol/L after72h of treatment(P<0.05).But the NSC-OST with final concentration of10-4mmol/L can obviously inhibit the proliferation of osteoblast(P<0.01).The NSC-OST with final concentration of10-6mmol/L and 10-5mmol/L can promote the OPG expression and inhibit the RANKL expression of osteoblast(P<0.05); The NSC-OST with final concentration of10-6mmol/L and10-5mmol/L can obviously raise the ratio of OPG/RANKL of osteoblast(P<0.01).③The cells shared some of the typical characteristics of osteoclasts.The cells were stained in red by TRAP.Numbers of osteoclasts of10-5mmol/L group and10-4mmol/L group were much lower than the control group (P<0.05) at48h.Areas of lacuna of10-6mmol/L group,10-5mmol/L group and10-4mmol/L group were much smaller than the control group (P<0.05) at48h.In bone resorption assay, area of lacuna of blank group was larger than those of groups (P<0.05);Numbers of osteoclasts and Areas of lacuna of all the NSC-OST groups were much smaller than the control group (P<0.05, P<0.01) at96h,,and no difference was found among of10-6mmol/L group,10-5mmol/L group and10-4mmol/L group(P>0.05).Conelusions①Using water soluble chitosan derivative(NSC) as a bone-targeted carrier, bone NSC-OST was prepared and characterized,which can easily dissolved in water,and the nature help NSC-OST to effective concentration of treatment and treated areas.②The proper concentration of NSC-OST can promote the proliferation and the OPG expression of and expression of osteoblast,while it can degrades the numbers of osteoclasts and areas of lacuna induced by NSC-OST.The research may be provid a valuable reference for a new anti osteoporosis drug of higher target selectivity and stronger local effect.