Synergic Effect Of Natrin Protein From Guangxi Cobra Venom And Biphasic Calcium Phosphate Ceramic (BCP) On Osteogenesis

Objective: To provide reference for the repair of cranial bone defect inclinic, this study investigated the synergic effect of bioactive factor-Natrinprotein from Guangxi cobra venom and biphasic calcium phosphate ceramic(BCP) on osteogenesis..Methods:(1) Different concentrations of bioactive Natrin protein (0g/ml,2.5g/ml,5g/ml and10g/ml) were added in the culture of primaryosteoblasts from3-7days’ SD rats. After2,5and8days, MTT, ALP activity andstaining, the relative expression of osteogenesis related genes (BMP2，RUNX2，ALP，BSP，OCN，COL1A1), cell viability assay, cell morphology analysis,calcium contents detection and immunohistochemical staining were performed;(2) Different concentrations of bioactive Natrin protein (0g/ml,1.25g/ml,2.5g/ml and5g/ml) were added in the culture of primary osteoblasts loadedin porous HA/β-TCP biphasic calcium phosphate ceramic (BCP, mass ratio=6:4, diameter=12mm, thickness=2mm, porosity>75%). After2,5and8days,MTT, ALP activity and staining, the relative expression of osteogenesis relatedgenes (BMP2， RUNX2，ALP，BSP，OCN，COL1A1), cell viability assay,cell morphology analysis, calcium contents detection and immunohistochemicalstaining were carried out;(3)40adult male SD rats (body weight=300±20g)were randomly divided into4groups (group A, B, C and D,10rats per group)and5mm diameters of calvarial bone defect was created at the upper rightquadrant. Group A and B were without BCP. Rats in group C and D, wereimplanted with BCP (mass ratio=6:4, diameter=5mm, thickness=2mm,porosity>75%) to repair the defect. After3days’ anti-inflammation, rats ingroup A and C were injected with0.5ml normal saline and those in group B andD were with equal amount of saline added with Natrin of5g/ml and2.5g/mlrespectively. After1month and2months respectively, the repaired tissue wereharvested for gross observation, histological analysis. Besides, cone beam CTscan was used for2months’ detection.Results:(1) In the monolayer culture, Natrin had a positive effect onosteogenesis and proliferation of osteoblasts, which was demonstrated by theresults of MTT, ALP activity, the expression of osteogenesis related genes andhistological findings. The recommended concentration of Natrin is5g/ml;(2)In the three-dimensional culture based on BCP, Natrin had a promotive effect onosteogenesis and proliferation of osteoblasts, which was demonstrated by theresults of MTT, ALP activity, SEM observation, the expression of osteogenesisrelated genes and and histological findings, especially at the concentration of2.5g/ml. The results suggested that Natrin and BCP have synergy effect onosteogenesis;(3) In the animal experiment, bone defects using Natrin and BCP showed the growth of new bone, obtaining a satisfactory interface with theoriginal bone inside the defect, as detected by gross observation, PCR detection,hard tissue section staining.Conclusion: Natrin and BCP have synergy effect on the repair of calvarialbone defect, which demonstrated the potential as satisfiable products for therestoration of bone defect.