Effects Of NELL-1 And BMP2 Combination On Rat Dental Pulp Repair And Regeneration

ObjectiveNel-like molecule-1(NELL-1)is a novel highly specific growth factor which can induce osteoblast differentiation and bone regeneration.Previous studies have suggested that NELL-1 can synergistically increase bone formation with bone morphogenetic protein 2(BMP2)and inhibit adverse effects induced by BMP2,including local inflammation and swelling.Dental tissue and craniofacial bone are all neural crest-derived tissues.Odontoblasts and osteoblasts are all differentiated from mesenchymal cells,sharing several similarities in genetic structure.Our previous studies have investigated that HELL-1 plays an important role in odontoblast differentiation as well as dentin formation.The aim of this study was to evaluate the combined effects of NELL-1 and BMP2 on pulp repair and regeneration.Methods1.Healthy non-carious upper first molars from Wistar rats were used for this investigation to establish rat pulp exposure model.Exposed pulps were capped with 400 ng/ml,500 ng/ml,600 ng/ml or 700 ng/ml NELL-1(sterile collagen sponge as carrier).After 2 weeks,Hematoxylin-Eosin(HE)staining was used to observe the formation of reparative dentin,and determined the minimum effective concentration of NELL-1.2.Upper first molars from 60 Wistar rats were used to establish rat pulp exposure model.The rats were randomly divided into five groups:(1)Control group;(2)Dycal group;(3)BMP2 group +collagen sponge;(4)NELL1 group +collagen sponge;(5)NELL1+BMP2 group.3.HE staining was used to observe the formation of reparative dentin,and immunohistochemical staining was used to detect the expression of dentin specific protein-dentin sialophosphoprotein(DSPP).4.Quantitative RT-PCR and immunohistochemical staining were used to investigate the expression of pro-inflammatory cytokines Interleukin-6(IL6)and Interleukin-8(IL8).Results1.The experiment showed that 700 ng/ml was the minimum effective NELL-1 concentration in reparative dentin formation in rat pulp compared with the other concentrations.2.The results showed that pulp capped with NELL-1 plus BMP2 had earlier and more obvious reparative dentin formation compared with the other groups by HE staining.At 1 week,the NELL-1 plus BMP2 group showed the short and irregular tubular dentine.At 2 weeks,the reparative dentin contained abundant dentinal tubules and appeared more like the normal surrounding dentin,At 4 weeks,NELL-1 plus BMP2 group exhibited more obvious dentine bridge formation beneath the site of pulp exposure compared with the other groups,3.The expression of DSPP in NELL-1 plus BMP2 group was higher than the control group(p<0.01),Dycal group(p<0.01 at 1 and 4 weeks,p<0.05 at 2 weeks)and BMP2 group(p<0.01 at 1 week,p<0.05 at 4 weeks).4.Pulp capping with NELL-1 plus BMP2 in rats showed lower inflammatory cell response.The immunohistochemical results showed that the expression of IL6 and IL8 were lower in the combination of NELL-1 plus BMP2 than the other groups.The qRT-PCR analysis provided similar results.The results demonstrated that the mRNA levels of IL6 and IL8 were remarkably down-regulated in NELL-1 plus BMP2 group compared with the other groups(p<0.01)at each time points.Conclusions1.The rat pulp exposure model was established successfully.This is the first study to demonstrate an additive effect of combined NELL-1 and BMP2 treatment on pulp repair and regeneration.NELL-1 plus BMP2 group had earlier and more obvious tubular reparative dentin formation,and the expression of DSPP in NELL-1 plus BMP2 group was higher compared with the other groups.2.This investigation has demonstrated that treatment with NELL-1 plus BMP2 suppressed the expression of IL6 and IL8 in the rat pulp exposure model,suggesting its potential usefulness as an anti-inflammatory agent for pulpal inflammation.