Compound Function Of Simvastatin With Demineralized Bone Matrix Act As A Pulp Capping Material Used In Rats

Objectives:Reparative dentin is one of the important manifestation after dental pulp-dentin complex injury,it depends on the formation of functional odontoblasts cells.During the recent years,the studies found that simvastatin promote dental pulp stem cells to differentiate into odontoblasts,but in vitro,local use can easily cause mass of odontoblast-like cells,but lack of mineralization space,so we consider use it composite with scaffolds.Decalcified bone matrix?demineralized ipads matrix,DBM?many used as bone tissue engineering scaffolds,and we prior experimentally found it can be used as pulp capping agent alone,which can provide a good mineralization sites and space,but we also observed less odontoblast-like quantity with few mineralization.So we designed the experiment that simvastatin compound DBM by freeze-drying technology,used as pulp capping agent and applied to pulptomy animal model,set DBM and clinical"gold standard"---calcium hydroxide,for contrast to compare them in formation of reparative dentin,explored whether simvastatin composite DBM can provide a better environment,to promote the formation of reparative dentin.Methods:?1?Using freeze drying technology composite simvastatin and DBM:1?M simvastatin soluble in anhydrous ethanol and DBM compound,samples pre cool to-25?in about 2?/min rate,maintain clean vacuum evaporation anhydrous ethanol solvent to 2 h,collection,airtight.?2?Pulptomy animal model establishment and specimens made:8 weeks of 75 male Wistar rats randomly divided into the Ca?OH?₂,DBM,and simvastatin compound with DBM group,each group randomly divided into 1d,3d,7d,14d and 28d in all.Set 5 subgroups,each subgroup has 5 rats.Under general anesthesia,maxillary first molars carried out pulptomy,corresponding pulp capping agent were placed.Under the glass ion cement bottom,3M flow resin filling.The other side of the maxillary first molars as blank control group,postoperative feed pap and free water.In postoperative 1 d,3d,7d,14d and 28d heart perfusion fixation method is executed in three groups.Complete separation of bilateral maxillary specimens,4?under 4%paraformaldehyde fixed liquid external fixation,2 to 3 h after filming X-ray,replacing 10%EDTA solution under 4?decalcified dewaxing dehydration embedding after 4 weeks,making paraffin section.?3?Histological observation:HE staining for dental pulp tissue,reparative dentine formation process was observed under optical microscope.Three colors dyeing to observe the reparative dentin bridge structures.Immunohistochemical detection of early reparative dentine formation correlation factor Runx2,OCN,COLI,and the expression of DSP.Results:?1?Simvastatin compound DBM material preparation:after vacuum 2 h closed vacuum pump,vacuum value at this time no change,super clean bearing units,sample quality no change before and after vacuum,fully volatiled solvent.?2?Pulp capping imaging observation:after all subjects tooth images,the reign of fillings,root tip not seen obvious abnormity.Simvastatin+DBM 7d group after a root canal orifice were visible high density calcified barrier earlier than in calcium hydroxide and DBM group,calcium hydroxide of 14d and 28d after subjects teeth suggest possible pulp cavity wall calcification is serious.?3?After pulp capping histological observation:?1?.HE staining to observe histological changes after pulp capping:calcium hydroxide pulp capping,coagulation necrosis tissue appeared,1d,3d dental pulp vasodilatation,14d coagulation necrosis layer is replaced by pink compound,28d dental bridge formation,dental bridge become thicker of the medullar cavity.DBM group pulp capping 1d,3d,7d appear inflammation,decrease in 14d,when cells can spread to the stent,but less odontoblast-like cells,28d formed thin dental bridge.Simvastatin compound DBM group,the early vascular no obvious reaction,does not appear inflammation,3d stent obviously increase the number of odontoblasts and 7d,odontoblast-like cells scattered within the stent and mineralization,14d mineralization of odontoblast-like densely connected to the start of the stent,28d form dental bridge.?2?.Three colors dyeing observe dental bridge structure:calcium hydroxide bridge structure was less number of dentin,with more calcium deposition.Simvastatin compound of DBM odontoblast-like cells along the DBM collagen scaffold distribution and mineralization,forming continuous three-dimensional mineralization of bridge structure.Prove that simvastatin compound DBM pulp calcification of bridge structures is formed by odontoblast-like cells mineralization,compared with calcium hydroxide closer to the physiological repair process.?4?Immunohistochemical detection of the expression of early mineralization related factors:select Runx2,OCN,COLI,and DSP four mineralization related factors.After three sets of pulp capping,mineralization related factor expression trend is roughly same,all appear the reparative dentine formation,the characteristics of compound DBM simvastatin group showed more earlier and better promote the expression of related factors,closer to the physiological characteristics of regenerative repair.Conclusions:Simvastatin compound DBM pulp capping,the effect of both is complementary,enhanced.To promote early reparative dentine formation of pulp capping effect,has a good application potential.