Effects Of Two Kinds Of Resin-Matrix Composites On The Biological Behavior Of HGFs

ObjectiveTo investigate the effects of two kinds of restoration material,ENAMIC and Ceramage on attachment,proliferation,synthetic of protein and gene expression of human gingival fibroblasts(HGFs). To know the biocompatibility of them and provide some experimental basic for selecting restoration material for dental implants.Method1.Groups of ExperimentsENAMIC,Ceramage and pure titanium were divided into three groups:EN, Ce and Ti.DMEM containing with 10% FBS was negative control group (Nc), and DMEM containing with 10% FBS and 0.64% phenol was positive control group(Pc).Ceramage was shaped and lighted curing as 10x12x1.5mm. ENAMIC and pure titanium were cut and ground as 10x12x1.5mm.Each kind of material were made 30 piece of specimens.Ground all specimens into the size about 10x8xlmm by water-resistant sandpaper with different particle size.Specimens were ultrasonically cleaned,dried and disinfected.Three piece of specimens of each group were used to observe the attachment of HGFs. The remaining specimens were leached at 37℃ for 72 h according to the standard 3cm2/mL of superficial area/valume of leaching solution.The leaching solution was filtrated by filter of 0.22μM.2. Cultivation and identification of HGFsHGFs were obtained and purified by gingival tissue cultivation.In order to identify whether the cells were HGFs,we performed immunocytochemical staining and observed the morphology of the cultured cells.To obtain the needed HGFs, We continuously cultured the primary cells.We counted HGFs and drew the growth curve for knowing the proliferation regular.3. Attachment of HGFs around specimen of group EN,Ce,TiHGFs were seeded on three piece of specimens of group EN,Ce,Ti for three days.And HGFs were also seeded in group Nc as control.Then we examined attachment of HGFs by microscope.4. Proliferation of HGFs affected by leaching solution of group EN Ce,TiHGFs were seeded in the leaching solution of group EN,Ce,Ti and group Nc,Pc culture medium for 1,3,5,7 days. We detected the proliferation of HGFs by MTT assay.5. Cytotoxicity evaluation of three kinds of materialWe calculated the relative growth rate (RGR) of each group,then turned RGR into grade of toxicity and evaluated the cytotoxicity of three kinds of material.6.Expression of COL-I,Fn gene mRNA affected by leaching solution of group EN,Ce,TiHGFs were seeded in the leaching solution of three kinds of material and group Nc culture medium for 3 days.Expression of COL-I,Fn gene mRNA of HGFs were measured by RT-qPCR.7. Synthesis and secretion of COL-I of HGFs affected by leaching solution of EN,Ce,TiHGFs were seeded in the leaching solution of EN,Ce,Ti and group Nc culture medium for 1,3 days.We measured COL-I in the supernate by ELISA.Result1.The cultivation and identification result of the HGFsPrimary cells crawled out from gingival tissue after 5 days and covered two thirds of the bottom at about 15 days.HGFs were fusiform or star shape,they had some variable cytoplasmic processes,the morphology was the same with fibroblasts.Distributed as radiated,circinate or funicular.Immunocytochemistry staining showed that HGFs were positive staining of Vimentin,but negative of Keratin.Positive staining particles distributed in the cytoplasm,the nuclei was not stained and it’s clear.It proved that the source of these cells were mesoderm.According to the tissue origin from human gingival, we can know it’s HGF.The growth curve was "S"shape,that’S to say HGFs experienced a slow growth phase,logarithmic growth phase and a plateau.2. Attachment of HGFs around three kinds of specimensHGFs grow well and the morphology were normal around three specimens of three groups as in group Nc.HGFs were fusiform or star shape, and distributed as radiate,circinate or funicular.3. Proliferation of HGFsThe OD was similar in group EN,Ce,Ti and increased from day 1 to day 7,they were in accordance with the regular of group Nc.The proliferation of group EN,Ce,Ti were lower than group Nc at each period(P<0.05).But there were no difference between group EN,Ce,Ti (P>0.05).Group EN,Ce,Ti and Nc were significantly more than Group Pc (P<0.05).4. Evaluation result of cytotoxicity of three kinds of materialRGR of each group at each period:There were no difference between group EN,Ce,Ti (P>0.05); group EN,Ce,Ti were significantly more than Group Pc (P < 0.05).Level of toxicity assessment:group EN,Ce,Ti were grade 1 at each period,grade 1 means no toxicity.Group Pc was grade 4 at each period,grade 4 means severe toxicity.5. Expression of COL-I, Fn gene mRNA of HGFsCompared with group Nc, expression of COL-I and Fn mRNA was decreased in group EN,Ce,Ti.The differences were statistically significant(P< 0.05).But,the differences between group EN,Ce,Ti were statistically insignificant (P>0.05).6.The synthesis and secretion of COL-IThe content of COL-I:There were statistically insignificant difference between group EN,Ce,Ti and group Nc(P>0.05).ConclusionThe toxicity evaluation of ENAMIC, Ceramage and pure titanium were the same, their toxicity were grade 1,means they have no toxicity,they are meet the requirement of clinical use of biological material according to national standard. ENAMIC, Ceramage,pure titanium had certain effect on proliferation of HGFs,and expression of COL-I,Fn gene mRNA were decreased,but the effects were weak.The synthesis and secretion of COL-1 were not affected.