Biocompatibility Evaluation Of Light-cured Nano-hydroxyapatite Composite For Tooth Restoration

Objective:The dental filling materials with the human body,especially the oral mucosa of long-term exposure, the ideal repair material should have good biocompatibility and biological activity.Nano-hydroxyapatite as a new type of biological material for its outstanding performance in dental and orthopedic fields demonstrated an attractive prospect.To evaluate the biocompatibility of the light-cured nano-hydroxyapatite composite for tooth restoration and to provide experiment and theoretical basis for the safety of clinical application, which provide the experimental basis on biosecurity for clinical application of the materials.Based on the relevant standards(Chinese National Standards GB/TI6886)of medical biomaterials(implantation materials)safety evaluation.Methods:Experiment1Prepare composite materials of the light-cured nano-hydroxyapatite composite for tooth restoration:The composite materials were composed of a visible light-curing monomer mixture as a matrix and NHA as a inorganic filler. The surface of the NHA particles was modified by using a silane coupling agent KH-570. Shading sealed in glass containers in the experimental material in the mold was prepared by the internal diameter is15.0±0.1mm,High0.30±0.1mm cylindrical specimen,light curing, set aside.Experiment2The extraction of material extract:Take20experimental specimen after measurement and calculation,the average surface area of4.95cm~2,in line with the requirements of the biological evaluation of test. Specimen average into two sterile jar after treatment with ultrasonic cleaning,drying and autoclave.In accordance with the requirements of the biological evaluation tests were added to normal saline49.50ml.Placed in37℃for extraction72h,filtration,packing,placed in37℃stored and slternated.Experiment3an cute toxicity test:Samples were applied to mice bytail vein injection.Immediately after injection,24h,48h,and72h,mice were observed respectively with general states,toxicity appearance,mortality,and the weight in72hExperiment4pyretogen test:The experimental material extract were injected lml dose to mice bytail vein. Interval30min to measure mice’s temperature, six times in a row,6times in the highest minus the increased value of the normal temperature was the Increased value of the test rats.The temperature of the test environment is22℃.Experiment5dermal sensitization test:Test animals divided into two group,the experimental group were injected the extrac of light-cured nano-hydroxyapatite composite for tooth restoration.The control group by the same point in time injected of the same quality of saline.Two steps induced and aroused were applied to guinea pigs.The erythema and edemawere observed and the indexes of response were computed.Experiment6oral mucous membrane irritation test:Experimental materials and control materials sutured to the buccal surface of the rat oral cavity on both sides after autoclave treatment,observed the oral mucosa whether have congestive erosion, ulceration and edema response after surgery14days. Experiment7skin irritation test:Selected three injection points in paravertebral bilaterally symmetrical position of the test rats. and and spacing of lcm.Respectively injected with the extract of the light-cured nano-hydroxyapatite composite for tooth restoration and saline,injection volume was1ml.Respectively to observe the skin irritation and grading respectively of the experimental group and control group of animals after injection6,24,48,72hours.Experiment8bone implantation test:Sterilized by test materials (nano-hydroxyapatite composite for tooth restoration) and control specimens (Carey Charisma) were implanted in the rats femur. At the specified time points (lw,2w,4w) drawn and conducted the observation of the gross specimen and take pathological tissue sections.To evaluate the local reactions and the histocompatibility of animal body after the implantation.Experiment9muscle implant test:Test samples and control samples were sterile implanted the right and left leg muscle.The implanted parts of the laboratory observation and pathological anatomy, and observe the results were analyzed,analysis of observations at the specified time points (1w,2w,4w).Experiment10cytotoxicity test in vitro(MTT):According to ISO standards,the use of in vitro cytotoxicity-test(MTT method) testing the absorbance value (OD value) of the human gingival fibroblast(HGF) in the selfmade light-cured nano-hydroxyapatite composite for tooth restoration extracts of1,3,5d,observed changes in cell morphology,to calculate the relative cell proliferation rate(RGR), and determine the cytotoxicity grade.Result:1.Symptoms and changes in body weight of experimental animals are within the acceptable range in acute toxicity test at each observation time point (24h,48h,72h) all animals were generally in good condition after injection. Symptoms of experimental animals and weight change were within the acceptable range, all no toxicity, no animal deaths. It can be considered that the light-cured nano-hydroxyapatite composite for tooth restoration can’t bring acute general toxicitywas, conformed the requirements.2.1n pyrogen experiments,no elevated temperature more than0.4℃in the six rats, and elevated temperature sum of each three rats were less than1.4℃.It can be considered that the light-cured nano-hydroxyapatite composite for tooth restoration were non-irritancy.3.In dermal sensitization test all the experimental groups and the control groups were generally in good condition after the last injection.Did not appear the situation like the vertical hair, convulsions,sneezing,breathing difficulties,claws scratching thenose,the reaction of shock, incontinence and death.It can be considered that the new materials were no pyretogen.4.In oral mucous membrane irritation test all test animals were no systemic adverse reactions, The experimental material samples were not loose,not fall off.On the buccal mucosa in contact with the experimental sample found no reactions such as swelling, rough,erosion and ulceration.It can be considered that the new materials were no oral mucous membrane irritation.5.After local injection in skin irritation test,material extract group and the saline group for each injection sites have not found erythema,edema,exudate, ulceration and found no obvious skin irritation.The results show that the light-cured nano-hydroxyapatite composite for tooth restoration were non-irritancy. had no irritation.6.1n the implantation experiments no obvious necrosis around the implant material and found mild inflammatory response in a week.As time goes by inflammatory reaction gradually subsided and appear fiber wrapped.The results suggested that the new nano-hydroxyapatite composite of tooth restoration have good histocompatibility after the local implantation.7.In cytotoxicity test in vitro,the experimental group and the negative control group have no significant morphological differences.Quantitative analysis of MTT test revealed that the cytotoxicity level of the experimental cell was0-1.Quantitative tests and qualitative tests all showed the result that the the new nano-hydroxyapatite composite of tooth restoration have no significant cytotoxicity.Conclusion:The preliminary experiments proved that the new nano-hydroxyapatite composite of tooth restoration were non-irritancy,no pyretogen,no significant cytotoxicity,no irritation and systemic acute toxicit and have good histocompatibility after the local implantation.The above experimental results show that the light-cured nano-hydroxyapatite composite for tooth restoration has a good biocompatibility,a high bio-security and no toxicity against the body.And this study may provide a reliable safety data for the further clinical trials of the new materials.