The Effect Of Different Doses Of Gamma Ray On Biocompatibility And Biodegradation Of Silk Fibroin

Objective: To evaluate the biocompatibility and biodegradation of silk fibroin withγ-irradiation， we test the mechanical properties and molecular structure and selectedthe dose range for the next experiments. The cells experiments and rats surgery wereused to evaluate the biocompatibility and biodegradation.Methods:1.Mechanical and molecular structure changes of gamma ray irradiatedsilk fibroin: The silk fibroin films were divided into different groups which were0,25,50,100,200,300,500,1000kGy, respectively.(1) Changes of break strength andelongation of silk fibroin: Silk fibroin films were made into special shape with mould ofinternational standard and tested the break strength and elongation in every group.(2)Molecular structure change: The silk fibroin were made into powder, tableting withKBr, and tested the structure change with FTIR.2. Biocompatibility of gamma rayirradiation silk fibroin films in vitro: We got the skin of back of newborn SD rat,detached the hypodermal cell from the skin, cultured and subcultured the cells to the3rdgeneration.(1) Growth curve of dermal cells on silk fibroin films: The dermal cellswere cultured into24-well plates,1×105cells per well. Cultured the cells for7days,and then, we tested the grown situation of cells everyday with CCK-8kit, testing theOD value of6wells of every group on the microplate reader at the wave of450nm.(2)The extract toxicity of silk fibroin: Dermal cells were cultured with the extracts of silkfibroin in the96-well plates,5000cells per well. After cells adherence, we exchangedthe normal culture media with extracts,100μL per well and tested the OD value of cellat the1st,3rd,6thday on the microplate reader at the wave of450nm.(3) hemolysis: Weget the blood of volunteer and diluted it. The experiments had4groups which werenegative group, positive group, extract group and the normal saline with silk fibroinfilms group. Every test tube was injected10ml normal saline, distill water, extracts ornormal saline with silk fibroin film and0.2ml diluted blood,37℃water bath for60min.We had the tubes centrifuged with1000r/min for5min, moved the supernate into 96-well plate and test the OD value at the wave of545nm on the micoplate reader.3.Biocompatibility and biodegradation of silk fibroin films in vivo: We implanted the silkfibroin films with different doses(25,50,100,200kGy) of gamma ray irradiation in theback of SD rat subcutaneously. At the date of7th,14th,28th,56th,84th, we got the skintissue where the silk fibroin films implanted, blood of experiment animals. We madeparaffin sections with the tissue and stained them with HE staining kit. We test theconcentration of IL-6and TNF-αwith the ELISA kit. The silk fibroin films gotten fromthe tissue were weight and compared with the mass weighted before, getting the massdecreasing curve.Results:1. Mechanical and molecular structure changes of gamma ray irradiatedsilk fibroin: The break strength and elongation of silk fibroin was decreased afterirradiated by gamma ray. And the extent of decrease was more with the increasing of thedose. The FTIR test results showed that the special peaks of Silk II, which is at1628cm-1,1526cm-1, had not moved significantly. It indicated that the gamma ray wouldnot change the secondary structure of silk fibroin.2. Biocompatibility of gamma rayirradiation silk fibroin films in vitro:(1)The dermal cells showed similar trend in growthcurve. We analyzed the OD value of cells in different groups with variance analysis andfound that there was no significant on their growth(p>0.05).(2)We tested the growthactivity of dermal cells cultured with extracts of silk fibroin with different dosesirradiated by gamma ray at the date of1st,3rdand6thday after cells adherence. Thetoxicity of extracts were within the range of the1stlevel of toxicity grades, which meansthat the RGRs of extracts were between75%-99%. And there was no significantdifferent among the groups after analyzed by SPSS17.0(p>0.05). The hemolysis testshowed there was no significant different among the groups(p>0.05).And the hemolysisrate in all these groups were less than5%, which is specified by national standard.3.Biocompatibility and biodegradation of silk fibroin films in vivo: The subcutaneousexperiment did not occurred obviously inflammatory responses in all these groups.Along the periods after implantation, silk fibroin films in the higher dose groupsfracture more obviously. The concentrations of IL-6and TNF-α in different groupshad no significant differences compared with the Blank(p>0.05). We could not find outthat the post-irradiated silk fibroin occurred obviously inflammatory responses. Thebiodegradation was showed by the fragments and mass decrease. They all showed obviously in the higher dose groups.Conclusions:1. The break strength and elongation of silk fibroin showed a significant downtrendand the molecular structures were not obviously changed in the0-1000kGy dose range.2． Silk fibroin films with0-200kGy dose of γ-irradiation had goodcytocompatibility and Blood compatibility.3. In the0-200kGy dose range, silk fibroin films in all these groups showed thegood biocompatibility and the biodegradation rate increased more significantly with theincrement of the absorbed doses and implantation time.