Study On Biosecurity Of The Silk Fibroin/biphasic Calcium/calcium Sulfate Composite

Objectives To evaluate the biosecurity of silk fibroin, biphasic calcium and calcium sulfate composite, which would provide reference for the development and research of material and clinical application in the future.Methods According to the standards of GB/T 16886-2008/ISO 10993:2003 and GB/T 16175-2008, the biosecurity of bone cement was evaluated both in vitro test (cytotoxicity test, hemolysis test) and in vivo test (acute toxicity test, pyrogen test, micronucleus test, sensitization test, intradermally stimulus test, implantation test).⑴In cytotoxicity test, L929 was made into unicell suspension after passage and resuscitation to inoculate and cultivate separately for continuous 2 days, 4 days, and 8 days cultivation. Cellular proliferation was evaluated with MTT method.⑵In hemolysis test, 2% fresh erythrocyte suspension was separately injected into experimental group, negative control group and positive control group, optical density and the haemolysis rate for different groups was calculated.⑶In acute toxicity test, lead acetate liquor, normal sodium and leaching liquor with three different concentrations were accordingly injected via the tail vein into experimental group and control group. The general situation was observed at 24 h, 48 h, 72 h respectively and body weight change was valued.⑷In pyrogen test, the leaching liquor and the control liquid were injected into rabbits through the vein, the chang of body temperature was observed.⑸In micronucleus test, leaching liquor with three different concentrations, normal saline and cyclophosphamide were separately injected via the tail vein into mice, the animals were sacrificed by using four exposure methods, the bone marrow was harvest to smear for genotoxicity evaluation through the bone marrow micronucleus frequency.⑹In intradermally stimulus test, leaching liquor and control liquid were intradermally injected. Their local skin irritation were immediately observed, the skin irritation reaction was classified.⑺In sensitization test, leaching liquor and control liquid were intradermally injected with the maximum dosage, the skin of irritating parts was observed after the induction and provocation phase.⑻In implantation test, PMMA and the composite were disinfected and implanted into sacrospinal muscle of the rabbits, two rabbits were killed at 1,4,8,12 weeks post-operation each time, histological change was observed.Results⑴In cytotoxicity test, under microscope, visible cytotoxicity was observed in positive control group, cell morphous was natural in negative control group and experimental group. Cytotoxicity of all three experimental groups were scored as classⅠ, negative control group was class 0, positive control group was classⅤ.⑵In hemolysis test, hemolytic crisis was observed in positive control group, in negative control group and experimental group, hemolytic crisis was't observed. The highest rate of hemolysis of experimental group was at 1.65%.⑶In acute systemic toxicity test, the mice in the negative control group and experimental group were generally in good condition and without weight loss. In positive control group, the weight of the mice decreased significantly.⑷Pyrogen test showed that the temperature rise was up to 0.06℃maximum, sum was less than 1.4℃, in control group and experimental group.⑸The results of micronucleus test showed that the highest incidence of micronucleus test group was 2.4‰, no statistic difference was found compared with the negative control group (P>0.05), statistic difference was found compared with the positive control group (P<0.05).⑹In intradermally stimulus test, skin stimulation index of the experimental group was 0.0-0.2, which belongs to a very mild stimulation. skin stimulation index of the positive control group was midrange and heavy.⑺In the sensitization test, no skin abnormalities were observed in the experimental group, erythema and edema were observed the positive control group.⑻In implantation test, no significant difference were observed at 1week, 4 weeks between experimental group and control group. While significant difference was observed in fibrous capsule at 8 weeks, 12 weeks, no significant difference was found in the area of inflammatory cells.Conclusions Silk fibroin/biphasic calcium phosphate/calcium sulfate composite bone cement met GB/T 16886-2008/ISO 10993:2003 and GB/T 16175-2008 standards, it had no cytotoxicity, no hemolysis, no acute systemic toxicity, no pyrogen, no stimulation, no genetoxic.It showed that the composite had certain degree of good biosecurity. It could be implanted in the body safely.