Research On Preparation And Stability Of Keratinocyte Growth Factor 2 Biologic Protein Composite Sponge

Keratinocyte growth factor-2 (KGF-2) has played a very significant role in many fields like damage repair, however, the application of KGF-2 is significantly influenced by biological characteristics of themselves and the external environment, when applied in the body the stability and biological activity of protein are not easy to maintain, as in 37℃in phosphate buffer liquid or physiological saline, the activity of KGF-2 is maintained less than 1h and can't effectively play the biological function. Therefore, the effective transmission means which can slow release growth factors is needed to solve this problem. The key is the ideal slow release carrier.Biological protein sponge has excellent biocompatibility, biodegradation, hemostasis, absorbability, slow release and the availability to be processed into various carrier shapes in mild conditions. And biological protein sponge can reversibly combine with growth factors in a simple and reliable way, namely fixed growth factors, which can effectively maintain growth factors' activity and control slow release, and exert the effect of promoting angiogenesis,inhibition of scar formation, damage repair and etc. This research design applies biological protein sponge as control-released carrier of KGF-2, which can, on the one hand, extend the half life of KGF-2 and the effective time of it in vivo, thus better performs its role. On the other hand, biological protein sponge compound KGF-2 does not prevent the biological protein sponge playing its own advantage so that both can be of very good effect.Objectives:To prepare KGF-2 biologic protein composite sponge and establish the biological activity measurement methods of KGF-2 biological proteins composite sponge in vitro and determination of KGF-2 biological proteins composite sponge stability and KGF-2 slow releasing circumstances from KGF-2 biological proteins composite sponge.Methods:This research under acidic conditions utilizes enzyme method and uses pigskin as raw material to make collagen protein solution, after the solution was identified and determined concentration by protein electrophoresis analysis, ultraviolet spectrophotometer analysis, ultraviolet absorption method, Kjeldah method, we preserved the solution under-80℃, then cryodesiccated it to make collagen sponge and KGF-2 biologic protein composite sponge. After that we respectively detect its biocompatibility, physical and chemical properties and biological activity in vitro. Then examine the stability of KGF-2 biological proteins composite sponge with the method of cell activity in experimental and calculate KGF-2 releases from KGF-2 biological proteins composite sponge with time changing by ELISA kit.Therefore, it's available to get knowledge of KGF-2 slow releasing circumstances from KGF-2 biological proteins composite sponge.The results showed that:SDS-PAGE gel electrophoresis chart indicates that the higher purity of the extracted collagen, ultraviolet spectrophotometry analysis shows that the maximum ultraviolet absorption of extracting pigskin collagen through pepsin is about 231.8 wavelengths which accords with the characteristics of collagen.Determination of collagen concentration is 4.507mg/ml. For frozen dried collagen sponge, cells grow well on it, which suggests that the biocompatibility of the material is fine. Appearance, surface structure, apparent density, pH, moisture content, wetting time, liquid absorption, water absorption rate, porosity and other physical and chemical indicators of KGF-2 biological protein composite sponge have no significant difference from those of collagen protein sponge. It means that put KGF-2 into the biological protein sponge does not change its physical and chemical properties. SDS-page analysis indicated that the KGF-2 biologic protein composite sponge has apparent strip in 20 kD. Observed by SEM we can see that the collagen protein sponge group and the KGF-2 biological protein composite sponge groups have no significant difference in the overall morphology.In vitro biological activities, it showed that both KGF-2 biologic protein composite sponge and KGF-2 plain liquid can promote NIH3T3 cells proliferation, and there does exist significant differences contrasting PBS control group(P<0.01, P<0.05); collagen sponge also can promote NIH3T3 cells proliferation, and there does not exist significant differences contrasting PBS control group(P>0.05). There is no significant difference in activity when KGF-2 biological protein composite sponge is preserved for six months in the condition of 25℃and 37℃(P>0.05), which shows that its stability is favorable. Finally the slow release experiment shows that the slow release cycle of prepared KGF-2 biologic protein composite sponge is long, releasing about 51.33±0.32% when in 14d.Conclusion:The KGF-2 biological protein composite sponge has been successfully prepared, the test results of its physical and chemical properties, in vitro biological activity and stability are favorable, KGF-2 biological proteins composite sponge has longer slow release cycle.