Basic Fibroblast Cell Growth Factor Micro And Nano-embedded Collagen Matrix Loading Technology Research

With the deep development of biotechnology,basic fibroblast growth factor (bFGF) is utilized more and more extensively.However,as a kind of basic polypeptides,bFGF is sensitive to heat and acid,diffuse quickly in vivo,and can be zymohydrolysised easily.And its half-decay period is short.So its biological effect can not be brought into full play.How to make full use of its biological effect,that restricts its further exploratory development in vivo.The technology of controlled release in pharmaceutics can solve this question.In this study,poly(lactic-co-glycolic acid )(PLGA) is used as a kind of relatively ideal carrier of drug controlled delivery to prepare the bFGF-PLGA microspheres which contain bFGF as the model drug,by the method of water in oil in water(w₁/o/w₂).The bFGF-PLGA microspheres can isolate bFGF from the outside enzymolysis environment,which can release drug in a relatively long time and keep the drug density in a steady level.However, microspheres' drug delivery experiment in vitro exists an initial burst release phase, which can be relieved by combining bFGF-PLGA microspheres with collagen sponge.The method of water in oil in water(w₁/o/w₂) was utilized to prepare bFGF-PLGA microspheres.The effect of PLGA species and concentration in oil phase(o),polyvinyl alcohol(PVA) concentration in external water phase(w₂), emulsifying speed of primary emulsion,volume proportion of innner water phase(w₁) to oil phase(w₁/o) and volume proportion of innner water phase to external water phase(w₁/w₂) on microspheres properties was further studied.Depending on the different preparation parameters,the effects of making microspheres were observed, and the mean diameters and certain diameter distributions were detected.Through scanning electron microscope(SEM) detection,the microspheres seemed smoothly and showed different degrees of porous structure.Varying with preparation conditions,the maximum and minimum bFGF encapsulation efficiency within the polymeric microspheres were 85.8 and 43.6%,respectively.The experiment of vitro release was used to validate the feasibility of controlled release of microspheres.The release mechanism of hydrophilic drug from microspheres depended on polymer degradation and drug diffusion,so that the process of drug release could be sorted to three successive phases:â‘ initial burst release phase.The drug which was located on the surface or near surface of microspheres released quickly from microspheres;â‘¡slow release phase.The drug released from microspheres by means of diffusion or transfer.This was due to the formation of porous structure;â‘¢fast release phase.The drug released largely from microspheres with the destruction of porous structure which was caused by the degradation of polymer.Owing to the long period degradability of PLGA,the release mechanism of bFGF from bFGF-PLGA microspheres which were prepared in our lab depended on drug diffusion.The process of drug release was initial burst release phase and slow release phase.The result of vitro release experiment showed the feasibility of microspheres which were used to be the carrier of long controlled release.According to the guide line of the mean diameters and certain diameter distributions,higher bFGF loading,higher bFGF encapsulation efficiency and smaller bFGF initial burst release,the better preparation parameters in our lab condition were Confirmed as follows:PLGA species:75/25,molecularweight:50k,PLGA concen -tration:10mg/ml,PVA concentraiton:2%,(w₁/o/w₂:15:2:50,emulsifying speed: 30krpm.The microspheres morphology,mean diamerer,drug loading,encapsulation efficiency,initial burst release rate,diameter distribution,vitro release were set as evaluative guide line to study the microspheres stability under 4℃state.The results indicated that all the properties of microspheres had no evident changes in 4℃state. Otherwise,considering the bFGF conserving- temperature,the microspheres should be conserved in 4℃state.The experiment of bFGF-PLGA microspheres activity detection indicated that there were about 60%bFGF from bFGF-PLGA microspheres remained active.The cell proliferation states showed:in initial cultivation phase,the A value of bFGF groups was higher than two other groups;in midanaphase,the A value of bFGF-PLGA microspheres groups was higher than bFGF groups and banks.The results indicated that the free bFGF in bFGF groups were more than that in bFGF-PLGA microspheres groups in initial cultivation phase.However,in cultivation midanaphase,most bFGF activity in bFGF groups was missing.At the same time,the bFGF-PLGA microspheres could release active bFGF sustained,which could promote cell division growth.Therefore,the bFGF-PLGA micro- spheres prepared in our lab condition can sustained release active bFGF,which can hold on an effective density.All the above showed that bFGF-PLGA microspheres could use to be an ideal carrier of a long-term bFGF controlled release.The collagen was manufactured from the skin of porcine with pepsin digestion and neutral salt extractions.By adding HAc into collagen gel extracted freshly,we obtained a homogenic diluted acid dispersion of this insoluble collagen gel.The collagen sponge was prepared by lyophilization after freezing.We carried out one-step method to EDC/ NHS crosslinking and one-step simultaneous method to EDC/NHS crosslinking and heparin immobilization for modifying collagen sponge, which could enhance its mechanics performance.By combinating bFGF-PLGA microspheres with collagen sponge,we prepared special collagen scaffold embedding bFGF-PLGA microspheres.And the vitro bFGF release state and collagen scaffold degradation state were further studied.The results indicated that special collagen scaffold embedding bFGF-PLGA microspheres could relieve fast release phase effectively in vitro release process,initial burst release from 19.8%down to 14.6%.The vitro release bFGF density was relatively stable.The special collagen scaffold embedding bFGF-PLGA microspheres had a longer time for drug controlled release than microspheres itself.The bFGF-PLGA microspheres were successively prepared by w₁/o/w₂.The mean diameter was about nanometer,and a certain diameter distribution was formed. The microspheres took on intact sphericity and little porous structure by the SEM analysis.The porous structure might be the passage of drug diffusion.Twelve batches of bFGF-PLGA microspheres were prepared according to the preparation conditons to validate the technics stability,the bFGF loading was(60.8±0.56) 10^-3 *%,and the encapsulation efficiency was(80.5±1.22)%.So,the technics of bFGF-PLGA microspheres was considered to be well stable.The bFGF initial burst release of microspheres was 19.8%,the cumulative release was about 80%up to 45 days.By combinating bFGF-PLGA microspheres with collagen sponge,fast release phase was relieved effectively in vitro release process,initial burst release only 14.6%.The experiment of bFGF-PLGA microspheres activity detection indicated that there were about 60%bFGF from bFGF-PLGA microspheres remained active.Finally,it is expected that the bFGF-PLGA microspheres could be an ideal drug controlled release carrier in clinical applications,and that the special collagen sponge embedding bFGF-PLGA microspheres could be an ideal kind of wound dressing or tissue engineering scaffolds for deep thickness injure,regulate microenvironments for accelerate tissue repairin in vivo.