Green Manufacture Of An Environmentally Friendlybiomedical Material And Its Application

Based on the hazards and disposal difficulty of medical waste,as well as elimination of the source,the usage of biodegradable materials can reduce medical waste yield and simplify the disposal.Collagen is an important biodegradable material with excellent biocompatibility and low immunogenicity.Three-dimensional(3D)collagen biomedical sponges act as substrates to promote cell adhesion,migration,proliferation and differentiation,further to manipulate cell function and guide new tissue formation.They play important roles in meidical application.In this paper,we prepared recombinant human collagen through an environmental friendly way,this safe material was selected as key material,combined with natural polysaccharide chitosan and natural cross-linking agent proanthocyanidin(PA)to fabricate 3D porous biomedical sponges through regulation of molecular interactions and phase transformation,then biological activity of these sponges as new tissue engineering materials was further detected.The full-text includs the gain of recombinant human collagen by high-density fermentation and purification,optimization of PA cross-linked recombinant human collagen-chitosan biomedical sponges preparation,and their potential in biomedicalapplication;finally the disposal of waste water andutilization of yeast residues for cleaner production;The results as follows:(1)Based on the recombinant human collagen gene engineering bacteria constructed by our laboratory,high-density fermentation was carried out on a 100L fermentor.The objective protein was efficiently accumulated through fermental regulation.Finally,the wet weight of bacteria could reach to 449.2±12.2g/L and the dry weight of bacteria was 150.2±2.9g/L,while the total protein concentration was about 32.17±1.55g/L and the dimer of recombinant human collagen(RHC)concentration achived 16.87±0.70g/L.RHC was obtained through separation and purification.Thalli-supernant separation of the fermentation broth was carried out by low temperature-high speed centigeration,followed by dialysis and afiltration to purificate RHC.The purity of prepared RHC could achieve 98.08%,while the recovery was about 96.19%through High Performance Liquid Chromatography(HPLC)detection.The molecular weight of RHC was 112 kD detected by Polyacrylamide Gelelectrophoresis(SDS-PAGE)while its fibril diameter was 3?m?5?m under Scanning Electron Microscope(SEM).Ultraviolet and visible spectrum(UV-vis)?Fourier Transform Infrared Spectroscopy(FT-IR)?Nuclear Magnetic Resonance(NMR)characterization provided evidences that the purified protein was the aim RHC,of whichthe structure was similar to that of type III collagen.Differential Scanning Calorimetry(DSC)analysis showed the denaturation temperature of RHC was 40.68?.(2)Cell viability of RHC was detected.Water Tetrazolium Salts(WST-1)detection showed that the human cells viability were about 90%under RHC culture..Acridine Orange/Ethidium Bromide(AO/EB)fluorescence staining indicated that HUVEC maintained cell membrane integrity and proliferated when co-cultured with RHC.These detection indicated the RHC was a biocompatible material.(3)RHC-chitosan biomedical sponges were assembledwith cross-linking agentProanthocyanidin(PA)in two cross-linking methods(immersing and premixing).? Immersing method:Biomedical sponges were fabricated through regulating RHC/chitosan ratio(1:4-4:1),materials concentration(1.0%-2.0%,wt%)and phase transition(freezing rate 0.7?/min-4.1?/min,phase transiton temperature-20??60?)followed by immersing cross-linking in Phosphate Buffered Saline(PBS)(0.3M,pH6.81)with 0.5%(wt%)PA concentration.? Premixing method:Biomedical sponges were fabricated through regulating premixed PA concentration(0.1?0.5%5 wt%)and the followed neutralization condition(PBS concentration 0.01M-0.2M,pH 5.59-6.81)under condition of 1.0%(wt%)materials concentration,RHC/chitosan ratio 1:1 and freezing rate 4.1?/min.The optimized premixing condition was 0.3%(wt%)PA concentration while neutralization condition was PBS of 0.2M concentration and pH6.24.The parameters of premixing cross-linked biomedical sponges as follows:mean pore size was 115.01±3.1?m porosity was 86.4±1.1%and swelling was 3 9.3±1.8;compressive modulus was 889±2KPa when thermal mass loss rate was 48.99%(heating up to 500?)that thermal stability improved;the biodegradation rate was about 30%after collagenase and lysozyme function for 7dindicating that the resistance to the enzyme degradation was enhanced.(4)The toxicity of biomedical sponges was evaluated on cellular level.HUVEC cell growth was detected via WST-1 detection and AO/EB fluorescence staining after three dimensional culture in sponges.The results showed these PA immersing cross-linked biomedical sponges with small pore size were more proper to promote cell proliferation.Better cell adhesion,faster cell proliferation and futher cell migration could be observed in PA premixing cross-linked biomedical sponges when compared to culture in PA immersing cross-linked sponges.Severalproteins of cells cultured in biomedical sponges were evaluated on molecular level.The expression of the genes coding proteins that relate to interactions between endothelial cells and biomedical sponges was detected via qRT-PCR experiment.As a result,three dimensional culture of HUVEC in PA premixing cross-linked biomedical sponges could improve the gene expression of CD31?VEGF?Integrin ?1?COL ??MMP1 when compared to two dimensional culture and chitosan biomedical sponges three dimensional culture.It indicated that PA premixing cross-linked biomedical sponges could promote cell proliferation and the expression of vascularization related genesin endothelial cell.(5)Process the waste water produced during fermentation and purification to achieve non-pollution manufacture.Waste water disposal was carried out via A202(anaerobic-anoxic-two stage aerobic)-flocculation,the water was detected to approach the requirement of standard of Yeast Industual Waste Water Pollution Emission and Ferment Pharmaceutical Industual Waste Water Pollution Emission(GB21903-2008).The waste water also identified by Ames experiment,it indicated that the fermentation supernatant and water after disposal presented no significant mutagenic activity tosalmonella typhimurium,which ensured its safety to environment.(6)Resource of the yeast residue produced in fermentation and purificationto promote the comprehensive development.Extract Chitin-Glucan Complex(CGC)which is a high value product in cosmetic or biomedicine with excellent biocompatibility,inoxidizability and a cetain degree of antibacterial,anti-inflammatory.The optimized extration condition based onBox-Behnken Design(BBD)was NaOH 0.03 M,material-ratio 0.07,extration temperature 65?,and the response value was 16.3%.