Investigation Of Accelerating Wound Healing Of Marine Collagen And Electrospun Bovine Collagen Nanofibers

Collagen has excellent physical and mechanical properties,good biocompatibility,safety and low immunogenicity.Collagen can promote cellular adhesion and proliferation,vascularization,granulation and epithelial growth,to be able to accelerate wound healing,can be used as good material.Electrospun collagen nanofibers have excellent biology characteristics of collagen and high specific surface area,high porosity,pore connectivity,three-dimensional structure and the bionic natural extracellular matrix of electrostatic spinning nanofibers,was expected to become the excellent biomedical tissue engineering material.Evaluation of biomedical tissue engineering material was the key of preclinical study.Collagen cooperate with polyethylene oxide(PEO)or chitosan,was prepared for electrospun collagen nanofibers.The efficiency of electrospun collagen nanofibers was evaluated,including accelerating wound healing,bacteriostatic and hemostasis,to provide research basis for electrospun collagen nanofibers as biological dressing.The first chapter introduces research progress of electrospun collagen nanofibers,including status of collagen,electrostatic spinning technology,advantages of electrospun collagen nanofibers,clinical application and wound healing and so on.(1)The second chapter studies antibacterial properties of collagen and electrospun collagen nanofibers.To explore antibacterial activity of bovine skin collagen(BSC),pepsin soluble collagen from tilapia skin(PSC)and acid soluble collagen from tilapia skin(ASC).Tilapia skin was extracted,centrifuged,dialyzed and freeze-dried to obtain BSC,PSC,ASC.E.coli,S.aureus,P.aeruginosa and C.albicans were cultured in tryptone soy broth medium containing BSC,PSC or ASC.Absorbance values of bacteria,the number of bacteria,electric conductivity and morphology characteristics of bacteria were evaluated regularly.The results indicated that BSC,PSC or ASC showed bacteriostasis on E.Coli,S.aureus,P.aeruginosa and C.albicans.Antibacterial rate was from 22.97%±2.35% to 65.21%±6.42%.The effect of PSC restrain P.aeruginosa was significant,antibacterial rate was 65.21%±6.42%;the antibacterial rate of ASC on E.coli was 22.97%±2.35%.The result of hemacytometer was similar to absorbance value.Electric conductivity of bacteria conducted by PSC or ASC was from 36.42±1.35?s/cm to 185.80±4.70 ?s/cm.Electric conductivity of P.aeruginosa treated by PSC was 36.42±1.35?s/cm.Electric conductivity of C.albicans treated by ASC was 185.80±4.70 ?s/cm.Deformation and fracture of bacteria were observed with scanning electron microscope.Conclusion Compared with the BSC,PSC and ASC showed high antimicrobial activities bacterial presented by antibacterial rate,electric conductivity and morphological characteristics.(2)BSC was dissolved in a mixture of hexafluoroiso-propanol(HFP)and acetic acid solution(volume ratio is 50:50)with the weight concentration of 4%.The weight ratio of bovine collagen and polyethylene oxide was 90:10.The polymer was then placed in a plastic syringe and inserted in a syringe pump(789100C,Cole-Parmer,USA).The voltage and flow rate during electrospinning were 12 kV and 1.0 m L/h,respectively.The distance from the needle to the aluminum foil collector was 20 cm.EBCN formed membranes were further crosslinked using glutaraldehyde vapor and stored in a vacuum-drying oven.The bovine collagen was preparated with polyethylene oxide(PEO)which is EBCN?.In contrast,the bovine collagen was preparated with PEO and chitosan which is EBCN?.The weight ratio of bovine collagen,PEO and chitosan is 150:17:2.Antimicrobial of EBCN ?,EBCN ?was test according to the above method.By measuring the absorbance values,the amount of bacteria,the result was that EBCN ?,EBCN ? had no bacteriostasis.The third chapter study wound healing of collagen and electrospun collagen nanofibers.In this investigation,BSC,PSC,ASC,EBCN ?,EBCN ? were used for wound dressing,to evaluate their accelerating wound healing and mechanism.Wound healing rate,healing time,total protein,hydroxyproline content,pathological biopsy and immunohistochemical analysis were determinated as indicators.The results show that PSC,ASC,EBCN ?,EBCN ? enhanced total protein and hydroxyproline level in the rats.It could induce fibroblasts proliferation and differentiation,accelerate the formation and deposition of collagen,granulation tissue formation and epithelization,resulting in accelerating wound healing.By raising the level of EGF,FGF and CD31,PSC,ASC,EBCN ?and EBCN ?improve wound healing and quality of SD rats.The fourth chapter studies hemostatic properties of collagen and electrospun collagen nanofibers.The ear vein,ear arteries,liver bleeding wounds were prepared and coagulation index in vitro was determinated,in order to explore hemostatic properties of BSC,PSC,ASC,EBCN?,EBCN ?.The bleeding time and blood losses of New Zealand rabbit ear vein in ASC,EBCN,EBCN ? was reduced distinctly compared with control group.There was no significant difference between BSC,PSC with control group.The bleeding time and blood losses of New Zealand rabbit ear arteries in EBCN?,EBCN ? was reduced distinctly compared with control group.In the study of New Zealand rabbit liver bleeding,PSC,EBCN ?,EBCN ? could significantly shorten the bleeding time,and ASC,EBCN ?,EBCN ? could obviously reduce blood losses compared with control group.The coagulation index in vitro of EBCN ?,EBCN ?was significantly lower than control group.EBCN?,EBSCN ?can shorten the bleeding time and reduce blood losses.PSC,ASC also can shorten the bleeding time and reduce the bleeding,but the effect is not obvious.