Isolation And Purification Of Collagen From Jellyfish And Its Effects In Hemostasis And Wound Healing

Collagen is an extracellular matrix protein,which has a specific amino acid sequence and is widely present in connective tissues such as skin,bone,and tendon of animals.Collagen shows a good biocompatibility and low immunogenicity,and is often used as a variety of biomedical materials,such as artificial skin,drug delivery carriers,dressings,hydrogels,and so on.Livestock and poultry-derived tissues are the main way for people to obtain natural collagen,but it was found that the collagen prepared from cattle and pig could spread some diseases(mad cow disease,aftosa,etc.),and might be subject to religion and regional culture.Therefore people are constantly developing new sources of collagen.The umbrella tissue of jellyfish was reported to contain a large amount of collagen,and the yield of collagen based on dry weight of jellyfish ranged from 13.0% to 46.4%.Considering the enormous biomass of jellyfish,they may be an important source of collagen for biomedical applications.Our research group has been focusing on jellyfishderived bioactive substances for many years,so this paper focuses on the purification and medical application of jellyfish collagen.Injuries with seawater immersion are common in the marine environment.Due to the low temperature,high permeability and pathogenic bacteria in seawater,it is difficult for wound healing after seawater immersion.Therefore,effective hemostatic and promoting healing measures will greatly reduce the risk of injury deterioration after seawater immersion.Common dressings on the market,such as gauze and chitosan dressings,can play a role in hemostasis,but their effects are not satisfactory in the process of seawater immersion injury.Jellyfish collagen dressings may help to improve this situation.In this study,we isolated and extracted collagen from jellyfish umbrella tissue and prepared a jellyfish-derived collagen dressing.A rat model with seawater immersion injury was established and the hemostatic and healing promoting properties of the jellyfishderived collagen dressing were evaluated.MethodsPart Ⅰ: Purification of collagen from jellyfish exumbrella tissueFirstly,soaked the pickled jellyfish umbrella tissue with pure water,and then used Na OH to remove the protein,isopropyl alcohol to remove fat,and tried to retain collagen.Then,the crude collagen was further extracted by the combination of acid and enzyme,and the extraction conditions were optimized by changing the dissolution time,the amount of enzyme and the acid concentration.Dialysis,salting out and ultrafiltration were used to obtain high-purity collagen.Subsequently,sirius red method,hydroxyproline detection,UV,FT-IR,SDS-PAGE and amino acid composition analysis were used to identify the purified jellyfish-derived collagen.Finally,the solubility,isoelectric point and other physical and chemical properties of the collagen were explored.Part Ⅱ: Preparation and properties of jellyfish collagen dressingA jellyfish collagen dressing,a cross-linked jellyfish collagen dressing and a jellyfish collagen-chitosan composite dressing were prepared by freeze-drying,respectively.The water absorption and porosity of the three types of dressings were then observed and compared by scanning electron microscopy.Finally,the dressings were evaluated for safety,including using L929 cells to detect the cytotoxicity and diluted rat blood to test the hemolytic activity.Part Ⅲ: Hemostatic and healing promoting properties of jellyfish collagen dressingThe blood clotting index(BCI)of the dressing was characterized by an in vitro coagulation experiment,and the platelet adhesion rate of the dressing was measured by a platelet adhesion experiment.Then a rat model with seawater immersion injury was established,and the jellyfish collagen dressing,gauze,and chitosan dressing were evaluated.On the 3rd,5th,7th,and 14 th days,respectively,parts of the rats were euthanized and immunohistochemical analysis was used to explore the histological changes during wound healing.ResultsPart Ⅰ: Purification of collagen from jellyfish exumbrella tissueIn the extraction of collagen,the results of orthogonal experiment indicated that the amount of enzyme acid played the most critical role,followed by the extraction time,the material-liquid ratio,and the amount of acid used.The optimized conditions were: 0.4 mol/L acetic acid,0.3% pepsin,the ratio of material to liquid was 1:2 g/m L,and the reaction was performed at 4 ℃ for 72 h.Sirius red method and hydroxyproline test showed that the purity of collagen was 97%.SDS-PAGE electrophoresis analysis showed that jellyfish collagen had few hetero-bands and molecular weight was above 116 k Da,which was in line with the characteristics of type I collagen.The characteristic absorption peak of ultraviolet spectrum of jellyfish-derived collagen was at 220 nm,similar to the characteristic absorption peak of the standard collagen from cattle.Fourier transform infrared spectroscopy confirmed that the triple-helix structure of jellyfish collagen was preserved intact during the extraction process.In the analysis of amino acid components,jellyfish-derived collagen contained 34.8% of glycine,8.2% of proline,and 6.3% of hydroxyproline,conforming to the characteristics of primary structure of the collagen ternary component Gly-X-Y.The physicochemical property analysis of jellyfish collagen showed that the isoelectric point(p I)was about 7,and its relative solubility decreased sharply when the Na Cl concentration reached 0.9 mol/L.Part Ⅱ: Preparation and properties of jellyfish collagen dressingThe sensory evaluation of the dressing prepared by vacuum freeze-drying showed that the jellyfish collagen dressing was excellent in elasticity at a concentration of 3 mg/m L.The dressing surface was flat without large pores and it could recover its shape after pressing without impurities and spots,and after being folded without breaking and without cracks at the crease.The screened out conditions for dressing preparation were: jellyfishderived collagen(3 mg/m L),composite dressing(chitosan: collagen 1: 1)and crosslinker ratio(EDC: NHS 4: 1).Scanning electron microscope observation showed that the composite dressing had more pores than the cross-linked dressing and the jellyfish collagen dressing.The results of chemical properties of the dressing showed that the cross-linked dressing had the highest water absorption,and the composite dressing had the largest porosity.The cytotoxicity of the dressings was grade 1 and the hemolysis was less than 5%,showing that the dressings had no obvious cytotoxicity and hemolysis and met the requirements for medical devices.Part Ⅲ: Hemostatic and healing promoting properties of jellyfish collagen dressingThe platelet adhesion ability and in vitro coagulation experiments showed that the cross-linked jellyfish collagen dressing adhered the most platelets,had the smallest BCI,and showed more significant hemostatic ability than other dressings.The results of the rat model with seawater immersion injury showed that the positive expression ratio of CD31 and TGF-β in the cross-linked jellyfish collagen dressing group and the composite dressing group increased on 3rd and 5th day after treatment;on 14 th day,the positive expression ratio of CD31 and TGF-β gradually decreased.These results showed that the cross-linked jellyfish collagen dressing and composite dressing could play a good role in the initial stage of seawater immersion wounds,thereby shortening the healing time and improving the healing ability of seawater immersion wounds.ConclusionThe extraction method of jellyfish collagen was optimized,and the high-purity jellyfish collagen was separated in line with the characteristics of type I collagen.Three types of dressings,including jellyfish collagen dressing,cross-linked jellyfish collagen dressing and jellyfish collagen-chitosan composite dressing,were prepared by vacuum freeze-drying.The cross-linked jellyfish collagen dressing had a stronger water absorption and the composite dressing had a larger porosity.The cross-linked jellyfish collagen dressing showed more significant hemostatic ability than other dressings.The safety evaluation showed that all the three types of dressings had no obvious cytotoxicity and hemolysis.The results of the rat model with seawater immersion injury showed that the cross-linked jellyfish collagen dressing and composite dressing could shorten the healing time and improve the healing ability of seawater immersion injuries.