Research On The Collagen Self-assembly Behavior In Vivo And The Thermal Stability Of Collagen Fibers By Fluorescent Probe

With the increased application of collagen in the biomedical tissue engineering collagen has become a hot spot in the global research Because the fish-derived collagen has the advantage of wide sources low price good security In addition the global outbreak of mad cow disease and foot and mouth disease zoonotic disease has caused mammal collagen security problems so the use of fish-derived collagen replaced the mammal collagen has become an inevitable trend in order to satisfy the demands of application in the field of biomedical materialsAt present, the study of the fish-derived collagen is lack of system and depth especially for the fish-derived collagen molecular behavior and self-assemble of fibril has not yet to grasp fully to a large extent this has affected the further development and application of fish-derived collagen products This study attempts to use the fluorescent probe tracking of collagen self-assembly behavior in vitro and thermal stability of conducting fiber which provides the theoretical foundation and new product development for the fish-derived collagenThe study is focused on the snakehead fish pepsin-soluble collagen as the main research object according to the characteristic changes by the molecular structure and the solvent in the process of collagen assemble micro probe fluorescence spectrum through the introduction of fluorescent probes the thioflavin-T and pyrene which caused by environment change tracking of collagen fiber behavior and compared with the traditional turbidity experimental method The turbidity experiments show that the thioflavin-T does not affect the self-assembly behavior and which lead to the change of the fluorescence intensity through the specific combination with collagen fiber Compared the turbidity experiments with fluorescence intensity experiments the results show that the fluorescence intensity change curve with thioflavin-T is the same with the turbidity curve which confirmed that the fluorescence probe method can effectively track the collagen assemble behavior in vitro At the same time in fluorescence curve the collagen fiber formation time is in advance than the turbidity experiments however the assembly balance time lag This shows that fluorescent probe thioflavin-T can be sensitive to subtle changes in the process of collagen assemble were characterized After adding the fluorescence probe pyrene the fluorescence spectrum characteristic is also change along with the collagen assembly process but the curves of fluorescence spectrum is not consistent with the curve of turbidity because its fluorescence characteristic changes mainly affected by the polarity solvent system change with the collagen fiber formation at the beginning of the assembly fluorescent characteristic ratio rose briefly but with the extension of the assemble process its fluorescence characteristic ratio decreasing Compared the turbidity experiment and the fluorescent probe thioflavin-T the fluorescence probe pyrene has good detection effect on the collagen fiber aggregation state of assembleResearching the dissociate behavior of collagen fiber with fluorescent probe thioflavin-T The research results show that the collagen fiber will dissociate when they are heated but its dissociation temperature lower than that of collagen denaturation temperature dissociation rate along with the increase of temperature The CD method is used to determine denaturation temperature for snakehead fish bullfrogs and pigskin collagen is 46 47 51 respectively and the fluorescence method was developed for the determination of the temperature of collagen fiber dissociation was 37 39 42 respectively The analysis of SEM TEM and DSC results show that when the collagen fibers was under the condition of heat treatment when the temperature above fiber dissociation temperature and below the collagen denaturation temperature the collagen fibers in dissociation behavior but exist the three helical molecular structureBased on the research above studying the thermal stability of collagen fibers under the condition of different assembly changes using fluorescent probe thioflavin-T By research the dissociation behavior with changing the assembly conditions and the quantitative comparison of activation energy in the different types of collagen The research results show that the different condition of assembly temperature(20 25 and 30) and different assembly time(30 min 1 h 8 h 12 h 24 h) the dissociate temperature of fish-derived collagens is 39 which shows that different assembly condition and assembly speed the dissociate temperature of the collagen is in consistent the chemical and physical bonds do not change By activation energy analysis experiment with the extension of assembly time the activation energy is increasing the activation energy of assembly 12 h is bigger than assembly 1 h but the assembly activation energy of the 12 h and 24 h assembly is almost the same that extend the time of assembly collagen fibers in the collagen molecules between the single molecule function more dense On this basis the activation energy of the collagen fibers in different species were determined and compared the results show that there exists significant difference between them is also different as follows pig > bullfrog > snakehead fish...