The Role Of Silk Fibroin In Controlling The Crystallization Of Calcium Phosphate

Over billions of years of evolution,organisms in nature,have developed specific biological structures to adapt to the harsh living environment.Taking lessons from these specific biological processes is helpful to develop new techniques,and fabricate materials with specific structures and functions.Biomineralization,as a typical bioprocess in nature,has been widely studied.Due to the participation of cellular metabolism and organic matrix in organisms,which is efficient to obtain the composite materials with exquisite structures and high performance.Learning from the biomineralization process,we have understood that the amorphous phase of mineral transformation and assembly in organisms is subjected to the control of macromolecules and cells.However,the preassembly of organic matrix effect on the formation process of minerals has not been fully understood.Hard tissue such as bones has been confirmed with the composition of collagens and hydroxyapatite.However,the intermediate state of the formation process has not been well understood.Silk fibroin is similar to that collagen in bone,as a structural protein.In addition,silk fibroin has a unique molecular recognition site and self-assembly characteristics,as well as a multilevel orientation arrangement structure,which makes silk fibroin be used as a substitute for collagen in bone tissue engineering.With the wide application of silk fibroin and calcium phosphate in bone tissue engineering,the mechanism of silk fibroin regulating the crystallization process of calcium phosphate remains unclear.Therefore,in this thesis,we discussed the regulation of silk fibroin on calcium phosphate formation process from three aspects as follows:Firstly,in specific environments,the effect of silk fibroin framework on calcium phosphate transformation was explored.Silk fibroin film as insoluble framework treated by SBF to induce the amorphous calcium phosphate（ACP）formation on the surface.The transformation of ACP was observed with three different biological environment treatments.In protease solution,degradation of silk fibroin of silk fibroin results the p H change of the solution can affect the crystallization of ACP.The degradation released silk segments can also regulate the transformation of ACP to HAp.However,in PBS solution,the change of p H and degradation of silk fibroin becomes moderate.Due to the ACP partial transformation,TCP and HAp mixture is found in PBS solution.The ACP also changed to HAp in the cell culture environment,but the morphology of minerals was different from that of the enzyme solution.The cell culture environment and the extracellular matrix secreted by osteoblasts can affect the mineralization process.This study not only provides deeper understanding of the biological environment influence on the transformation of amorphous calcium phosphate,but also inspires the material fabrication for bone tissue engineering.We further discuss the process of silk fibroin matrix interferes with calcium phosphate formation process and regulate the assembly structure of the mineral.The formation of calcium phosphate with the specific structure was induced by using silk film as an insoluble template.The existence of silk film changes the mineralization pathway of calcium phosphate.The ammonia induced mineralization process and forms a composite structure of monetite（DCPA）and silk fibroin,and the silk fibroin inside acts as a template to stabilize the crystal structure and induce mineral oriented crystallization.Finally,the oriented DCPA nanocrystals were assembled to form the hierarchical structure under the regulation of silk fibroin.Finally,we explored the influence of silk fibroin composition and self-assembly behavior on calcium phosphate crystallization.The silk fibroin film was prepared from the silk solution（SF）,and the water-soluble part（WSF）was obtained by dissolving the film in di H₂O.The insoluble part of the silk film was redissolved in Li Br solution as ISF.The crystallization process of brushite（DCPD）was regulated by SF,WSF and ISF solution.Results show that WSF first form insoluble clusters and combine with DCPD for further regulation.The ISF and DCPD initially formed hybrid structure in the solution and then ISF inside the structure mediate the growth of the crystal.Both regulation modes are found in the DCPD growth in SF solution.These regulation methods of silk fibroin on mineral growth are the same as the growing process in which proteins regulate mineral formation in nature.Studying these mineralization processes can provide a new way for materials fabrication.