| Chitosan and its derivates have been studying systematically as tissue repairmaterials, especially the biological functionality are focused in current chitosan research.A series of novel chitosan derivates were prepared by innovative cholesterol modificationin this study. The biocompatibility and mineralization of the cholesterol-g-chitosan(CHCS) were explored in detail. The obtained data and theories are hoped to be used forR&P of new bone repair materials.To investigate the effects of the cholesterol and lecithin contents on themicrostructure of hydroxyapatite, cholesterol and lecithin with certain ratio were added tochitosan and hydroxyapatite mix water solution. Then membranes were prepared by thecommon solution cast method and ageing in the lye. The microscopic structure ofhydroxyapatite in the composite membrane was investigated by SEM and TEM. Theresults show that with the increasing proportions of cholesterol and lecithin in thecomposites, the hydroxyapatite morphology changed from the spherical to the rod-likeform. As the mass ratio of lecithin and cholesterol was5:1, the morphology and size ofthe hydroxyapatite in composite materials were close to nature bone tissue. But in thecontrol system without lecithin and cholesterol, the microscopic structures ofhydroxyapatite were almost lamellate. The results revealed that the cholesterol andlecithin would affect the microscopic structure of hydroxyapatite.Cholesterol-g-chitosan (CHCS) was synthesized with succinyl linkages and thechemical composition was verified by Fourier transforms infrared (FTIR) and protonnuclear magnetic resonance (1~H NMR) spectra. The grafting ratio of cholesterol wascharacterized by elemental analysis. The results show that the grafting ratio ofcholesterol on chitosan chain was increased slightly with the quantity raw material ofcholesteryl hemisuccinate (CHS) increasing, but there were no linear correlation.When the raw molar ratio (the number of chitosan sugar ring compare to the numberof cholesterol molecule) were15:1,10:1and5:1, the grafting ratio (the number ofcholesterol molecule connected on100chitosan sugar ring) were3.71,4.02and5.94.The hydrophobility of CHCS was increased with the grafting ratio of cholesterol increasing as well. The contact angle of chitosan membrane was72.1°, but thecontact angles of the CHCS membranes were76.7°,81°and85.8°respective withthe grafting ratio increasing. The mass of BSA absorption on membrane materialsincreased with the grafting ratio increasing. The mass of BSA absorption on chitosanmembrane was0.354μg, but the value increased from7.664μg to16.815μg on CHCSmembrane with the grafting ratio increasing. The results also demonstrate that3T3cells adhesion and proliferation on CHCS were quicker than that on chitosanmembrane. Furthermore lamellipodia and filopodia of fibroblast3T3cells on CHCSmembrane spread out more fully than that on chitosan membrane after cultured1day.Extracellular matrix express on CHCS scaffold more than on chitosan scaffold after3days and the threadiness extracellular matrix increasingly with the graft ratioincreasing.Two biomimetic mineralization systems were performed in this study tocharacterize the mineralize ability of the products: in simulated body fluids (SBF) andrapid mineralization. In the first system, the calcium phosphate apatite precipitated onCHCS membrane was more stable than that on chitosan membrane. In the latter system,spherical granulates were observed on different graft ratio CHCS scaffold at24h bySEM, and on chitosan scaffold was amorphous. After rapid mineralization48h, theshape of calcium phosphate apatite were island needle-like on low graft ratio CHCSscaffold, and ordering lamellar accumulate on high graft ratio CHCS scaffolds. Theresults of the interaction between CHCS and osteoblast show that osteoblast attach andproliferation more ordering, easily and quickly on CHCS membrane than that onchitosan membrane. The feature morphology of osteoblast on CHCS membranemaintain better, and there are more osteoblast pseudopodia stretch out on CHCSmembrane than on chitosan membrane. Especially, high graft ratio CHCS have thetendency of promoting osteoblast proliferation and restraining osteoblast differentiationin certain time, and facilitating osteoblast differentiation after7days.
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