Biomimetic Synthesis And Separating Protein With Histidine Grafted Chitosan Materials

The research and development of tissue engineering in medicine has attractedpolymer scientists and the likers in pursuit of new biomaterials as scaffold. Both ofchitosan (CS) and poly(L-lactide)(PLLA) are well-known biomaterials. Histidine hasa good pH-responsive group of imidazole. The groups of hydroxyl and unsubstitutedamino are retained on histidine grafted chitosan, and the imidazole group is presentedon histidine grafted chitosan. Therefore, histidine grafted chitosan have borne bothcharacteristics of good bioactivity and pH-sensitive behavior. Blending two polymersis an approach to develop new biomaterials exhibiting combinations of properties thatthey can not be obtained by individual polymers. A series of histidine graftedchitosan (NHCS) with different substitution degrees were prepared in this paper,which will make chitosan and PLLA use in biomedical materials. The novelbiomaterials of NHCS and NHCS/PLLA were prepared, and were applied inbiomimetic synthesis and separating protein in this paper.Chitosan was used as target polymer to be modified by histidine,1-(3-dimethylamino propyl)-3-ethyl carbodiimide hydrochloride andN-hydroxysuccinimide. The histidine was modified to chitosan, and the histidinegrafted chitosan (NHCS) was synthesized finally. NHCS scaffolds (Ns) and NHCSpowers (Np) were prepared by freeze-drying and concentration-drying respectively. Aseries of NHCS with different substitution degrees were prepared by changing themolar ratio of histidine to chitosan and the molecular of chitosan. Chemical structureand morphology of NHCS were characterized through Fourier transform infraredspectrometer (FT-IR),1H NMR spectroscopy, elemental analysis, thermal gravimetricanalyzer and scanning electron microscope. The results indicated that: chitosan weregrafted successfully. The degree of substitution and the pore size of NHCS wereaffected by the molar ratio of histidine/chitosan and the molecular of chitosan. Thedegree of substitution was between3~11%, which increased with increasing themolar ratio of histidine/chitosan and decreasing the molecular of chitosan. The poresize distribution was5~120μm, the porosity was more than85%, which could beapplied in the fibroblasts, skin tissue reconstruction and bone tissue engineering. The porous composite scaffolds of NHCS/PLLA were prepared through the dualphase separation technique by changing the mass ratio of NHCS/PLLA and the typeof NHCS. Chemical structure, morphology and mechanical properties ofNHCS/PLLA composite scaffolds were characterized through FT-IR, X-raydiffraction (XRD), thermal gravimetric analyzer (TGA) and field-emission scanningelectron microscope (FESEM). The pore size of NHCS/PLLA composite scaffoldsdecreased with the decrease of the mass ratio of NHCS/PLLA, making the density ofNHCS/PLLA scaffolds larger. The pore size of NHCS/PLLA composite scaffoldswas about13~18μm, the porosity was greater than92%. The comprehensive strengthand the comprehensive modulus were0.33~0.78MPa and1.75~5.28MParespectively, which could be applied in cartilage tissue engineering.NHCS powder was used as the organic matrix for biomimetic synthesis ofhydroxyapatite (HAP) in simulated body fluid system. The effects of molar ratio ofhistidine/chitosan, molecular of chitosan, initial concentration of Ca2+ions,temperature and aging time on morphology and polymorph of hydroxyapatite wereinvestigated. Hydroxyapatite was synthesized in simulated body fluid system andaqueous system respectively, which were used as a control without addition of NHCS.The products were characterized by FT-IR, XRD and FESEM. Micro-hydroxyapatitewas formed in absence of NHCS. However, spindle and sphericalnano-hydroxyapatite were obtained in presence of NHCS. The result indicated thatNp7(the molecular of the chitosan was50kD, the molar ratio of histidine to chitosanwas2:1) was the best for controlling the morphology and size of hydroxyapatite,when the dosage of Np7was0.01g, the initial concentration of Ca2+was0.01mol·L-1,the reaction temperature was37.0℃, the aging time was24h. The size of theobtained spindle HAP was about200nm×40nm. It was similar as bone because theobtained HAP contained a few carbonate hydroxyapatite. In addition, the possiblemechanisms of formation of spindle and spherical nano-hydroxyapatite wereproposed, which could be helpful to offer a new method for biomimetic synthesis ofhydroxyapatite, and bone repair.NHCS powder was used as the organic matrix for biomimetic synthesis of calciumcarbonate in aqueous system. The effects of molar ratio of histidine/chitosan,molecular of chitosan, initial concentration of Ca2+ions, pH value, temperature and aging time on morphology and polymorph of calcium carbonate were investigated,and used as a control in absence of NHCS. The products were characterized by FT-IR,XRD and FESEM. The result showed the sole rhombohedral calcite phase wasformed in absence of NHCS, whereas the coexistence of vaterite-calcite phases wasgained in presence of NHCS. The content of vaterite reached93.7wt%at pH8.0.However, it was only62.2wt%at pH6.5. The flaky-floret and multilayered vateritecould slowly convert to calcite with increasing the aging time, but the content ofvaterite could maintain86.2wt%stably after24h. In addition, the possiblemechanisms of formation and stabilization at different pH values and different agingtime were proposed to explain the formation of coexistence of vaterite-calcite phaseswas discussed in the study. The result indicated that NHCS was an effective softtemplate and pH responsive matrix to control the morphology and polymorphs ofcalcium carbonate. This study could be helpful to offer new method for the controlledsynthesis of novel biomaterials and understand the mechanism of biomimeticsynthesis.Adsorption behavior of bovine serum albumin (BSA) by NHCS and NHCS/PLLAscaffolds in simulated body fluid (SBF) were investigated, while the adsorptionisotherm, adsorption kinetics and thermodynamics of behavior were studied too. Theresults indicated that these scaffolds were effectively to adsorb BSA, which theadsorption capacity were about335.84~1048.64mg·g-1, Ns10and NPs3were the bestfor adsorbing BSA of all NHCS scaffolds and NHCS/PLLA composite scaffoldsrespectively. The adsorption capacity of them were820.90mg·g-1and928.53mg·g-1respectively. Ns10and NPs3could be used repeatedly. The adsorption capacity ofthem were found to decrease by~1.00%after recycled for five times NHCS scaffoldsand NHCS/PLLA composite scaffolds could be reused for separation and purificationof BSA, which could provide a carrier for other protein and tissue engineering.