Preparation And Crosslinking Of Nano-Hydroxyapatite/Collagen Bio-Composite In Carbodiimide

Hydroxyapatite is the main inorganic component of natural bone, and has goodbiocompatibility, bioactivity and osteoconduction. Hydroxyapatite has the calcium andthe phosphonium, the essential elements of the human body. When the hydroxyapatiteis implanted in the body, the calcium and the phosphonium could be educed in thehumoral and absorpted by the organism, forming chemical bond with the bone tissuefollwed by new bone formation. Thus, hydroxyapatite as the bone substitutes has beenused in periodontal regeneration widely. Nano-hydroxyapatite has better biocompatibilitythan dense hydroxyapatite. Nano-hydroxyapatite can promote the attachment,multiplication and Osteoblast phenotype differentiation of the periodontal ligamentcells, compared with the dense hydroxyapatite. Nano-hydroxyapatite is different in thephysicochemistry and the structure with the other form of hydroxyapatite, and has betterdissolvability, regeneration and bone conduction.Collagen is the main constitutive protein of the extracellular matrix, and the majorstructural proteins of connective tissue such as the skin, bone, cartilage, tendons,ligaments and blood vessel. There are more than nineteen types collagen, and thetype-Ⅰ～Ⅴ-collagen have been studied constantly. The type-â… -collagen has beenused in periodontal regeneration extensively. The collagen has been used as a barriermembrane in the guided tissue regeneration in the primal research. It has manyadvantages, such as participating in the metabolism of the periodontal tissue,conducting the periodontal ligament cells, restraining the locomotion of the crownepithelial cells.Hydroxyapatite and collagen are main inorganic and organic componen of naturalbone, widely applied in periodontal regeneration for both of them have good biologicalproperties.Nevertheless, the application of single material is very limitated to satisfy theneeds of tissue engineering. A bundle formation of collagen and c-axis orienteddeposition of HAp nanocrystals on it occurs automatically. HAp particles are uniformlydispersed in a collagen matrix, the COO- groups of the triple-helical conformation react with the Ca ions in the HAp surface via the electrostatic interactions. Ca²⁺ coordinatewith two PO₄^3- groups in the HAp surface and the COO- groups in the collagen surface.It is generally considered that heterophase nucleation initiate the HAp formation, and theCOO- groups in the collagen surface are the active situs to coordinate with the Ca ions,then the PO₄^3- groups form the threshold scale nucleation.This mechanism obviouslyresults in paralleling of the long axis of a collagen molecule to the c-axis of HAp.In this paper we study the derivation of the collagen, the preparation of the nanohydroxyapatite/collagen and the crosslinking of the composite, to provide a theoreticalbasis for the preparation and the application of the composite.The collagen which is extracted through the acetic acid hydrolysis and pepsindigestion from bovine tendon is studied in this paper. Collagen dispersion is purified bythe way of step-by-step depositon. The relative molecular weight of the collagen isstudied by the sodium dodecylsulphate- polyacrylamide gel electrophoresis(SDS-PAGE), and the structural analysis is studied by fourier transform infraredspectrum. The results indicate that the acetic acid hydrolysis and pepsin digestion canextract relatively high-purity collagen from bovine tendon. The relative molecular weightand the structural property of the collagen are coincidenced with the typeâ… collagen,and the triple helix of collagen is maintained successfully.On the basic of the methodology and its mechanism for the biomimeticself-assembly method, we perparaed the nano-HAp/Col composite, with the collagenextracted from bovine tendon, ammonium phosphate dibasic and calcium nitratetetrahydrate.These amounts of the starting materials were determined from thecalulation that HAp/Col obtained expect to have a weight ratio of 40/60, 50/50, 80/20under assumption that HAp forms ideally. The Crystalline structure,the crystallizationdegree, the chemical bond, microstructure and the composition of HAp/Col wereanalyzed by X-ray diffraction, fourier transform infrared spectra, scanning electronmicroscope, thermogravimetric analysis. The results indicated that the nanosizedinorganic phase of the newly synthesized material has low crystallinity and the size was10nm which resembles that of natural bone. There are chemical bonds for HAp and Colinteracting, and the carboxy group and the carbonyl group are the major nucleation sitefor HAp to precipitate on Col. The composite genesised carbonation during the preparation. Col was as the matrix and HAp precipitated on it uniformly, confluencingporosity structure. In the ratio of 80/20, the composite had the similar microstructure withthe natural bone. The composite had bound water, Col and HAp, the ratio wasconformity with the design.To improve the thermal stability, the swelling property and the anticollagenaseproperty of the composite, the composite was cross-linking by carbodiimide andsuccinimide in different buffer and different reagent concentration. The effect wereevaluated by fourier transform infrared spectra,differential scanning calorimetryanalysis,swelling ratio and anticollagenase in vitro. The results indicated that thecross-linking reacted between the Col molecules, and theα-amino group of the Colmolecules decreased, accompanied by the amide bond increasing, not effect thestructure of HAp. After the cross-linking, the glass transition inversion temperature of thecomposite increased, and the swelling ratio decreased, moreover, the property ofanticollagenase increased. When the pH value was 5.5, the thermal stability, theswelling ratio and the anticollagenase property of the composite were better than thetwo other conditions. Crosslinked the composite in the condition that the concentrationof the carbodiimide and succinimide were 0.08mol/L respectly, the composite gained thebetter property than in other conditions.