Controlled Crystallization Of Tooth-like Hydroxyapatite Under Organic Polymer Monolayer

The biogenetic formation of mineralized tissues such as enamel is a complex multi-step process including elements or compounds such as potassium, calcium, or phosphate and leading from precursor soft tissue, formed by extracellular matrix proteins (mainly collagen typeâ… ), to a mineralized tissue, mainly composed by hydroxyl apatite and some residues of the organic extracellular matrix. As adult tooth enamel is not living tissue, it hardly can remineralize after the substantial mineral loss. Many in vitro methods has been developed to produce the artificial enamel lesions for the use in de- or remineralization studies, which include the acidified gels, buffered solutions, exposure to acid vapor, or incubation with natural plaque. In addition, various surface-protective agents have been used to obtain lesions with characteristic surface zones.The formation of Calcium matrix proteins(mainly collagen typeâ… ) in bones and teeth are associated with the nucleation and growth of hydroxyapatite crystals. As the hydrolysate of collagen, gelatin shows this characteristics both in vivo an vitro. Based on the theory of "molecular recognition", we designed a cross-link reaction to get a bounding layer between gelatin/silk protein and tooth tissue to enhance the intensity. This organic molecules model can be used to induce the crystallization of hydroxyapatite, which is usefull to build a tooth-like calcium phosphate/hydroxyapatite under a controllable way in vitro.Human molars were sliced into 2mm thickness disks and etched with phosphoric acid/EDTA to reveal the different orientations of the enamel rod/dentinal tubule. Surface modification of decalcified enamel was conducted by low temperature plasma processing. Peptide bond compound: N,N-(3-dimethylaminopropyl) -N'-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuecinimide (NHS) were purchased from Medpep(Shanghai, China). The molars disks were incubated with EDC and NHS in 0.02M Phosphate Buffer Solution(0.02 M EDC, 0.05M NHS) for 30min at 37℃. Gelatin/silk protein was added into the suspension at concentration of 0.1M, and the mixture was incubated at 37℃for 2 h with gentle shaking. The variation of elements on the surface of sample were investigated by X-ray photoelectron spectroscopy (XPS, Kratos Analytical Ltd., Surface Analysis Product Group, United Kingdom).Disks of molar after crosslinking were soaked in calcification solution (SCS, 5mM CaCl₂, 5mM NaH₂PO₄, 1.5mM NaHCO₃, pH 6.31) for 24h at 37℃. Other group were soaked in artificial saliva(NaCl 1.71mmol/L, KCL 1.32mmol/L, CaCl₂ 1.02mmol/L, NaH₂PO₄ 0.90 mmol/L, Na₂S 0.004 mmol/L, CO(NH₂)₂ 4.16 mmol/L) for one month, supersede the environmental fluid every day. All the samples were studied by X-ray diffraction (XRD, X'Pert Pro MPD, Philips, Holland), SEM and XPS. The microhardness of the neonatal crystal were tested through Knoop hardenss testing. Periodontal fibroblast were cultivated with samples to evaluate the biological reaction of the neonatal crystal. Oral pathogenic microbes(Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Actinomyces viscosus, Candida albicans) were co-incubated with neonatal crystal to test the adhesion of these pathogenic microbes to neonatal HA.XPS analysis of gelatin/silk protein monolayer for samples cycle group revealed that the surface organic compositions is higher than that of normal dentin and decalcified dentin surface. We speculated that-NH, -NH₂,-NH₃ and -COOH were grafted to the surface of tooth.The XRD showed that the precipitation was calcium fluoride phosphate and Ca:P was 1.6. At the same time, SEM micrographs of biomaterial showed this composite a continuous structure of columns crystal with size of 10-40nm. Furthermore, there were column crystal with parallel direction inside, as same as the crystal array in the top of enamel rod. The hardness of neonatal crystal close to the normal dentin. The binding affinity between neonatal crystal and normal tooth is 20N.There are well bio-compatibility of neonatal crystal which confirmed by MTT test. There aren't differential adhesion between oral pathogenic microbes and neonatal crystal.According to the guide of block copolymer solution, the enamel-like calcium phosphate/hydroxyapatite compound material can be mineralized by biomimetic method. The specific functions of the copolymers can be used as a potential effective crystal growth modifiers.