| Part 1 The study on mineralization of collagen from eggshell membrane induced by different acidic amino acidsObjectives:To evaluate the effect of different acidic amino acids inducing mineralization of type ? collagen from eggshell membrane--outer membrane in the mineralization system containing mesocrystal (HAP coated by ACP) in vitro.Materials and methods:Type ? collagen from outer membrane of eggshell membrane was prepared, digested, de-crosslinked, and phosphorylated for use. Nanospheres containing hydroxyapatite coated by amorphous calcium phosphate were synthesized under high speed centrifugation, then dispersed in buffer solutions to generate mesocrystal mineralization system in vitro. Glycine, glutamate acid and aspartic acid were used to induce mineralization of blank and phosphorylated outer membrane from eggshell membrane in this mineralization system for one week respectively, and the effect of mineralization of each group was evaluated by scanning electron microscope (SEM).Results:Fourier transform infrared spectrum (FTIR) and SEM show that the model of blank and phosphorylated type ? collagens has been created successfully. The results of TEM manifested that the mesocrystal mineralization system has been set up, in which the aggregates (HAP coated by ACP) were synthesized. No matter phosphorylated or not, glycine could not induce mineralization of type ? collagen from eggshell membrane in one week, while needle-like crystal similar to enamel was induced by glutamate acid, and rod or plate-like crystal which elongated along the long axis of collagen and was induced by aspartic acid. Furthermore, in the last two groups, there were more minerals produced after type I collagen was phosphorylated.Conclusions:Glutamate acid and aspartic acid could induce mineralization of type I collagen from eggshell membrane in the mesocrystal mineralization system in vitro. Most of the crystal induced by glutamate acid is needle-like which is similar to enamel crystal, while rod or plate-like crystal deposited in aspartic acid group and elongated along the long axis of collagen. Besides, phosphorylation could facilitate mineralization of both glutamate acid and aspartic acid groups.Part 2 The study of functional peptide inducing mineralization of reconstituted type I collagenObjective:To design and synthesize amphiphilic functional peptide DSESSEEDRSVSVGMKPSPRP, and evaluate the capacity of which to induce the mineralization of reconstituted type 1 collagen.Materials and methods:TEM grid was soked in acidic collagen solution and neutralized carefully using the ammonium hydroxide to promote collagen cross-linking and reconstitution. The effect of collagen recombination was evaluated through TEM. An amphiphilic functional peptide DSESSEEDR-SVSVGMKPSPRP was designed and synthesized. TEM grid fixed the peptide was immersed into calcium, phosphorus solution alternatively to make it mineralized, and the ability of which to absorb the inorganic ions and to induce the calcium phosphate crystals to nucleate, grow and crystallize was assessed by TEM. For another side, the capacity of specific affinity of peptide to dentine surface was evaluated by fluorescence microscopy, and depth of the peptide penetrated into the interior of dentin was assessed by CLSM after FITC labeled peptide incubated on the surface of dentin. TEM grid with reconstituted type I collagen was soked into the mineralization solution with functional peptide at 37? for 10 days and the mineralization effect was assessed from morphology and crystallographic characteristics by TEM.Results:Banded reconstituted type I collagen was observed by TEM, and it extended with a period of 67 nm, light and dark, form an overlapping region (overlap) along the long axis of collagen, and leave a gap in the terminal of molecular (gap), which is in line with the periodic structure of self-assembly collagen fibers. SAED results showed that it had no crystallographic characteristics. After alternating mineralization, functional peptide can induce a large amount of deposition with high electron density, and showing a significant growth orientation, SAED indicated the typical crystal ring structure, with crystallographic characteristics. Fluorescence microscopy suggested specific and high affinity of functional peptide to dentine. Confocal laser scanning microscope showed the peptide can even penetrate into the interior of dentin, and the depth is at least 5 um. After the mineralization of reconstituted type I collagen induced by peptide for 10 days, a lot of deposition with high electron density was visible around collagen periodically, the collagen structure is misty. SAED suggested crystal with low crystallinity was formed.Conclusions:Type I collagen was reconstituted successfully, with clear structure, no stacking, which is in favor of study; functional peptide was designed and synthesized which is capable of binding to type I collagen specifically, locating nucleation sites, on the other hand, absorbing minerals ions, and guiding their nucleation, growth, crystallization; besides, this peptide has the ability to induce the mineralization of reconstituted type I collagen.Part 3 The study of functional peptide on remineralization of partially demineralized dentin collagenObjectives:To evaluate the ability of amphiphilic functional peptide DSESSEEDR-SVSVGMKPSPRP on inducing the remineralization of demineralized dentin.Materials and methods:Intact extracted caries-free human third molars were collected and used within 1 month. The root and occlusal enamel of the teeth was removed, dentine discs were prepared and wet-grinded step-by-step, following by ultrasonic cleaning. The dentine surfaces were etched with 37%phosphoric acid for 15 sec, simulating the etching procedure of clinical dentin bonding and preparing partial demineralized dentin model. The dentine discs were placed in peptide solution and incubated for 24h, then soaked in the mineralization solution containing peptide to mineralize at 37? for 1,2 and 4 weeks, respectively. The as-mineralized specimens were dehydrated, dried and gold-sprayed, the remineralization condition was observed by FE-SEM.Results:After mineralizing the demineralized dentin by mineralization solution containing peptide for 1 week, a few mineral deposition was found on collagen fibers periodically, leading to bead-like changes, and resemble deposition was appeared on the intratubular collagen of the deep dentin as well. After mineralizing for 2 weeks, dentine surfaces were deposited by evident minerals which were focused mainly on the surface of the peritubular dentin, the tubules still opened and certain regions were half-covered. At high magnification, newborn minerals were primarily needle-like crystal, mineral deposition was also observed in deep dentinal tubules. After mineralizing for 4 weeks, dentine surfaces were covered by a mount of newborn minerals, the dentinal tubules and other special structure of dentine were completely covered, newborn crystals were piled up. At high magnification, newborn crystals were sheet-like or plate-like, which is in according with the morphology of mature hydroxyapatite crystal.Conclusions:Peptide DSESSEEDR-SVSVGMKPSPRP could induce both of the gap mineralization and surface mineralization. After interaction with collagen, the peptide locates the nucleation site and forms an ordered structure. It is beneficial for regulating the growth of the absorbed minerals along the c-axis which is paralleled with the long-axis of collagen fibers, facilitating the conversion of the minerals from calcium phosphate precursor to HAP. |
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