The Effect Of Phosphorylated Chitosan On Type 1 Collagen Biomineralization

Background:With the continuous development of the human economy,oral and maxillofacial severe trauma,cancer,inflammation and other diseases increasing,bone and soft tissue damage and loss not only affect the patient's chewing function and facial beauty,but also become a major threat to human life and health threats.In the field of dental restoration,due to the depth of the etching and resin monomer penetration depth does not match,the adhesive interface in the collagen fibers have been demineralized exposed and resin can not penetrate the wrapped water-rich areas.Lack of resin and mineral protection of the exposed collagen mechanical properties of poor,easily by matrix metalloproteinases(Matrix Metal Proteinases,MMPs)degradation,further damage the adhesive interface affect the resin-dentin bonding durability.Therefore,the repair and regeneration of bone,dentin and other hard tissue become the important issue of oral research.Objective:Based on the good biocompatibility of phosphorylated chitosan and the properties of chelating calcium ions,this study was to investigate the effect of phosphorylated chitosan on the progress of type 1 collagen in biomineralization.The particle size and phase transition time of ACP particles and the mineralization of type 1 collagen were characterized by transmission electron microscopy(TEM)and Fourier Transform Infrared(FTIR).To explore the mechanism of phosphorylated chitosan to promote the mineralization of type 1 collagen and its influence on the kinetics of calcium phosphate mineral transformation in the system.Biomimetic mineralization is a conceptual validation strategy that uses nanotechnology to mimic the process of biomineralization.The amorphous phase is widely present in the biomineralization process and acts as a precursor to the formation of hard tissue in the formation of dentin.The mineralization-related proteins regulate the size and phase of ACP to penetrate into the interior space of the collagen fibers to form highly complex organic-inorganic biomaterials.This mineralization strategy is a promising hard tissue repair method.In the study,we simulated the structural characteristics of non-collagenous proteins(NCPs)related to biomineralization,and use polyacrylic acid(PAA)stabilized metastable ACP to convert hydroxyapatite(Hydroxyapite,HAP)and to promote the demineralized dentin bionic remineralization,and re-mineralization dentin structure and microscopic mechanical properties and natural dentin similar.But there is a long time-consuming problem.Therefore,it is an urgent problem to explore a more time-consuming mineralization system.Chitosan,chitin the form of complete or partial deacetylation,is a necessary support structure in nature as several living organisms.Unique macromolecules have the advantages of non-toxic,antibacterial,antifungal and analgesic.In addition,it can support mucosal adhesion,show hemostatic properties,and is enzymatically biodegradable.The synthesis of new biologically functional chitosan derivatives by chemical modification has been extensively studied.This method preserves both the original physicochemical and biochemical properties of chitosan,and the newly introduced functional groups confer new properties.Due to the inherent biocompatibility and biodegradable behavior,this natural polymer composite material tends to synthesize composite biomaterials extensively.Methods:1.type 1 collagen solution preparation:the calf skin freeze-dried powder dissolved in 0.1 mol/L acetic acid solution(pH = 3.0)preparation of 0.2g/L collagen solution,4 degrees storage 24h stand by.Preparation of biomimetic remineralization medium.Acquiring remineralization2.Type 1 collagen self-assembly:0.2g/L collagen solution into the carbon-containing transmission of nickel mesh soak,placed in a container containing 1%ammonia,and 4h after the transfer to continue to culture 21h.To stabilize the collagen structure,with 0.5%glutaraldehyde cross-linking 1h,prepared by observing the sample 60 solution of certain concentration,PAA and Tris were added into calcium solution.3.Phosphorylated Chitosan Preparation:1 g of chitosan powder,5 g of urea and 10 mL of phosphoric acid were added to 40 mL of dimethylformamide(DMF),and the mixture was stirred at 150? for 1 hour in an oil bath.After cooling to room temperature,the solution was filtered and the precipitate was sufficiently washed with distilled water and absolute ethanol.The dried Pchi was then dried in vacuum to give 50 mg/ml of the solution.4.Preparation of biomimetic remineralization medium.Acquiring remineralization medium by blending calcium chloride solution and disodium hydrogen phosphate solution of certain concentration,PAA and Tris were added into calcium solution.The final concentrations of calcium,phosphorus,PAA in the experimental group were 10 mM,6mM,350ug/ml respectively.Finally,the prepared Pchi solution was added to the experimental group at a final concentration of 0.2 mg/ml.Specis were randomly divided into two groups:the reaction group was placed in the experimental group and the blank group.The reaction was carried out at a specific time point.Results:1.Type 1 collagen mineralization process and microstructure.The results of TEM showed that the mineralization was started after 36 hours of treatment,and the mineralization was completed after 72 hours.The density was higher and the collagen was thickened,and the mineralization process was accelerated obviously compared with the control group.2.Infrared spectrum results.In the experimental group,Pchi could form functional groups with the collagen-modified collagen surface.3.Phase change process.FT-IR results showed 36h,the experimental group and the control group ACP were phase change,the experimental group of mineral acid in the calcium phosphate in a relatively short period of time phase transition occurred,while the blank group of mineralized calcium phosphate Phase change in 48h.4.mineral morphology.The results of TEM electron microscopy showed that the inorganic particles formed in the experimental group were aggregated and adhered,while the control groupconclusion:Pchi can effectively promote the biomimetic mineralization of type 1 collagen.Infrared spectroscopy confirmed that pchi could modify the collagen surface and react with collagen.The mineralization of type 1 collagen was accelerated by transmission electron microscopy(TEM),and the content of inorganic matter on collagen surface was increased.The phase change of ACP was accelerated and the grade of collagen was formed in short time.The collagen was consistent with natural mineralized collagen.According to the structural characteristics of bio-mineralization-related proteins,the effect of NCP regulation on mineralization was simulated in vitro.Through the Pchi on the collagen modification and good calcium chelating effect,combined with its good antibacterial and good biocompatibility,in a short time to complete the re-mineralization of demineralized collagen,in order to improve the dentin resin bonding The durability of the complex and the construction of bone tissue engineering provide a meaningful biomimetic strategy.