Study On The Mechanism Of Collagen Mineralization In Vitro And Construction Of Collagen Mineralization Model

Biomineralization is carried out on the basis of organic matrix(osteoid),which is mainly composed of collagen secreted by organisms.Pre-organized organic matrix is mainly composed of water-soluble collagen fibers and a small amount of water-soluble noncollagenous proteins(NCPs).Recent studies have shown that type I collagen fiber is a passive template for mineral deposition,which plays an active regulatory role in the mineralization process.However,the collagen fiber itself cannot induce mineralization independently.NCPs are mainly charged proteins,especially negatively charged proteins.It can not only combine with collagen fibers,but also induce the mineralization of collagen fibers by regulating the form,phase transformation,nucleation and growth of inorganic mineral calcium and phosphorus.At present,in a large number of studies on biomimetic mineralization of collagen,charged macromolecular polyelectrolytes such as polyacrylic acid(PAA)and poly-aspartic acid(PAsp)are usually used to simulate the function of NCPs to regulate the mineralization of collagen fibers.However,there are also contradictions and defects between various theories of collagen fiber mineralization at present,and the specific mechanism of collagen fiber mineralization still needs to be further explored.1.Objective.In view of the current problems in the biomimetic mineralization mechanism of collagen,the purpose of this study is to explore the biomimetic mineralization mechanism of collagen furtherly,so as to supplement or add new insights to the whole biomimetic mineralization system of collagen and deepen the understanding of the mineralization process of collagen fibers.In addition,the research on biomimetic mineralization of collagen has not paid enough attention to the extrafibrillar mineralization,description of the characteristics of extrafibrillar mineralization is less,and the theory of extrafibrillar mineralization is missing.In view of this problem,we further explore the mechanism of extrafibrillar mineralization under the guidance of the model of mineralization intrafibrillar mineralization.The two processes of extrafibrillar mineralization and intrafibrillar mineralization are connected as a continuous process,and a complete collagen fiber mineralization mechanism is proposed.We expect to build an ideal model of collagen fiber mineralization in vitro through this mechanism,so as to perfectly replicate the collagen fiber mineralization in vivo.2.Methods.(1)Construction and characterization of polyelectrolyte-calcium phosphate complex.By stabilizing supersaturated calcium phosphate solution with polyanionic polyelectrolytes(polyacrylic acid,PAA;poly-aspartic acid,PAsp;molecular weight less than 6 kDa),polyelectrolyte-calcium phosphate precursor clusters(PAA-CaP and PAspCaP)were constructed for the study of collagen fiber mineralization.The construction of PAA-CaP and PAsp-CaP calcium phosphate precursor clusters was detected by transmission electron microscopy(TEM),Fourier transform infrared spectroscopy(FIRT)and X-ray diffraction(XRD);The particle size of calcium phosphate precursor clusters was analyzed by dynamic light scattering(DLS);Zeta potential was used to detect the surface charge of calcium phosphate precursor clusters;The secondary structures of PAA,PAsp,PAA-CaP and PAsp-CaP were characterized by circular dichroism(CD).The interaction model of polymer PAA and PAsp with Ca2+ion and HPO42-ion was built by molecular dynamics(MD)simulation to simulate the adsorption process of PAA and PAsp polymer with calcium and phosphorus ion.(2)Exploration of the mechanism of intrafibrillar mineralization.Collagen molecules were self-assembled into monolayer collagen fiber model loaded on the nickel mesh and prepared into collagen membrane as unmodified collagen model.Poly-lysine(PL,molecular weight Mw:3 kDa)and polyaspartic acid(PAsp,molecular weight Mw:4-5 kDa)were covalently grafted onto the surface of self-assembled collagen by carbodiimide hydrochloride(EDC)and N-hydroxysuccinimide(NHS)to construct the positively charged collagen model:PL-collagen and the negatively charged collagen model:PAsp-collagen.The constructed PAA-CaP and PAsp-CaP mineralization systems were used to explore the intrafibrillar mineralization of unmodified collagen model,positively charged modified collagen model and negatively charged modified collagen model.Attenuated total reflection-Fourier transform infrared spectroscopy(ATR-FTIR)was used to analyze the changes of functional groups on the surface of collagen before and after grafting;Zeta potential of solid surface was used to detect the change of collagen surface potential before and after grafting;The mineralization of collagen fibers in various collagen models was observed by TEM;Quartz crystal microbalance(QCM)was used to analyze the difference in the adsorption capacity of unmodified collagen to PAA/PAA-CaP and PAsp/PAsp-CaP,as well as the difference in the adsorption capacity of positive and negative charged collagen models to PAA-CaP;MD simulated the interaction process between PAA-CaP and PAsp-CaP mineralized system and unmodified collagen,as well as the interaction process between positive and negative charged collagen model and PAA-CaP mineralized system.(3)Exploration of the mechanism of extrafibrillar mineralization.The mineralization system and collagen model of intrafibrillar mineralization were used to explore the mechanism of extrafibrillar mineralization.The extrafibrillar mineralization was observed by SEM.XRD was used to demonstrate mineral crystal composition on the surface of mineralized collagen fibers.Collagen was grafted with high molecular weight polycationic electrolyte polyacrylamide hydrochloride(PAH;Mw:15 kDa)and small molecules with low charge(aspartic acid(Asp),arginine(Arg)and tri(2aminoethyl)amine(TREN))were used to construct collagen models with different charge gradients.In addition,different amounts of PAsp were added to silk fibroin(SF)by electro spinning technology,and PAsp-SF fibers with charge gradient increase were spun.Thus,the influence of the charge load on the fiber surface on the fiber mineralization was explored.(4)Construction and mineralization of polyphenol-collagen mineralization model.The EGCG-collagen and NE-collagen mineralization models were constructed by epigallocatechin gallate(EGCG)and norepinephrine(NE)under alkaline conditions(pH=9).The changes of collagen surface functional groups before and after EGCG and NE modification were detected by ATR-FTIR.Solid surface Zeta potential was used to detect the changes of collagen surface potential before and after EGCG and NE modification.The distribution of polyphenols on collagen surface was detected by ruthenium red/uranyl acetate double staining.Observation of calcium fluorescence on collagen surface by confocal laser scanning microscope(CLSM)and detection of the calcium content of collagen surface by X-ray photoelectron spectroscopy(XPS)to evaluate the calcium binding ability of EGCG-collagen and NE-collagen.The intrafibrillar mineralization of EGCG-collagen and NE-collagen mineralization models was observed by TEM.The extrafibrillar mineralization of EGCG-collagen and NE-collagen fibers was observed by SEM.XRD was used to analyze the mineral crystals on the surface of mineralized EGCG collagen and NE collagen fibers.(5)Study on the durability of resin-dentine bonding using dopamine-acrylamide(DAA)-collagen model.The DAA-collagen mineralization model was constructed using N-2-(3,4dihydroxyphenyl)ethyl acrylamide(DAA)designed and synthesized by our research group(reaction condition:anhydrous ethanol,6 mol/L NaOH adjusted pH to 9).The above conventional intrafibrillar and extrafibrillar mineralization analyses were performed to demonstrate the mineralization capacity of the DAA-collagen mineralization model.After evaluating the biocompatibility of DAA,it was used as a resin-dentin preconditioning agent,and then the in vitro remineralization of bonding interface was studied by PAA-CaP mineralization system,and the remineralization of bonding interface was observed by TEM ultra-thin section.In addition,a resin-dentin restoration model was prepared on the tooth surface of rats to evaluate the remineralization of the bonding interface in vivo.The remineralization of the bonding interface was observed by TEM.3.Results.(1)Construction and characterization of polyelectrolyte-calcium phosphorus complex.The particle sizes of immediate PAA-CaP and PAsp-CaP were 2.5476±0.13 nm and 22.7933±0.23 nm,respectively.In terms of morphology and structure,PAA-CaP was homogeneous and compact spherical aggregate particles,while PAsp-CaP was loose chain arranged aggregate particles.PAA-CaP and PAsp-ACP nanoparticles were analyzed by XRD and SAED as amorphous mineralization precursors.The Zeta potential of PAA-CaP and PAsp-CaP were-26.95±3.15 mV and-19.40±1.37 mV,respectively.CD analysis showed that there was no secondary structure of PAA molecule,and PAACap showed random curling conformation.PAsp/Pasp-Cap exists in solution in the form of secondary structure β-angle.MD simulations showed that PAA interacts with calcium and phosphate ions more strongly.The mean binding energy of PAA and PAsp with calcium and phosphorus ions were-21297.20±171.77 kJ/mol and-20926.20±159.70 kJ/mol,respectively.(2)Exploration of the mechanism of intrafibrillar mineralization.ATR-FIRT results showed that compared with unmodified collagen,the absorption peaks of amide A,B,Ⅰ,Ⅱ and Ⅲ of PL-collagen and PAsp-collagen were increased.The Zeta potential on the surface of unmodified collagen,PL-collagen and PAsp-collagen were-4.60±0.17 mV,6.35±0.95 mV and-11.81±1.10 mV,respectively.TEM observation showed that PAA-CaP could not induce intrafibrillar mineralization of unmodified collagen,while PAsp-CaP could induce intrafibrillar mineralization of unmodified collagen,but both of them could induce intrafibrillar mineralization of positively and negatively modified collagen.The results of MD showed that the average binding energy of collagen and ACP in collagen/PAsp-CaP and collagen/PAA-CaP systems were-106942.36±1798.94 kJ/mol and-46539.10±1045.02 kJ/mol,respectively.On the whole,ACPs formed by PAsp and calcium and phosphorus ions have a stronger affinity with collagen fibers.In the two systems of PAsp-collagen/PAsp-CaP and PLcollagen/PAsp-CaP,the adsorption of Ca2+ ions in solution by PAsp-collagen was stronger,and the adsorption of HPO42-ions in solution by PL-collagen was stronger.The average binding energies of collagen and ACP in the two systems were-112513.03±5493.92 kJ/mol and-146448.05±5571.16 kJ/mol,respectively.In general,ACPs formed by PAsp have stronger affinity with PL-collagen than PAsp-collagen.(3)Exploration of the mechanism of extrafibrillar mineralization.SEM results showed that the mineralized HA crystals outside the fibers of the unmodified collagen surface were disordered and grew without orientation.In contrast,the positive and negative charge collagen model(PL-collagen and PAsp-collagen)has HA crystals growing along collagen fibers,and the c-axis of HA crystals was parallel to the long axis of collagen fibers.The Zeta potential on the surface of PAsp-collagen,PAHcollagen,TREN-collagen and Arg-collagen was-6.57±0.34 mV,16.13±0.61 mV,0.68±0.12 mV and-2.47±0.41 mV by changing the amount of charge on the surface of collagen fibers.The results showed that HA crystals on the surface of collagen fibers gradually grew along the collagen fibers and were arranged in order with the increase of the load charge of collagen fibers.To construct PAsp-SF electro spinning fibers with increasing charge,the Zeta potentials on the surfaces of SF,1%PAsp-SF,5%PAsp-SF,10%PAsp-SF and 20%PAsp-SF are as follows:-3.76±0.04 mV,-10.14±1.04 mV,20.48±0.52 mV,-22.31±0.03 mV and-26.49±1.28 mV;The results showed that with the increase of the load charge of PAsp-SF fiber,the content of HA on the fiber surface increased,and HA gradually grew and arranged along the collagen fiber.(4)Construction and mineralization of polyphenol-collagen model.ATR-FIRT results showed that compared with unmodified collagen,the characteristic peaks of the amide A band and the amide I band of EGCG-collagen and NE-collagen moved slightly from the low band to the high band,and the intensity of all characteristic peaks increased significantly.EGCG-collagen and NE-collagen showed high electronegativity with Zeta potential of-27.5749±0.42497 mV and-42.0484±0.49027 mV respectively.TEM observation showed that no ruthenium red development was observed on the surface of the unmodified collagen after ruthenium red/uranyl acetatedouble staining,and the surface of EGCG-collagen and NE-collagen modified with EGCG and NE showed obvious high-density clusters of shadow electron regions.CLSM showed that green fluorescence was not observed on the surface of the unmodified collagen membrane,while the EGCG-collagen and NE-collagen membrane showed intensive calcium ion fluorescence under the microscope.The XPS results showed that calcium ions could not be detected in unmodified collagen,while the atomic percentage of calcium in EGCG-collagen and NE-collagen membranes reached 0.90%and 1.36%,respectively.TEM and SEM results showed that the EGCG-collagen and NE-collagen models could rapidly realize the intrafibrillar mineralization of collagen fibers,and the HA crystals mineralized outside the fibers were highly ordered on the surface of collagen fibers.The mineralized collagen fiber structure was highly similar to the intrafibrillar and extrafibrillar mineralization of natural hard tissue structure.(5)Study on the durability of resin-dentine bonding using dopamine-acrylamide(DAA)-collagen model.ATR-FIRT results showed that,compared with unmodified collagen,DAA-collagen’s amide Ⅰ band peak shifted slightly from 1634 to 1653cm-1 and amide Ⅱ band shifted slightly from 1548 to 1552cm-1,and the absorption peak intensity of all characteristic peaks of collagen increased.The Zeta potential of DAA-collagen was-54.09±1.65 mV.After the DAA-collagen was dyed by ruthenium red/uranyl acetate,the surface of collagen showed obvious dark electron regions with high density clusters.CLSM observed that DAA-collagen membrane showed dense green fluorescence of calcium ions.The atomic percentage of calcium in DAA-collagen was 0.83%detected by XPS.The mineralization of DAA-collagen was observed by TEM after 24 h,the intrafibrillar mineralization of collagen fibers was almost complete.SEM showed that on the mineralized DAA-collagen surface,HA crystals grew in an orderly arrangement along collagen fibers,and the c-axis direction of HA followed the parallel arrangement of collagen fibers along the long axis.The XRD patterns of DAA-collagen surface minerals were well matched with the standard peaks of HA(Ca10(PO4)6(OH)2).Thermogravimetry showed that the mineralized mineral content of unmodified collagen membrane and DAA-collagen membrane reached 43.52%and 67.21%,respectively,after 7 days of collagen membrane mineralization.After 7 days of remineralization of DAA-treated demineralized dentin,there was substantial HA growth on the demineralized collagen fibers(dentin surfaces and dentin tubules).The c axis of HA followed the parallel arrangement of collagen fibers along the long axis);The results of CCK-8 cell viability test,TRITC-phalloidin cell staining and SEM cell morphology observation showed that DAA has no cytotoxicity.The remineralization results of resin-dentin bonding interface in vitro showed good remineralization effect,and the remineralization results of resindentin bonding interface in vivo showed that DAA had the potential to induce remineralization of the bonding interface.4.Conclusion.This study showed that the composite of polyelectrolyte and amorphous calcium phosphate prenucleation cluster induced collagen mineralization mainly through the interaction between amorphous calcium phosphate prenucleation cluster and collagen,which was assisted by the interaction between polyelectrolyte and collagen.This interaction is determined by the mineralizing activity of the polyelectrolyte with amorphous calcium phosphate prenucleation clusters and the charge load on the surface of the collagen fibers.The key role of polyelectrolytes is to stabilize amorphous calcium phosphate prenucleation clusters and determine their structure and morphology related to mineralization activity.In this study,we reveal the behavior of collagen intrafibrillar/extrafibrillar mineralization for the first time and propose a general principle for the design of in vitro collagen mineralization model.According to this principle we propose the closest to the organism and the most suitable in vitro collagen mineralization model,the polyphenol-collagen mineralization model,which can realize the natural structure of mineralized collagen fibers.Combining the advantages of the polyphenolcollagen model and the characteristics of mussel adhesion protein,the DAA-collagen mineralization model was further constructed.And DAA was applied as a primer to promote the remineralization of resin-dentin adhesive interface,which is expected to achieve the durability of the resin-dentin adhesive interface.