| Objectives:Biomimetic remineralization improves the longevity of resin-dentin bonds by intrafibrillar mineralization of denuded collagen within resin-sparse, water-rich regions of hybrid layers with polyacid-stabilized amorphous calcium phosphate (Pa-ACP). For clinical translation of this technology, direct delivery of Pa-ACP (end-product) is more effective than delivery of remineralization components (Ca, P sources and biomimetic analogs). This work examined the use of amine-functionalized mesoporous nanosilica as delivery devices for Pa-ACP prenucleation clusters, the stability of the latter that were encapsulated in the nanoconfinements of mesoporous silica was examined by SAED (selected area electron diffraction), the release kinetics of the Pa-ACP loaded mesoporous silica were investigated under acidic, neutral and basic solution by spectrophotometric method, the potential use of this control led-r el ease delivery system for intrafibrillar mineralization of collagen was further examined by choosing reconstitued type-I collagen as mineralization model.Materials and methods:Part-Ⅰ. Mesoporous silica nanoparticles (MSNs) was firstly synthesized using sol-gel process, tetraethyl orthosilicate (TEOS) as Si source, ammonium as catalyst, a kind of cationic surfactant (cetyltrimethyl ammonium bromide, CTAB) was used as structure-directing agents, the size of the particles were controlled according to Stober method by modified the TEOS:NH40H:CTAB:H2Omolar ratio as1:9.14:0.128:3969; the surfactant was removed by calcination after nanosilica formation. MSNs were characterized with TEM (Transimission Electron Microscopy), TGA (thermogravimetric analysis), XRD (Powder X-Ray Diffraction), XPS (X-ray Photoelectron Spectroscopy), ATR-FTIR (Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy), zeta potential and29Si CP-MAS NMR (Solid-State Nuclear Magnetic Resonance Spectroscopy) Because both of Pa-ACP and MSNs have negative zeta-potentials, MSNs were post-functionalized with aminosilane that formed into amine-functionalized MSNs (AF-MSNs) with net positive charge for storage of Pa-ACP prenucleation clusters. AF-MSNs were characterized with TEM, STEM-EDX (Scanning Transmission Electron Microscopy-Energy Dispersive X-ray Analysis), AFM (Atomic force microscopy), TGA, ATR-FTIR,29Si CP-MAS NMR, XPS and N2sorption/desorption (specific surface area, pore size and volume).Part-Ⅱ Polyacylic acid stablized amorphous calcium phosphate (Pa-ACP) were generated in supersaturation solution using PILP method. After Pa-ACP prenucleation clusters were diffused into the inner pores of AF-MSNs or immobilized onto the external surface of the silica nanoparticles, the Pa-ACP prenucleation cluster-loaded AF-MSNs were then characterized with TEM, STEM-EDX, AFM. The amorphous phase of Pa-ACP was characterized by SAED.Part-Ⅲ The total amount of Ca, P and Si in per mg of MSNs and AF-MSNs powder were quantified using ICP-AES (Inductive Coupled Plasma-Atomic Emission Spectroscopy). Morphology of Pa-ACP that released from AF-MSNs at acidic, neutral and alkaline pH was examined using TEM. The release kinetics of Pa-ACP conducted under above conditions were investigated by spectrophotometric method.Part-IV The use of Pa-ACP loaded AF-MSNs to mineralize collagen fibrils was examined using a2-D collagen mineralization model. Bovine skin-derived type I collagen solution was allowed to self-assemble into fibrillar collagen on nickel TEM grids by interacting with ammonia vapor. Reconstituted collagen fibrils were cross-linked with0.3M carbodiimide prior to the mineralization experiments. Pilot work showed that direct application of Pa-ACP loaded AF-MSNs dispersed in acidified deionized water (pH2) to the TEM grids resulted in AF-MSNs adhering to the fibrils’surface, which made it impossible for ultrastructural examination of intrafibrillar mineralization. Thus, an indirect application method was used in which Pa-ACP loaded AF-MSNs were first dispersed in acidified deionized water (pH2) for5days to release Pa-ACPs; centrifuging was performed for4min at4000rpm to precipitate the majority of AF-MSNs, while allowing Pa-ACPs to remain in the solution. Mineralization of collagen fibrils was performed by floating TEM grids over droplets of Pa-ACP containing supernatant at37°C for1-4days.Results:MSNs had a mean diameter of50nm and hexagonal pore arrangement. Zeta-potentials of Pa-ACP and AF-MSNs were-33.8eV and17.5eV, respectively. Amine-functionalization was confirmed using29Si CP-MAS NMR and XPS. AF-MSN had specific surface area of953m2/g, pore volume of0.44cm3/g and mean pore width of2.1nm. STEM-EDX and AFM showed that Pa-ACPs were loaded both within the mesopores and on the external surface of AF-MSNs. Release of Pa-ACP prenucleation clusters from AF-MSN was observed in acidic, neutral and alkaline medium, intensive release of the prenucleation clusters were found in acidic and alkaline conditions, compared to the dissolution of silica framework under pH10, ordered mesostructures of silica nanoparticles remained under pH2. With the sustain release of Pa-ACP from AF-MSNs under pH2and effective elimination of silica nanoparticles, the mineralization precursors could diffuse into the inner compartments of collagen and ultimately formed into apatite crystallites arranged along the fibrils’longitudinal axis. |
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