Large Pore Mesoporous Silica Nanoparticles As Delivery Devices For Biomimetic Intermediate Precursors Of Intrafibrillar Mineralization

Objectives:Resent study showed that intrafibrillar mineralization of collagen could be conducted by polymer-induced liquid like precursor. Polymer such as polyaspartic acid, polyacrylic acid could act as a nucleation inhibitor to form stable amorphous calcium phosphate precursor, the latter infiltrate into the collagen to conduct the intrafibrillar mineralization subsequently.In some condition such as bone defect filling and teeth decay repair, mineral ion release has been studied a lot by adding different mineral containing materials, but none of these researches directly use precursor clusters to induce mineralization. Moreover, in some condition such as teeth decay, simply release of the calcium and phosphate does not lead to remineralizaiton of the hypomineral tissue due to the lack of the nucleation inhibitor. So a pathway that could store and deliver the polymer stabilized precursor into the targeted area directly will be of great help to the remineralization process.Mesoporous silica nanoparticles have been synthesized for the biomedical application such as drug and biomolecule delivery due to their unique physical and chemical features. Large pore mesoporous silica has been reported to be able to encapsulate abundant cargoes inside the pore rather than tethering them on the surface due to the large pore and pore volume.Here we reported a process that generated large pore mesoporous silica with amine-functionalization for the storage and sustained release of biomimetic analog stabilized amorphous calcium phosphate precursor. Polymer stabilized amorphous calcium phosphate precursor was prepacked, stored and sustained released from the large pore mesoporous silica. This system was used to perform intrafibrillar mineralization of single layer reconstitute collagen model to see the feasibility of the system in biomineralization.Materials and methods:PART1Large pore mesoporous silica nanoparticles (LPMSN) was synthesized by using1,3,5-trimethylbenzene (TMB) as pore swollen agent after the sol-gel synthesis of small pore mesoporous silica.3-aminopropytriethoxylsilane (APTES) was used to modify LPMSN under toluene reflux condition to get amine-functionalized mesoporous silica nanoparticles (AFMSN) with covalent bonded aminopropyl group and positively charged particle surface. LPMSN and AFMSN were characterized by transimission electron microscopy (TEM), field emission transmission electron microscopy (FTEM), powder X-Ray diffraction (XRD), thermogravimetric analysis (TGA), fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectrograph (XPS), solid-state nuclear magnetic resonance spectroscopy (SSNMR), zeta potential test and nitrogen adsorption and desorption experiment.PART2Polyacrylic acid (PAA) was used to fabricate the biomimetic analogue stabilized amorphous calcium phosphate precursor (PAA-ACP) solution with calcium solution and phosphate solution. AFMSN powder was soaked inside the solution subsequently to adsorb the precursor to get PAA-ACP@AFMSN powder.The PAA-ACP@AFMSN nanoparticles was examined by TEM and FTIR. The PAA-ACP@AFMSN was examined by FTEM and TEM-elemental mapping.The drug release profile of stabilized amorphous calcium phosphate precursor from PAA-ACP@AFMSN was conducted by determining the calcium, phosphorous ion, and silicon release in HEPES solution by spectrophotometric method in different time interval from0.5h to120h. PART3Single layer collagen was reconstituted with calf skin derived typeⅠcollagen by neutralized with ammonia vapor.To examine the ability to store and release amorphous calcium phosphate precursor, PAA-ACP@AFMSN was immersed in HEPES buffer solution. Single layer collagen was co-cultured with the solution to conduct the remineralization of the collagen by immersing the collagen grid in the solution for4days. The grids were examined by TEM every day to inspect intrafibrillar remineralizion of the collagen. This collagen remineralization strategy was an indirect method to test the release profile of amorphous calcium phosphate precursor and a direct method to prove the feasibility of PAA-ACP@AFMSN in biomineralization application.Results:1. With the use of swollen agent TMB, large pore mesoporous silica nanoparticles (LPMSN) with an average pore diameter of14.56nm was synthesized. Aminopropyl groups were covalent bonded to the surface of the LPMSN to get positively charged nanoparticle. The modification facilitates the adsorption of the negatively charged PAA-ACP.2. PAA-ACP@AFMSN was fabricated by adsorbing PAA-ACP into AFMSN. The sustained release of amorphous calcium phosphate precursor from PAA-ACP@AFMSN was proved from the release profile of calcium and phosphorous ion.3. Reconstitute single layer collagen was able to be remineralized in PAA-ACP@AFMSN solution, which proved that the PAA-ACP@AFMSN system had great potential in the application of biomimetic remineralization.Conclution:In summary, we developed an approach to directly load intermediate precursors of calcium phosphate in amine functionalized large pore mesoporous silica, with the aim that this pre-fabricated precursor could remineralize the collagen by sustained release from the nanoparticle carrier.The fact that the reconstitute collagen fibrils can be mineralized using PAA-ACP@AFMSNs provides the proof of concept that intermediate precursors of calcium phosphate may be pre-fabricated for loading and release. This experiment showed that continuous replenishment of the mineralization medium is possible for in-situ remineralization of hypomineralized body tissues, which represents an important advance in the translation of biomineralization concepts into regimes for in-situ remineralization of bone and teeth.