| Objectives Bone defect is often caused by tumor,trauma,infection and other diseases.As one of the most common clinical diseases,large bone defects are often treated with autogenous or allogeneic bone grafts.However,being limited by the source of autogenous bone and the absorption and rejection of allogeneic bone,the development of better scaffold materials for artificial bone has become an urgent problem to be solved.In bone tissue engineering,scaffolds are often used to provide a 3D environment for cell growth.Bone marrow mesenchymal stem cells and growth factors are used as osteoblast-inducing supplements to optimize bone regeneration.However,the extensive use of growth factors,such as bone morphogenetic proteins,has been proved to have adverse effects such as ectopic osteogenesis,fat liquefaction and so on.Therefore,it is of great significance to explore a non-growth factor-like material as an osteogenic supplement.With the increasing understanding of bone tissue formation process,collagen fiber-based biomaterials have been widely used in bone tissue engineering.Bone tissue is a mixture of protein,mineral and collagen fibers.The intrafibrillar mineralized collagen fibrils are the second level of the bone hierarchy,which provides the physical stress structure basis for the bone tissue.Polymer-induced liquid-precursor process generate amorphous calcium phosphate precursor(ACP),which is able to achieve intrafibrillar mineralization in the case of bone biomineral in vitro.In the process of biomineralization,the polymer forms amorphous calcium phosphate biomimetic precursor by chelating calcium ions and phosphorus ions in calcium phosphate solution.This amorphous calcium phosphate biomimetic mineralized precursor infiltrates into the collagen fibril through a specific site,crystallizes and solidifies,resulting in the formation of hydroxyapatite mineral crystallites arranged along the long axis of the collagen fiber.This amorphous calcium phosphate,as the basic material in the process of osteogenesis,not only has the ability to promote the mineralization of collagen fibrils,but also can regulate the function of cells and tissue differentiation.Therefore,this amorphous calcium phosphate biomimetic precursor has the potential to be used as an osteogenic supplement.However,the liquid characteristics of ACP precursors limit the local use of in situ intrafibrillar mineralization.So a transport system is necessary to transport them to specific sites in order to develop more potential clinical application.Mesoporous silica nanoparticles possess good biocompatibility and mass loading properties.It has been widely used for bioactive agent transport system,and also provides an excellent resolution to the transport of biomimetic mineralized precursors.In this experiment,the transport system for biomimetic mineralized precursor was constructed by the synthesis of carboxyl-functionalized mesoporous silica.To explore the potential application of the nanoparticles as an osteogenic supplement,the physical and chemical properties,the ability of intrafibrillar mineralization,and osteogenic induction in vitro of the prepared materials were studied.Methods:The experiment was summarized as follows:1.The monodisperse ultra large pore mesoporous silica(LMSN)was prepared by expanding the pore of the traditional MCM-41 type mesoporous silica by trimethylbenzene(TMB).Amine functional groups were grafted onto mesoporous silica surface by 3-aminopropyl triethoxysilane(APTES),and then carboxyl group(-COOH)was grafted onto mesoporous silica by ring opening reaction of succinic anhydride to get carboxyl functionalized large pore mesoporous silica(CLMSN).Poly allylamine hydrochloride(PAH)induced amorphous calcium phosphate(PAH-ACP)was adsorbed into CLMSN to obtain PAH-ACP@CLMSN.The synthesized nanoparticles LMSN,CLMSN and PAH-ACP@CLMSN were characterized by physical and chemical characterization.The energy dispersive x-ray spectroscopy(EDS),transmission electron microscopy(TEM),powder x-ray diffraction(XRD),thermogravimetric analysis(TGA),Zeta potential test,Fourier transform spectroscopy(FTIR),photoelectron spectroscopy(XPS),and Nitrogen adsorption desorption experiment were used to comprehensively verified the functionalization of carboxyl groups and the loading of PAH-ACP.2.The release kinetics of Si,P,Ca ions from nanoparticles was measured by inductively coupled plasma atomic emission spectrometry(ICP-OES)to obtain the release kinetics of biomineralization precursors in nanoparticles.At the same time,TEM was used to detect the biodegradability of the nanoparticles.A 2D collagen model was mineralized by PAH-ACP@CLMSN system,to explore the mechanism of precursor release,adhesion and nucleate reaction into collagen fibrils.The intrafibrillary mineralization ability of PAH-ACP@CLMSN was further verified by the mineralization of 3D collagen sponge.3.Rat bone marrow mesenchymal stem cells(r BMSCs)and macrophage cell line(RAW264.7)were used to evaluate the biocompatibility of LMSN and PAH-ACP@CLMSN at different concentrations(CCK-8 method).Flow cytometry was used to detect the effect of PAH-ACP@CLMSN on apoptosis and necrosis of rBMSCs.At different time interval,Col I,OPN,OCN,RUNX 2 gene expression,alkaline phosphatase activity and deposition of extracellular calcified nodules were examined to determine if LMSN and PAH-ACP@CLMSN had osteogenic induction potential.FITC-labeled mesoporous nanopaticles(PAH-ACP@FITC-CLMSN)were synthesized by incorporating FITC into the C LMSN network.Immunofluorescence analysis and ICP-OES were conducted to investigate the internalization of PAH-ACP@FITC-CLMSN particles and its effects on the sytoskeleton of r BMSCs.Results The large pore mesoporous silica nanoparticles with average pore diameter of about 14.88 nm was obtained effectively under the reaction of pore expansion.The carboxyl functionalization of the mesoporous silica has been successfully carried out.The carboxyl groups was not only beneficial to the loading of PAH-ACP precursors but also improved the biocompatibility of the nanoparticles.The PAH-ACP biomineralization precursors could be effectively loaded into CLMSN,which will be beneficial to the subsequent application of intrafibrillar mineralization and osteogenesis application.The release kinetics of biomineralization precursors indicated that CLMSN can effectively transport and release biomineralization precursor PAH-ACP in solution.The nano-delivery system showed a rapid release profile in the early10 days,followed by a declination in the next 10-30 days.These precursors were continuously released from PAH-ACP@CLMSN in solution to bind to collagen fibrils,infiltrated to the fibrils from special regions,diffused through the spaces and subsequently transformed into apatite crystallites along the axis of the collagen fibrils.This biomimetic mineralization transport system was effective for both 2D and 3D collagen models.The degradation of PAH-ACP@CLMSN began at the early stage,completed in about 60 days and had no adverse effect on the laden of the biomineralizaiton precursors.PAH-ACP@CLMSN exihbited excellent biocompatibility to r BMSCs and macrophages,which provides the biological application foundation for further application.The internalization of PAH-ACP@CLMSN by macrophages did not require special mediators.The internalization of nanoparticles by macrophages had no adverse effect on the morphology of macrophages,which is beneficial to the clearance of nanoparticles in vivo.The internalization of PAH-ACP@CLMSN by r BMSCs had no adverse effects on the cytoskeleton and cell function.Comparing with LMSN and the blank group without nanoparticles,PAH-ACP@CLMSN was more effective in increasing the ALP activity,promoting the formation of extracellular mineralized nodules,and elevating the expression of osteogenic related genes.This results indicated that PAH-ACP@CLMSN could effectively induce the osteogenic differentiation of bone marrow mesenchymal stem cell.Conclusion In summary,the successful translational strategy for biomineralization precursors shows that PAH-ACP@CLMSN,as a biocompatible and biodegradable precursor carrier system,has the properties of inducing the intrafibrillar mineralization of collagen fibrils and osteogenic differentiation of bone marrow mesenchymal stem cells.The nanoparticles has the potential to be blended with any osteoconductive scaffold as a versatile scaffold supplement for in-situ remineralization of bone,such as localized osteoporotic bone defects.However,it needs further studies on the mechanism in vitro and in vivo. |
