Regulation Of Stem Cell Osteogenic Differentiation By Mesoporous Silica Nanoparticles

The treatment of bone-related diseases is gradually becoming a hot topic of scientific research due to the limited self-repair ability of bone.Research based on biomaterials and stem cell osteogenic differentiation has provided a new direction for bone repair and regeneration.Silica element is considered as a candidate for enhancing the efficiency of bone regeneration because of its good biocompatibility and osteogenic capacity.Mesoporous silica nanoparticles(MSNs)with uniform size,high porosity,high specific surface area,and appropriate pore size are widely applied in drug delivery and stem cell fate modulation.For the effects of MSNs on stem cell fate,plenty of studies have been carried out based on cell population-level performance.Multiple replicate experiments can verify the regulatory effect of the material on cell differentiation.However,interactions between cells,especially the effect of cytokines secreted during osteogenic differentiation on the osteogenic differentiation of the remaining cells,make it difficult to assess the efficiency of material regulation.Therefore,the design of an advanced cellular assay platform with integrated massively parallel experiments may provide a solution to the above problem.In addition,previous studies on osteogenic differentiation mostly focus on stem cells with spontaneous osteogenic differentiation tendency such as bone marrow-derived mesenchymal stem cells(BMSCs).Studying the regulation of silica element on other stem cells with non-spontaneous osteogenic differentiation tendency,such as human adipose-derived mesenchymal stem cells(hADSCs),will facilitate a comprehensive discussion of its role in stem cell osteogenic differentiation.It is well known that bone repair in tumor patients can be affected and hindered by tumor cells.Therefore,in the treatment strategy of bone injury repair for tumor patients,it is necessary to consider not only the postoperative repair and regeneration of bone tissue,but also the inhibition of possible tumor cell migration and proliferation at the injury site,the effective killing of tumor cells.The high porosity of mesoporous silica nanoparticles guarantees their excellent antitumor drug loading capacity.However,commonly used antitumor drugs such as doxorubicin(DOX)are mostly strong destructive molecules that have a damaging effect on stem cells and surrounding normal cells and tissues.Epigallocatechin gallate(EGCG)is an active substance extracted from green tea that exhibits immunomodulatory,antitumor,antibacterial and stem cell differentiation modulating properties.Therefore,using EGCG,which is friendly to tissue cells and toxic to tumor cells,to conduct stem cell bone repair studies,not only maintains the osteogenic advantage of stem cells but also inhibits the survival of tumor cells at the site of bone damage.Based on this,the main research carried out in this thesis includes the use of high-throughput microfluidic technology to analyze and validate the effect of mesoporous silica nanoparticles on the osteogenic differentiation of hADSCs.As well as exploiting the high drug loading properties of mesoporous silica nanoparticles to address the effects of tumor cells during stem cell bone repair.Detailed research is as follows:(1)Mesoporous silica nanoparticles promote osteogenesis in human adipose-derived stem cells identified by a high-throughput microfluidic assayMagnetic mesoporous core-shell nanoparticles(MNPs)with iron tetraoxide nanoparticles as the core and mesoporous silica as the shell were prepared.MNPs had a uniform size distribution with an average particle size of 81.5±0.5 nm and can be endocytosed into lysosomes of stem cells.The metabolism of MNPs were studied in a simulated lysosomal environment,and it was demonstrated that MNPs were able to release silicon element by slow decomposition in cellular lysosomes.At the population level of cell culture,it was confirmed that MNPs could effectively upregulate the expression of osteogenic-related genes runt-related transcription factor 2(RUNX2),osteopontin(OPN),osteocalcin(OCN)and bone morphogenetic protein-2(BMP-2)by real-time quantitative fluorescence polymerase chain reaction(RT-qPCR).It was further demonstrated that MNPs promoted the expression of osteogenic-related OPN and OCN proteins by immunofluorescence staining.In addition,the alkaline phosphatase activity and the mineralization in vitro of cells were increased in the presence of MNPs.The above results basically confirm that MNPs can promote the differentiation ability of hADSCs toward osteogenesis.Additionally,a high-throughput assay platform that combines microfluidic detection with cell culture was proposed and established,using the more than 7000 microchambers it contains to carry out thousands of effective parallel experiments simultaneously.The extracellular secretion of the osteogenic-specific protein OCN expressed during cell differentiation was analyzed by fluorescent signals.Statistical analysis revealed that the average fluorescence intensity of cells cultured with MNPs of 75 μg/mL secreting OCN proteins was enhanced approximately 2.5-fold compared with the average fluorescence intensity detected by cells cultured with 0 μg/mL MNPs,confirming that MNPs have a promotional effect on osteogenic differentiation of hADSCs.The application of this microfluidic assay technique in cell analysis and detection effectively avoids the effect of cytokines secreted by osteogenic differentiation of some cells on the remaining cells osteogenic differentiation,which can provide a more reliable and accurate result for MNPs to enhance osteogenic differentiation of hADSCs.(2)EGCG/MSNs nanocomposites promote osteogenesis of mesenchymal stem cells and inhibit tumor cell growthMesoporous silica nanoparticles(MSNs)were prepared and synthesized based on the cetyltrimethylammonium bromide(CTAB)template method,with an average diameter between 50-70 nm and a homogeneous and stable spherical structure.The BET and Langmuir surface areas of MSNs are around 1004 m2/g and 3924 m2/g,respectively.The mesoporous pore size of MSNs is about 3 nm.Vitamin C was used to inhibit the oxidative failure process of EGCG in the solution.And the strong adsorption property of MSNs was applied to construct the vitamin C-protected EGCG/MSNs nanocomposites(MEV).UV-Vis absorption spectroscopy and thermogravimetric analysis showed that the loading of vitamin C-protected EGCG on MSNs was up to～5 wt%.MEV was added to the cell culture environment to study their cytocompatibility and regulation of osteogenic differentiation of BMSCs.The good cytocompatibility of EGCG,vitamin C,MSNs,and MEV was confirmed using live/dead staining and cell activity assays.The results of RT-qPCR,immunofluorescence staining,and alizarin red staining showed that MEV upregulated osteogenesis-related gene and protein expression and promoted the generation of calcium nodules.That is,MEV promoted the differentiation of BMSCs toward osteogenesis.Cell activity assay after culturing human breast cancer cells(MCF-7)with different concentrations of MEV showed that cellular activity was decreased to～0.17-fold of the 0 μg/mL control when cultured with 800 μg/mL of MEV.The apoptosis of tumor cells induced by MEV was analyzed by apoptosis staining,and it was confirmed that MEV nanoparticles could produce certain antitumor effects by inducing apoptosis of MCF-7.The apoptosis of MEV-induced cancer cells was analyzed by apoptosis staining.Thus,the vitamin C-protected EGCG/MSNs nanocomposites not only inhibited the growth of tumor cells but also effectively promoted the osteogenic differentiation of MSCs,providing an effective solution for the repair of bone damage with tumor cells.In summary,this thesis investigated the effect of mesoporous silica nanoparticles on osteogenic differentiation of human adipose-derived mesenchymal stem cells,and confirmed the regulation of cell fate by the material when combined with microfluidic multi-parallel experimental assays.Meanwhile,the use of mesoporous silica nanoparticles loaded with vitamin C-protected EGCG,which inhibits the growth of tumor cells while achieving the promotion of stem cell osteogenesis,demonstrates the multiple biomedical prospects of mesoporous silica nanoparticles.