Mesoporous Silica Promotes Osteogenesis of Human Adipose-Derived Stem Cells Identified by a High-Throughput Microfluidic Chip Assay

Abstract

Silicon-derived biomaterials are conducive to regulating the fate of osteo-related stem cells, while their effects on the osteogenic differentiation of human adipose-derived stem cells (hADSCs) remain inconclusive. Mesoporous silica (mSiO₂) is synthesized in a facile route that exhibited the capability of promoting osteogenic differentiation of hADSCs. The metabolism of SiO₂ in cells is proposed according to the colocalization fluorescence analysis between lysosomes and nanoparticles. The released silicon elements promote osteogenic differentiation. The detection of secretory proteins through numerous parallel experiments performed via a microfluidic chip confirms the positive effect of SiO₂ on the osteogenic differentiation of hADSCs. Moreover, constructed with superparamagnetic iron oxide (Fe₃O₄), the magnetic nanoparticles (MNPs) of Fe₃O₄@mSiO₂ endow the cells with magnetic resonance imaging (MRI) properties. The MNP-regulated osteogenic differentiation of autologous adipose-derived stem cells provides considerable clinical application prospects for stem cell therapy of bone tissue repair with an effective reduction in immune rejection.

Keywords: human adipose-derived stem cells; mesoporous silica; microfluidic detection; osteogenic differentiation.

Figure 1

Synthesis and characterization of MNPs. (a) Schematic illustration of the preparation process of MNPs. (b,c) Transmission electron microscopy (TEM) images of Fe₃O₄ and MNPs, respectively. (d) N₂ adsorption–desorption isotherms and mesopore size distribution (the inset) of MNPs. (e) Scanning electron microscope (SEM) image and particle diameter distribution (the inset) of MNPs. (f) The X-ray diffraction (XRD) patterns and (g) the Fourier transform infrared spectra (FTIR) images of Fe₃O₄ and MNPs. (h) Saturation magnetization curve of MNPs at room temperature. (i) T₂-weighted magnetic resonance imaging (MRI) images of different samples. Samples 1 to 5 represent water, silica of 75 and 150 μg/mL, and MNPs of 75 and 150 μg/mL, respectively.

Figure 2

Viability and morphology of hADSCs cultured with MNPs. (a) Live/dead cellular staining images of hADSCs after culture with 0, 50, 100, and 150 μg/mL MNPs for 3 days. The live cells were green, and the dead cells were red. (b) Survival rate of hADSCs cultured with increasing concentrations of MNPs for 3 days. Data represent mean ± standard deviation (n = 3). Significance was determined by one-way ANOVA (ns: not significant vs. the 0 μg/mL group). (c) Relative cell viability of hADSCs after incubation with 0, 50, 100, and 150 μg/mL MNPs for 1, 2, and 3 days. Data represent mean ± standard deviation (n = 3). Significance was determined by two-way ANOVA (* p < 0.05, ** p < 0.01, ns: not significant compared with the 0 μg/mL group on Day 1). (d) Cytoskeleton staining images of hADSCs cultured with 0, 25, 50, and 75 μg/mL MNPs for 3 days. F-actin was stained red, and nuclei were stained blue.

Figure 3

Colocalization between MNPs and lysosomes. Fluorescence images of hADSCs without (a) and with MNPs (b) for 4 h and cultured without (c) and with MNPs (d) for 20 h. Lysosomes were stained with LysoTracker (red), MNPs were tagged with FITC (green), and the nucleus was stained with Hoechst (blue). (e) Silicon release from MNPs at pH 5 for 14 days. (f) Schematic illustration displaying the intracellular processes of MNPs in hADSCs.

Figure 4

Osteogenic-specific gene expression by real-time quantitative polymerase chain reaction (RT–qPCR) analysis. RUNX2 (a), OPN (b), OCN (c) and BMP-2 (d) mRNA expression on Day 7, Day 14, and Day 21. Data represent mean ± standard deviation (n = 3). Significance was determined by one-way ANOVA (* p < 0.05, *** p < 0.001, **** p < 0.0001, ns: not significant vs. the 0 μg/mL group).

Figure 5

Immunofluorescence staining of OPN and OCN protein. (a) OPN (green), F-actin (red), and nuclear (blue) staining images for 7 days. (b) Quantitative analysis of the mean fluorescence intensity of immunofluorescence-stained images of OPN expression at 7 days. (c) OPN (green), F-actin (red), and nuclear (blue) staining images for 14 days. (d) Quantitative analysis of the mean fluorescence intensity of immunofluorescence-stained images of OPN expression at 14 days. (e) OCN (green), F-actin (red), and nucleus (blue) staining images for 14 days. (f) Quantitative analysis of the mean fluorescence intensity of immunofluorescence-stained images of OCN expression at 14 days. Data represent mean ± standard deviation (n = 10). Significance was determined by one-way ANOVA (**** p < 0.0001, vs. the 0 μg/mL group).

Figure 6

Identification of alkaline phosphatase (ALP) and mineralized nodules in osteoblasts. (a) ALP staining images on Days 7 and 14 after being cultured with 0, 25, 50, and 75 μg/mL MNPs. (b) Quantitative analysis of ALP staining intensity after being cultured with different concentrations of MNPs. Data represent mean ± standard deviation (n = 3). Significance was determined by two-way ANOVA (** p < 0.01, *** p < 0.001, ns: not significant vs. the 0 μg/mL group at 7 days). (c) Relative ALP activity after 7 and 14 days when cultured with 0, 25, 50, and 75 μg/mL MNPs. Data represent mean ± standard deviation (n = 3). Significance was determined by two-way ANOVA (** p < 0.01, **** p < 0.0001, vs. the 0 μg/mL group at 7 days). (d) Alizarin Red S staining images of hADSCs cultured with 0, 25, 50, and 75 μg/mL MNPs after 14 days.

Figure 7

(a) Schematic of hADSC-secreted OCN protein detection by a polydimethylsiloxane (PDMS) microfluidic chip and a functionalized glass slide with antibody barcodes. (b) Pure PDMS chip in bright field. (c) Cell seeded on the PDMS chip at 0 h. (d) Cell morphology after being seeded on the PDMS chip for 24 h. Detection of capture antibody spreading on the glass slide (e,g) and fluorescence signal image of the antibody-barcoded glass slide (f,h) cultured with 0 and 75 μg/mL MNPs, respectively. (i) Scatter diagram showing the distribution of fluorescence intensity after 7 days of cultivation on hADSCs with 0 and 75 μg/mL MNPs. (j) Violin plot showing the average fluorescence intensity after 7 days of culture with 0 and 75 μg/mL MNPs on hADSCs.