Stimulation of osteogenesis of rat bone marrow mesenchymal stem cells by modified large pore mesoporous silica nanoparticles

doi:10.3877/cma.j.issn.1674-1366.2018.03.001

Abstract

Abstract:

Objective

To study the material properties and effect of the synthetic large pore mesoporous silica nanoparticles (LPMSNs) on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats.

Methods

Mesoporous silica nanoparticles (MSNs) was synthetised with the method of sol gel. The expanding agent 1, 3, 5-Trimethylbenzene was applied to expand the porous of the initial synthesis MSNs with high temperature. Transmission electron microscopy (TEM) , N₂ adsorption-desorption, pore size distribution, pore volume and specific surface area analysis, Fourier infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) were used to detect the morphological structure as well as physical and chemical properties of LPMSNs. BMSCs were cultured in vitro and their proliferative capacity and multidirectional differentiation potential were identified. The third generation of the cultured cells were selected for subsequent experiment. Different concentrations of LPMSNs were configured and a CCK-8 kit was applied to detect the cytotoxicity of LPMSNs. The osteogenic induction cell medium contained LPMSNs (10 μg/mL) was incubated with the BMSCs cells. After 21 days, alizarin red staining was used to detect the cell mineralized nodules and compared with the control group. The expression of bone related genes ALP, Runx2 and OCN were detected by qPCR method after 7 and 14 days incubation. After 14 days of cultivation, the expression of bone related gene Runx2 and OCN at protein level was detected by Western blot. The data were analyzed by One-Way ANOVA and t test by SPSS 20.0 software package. The difference was statistically significant with P<0.05.

Results

TEM showed that LPMSNs particle diameter was about 200 nm, and the Nitrogen sorption isotherms of LPMSNs exhibited typical type Ⅳ isotherm and a hysteresis loop. The pore width, Brunauer-Emmett-Teller (BET) specific surface area and Barrett-Joyner-Halenda (BJH) pore volume were 7.39 nm, (340.5 ± 2.8) m²/g and 1.8 cm³/g respectively. As shown in FTIR spectra of LPMSNs, 460 cm^-1 represents the transverse optical rocking motions of Si-O-Si, 800 cm^-1 represents the symmetric stretching of Si-O-Si; 1070 cm^-1 represents the antisymmetric stretching of Si-O-Si. The peak of 940-960 cm^-1 represents the Si-OH bond, 3000-3700 cm^-1 represents the-OH bond. TGA showed the weight loss percentage of LPMSNs in 28-1000 ℃ was about 1.61%. The results indicated that low concentration (<20 μg/mL) LPMSNs had no significant effect on the cell viability. Alizarin red staining showed more mineralized nodules in LPMSNs group compared with the control group. PCR results showed that at the 7th day, the expression of ALP (6.2 ± 1.9) and Runx2 (4.3 ± 0.6) in LPMSNs group were significantly higher than the control group (t_ALP= 4.83, P_ALP = 0.0085; t_Runx2 = 9.76, P_Runx2 = 0.0006) respectively. At the 14th day, the expression of ALP (19.2±1.4) , Runx2 (4.9 ± 0.7) and OCN (17.2 ± 3.6) in LPMSNs group were significantly higher than control group (P<0.05) . The results of Western blot showed that the expression of Runx2 in LPMSNs group (1.7 ± 0.3) was significantly higher than control group (t = 3.48, P = 0.0254) together with the expression of OCN (1.69±0.21) in LPMSNs group (t = 5.71, P = 0.0047) at the 14th day.

Conclusions

The modified LPMSNs had large specific surface area, pore volume and hydrophilic surface, which is suitable for loading large molecular weight proteins or cytokines. LPMSNs could induce the osteogenic differentiation of rat BMSCs without cytotoxicity. Therefore, LPMSNs is a promising biomaterial for bone regeneration.

Key words: Mesenchymal stem cells, Osteogenesis, Nanoparticles, Large pore mesoporous silica nanoparticles

Huimin Chen, Qizhi Zhong, Zihua Huang, Jiaxin Guo, Ruoxun Wang, Sui Mai. Stimulation of osteogenesis of rat bone marrow mesenchymal stem cells by modified large pore mesoporous silica nanoparticles[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2018, 12(03): 135-143.

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References 23

[1]	Shi M，Zhou Y，Shao J，et al. Stimulation of osteogenesis and angiogenesis of hBMSCs by delivering Si ions and functional drug from mesoporous silica nanospheres[J]. Acta Biomater，2015（21）：178-189.
[2]	Neumann A，Christel A，Kasper C，et al. BMP2-loaded nanoporous silica nanoparticles promote osteogenic differentiation of human mesenchymal stem cells[J]. RSC Advances，2013，3（46）：24222-24230.
[3]	Gan Q，Zhu J，Yuan Y，et al. A dual-delivery system of pH-re-sponsive chitosan-functionalized mesoporous silica nanoparticles bearing BMP-2 and dexamethasone for enhanced bone regenera-tion[J]. J Mater Chem B，2015，3（10）：2056-2066.
[4]	Kaiser C，Buchel G，Ludtke S，et al. Processing of microporous/mesoporous submicron-size silica spheres by means of a template-supported synthesis[J]. Royal Socienty of Chemistry，1997，213（7）：406-412.
[5]	Colon G，Ward BC，Webster TJ. Increased osteoblast and decreased Staphylococcus epidermidis functions on nanophase ZnO and TiO₂[J]. J Biomed Mater Res A，2006，78（3）：595-604.
[6]	Webster TJ，Schadler LS，Siegel RW，et al. Mechanisms of enhanced osteoblast adhesive on nanophase alumina involve vitronectin[J]. Tissue Eng，2001，7（3）：291-301.
[7]	Guo X，Gough JE，Xiao P，et al. Fabrication of nanostructured hydroxyapatite and analysis of human osteoblastic cellular response[J]. J Biomed Mater Res A，2007，82（4）：1022-1032.
[8]	Zhao G，Zinger O，Schwartz Z，et al. Osteoblast-like cells are sensitive to submicron-scale surface structure[J]. Clin Oral Implants Res，2006，17（3）：258-264.
[9]	Zhao Y，Zhang Y，Ning F，et al. Synthesis and cellular biocompatibility of two kinds of HAP with different nanocrystal morphology[J]. J Biomed Mater Res B Appl Biomater，2007，83（1）：121-126.
[10]	Li B，Chen X，Guo B，et al. Fabrication and cellular biocompati-bility of porous carbonated biphasic calcium phosphate ceramics with a nanostructure[J]. Acta Biomater，2009，5（1）：134-143.
[11]	Hornez JC，Chai F，Blanchemain N，et al. Biocompatibility improvement of pure hydroxyapatite（HA）with different porosity[J]. Key Eng Mater，2007（330）：927-930.
[12]	Veiseh O，Gunn JW，Zhang M. Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging[J]. Adv Drug Deliv Rev，2010，62（3）：284-304.
[13]	Harris JM，Chess RB. Effect of pegylation on pharmaceuticals[J]. Nat Rev Drug Discov，2003，2（3）：214-221.
[14]	Zhang Y，Hu L，Yu D，et al. Influence of silica particle internal-ization on adhesion and migration of human dermal fibroblasts[J]. Biomaterials，2010，31（32）：8465-8474.
[15]	Napierska D，Thomassen LC，Rabolli V，et al. Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells[J]. Small，2009，5（7）：846-853.
[16]	He Q，Zhang Z，Gao Y，et al. Intracellular iocalization and cyto-toxicity of spherical mesoporous silica nano-and microparticles[J]. Small，2009，23（5）：2722-2729.
[17]	Tao Z，Morrow MP，Asefa T，et al. Mesoporous silica nanoparticles inhibit cellular respiration[J]. Nano Letters，2008，8（5）：1517-1526.
[18]	Nielsen FH，Poellot R. Dietary silicon affects bone turnover differently in ovariectomized and sham-operated growing rats[J]. J Trace Elem Exp Med，2004，17（3）：137-149.
[19]	Reffitt DM，Ogston N，Jugdaohsingh R，et al. Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentia-tion in human osteoblast-like cells in vitro[J]. Bone，2003，32（2）：127-135.
[20]	Han P，Wu C，Xiao Y. The effect of silicate ions on proliferation，osteogenic differentiation and cell signalling pathways（WNT and SHH）of bone marrow stromal cells[J]. Biomaterials Science，2013，1（4）：379-392.
[21]	Zhou X，Moussa FM，Mankoci S，et al. Orthosilicic acid Si（OH）₄ stimulates osteoblast differentiation in vitro by upregulating miR-146a to antagonize NF-κB activation[J]. Acta Biomater，2016（39）：192-202.
[22]	Wu C，Chang J，Ni S，et al. In vitro bioactivity of akermanite ceramics[J]. J Biomed Mater Res A，2006，76（1）：73-80.
[23]	Guo Z，Liu XM，Ma L，et al. Effects of particle morphology，pore size and surface coating of mesoporous silica on Naproxen dissolution rate enhancement[J]. Colloids Surf B Biointerfaces，2013（101）：228-235.

基因	引物序列
ALP	F:5′-GAAGTCCGTGGGCATCGT-3′
?	R:5′-CAGTGCGGTTCCAGACATAG-3′
Runx2	F:5′-CCCAACTTCCTGTGCTCC-3′
?	R:5′-AGTGAAACTCTTGCCTCGTC-3′
OCN	F:5′-GGCAGTAAGGTGGTGAATAG-3′
?	R:5′-GTCCTGGAAGCCAATGTG-3′
GAPDH	F:5′-TGCTGAGTATGTCGTGGAG-3′
?	R:5′-GTCTTCTGAGTGGCAGTGAT-3′

基因	引物序列
ALP	F:5′-GAAGTCCGTGGGCATCGT-3′
?	R:5′-CAGTGCGGTTCCAGACATAG-3′
Runx2	F:5′-CCCAACTTCCTGTGCTCC-3′
?	R:5′-AGTGAAACTCTTGCCTCGTC-3′
OCN	F:5′-GGCAGTAAGGTGGTGAATAG-3′
?	R:5′-GTCCTGGAAGCCAATGTG-3′
GAPDH	F:5′-TGCTGAGTATGTCGTGGAG-3′
?	R:5′-GTCTTCTGAGTGGCAGTGAT-3′

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