Article Cite This: ACS Biomater. Sci. Eng. 2019, 5, 3352−3360
pubs.acs.org/journal/abseba
Insights into the Osteogenic Differentiation of Mesenchymal Stem Cells on Crystalline and Vitreous Silica Guohui Shou,† Suya Lin,† Shuxian Shen,† Xuzhao He,† Lingqing Dong,†,‡ Kui Cheng,† and Wenjian Weng*,† †
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School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Zhejiang University, Hangzhou 310027, China ‡ The Stomatologic Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China S Supporting Information *
ABSTRACT: Cell responses to oxide biomaterials depend on the protein adsorption behavior of the biomaterial surface. Thus, the inherent properties of oxide biomaterial surfaces play a key role in this process. However, commonly used biomaterials, such as calcium phosphate and titanium dioxide, have surfaces with strong mineralization, which may interfere with the ability to clarify the key aspects of the oxide biomaterial regarding protein adsorption and cellular processes. Here, nonmineralized crystalline and vitreous silica were selected as model oxide biomaterials to explore the inherent properties of these materials on the absorption behavior of the functional protein fibronectin (Fn) and on the osteogenic differentiation of mesenchymal stem cells (MSCs). We demonstrated that due to the smaller O1s binding energy, the weaker polarization of oxygen atoms in vitreous silica produced a greater amount of acidic hydroxyls after hydration compared to crystalline silica. These distinct features significantly upregulated the exposure of arginylglycylaspartic acid (RGD) and synergy sites (PHSRN) of Fn and eventually enhanced the osteogenic differentiation of MSCs on vitreous silica surfaces through activation of the integrin-linked kinase (ILK) signaling pathway. Our results highlight the key role of inherent oxide biomaterial crystallinity in protein adsorption and cell behavior. KEYWORDS: mesenchymal stem cells, α-quartz, vitreous silica, nonmineralization, osteogenic differentiation
1. INTRODUCTION Cellular responses on material surfaces can be significantly affected by adsorbed proteins, which can be regulated by the inherent properties of the material, such as matrix stiffness,1 surface roughness,2 surface chemistry,3 and surface atomic structure.4 When biomaterials are exposed to a physiological environment, protein adsorption occurs instantaneously.5,6 The cells perceive the materials via the proteins that are adsorbed onto the surfaces.7 Hence, cellular responses are directly modulated by the composition, distribution, and conformation of the adsorbed proteins.8−10 There are many surface features of materials that can affect the behavior of protein adsorption, such as hydrophobicity, surface charge, morphology, and functional groups.11,12 The wettability of the material surfaces can also affect the protein adsorption,13 and hydrophobic surfaces are more conducive to protein adsorption.14 Some studies have also shown that excessive hydrophobic surfaces may change protein structures, which results in a downregulating of protein activity.15 Charged surfaces are more attractive to proteins than uncharged surfaces,16 and the surface charge can even change the conformation of proteins on the surface.17 Regarding © 2019 American Chemical Society
atomic structures, the protein adsorption on different crystal facets of the same nanocrystal can be significantly different.18 For example, the amorphous and anatase TiO2 leads to different protein adsorption characteristics.19 Because the surface features of a material are determined by the atomic arrangement of the component molecules, it is meaningful to study the biological properties of different crystal facets. Studying the interactions between serum proteins and biomaterial surfaces is fundamental to understanding and regulating the performance of oxide biomaterials.6 However, in the culture medium or simulated body fluid, mineralization takes place very easily on the surface of typical oxide biomaterials such as calcium phosphate and titanium dioxide.20,21 This surface mineralization may interfere with observations of the intrinsic cellular responses taking place on the oxide surfaces. Biomaterials based on silicon and silica have been widely applied in biomedicine, especially in the area of bone Received: May 15, 2019 Accepted: June 10, 2019 Published: June 10, 2019 3352
DOI: 10.1021/acsbiomaterials.9b00679 ACS Biomater. Sci. Eng. 2019, 5, 3352−3360
Article
ACS Biomaterials Science & Engineering biology.22 Because almost no mineralization on pure SiO2 surfaces in SBF has been reported,23 quartz was selected as the model oxide in this study. The {110} α-quartz and vitreous silica terminal layers, originally formed by an oxygen atom layer, were utilized as ordered and disordered surfaces to investigate the intrinsic cellular responses when grown on oxide surfaces. The physicochemical properties of the {110} quartz and vitreous silica surfaces were characterized, and MSCs were used to evaluate the osteogenic differentiation response to the two surfaces. A possible mechanism is also proposed.
streptomycin (Gibco, USA)) and 10% fetal bovine serum (FBS, Cellmax, China) in a humidified atmosphere of 5% CO2 at 37 °C. MSCs in the third to fifth passage were used in the experiments. The related experiments in this work were all approved by the Institutional Animal Care and Use Committee of Zhejiang University, Hangzhou, China. 2.4. Cell Viability. The cell viability was determined by a cell counting kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) assay.29 The MSCs were cultured in an incubator with a seeding density of 25 000 cells/cm2. After 1, 4, and 7 days, substrates were transferred to new plates and washed with PBS. Next, 10% CCK-8 solution was added into fresh culture media and incubated for 3 h at 37 °C. Finally, the absorbance of the samples was read at 450 nm using a microplate reader. 2.5. Immunofluorescence Staining. The MSCs were seeded on different substrates at a density of 10 000 cells/cm2. After 24 h of cultivation, the samples were washed twice with 0.05% Tween/PBS and fixed with 4% paraformaldehyde. They were then washed again and permeabilized with 0.4% Triton X-100 in PBS for 15 min and then blocked in PBS solution. Nuclei, F-actin and focal adhesion were stained by DAPI (Sigma, USA), Alexa-Fluor 594 phalloidin (Sigma, USA) and antivinculin polyclonal antibody (Abcam, UK), respectively, according to the manufacturers’ protocols. The fluorescent secondary antibody used in the experiment was FITC-conjugated secondary antibody. After staining, the samples were examined by a CLSM (LSM780, ZEISS, Germany). The MSC spread area and the number of FAs were quantitatively analyzed using the ImageJ software. The area of each cell on the two surfaces was measured with ImageJ, and the average of all cell areas was calculated. The measurements were made on more than 30 randomly selected cells. FA sites were defined using the ImageJ freehand tool, and the other steps were as in the above method.30 2.6. Alkaline Phosphatase (ALP) Activity. The MSCs were seeded on different substrates at a density of 25 000 cells/cm2. After 4 days of culture, the ordinary medium was replaced by osteogenic induction medium, and the cells were cultured for an additional 7 or 14 days. The MSCs were then washed, lysed with cell lysis buffer (Sigma, St. Louis, USA), and transferred to a new 24-well culture plate. The ALP activity was measured with LabAssay ALP (Wako Pure Chemical Industries, Ltd. (Japan)). The ALP quantity was evaluated by the absorbance OD value at 405 nm and normalized to the total protein content, which was assessed by a BCA protein assay kit (Thermo Scientific, USA). The BCIP/NBT ALP color development kit (Beyotime, Shanghai, China) was used to stain the ALP product after 14 days. The stained samples were then imaged by an optical microscope to show the area of the ALP products. 2.7. Extracellular Matrix Mineralization. Alizarin red staining was used to evaluate the mineral deposition of MSCs on the different substrates. The MSCs were cultured in an incubator at a seeding density of 25 000 cells/cm2. On the fourth day, osteogenic induction media was used to replace the culture medium. After a further 21 days of cultivation, the samples were washed and then fixed in 4% paraformaldehyde and stained with 1% Alizarin Red S solution (Sigma, USA). The stained samples were imaged with an optical microscope to show the area of calcium deposition. Subsequently, 10% cetylpyridinium chloride was used to desorb the staining, and the absorbance was detected at 560 nm. 2.8. Quantitative Real-time (RT) Polymerase Chain Reaction (PCR) Assay. The expression of osteogenic genes was examined through a RT-PCR assay. The MSCs were cultured in an incubator at a seeding density of 25 000 cells/cm2. On the fourth day, osteogenic induction media was used to replace the culture medium. After 7 and 14 days of cultivation, the total RNA was extracted using TRIzol reagent. RT-PCR was conducted with an Eppendorf Mastercycler RealPlex and SYBR Green I master mix. The relative expression of osteogenic genes was normalized to that of the reference gene glyceraldehyde-3-phosphate dehydrogenase. The primers for the target genes are provided in the Supporting Information. 2.9. Western Blotting Assays. Western blotting was used to analyze integrin β1, integrin β3, ILK, p-Akt and β-catenin. The MSCs
2. EXPERIMENTAL SECTION 2.1. Substrate Preparation and Characterization of Surface Features. Single-crystal {110} α-quartz and vitreous silica (10 × 10 × 1 mm3, Hefei Kejing Co., Ltd., China) were washed with water and ethanol, and then dried at 60 °C for 24 h. The surface topography features of the two substrates were investigated by atomic force microscopy (AFM, NTEGRA Spectra, NTMDT, Russia) in the noncontact mode. The water contact angle (WCA) was investigated by a contact angle meter (Dataphysics, OCA20, Germany) with the sessile drop method. The surface composition of the two substrates was determined by X-ray photoelectron spectroscopy (XPS, Kratos AXIS Ultra DLD, Al Kα, 1486.6 eV, UK). The XPS spectrum in this work was calibrated to the binding energy of C1s at 284.6 eV. The morphology was observed by a scanning electron microscope (SEM, Hitachi SU-70, 3 kV, Japan). The phase structure of the two substrates was investigated by an X-ray diffractometer (XRD, Thermo ARL X’TRA, Cu Kα, 35 kV, USA). The surface zeta potential of the two substrates at various pH values (6−10) was determined using a SurPASS electrokinetic analyzer (Anton Paar GmbH, Austria) with 0.001 mol/L aqueous KCl solution. 2.2. Adsorption and Conformation of Fibronectin (Fn). To investigate the adsorption and conformation of a cell adhesion-related protein, human plasma Fn was chosen as a model protein in this work. The amount of Fn adsorption on the two surfaces was measured by a micro BCA protein assay kit (Beyotime Biotechnology, China). The samples were immersed in a 10 μg/mL Fn solution for 2 h at 37 °C. The adsorbed Fn was then extracted by a 2 mg/mL SDS (sodium dodecyl sulfate) elution buffer under agitation, after which the nonadsorbed proteins were removed by PBS.24,25 The optical density at 562 nm was measured by a microplate reader (Multiskan MK3, Thermo Scientific, USA). The conformation of the Fn that adsorbed on the different substrates was investigated by enzyme-linked immunosorbent assay (ELISA). First, the different substrates were immersed in excess BSA solution for 24 h at 37 °C. The samples were then incubated in 10 μg/mL Fn protein solution for 24 h at 37 °C. Next, 1% BSA/PBS blocking buffer was added and incubated for 30 min at 37 °C, followed by washing with PBS. The primary monoclonal antibodies HFN7.1 (Developmental Studies Hybridoma Bank), which binds to the flexible linker between the 9th and 10th type-III repeat, and mAb1937 (Millipore, USA), which binds to the 8th type-III repeat,26 were used in this work. The samples were incubated with HFN 7.1 and mAb1937 for 1 h at 37 °C and then rinsed with 0.1% Tween-20 (in PBS). Next, the samples were incubated with ALP-conjugated antimouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) as the secondary antibody for 1 h at 37 °C. The samples were again rinsed, and 4MUP was added and incubated for 45 min at 37 °C. The reaction products were quantified by using a fluorescence microplate reader at 365 nm excitation/460 nm emission. The detailed experimental procedures were based on a previous report.17 2.3. Cell Culture. MSCs were isolated and cultured based on previous reports.27 Briefly, MSCs were isolated from the bone marrow of femurs and tibias of male Sprague−Dawley (SD) rats that were approximately 3 weeks old.28 The MSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Cellmax, China) containing 0.272 g/L L-glutamine (Sigma, USA), 1% antibiotic solution (penicillin and 3353
DOI: 10.1021/acsbiomaterials.9b00679 ACS Biomater. Sci. Eng. 2019, 5, 3352−3360
Article
ACS Biomaterials Science & Engineering
Figure 1. Characterization of the {110} quartz and vitreous silica substrates. (a) XRD spectrum of the {110} α-quartz substrate. (b) XRD spectrum of the vitreous silica substrate. (c) Surface roughness, (d) O1s spectra from XPS, (e) contact angles, (f) zeta potential against pH on the two surfaces. (g) SEM images and the XPS survey after soaking in SBF at 37 °C for 7 days: (h) the {110} quartz surface and (i) the vitreous silica surface. were cultured in an incubator at a seeding density of 25 000 cells/cm2. After 4 days of cultivation, the MSC total protein was collected using RIPA lysis buffer that included 1 mM phenylmethanesulfonyl fluoride (Kangchen, China). The extracted proteins were analyzed by electrophoresis. Chemiluminescence immunodetection was carried out using ECL plus reagent (Thermo Scientific, USA) and the intensity was quantified by the ImageJ software. 2.10. Statistical Analysis. All measurement data were tested in triplicate and expressed as the mean ± standard deviation (SD). Statistical significance was determined via Tukey’s post hoc test and one-way analysis of variance. The data were analyzed by the SPSS software. A p value
