Article pubs.acs.org/journal/abseba
In Situ Spectroscopic Screening of Osteosarcoma Living Cells on Stoichiometry-Modulated Silicon Nitride Bioceramic Surfaces Giuseppe Pezzotti,*,†,‡ Bryan J. McEntire,§ Ryan Bock,§ Wenliang Zhu,∥ Francesco Boschetto,†,⊥ Alfredo Rondinella,†,⊥ Elia Marin,† Yoshinori Marunaka,‡ Tetsuya Adachi,⊥,# Toshiro Yamamoto,⊥ Narisato Kanamura,⊥ and B. Sonny Bal§,¶ †
Ceramic Physics Laboratory, Kyoto Institute of Technology, Sakyo-ku, Matsugasaki, 606-8126 Kyoto, Japan Department of Molecular Cell Physiology, ⊥Department of Dental Medicine, and #Department of Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan § Amedica Corporation, 1885 West 2100 South, Salt Lake City, Utah 84119, United States ∥ Department of Medical Engineering for Treatment of Bone and Joint Disorders, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0854, Japan ¶ Department of Orthopaedic Surgery, University of Missouri, Columbia, Missouri 65212, United States ‡
ABSTRACT: Osteosarcoma cell viability, proliferation, and differentiation into osteoblasts on a silicon nitride bioceramic were examined as a function of chemical modifications of its as-fired surface. Biological and spectroscopic analyses showed that (i) postsintering annealing in N2 gas significantly improved apatite formation from human osteosarcoma (SaOS-2) cells; (ii) in situ Raman spectroscopic monitoring revealed new metabolic details of the SaOS-2 cells, including fine differences in intracellular RNA and membrane phospholipids; and (iii) the enhanced apatite formation originated from a high density of positively charged surface groups, including both nitrogen vacancies (VN3+) and nitrogen N−N bonds (N4+) formed during annealing in N2 gas. At homeostatic pH, these positive surface charges promoted binding of proteins onto an otherwise negatively charged surface of deprotonated silanols (SiO−). A dipole-like electric-charge, which includes VN3+/N4+ and SiO− defective sites, is proposed as a mechanism to explain the attractive forces between transmembrane proteins and the COO− and NH2+ termini, respectively. This is analogous to the mechanism occurring in mineral hydroxyapatite where protein groups are specifically displaced by the presence of positively charged calcium loci (Ca+) and off-stoichiometry phosphorus sites (PO42−). KEYWORDS: silicon nitride bioceramic, surface treatment, nitrogen vacancies, hydroxyapatite formation, SaOS-2 cells
1. INTRODUCTION As scientific research continues to reveal fine details of the ultrastructural organization of human cells, patients suffering from bone diseases are benefiting from new repair strategies that utilize materials with improved bioactivity.1 Hydroxyapatite is the most widely used bioceramic clinically because it is among a group of biomaterials that promote faster healing. It has been the object of intense research for its ability to promote bone integration through rapid cell proliferation.2−5 Recent electrical polarization studies performed on hydroxyapatite substrates have shown that a permanent residual charge of an opposite sign can positively influence the response and activity of cells.6−8 This effect is closely interfaced with crystal stoichiometry, which has been shown to play a pivotal role in overall cell activity. Restricting dehydration of OH− ions in the apatite lattice improves protonic conductivity, whereas partial dehydration produces an increased concentration of vacancies that detrimentally affects protein adhesion.9−11 In the chemistry of natural calcium phosphates, a new concept of “nomadic © 2016 American Chemical Society
polarization” (i.e., the electrical charge caused by protonic mobility due to partial dehydration of the OH− sublattice) is key in elucidating the positive effect of an electrical dipole on the biological response of cells. However, stoichiometric stabilization of synthetic apatites represents a formidable challenge as demonstrated by the number of contradictory in vitro and in vivo results.12−17 Localized charge fluctuations continuously occur at the surface of hydroxyapatite in the biological environment in response to its ongoing chemical transitions. Indeed, the persistent swing in the formation of positively- and negatively charged sites is the main driving force in promoting hydroxyapatite formation at this biomaterial’s surface. Nevertheless, it is also the physical reason behind the difficulty in obtaining stable chemical functionalization of synthetic apatite surfaces.18 Received: March 3, 2016 Accepted: June 16, 2016 Published: June 16, 2016 1121
DOI: 10.1021/acsbiomaterials.6b00126 ACS Biomater. Sci. Eng. 2016, 2, 1121−1134
Article
ACS Biomaterials Science & Engineering
employed to maximize the surface density of amine relative to hydroxyl groups. All samples were prepared as disks with diameters and thicknesses equal to 12 and 1 mm, respectively. For comparison testing, disk samples were also made of biomedical Ti with the same dimensions and similar surface roughness characteristics. The overall roughness, as measured by laser profilometry, differed within a relatively small range (Ra = 2.5 ± 0.22 μm) among all of the studied surfaces. Therefore, the influence of Ra among different samples was not considered to be a significant uncontrolled variable within this study. Cell Culture and Characterizations. The SaOS-2 human osteosarcoma cell line was selected for this study because it has been widely used in other bone cell differentiation, proliferation, and metabolism research and because these cells are known to be capable of rapid bone production.23,24 The cells were first cultured and incubated in an osteoblast-inducer medium consisting of 4.5 g/L of glucose DMEM (D-glucose, Lglutamine, phenol red, and sodium pyruvate) supplemented with 10% fetal bovine serum. They were allowed to proliferate within Petri dishes for 24 h at 37 °C. The final SaOS-2 concentration was equal to 5 × 105 cell/ml. The cultured cells were then deposited on the top surface of each of the variously treated Si3N4 and Ti disks, all of which were previously sterilized by exposure to UV light. In the cell adhesion tests, cell seeding took place in an osteogenic medium, which consisted of DMEM supplemented with the following nominal amounts: 50 μg/ mL of ascorbic acid, 10 mM β-glycerol phosphate, 100 mM hydrocortisone, and 10% fetal bovine calf serum. All samples were incubated for 7 days at 37 °C. The medium was changed twice during the incubation period. Cell attachment tests were repeated three times (n = 3) for each investigated sample. The arithmetic average of the three tests were then plotted to compare differences. The presence or deficiency in the receptor activator of NF-kB ligand (RANKL), a membrane-bound protein cleaved into soluble sRANKL by metalloproteinase 14, was used as a probe to test for the propensity to form osteoclasts. sRANKL and OPG were quantified in cell conditioned media using the R&D System ELISA kits MTR00 and MOP00, respectively, according to the manufacturer’s instructions. Titration of free sRANKL was computed by the difference between the equivalent weight of sRANKL and OPG obtained from the ELISA assays, assuming 1:1 as the reactive normality of sRANKL:OPG ratio. Human recombinant sRANKL (Peprotech, Cat. #310-01) was used for the in vitro osteoclastogenic assay.25 Insulin-like growth factor 1 (IGF-1) is essential for apatite growth and differentiation processes,26,27 and it is a modulator of bone growth through endocrine/paracrine and autocrine mechanisms. IGF-1 signaling was used as a probe for measuring cell proliferation and differentiation efficiency. Human IGF-1 ELISA (RayBio; RayBiotech, Inc., Norcross, GA) was employed to measure IGF-1 in the osteosarcoma cell culture supernates. Prior to measuring, samples were incubated for 1 h at room temperature. Then, 100 μL of streptavidin solution was added followed by 45 min of incubation at room temperature. This was followed by the addition of 100 μL of TMB one-step substrate reagent and incubation for 30 min at room temperature. Finally, 50 μL of stop solution was added. Optical density results at 450 nm were read immediately after these preparation procedures. sRANKL and IGF-1 tests were both repeated twice on each individual sample, and their arithmetic averages were plotted. Because two identically prepared samples for each type of material were tested, the total number of tests per each type of material was n = 4. Data for hydroxyapatite formation, sRANKL, and IGF-1 experiments were expressed as means ± one standard deviation. Statistical analyses were performed according to the unpaired Student’s t test or according to one-way analysis of variance (ANOVA). A value of p < 0.05 was considered statistically significant. Surface Characterizations. Scanning electron microscopy (SEM) was carried out on the as-fired Si3N4 surfaces before and after chemical/heat treatments using a field emission gun scanning electron microscope (FEG-SEM) (Quanta, FEI, Hillsboro, OR). Micrographs were collected using secondary and backscattered electron detectors. The microscope was equipped with an energy dispersive X-ray spectroscopy (EDS) device for elemental mapping. All samples were
In a previous paper, an alternative synthetic bioceramic, silicon nitride (Si3N4), was proposed as a primary candidate for bone restoration.19 Chemical modifications of its surface led to the favorable and stable formation of a balance of positively and negatively charged off-stoichiometric sites (i.e., N vacancies and N−N bonds versus silanol groups (SiO−), respectively), leading to enhanced in vitro cell adhesion and apatite formation. This phenomenon is analogous to the way defective Ca+ and PO42− sites perform in synthetic hydroxyapatite.20 However, the covalent nature of the Si−N bond in Si3N4 bestows an environmental stability that synthetic apatites cannot achieve because of the strong ionic bonding of the phosphate and hydroxyl groups to calcium ions. This paper describes the in vitro response of osteosarcoma (SaOS-2) cells to chemically modulated Si3N4 surfaces after long-term (7 day) exposure. Cell attachment and proliferation were quantitatively monitored by conventional optical microscopy, and cellular metabolic activity was screened in situ by Raman spectroscopy. Prior studies revealed that annealing of polished Si3N4 samples in a high-temperature nitrogen atmosphere (i.e., N2-annealed) greatly enhanced cell adhesion and hydroxyapatite mineralization.19 The level of apatite formation on the N2-annealed discs was found to be more than 6 times the amount observed on untreated samples. Given the results of this prior research, the aim of the present study was to examine the same Si3N4 series of samples previously investigated but now using rougher as-fired versus polished surfaces. As-fired surfaces are representative of actual orthopedic devices currently used as intervertebral spacers in spinal fusion surgery.21 This added investigation was undertaken with two specific goals: (i) to confirm improved osteoblast differentiation and proliferation on as-fired Si3N4 due to the N2 treatment and (ii) to elucidate the mechanism(s) behind the observed improvements. These objectives were realized by employing in situ Raman spectroscopy to examine changes in cellular metabolism. Cataloging specific spectroscopic signatures or markers of metabolic activity and developing a mechanistic understanding of cellular responses are essential to improving clinical treatment strategies for advanced spinal or bone graft implants.
2. EXPERIMENTAL PROCEDURES Materials and Surface Treatments. The Si3N4 samples used in the present study were produced by Amedica Corp., (Salt Lake City, UT, USA) using conventional ceramic fabrication techniques. The material contained minor fractions of Y2O3 and Al2O3 as sintering aids. The resulting microstructure exhibited a bimodal granular population, consisting of a minor fraction of relatively large acicular ß-Si3N4 grains embedded in a finer grain matrix. The sintering additives resulted in thin grain boundaries and multiple grain junctions composed of either amorphous or crystalline silicon-yttrium-aluminum-oxynitride (SiYAlON), respectively. Details on the processing of this Si3N4 have been published elsewhere.22 Surfaces of as-fired Si3N4 samples were subjected to one of the following treatments: (i) wet chemical etching by hydrofluoric acid (5% HF solution for 45 s), which was intended to etch out surface SiO2 stemming as a passivation layer, (ii) oxidation treatment upon exposure to ambient atmosphere at 1070 °C for 7 h, and (iii) nitrogen heat treatment at 1400 °C for 30 min under 1−2 psi of filtered N2 gas. The chemical etching and oxidation treatments could be considered as antithetical to each other, conspicuously eliminating or greatly enhancing the amount of glassy silica at their respective sample surfaces. In terms of surface termination species, the concentrations of amine and hydroxyl groups at the Si3N4 surface were pushed to the nitride and oxide ends of the nitride-oxide spectrum, respectively. The high-temperature N2-atmosphere treatment was 1122
DOI: 10.1021/acsbiomaterials.6b00126 ACS Biomater. Sci. Eng. 2016, 2, 1121−1134
Article
ACS Biomaterials Science & Engineering
Figure 1. Backscattered FEG-SEM micrographs of (a) as-fired, (b) HF-etched, (c) thermally oxidized, and (d) N2-gas annealed Si3N4 samples (note the brighter areas emphasized by white contours). (e, f) Elemental analyses by EDS at spots A and B, respectively, as labeled in (d). at 785 nm, and the spectral resolution was 1.2 cm−1 (spectral pixel resolution equal to 0.3 cm−1/pixel). As the software attached to this latter equipment automatically provided average spectra on selected areas, Raman spectra from SaOS-2 cells on different substrates were compared by averaging them over ∼103 measurements per each sample.
sputter-coated (108auto, Cressington, Watford, UK) with a thin (∼20−30 Å) layer of gold. Samples were imaged using an accelerating voltage of 10 kV at working distances of 7−10 mm and spot sizes of 4−4.5 mm. Laser-scanning micrographs of the sample surfaces after 7 days of exposure to SaOS-2 cells were collected by means of a 3D laser-scanning microscope (VK-X200 K Series, Keyence, Osaka, Japan) using a 150× objective lens with a numerical aperture of 0.9. The supplied software allowed for the calculation of the surface roughness in accordance with ISO 4287:1997. Raman Spectroscopy. Three types of Raman instruments were used in the study. The Raman spectra of the Si3N4 samples before the cell adhesion tests were recorded in backscattering mode using a triple monochromator (T-64000, Jobin-Yvon, Horiba Group, Kyoto, Japan) and an excitation source emitting at 532 nm (Nd:YVO4 diode-pumped solid-state laser; SOC JUNO, Showa Optronics Co. Ltd., Tokyo, Japan) operating with a power of 200 mW. An objective lens with a numerical aperture of 0.5 was used to both focus the laser beam on the sample surface and collect the scattered Raman light. A pinhole aperture of 100 μm was adopted while employing an objective lens with a magnification of 100×. Averages of 30 Raman spectra collected at random locations were used to compare the treated Si3N4 samples. The collected Raman bands were analyzed and compared with respect to their spectral location and full width at half-maximum (FWHM). Raman analyses of the grown apatite and osteoblast metabolism before and after 7 days of exposure to the various disk surfaces were performed by means of a LabRAM HR800 (Horiba/Jobin Ivon, Kyoto, Japan) operated in microscopic measurement mode with confocal imaging capability in two dimensions. The light source was a HeNe laser operating at 633 nm with a power of 10 mW. A holographic notch filter was incorporated into the system to enable acquisition of Raman spectra with conditions optimized by the manufacturer for high sensitivity. The Raman emission was monitored by a single monochromator connected to an air-cooled charge-coupled device (CCD) detector (Andor DV420-OE322; 1024 × 256 pixel). The acquisition time was fixed at 10 s. Spectral deconvolution into Lorentzian bands was performed by means of commercially available software (Origin 9.1, OriginLab Co., Northampton, MA, USA). An average of 20 Raman measurements was obtained at different random locations from as-cultured SaOS-2 cells and from SaOS-2 cells deposited onto various disk surfaces. In situ Raman microscopy images were collected on living SaOS-2 cells using a dedicated instrument (RAMANtouch, Nanophoton Co., Osaka, Japan) with a 20× immersion-type optical lens. This spectroscope allowed ultrafast imaging of up to 400 spectra simultaneously, thus collecting maps in a time faster than the movement of the cells. The excitation source was
3. RESULTS AND DISCUSSION Characterizations of Modulated Si3N4 Surfaces and SaOS-2 Cell Adhesion. Panels a−d in Figure 1 show FEGSEM micrographs of as-fired, HF-etched, thermally oxidized, and N2-annealed samples, respectively. A comparison among the differently treated samples revealed that acicular grains abundantly grew over the free surfaces of all samples. Moreover, the morphologies of their surfaces did not significantly differ among the samples. However, one peculiar feature that could be noted in the N2-annealed sample was the presence of brighter areas in the backscattered FEG-SEM micrographs typically 1 in PC and PI, whereas it distinctly displayed values
