Stability of Silicon Nitride Again Hf Acid

Abstract

The remarkable stoichiometric flexibility of hydroxyapatite (HAp) enables the formation of a variety of charged structural sites at the material's surface which facilitates os remodeling due to binding of biomolecule moieties in zwitterionic mode. In this paper, we report for the first fourth dimension that an optimized biomedical grade silicon nitride (Si₃N₄) demonstrated jail cell adhesion and improved osteoconductivity comparable to highly defective, non-stoichiometric natural hydroxyapatite. Si₃Due north₄'s zwitterionic-similar behavior is a function of the dualism between positive and negative charged off-stoichiometric sites (i.east., Due north-vacancies versus silanols groups, respectively). Lattice defects at the biomaterial'southward surface profoundly promote interaction with positively- and negatively-charged functional groups in biomolecules and issue in the biologically effective characteristics of silicon nitride. These findings are predictable to be a starting point for further discoveries of therapeutic bone-graft substitute materials.

Introduction

The major claiming facing mod orthopaedic devices is their conspicuous inability to suit or respond to irresolute physiologic conditions because they are fabricated from non-living bioinert substances. Conversely, Nature took a peculiarly different approach when information technology chose hydroxyapatite (HAp) as its fundamental edifice material for vertebrates^one,two. While biologic HAp is also non-living, it is not bioinert. Indeed, HAp'southward unique crystal structure is capable of being greatly distorted to accommodate a wide range of cation/anion sizes, with upward to 20% of these sites substituted with other elemental groups (e.g., Na⁺, Mg²⁺, Thou⁺, Sr^two+, CO_three ²⁻, HPO_iv ²⁻, Cl⁻, F⁻, etc.)³. This remarkable stoichiometric flexibility enables the formation of a diverseness of charged structural sites at the fabric'south surface which facilitates bone remodeling due to binding of biomolecule moieties in zwitterionic fashion^iv. HAp's natural surface chemistry is difficult to synthetically replicate. Equally a result, man-made HAp formulations are suboptimal in terms of osteoconductivity and cell-adhesion^five. Natural HAp'due south unique defect chemistry inspired the current investigation of an alternative constructed ceramic, silicon nitride (Si_threeN_four)⁶, to see if it as well exhibits zwitterionic-similar functionality. Prior studies investigated modifications of its surface chemistry to introduce both positively- and negatively-charged defects then classified these sites according to basic concepts of physical chemistry^7,eight. In contrast to other more common biomaterials (i.east., alumina and Ti-alloys), we written report for the first time that an optimized biomedical course Si_threeN₄ demonstrated cell adhesion and improved osteoconductivity comparable to highly lacking, non-stoichiometric natural HAp. Si₃Northward₄'s zwitterionic-like behavior is a function of the dualism between positive and negative charged off-stoichiometric sites (i.eastward., N-vacancies versus silanols groups, respectively)^eight, coordinating to the way lacking Ca^two+ and PO₄ ³⁻ sites perform in HAp². These findings are predictable to be a starting point for further discovery of therapeutic bone graft substitute materials.

The Si₃N₄ of this study (Amedica Corporation, Salt Lake City, UT) contained half dozen weight pct (wt.%) yttrium oxide (Y_iiO_three) and 4 wt.% aluminum oxide (Al_twoO_three) as densification additives. It was prepared by sintering in nitrogen at a temperature in backlog of 1700 °C for ~3 hours, followed by hot-isostatic pressing above 1650 °C under a nitrogen pressure of >200 MPa for ~two hours. The resulting two-phase microstructure consisted of anisotropic ß-Si₃N₄ grains intermixed with a partially crystallized burnished grain-boundary phase of silicon-yttrium-aluminum-oxynitride (Si(Y)AlON)^ix. Preliminary energy dispersive X-ray analyses revealed a composition of the Si(Y)AlON glass 20.8 at.% Si, 20.viii at.% Y, nine.five at.% Al, 44.3 at.% O and 4.v at.% North.

Circular deejay samples of this base material (12.seven mm and 1 mm in diameter and thickness, respectively), designated "as sintered" (Equally), were later on polished and lapped to less than xx nm Ra surface terminate and cleaned in deionized h2o to remove contaminants. Iii separate surface treatments were then performed on these samples including wet-chemical etching in hydrofluoric acid (HF), loftier-temperature nitrogen atmosphere annealing (NA) and loftier-temperature thermal oxidation (TO). HF etching of the AS Si_iiiNorth_iv removed the amphoteric silica (SiO_two) passivation layer without significantly affecting the underlying nitride grains. This treatment was expected to maximize the concentration of surface amine groups, biasing the composition as far every bit possible to the nitride finish of the nitride-oxide spectrum. The NA treatment (1400 °C for thirty min. under filtered N₂ at ~115 kPa) was adopted with the intent of increasing the density of surface amines relative to hydroxyl groups. The TO handling (seven h at 1070 °C in an open up-air kiln) was aimed at completely oxidizing the surface, maximizing the concentration of hydroxyl groups and biasing the composition as far equally possible to the oxide end of the nitride-oxide spectrum. X-ray photoelectron spectroscopy information, which nosotros extensively presented in a previous paper^seven, showed a surface composition for the NA samples with N/Si and O/Si diminutive ratios equal to 1.02 and 0.5, respectively. On the other hand, the TO samples showed N/Si and O/Si atomic ratios equal to 0.09 and 1.98, respectively. Characterizations of the mechanical properties of the surface treated materials are nonetheless ongoing. However, preliminary data showed minimal variation of bulk backdrop for strength and toughness as compared to the Every bit sample. Each sample grouping was then spectroscopically examined and subsequently subjected to biologic testing and characterization.

The Raman spectra (RS) were recorded in backscattering using a triple monochromator (T-64000, Jobin-Yvon, Horiba Grouping, Kyoto, Japan) and an excitation source at 532 nm. Cathodoluminescence (CL) spectra were nerveless in a field-emission gun scanning electron microscope (FEG-SEM, SE-4300, Hitachi Co., Tokyo, Nippon). For all samples, exactly the same experimental conditions were applied (acceleration voltage and beam current fixed at half-dozen kV and 180 pA, respectively). The electron-stimulated luminescence was analyzed past a high spectrally resolved monochromator (Triax 320, Jobin-Yvon, Horiba Group, Tokyo, Japan).

Prison cell proliferation and osteoconductivity were assessed using homo osteosarcoma cells (SaOS-2) with a density of 5 × ten^v prison cell/ml seeded onto the treated Si₃N₄ disks within petri-dishes. In the cell-proliferation experiments, cells were incubated in four.5 yard/L glucose DMEM (D-glucose, L-Glutamine, Phenol Crimson and Sodium Pyruvate) supplemented with 10% fetal bovine serum and allowed to proliferate within each petri-dish for about 24 h at 37 °C. The proliferation time was selected according to a previously published report by other authors¹⁰. Afterward, the cells were stained for fluorescence microscopy with Phalloidin (green; F-actin) and Hoechst 33342 (blue; nuclei) for i hour and washed three times with i mL TBST solution.

Cell counts were and so performed on more than 30 fluorescence micrographs for each of the samples. 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 ELISA, assuming 1:1 equally reactive normality of sRANKL:OPG ratio. Human being recombinant sRANKL (Peprotech, True cat. #310-01) was used for the in vitro osteoclastogenic assay¹¹. In the osteoconductivity tests, cell seeding took place in an osteogenic medium which consisted of DMEM supplemented with almost fifty μg/mL ascorbic acrid, well-nigh x mM β-glycerol phosphate, 100 mM hydrocortisone and nigh 10% fetal bovine dogie serum. The samples were incubated for 7 days at 37 °C. Results were assessed by laser microscopy and 3D image analyses. All experiments were repeated in triplicate, with data expressed as means ± i standard deviation. Statistical analysis was performed co-ordinate to the unpaired Educatee's t-test or to i-way Assay of Variance (ANOVA). A p value < 0.05 was considered statistically significant.

Effigy 1(a–c) are fluorescence micrographs of SaOS-2 cells after 24 h proliferation on the NA, TO and Equally Si_iiiN₄ samples, respectively. Upon visual inspection, the NA sample showed the highest affinity for jail cell adhesion and proliferation amidst the series of tested Si_iiiN_iv samples. Looking at the CL spectra from differently treated Si₃N₄ materials in (d), its biological affinity was establish to be directly proportional to the population of positively charged defects (i.eastward., nitrogen vacancies and N-N bonds; cf. labels). Effigy 2(a–c) show three-dimensional laser micrographs of the HAp structure developed on the NA, TO and AS Si_iiiDue north₄ samples after one calendar week'south activity of the SaOS-ii cells, respectively. The NA sample showed the most significant HAp growth, with locations as high as 0.4 ~ 0.5 mm. In (d), a summary is shown of the Raman characterization of the surfaces of these various samples. These analyses confirmed both the germination of HAp (cf. PO_four ³⁻stretching at ~960 cm^−one and labels) and a college average amount of HAp on the NA samples. Results from the statistical evaluation of cell proliferation and HAp formation for all Si₃Northward₄ samples in comparison with commercially available alumina and Ti-alloy biomaterials are given in Fig. 3(a,b), respectively. Again, the highest activity was plant on the NA Si₃North₄ samples. Their improvement was significant in terms of cell proliferation (cf. plot of number of cells per unit area in Fig. three(a)) and HAp growth (cf. plot of formed HAp volume per unit of measurement area in Fig. iii(b) as obtained by three-dimensional laser microscopy), not only with respect to the AS Si_threeN₄ samples, just also in comparing to the alumina and Ti-alloy biomaterials. Photographs of water contact angles, shown at the summit of Fig. 3, demonstrate that the improved hydrophilicity of the tested samples matches their biological affinity. Thermal treatments of the Every bit Si_iiiN_four samples produced extremely low contact angles (cf. Fig. 3), measured using a goniometer technique, as previously reported⁷. The analogousness of the various Si₃N₄ samples for osteoblast cell attachment in comparison with other biomaterials was confirmed by results from the sRANKL experiments shown in Fig. 3(c). The observed deficiency in the Receptor Activator of NF-kB Ligand (RANKL), a membrane-bound poly peptide cleaved into soluble sRANKL by metalloproteinase 14, revealed a depression propensity for osteoclast formation, suggesting that Si₃N₄ presents a biologically friendly surface. Considering the physical chemistry characteristics of the Si₃N₄ surface and its favorable cell adhesion/osteoconduction performance, it is hypothesized that the thermal treatments produce a mixture of negatively- and positively-charged functional surface groups (i.e., silanols, silicon-amines and N-vacancies, etc.) that are capable of binding biomolecules in zwitterionic fashion.

While streaming potential measurements yield a net negative surface charge at homeostatic pH^seven, positive charges are also present. Besides amine groups, Y-OH and Al-OH groups (i.e., from sintering additives) likely protonate and become positively charged at physiologic pH. They can substitute for silanols, thereby increasing the zwitterionic-similar character of the surface. Another significant finding was the formation of a peculiar Si(Y)AlON stage on the surface of the NA sample, which increased the positively charged lacking sites equally observed by CL. These defects intimately mix at the atomic level with the silanol-dominated (i.eastward., negatively-charged) Si_threeNorthward_four surface. Due to their predominantly positive charge, Due north-vacancies and defects associated with N-N bonds further the zwitterionic-like character of the surface. A significant corporeality of differential surface charge is therefore present on Si₃North₄ in comparison to oxide biomaterials (e.g., alumina, which exhibits only one dominant functional grouping). Evidence for this assertion is the hitting similarity found for the distribution and morphology of the newly formed Si(Y)AlON phase and the proliferated cells on the surface of the NA Si₃Northward₄ sample (cf. Fig. 4). The morphology of the Si(Y)AlON phase formed on the surface, which is shown in the laser micrograph in Fig. 4(a), resembled the location of proliferating cells on the surface of the NA Si₃N₄ sample after 24 h of SaOS-2 proliferation (given in the scanning electron micrograph on fixed sample in Fig. 4(b)).

A significant amount of interest has been generated over the past decade in functionalizing biomaterial surfaces to be zwitterionic for anti-fouling purposes¹². A surface where positive and negative charges are intimately mixed allows for extreme hydrophilicity (as observed on the current surface-treated Si₃N₄ materials); it also prevents bacterial adhesion (as previously reported for Every bit Si_threeN₄ samples)^thirteen. Moreover, zwitterated surfaces accept also been observed to induce greater apatite germination on titanium alloys^xiv. The zwitterionic-like backdrop of the NA Si₃N₄ samples are derived from: (i) a significant but minority population of amine sites that are positively-charged; (ii) substitutional Y and Al on Si sites yielding positively-charged Y-OH_ii ⁺ and Al-OH_two ⁺ groups; (iii) an increased concentration of positively-charged lattice defects; and (iv) an intermixed layer of negatively-charged Si-O⁻ groups. Equally more than positive charges go admixed into a mostly negative surface, it exhibits greater zwitterionic, hydrophilic and osteopromotive characteristics. Consequently, lattice defects in the biomaterial's surface profoundly promote interaction with positively and negatively charged functional groups in biomolecules. Further studies, presently ongoing, seem too to confirm a favored osteoblast differentiation for the NA sample in a higher place other surface treatments. Like to naturally occurring HAp, Si_iiiDue north_four illustrates the concept that atomically lacking materials exhibit biologically effective characteristics.

Methods

Cell proliferation experiments

The SaOS-2 cells used in this research were cultured in DMEM medium consisting of 4.5 g/L D-glucose, L-Glutamine, Phenol Cherry and Sodium Pyruvate, in addition to 10% of fetal bovine serum. Cell proliferation and osteoconductivity were assessed using an initial prison cell density of v × ten^v prison cell/ml seeded onto the treated Si₃N₄ disks within conventional petri dishes. Cells were allowed to proliferate within the petri dish for 24 h at 37 °C. The cells were and then fixed with 4% paraformaldehyde (PFA) for x min and permeabilized in TBS-0.1% Triton 10 for v min. Before each of these two steps, the cells were washed with 1 mL TBS (twenty mM Tris-HCl pH vii.5 and 150 mM NaCl). Finally, the cells were washed with i mL TBST (TBS, 0.05% tween 20 and 0.05% NaN₃) for i hour.

For visualization, the cells were stained for fluorescence microscopy with Phalloidin (green; F-actin) and Hoechst 33342 (blue; nuclei) for i hour and washed three times with 1 mL TBST solution (mixture of Tris-Buffered Saline and Tween 20).

Osteoconductivity experiments

In osteoconductivity tests, cell seeding took place in an osteogenic medium, which consisted of DMEM supplemented with about 50 μg/mL ascorbic acid, about ten mM β-glycerol phosphate, 100 mM hydrocortisone and virtually 10% fetal bovine calf serum. The samples were incubated for 7 days at 37 °C. Results were assessed by light amplification by stimulated emission of radiation microscopy and 3D image analyses, equally explained later on.

Raman experiments

The Raman spectra were recorded in backscattering using a triple monochromator (T-64000, Jobin-Yvon, Horiba Grouping, Kyoto, Nippon) equipped with liquid nitrogen-cooled charge coupled device (CCD), a confocal pinhole and polarization filters. The excitation source in the present experiments used a 532 nm Nd:YVO_four 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.v was used both to focus the laser beam on the sample surface and to collect the scattered Raman light. Confocal experiments were conducted with a pinhole aperture of 100 μm and by employing an objective lens with a magnification of 100x. Spectral lines were analyzed with the aid of a commercially available software parcel (Labspec 4.02, Horiba/Jobin-Yvon, Kyoto-Nippon).

Cathodoluminescence experiments

Cathodoluminescence spectra were nerveless in a field-emission gun scanning electron microscope (FEG-SEM, SE-4300, Hitachi Co., Tokyo, Japan). All samples were analyzed during the same experimental session, with exactly the same experimental conditions being applied (acceleration voltage and axle current fixed at 6 kV and 180 pA, respectively). The nominal spatial resolution of the electron axle at the sample surface was ane.5 nm. The microscope was equipped with a CL device consisting of an oblong mirror and a bundle of optical fibers, used to collect and to focus, respectively, the electron-stimulated luminescence emitted by the sample into a loftier spectrally resolved monochromator (Triax 320, Jobin-Yvon/Horiba Group, Tokyo, Japan). The obtained spectra were deconvoluted into Gaussian sub-bands using commercially available software (Origin nine.one, OriginLab Co., Northampton, MA, USA).

Laser microscopy experiments

The optical morphologies of the samples were characterized in a back-scattered confocal laser microscope (Keyence, VK-X210, Osaka, Nippon). The excitation source in the experiments used a 408 nm violet semiconductor light amplification by stimulated emission of radiation operating with an output of 0.95 mW. An objective lens with a numerical aperture of 0.55 was used both to focus the laser beam on the sample surface and to collect the reflected light. Taking advantage of an automated phase, whose movement can be controlled in xyz directions, a three dimensional colored paradigm of the sample surface tin can be caused by joining the 3D profile with the optical microscope image.

Boosted Data

How to cite this commodity: Pezzotti, G. et al. Silicon Nitride: A Synthetic Mineral for Vertebrate Biology. Sci. Rep. 6, 31717; doi: 10.1038/srep31717 (2016).

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Acknowledgements

The authors profoundly thank Prof. M. N. Rahaman, Dr. Hiroaki Ichioka and Dr. E. Marin for their contribution to this piece of work.

Author information

Affiliations

Ceramic Physics Laboratory, Kyoto Found of Technology, Sakyo-ku, Kyoto, 606-8126, Matsugasaki, Nippon

Giuseppe Pezzotti, Marco Boffelli & Eleonora Vitale
Amedica Corporation, 1885 West 2100 South, Salt Lake City, 84119 Utah, The states

Bryan J. McEntire, Ryan Bock & B. Sonny Bal
Department of Medical Engineering for Treatment of Bone and Joint Disorders, Osaka University, 2-ii Yamadaoka, Suita, 565-0854, Osaka, Nihon

Wenliang Zhu
Section of Molecular Jail cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kyoto, 602-8566, Kamigyo-ku, Nippon

Leonardo Puppulin
Department of Dental Medicine, Graduate School of Medical Science, Kyoto Prefectural Academy of Medicine, Kyoto, 602-8566, Kamigyo-ku, Japan

Tetsuya Adachi, Toshiro Yamamoto & Narisato Kanamura
Department of Orthopaedic Surgery, University of Missouri, Columbia, 65212, Missouri, United states

B. Sonny Bal

Contributions

K.P. conceived the logic of the paper and wrote the manuscript. B.J.M. prepared the silicon nitride samples and helped write the manuscript. R.B. performed the mail service-sintering treatments on the silicon nitride samples and measured the contact wetting angles. Chiliad.B. performed the cathodoluminescence experiments. W.Z. performed the theoretical treatment of the cathodoluminescence spectra leading to their Gaussian deconvolution and provided the interpretation of the deconvoluted sub-bands. E.Five. performed the cell proliferation counting and the assessment of hydroxyapatite volume fraction by image analysis. Fifty.P. performed the Raman spectroscopy experiments. T.A. cultivated the SaOS-ii cells and made the cell proliferation and apatite growth tests. T.Y. conceived the cell experiments and assessed their statistical reliability. Due north.K. conceived and supervised the biological function of this study. B.Due south.B. suggested the idea of testing silicon nitride with respect to its osteoconductivity, realized the importance of the findings in the medical field and contributed to the training of the manuscript.

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Pezzotti, G., McEntire, B., Bock, R. et al. Silicon Nitride: A Synthetic Mineral for Vertebrate Biological science. Sci Rep half-dozen, 31717 (2016). https://doi.org/10.1038/srep31717

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Received: 02 Nov 2015
Accepted: 26 July 2016
Published: 19 Baronial 2016
DOI : https://doi.org/10.1038/srep31717

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