orthosilicic acid, si(oh)4, stimulates osteoblast differentiation in … · 2019. 2. 13. ·...
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Accepted Manuscript
Orthosilicic acid, Si(OH)4, stimulates osteoblast differentiation in vitro by up-regulating miR-146a to antagonize NF-κB activation
Xianfeng Zhou, Fouad M. Moussa, Steven Mankoci, Putu Ustriyana, NianliZhang, Samir Abdelmagid, Jim Molenda, William L. Murphy, Fayez F. Safadi,Nita Sahai
PII: S1742-7061(16)30216-1DOI: http://dx.doi.org/10.1016/j.actbio.2016.05.007Reference: ACTBIO 4239
To appear in: Acta Biomaterialia
Received Date: 29 December 2015Revised Date: 28 April 2016Accepted Date: 5 May 2016
Please cite this article as: Zhou, X., Moussa, F.M., Mankoci, S., Ustriyana, P., Zhang, N., Abdelmagid, S., Molenda,J., Murphy, W.L., Safadi, F.F., Sahai, N., Orthosilicic acid, Si(OH)4, stimulates osteoblast differentiation in vitroby upregulating miR-146a to antagonize NF-κB activation, Acta Biomaterialia (2016), doi: http://dx.doi.org/10.1016/j.actbio.2016.05.007
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Orthosilicic acid, Si(OH)4, stimulates osteoblast differentiation in vitro by
upregulating miR-146a to antagonize NF-κB activation
Xianfeng Zhou1,*, Fouad M. Moussa2, Steven Mankoci1, Putu Ustriyana1,Nianli Zhang3, Samir
Abdelmagid2, Jim Molenda4, William L. Murphy4, Fayez F. Safadi2, and Nita Sahai1,5,6,*
1Department of Polymer Science, University of Akron, Akron, OH 44325, USA
2Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown,
Biomedical Science Graduate Program, Kent State University, Kent, OH, USA
3Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor,
MI 48109, USA
4Department of Biomedical Engineering, Department of Orthopedics and Rehabilitation,
University of Wisconsin, Madison, WI 53705, USA
5Integrated Bioscience Program, University of Akron, OH 44325, USA
6Department of Geology, University of Akron, OH 44325, USA
Correspondence should be addressed to*X.Z. and *N.S.
E-mail:[email protected]; [email protected]
Fax: +1-330-972-7001
Phone: +1-330-972-5795
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ABSTRACT
Accumulating evidence over the last 40 years suggests that silicate from dietary as well as
silicate-containing biomaterials is beneficial to bone formation. However, the exact biological
role(s) of silicate on bone cells are still unclear and controversial. Here, we report that
orthosilicic acid (Si(OH)4) stimulated human mesenchymal stem cells (hMSCs) osteoblastic
differentiation in vitro. To elucidate the possible molecular mechanisms, differential microRNA
microarray analysis was used to show that Si(OH)4 significantly up-regulated microRNA-146a
(miR-146a) expression during hMSC osteogenic differentiation. Si(OH)4 induced miR-146a
expression profiling was further validated by quantitative RT-PCR (qRT-PCR), which indicated
miR-146a was up-regulated during the late stages of hMSC osteogenic differentiation. Inhibition
of miR-146a function by anti-miR-146a suppressed osteogenic differentiation of MC3T3 pre-
osteoblasts, whereas Si(OH)4 treatment promoted osteoblast-specific genes transcription, alkaline
phosphatase(ALP) production, and mineralization. Furthermore, luciferase reporter assay,
Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence
showed that Si(OH)4 decreased TNFα-induced activation of NF-κB, a signal transduction
pathway that inhibits osteoblastic bone formation, through the known miR-146a negative
feedback loop. Our studies established a mechanism for Si(OH)4 to promote osteogenesis by
antagonizing NF-κB activation via miR-146a, which might be interesting to guide the design of
osteo-inductive biomaterials for treatments of bone defects in humans.
Key words: Silicic acid, osteoblastic differentiation, osteoclastic differentiation, microRNA, NF-
κB.
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1. INTRODUCTION
The earliest work involving silicate and bone dates back to the early 1970s, when it was
suggested that silicate may play an important role in skeletal development and repair [1-3].
Subsequently, the positive effect of silicate on bone has raised the interest of multiple research
groups working on bone graft substitutes [4]. Silicate-based bioactive glasses [4-9], silicate-
substituted ceramics [10-15], and natural silicate bioceramics [16-20] have been developed and,
in most of cases, have shown better biological properties in orthopaedic applications when
compared to their silicate-free counterparts. Systematic studies by Hench et al. [21-23] and De
Aza [16, 17, 24] have identified silicate in bioactive glasses or glass-ceramics could be released
as Si(OH)4 by hydrolyzing Si-O-Si bridges and re-precipitate to form surface silanol groups. The
concentration of silanol groups could be critical because they act as a nucleation agent for apatite
formation [25, 26]. A “passive” role for silicate has been proposed in which the presence of
silicate in the materials results in changes of grain size, surface topography, etc., leading
ultimately to a change of biological response [4, 8, 9, 27]. There is evidence in the literature
which indicates that inorganic dissolution products (Si(OH)4, Ca2+, PO43-, etc.) from inorganic
silicate materials can influence and control the cell cycle of osteogenic precursor cells and
ultimately control the differentiated cell population [28, 29]. An “active” role, as claimed by
researchers and companies, is that silicate can affect bone cell metabolism [30]. Thus, the silicate
materials are not simply bone graft substitutes but, effectively, can be cell- and gene-activating
biomaterials [31]. However, none of the studies focused on the active roles have demonstrated
yet that the positive biological effects can be attributed solely to soluble silicate, since Ca2+ and
PO43-, which are released from silicate-based materials, are known to have osteo-inductive
effects on bone cells [32, 33]. A number of in vitro studies using osteoblast-like cells have added
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to the evidence that silicate may be beneficial for bone formation. For example, silicic acid
addition to MG63 osteosarcoma cells has been shown to increase collagen type I (Col I) levels
1.75-fold [34]. The authors proposed a structural role (stabilization of collagen) or a metabolic
role (a co-factor for prolyl hydroxylase) for silicate [35]. In osteoblasts and mesenchymal stem
cells, silicic acid also increased the gene expression of osteocalcin (OCN) and alkaline
phosphatase (ALP) [36-38], which are excellent indicators of osteogenic differentiation and
mineralization. Recently, silica (SiO2) nanoparticles and nanoplatelets were shown to mediate
potent stimulatory effects on osteoblast differentiation [39, 40]. The spherical silica nanoparticles
of 50 nm were shown to enter the cells through caveolae-mediated endocytosis, which triggered
the stimulation of the mitogen activated protein kinase (MAPK) ERK1/2 (p44/p42) pathway.
These nanoparticles were likely released from the endosome to stimulate autophagy, which
suggested a cellular mechanism for the stimulatory effects of silica nanoparticles on osteoblast
differentiation and mineralization [41]. It should be noted, as the authors themselves point out,
that subtle changes in surface charge, surface area, shape, size, and/or surface chemistry of the
particles may play direct roles in determining specific cellular responses [39]. For example,
hexagonal silica nanoparticles with size about 100 nm did not show any effect on differentiation
of MSCs in vitro [42]. It is possible that not silicate chemistry but the nano-size particles could
be responsible for the positive outcome in these studies. Therefore, the exact biological roles of
soluble silicate on bone cells remain unclear and highly controversial [31].
Bone marrow-derived human mesenchymal stem cells (hMSCs) are a population of self-
renewing multipotent cells and are excellent candidates for cell-based therapeutic strategies to
regenerate injured or damaged tissue. They can differentiate into multiple lineages, including
osteogenic lineage, in response to appropriate stimuli [43]. It is now evident that osteogenic
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differentiation of MSCs is tightly controlled by several regulators including microRNAs
(miRNAs) [44, 45]. MiRNAs are small endogenous RNA molecules that govern gene expression
by targeting mRNA at post-transcriptional level. miRNAs have emerged as key regulators of
diverse cellular processes, including growth, apoptosis, development, metabolism, stress
adaption, differentiation, etc. [46-48]. For example, miR-138 was shown to inhibit osteoblast
differentiation of hMSCs by targeting FAK (focal adhesion kinase) translation. Consequently,
reduced phosphorylation of FAK and ERK1/2 (Extracellular signal-related kinase) resulted in a
decrease of Runt-related transcription factor 2 (Runx2) phosphorylation [49]. miR-29b has been
reported to promote osteogenesis by directly down-regulating known inhibitors of osteoblast
differentiation, HDAC4, TGFβ3, ACVR2A, CTNNBIP1, and DUSP2 proteins, through binding
to target 3’-UTR sequences in their mRNAs [50]. Various miRNAs that positively or negatively
regulate osteoblast differentiation were summarized by Vimalraj and Selvamurugan [51].
However, no miRNAs have been identified to bridge the biological functions of silicate with
bone formation.
In the physiological pH and at concentration below 2 mM (56 ppm Si), silicate exists
predominantly as orthosilicic acid (Si(OH)4). This tetrahedral Si(OH)4 is uncharged at neutral pH,
but has the tendency to polymerize to polysilicate at silicate concentration above 2~3 mM
(56~84 ppm Si) [52]. To clarify the direct effects of silicate on bone formation, we examined the
effect of (Si(OH)4) on bone cell proliferation, differentiation, and intracellular signaling events.
We demonstrated that soluble Si(OH)4 stimulated hMSCs to proliferate in growth medium and
enhance osteogenic differentiation in osteogenic medium. We also sought to ascertain at least one
mechanism by which Si(OH)4 accomplishes these bioactivities by regulating microRNAs
expression.
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2. MATERIALS AND METHODS
2.1. Si(OH)4 preparation
Final concentrations of Si(OH)4 solution with 0-50 ppm Si were prepared by adding sodium
metasilicate (Na2SiO3.9H2O) in αMEM and then adjusting pH to 7.2 ~ 7.4 with 1M HCl before
adding FBS or any other supplements (Equation below). Inductively coupled plasma optical
emission spectrometry (ICP-OES) analysis was performed to establish the ion concentration for
Si in the medium using an Agilent Technologies 710 ICP-emission spectrometer. Standard
solutions (SQCSTD27-100B) were purchased from RICCA Chemical Company for the
calibration of the instrument. All the reagents, unless stated specially, were obtained from Sigma-
Aldrich and were of the highest available purity.
Si
O
O O
3H2O Si
OH
OHHO
OH
2OH-++
2.2. Cell Culture
Bone marrow-derived hMSCs (Lonza®) at passage 4 were grown in regular growth medium
(αMEM, 10% FBS, and 1% penicillin-streptomycin). The cells were cultured until 70~75%
confluence. Then, cells were trypsinized and seeded onto 24-well culture plates at the density of
3 × 103 cells/cm2. After 24 hours, the medium was replaced with Si-containing (0, 10, 50 ppm)
medium.
MC3T3-E1calvarial cells and mouse monocytic cell line RAW264.7 were purchased from
American Type Culture Collection (ATCC, Manassas, Virginia) and cultured in DMEM + 10%
FBS. All cultures were grown at 37 °C and 5% CO2.
2.3. The effects of Si(OH)4 on hMSC cytotoxicity and proliferation
Cytotoxicity of Si(OH)4 was determined after 24 hours incubation using Live/Dead staining
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(Invitrogen®) according to the manufacturer’s protocol.
Cell proliferation was measured with the CyQuant Cell Proliferation Assay kit (Invitrogen®). At
the end of incubation time (Day 1, 7, 14, and 21), the cells were washed with PBS to remove
non-adherent cells and frozen at -80 °C until being assayed. After thawing, 100 µL of lysis buffer
was added to the cell pellet at room temperature for 1 h and then 100 µL of CyQuant GR dye
was mixed with lysis buffer in 96-well plate and incubated for 5 min. Fluorescent signals were
detected with excitation at 480 nm and emission at 520 nm using a fluorometricplate reader
(Synergy H1, Biotek®). Values were determined against a standard curve and displayed as ng/mL
DNA. Five replicates were performed.
2.4. The effects of Si(OH)4 on osteogenic differentiation of hMSCs
Osteoblastic differentiation was induced using osteogenic medium (growth medium
supplemented with 0.1 µM dexamethasone, 50 µM ascorbic acid 2-phosphate, and 10 mM β-
glycerol phosphate) with or without Si(OH)4. The medium was changed twice weekly. The
osteoblast phenotype was evaluated by determining ALP activity and corrected by the total DNA
content [19]. ALP activity was measured using a fluorometric endpoint assay (ab83371), which
quantified ALP enzyme by cleaving the phosphate group of non-fluorescent 4-
methylumbellifferyl phosphate disodium salt (MUP) substrate resulting in an intense fluorescent
signal. Alizarin Red S staining (ARS) was performed to detect matrix mineralization. On day 28,
cells were washed three times with PBS and then fixed with 10% formalin (10 minutes). The
fixed cell were further washed with distilled water in order to remove any salt residues and then
a solution of ARS (40 mM) with a pH adjusted to 4.2 was added to the wells. After an incubation
of 20 minutes at room temperature, the excess of ARS was washed with distilled water.
Mineralization was quantified by measuring the absorbance of orange-red ARS. After staining,
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10% (v/v) acetic acid was incubated with the cells overnight at -20 °C. Then, the cells with the
acetic acid were transferred to tubes and centrifuged for 15 minutes at 20,000 g. The supernatant
was removed to other tubes and neutralized with 10% (v/v) ammonium hydroxide. 100 µL of
each sample was added to 96-well plates and the OD405 was read using microplate reader.
2.5. The effects of Si(OH)4 on osteoclast differentiation of mHSCs
Mouse hematopoietic stem cells (mHSCs) were isolated and cultured as described: 4 - 8 weeks
old male C57/Blk-6 mice were dissected and both tibiae and femur marrow cavities were flushed
with 10 ml media (αMEM 10% FBS, 1% penicillin-streptomycin, and 0.1% Amphotericin) into a
50 ml centrifuge tube using a sterile 20-gauge needle. All flushed bone marrow was collected
and centrifuged at 1200 rpm for 12 minutes at 4 °C. The supernatant was discarded and the pellet
was re-suspended with fresh media (αMEM, 10% FBS, 1% penicillin-streptomycin, and 0.1%
amphotericin). The re-suspended mix was plated on 10 cm dish to incubate for 24 hours and non-
adherent cells were cultured at a density of 1 × 105 cells/cm2 for 2 days more in the presence of
20 ng/ml of macrophage colony-stimulating factor (MCSF). The resultant preosteoclasts were
further cultured in medium supplemented with 20 ng/ml of MCSF and 20 ng/ml of receptor
activator of nuclear factor-kappa B ligand (RANKL). The osteoclast phenotype was evaluated by
determining tartrate resistant acid phosphatase (TRAP) activity using a commercially available
kit. The number of mature osteoclasts (TRAP positive with ≥ 3 nuclei) was counted under light
microscopy. Pit formation assays were conducted using BD BioCoatOsteologic bone cell culture
system (BD Biosciences®). Resorption areas were examined under normal light microscope and
pictures were analyzed using Image J and Photoshop.
2.6. RNA extraction, cDNA synthesis and quantitative real-time RT-PCR.
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The cell culture medium was removed and, after washing cells with RNase-free PBS, total RNA
was extracted using Sure PrepTM RNA purification kit (Fisher Bioreagents®). The purity and
quantity of RNA was determined spectrophotometrically using a NanoDrop 1000 (Thermo
Fisher Scientific®). The ratio of sample absorbance at 260 and 280 nm was used to assess the
purity of the RNA. A ratio of ~ 2.0 is acceptable as pure RNA. The RNA samples were treated
with DNAse and reverse-transcribed using the SuperScript first-strand synthesis system for real-
time PCR (Invitrogen®). SYBR (N’, N’-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-
ylidene) methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine) green mRNA
qRT-PCR was performed using ABI PRISM 7700 Real PCR system for analyzing expression of
Runx2, OCN, OPN, Col 1, NFATc1, CALCR, DC-STAMP, and TRAP genes. GAPDH served as
the house-keeping gene and the expression for the gene of interest was normalized to the
GAPDH expression. The miRNA qRT-PCR primer specific for miR-146a was purchased from
Applied Biosystem®. Each sample was run in triplicate. The relative target mRNA expression
was calculated by using the ∆∆Ct method.
2.7. Microarray.
Total RNA with small RNA (< 200 nucleotides) from cultured cells was isolated, immediately
frozen in dry ice, and submitted to the Case Comprehensive Cancer Center in Case Western
Reserve University/University Hospital of Cleveland. 800 ng of RNA was used for RNA labeling
with FlashTag® HSR RNA Labeling Kit (Genisphere®). The biotin-labeled RNAs were
hybridized with miRNA 2.0 arrays (containing 633 human miRs) at 48 °C for 16 hours, stained
using theGeneChip Fluidics Station 450, and scanned with GeneChip Scanner 3000.
2.8. Transfection of oligonucleotides.
Synthetic mmu-miR-146a mimic (Qiagen®) is a small, double-stranded RNA molecule designed
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to mimic endogenous mature miR-146a molecule when introduced into cells. The mature
sequence is 5’UGAGAACUGAAUUCCAUGGGUU3’. Synthetic mmu-miR146a inhibitor
(MISSION®), also a double-stranded RNA molecule, was designed to inhibit the mature miR-
146a. Transfection of 5-20 nMmiR mimic (or inhibitor) was performed according to the
manufacturer’s instructions using siLentFectTM Lipid reagent (Bio-Rad®). In brief, MC3T3-E1
preosteoblasts were plated onto 24 wellplates for 24 hours prior to miRNA transfection.
Transfection mix was prepared in serum-free medium and transfection was done in antibiotic-
free medium. Transfection efficiency was monitored by using 5’ carboxyfluorescein-labeled
oligonucleotides. After 4 hour’s incubation, the medium was changed to normal medium with or
without Si(OH)4. Cells were harvested for RNA isolation 72 hours post transfection. miR-146a
levels were confirmed by qPCR. Transcription levels of the target proteins in transfected cells
were analyzed by qPCR using the corresponding primers. The osteoblast phenotype was
evaluated by ALP staining at day 10 and ARS staining at day 21.
2.9. NF-κB Reporter Transfection and Luciferase Assays.
Cells (MC3T3-E1 or RAW264.7) were seeded in 12-well culture plates until ~ 70% confluency
and transfected with 1 µg of commercial NF-κB responsive reporter pNF-κB-Luc plasmid (a
kind gift from Kent State University, Ohio, USA), using Lipofectamine 2000 reagent
(Invitrogen®) as per the manufacturer’s protocol. For miRNA inhibition experiments, cells were
co-transfected with reporter clone of NF-κB plasmid and anti-miR-146a and harvested 72 hours
post transfection. NF-κB activation was induced in MC3T3 cells by addition of TNFα (10 ng/ml).
NF-κB activation was stimulated in RAW264.7 cells by treatment with RANKL (50 ng/ml).
Luciferase activity was measured using the Luciferase Assay kit (Promega®) and normalized
with total protein content (Pierce BCA Protein assay). Data are expressed as Relative Light Units
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(RLU) and represent the mean ± SD of four replicate wells for each data point.
2.10. Nuclear protein extraction and NF-κB Transcription Factor Assay.
MC3T3 cells were treated with TNFα (10 ng/ml) and miR-146a mimic in presence or absence of
Si(OH)4 to assess the effect of silicate exposure on NF-κB expression.RAW264.7 cells were
stimulated with RANKL (50 ng/ml) and miR-146a (10 nM) in presence or absence of Si(OH)4 to
evaluate the NF-κBactivation. The EpiSeeker Nuclear Extraction kit (abcam®) was used to
extract nuclear proteins from cells 72 hours post transfection. NF-κB level in the nuclei was
analyzed by western blot with the corresponding antibody. DNA-binding ELISA for NF-κB p65
Transcriptional Factor Assay kit (abcam®) was used to quantify the nuclear NF-κB levels,
according to the instructions of the manufacturer. Briefly, nuclear extractions were incubated in
96-well plates coated with immobilized oligonucleotide containing a binding site for p65 subunit
of NF-κB. NF-κB p65 was detected by incubation with the primary antibody specific for the
activated form of p65, visualized by anti-IgG horseradish peroxidase (HRP)-conjugate and
developing solution and quantified by spectrophotometry at 450 nm with a reference wavelength
of 655 nm. Nuclear NF-κB expression was normalized by total nuclear fraction protein and fold
changes were calculated with respect to the control.
2.11. Immunofluorescence of Runx2.
MC3T3 cells were treated with TNFα (10 ng/ml) in presence or absence of Si(OH)4 to assess the
effect of Si(OH)4 exposure on Runx2 expression. After 14 days of culture withSi(OH)4, cells
were fixed with 10% formalin for 10 min and rinsed with PBS. Then, cells were permeabilized
with 0.1% Triton X100 for 5 min and nonspecific binding was blocked with 10% (m/v) BSA.
Cells were incubated overnight at 4 °C with primary antibody (mouse monoclonal to Runx2,
abcam®, ab76956). After incubation, cells were rinsed 3 times with PBS for 5 min and incubated
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with secondary antibody (Alexa Fluor goat-anti-mouse IgG). Actin was counterstained with
phalloidin-rhodamine and then rinsed 3 times. Immunolabeling was qualitatively analyzed using
an IX51 Epifluorescence Microscope (Olympus Co., Japan) equipped with fluorescent light
source and filters.
2.12. Statistical Analyses.
Data are represented as mean ± SD from ≥ 3 replicates for each condition/time point, and each
experiment was repeated at least three times independently. Comparisons were made by using
one-way ANOVA with post-hoc testing or Student’s t test. All the comparisons and statistical
analyses have been done in Microsoft excel. Observations were considered to be significantly
difference for p < 0.05.
3. RESULTS
3.1. Effects of Si(OH)4 on hMSC Osteoblastic Differentiation.
Using the CyQuant® cell proliferation assay, we found the steady increase of cell proliferation
with time (Fig.1a), which indicated that the cells were proliferating and the Si(OH)4
concentration in this range had no overtly toxic effect to the hMSCs. This result was confirmed
by Live/Dead cell staining, which showed that in the presence of Si(OH)4, the percentage of
fluorescent green cells (living cells) did not drop below 98% (Fig.1b). We also found that
Si(OH)4 significantly increased the proliferative activity of hMSCs between 3 and 7 days of
incubation in growth medium (Fig. 1a). However, no significant difference in cell proliferation
was shown for different doses of Si(OH)4 after 7 days culture. To study the impact of Si(OH)4 on
the osteogenic differentiation of hMSCs, the hMSCs were cultured in osteogenic medium in
different doses of Si(OH)4. A convenient fluorogenic assay was used to detect the activity of ALP,
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an enzyme involved in bone mineralization produced by osteoprogenitors and osteoblasts (Fig.
1c). hMSCs cultured for 7-14 days in osteogenic medium typically differentiate into ALP-
producing osteoblasts. In the Si(OH)4-supplemented medium, the cells revealed higher levels of
ALP production compared to control cells cultured without Si(OH)4 at days 7, 14 and 21. An
extended culture time of 28 days allowed positive identification of mineralized nodules, detected
by ARS staining for calcium deposition. Mineralization was quantified by extracting the ARS
from the stained wells. In contrast to the results of ALP activity, increasing Si(OH)4 in medium
decreased the matrix mineralization , shown in Fig. 1d.
To further evaluate the effect of Si(OH)4 on the osteogenic differentiation of hMSCs, the
transcription levels of osteoblast-specific differentiation markers were assayed by qRT-PCR
under osteogenic condition. As shown in Fig. 2, Si(OH)4 treatment increased mRNA
transcription levels of Col 1, the major structural component of the organic matrix. This increase
was maximal on day 7 after Si(OH)4 treatment, consistent with an early role of Col 1 in the
osteoblast maturation sequence. Another notable finding is the up-regulation of mRNA levels of
Runx-2, the master transcription factor responsible for osteogenic differentiation [53].
Transcription of OCN and osteopontin (OPN), major osteoblast proteins downstream of Runx-2,
were also induced by addition of Si(OH)4 (Fig. 2). However, treatment with Si(OH)4 did not alter
the mRNA transcription of the house-keeping gene GAPDH (Fig. S1). Together, these findings
suggest that Si(OH)4may act as a positive regulator of osteogenic differentiation. Based on the
cytotoxicity, proliferation, and osteogenic differentiation studies, we selected 50 ppm of
Si(OH)4to gain a better understanding of the mechanism of action.
3.2. Identification of Differentially Expressed miRNA in Si(OH)4-treated hMSCs.
To identify differentially expressed miRNAs during osteogenic differentiation of hMSCs treated
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with Si(OH)4, total RNA from the hMSCs were labelled using FlashTag® HSR RNA labelling kit
for miRNA microarray analyses. A total of 663 human miRNAs were analyzed (Fig. S2). Of the
663 miRNAs, 26 were consistently altered by Si(OH)4 treatment (50 ppm). Among them, eleven
miRNAs were down-regulated (blue color), and fifteen miRNAs were up-regulated (red color).
MiR-146a wasthe most robustly upregulated miRNA by Si(OH)4treatment during hMSC
osteogenic differentiation (Fig. 3a). To further validate that miR-146a is indeed upregulated by
Si(OH)4, qRT-PCR was performed and showed miR-146a was significantly up-regulated during
the later stages of hMSC osteogenic differentiation(day 14 and day 21, Fig. 3b).
Pre-osteoblasts are the late-stage cell lineage of MSCs during osteo-differentiation. To evaluate
whether the biological effects of miR-146a is of critical importance in later stages of osteogenic
differentiation, we inhibited miR-146a levels in MC3T3-E1 pre-osteoblasts using synthetic
antisense miRNA inhibitor (anti-miR-146a, Mission®). Also, in determining the role of miR-
146a, it is advantageous to use MC3T3-E1 cells because MSCs are more difficult to transfect
successfully with commercial transfection reagents. The degree of miRNA inhibition was
monitored by qRT-PCR after transfection of anti-miR-146a. Mature miR-146a levels were
decreased ~ 1.6 fold relative to control cells 72 h post-transfection (Fig. S3).
MC3T3-E1 pre-osteoblasts were then induced to differentiate into osteoblasts after transfection
with anti-miR-146a, in the presence or absence of Si(OH)4. Inhibition of miR-146a decreased
osteoblastic differentiation, which was indicated by lower transcription levels of the osteoblast-
specific genes Runx2, Col 1, ALP, and OCN (Fig. 3c), as well as by decreased ALP production
(visualized by ALP staining) and mineralization (Fig. 3d). However, this suppression of
osteoblast marker gene transcription, ALP production, and mineralization could be reversed by
Si(OH)4 treatment (Fig. 3c&3d). Therefore, our results suggest that Si(OH)4may be a positive
15
regulator of osteoblast differentiation via miR-146a.
3.3. Si(OH)4 targets NF-κB signaling via miR-146a.
Since miR-146a has been reported to be NF-κB dependent and directly down-regulate NF-
κBsignaling[54], the knockdown studies of miR-146awere performed to determine the direct
regulatory role of Si(OH)4on the NF-κB activation by regulating miR-146a expression. MC3T3
pre-osteoblasts were transfected with a luciferase reporter driven by tandem NF-κB consensus
motifs and were treated with anti-miR-146a (co-transfection) in the presence or absence of
Si(OH)4. TNFα-induced NF-κB promoter activity was increased upon anti-miR-146a
transfection, as shown by increase in luciferase activity (Fig. 4a), which indicated the inhibitory
role of miR-146a on NF-κB promoter activation. This result is in agreement with the previous
studies in which the inhibition of miR-146a increased the activity of NF-κB[55]. Treatment of
MC3T3-E1 cells with Si(OH)4 was found to significantly decrease TNFα-induced NF-κB
transactivation even in presence of anti-miR-146a (Fig. 4a), which means the up-regulation of
NF-κB can be effectively counter-balanced by increasing the cellular levels of miR-146a through
Si(OH)4 treatment.
We further validated the capacity of Si(OH)4 to suppress NF-κB signaling in MC3T3-E1 cells by
extracting cell nuclear proteins and subsequently performing western blot analysis.These data
showed that the nuclear NF-κB protein level in miR-146a-transfected cellswas less at day 3 when
compared with TNFα-treated control cells.Treatment with Si(OH)4to miR-146a-transfected cells
further markedly decreased NF-κB translocation to the nucleus (Fig. 4b).To quantify the Si(OH)4
regulating NF-κBactivation, we also exposedthe nuclear extract of MC3T3-E1 cells towells
coated with NF-κB binding DNA (abcam®) and performed anELISA(Fig. 4c). TNFα induced
significant binding to the specific double-stranded DNA containing the NF-κB consensus, shown
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from the about200% increase of NF-κB p65 trapped in the wells. MiR-146a transfection to
MC3T3-E1 cellsdecreased NF-κB binding to its consensus binding site (~28% decrease after
transfection). Si(OH)4 treatment to the miR-146a transfected cells further potently diminished
NF-κB translocation (p < 0.05) to the nucleus and decreased DNA binding (~50% decrease with
Si(OH)4 treatment). These observations support the inhibitory role of Si(OH)4 on NF-κB activity
and indicate miR-146a as an important component to the effect of Si(OH)4.
3.4. Si(OH)4 increased Runx2 expression via NF-κB signaling
To further understand the roles of Si(OH)4 regulating NF-κB activity to influence osteoblastic
differentiation, immunofluorescence was used to detect expression of the bone-specific
transcription factor Runx2, which is known to be downstream of NF-κB and essential for
osteoblastic differentiation (Fig. 5). All MC3T3-E1 cells cultured for 14 days in osteogenic
medium exhibited good cell density. Cells treated with TNFα remained bipolar and
predominantly fibroblast-likein appearancebut showed negligible Runx2 production. However,
the cells cultured in Si(OH)4-containing medium were observed to raise the level of Runx2
expression. Furthermore,the TNFα-induced Runx2 inhibition could be rescued by treating the
cells with Si(OH)4.This observation demonstrated that Si(OH)4could mediate potent stimulatory
effects on osteoblastic differentiation by suppressing TNFα-induced activity ofNF-κB.
4. DISCUSSION
Our previous studies [20] identified that β-CaSiO3 is more bioactive than α-CaSiO3 in inducing
hMSCs osteogenic differentiation. The two polymorphs of CaSiO3 have identical chemical
composition and stoichiometry but different crystal structures. This difference in crystal structure
results in the greater dissolution rate of silicate from β-CaSiO3 compared to α-CaSiO3 [19, 56].
17
Soluble silicate concentrations peaked at higher levels and earlier time points for β-CaSiO3 than
for α-CaSiO3, which may address the issue whether soluble silicate is bioactive to regulate bone
cell metabolism and bone formation. However, as pointed out in a critical review, it is not clear if
the positive effects can be purely attributed to silicate [31]. The aim of the present study was
therefore to test the hypothesis that Si(OH)4, the simplest soluble form of silicate, has a direct
effect on osteogenesis and bone formation in vitro and investigate the potential cellular
mechanisms by which Si(OH)4 might influence bone cell behaviors.
First, our data suggested that Si(OH)4 stimulated hMSCs proliferation and osteogenic
differentiation (Fig. 1 & 2). However, our data seemed to indicate an inhibitory role of the
soluble silicate in mineralization, which might be contrary to Carlisle, Schwarz, and Milne’s
dietary silicon deprivation studies in chickens and mice [1-3]. In the animals, silicon was taken
up via gastrointestinal absorption and finally concentrated and bonded within the osteoid layer of
growing bone. Therefore, the silanol groups, which have been known as nucleation sites for
apatite formation, could be successfully formed. The apatite is formed by the co-precipitation of
phosphate and Ca2+ in the matrix of collagen. Since soluble silicate is uncharged in neutral cell-
medium environment, perhaps, oligomers of soluble silicate [52] which have lower pKa than
Si(OH)4 [57], could de-pronate and attract Ca2+ in the solution to form ion clusters and decrease
the Ca2+ concentration close to collagen network (Fig. 5b). Although, Si(OH)4 has been shown in
previous work [34] and in the current study to increase the production of collagen matrix (Fig. 6a)
the limited concentration of Ca2+ might result in the lower apatite formation (Fig. 1). However, at
this point, further experimental evidence is needed to gain a complete understanding of all the
roles of the various silicon species in the process of apatite formation.
Additionally, within the current study, a mechanism has been described by which Si(OH)4
18
regulates the expression of miR-146a, which ultimately suppresses the activity of NF-κB in bone
cells to enhance osteoblastic differentiation.Several recent studies have shown that NF-
κBinfluences both osteoblast differentiation and activity. In one of the studies, it was shown
thatthe inhibition of NF-κB p65 by a mutant IKKγ (IKK-DN) and a super IκBα repressor (SR-
IκBα) enhanced osteoblastic differentiation of mesenchymal C2C12 cells in vitro[58]. NF-κB
p65 can interact with the Smad1-Smad5 complex in the nucleus and disrupt its binding to target
promoters [59]. Inhibiting this interaction of NF-κB with TGFβ-mediated Smad signaling can,
therefore, potently stimulate the differentiation and mineralization of primary murine bone
marrow stromal cells [60]. Runx2, the primary transcription factor required for commitment to
the osteoblast lineage, is NF-κB dependent [45]. In our studies, Si(OH)4 significantly
antagonized TNFα-induced NF-κB activity(Fig. 4) and activated Runx2 (Fig. 5) in MC3T3 cells.
Therefore, the effects of Si(OH)4 in osteoblasts could be suggested to block TNFα-dependent
canonical NF-κB signaling to activate Runx2pathway.
MiR-146a was previously identified as an immune system regulator that influences the
mammalian response to microbial infection. Further characterization of miR-146a revealed that it
is also NF-κB dependent, which has been shown to base-pair with sequences in the 3’ UTR of
TRAF6 (TNF receptor associated kinase) and IRAK1 (IL-1 receptor associated kinase) genes,
the key adaptor molecules downstream of toll-like and cytokine receptors in the NF-κB pathway
[61]. In the present study, miR-146a was found as a miRNA signature of osteogenic
differentiation of hMSCs treated with Si(OH)4.Data obtained from our in vitro experiments
revealed that inhibition of miR-146a function decreased osteogenic potential of pre-osteoblasts,
whereas Si(OH)4 treatment enhanced osteoblast differentiation (Fig. 3).Therefore, these findings
suggested a regulatory role of Si(OH)4, in which Si(OH)4 up-regulates the expression of miR-
19
146a gene that, upon processing and maturation, down-regulates NF-κB to reduce the activity of
Runx2 (Scheme 1). However, how Si(OH)4 up-regulates miR-146a in bone cellsis presently
unknown and remains to be investigated in our lab.
Recently, some studies have shown that soluble silicate inhibited osteoclast phenotypic gene
expression, osteoclast formation and function in vitro[62]. In agreement with their study, we also
demonstrated the inhibitory effects ofSi(OH)4 on osteoclast formation of hematopoietic stem
cells from 4-8 week old male C57/BLK-6 mice (Fig. 7a). MCSF/RANKL induced the
differentiation of mHSCs into osteoclasts[63], which were distinguished by their TRAP-positive
and multinucleated phenotype (Fig. 7a). Si(OH)4 treatment resulted in a significantly reduced
number of both osteoclasts and TRAP+ pre-osteoclast formation (Fig. 7a). In addition, the cells
treated with Si(OH)4 showed functional defects in osteoclast function, shown by decreased
number and average size of pits on BD BioCoatOsteologic bone cell culture system (Fig. 7b).
Cell viability assay demonstrated that this concentration of Si(OH)4 failed to mediate any toxic
effects on the viability or proliferation rates of mHSCs (Fig. S4). This suggests that the anti-
osteoclastogenic activities are related to suppression of differentiation along the osteoclast linage
rather than as a consequence of cytotoxicity. Furthermore, Si(OH)4 was shown to down-regulate
mRNA expression levels of osteoclast marker genes in mHSCs (Fig. 7c). NFATc1, the master
regulator of osteoclastogenesis[64], is induced potently by RANKL at early stage (day 2).
Notably, Si(OH)4 decreased mRNA expression levels of NFATc1 at both early stage (day 2) and
late stage (day 4). The transcription of DC-STAMP, a downstream target of NFATc1 to promote
cell-cell fusion of osteoclasts [65], was suppressed by the addition of Si(OH)4. The other
osteoclast-specific genes such as TRAP and CALCR, activated by transcriptional complex
containing NFATc1 [64], were also down-regulated by Si(OH)4 treatment.By using a Boyden
20
chamber co-culture system, various research groups concluded that the inhibition of
osteoclastogenesis of RAW264.7 pre-osteoclasts was due to the increase the level of
osteoprotegerin (OPG) released by SaOS-2 osteoblasts after exposed to silicate. They
suggestedan intercellularmechanism, by which silicate increased the levels of OPG, a soluble
decoy receptor for RANKL and inhibits the RANK-RANKL signaling pathway [62,
66].However, it has been recognized that silicate should have direct inhibiting effects on
differentiation and fusion of osteoclast precursors by interacting with important intracellular
signaling pathway [62].Since NF-κB is well known as one of the most important molecules in
osteoclastogenesis to activate osteoclast precursor cells, we hypothesized Si(OH)4 treatment
could apply the same pathway to up-regulation of miR-146a and potentially lead to suppression
of osteoclast formation and function. To explore this, we transfected miR-146a mimic into pre-
osteoclast RAW264.7 cells and osteoclastogenesis was induced by MCSF/RANKL treatment.
The number of TRAP-positive multinucleated cells was significantly reduced by transfection of
miR-146a mimic (Fig. 8a&b). Furthermore, we transfected RAW264.7 cells with the luciferase
reporter driven by tandem NF-κB consensus motifs, and then treated the cells with Si(OH)4 and
miR-146a mimic. Both Si(OH)4and miR-146a were found to suppress RANKL-induced NF-κB
translocation from cytosol to nucleus in RAW264.7 cells (Fig. 8c).These results clearly indicated
that Si(OH)4 might negatively regulate NF-κB via miR-146a to affect osteoclast formation, and
could play a role in the remodeling process of bone by inhibiting the bone physiological
resorption process [67, 68].
Taken together, Si(OH)4 may reveal a considerable biomedical potential with therapeutic and
prophylactic implementations, e.g. concerning osteoporosis. This disease is due to an imbalance
between bone formation and resorption, which is most common in women after menopause and
21
older men [69]. Therefore, Si(OH)4 could be used as a nutritional supplement in osteoporotic
persons [35, 70]. Until now, no reports are available that indicate either a passive or an active
influx of uncharged or charged Si(OH)4 into bone cells. In siliceous sponges and diatoms, silicate
may be transferred into cells through the membrane [71]. Experimental evidence indicates that
the membrane protein may use Si(OH)4 instead of HCO3- for the co-transport of Na+ (Na+-HCO3
-
co-transporter, NBC-1) [66, 71, 72]. The lack of Si-labelling technique makes it hard to
identifyhowSi(OH)4 enters bone cells and up-regulates the expression of miR-146a, which would
be investigated in the future.
5. CONCLUSION
We found that soluble Si(OH)4 can contribute to bone-formationin vitrobut reduce the activity
and number of osteoclasts. Si(OH)4 induces the expression of endogenous miR-146a to
antagonize activation of NF-κB. This inhibition of NF-κB could result in activation of Runx2,
the master transcription factor for osteoblast precursor differentiation, and suppresses the
expression of NFATc1, the key transcription gene for osteoclast precursor differentiation. To the
best of our knowledge, this is the first study showing soluble silicate’s possible cellular
mechanism. These data suggest that soluble Si(OH)4, along with miR-146a, can be used as
pharmaceuticals for bone fracture prevention and therapy in conditions such as localized bone
loss or generalized osteoporosis.
SUPPLEMENTARY INFORMATION AVAILABLE:The microRNA microarray heat map,
qRT-PCR of GAPDH and miR-146a, and viability of mHSCs to Si(OH)4.
ACKNOWLEDGMENTS
22
We are grateful to Dr. Kan He, Department of Pathology, University of Pittsburgh School of
Medicine, for excellent technical assistance and helpful discussion. Mr. Weilong Zhao in
University of Akron is gratefully thanked for assistance with illustration of the mechanism. The
Gene Expression and Genotyping Facility at Case Western Reserve University are acknowledged
for the help with microRNA microarray and data analysis. NF-κB Luc plasmid is the gift from Dr.
Thomas in Kent State University. Financial support was provided by NSF DMR (0906817) grant
and start-up funds from University of Akron, and Ohio Research Scholar Program to Dr. Nita
Sahai, PI.
23
FIGURE CAPTIONS
Figure 1. Effects of Si(OH)4 on the proliferation and osteoblastic differentiation of hMSC. (a)
hMSCs were grown in growth medium treated with different doses of Si(OH)4. Cell proliferation
was measured by the CyQuant proliferation assay kit and displayed as ng/mL DNA (n = 5 for all
experiments). (b) Cell viability and morphology of hMSCs after incubation with different doses
of Si(OH)4 for 24h. Live cells are stained fluorescent green and dead cells appear red. (c)
Osteoblastic different iation of hMSCs in osteogenic medium supplemented with different doses
of Si(OH)4. ALP activity was measured during the course of differentiation by normalizing to
total DNA (n = 4 for all experiments), *p < 0.05, **p < 0.01. (d) Alizarin Red staining were
performed at day 28. Mineralization was quantified by measuring the absorbance of orange-red
ARS (insert value). Data presented are from one of three independent experiments, with all three
exhibiting comparable results.
Figure 2. The specific osteogenic differentiation markers (Col1, Runx2, OCN, and OPN) were
assayed by qRT-PCR. Data presented are from one of three independent experiments, with all
three exhibiting comparable results. Values are presented as mean ± SD by normalized to
GAPDH (n = 3 for all experiments), **p < 0.01.
Figure 3. Expression of miR-146a at day 14 & day 21 in osteoblastic differentiation of hMSCs
treated with or without Si(OH)4. (a) The heat map was generated with the use of hierarchical
cluster analysis to show distinct miRNA expression patterns in hMSCs treated with or without
Si(OH)4 in osteogenic medium. The color bar was extracted to show the color contrast level of
the heat map. Red and blue indicate high expression levels and low expression levels,
respectively. (b) miR-146a transcription levels were determined by qRT-PCR during hMSCs
osteogenic differentiation treated in the presence or absence of Si(OH)4. Values are presented as
24
the mean ± SDs (n = 3). (c) Anti-miR-146a inhibited osteoblast differentiation on day 3, as
shown by qRT-PCR analysis of osteoblast-specific genes (Runx2, Col1, ALP,
andOCNnormalized to GAPDH). However, Si(OH)4 treatment promoted the transcription of
these genes even in the presence of anti-miR-146a. *p < 0.05;**p < 0.01, (n = 3 for all
experiments). (d) ALP staining at day 10 and Alizarin Red staining at day 21 were performed.
Data presented are from one of three independent experiments, with all three exhibiting
comparable results.
Figure 4. Si(OH)4 treatment suppressed NF-κB activation in MC3T3 pre-osteoblast by
increasing miR-146a expression. (a) MC3T3 cells were transfected with NF-κB-responsive
luciferase reporter, and then co-transfected with anti-miR-146a in presence or absence of
Si(OH)4. TNFα was used to induce NF-κB activation in cells and luciferase activity was
quantitated 48 hours later. Data expressed as relative light units (RLU) normalized to total
protein (n = 4). (b) MC3T3 cells stimulated with TNFα, and then treated with miR-146a in
presence or absence of Si(OH)4 for 48 hrs. Western blot analysis was performed for NF-κB in
nuclear extract. (c) NF-κB contained in nuclear extract can bind specifically to the NF-κB
response dsDNA immobilized onto the wells. NF-κB subunit p65 of mouse was detected by
addition of a specific primary antibody and a secondary antibody conjugated to HRP. The data
represent the means ± SDs (n = 4). Data presented are from one of three independent
experiments, with all three exhibiting comparable results.
Figure 5. Runx2 staining of MC3T3 preosteoblasts after 14 days of culture with Si(OH)4.
MC3T3 cells treated with TNFαlacked the positive Runx2 stain; and Si(OH)4 can stimulated
Runx2 expression, even under the influence of TNFα. Data presented are from one of three
independent experiments, with all three exhibiting comparable results.
25
Figure 6. Soluble silicic acid increased extracellular matrix formation but decreased
mineralization in vitro. (a) Silicic acid increased the expression of collage, shown by Sirius
Red/Fast Green staining. Sirius Red specifically binds to the [Gly-X-Y]n helical structure of
fibrillar collagens and Fast Green binds to non-collagenous proteins. (b) The proposed
mechanism why calcium deposition was inhibited by soluble silicate. The soluble
silicateoligomers could be de-protonated in neutral buffer and attract Ca2+ to form ion cluster,
which could decreased Ca2+ concentration close to collagen network to suppress the
Ca2+deposition.
Figure 7. Si(OH)4 suppressed RANKL-inducedmousehematopoietic stem cells (mHSC)
differentiation to osteoclasts in vitro. 10 ppm of Si(OH)4 was used to evaluate osteoclast
formation of mHSCs. (a) Microphotographs are representative TRAP staining of cells treated
without or with Si(OH)4. Scale bars represent 100 µm. Top right is mature multinucleated
TRAP+ osteoclasts quantitated in the presence of M-CSF and RANKL. Value presented as the
mean ± SDs (n = 5). (b) Si(OH)4 decreased pit numbers and reduced pit areas in differentiated
osteoclasts from mHSCs. Scale bars represent 200 µm. Middle right is quantitative analysis of
pits areas. (c) The specific osteoclastic differentiation markers (NFATc1, DC-STAMP, TRAP, and
CALCR) were assayed by qRT-PCR. Values are presented as the mean ± SDs and are normalized
to GAPDH (n = 3) and representative of three independent experiments, **p < 0.01. Data
presented are from one of three independent experiments, with all three exhibiting comparable
results.
Figure 8. MiR-146a suppressed RANKL-induced osteoclastogenesis in RAW264.7 pre-
osteoclasts. (a,b) TRAP staining 5 days after induction of osteoclatogenesis. The number of
TRAP-positive multinucleated cells was decreased by miR-146a mimic transfection. (c)
26
RAW264.7 cells were transfected with NF-κB-responsive luciferase reporter, and then treated
with miR-146a or Si(OH)4. RANKL was used to induce NF-κB activation in cells and luciferase
activity was quantitated 48 hourspost transfection. Data are expressed as relative light units
(RLU) normalized to total protein (n = 4), **p < 0.01. Data presented are from one of three
independent experiments, with all three exhibiting comparable results.
Scheme 1: Suggested mechanism for the effect of Si(OH)4 on bone cells.The dashed red line
indicates that it is not known whether and how Si(OH)4 enter the cell through Na+-HCO3- co-
transporter (NBC-1).Within the cell Si(OH)4may induce the expression of endogenous miR-146a
to reduce activation of NF- κB. The inhibition of NF-κB results in activation of Runx2, the
master transcription factor necessary for osteoblast precursor differentiation. This deactivation of
NF-κB also inhibits the expression of NFATc1, the key transcription gene for osteoclast
precursor differentiation. Thus, Si(OH)4 can stimulate osteoblast differentiation and inhibit
osteoclast differentiation by antagonizing NF-κB activation via miR-146a, which implies a
potential role in bone remodeling.
27
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Scheme-1
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Graphical abstract
SIGNIFICANT STATEMENT
Accumulating evidence over 40 years suggests that silicate is beneficial to bone formation.
However, the biological role(s) of silicate on bone cells are still unclear and controversial. Here,
we report that Si(OH)4, the simplest form of silicate, can stimulate human mesenchymal stem
cells osteoblastic differentiation. We identified that miR-146a is the expression signature in bone
cells treated with Si(OH)4. Further analysis of miR-146a in bone cells reveals that Si(OH)4 up-
regulates miR-146a to antagonize the activation of NF-κB. Si(OH)4 was also shown to de-
activate the same NF-κB pathway to suppress osteoclast formation. Our findings are important to
the development of third-generation cell-and gene affecting biomaterials, and suggest silicate and
miR-146a can be used as pharmaceuticals for bone fracture prevention and therapy.