Biocompatibility assessment of rice husk-derived biogenic silica nanoparticles for biomedical

applications

Abstrak

Synthetic forms of silica have low biocompatibility, where as biogenic forms have myriad beneficial
effects incurrent toxicological applications. Among the various sources of biogenic silica, rice husk is
considered a valuable agricultural biomass material and a cost-effective resource that can provide
biogenic silica for biomedical applications. In the present study, highly pure biogenic silica
nanoparticles (bSNPs) were successfully harvested from rice husks using acid digestion under
pressurized conditions at 120 C followed by a calcination process. The obtained bSNPs were
subjected to phase identification analysis using X-ray diffraction, which revealed the amorphous
nature of the bSNPs. The morphologies of the bSNPs were observed using transmission electron
microscopy (TEM), which revealed spherical particles 10 to 30 nm in diameter. Furthermore, the
biocompatibility of the bSNPs with human lung fibroblast cells (hLFCs) was investigated using a
viability assay and assessing cellular morphological changes, intracellular ROS generation,
mitochondrial transmembrane potential and oxidative stress-related gene expression. Our results
revealed that the bSNPs did not have any significant incompatibility in these in vitro cell-based
approaches. These preliminary findings suggest that bSNPs are biocompatible, could be the best
alternative to synthetic forms of silica and are applicable to food additive and biomedical applications.

1. Introduction

Nanomaterials, defined as particles with diameters of less than 100 nm, have revolutionized various
industries, including the electronics, textile, medicine, cosmetics, agriculture and food industries, due
to their unique physiochemical properties and tunable characteristics. Silica nanoparticles have
received attention for a variety of applications, such as in catalysis, pigments, thin-film substrates,
thermal insulators, pesticides, food additives, drug delivery, gene therapy, molecular imaging and
additivesin plastics [13]. Silicon is ubiquitous and is the second most abundant element on Earth; it
is commonly present in both crystalline and amorphous states as silicon dioxide (silica; SiO 2 ). Silica
plays an important physiological role in plants as an alleviator of biotic and abiotic stress [4].
Moreover, silicon is an essential contributor to bone health, as silicon deficiency is associated with
poor skeletal development [5]. According to Eisinger and Clairet, silicon supplementation has
increased bone mineral density in women[6]. Reffittetal. Suggested that silica in the form of
orthosilicic acid stimulates type I collagen synthesisin human osteoblast like cells and induces
osteoblastic differentiation [7]. Earlier studies suggested that biosilica inhibits osteoclast
differentiation through osteoprotegerin induction for the treatment of osteoporotic disorders [8].
Furthermore, nanoscale silica particles have the potential to induce osteoblast differentiation and
mineralization and also to inhibit osteoclast differentiation in vitro [9]. In chicks, silica deficiency
causes defects in the connective skeletal tissue [10]. Following the ingestion of nutrients, the
concentration of silicon was increased in the connective tissue, bone and blood vessels [11]. Silica
inhibits aluminum uptake in the gastrointestinal tract due to the interaction between aluminum and
silica, subsequently decreasing aluminum bioavailability [12]. Notably, amorphous silica has been
approved by the FDA as an anti-caking agent for use in the food industry [13]. A three-dimensional
lungaggregate modelshows similarity to in vivo tissue and the model is suitable to investigate
infectious disease. For instance, pathogenesis of Pseudomonas aeruginosa and Francisella tularensis
was studied using 3-D aggregates of lung cells [14,15]. More over,3-D aggregates of mono and triple
co-cultureoflungcells is an appropriate model to analyze the engineered nanoparticle toxicity[16,17].
Rice husk (RH) is a major agricultural waste product, with approximately 600 million tons produced
annually around the world[18]. Considerable amounts of RHare generated each yeardue to rice
production and processing activities. However, rice husk waste utilization is extremely low due to its
undesirable properties, such as resistance to degradation, low nutritive value and high ash content
[19]. In addition, RH is often disposed of using open burning, leading to environmental pollution [20].
However, rice husk contains 6575% organic (lignin, cellulose and hemicellulose) and 1520%
inorganic substances (SiO 2 ) [21]. Recently, RH was exploited in various applications, being used,
for instance, as a feedstock for bio-fuel production, fuel for boilers, in electricity generation, and as a
bulking agent for the composting of animal manure [22]. In particular, the high silica content in RH
has attracted the attention of researchers for its potential use as a raw material for the production of
silicon-based materials, including silicon carbide, silica, silicon nitride, silicon tetrachloride, pure
silicon, and zeolite [19]. A variety of approaches are used for preparing silica nanoparticles from RH,
such as microwave hydrothermal processes [23], flame synthesis [24], solgel processes [25],
microemulsion methods [26], and combustion synthesis [27]. In the large-scale production of silica,
quartz sand is treated with sodium carbonate at 1300 C[28]. However, this method is hazardous to
the environment because it emits large quantities of CO 2 gas[29]. Ingeneral, most silica production
methods are time consuming and produce low-purity silica. Moreover, the biological aspects of RH-
derived silica nanoparticles remain poorly understood. In consideration of prepared silica
nanoparticles future application in human therapeutics needs to complete investigation of their
biocompatibility. Therefore, in the present investigation, we propose a novel and simple method to
produce high-purity biogenic silica nanoparticles (bSNPs) from RHs and analyze their biocompatible
properties using in vitro cell-based approaches. The bSNPs were well characterized by X-ray
diffraction, Fourier infrared spectroscopy, EDX and TEM. Furthermore, the biocompatibility of the
rice husk-derived bSNPs was evaluated at the cellular and molecular levels by the MTT assay and by
assays for mitochondrial membrane potential, cell cycle, gene expression and ROS generation in
human lung fibroblast cells. These studies suggested that the bSNPs can be utilized for three
dimensional cell culture and bone tissue engineering.

2. Materials and methods

2.1. Chemicals

Rice husks were obtained from rice mills in India. Analytical reagent-grade hydrochloric acid (HCl)
was purchased from Merck, Saudi Arabia. MilliQ water was used throughout the experiment. All
chemicals were used as supplied without further purification. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), 5,5,6,6-tetrachloro-1,1,3,3
tetraethylbenzimidazolylcarbocyanineiodide (JC-1), dimethyl sulfoxide (DMSO) and fetal bovine
serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). Propidium iodide (PI) was
purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Synthesis of biogenic silica nanoparticles (bSNPs) from rice husk

Approximately 20 g of rice husk was mixed with 100 mL of 1 N HCl under magnetic stirring. These
mixtures were transferred to an autoclave and maintained at 120 C for 2 h under pressurized (15 lbs)
conditions. Subsequently, the acid-digested rice husk was washed with MilliQ water to remove the
hydrochloric acid. The obtained sample was dried at 85C for 5 h in a hot air oven. Consequently, the
dried samples were calcinated at different temperatures (500 C, 600 C and 700 C) for 1 h using a
muffle furnace. The brown residue became white in color, indicating silica formation.
2.3. Characterization of the silica nanoparticles

The crystallinity of the samples was investigated using powder XRD (JEOL model). The obtained
silica samples from rice husk were ground with KBr to prepare pellets for Fourier transfor min frared
characterization, which was performed on a Perk in Elmer Spectrum One FT-IR spectrophotometer.
Dynamic light scattering (DLS) analysis of prepared samples in aqueous solution was performed
using a Zetasizer Nano ZS-90 analyzer (Malvern, UK). The average size was calculated applying the
software based on the measured intensity, volume, and number distributions. The obtained silica
powders were dispersed in absolute ethanol and ultrasonicated before TEM characterization. The
morphologies, sizes and elemental compositions of the samples were examined using a JEOL
transmission electron microscope (TEM) at an accelerator voltage of 200 kV.

2.4. Cell culture

The WI-38 cell line (human lung normal fibroblasts) was obtained From ATCC(USA). The cells were
cultured in Eagle minimum essential medium (EMEM) with fetal bovine serum (FBS), 100 U/mL
penicillin and 100 g/mL streptomycin in 25 cm 2 or 75 cm 2 flasks or 6-, 12-, 24- and 96-well
culture plates at 37 C in a humidified atmosphere containing 5% CO 2 . All experiments were
performed using cells from passage 15 or less.

2.5. Cell viability assay

The MTT assay was performed as previously described by Blagosklonny and El-Diery [30]. Briefly,
WI-38 cells were plated at a density of 1 10 4 cells per well in 200 L of fresh culture medium.
After growth overnight, thecells were treated withdifferent concentrations (25400 g/mL) of well
characterized bSNPs for 24 and 48 h. After incubation, 20 L of MTT solution [5 mg/mL in
phosphate buffered saline (PBS)] was added to each well. The plates were wrapped with aluminum
foil and incubated for 4 h at 37 C. The plates were centrifuged, and the purple formazan product was
dissolved by the addition of 100 L of DMSO to each well. The absorbance was monitored at 570 nm
(measurement) and 630 nm (reference) using a 96-well plate reader (Bio-Rad, CA, USA). Data were
collected for tetraplicates of each concentration of bSNPs, and these data were used to calculate the
mean. The percent inhibition was calculated from these data by the following formula:

Cellviability

Mean OD of untreated cells control Mean OD of treated cells Mean OD of untreated cells
control ?100:

2.6. Cellular morphology analysis

The cell morphology of bSNPs-treated WI-38 cells was analyzed by fluorescence microscopy, after
24 h. The nuclear and cytoplasmic morphology of the WI-38 cells was analyzed after treatment with
bSNPs for 24 h. Control cells were grown in the same manner in the absence of bSNPs. Then, the
cells were stained by AO/EB (Sigma) at 37 C for 5 min in the dark. The stained cells were examined
under an inverted fluorescence microscope (Carl Zeiss, Jena, Germany).

2.7. Mitochondrial membrane potential assay

The mitochondrial trans membrane potential was assessed using the fluorescent probe JC-1, which
produces green fluorescence in the cytoplasm and red-orange fluorescence in healthy mitochondria. If
the mitochondrial membrane potential is affected, JC-1 staining will be limited to the cytoplasm, and
the whole cell will fluoresce green. WI-38 cells were grown in 12-well plates and treated with bSNPs.
After exposure for 24 h,thecells were stained for 30 min with JC-1 (2 g/mL) in the culture medium.
The cells were then washed three times with PBS and lifted using 250 L of trypsinEDTA. The cells
were collected in PBS, washed by centrifugation, resuspended in 0.3 mL of PBS, mixed gently and
analyzed by a flow cytometry (BD FACSCantoII, San Jose, CA, USA). The presence of JC-1
monomers or dimers was determined using a fluorescence microscope with filter pairs at 530 nm/590
nm (dimers) and 485 nm/538 nm (monomers).

2.8. Measurement of ROS

Intracellular reactive oxygen species (ROS) were determined using the fluorescent probe 6-carboxy-
2, 7-dichlorodihydro fluoresce in diacetate, di(acetoxymethyl ester) according to the manufacturer's
instructions (Invitrogen, USA). The probe readily diffuses through the cell membrane and is
enzymatically hydrolyzed by intracellular esterases to form nonfluorescent 2,7-dichlorofluorescein
(DCFH), which then rapidly oxidizes to form highly fluorescent2,7 dichlorofluorescin (DCF) in the
presence of ROS. The DCF fluorescence intensity is indicative of the amount of ROS formed intra
cellularly. To determine the total intracellular ROS levels in WI-38 cells that were not treated
(control) or treated with D1 and D2 of bSNPs (g/mL) for 24 and 48 h, cells were seeded on 12-well
plates on the day prior to the assay. After treatment for 24 and 48 h, the medium was removed, and
the cells were washed with PBS and then incubated with 10 M probe in the loading medium. After
the probe was removed, the cells were washed and incubated with PBS. The intracellular fluorescence
intensity of 10,000 events was measured using a BD FACScalibur (BD biosciences, USA) at FL-1
(green) channel. The acquired data were analyzed using BD CellQuest Pro software (USA).

2.9. Cell cycle analysis

WI-38 cells were plated at 2 10 5 cells/mL in a 6-well plate. After a 24-h culture (37 C, 5% CO 2 ),
the cells were treated with D1 and D2 of bSNPs for 24 and 48 h. Following trypsinization, the cells
were centrifuged at 1000 g for 10 min, and the pellet was resuspended in 0.5Ml of PBS. Fixation
was completed by adding 4.5mL of cold, 70% ethanol for at least 16h at4 C.The fixed cells were
centrifuged at 1000 g for 10 min, and the pellet was suspended in 5 mL of PBS. After being washed
with PBS, the pellet was resuspended in 1 mL of propidium iodide(PI) stainingsolution.After
incubation at37 Cfor15min,the cells were analyzed to determine the cell cycle stage by a flowcy to
meter (BDFACSC anto TM II, San Jose, CA, USA) with an excitation wavelength of 488 nm and
emission at 670 nm. The presented data are representative of those obtained in at least three
independent experiments conducted in triplicate.

2.10. Quantitative real-time PCR analysis of antioxidant enzyme genes

The expression of antioxidant enzyme genes was analyzed by reverse transcription PCR (RT-PCR;
Applied Biosystems 7500 Fast, Foster City, CA) using a real-time SYBR green/ROX gene expression
assay kit from QI AGEN(Hilden,Germany). cDNA was directly prepared from cultured cells using a
Fastlane Cell cDNA kit from QIAGEN (Hilden, Germany), and the mRNA levels of catalase
(CAT), glutathione Stransferase A4 (GSTA4), tumor necrosis factor (TNF), cytochrome P450 1A
(CYP1A), cytochrome P450 reductase (POR), superoxide dismutase (SOD), glutathione S-transferase
M3 (GSTM3), glutathione peroxidase (GPX), glutathione reductase (GSR) and the reference gene,
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were assayed using gene-specific SYBR
green-based QuantiTect Primer assays from QIAGEN (Hilden, Germany). Quantitative real-time
RT-PCR was performed in a reaction volume of 25 L in accordance with the manufacturer's
instructions. Briefly, 12.5 L of the master mix, 2.0 L of assay primers (10) and 10 L of template
cDNA (100 ng) were added to each well. After being centrifuged briefly, the plate was subjected to
PCR under the following conditions: (i) activation at 95 C for 5 min, followed by 40 cycles of (ii)
denaturation at 95 C for 5 s and (iii) annealing/extension at 60 C for 30 s. All samples and controls
were run in triplicate on an AB7500 Fast Real-time PCR system. The quantitative RT-PCR data were
analyzed using a comparative thre shold (Ct) method, and the fold inductions of the samples were
compared With those of the untreated samples. GAPDH was used as an internal reference gene to
normalize the expression of the antioxidant enzymegenes. The Ct cycle was used to determine the
expression level in the control and in WI-38 cells treated with bSNPs for 24 and 48 h. The gene
expression level was then calculated as previously described by Yuan et al. [31]. The results are
expressed as the ratio of the reference gene to the target gene as calculated by the following formula:
Ct = Ct (anti oxidantgenes) Ct(GAPDH). To determine the relative expression
level,thefollowingformula was used: Ct =Ct(treated) Ct (untreated control). Thus, the
expression levels are expressed as n-fold differences relative to the reference gene. This value was
used to plot the expression of antioxidantenzyme genes using the mathematical expression 2 Ct .

3. Results

3.1. Biogenic silica nanoparticle synthesis

Agricultural wastes are a remarkable bio-precursor for several products, including bio-fuel, animal
feed, cellulose, activated carbon and silica. A schematic diagram of the bSNP formation mechanism is
illustrated in Fig. 1. Rice husk (RH) contains cellulose, hemicellulose, lignin, silica, and trace amount
of metal ions. RH was pretreated with hydrochloric acid that process eliminated metal ion impurities
and hydrolyzed the cellulose, hemicellulose and lignin of the rice husk. The acid pretreated residues
were calcinated that eliminated organic substances and generated silica nanoparticles with high purity
and amorphous.

3.2. Characterization of the biogenic silica nanoparticles

The XRD pattern soft he prepared biogenic silica nanoparticle sare illustrated in Fig. 2A. The
prepared samples displayed a peak with a 2 value of 22 corresponding to amorphous silica
nanoparticles, and no other impurities were detected. The elemental profile of the prepared bSNPs
was analyzed using EDX (Fig. 2B), and the observed peaks revealed the presence of C, Cu and silica
elements. According to EDX, the copper and carbon elements corresponded to the carbon-coated
copper grids used to support the samples. Therefore, the results confirmed that the prepared samples
were highly pure silica nanoparticles. The FT-IR spectrum (Fig. 2C) of the bSNPs revealed the
presence of silica. Further more, the prepared silica displayed peaks at 10561078 cm 1 , 790800
cm 1 and 440450 cm 1 corresponding to the O\Si\O asymmetric stretching, O\Si\O symmetric
stretching and Si\O bending vibration modes, respectively. Average particle size of prepared bSNPs
was estimated by DLS (Fig. 3); results showed that particle size of bSNPs is found to be around 121,
114 and 101 nm of 500, 600 and 700 C respectively. This analysis shows that size of particle can be
affected by the trend of the samples to form agglomerates, which maybe due to the influence of
Brownian motion. TEM images (Fig. 3) of the biogenic silica nanoparticles showed aggregation of
primary particles 1030 nm in size that were spherical in shape. However, the primary particles
aggregated with amorphous structure. The result confirmed that the primary particle size decreased
with increasing calcination temperature. The TEM images suggested that the calcination temperature
plays an important role in the formation of bSNPs.
3.3. Cytocompatibility of biogenic silica nanoparticles

An MTT assay was used to evaluate the biocompatibility of the bSNPs (Fig. 4). The hLFCs were
exposed to various concentrations (25, 50, 100, 200 and 400 g/mL) of bSNPs for 24 and 48 h.
Ultimately, there was no difference between the control and the low concentration groups, Where as
slight changes were observed at the high concentration group. Because the cell viability was greater
than 85% at the high concentration groups,the results indicate thatthe bSNPs are biocompatible with
hLFCs.

3.4. Biogenic silica nanoparticle effects on hLFC cell morphology

The effects of the bSNPs on the hLFC cell morphology were evaluated using AO/EB staining under
fluorescence microscopy. The images revealed the presence of healthy, round nuclei cells, and no
significant changes in cell morphology were observed upon treatment of the hLFCswith
thebSNPs[Dose-1(D-1) and Dose-2 (D-2)]for24 h(Fig.5A, B and C). The observations confirmed that
the bSNPs are non-toxic and biocompatible with hLFCs.

3.5. Effect of bSNPs on the mitochondrial membrane potential (M)

We evaluated the effects of the bSNPs on the mitochondrial membrane depolarization using flow
cytometry after staining with the mitochondrial potential-sensitive dye JC-1, which emits green
fluorescence when the M is disrupted. As shown in Fig. 6A, the treatment of the hLFCs with the
bSNPs (D1 and D2) for 24 h resulted in non-significant increases in the proportion of green
fluorescence-positive cells. For instance, the percentages of green fluorescent cells in the cultures
treated with the bSNPs at D1 and D2 for 24 h increased to 7.6% and 10.4%, respectively, compared
with 4% in the untreated control. However, the green fluorescent cells decreased to 6.1% and 8.4%
after treatment with the bSNPs at D1 and D2, respectively, for 48 h (Fig. 6B). Overall, the results
revealed no significant changes in the bSNP-treated cells, indicating that the bSNPs are biocompatible
and may be applicable as anti-caking agents in the food industry and as bone regeneration materials in
tissue engineering.

3.6. Effect of bSNPs on intracellular ROS

We analyzed the intracellular ROS levels in the bSNP-treated hLFCs (Dose-D1 and Dose-D2) for 24
and 48 h (Fig. 7). No significant changes were found in the intracellular ROS levels in the treated
hLFCs compared with the control. This result indicates that the bSNPs did not increase the ROS level
in the treated cells; therefore, they may be suitable for biological applications.

3.7. Effect of bSNPs on the cell cycle

After 24 and 48 h of incubation, the cellcycles of both the treated and untreated hLFCs were assessed
using flow cytometry (Fig. 8). The cell cycle results revealed that the number of cells in the G1
phased ecreased for all bSNP-treated cells. Compared with the control, the number of cells increased
in the S and G2 phases. These results clearly indicate that the bSNPs promoted cell proliferation.

3.8. Quantitation of the mRNA levels of stress and antioxidant response genes

RT-PCR was used to investigate the effects of the bSNPs on the mRNA levels of several antioxidant
enzymes and of the stress-related genes in WI-38 cells. The relative mRNA levels of CAT, GSTA4,
TNF, CYP1A, POR, SOD, GSTM3, GPX and GSR were quantified with a SYBR green-based assay
on a 7500 Fast Real-time System (Applied Biosystems). Fig.9 summarizes the expression levels of
GSR1, TNF, GSTA4, CYP1A and POR, which were upregulated compared with the untreated cells.
The expression levels of POR and TNF were significantly higher in the WI-38 cells that were treated
with bSNPs relative to the untreated control. There were no significant differences in the expression
levels of the CAT, GSTM3, GPX, and SOD1 genes in the WI-38 cells treated with the bSNPs at D1
and D2 compared with the untreated cells at 24 and 48 h.

4. Discussion

Rice husk is an agricultural biomass that contains organic (cellulose, hemicellulose and lignin) and
inorganic substances (SiO 2 ,Na,Ca,Mg,Fe,K,MnandAl)[21]. Annually,127 milliont ons of rice husk
are generated world wide, polluting the evironment [19]. Our current study developed highly pure
bSNPs from rice husk using low-concentration hydrochloric acid under pressurized temperatures, and
during this process, metalion impurities were also sufficiently eliminated (Fig. 1). Earlier reports
demonstrated that a variety of acids can be exploited for the removal of metal ion impurities
[29,32,33]. Our present physicochemical analysis confirmed that the bSNPs were amorphous in
nature, representing aggregates of 1030 nm primary particles. The FTIR spectrum showed peaks at
10561078 cm 1 , 790800 cm 1 and 440450 cm 1 corresponding to silica. Electron microscopy
images showed that ultrafine primary particles agglomerated. Interestingly, the TEM images
suggested that the calcination temperature plays a vital role in the formation of bSNPs. Previous
studies have suggested that nanoparticle formation occurs in a similar manner [34].

The biomedical and food industries require materials that are biocompatible and capable of
maintaining the natural morphologies of cells and tissues. Preliminary studies of the biocompatibility
of the rice husk-derived, highly pure bSNPs with hLFCs were performed using an in vitro model. The
cell viability results suggested that the bSNPs did not harm the hLFCs. Lin et al. Have reported that
15nm and 46nm diameter synthetic silica particles exhibited significant cytotoxic effects at low
concentrations (50 g/mL) [35]. In this study, the bSNPs allowed more than 85% cell viability even at
concentrations as high as 400 g/mL, suggestingthat the bSNPs are biocompatible. However, previous
studies have shown that the physicochemical properties of nanomaterials, such as the particle size,
shape, surface area and structure, play an important role in biocompatibility [36]. The collapse of the
M is a critical step in the mitochondrial apoptotic pathway. In this study, we sought to determine
whether bSNP-induced apoptosis was associated with a disruption of the mitochondrial membrane
potential. Reactive oxygen species (ROS) are a vital biomarker of intracellular oxidative stress, which
is generated by a variety of cellular biochemical processes. Increases in intracellular ROS contribute
to a number of human diseases via cell membrane damage, mitochondrial dysfunction and DNA
damage [37]. Biocompatible materials are either free radical scavengers or do not alter the ROS level
in biological systems. In the present investigation, bSNPs did not increase the ROS level in hLFCs.
Grant et al. demonstrated that biocompatible gold nanoparticles act as anti-oxidants by scavenging
free radicals [38]. Furthermore, earlier studies provided evidence that synthetic silica nanoparticles
induce ROS generation, which leads to cell death [39]. Nevertheless, the bSNPs did not have any
adverse effects on the ROS level, which indicates that they are suitable for biological systems. Our
results demonstrate that bSNPs modulate cell cycle distribution and progression in hLFCs by
triggering cell proliferation. Using RT-PCR, we analyzed the stress-related gene expression in hLFCs
treated with bSNPs for 24 and 48 h. Our results indicated that the bSNPs induced the upregulation
ofthePOR andTNFgenes. Similarly, Barrettet al. demonstrated that crystalline silica induced the
upregulation of TNF gene expression and led to oxidative stress [40]. By contrast, our present
investigation indicated that bSNPs do not trigger free radicals in hLFCs. Inconclusion,our physico
chemical results suggested that the harvested bSNPs were amorphous and spherical in shape with
diameters of 10 30 nm for the primary particles, which aggregated into clusters. Preliminary studies
of the biocompatibility of the bSNPs with hLFCs suggested that the bSNPs could be safely applied as
an anti-caking agent in the food industry and for bone tissue engineering in biomedical applications.
Further studies are underway to investigate the effects of bSNPs on bone tissue engineering and three
dimensional cell culture at the molecular level in human mesenchymal stem cells and lung fibroblast
cells respectively.