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Bioactive Silicon Nitride Implant Surfaces with Maintained Antibacterial Properties

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Silicon nitride (Si3N4) is a promising biomaterial, currently used in spinal fusion implants. Such implants should result in high vertebral union rates without major complications. However, pseudarthrosis remains an important complication that could lead to a need for implant replacement. Making silicon nitride implants more bioactive could lead to higher fusion rates, and reduce the incidence of pseudarthrosis. In this study, it was hypothesized that creating a highly negatively charged Si3N4 surface would enhance its bioactivity without affecting the antibacterial nature of the material. To this end, samples were thermally, chemically, and thermochemically treated. Apatite formation was examined for a 21-day immersion period as an in-vitro estimate of bioactivity. Staphylococcus aureus bacteria were inoculated on the surface of the samples, and their viability was investigated. It was found that the thermochemically and chemically treated samples exhibited enhanced bioactivity, as demonstrated by the increased spontaneous formation of apatite on their surface. All modified samples showed a reduction in the bacterial population; however, no statistically significant differences were noticed between groups. This study successfully demonstrated a simple method to improve the in vitro bioactivity of Si3N4 implants while maintaining the bacteriostatic properties.
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Citation: Katsaros, I.; Zhou, Y.;
Welch, K.; Xia, W.; Persson, C.;
Engqvist, H. Bioactive Silicon Nitride
Implant Surfaces with Maintained
Antibacterial Properties. J. Funct.
Biomater. 2022,13, 129. https://
doi.org/10.3390/jfb13030129
Academic Editor: Yuqin Qiao
Received: 4 August 2022
Accepted: 25 August 2022
Published: 27 August 2022
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4.0/).
Journal of
Functional
Biomaterials
Article
Bioactive Silicon Nitride Implant Surfaces with Maintained
Antibacterial Properties
Ioannis Katsaros 1, Yijun Zhou 2, Ken Welch 3, Wei Xia 1, Cecilia Persson 2and Håkan Engqvist 1,*
1Division of Applied Materials Science, Department of Materials Science and Engineering, Uppsala University,
75103 Uppsala, Sweden
2Division of Biomedical Engineering, Department of Materials Science and Engineering, Uppsala University,
75103 Uppsala, Sweden
3Division of Nanotechnology and Functional Materials, Department of Materials Science and Engineering,
Uppsala University, 75103 Uppsala, Sweden
*Correspondence: hakan.engqvist@angstrom.uu.se
Abstract:
Silicon nitride (Si
3
N
4
) is a promising biomaterial, currently used in spinal fusion implants.
Such implants should result in high vertebral union rates without major complications. However,
pseudarthrosis remains an important complication that could lead to a need for implant replacement.
Making silicon nitride implants more bioactive could lead to higher fusion rates, and reduce the inci-
dence of pseudarthrosis. In this study, it was hypothesized that creating a highly negatively charged
Si
3
N
4
surface would enhance its bioactivity without affecting the antibacterial nature of the material.
To this end, samples were thermally, chemically, and thermochemically treated. Apatite formation
was examined for a 21-day immersion period as an in-vitro estimate of bioactivity. Staphylococcus
aureus bacteria were inoculated on the surface of the samples, and their viability was investigated. It
was found that the thermochemically and chemically treated samples exhibited enhanced bioactivity,
as demonstrated by the increased spontaneous formation of apatite on their surface. All modified
samples showed a reduction in the bacterial population; however, no statistically significant dif-
ferences were noticed between groups. This study successfully demonstrated a simple method to
improve the in vitro bioactivity of Si3N4implants while maintaining the bacteriostatic properties.
Keywords: bioactivity; silicon nitride; surfaces; antibacterial; biomedical
1. Introduction
Silicon nitride (Si
3
N
4
) is a ceramic material that has a long history of being utilized in
applications where components are to be exposed to thermally and mechanically demand-
ing environments, such as combustion engines and gas turbines [
1
]. The main driving
forces behind its use are the mechanical properties of the material, resulting from its unique
microstructure [
2
], in combination with its refractory nature as a ceramic material. Even-
tually, silicon nitride was evaluated as a potential biomaterial. Silicon nitride has indeed
proven to be non-toxic both
in vitro
and
in vivo
[
3
,
4
] and this, in combination with its
mechanical properties, has led to its use as an orthopedic implant material [
5
7
]. Today,
spinal fusion devices made from silicon nitride have been approved by regulatory agencies
for human use [
8
]. Compared to other common spinal implant materials such as Ti6Al4V
and poly(ether ether ketone) (PEEK), it has been found to be more osteogenic both
in vitro
and
in vivo
[
9
12
] and to have comparable clinical results [
13
,
14
]. Furthermore, the surface
of silicon nitride materials has been found to be inhibitory to bacterial attachment [
15
17
],
and even induce lysis in specific bacterial strains through the formation and elution of
ammonia ions when in the presence of water [18].
The ideal spinal fusion material should rapidly form a strong bond with the native
bone while shielding the affected area from bacterial infection. Failure to do so might lead
to pseudarthrosis or non-union of the affected vertebrae, requiring revision surgery [
19
].
J. Funct. Biomater. 2022,13, 129. https://doi.org/10.3390/jfb13030129 https://www.mdpi.com/journal/jfb
J. Funct. Biomater. 2022,13, 129 2 of 14
Pseudarthrosis is responsible for almost a quarter of revision surgeries after attempted
fusions in the lumbar spine [
20
]. Its diagnosis can be challenging, and it can lead to
disability, pain, and discomfort for affected patients. While there are many risk factors for
pseudarthrosis including patient health, age, and construct length [
21
] among others, its
incidence can be reduced by way of implant material selection.
Indeed, increasing the bioactivity of spinal implant materials can be a way to increase
the fusion rate and reduce the incidence of pseudarthrosis [
22
,
23
]. The term “bioactivity”
has been used to describe the ability of a material to spontaneously form a layer of apatite
on its surface after immersion in simulated body fluid [
24
]. Bioactive biomaterials are
hypothesized to more rapidly form a bond with the native bone after implantation, resulting
in enhanced implant stability. Researchers have identified a variety of properties that are
correlated with bioactivity where the surface charge [
25
] and roughness [
26
] of the material
are key factors. A negative surface charge at physiological pH leads to a stronger attraction
of Ca
2+
ions to the surface of the material, while rougher materials provide more nucleation
sites for apatite.
In Si
3
N
4
, these properties can be affected by thermal and/or chemical surface treat-
ments [
27
]. Bock et al. [
28
] utilized a variety of thermochemical surface modifications
to alter the surface charge and chemistry of silicon nitride materials in order to examine
their effect on properties that directly affect the biological behavior of implants such as
surface roughness, charge, and wettability. The authors speculated that from the chosen
modifications, heat treatment of silicon nitride samples at 1070
C for 7 h was particularly
promising, as it created a highly negatively charged and hydrophilic surface. Bock et al. [
15
]
compared different modulations of Si
3
N
4
, Ti6Al4V, and PEEK in terms of their bacteriostatic
behavior. They found that silicon nitride was clearly bacteriostatic, with as-fired, oxidized,
nitrogen annealed and SiYAlON coated samples retaining that property at comparable
levels. Finally, Hnatko et al. [
29
] successfully increased the bioactivity of silicon nitride
materials through an oxyacetylene flame treatment that oxidized their surface and created
a porous surface layer that promoted apatite formation and cell attachment.
However, a cost-effective and easy-to-apply modification that could be used to en-
hance the bioactivity of currently-used silicon nitride implants, without affecting their
antibacterial behavior, has yet to be identified. The aim of this study was to enhance the
bioactive nature of silicon nitride materials through different surface modifications that
could potentially be used post-manufacturing before implants reach the clinic. A secondary
aim was to ensure that the surface modifications do not affect the inhibitory bacterial
environment the material creates. To achieve these aims, commercially-produced silicon
nitride samples were thermally, chemically, and thermochemically treated. Afterwards, the
chemical or morphological changes on the silicon nitride surfaces were studied. Finally,
the effectiveness of each treatment in terms of increasing the bioactivity and inhibiting
bacterial proliferation was evaluated in vitro.
2. Materials and Methods
2.1. Materials
Two types of Si
3
N
4
samples were used in this study. Bulk non-porous bars (
length = 47 mm
,
height = 2.9 mm, width = 4 mm) and porous cylinders (ø12.6 mm, height = 10 mm, approximate
porosity 70%) were both produced by SinTX Technologies (Salt Lake City, UT, USA) using
alumina (6% wt) and yttria (4% wt) as sintering additives. This composition is currently used
for the production of Si
3
N
4
spinal implants [
30
]. The bulk non-porous samples were used
for surface characterization after the surface modifications and the assessment of antibacterial
behavior, while the porous samples were utilized to evaluate bioactivity after having their
morphology studied.
2.2. Surface Modifications
Three different surface modification processes were utilized to enhance the bioactive
nature of the material. Before being treated, all samples were washed in consecutive 15-min
J. Funct. Biomater. 2022,13, 129 3 of 14
steps in distilled water and ethanol, and then sonicated and thoroughly dried in a desiccator.
The samples were divided into 4 groups.
The control group consisted of the non-treated (NT) samples. The chemically treated
(CT) group consisted of samples that were immersed in falcon tubes containing 50 mL of a
10 M sodium hydroxide (NaOH) solution (pellets, Sigma-Aldrich, St. Louis, MI, USA) for
24 h in an environment heated to 60
C. To ensure homogenous surface treatment, samples
were suspended using fishing line, ensuring they were not in contact with the falcon tube
walls. The thermally treated (TT) samples were placed in an induction furnace (Gero CWF,
Carbolite Furnaces, Sheffield, UK) and heat treated at a temperature of 1070
C with a
ramping rate of 12
C/min for 4 h, then left to cool overnight. Finally, the thermochemically
treated samples (TCT) were thermally and then chemically treated as described above.
2.3. Material Characterization
To further understand the effect each type of modification had on the materials, a
variety of characterization methods were employed. The detailed characterization of the
samples post-modification also aimed to ensure that any differences in
in vitro
bioactivity
and antibacterial behavior would not be attributed to any differences between samples
other than the ones brought on by the modifications.
2.3.1. X-ray Diffraction (XRD)
X-ray diffraction (D500, Bruker, Billerica, MA, USA) was used to identify whether
any new crystalline phases were formed on the surface of the material after the treatments.
The samples were scanned from 10
to 80
with a scanning rate of 0.02
/s using a Bragg-
Brentano configuration and CuKa radiation (λ= 0.15418 nm) [31].
2.3.2. X-ray Photoelectron Spectroscopy (XPS)
X-ray photoelectron spectroscopy (Quantera II, Physical Electronics, Chanhassen,
MN, USA) was used to further elucidate the chemical composition of the surface of the
samples. Survey scans were taken at a pass energy of 140 eV, after which atomic percentage
concentrations were calculated from the elemental peak area. Results were averaged after
10 measurement cycles. Prior to measurements, samples were sputtered at 500 V for one
minute to remove surface contamination. Ion and electron guns were turned on during
measurements to neutralize the surface charge build-up of the non-conductive samples.
Data analysis was performed using the MultiPak software (Version 9.6, Physical Electronics,
Chanhassen, MN, USA).
2.3.3. Scanning Electron Microscopy (SEM)
Scanning electron microscopy (SEM 1530, Zeiss, Jena, Germany) was utilized in order
to examine the surface of the porous samples after the treatments. Samples were coated
with a layer of conductive Au/Pd with a thickness of approximately 10 nm to avoid sample
charging. Images of the samples were taken after the surface treatments at an accelerating
voltage of 8 kV, and a working distance of approximately 5 mm. Images of the samples
after the immersion assay were taken at an accelerating voltage of 5 kV, and a working
distance of approximately 5 mm. The adjustment in the accelerating voltage was made as
apatite formation increased charging.
2.3.4. Computed Microtomography Scans (µCT)
Computed microtomography scans (Skyscanner, Bruker) of the porous samples were
taken in order to visualize the porous network of the samples and to identify the effect,
if any, the surface treatments had on it. Scans were taken at a resolution of 9
µ
m, with a
voltage of 100 kV, a current of 100
µ
A, an exposure time of 2070 ms and a rotation step of 0.4
.
The
µ
CT images were reconstructed with NRecon (Bruker) and post-processed in MATLAB
(Version 2021b, MathWorks, Natick, MA, USA). The 3D-models were thresholded with
respect to the histogram of grey level value distribution and the porosity was calculated
J. Funct. Biomater. 2022,13, 129 4 of 14
in MATLAB (Version 2021b, MathWorks, Natick, MA, USA) by counting the number of
remaining voxels.
2.4. Immersion Assay
Porous samples (n = 4) from each group were suspended using a fishing line in
Dulbecco’s phosphate-buffered saline (DPBS) (Sigma-Aldrich) in order to avoid preferential
apatite deposition on sample surfaces, for a total of 21 days. This method was previously
used to evaluate the surface bioactivity of biomaterials [
32
]. DPBS supplemented with
CaCl2and MgCl2was used, as it has a similar ionic concentration to that of blood plasma
and can simulate physiological fluids [
33
]. During the first two weeks of immersion, the
solution was not replenished in order to facilitate inductively coupled plasma optical
emission spectrometry (ICP-OES) (PerkinElmer ICP-OES, Avio 200, PerkinElmer, Waltham,
MA, USA) measurements of calcium ions in the solution. These measurements were taken
after 1, 5, 7, and 14 days of immersion as a way to pinpoint both the start and the rate
of precipitation. A decrease of calcium in the solution would indicate the formation of
precipitates. After the first two weeks of immersion, the DPBS solution was replenished
in order to provide a fresh supply of ions to support potential apatite formation. After
21 days of immersion, the samples were removed, washed gently with deionized water,
and dried in a desiccator. Cross-sections of the porous samples were examined through
SEM to evaluate apatite formation.
2.5. Optical Profilometry
Bacterial and cell attachment have been shown to be significantly affected by the
surface roughness of the material on which they are seeded [
34
36
]. The samples used
for the bacterial attachment assay were initially polished down to an average surface
roughness (Sa) of approximately 5nm. Optical profilometry (ZYGO, Middlefield, CT, USA)
was used in order to study the effect of the treatments on the average surface roughness of
the samples. A 50
×
magnification was used to scan a square area (
α
= 167
µ
m), with five
replicates per measurement. Three measurements were taken per sample, after which the
results were averaged.
2.6. Bacterial Testing
2.6.1. Bacterial Culture
Gram-positive Staphylococcus aureus bacteria were used for this study. 10
µ
L of
a bacterial suspension was added to 10 mL of sterilized tryptic soy broth (TSB) (Sigma-
Aldrich), and the mixture was incubated overnight at 37
C. Following this, the suspension
was centrifuged to isolate the bacteria, which were then resuspended in 10 mL of TSB.
Finally, the bacterial solution was diluted to an OD
600
= 0.2, measured using a UV-VIS
spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan).
2.6.2. Bacterial Attachment Assay
Polished bulk silicon nitride samples were autoclaved at 120
C for 20 min to avoid
cross-contamination with other environmental bacterial strains. The samples were then
placed in sterile ø10 cm Petri dishes (Sigma-Aldrich). 10
µ
L of the diluted bacterial solution
was then placed on the surface of each sample for two hours at room temperature so the
bacteria could come in contact and interact with the surface of the material. As a negative
control, the same volume of bacterial solution was placed into a 2 mL polypropylene micro-
centrifuge tube for the same amount of time. Both the petri dishes and the microcentrifuge
tube were kept closed, and the bacterial solutions were monitored to ensure the bacterial
suspension did not evaporate during the incubation period. When the incubation time
elapsed, the samples were placed in 1 mL of DPBS (Sigma-Aldrich) and vortexed for 1 min
each so the adherent bacteria could be detached from the surface of the samples. One
ml of PBS was added to the bacterial solution being used as a control, which was then
also vortexed for 1 min. These bacterial solutions were then diluted tenfold in four steps,
J. Funct. Biomater. 2022,13, 129 5 of 14
with 100
µ
L of each dilution then plated onto TSB-agar plates. The agar plates were then
incubated overnight so bacterial colonies could form and then be counted.
2.7. Statistical Analysis
Quantitative data is reported as means
±
standard deviations. IBM SPSS Statistics
(Version 26, IBM Corp, New York, NY, USA) was used to perform a one-way analysis of
variance (ANOVA) with Tukey post-hoc tests to identify significant differences between
groups. Welch’s robust test of equality of means combined with Tamhane’s post-hoc test
was used when the assumption of homogeneity of variance was violated. A significance
level of p= 0.05 was set for all tests.
3. Results and Discussion
3.1. XRD
The XRD pattern of all samples can be seen in Figure 1. All samples had the typical
peak distribution of
β
-phase silicon nitride. The samples used in this study, both bulk and
porous, were manufactured using thermal processing which ensured the transformation
from the
α
-Si
3
N
4
to the
β
-Si
3
N
4
phase, which is favorable to the mechanical properties of
the material [
37
]. No other crystalline phases were detected in any of the treated samples.
However, the potential formation of amorphous sodium silicates in NaOH treated samples,
or amorphous silicon dioxide in heat-treated samples, cannot be excluded.
J. Funct. Biomater. 2022, 13, x FOR PEER REVIEW 6 of 15
Figure 1. The XRD pattern from samples of all groups are displayed in comparison with the refer-
ence pattern of β-Si
3
N
4
(PDF 01-078-2963), indicating that this was the main crystalline phase present
in all groups.
3.2. XPS
Figure 2 shows that minuscule amounts of sodium were detected on the surfaces of
bulk samples. Looking at the atomic concentrations of the surfaces of all groups (Table 1),
the thermally treated samples stand out. It is clear that a layer of silicon dioxide was
formed, while a very small amount of nitrogen remained present on the surface of the
material, as expected from the literature [38]. More interestingly, in the thermochemically
treated samples, the NaOH treatment seemed to have etched the samples, removing the
oxide layer formed through the heat treatment.
Figure 2. XPS survey spectra, in (a), of all samples post-modification showed that the main elements
on their surface were mostly the same. However, the difference in peak intensities indicates differ-
ences in their amounts. Most notably in thermally treated samples, oxygen peaks were intensified
Figure 1.
The XRD pattern from samples of all groups are displayed in comparison with the reference
pattern of
β
-Si
3
N
4
(PDF 01-078-2963), indicating that this was the main crystalline phase present in
all groups.
3.2. XPS
Figure 2shows that minuscule amounts of sodium were detected on the surfaces of
bulk samples. Looking at the atomic concentrations of the surfaces of all groups (Table 1),
the thermally treated samples stand out. It is clear that a layer of silicon dioxide was
formed, while a very small amount of nitrogen remained present on the surface of the
material, as expected from the literature [
38
]. More interestingly, in the thermochemically
treated samples, the NaOH treatment seemed to have etched the samples, removing the
oxide layer formed through the heat treatment.
J. Funct. Biomater. 2022,13, 129 6 of 14
J. Funct. Biomater. 2022, 13, x FOR PEER REVIEW 6 of 15
Figure 1. The XRD pattern from samples of all groups are displayed in comparison with the refer-
ence pattern of β-Si
3
N
4
(PDF 01-078-2963), indicating that this was the main crystalline phase present
in all groups.
3.2. XPS
Figure 2 shows that minuscule amounts of sodium were detected on the surfaces of
bulk samples. Looking at the atomic concentrations of the surfaces of all groups (Table 1),
the thermally treated samples stand out. It is clear that a layer of silicon dioxide was
formed, while a very small amount of nitrogen remained present on the surface of the
material, as expected from the literature [38]. More interestingly, in the thermochemically
treated samples, the NaOH treatment seemed to have etched the samples, removing the
oxide layer formed through the heat treatment.
Figure 2. XPS survey spectra, in (a), of all samples post-modification showed that the main elements
on their surface were mostly the same. However, the difference in peak intensities indicates differ-
ences in their amounts. Most notably in thermally treated samples, oxygen peaks were intensified
Figure 2.
XPS survey spectra, in (
a
), of all samples post-modification showed that the main elements
on their surface were mostly the same. However, the difference in peak intensities indicates differences
in their amounts. Most notably in thermally treated samples, oxygen peaks were intensified while
nitrogen peaks were diminished. Also, both TCT and TT samples had higher amounts of carbon
contamination, possibly as a result of the heat treatment. (
b
,
c
) corresponds to the area where the
sodium peaks were noticed in thermochemically (b) and chemically (c) treated samples.
Table 1.
Atomic concentration of the main elements comprising the surface of the samples of each
group (all atomic concentration values have been rounded to integers).
Atomic Concentration (%)
Group Si N O Si/N Si/O
Non-treated 40 39 16 1 2.5
Chemically treated 40 44 7 0.9 5.7
Thermally treated 15 2 38 7.5 0.4
Thermochemically treated
35 38 12 0.9 2.9
3.3. SEM before the Immersion Assay
SEM images of the samples after the treatments did not show differences in morphol-
ogy (Figure 3). All samples showcased an extended porous network with pores of varying
sizes. No shrinkage was noticed; the heat treatment at 1070
C without external pressure
did not create the necessary diffusion conditions for further consolidation of Si3N4.
3.4. Computed Microtomography Scans
As evidenced in Table 2, no statistically significant differences (p= 0.858) were found
in terms of porosity of the samples, which was approximately 70%. The same homogeneity
was noticed when examining the CT scans of all groups. The slice (
thickness = 270 µm
) dis-
played in Figure 4confirmed the porous structure observed in the SEM images. Porosity and
pore interconnectivity have been found to affect the bioactivity of porous
materials [39,40]
,
however, the non-significant differences between groups indicate that any differences in
apatite formation do not stem from differences in the porous structure of the samples.
Approximating microporosity using
µ
CT is limited in regards to resolution. The resolution
of these measurements was around 9.1
µ
m, meaning that pores below that size could not be
accounted for. However, in the case of the presented materials, SEM images confirmed that
the material was mainly comprised of pores that, at the very least, were ten times greater
that the lowest resolution of the material.
J. Funct. Biomater. 2022,13, 129 7 of 14
J. Funct. Biomater. 2022, 13, x FOR PEER REVIEW 7 of 15
while nitrogen peaks were diminished. Also, both TCT and TT samples had higher amounts of car-
bon contamination, possibly as a result of the heat treatment. (b,c) corresponds to the area where
the sodium peaks were noticed in thermochemically (b) and chemically (c) treated samples.
Table 1. Atomic concentration of the main elements comprising the surface of the samples of each
group (all atomic concentration values have been rounded to integers).
Atomic Concentration (%)
Group Si N O Si/N Si/O
Non-treated 40 39 16 1 2.5
Chemically treated 40 44 7 0.9 5.7
Thermally treated 15 2 38 7.5 0.4
Thermochemically treated 35 38 12 0.9 2.9
3.3. SEM before the Immersion Assay
SEM images of the samples after the treatments did not show differences in morphol-
ogy (Figure 3). All samples showcased an extended porous network with pores of varying
sizes. No shrinkage was noticed; the heat treatment at 1070 °C without external pressure
did not create the necessary diffusion conditions for further consolidation of Si3N4.
Figure 3. SEM images of the samples after the surface modifications, showing no significant differ-
ences in pore size and morphology between (a) non-treated, (b) chemically, (c) thermally, and (d)
thermochemically treated samples.
3.4. Computed Microtomography Scans
As evidenced in Table 2, no statistically significant differences (p = 0.858) were found
in terms of porosity of the samples, which was approximately 70%. The same homogene-
ity was noticed when examining the CT scans of all groups. The slice (thickness = 270 μm)
displayed in Figure 4 confirmed the porous structure observed in the SEM images. Poros-
ity and pore interconnectivity have been found to affect the bioactivity of porous materials
[39,40], however, the non-significant differences between groups indicate that any differ-
ences in apatite formation do not stem from differences in the porous structure of the
samples. Approximating microporosity using μCT is limited in regards to resolution. The
resolution of these measurements was around 9.1 μm, meaning that pores below that size
could not be accounted for. However, in the case of the presented materials, SEM images
Figure 3.
SEM images of the samples after the surface modifications, showing no significant dif-
ferences in pore size and morphology between (
a
) non-treated, (
b
) chemically, (
c
) thermally, and
(d) thermochemically treated samples.
Table 2. The average overall porosity of the porous samples of all groups after the modifications, as
estimated by µCT.
Average Porosity (%)
Non-treated 68.7 ±5.1
Chemically treated 70.2 ±3.3
Thermally treated 70.3 ±0.8
Thermochemically treated 69.2 ±1.5
J. Funct. Biomater. 2022, 13, x FOR PEER REVIEW 8 of 15
confirmed that the material was mainly comprised of pores that, at the very least, were
ten times greater that the lowest resolution of the material.
Figure 4. MicroCT 3D reconstructions of (a) a longitudinal cross section and (b) a transverse slice,
visualizing the porous network of samples of all groups. (a) has been cropped to exclude artefacts
due to sample fixation during scanning and (b) has been slightly rotated to enhance visibility.
Table 2. The average overall porosity of the porous samples of all groups after the modifications, as
estimated by μCT.
Average Porosity (%)
Non-treated 68.7 ± 5.1
Chemically treated 70.2 ± 3.3
Thermally treated 70.3 ± 0.8
Thermochemically treated 69.2 ± 1.5
3.5. ICP-OES
As can be seen in Figure 5, the samples that were thermochemically treated showed
a decrease in calcium concentration compared to that of non-treated samples as early as
the fifth day of immersion. A similar trend was seen in the chemically treated samples,
with the decrease in calcium in the solution being more gradual. These results were an
indication that the sodium hydroxide treatment was effective in enhancing the bioactivity
of porous silicon nitride samples. Thermally treated samples did not show a reduction,
and did not significantly diverge from the range of values of DPBS.
Figure 5. The results of the ICP measurements showing the calcium concentration during the first
14 days of immersion in PBS. A clear decreasing trend was detected for CT and TCT samples
throughout the immersion period, indicating calcium precipitation.
3.6. Apatite Formation
Figure 4.
MicroCT 3D reconstructions of (
a
) a longitudinal cross section and (
b
) a transverse slice,
visualizing the porous network of samples of all groups. (
a
) has been cropped to exclude artefacts
due to sample fixation during scanning and (b) has been slightly rotated to enhance visibility.
3.5. ICP-OES
As can be seen in Figure 5, the samples that were thermochemically treated showed
a decrease in calcium concentration compared to that of non-treated samples as early as
the fifth day of immersion. A similar trend was seen in the chemically treated samples,
with the decrease in calcium in the solution being more gradual. These results were an
indication that the sodium hydroxide treatment was effective in enhancing the bioactivity
of porous silicon nitride samples. Thermally treated samples did not show a reduction, and
did not significantly diverge from the range of values of DPBS.
J. Funct. Biomater. 2022,13, 129 8 of 14
J. Funct. Biomater. 2022, 13, x FOR PEER REVIEW 8 of 15
confirmed that the material was mainly comprised of pores that, at the very least, were
ten times greater that the lowest resolution of the material.
Figure 4. MicroCT 3D reconstructions of (a) a longitudinal cross section and (b) a transverse slice,
visualizing the porous network of samples of all groups. (a) has been cropped to exclude artefacts
due to sample fixation during scanning and (b) has been slightly rotated to enhance visibility.
Table 2. The average overall porosity of the porous samples of all groups after the modifications, as
estimated by μCT.
Average Porosity (%)
Non-treated 68.7 ± 5.1
Chemically treated 70.2 ± 3.3
Thermally treated 70.3 ± 0.8
Thermochemically treated 69.2 ± 1.5
3.5. ICP-OES
As can be seen in Figure 5, the samples that were thermochemically treated showed
a decrease in calcium concentration compared to that of non-treated samples as early as
the fifth day of immersion. A similar trend was seen in the chemically treated samples,
with the decrease in calcium in the solution being more gradual. These results were an
indication that the sodium hydroxide treatment was effective in enhancing the bioactivity
of porous silicon nitride samples. Thermally treated samples did not show a reduction,
and did not significantly diverge from the range of values of DPBS.
Figure 5. The results of the ICP measurements showing the calcium concentration during the first
14 days of immersion in PBS. A clear decreasing trend was detected for CT and TCT samples
throughout the immersion period, indicating calcium precipitation.
3.6. Apatite Formation
Figure 5.
The results of the ICP measurements showing the calcium concentration during the first
14 days of immersion in PBS. A clear decreasing trend was detected for CT and TCT samples
throughout the immersion period, indicating calcium precipitation.
3.6. Apatite Formation
SEM images after immersion confirmed the results of the ICP measurements
(Figure 6). As far as the samples of group TCT were concerned, a large number of spheres
showcasing the characteristic apatitic plate-like structures were developed throughout the
surface of the samples. Looking at the CT samples, their surface was similar, but with
less pronounced apatite formation. Those structures were not noticed in the NT and TT
samples, in accordance with the ICP-OES results.
J. Funct. Biomater. 2022, 13, x FOR PEER REVIEW 9 of 15
SEM images after immersion confirmed the results of the ICP measurements (Figure
6). As far as the samples of group TCT were concerned, a large number of spheres show-
casing the characteristic apatitic plate-like structures were developed throughout the sur-
face of the samples. Looking at the CT samples, their surface was similar, but with less
pronounced apatite formation. Those structures were not noticed in the NT and TT sam-
ples, in accordance with the ICP-OES results.
Figure 6. SEM images taken at 200× magnification for samples of all groups after 21 days of immer-
sion. Apatitic flakes were evident for CT (b) and TCT (d) samples but not in the NT (a) and TT (c)
ones. In (c) some remaining debris from sample preparation can be seen.
SEM analysis indicated that the chemical and thermochemical treatment did have the
expected effect. Between the two, the TCT samples had a surface with a denser network
of the characteristic plate-like structures of apatite (Figure 7). In the non-treated and ther-
mally treated samples, no apatitic flakes were detected. The heat treatment resulted in an
oxidized surface that did not seem to have any enhancing effect in terms of apatite for-
mation. The only indication of precipitation were amorphous layers in their forming
stages in parts of the material.
Figure 7. EDS analysis confirmed that the apatite-like structures mainly consisted of Ca, P, and O.
Traces of sodium were identified throughout the surface of the thermochemically treated materials.
Figure 6.
SEM images taken at 200
×
magnification for samples of all groups after 21 days of
immersion. Apatitic flakes were evident for CT (
b
) and TCT (
d
) samples but not in the NT (
a
) and TT
(c) ones. In (c) some remaining debris from sample preparation can be seen.
J. Funct. Biomater. 2022,13, 129 9 of 14
SEM analysis indicated that the chemical and thermochemical treatment did have the
expected effect. Between the two, the TCT samples had a surface with a denser network of
the characteristic plate-like structures of apatite (Figure 7). In the non-treated and thermally
treated samples, no apatitic flakes were detected. The heat treatment resulted in an oxidized
surface that did not seem to have any enhancing effect in terms of apatite formation. The
only indication of precipitation were amorphous layers in their forming stages in parts of
the material.
J. Funct. Biomater. 2022, 13, x FOR PEER REVIEW 9 of 15
SEM images after immersion confirmed the results of the ICP measurements (Figure
6). As far as the samples of group TCT were concerned, a large number of spheres show-
casing the characteristic apatitic plate-like structures were developed throughout the sur-
face of the samples. Looking at the CT samples, their surface was similar, but with less
pronounced apatite formation. Those structures were not noticed in the NT and TT sam-
ples, in accordance with the ICP-OES results.
Figure 6. SEM images taken at 200× magnification for samples of all groups after 21 days of immer-
sion. Apatitic flakes were evident for CT (b) and TCT (d) samples but not in the NT (a) and TT (c)
ones. In (c) some remaining debris from sample preparation can be seen.
SEM analysis indicated that the chemical and thermochemical treatment did have the
expected effect. Between the two, the TCT samples had a surface with a denser network
of the characteristic plate-like structures of apatite (Figure 7). In the non-treated and ther-
mally treated samples, no apatitic flakes were detected. The heat treatment resulted in an
oxidized surface that did not seem to have any enhancing effect in terms of apatite for-
mation. The only indication of precipitation were amorphous layers in their forming
stages in parts of the material.
Figure 7. EDS analysis confirmed that the apatite-like structures mainly consisted of Ca, P, and O.
Traces of sodium were identified throughout the surface of the thermochemically treated materials.
Figure 7.
EDS analysis confirmed that the apatite-like structures mainly consisted of Ca, P, and O. Traces
of sodium were identified throughout the surface of the thermochemically treated materials.
The initial hypothesis of the study was that surface charge would be the main mecha-
nism behind any increase in bioactivity, however, the results indicate that another mecha-
nism was at play. The surface of silicon nitride materials in wet environments is comprised
of silanol (Si-OH) and amine (Si-NH
2
) groups. The ratio of silanol to amine groups is
inversely proportional to the surface charge of the material at physiological pH. The ther-
mally treated samples had a surface dominated by silicon oxynitride and, consequently,
a highly negative charge at physiological pH [
28
]. Surprisingly, no specific enhancement
resulted from the thermal treatment. Instead, the chemical and thermochemical treatments
clearly enhanced bioactivity. The results of the ICP-OES and qualitative assessment of
the SEM images showed that the thermochemically treated samples had more calcium
phosphates precipitated on their surface. This led to the conclusion that sodium hydroxide
was a significant factor in the increase of bioactivity. While sodium has not been found to
be essential for apatite formation in bioactive glasses [41], this study clearly indicates that
NaOH treatment enhances apatite formation. In their study on the mechanism of apatite
formation on sodium silicate glasses, Hayakawa et al. [
42
] identified sodium binding in
silicon tetrahedra as being crucial to the formation of negatively charged sites. They found
that the dissolution of calcium silicates creates sites for the nucleation and crystallization
of apatite. XPS and EDS analysis showed the clear retention of sodium throughout the
porous structure of the materials. Thus, the effectiveness of the NaOH treatments seems
to stem from the dissolution of calcium silicates creating negatively charged vacancies,
suitable for calcium precipitation. It could be hypothesized that the general surface charge
of the material plays a lesser role in the nucleation of apatite than localized negatively
charged sites.
Finally, the increased bioactivity of TCT samples in comparison to CT samples may
be explained by the grain growth noticed in the TCT samples, due to the heat treatment
(Figure 8). The elongation of the needle-like
β
-Si
3
N
4
grains created a rough surface that
created a surface morphology favorable for the precipitation of calcium phosphates.
J. Funct. Biomater. 2022,13, 129 10 of 14
Figure 8.
SEM images taken at a 10,000
×
magnification revealed an increase in grain size in the
thermochemically treated samples (
b
) when compared to the non-treated samples (
a
), as a result of
the heat treatment.
The results of the immersion assay should be interpreted with caution.
Kokubo et al. [32]
reported that a surface layer of calcium phosphate formed on Bioglass, which was speculated
to be the cause of a faster and stronger bond with the natural bone after
in vivo
implantation
through preferential osteoblast attachment and proliferation on that apatite layer. Conse-
quently, it was proposed that the occurrence of spontaneous apatite formation on materials
immersed in solutions with ion concentrations similar to that of the human blood plasma is
a valid indication of their
in vivo
behavior. Since then, there has been an interesting debate
on the validity of these immersion assays as predictors of
in vivo
behavior. Bellucci et al. [
43
]
examined the biological behavior of commercial Bioglass and Bioglass composites using
immersion in SBF as well as cell attachment, viability, and proliferation assays, finding that the
two sets of methods could give contradicting results and, thus, immersion studies could be
misleading. Furthermore, Bohner and Lemaitre [
44
] also stated that the results of such assays
cannot be standalone predictors of natural bone-bonding as they can indicate false-positive or
negative results. With this debate in mind, an immersion in DPBS was utilized in this study
as a first estimation of bioactive behavior, with further studies needed to validate these results
and approximate in-vivo behavior.
3.7. Optical Profilometry
Heat treating the samples led to grain growth that due to the needle-like morphology
of the
β
-grains of the material increased surface roughness in Figure 9. As expected, heat-
treated samples showed an almost ten-fold increase in average surface roughness compared
to the non-heat-treated ones. Differences between the TT and TCT groups versus the NT
and CT groups were statistically significant (* p
0.001 for all). No statistically significant
differences were found between the NT and CT groups (* p= 0.966), nor between the TT
and TCT groups (* p= 1.0).
3.8. Colony Forming Unit Assay
The samples that were thermochemically treated showed the highest bioactivity and
were therefore selected to be compared with the non-treated ones in terms of their an-
tibacterial behavior. As the difference between the surface roughness of the two groups
was significant, samples of the TT group were tested as well in an effort to account for
the effect of the increased surface roughness. Figure 10 displays the amount of viable
colony-forming units after contact with the tested groups and the controls. The results
show a clear inhibitory effect by the material, with the number of colony-forming units
developed on the samples being approximately 15% of the negative control. However,
no statistically significant difference (p= 0.879) was detected between the untreated and
treated silicon nitride samples.
J. Funct. Biomater. 2022,13, 129 11 of 14
J. Funct. Biomater. 2022, 13, x FOR PEER REVIEW 11 of 15
and, thus, immersion studies could be misleading. Furthermore, Bohner and Lemaitre [44]
also stated that the results of such assays cannot be standalone predictors of natural bone-
bonding as they can indicate false-positive or negative results. With this debate in mind,
an immersion in DPBS was utilized in this study as a first estimation of bioactive behavior,
with further studies needed to validate these results and approximate in-vivo behavior.
3.7. Optical Profilometry
Heat treating the samples led to grain growth that due to the needle-like morphology
of the β-grains of the material increased surface roughness in Figure 9. As expected, heat-
treated samples showed an almost ten-fold increase in average surface roughness com-
pared to the non-heat-treated ones. Differences between the TT and TCT groups versus
the NT and CT groups were statistically significant (p 0.001 for all). No statistically sig-
nificant differences were found between the NT and CT groups (p = 0.966), nor between
the TT and TCT groups (p = 1.0).
Figure 9. The results of the optical profilometry measurements showed a statistically significant
difference between heat-treated (TT, TCT) and non-heat-treated samples (NT, CT). Compared to the
(a) non-treated and (b) chemically treated ones, evidenced by the larger red peaks on the surface
reconstructions, the (c) thermally treated and (d) thermochemically treated samples had rougher
surfaces
3.8. Colony Forming Unit assay
The samples that were thermochemically treated showed the highest bioactivity and
were therefore selected to be compared with the non-treated ones in terms of their anti-
bacterial behavior. As the difference between the surface roughness of the two groups was
significant, samples of the TT group were tested as well in an effort to account for the
effect of the increased surface roughness. Figure 10 displays the amount of viable colony-
forming units after contact with the tested groups and the controls. The results show a
clear inhibitory effect by the material, with the number of colony-forming units developed
on the samples being approximately 15% of the negative control. However, no statistically
significant difference (p = 0.879) was detected between the untreated and treated silicon
nitride samples.
Figure 9.
The results of the optical profilometry measurements showed a statistically significant
difference between heat-treated (TT, TCT) and non-heat-treated samples (NT, CT). Compared to
the (
a
) non-treated and (
b
) chemically treated ones, evidenced by the larger red peaks on the
surface reconstructions, the (
c
) thermally treated and (
d
) thermochemically treated samples had
rougher surfaces.
J. Funct. Biomater. 2022, 13, x FOR PEER REVIEW 12 of 15
Figure 10. Top: The colonies formed on samples expressed as a percentage of the negative control.
Bottom: Indicative images of colonies formed by bacteria after being in contact with: (a) Polystyrene,
(b) Non-treated Si
3
N
4
, (c) Thermally treated Si
3
N
4
, (d) Thermochemically treated Si
3
N
4
. A clear re-
duction in bacterial population can be noted for all silicon nitride materials.
Nitrogen, through the surface groups formed by it, plays a significant role in the an-
tibacterial behavior of the material [45]. Thus, it is not surprising that all materials retained
bacteriostaticity as nitrogen was present on all of their surfaces, though in different
amounts. Again, the behavior of the thermally treated samples is interesting. XPS analysis
(Table 1) showed that the surface of these samples contains very low amounts of nitrogen.
Nevertheless, the samples did not significantly diverge from other groups in terms of their
interaction with the bacteria. This could be an indication that even surfaces with low
amounts of nitrogen can exhibit antibacterial behavior. The results of the in vitro CFU
assay are in agreement with a study by Bock et al. [15] that examined the effect of surface
modifications on the bacteriostatic properties of silicon nitride, showing that heat-treated
samples showed comparable antibacterial behavior to non-heat-treated ones. Further-
more, in a recent study, Kushan Akin et al. [46] investigated the effect of the amount of
nitrogen in oxynitride glasses on their antibacterial behavior. They found that increasing
nitrogen content did not result in enhanced antibacterial behavior, as there are a plethora
of factors governing the material-pathogen interaction. Such factors can include surface
topography and roughness, as well as surface charge and wettability.
4. Conclusions
This study successfully demonstrated a simple way through which Si
3
N
4
implants
can become more bioactive in vitro. A thermochemical surface treatment resulted in more
apatite nucleation sites without influencing the bacteriostatic nature of the surface of the
material. A higher rate of osteointegration could be especially important for spinal fusion
implants, the current main biomedical application of Si
3
N
4
, as it would improve implant
stability and increase fusion rates.
Author Contributions: Conceptualization, I.K., W.X., C.P., H.E.; methodology, I.K. and K.W.; soft-
ware, Y.Z.; validation, I.K. and Y.Z.; formal analysis, I.K. and Y.Z.; investigation, I.K.; resources,
H.E.; data curation, I.K and Y.Z.; writing—original draft preparation, I.K.; writing—review and ed-
iting, K.W., C.P., W.X. and H.E.; visualization, I.K. and Y.Z; supervision, C.P., W.X. and H.E.; project
administration, I.K.; funding acquisition, C.P. All authors have read and agreed to the published
version of the manuscript.
Figure 10.
Top: The colonies formed on samples expressed as a percentage of the negative control.
Bottom: Indicative images of colonies formed by bacteria after being in contact with: (
a
) Polystyrene,
(
b
) Non-treated Si
3
N
4
, (
c
) Thermally treated Si
3
N
4
, (
d
) Thermochemically treated Si
3
N
4
. A clear
reduction in bacterial population can be noted for all silicon nitride materials.
Nitrogen, through the surface groups formed by it, plays a significant role in the
antibacterial behavior of the material [
45
]. Thus, it is not surprising that all materials
retained bacteriostaticity as nitrogen was present on all of their surfaces, though in different
amounts. Again, the behavior of the thermally treated samples is interesting. XPS analysis
(Table 1) showed that the surface of these samples contains very low amounts of nitrogen.
Nevertheless, the samples did not significantly diverge from other groups in terms of
their interaction with the bacteria. This could be an indication that even surfaces with
low amounts of nitrogen can exhibit antibacterial behavior. The results of the
in vitro
CFU
J. Funct. Biomater. 2022,13, 129 12 of 14
assay are in agreement with a study by Bock et al. [
15
] that examined the effect of surface
modifications on the bacteriostatic properties of silicon nitride, showing that heat-treated
samples showed comparable antibacterial behavior to non-heat-treated ones. Furthermore,
in a recent study, Kushan Akin et al. [
46
] investigated the effect of the amount of nitrogen
in oxynitride glasses on their antibacterial behavior. They found that increasing nitrogen
content did not result in enhanced antibacterial behavior, as there are a plethora of factors
governing the material-pathogen interaction. Such factors can include surface topography
and roughness, as well as surface charge and wettability.
4. Conclusions
This study successfully demonstrated a simple way through which Si
3
N
4
implants
can become more bioactive
in vitro
. A thermochemical surface treatment resulted in more
apatite nucleation sites without influencing the bacteriostatic nature of the surface of the
material. A higher rate of osteointegration could be especially important for spinal fusion
implants, the current main biomedical application of Si
3
N
4
, as it would improve implant
stability and increase fusion rates.
Author Contributions:
Conceptualization, I.K., W.X., C.P., H.E.; methodology, I.K. and K.W.; soft-
ware, Y.Z.; validation, I.K. and Y.Z.; formal analysis, I.K. and Y.Z.; investigation, I.K.; resources,
H.E.; data curation, I.K and Y.Z.; writing—original draft preparation, I.K.; writing—review and
editing, K.W., C.P., W.X. and H.E.; visualization, I.K. and Y.Z; supervision, C.P., W.X. and H.E.; project
administration, I.K.; funding acquisition, C.P. All authors have read and agreed to the published
version of the manuscript.
Funding:
This project has received funding from the European Union’s Horizon 2020 research and
innovation program under the Marie Sklodowska-Curie grant agreement No 812765.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors would like to acknowledge SinTx Technologies for providing the
silicon nitride samples used in the study.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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