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Research Article
Layered Antimicrobial Selenium Nanoparticle−Calcium Phosphate
Coating on 3D Printed Scaffolds Enhanced Bone Formation in
Critical Size Defects
Cedryck Vaquette, Nathalie Bock, and Phong A. Tran*
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ABSTRACT: Preventing bacterial colonization on scaffolds while supporting tissue formation is
highly desirable in tissue engineering as bacterial infection remains a clinically significant risk to
any implanted biomaterials. Elemental selenium (Se0) nanoparticles have emerged as a promising
antimicrobial biomaterial without tissue cell toxicity, yet it remains unknown if their biological
properties are from soluble Se ions or from direct cell−nanoparticle interactions. To answer this
question, in this study, we developed a layered coating consisting of a Se nanoparticle layer
underneath a micrometer-thick, biomimetic calcium phosphate (CaP) layer. We showed, for the
first time, that the release of soluble HSe− ions from the Se nanoparticles strongly inhibited
planktonic growth and biofilm formation of key bacteria, Staphylococcus aureus. The Se-CaP
coating was found to support higher bone formation than the CaP-only coating in critical-size
calvarial defects in rats; this finding could be directly attributed to the released soluble Se ions as
the CaP layers in both groups had no detectable differences in the porous morphology, chemistry,
and release of Ca or P. The Se-CaP coating was highly versatile and applicable to various surface
chemistries as it formed through simple precipitation from aqueous solutions at room temperature and therefore could be promising
in bone regeneration scaffolds or orthopedic implant applications.
KEYWORDS: selenium, coating, antimicrobial, bone formation, calcium phosphate, scaffolds
1. INTRODUCTION
Infection control is important in tissue engineering because
bacterial infection would delay or completely inhibit tissue
regeneration. An important consideration in any antimicrobial
strategies is the potential cytotoxicity. In tissue regeneration
applications, there is a substantial need for antimicrobial
treatment that is also supportive to mammalian cell functions.
Compared to modifying the bulk biomaterials with antimicrobial agents, antimicrobial coatings of biomaterials (scaffolds,
implants) have been a particularly attractive approach due to
their versatility in antimicrobial loading and elution control
without affecting the biomaterial’s bulk properties. A
straightforward approach is coating and delivery of antimicrobials (such as antibiotics, antimicrobial peptides) and
osteogenic/osteoconductive agents (such as bone morphogenic protein-2) from tissue engineering scaffolds. Qian et al.1
coated antimicrobial silver (Ag) and collagen type I on
electrospun biodegradable polymer [poly(lactic−co−glycolic
acid)/polycaprolactone (PLGA/PCL)] scaffolds and demonstrated the antimicrobial activity of the scaffolds against
Staphylococcus aureus and Streptococcus mutans and the
increased in vitro adhesion, proliferation, and differentiation
of osteoblastic cells. The authors also used the scaffolds in a
guided tissue regeneration approach in a mouse periodontitis
model and showed significant alveolar bone regeneration. In
another study, Chen et al.2 coated three-dimensional (3D)
© 2020 American Chemical Society
PLGA scaffolds with BMP-2 and an antimicrobial peptide
(ponericin G1) using polydopamine (PDA) as a priming layer.
The authors demonstrated the enhanced release duration of
the growth factors and the antimicrobial peptide compared to
the coating without PDA and that the coated scaffolds
maintained the antimicrobial and osteogenic properties of
these agents in vitro. Another example involved the deposition
of an immunomodulatory antibiotic, azithromycin, in the
porous network of a polycaprolactone electrospun membrane.
This resulted in a prolonged antimicrobial activity and
enhanced bone regeneration in a rodent calvarial model.3 As
the task of achieving optimal antimicrobial activity and tissue
formation without cytotoxicity remains challenging, research
groups are motivated to explore new antimicrobial agents that
have low or no cytotoxicity.
Recently, there has been significant interest in selenium (Se)
as a biomaterial for antimicrobial and tissue regeneration
applications. As a trace element in the body, selenium is
Received: September 22, 2020
Accepted: November 20, 2020
Published: December 3, 2020
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important in a number of redox processes and therefore has
been suggested to play key roles in protection mechanisms
against oxidative stress. 4−6 Selenium in the form of
methylseleninic acid was shown to dampen the inflammatory
stress response of osteoblasts to breast cancer cells.7 The
specific metabolisms of Se in bone cells are still the subject of
intensive investigation, yet it has been strongly suggested that
Se is metabolized into the three major biologically active forms
(H2Se, CH3SeH, and SeMet), which exert various effects (cell
signaling, immune functions, osteoclast apoptosis) via the
mechanisms such as redox recycling or modifying protein
thiols.5
In the last several years, selenium in the form of zero
oxidation state Se0 nanoparticles has emerged as a promising
biomaterial with unique anticancer and antimicrobial activity
while having significantly lower toxicity than the commonly
used selenite form. Our group pioneered the research into the
antimicrobial properties of these Se nanoparticles and have
applied them as coatings onto a range of substrate
chemistries.8−10 Our groups and others have shown that
these nanoparticles were nontoxic to a number of mammalian
cells at concentration as high as 250 μg/mL, while effective
against several key opportunistic pathogens such as and S.
aureus, Escherichia coli at concentration as low as 16 μg/
mL.11−13 We also demonstrated that Se nanoparticle coatings
on titanium substrates significantly inhibited the biofilm
formation of methicillin-resistant Staphylococcus aureus and
methicillin-resistant Staphylococcus epidermidis in vivo,8 in
agreement with the Se nanoparticle’s anti-biofilm activity as
reported by other groups.13,14
Despite the promising properties of Se nanoparticles, it
remains largely unknown how and to what extent they can be
combined with other biomaterials to enhance bone tissue
regeneration. Although research groups have used Se to modify
biomaterials such as bioglass15,16 or chitosan,17,18 most are
focused on doping calcium phosphate materials with selenite.
Nie et al. doped selenite ions into calcium phosphate particles
before coating them with Ag nanoparticles to create a complex
nanostructure that was shown to kill several Gram-positive and
Gram-negative bacteria.19 Selenium-substituted hydroxyapatite
materials have also been developed and showed antimicrobial
and anticancer properties and nontoxicity to osteoblasts.20−22
However, no studies have investigated the in vivo bone
formation of these materials. In addition, the material
preparation methods in these studies required high processing
temperatures and therefore are not suitable for applying onto
many polymeric substrates.
In the current study, we developed a novel combination of
selenium nanoparticles and biomimetic calcium phosphate
material as a coating on 3D printed macroscale porous
scaffolds for antimicrobial bone tissue regeneration. Using
precipitation chemistry occurring at room temperature (RT),
we first deposited elemental selenium as nanoparticles onto the
surface of polycaprolactone (PCL) scaffolds before formation
of a micrometer thick CaP coating on top. This synthesis route
is mild and suitable for substrates such as PCL that melts at 60
°C. This novel design of layered coatings also allowed us to
specifically investigate the roles of eluted soluble Se ions in the
antimicrobial and bone-forming properties of Se nanoparticles.
polycaprolactone (PCL) pellets (Purasorb PC12, Mw of 120 kDa,
Purac) were heated to 100−110 °C for 30 min and then layer-by-layer
extruded through a Gauge 22 nozzle with a 0/90° laydown pattern to
create scaffolds with a dimension of 40 mm × 40 mm × 1.5 mm
having a strut-to-strut distance of 1 mm. Subsequently, 5 mm biopsy
punches were utilized to section 3D printed PCL scaffold discs, which
were of use for all testing in this study. The porosity of the scaffolds
was approximately 67%.23 A polymer film was also prepared for
certain experiments (as specified below) by casting the PCL solution
in chloroform (10% w/v) on a glass Petri dish; after the solvent
completely evaporated, a film (∼80 μm thick) was obtained.
2.2. Surface Coatings. The selenium coating was prepared using
a surface nucleation−precipitation chemistry as reported before by
our group.8,9 Briefly, the PCL scaffolds were first treated in 2M NaOH
for 30 min to enhance surface wetting. The scaffolds were then rinsed
in ultrapure water and immersed in 3 mL of 10 mM Na2SeO3 solution
to which 40 mL of 100 mM ascorbic acid was added dropwise to
induce the reduction of SeO 3 to Se 0 and formation of Se 0
nanoparticles on the PCL surface. The scaffolds were then coated
one more time with Se using the same procedure. Total Se coating
was determined by dissolving the coating in nitric acid and measuring
the Se concentration by inductively coupled plasma optical emission
spectroscopy (ICP-OES) (n = 6).
The calcium phosphate (CaP) coating was applied on PCL
scaffolds as described before.24,25 Briefly, after NaOH etching, the
scaffolds were rinsed with a copious amount of ultrapure water before
being immersed in a supersaturated simulated body fluid (10× SBF)
adjusted to pH 6 using NaHCO3 for 30 min at 37 °C. The scaffolds
were rinsed with ultrapure water and coated with CaP one more time
to obtain a homogeneous coating.
The scaffold’s morphology was analyzed using scanning electron
microscopy (Zeiss FESEM, Carl Zeiss) combined with energydispersive X-ray spectroscopy (EDX) after being gold or carboncoated, respectively. Fourier transform infrared spectroscopy (FTIR)
analysis was performed in attenuated total reflection (ATR) mode
with a KBr beam splitter (Nicolet iS50 FTIR, Thermo scientific)
using a setting of 32 scans per sample and a resolution of four
wavenumbers. CaP coatings were also detached by bending the
scaffolds in liquid nitrogen and collected on a transmission electron
microscopy (TEM) grid before being imaged on a Jeol 1400 TEM
microscope. To study the release of Ca, P, and Se from the coatings,
the scaffolds (at least three replicates) were immersed in ultrapure
water, and extracted media were collected after predetermined time
points and analyzed using ICP-OES (PerkinElmer 8300DV).
Absorption spectra of the medium from PCL-Se-CaP scaffolds were
also obtained using a spectrophotometer (xMar, Biorad) to identify
the soluble Se species.
2.3. In Vitro Antimicrobial Test. S. aureus (ATCC 29213) was
used as a model bacterium. Fresh colonies were prepared by streaking
the bacteria on a Mueller−Hinton (MH) agar and incubated
overnight. A bacterial suspension in MH broth was prepared from
several fresh colonies and its absorbance at 600 nm was adjusted to
0.1 (corresponding to McFarland 0.5 and approximately 108 CFU/
mL) before being diluted 100 times to submerge the scaffolds. PCLCaP or PCL-Se-CaP scaffolds (eight replicates) were immersed in 0.5
mL of the 100× diluted bacterial suspension and placed in an
incubator. Typically, 100 μL of the bacterial suspension was sampled
at 6 and 48 h to evaluate planktonic bacterial growth by measuring its
absorbance at 600 nm (xMark, Biorad). Biofilm formation on the
scaffolds was evaluated at 48 h after incubation, and the scaffolds
(eight replicates) were rinsed with ultrapure water and crystal violet
was used to stain the biofilm; some samples were processed for SEM
imaging of the microscopic morphology of the biofilm on the scaffolds
as described before.26
2.4. In Vitro Osteoblast Adhesion and Proliferation. Primary
human osteoblasts (hOB) were isolated from the trabecular tibial
bone from healthy donors as approved by the Ethics Committee of
the Queensland University of Technology and the Prince Charles
Hospital as described before.27 The cells were cultured in complete
αMEM culture medium supplemented with 10% fetal bovine serum
2. MATERIALS AND METHODS
2.1. Scaffold Preparation. Scaffolds were prepared using a screwextrusion 3D printer as described before. 23 Medical grade
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Figure 1. Coating characterization. Microscopic surface morphology of the 3D printed PCL scaffold surface without (a) and with (d) Se
nanoparticle coating; insets are the gross morphology of the scaffolds. Microscopic morphology of the scaffolds after CaP coatings at low and high
magnifications (b, c for PCL-CaP; e, f for PCL-Se-CaP) showing the porous CaP layer. Insets (c1) and (f1) show the characteristic Ca and P peaks
for the PCL-CaP scaffold and Ca, P, and Se peaks for the PCL-Se-CaP scaffold.
reagent were added to each well and incubated for 4 h at 37 °C and
5% CO2. Next, the plate was brought back to ambient temperature
(20 °C) for 5 min, and fluorescence (excitation 544 nm, emission 590
nm) was measured using a POLARStar OPTIMA plate reader (BMG
LABTECH).
2.5.2. Immunofluorescence Staining and Imaging. After 3 and 7
days, the media was aspirated and the samples were washed with
phosphate-buffered saline (PBS) twice before fixing with 4%
paraformaldehyde (PFA, Sigma-Aldrich) for 20 min at room
temperature (RT) and washed in PBS two times (10 min each),
before permeabilization in 0.2% Triton X-100 in PBS for 5 min at RT.
After two washes in PBS (5 min each), samples were transferred into
0.5% bovine serum albumin (BSA) in PBS for at least 10 min. Then
samples were incubated with Alexa Fluor 488 phalloidin (A12379,
Invitrogen, 1:40) and 4′,6-diamidino-2-phenylindole (DAPI, 5 μg/
mL) for 2 h. After three washes in PBS (10 min each), fresh PBS was
added. The plate was covered by foil and kept at 4 °C until imaging.
Imaging was done using a Nikon spectral spinning disc confocal
microscope (SDC, X-1 Yokogawa spinning disc with Borealis
modification) fitted with a Plan Fluor ELWD 20× objective. Images
are shown as maximum projections following z-stack imaging (25 μm,
2 μm step size).
2.6. In Vivo Experiments. A guided bone regeneration model was
used, in which the macroscale porous scaffolds (n = 6 for each group
PCL-CaP and PCL-Se-CaP) were placed into calvarial defects that
were protected from soft tissue infiltration using occlusive
membranes.3 Ethic approval was obtained from the University
Animal Ethics Committee (UAEC) of Queensland University of
Technology (approval number 16−821). The 5 mm diameter
scaffolds were sterilized by 70% ethanol for 1 h and UV-irradiated
for 20 min on each side. Six Sprague-Dawley (SD) male rats (the
Animal Resources Centre, WA, Australia) were used to create a 5 mm
calvarial defect on each side of the skull midline, which was then fitted
with a scaffold as described before.3 Briefly, this guided bone
regeneration model was formed using an occlusive membrane (made
of PCL by electrospinning, ∼0.5 mm thick, mean pore size of 0.5−1
μm, 7−8 mm in diameter), which was placed on top of the exposed
dura mater to prevent soft tissue infiltration. Afterward, the scaffolds
were gently press-fitted into the defects, another occlusive membrane
was used to cover the scaffold, and the defect before the wound was
closed in the layer using Vicryl 5.0/4.0 resorbable sutures (Ethicon,
Germany). The animals were sacrificed at week 8 after implantation
and the calvaria collected, fixed in 4% paraformaldehyde for 24 h at 4
°C, and stored in PBS at 4 °C.
μ-CT scanning (μ-CT40 Scanco Medical AG, Switzerland) was
used to evaluate new bone formation within the defect using the
setting of 55 kWp, 145 μA, 8W, and a voxel size of 30 μm. Samples
(FBS) and 1% penicillin and streptomycin (PS) (Sigma-Aldrich) and
used at a passage number between 2 and 5 to seed onto 1.5 mm
(height) × 5 mm (diameter) scaffolds at a density of 10 000 cells per
scaffold. The cell-seeded scaffolds were cultured in complete αMEM
culture medium in a CO2 incubator (37 °C, 5% CO2), and the
medium was changed every 2 days. Cell adhesion was evaluated at 4 h
post-seeding by nuclei (4′,6-diamidino-2-phenylindole, DAPI)
staining and imaging with a confocal fluorescence microscope,
followed by counting the number of cells in the images using ImageJ.
Cell proliferation was evaluated by DNA quantification at day 3 and
day 7 of culture using a PicoGreen assay following the manufacturer’s
instruction. At least six independent samples were used for each
experiment.
2.5. In Vitro Osteogenesis of Mesenchymal Stem Cells
(MSCs). In this experiment, two-dimensional (2D) film samples were
used to avoid the low seeding efficacy of the macroscale porous 3D
printed scaffolds.28 Samples of diameter 5 mm were sectioned using a
biopsy punch from the PCL film and sterilized by ethanol 80% and
UV irradiation. The coated samples were placed in 96-well plates and
seeded with bone marrow MSCs at a density of 20 000 cells/cm2 in
100 μL of Dulbecco’s modified Eagle’s medium (DMEM). The plates
were kept in a CO2 incubator (37 °C, 5% CO2) for 3 and 7 days, and
the medium was changed every 2 days. Two sets of samples were
used: one set was cultured in basal medium (DMEM supplemented
with 12% FBS and 1% P/S) and the other set with osteogenic
medium (DMEM supplemented with 12% FBS, 1% P/S, ascorbic acid
0.17 mM, dexamethasone 100 nM, and β-glycerophosphate 10 mM).
2.5.1. Alkaline Phosphatase (ALP) Activity. Alkaline phosphatase
(ALP) activity was measured from the media at 7 days, after a 24 h
release period as follows. Briefly, samples (8 replicates) grown with
MSCs for 7 days were incubated in DMEM medium without phenol
red two times (10 min each). Samples were then transferred to a new
96-well plate, and 220 μL of this medium was added before the
samples were placed back in the incubator for 24 h. The ALP activity
was measured using the SigmaFASTTM kit (Sigma, Australia), as per
the manufacturer’s instructions. A volume of 100 μL of p-nitrophenyl
phosphate in Tris-base buffer was added to 100 μL of the culture
medium in a 96-well plate (two technical replicates per sample) and
incubated at 37 °C and 5% CO2 for another 24 h. At the end of the
second incubation period, the plate was brought back to ambient
temperature (20 °C) for 5 min and the absorbance was read at 405
nm using a plate reader (Benchmark Plus microplate spectrophotometer, BIO-RAD). The signal of the control medium was
subtracted from those of all samples. The ALP absorbance was
further normalized to the metabolic activity taken at the same time
point (7 days), as measured by a Prestoblue assay (Invitrogen,
Australia). Briefly, 90 μL of DMEM medium and 10 μL of Prestoblue
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Figure 2. FTIR and TEM analysis of CaP coatings showing similar chemical groups (a) and multicrystalline structures (b1−c2) on PCL-CaP and
PCL-Se-CaP.
were then decalcified, paraffin-embedded, sectioned, deparaffinized,
and stained with hematoxylin−eosin (H&E) and Masson’s trichrome.
Histomorphometry analysis (Osteometrics) was used to measure the
new bone area on at least three representative tissue sections per
animal, and each was at least 10 sections apart from each other.
Immunohistochemistry was also implemented as described before.23
Data were reported as mean ± standard error of means (SEM), and
the difference was analyzed using the Student’s t-test.
The gross morphology of the PCL scaffold after coating with
Se nanoparticles clearly showed the characteristic reddish color
of amorphous selenium (inset in (d)).9,11,29,30 Calcium
phosphate (CaP) coating was then applied using a
precipitation method from a highly concentrated simulated
body fluid solution, 10× SBF. The CaP coating appeared as a
micrometer-thick porous layer (1.6 ± 0.2 μm for PCL-CaP
samples and 1.7 ± 0.2 μm for PCL-Se-CaP samples) consisting
of vertically oriented plates, which are typical morphological
features for CaP formed through precipitation from supersaturated solutions25,31 (Figures 1c,f and S1c,d in the
Supporting Information). The random large pores on the
strut surfaces (Figure 1b,e) were the gaps between PCL
spherulites created during 3D printing. EDX analysis
confirmed the Ca−P chemistry of the coatings and the peaks
of Se in the PCL-Se-CaP samples (insets of Figure 1c1,f1).
3. RESULTS
3.1. Selenium−Calcium Phosphate Coating. Selenium
nanoparticles were first deposited onto the scaffold surface
using an in situ precipitation method where selenite salt was
reduced to generate surface-bound elemental selenium nanoparticles.8,9 The particles were about 70−120 nm in diameter
with some aggregates of larger size (∼200−600 nm) (Figure
1d) and of zero oxidation state (i.e., elemental Se0) as expected
from previously reported studies11 (Figure S1a).
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FTIR and TEM were used to compare the CaP coatings in
the two groups. FTIR spectra of the CaP coating formed
directly on PCL and on Se-coated PCL exhibited the same
phosphate group peak location (562 and 601 cm−1), suggesting
no chemical bonding with the PCL or Se underneath (Figure
2a). TEM analysis showed clearly the platelike structures of the
CaP coating (Figure 2b1,c1). Combined with selected area
electron diffraction (SAED, Figure 2b2,c2), this TEM analysis
indicated that the CaP in both groups was composed of
randomly oriented crystalline CaP plates.
Next, we analyzed the release of the key elements Ca, P, and
Se from the coatings into ultrapure water. Overall, the release
of Ca and P was consistently similar between PCL-CaP and
PCL-Se-CaP scaffolds over the testing period of 3 weeks
(Figure 3a,b). Together with the spectroscopy data above, this
release result strongly suggested the same chemical properties
of the CaP layers in both groups.
CaP coating, which allows us to specifically evaluate the
inhibitory effects of the soluble Se ions released from the
coating. The coated scaffolds were challenged with S. aureus by
immersing the scaffolds in a suspension of bacteria. These are
among the most found bacteria in infected implants or bones.
The bacterial growth in the suspension was determined by
measuring its absorbance at 600 nm and the biofilm formation
on the scaffold surface determined by measuring the staining of
crystal violet. The bacterial growth was significantly lower
when treated with PCL-Se-CaP samples compared to PCLCaP samples at 6 h of incubation (Figure 4c). Importantly, the
bacteria growth appeared to be completely inhibited on PCLSe-CaP samples as the absorbance did not increase at 48 h
after incubation.
The biofilm formation was also inhibited on the PCL-SeCaP samples as indicated from the significantly lower staining
with crystal violet. Microscopically, the biofilm was clearly
present as a dense layer on the PCL-CaP scaffolds while only
small patches on the PCL-Se-CaP scaffolds (Figure 4a,b). The
antimicrobial effects were consistent with the results of Se
release from the PCL-Se-CaP samples (Figure 3c,d), which
showed a strong release at day 1 (ca. 0.25ug/mg scaffold) and
a continuous release afterward. The release of Se was possible
because of the porous nature of the top CaP layer.
3.3. In Vitro Mammalian Cell Culture. We next
investigated the adhesion and proliferation of bone-forming
cells, osteoblasts on the coated scaffolds. Human primary
osteoblast (hOB) cells were seeded onto the scaffolds, and cell
adhesion was evaluated at 4 h (by DAPI staining and imaging)
and cell proliferation assessed on days 3 and 7 (by measuring
total cellular DNA content using a Picogreen assay). Cell
adhesion was found to be similar in both groups (Figure 5a−
c).
In terms of cell proliferation, DNA content measurements
indicated that there was no significant difference between
PCL-CaP and PCL-Se-CaP groups (compared within the same
time point) and that the cells appeared proliferating normally
as evident from the increase in DNA content from day 3 to day
7 in each group. Similar cellular adhesion and proliferation
were expected because of the similarity in the CaP layers
described above.
Next, we investigated the osteogenesis of mesenchymal stem
cells (MSCs) on the coatings. The cells were cultured on the
coatings for 3 and 7 days in basal culture media and osteogenic
media, and their alkaline phosphatase activity and morphology
were investigated. MSC cultured on PCL-Se-CaP samples
exhibited larger cell areas in basal media (Figure 6a,b,e,f) with
the characteristic elongated spindlelike morphology on day 3,
which clearly became a more cuboidal morphology of
osteoblasts at day 7. Similar morphological changes were also
observed in osteogenic media (Figure 6c,d,g,h).
The cells on PCL-Se-CaP samples showed significantly
higher alkaline phosphate (ALP) activity than those on PCLCaP in basal media (p < 0.05). In osteogenic media, no
significant difference in ALP activity was found (Figure 6i).
3.4. In Vivo Study. The scaffolds were implanted into
critical-size calvarial defects in rats to investigate bone
formation. After the animals were sacrificed at week 8, the
specimens were retrieved, fixed, and analyzed for new bone
formation. μCT scanning showed strong bone formation in
both scaffold groups with new bone appearing to form from
the defect edge toward its center (Figure 7a,b,e,f).
Figure 3. Ca, P, and Se release from the PCL-CaP and PCL-Se-CaP
scaffolds measured by ICP-OES. The two groups showed similar
release of Ca and P (a, b). Se was found to release continuously over
the 3-week testing period (c). Extracted medium from PCL-Se-CaP
scaffolds showed absorption at 250 nm (d, d1), characteristic of HSe−
ions.32
The release of Ca and P was also relatively monotonic at the
levels of about 0.07 μg/mg/day for Ca and 0.3 μg/mg/day for
P over the course of 3-week immersion in ultrapure water. Se
also exhibited similar monotonic, relatively linear (R2 = 0.95)
release except at a much lower rate of approximately 0.02 μg/
mg/day. Considering the total Se loading of 4.1 ± 0.5 μg/mg
scaffold (average ± standard deviation, n = 6), the cumulative
release of Se was only about 15% in the first 3 weeks. It is,
therefore, expected that a complete Se release will only occur
after about 6 months. Absorption spectra of the extracted
medium showed a characteristic absorption of HSe− at around
250 nm32 (Figure 3d,d1), which was not present in a freshly
prepared Se nanoparticle suspension (Figure S1b,b1).
3.2. In Vitro Bacterial Studies. Se nanoparticle
suspension and Se nanoparticle coatings have been previously
demonstrated by our groups and others to strongly inhibit the
growth and colonization of key bacteria such as S. aureus and S.
epidermidis.8,10−12,14,33 Therefore, in this study, we studied the
antimicrobial effects of this coating when combined with the
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Figure 4. In vitro antibacterial properties. (a, b) SEM images of scaffold surfaces after 48 h of culture with S. aureus showing extensive biofilm
formation on PCL-CaP scaffolds compared to PCL-Se-CaP scaffolds. (c) Growth of planktonic bacteria was significantly inhibited in the presence
of PCL-Se-CaP scaffolds. (d) Quantitative comparison using crystal violet staining showed significant lower biofilm formation on PCL-Se-CaP.
Figure 5. In vitro osteoblast adhesion and proliferation were found to be similar on PCL-CaP and PCL-Se-CaP. (a, b, c) Fluorescence images of
DAPI-stained scaffolds. (d) Quantification of DNA content in cells on the scaffolds using a Picogreen assay.
Measurements using μCT showed a significantly higher new
bone volume in the PCL-Se-CaP groups (Figure 7c, p = 0.04).
The bone volumes were calculated to be approximately 40−
50% of the defect volume. Bone mineral density determined by
thresholding intensity in μCT scans against a hydroxyapatite
standard showed no significant differences between the two
groups, indicating a somehow similar level of bone maturity
(Figure 7d). Analysis of tissue sections showed clearly the new
bone formation in the macroscale pore of the scaffolds in both
groups (Figures 7e,f and S2). The new bone formed a tight
bond with the CaP layer, which appeared to retain some of its
original thickness, indicating that the CaP coating had not fully
resorbed during the 8-week implantation as expected with
crystalline calcium phosphate materials (Figure S2). The
morphology of the new bone appeared less dense and under
remodeling compared to the resident host bone outside the
defect area (Figure 7e,f). Bone fill (reported as % of defect
area) measured from the tissue sections showed a significant
increase (p < 0.05) in the PCL-Se-CaP group (Figure 7g).
Analysis of fibrous tissue area within the defect region also
showed a significantly lower value in the PCL-Se-CaP group
(Figure 7h, p < 0.05). New bone formation was also validated
by Masson’s trichrome staining (Figure 7i,j) and immunohistochemistry (Figure S3).
4. DISCUSSION
As bacterial infection to implanted biomaterials remains a
clinically significant risk, there is a strong research interest in
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Figure 6. In vitro osteogenesis experiments on PCL-CaP and PCL-Se-CaP. (a−h) Fluorescence imaging of MSCs on the coatings. (i)
Quantification of alkaline phosphatase activity (*p < 0.05).
Figure 7. Guided bone regeneration in vivo. Scaffolds were fit to critically sized defects (5 mm in diameter) and sandwiched between two occlusive
membranes to investigate bone formation originating from the defect edges. μCT scanning (a, b) allowed for measurement of new bone volume
and bone mineral density (c, d). Histological analysis on H&E-stained tissue sections (e, f) showed a new bone within the macroscale pores (white
areas are the void after the polymer dissolved during tissue embedding) and the quantification of bone and fibrous tissue areas (g, h). Masson’s
trichrome staining highlights the new bone (red) and collagen (blue) around the scaffolds (i, j).
suggested that antimicrobial surfaces should be designed to
actively kill bacteria in the surrounding environment through
antimicrobial agent release. In the current study, we aimed to
develop a “bifunctional” coating for applications in bone tissue
regeneration that had a top layer for stimulating osteoblast cell
functions and a bottom layer for releasing an antimicrobial
agent.
multifunctional coatings that can impart both antimicrobial
properties and enhanced tissue regeneration capacity. Recently,
we used a surface that supported osteoblastic cell adhesion
while repelling S. aureus and demonstrated in a coculture that
the bacteria were more prone to the osteoblastic cells rather
than on the biomaterial surface and formed a biofilm-like
community that was resistant to gentamicin.34 We thus
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We created a layered coating that has Se nanoparticle
coating underneath a micrometer-thick biomimetic CaP
coating on 3D printed scaffolds. This innovative design
allowed us to examine specifically the effects of the released
soluble Se ions on bacteria and bone-forming cells as these
cells were never in direct contact with the Se nanoparticles. We
used 3D printed polycaprolactone (PCL) as a model scaffold
because of PCL’s slow degradation (∼2−3 years for complete
degradation at physiological pH) and its well-investigated
properties in the regeneration of tissues including bones.35
Calcium phosphate (CaP) coating was formed on top of the Se
nanoparticle coating via in situ precipitation from a supersaturated 10× SBF solution. This CaP coating has been
routinely used as a biomimetic coating to a number of implant
surfaces and bone tissue engineering scaffolds.25 With this
design, the CaP coating provided a biomimetic, microscopically rough surface for interacting with osteoblasts and its
porous structure allowed the release of antimicrobial ions from
underneath.
The Se nanoparticle coating underneath appeared to have
no detectable effects on the microscopic morphology or the
chemical properties of the CaP coating on top based on the
results from SEM, FTIR, and TEM analyses. Previous studies
by our group have shown that Se nanoparticles have a small
negative charge;11 thus it could be that the slightly charged Se
coating was quickly neutralized by the excess of counterions in
the high ionic strength 10× SBF solution as previously
suggested.36 The CaP coating therefore showed no significant
morphological and compositional differences when formed on
the PCL surface or on the Se-coated PCL surface. The
similarity in the CaP coating layers in both groups then
explained the observed similar adhesion and proliferation of
osteoblasts.
The inhibition of S. aureus biofilm formation on the PCL-SeCaP coating and planktonic bacterial growth strongly
suggested that these effects were a result of the released
soluble Se species. This result was supported by the ICP results
that showed a significant release of Se in the first day and a
steady release over 3 weeks. Our group and others have shown
that the selenium nanoparticles prepared using the same
reduction−precipitation method were amorphous11,37 and thus
their relatively high release at physiological pH (−log K0 ∼
6)32 could be attributed to their gradual dissolution. Despite
the initial high release of Se, the total release of this ion was
only 15% after 3 weeks, suggesting that the antibacterial
activity could be sustained for a prolonged time period.
The in vivo model used in our study was chosen to evaluate
specifically the bone formation induced by the selective cellular
migration from the bone and vasculature located at the edge of
the defect into the implanted scaffolds. The membranes on top
and at the bottom of the scaffolds were used as a barrier
occlusive to cellular migration and tissue infiltration according
to the guided bone regeneration principles.38 Upon implantation into the defects, blood clots would form onto the
scaffolds, establishing a temporary matrix for cellular migration
from the defect edge. The scaffolds in our study had an
interconnected macroscale pore network created by the layerby-layer additive manufacturing methodology with the smallest
pore size of approximately 0.3−0.4 mm. In vivo, blood would
readily infiltrate into this network upon scaffold implantation
and form clots that allowed cell migration to the center of the
scaffolds. The in vivo results of bone formation from the defect
edge were in good agreement with this.
In vivo results showed an increase in bone formation in the
PCL-Se-CaP groups compared to the PCL-CaP groups. The
scaffolds in both groups had the same architecture and
macroscale 3D porous morphology. The CaP layers in both
groups presented similar surface chemistry and microscale
morphology to the osteoblasts. The different bone formation
level observed could then only be attributed to the release of
Se ions from the Se0 nanoparticle coating through the pores in
the CaP layer. It is established in the literature that Se is
important in several redox enzymes in mammalian cells
including osteoblasts.4−7 In the current study, soluble Se
ions were shown to release through the porous CaP coating in
the form of HSe− and therefore could be attributed to the
observed higher bone formation in vivo, likely through
mechanisms related to osteoblast differentiation or metabolism. The enhanced bone formation in vivo is also supported by
the in vitro results that suggested higher osteogenesis of MSCs
when grown on the Se-CaP coating. The osteogenic properties
of Se have been reported by other groups,39−42 which showed
the increased expression of osteogenesis genes (e.g., alkaline
phosphatase, osteocalcin, and collagen-I) and osteogenesis
activity with Se treatment.
The bone formation in our antimicrobial scaffolds was
similar compared to that in other, albeit a small number of
studies using antimicrobial scaffolds. For example, Qian et al.1
demonstrated in vivo bone formation for Ag-coated electrospun scaffolds with a bone volume/total volume of about 35%
(compared to about 40% in our current study). It should also
be noted that our study used a different animal model and
longer time point (8 vs 6 weeks), but our study also used Se
nanoparticles that have much lower mammalian cell toxicity
compared to Ag nanoparticles.8,11 Another interesting study
concerning antimicrobial tissue regenerating scaffolds is by
Mathew et al.3 who loaded azithromycin onto calcium
phosphate-coated electrospun membranes and tested bone
formation in a skull defect model similar to the one in our
current study. They showed nearly complete bone filling of
some of the defects at 8 weeks and attributed this result to the
immunomodulatory effects of the antibiotic. Their antibiotic
coating was based on physical adsorption and surface tension
caused by the microscale pores (e.g., in their electrospun
membranes or in a new type of macroscale porous scaffold that
has microscale intra-strut pores reported by Dang et al.43,44 or
Visscher et al.45). In comparison, the surface modification in
this present study is based on surface-induced nucleation and
surface-bound growth of particles in solution and therefore
could be applied to even scaffolds of large macroscale porosity
as demonstrated in the current study.
Compared to other studies that use different scaffold designs
or scaffolds such as those using cell constructs for bone
regeneration, the bone formation in our animal study was also
comparable. For example, mesenchymal stem cell (MSC)
sheets implanted in a calvarial defect in rabbits showed 20−
30% of the defect volume filled with a new bone at 8 weeks46
or 10−20% in a rat femur defect model by scaffolds derived
from a cell-seeded decalcified bone matrix47 and 15−25% in
periodontal defects in sheep at 10 weeks by a multiphasic 3D
construct that deliver in vitro matured cell sheets.48 A study by
Probst et al. reported roughly 40% filling of mandibular defect
volume after 12 weeks in minipigs using tri-calcium phosphate
(TCP)-poly(D,L-lactide-co-glycolide) scaffolds seeded with
MSC.49
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Our findings of enhanced bone formation in vivo in Secoated scaffolds were also consistent with a study by Wang et
al.50 who developed a complex composite of selenite−
hydroxyapatite−chitosan. They showed that the composite
generated more bone than the control without selenite in rat
calvarial defects. Even though these authors used a similar
model and reached a similar conclusion, their composite with
SeO3− ions was fundamentally different from our coating that
has a much lower solubility of the zero oxidation state Se0.
Importantly, the Se-CaP material in our study was designed as
a coating and the versatile surface nucleation and precipitation
synthesis could be readily applied to a range of scaffold
materials or implants. We have demonstrated that Se coatings
can be applied on titanium, stainless steel, glass, and polymers
(PVC, PU, silicone),8,9,51 and CaP coating from 10× SBF has
been shown to form on a range of surface chemistries.25,36,52 In
our labs, we have successfully applied the Se-CaP layered
coating on titanium, chitosan, and PLGA substrates and are in
the process of in-depth testing of its effects on tissue cells and
bacteria. This coating is envisioned to be applicable to a
number of scaffolds or implant materials and to enhance their
antimicrobial properties and support bone formation.
The main limitation of our study is that the antimicrobial
efficacy was tested only in vitro. Despite the fact that selenium
coatings on implant surfaces had been shown to inhibit biofilm
formation of methicillin-resistant S. aureus (MRSA) and
methicillin-resistant S. epidermidis (MRSE) in vivo,8 the
selenium−CaP coating in our current study needs to be
further tested for its antimicrobial activity in vivo using a model
that has bacterial challenge during tissue regeneration. Future
work should also focus on assessing bone formation in such a
model to correlate successful tissue regeneration and effective
infection control.
Engineering Group, Queensland University of Technology,
Brisbane, QLD 4000, Australia; orcid.org/0000-00032820-7399; Email: phong.tran@qut.edu.au
Authors
Cedryck Vaquette − School of Dentistry, The University of
Queensland, Herston, QLD 4059, Australia; orcid.org/
0000-0001-7937-4432
Nathalie Bock − School of Biomedical Sciences, Faculty of
Health, Queensland University of Technology, Brisbane, QLD
4059, Australia; Translational Research Institute,
Woolloongabba, QLD 4102, Australia
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsami.0c17017
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
P.A.T. acknowledges the support from his Advance Queensland Research Fellowship, C.V. and P.A.T. acknowledge the
support from QUT’s Vice-Chancellor Research Fellowship and
Australian Dental Research Foundation. N.B. acknowledges
the TRI microscopy facility. The Translational Research
Institute is supported by grants from the Australian Government. Dr. Hoang Phuc Dang is acknowledged for his assistance
with animal surgeries, and Thao Tran is acknowledged for her
assistance with ICP-OES and histological analysis.
■
ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsami.0c17017.
Further physicochemical characterization of the coatings;
electron microscopy analysis of resin-embedded tissue
sections; and validation of new bone formation via
immunohistochemistry staining (PDF)
■
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5. CONCLUSIONS
In this study, we developed a novel layered coating consisting
of a Se nanoparticle coating underneath a porous CaP layer
and applied it on 3D printed scaffolds for antimicrobial bone
tissue engineering applications. Using this innovative design,
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■
Research Article
AUTHOR INFORMATION
Corresponding Author
Phong A. Tran − Centre for Biomedical Technologies,
Queensland University of Technology (QUT), Brisbane,
QLD 4000, Australia; School of Mechanical, Medical and
Process Engineering, Interface Science and Materials
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