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Citation: Hassan, M.; Khaleel, A.;
Karam, S.M.; Al-Marzouqi, A.H.; ur
Rehman, I.; Mohsin, S. Bacterial
Inhibition and Osteogenic Potentials
of Sr/Zn Co-Doped
Nano-Hydroxyapatite-PLGA
Composite Scaffold for Bone Tissue
Engineering Applications. Polymers
2023,15, 1370. https://doi.org/
10.3390/polym15061370
Academic Editors: Ju Fang and
Huijie Zhang
Received: 18 January 2023
Revised: 8 February 2023
Accepted: 12 February 2023
Published: 9 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
polymers
Article
Bacterial Inhibition and Osteogenic Potentials of Sr/Zn
Co-Doped Nano-Hydroxyapatite-PLGA Composite Scaffold for
Bone Tissue Engineering Applications
Mozan Hassan 1, Abbas Khaleel 2, Sherif Mohamed Karam 1, Ali Hassan Al-Marzouqi 3,
Ihtesham ur Rehman 4and Sahar Mohsin 1, *
1Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University,
Al Ain P.O. Box 15551, United Arab Emirates
2Department of Chemistry, College of Science, United Arab Emirates University,
Al Ain P.O. Box 15551, United Arab Emirates
3Department of Chemical and Petroleum Engineering, College of Engineering, United Arab Emirates
University, Al Ain P.O. Box 15551, United Arab Emirates
4School of Medicine, University of Central Lancashire, Preston PR1 2HE, UK
*Correspondence: smohsin@uaeu.ac.ae; Tel.: +971-3-713-7516
Abstract:
Bacterial infection associated with bone grafts is one of the major challenges that can
lead to implant failure. Treatment of these infections is a costly endeavor; therefore, an ideal bone
scaffold should merge both biocompatibility and antibacterial activity. Antibiotic-impregnated
scaffolds may prevent bacterial colonization but exacerbate the global antibiotic resistance problem.
Recent approaches combined scaffolds with metal ions that have antimicrobial properties. In our
study, a unique strontium/zinc (Sr/Zn) co-doped nanohydroxyapatite (nHAp) and Poly (lactic-co-
glycolic acid) -(PLGA) composite scaffold was fabricated using a chemical precipitation method
with different ratios of Sr/Zn ions (1%, 2.5%, and 4%). The scaffolds’ antibacterial activity against
Staphylococcus aureus were evaluated by counting bacterial colony-forming unit (CFU) numbers after
direct contact with the scaffolds. The results showed a dose-dependent reduction in CFU numbers
as the Zn concentration increased, with 4% Zn showing the best antibacterial properties of all the
Zn-containing scaffolds. PLGA incorporation in Sr/Zn-nHAp did not affect the Zn antibacterial
activity and the 4% Sr/Zn-nHAp-PLGA scaffold showed a 99.7% bacterial growth inhibition. MTT (3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay showed that Sr/Zn
co-doping supported osteoblast cell proliferation with no apparent cytotoxicity and the highest doping
percentage in the 4% Sr/Zn-nHAp-PLGA was found to be ideal for cell growth. In conclusion, these
findings demonstrate the potential for a 4% Sr/Zn-nHAp-PLGA scaffold with enhanced antibacterial
activity and cytocompatibility as a suitable candidate for bone regeneration.
Keywords:
strontium; zinc; nano-hydroxyapatite; PLGA; antibacterial; bone scaffolds; cell proliferation
1. Introduction
During a lifetime, skeletal bones are subjected to massive physical stress that can
induce bone remodeling and affect bone function and structure [
1
]. To withstand this
physical stress, the bone is a highly dynamic organ composed of ~60% inorganic matter,
~30% organic matter, and ~10% water [
2
]. The inorganic phase is represented mainly
by calcium phosphate present in the form of hydroxyapatite (HA) crystals permeated
through the gaps of the parallelly oriented type I collagen fibers which represent the
organic extracellular matrix. HA is responsible for bone stiffness, while collagen provides
elasticity and tensile strength [3].
Bone fractures have been imitatively restored using autogenous bone grafts which can
avoid immune rejection but have shown numerous drawbacks such as pain, long recovery
Polymers 2023,15, 1370. https://doi.org/10.3390/polym15061370 https://www.mdpi.com/journal/polymers
Polymers 2023,15, 1370 2 of 18
periods, infections, and limited quantity. Another possible treatment for bone defects is
allogenic grafts and xenografts, but their use has been limited due to biological issues such
as immune rejection and post-implantation infections, not to mention ethical issues [
4
].
Nowadays, the whole globe is facing a growing demand for bone grafts due to the increase
in the numbers of the elderly population, with over 2 million bone replacement surgeries
performed per annum [5].
Bone tissue engineering (BTE) using synthetic bone substitutes has been investigated
extensively as a promising approach for the treatment of bone disorders. The approach is
anticipated to fully supersede the currently used bone grafts in clinical applications and
augment bone repair and regeneration [
6
]. To achieve this, the grafted biomaterial should
fulfill certain criteria such as (i) biocompatibility, (ii) biodegradability that is tailored to the
new bone formation, (iii) the provision of proper mechanical support similar to the injured
bone, (iv) the enablement of cell attachment and vascularization, and (v) the promotion
of osteogenesis and allowance of osteointegration [7–10]. Biomaterials of different origins
have been used to bridge the gap and restore the strength of weak, broken, or deficient
bones. These include natural polymers [11], synthetic polymers [12], and ceramics [13].
Synthetic calcium phosphate scaffolds, mainly in form of HA, are widely used in BTE
due to their osteoconductivity and bioactivity. HA is a natural bone component known to
enhance cell attachment due to its ability to adsorb more cell-adhesive plasma proteins,
such as fibronectin and vitronectin, resulting in more adherent cells [
14
]. However, its
sole use has been limited due to its poor mechanical properties [
15
]. Recent technologies
have adopted synthetic calcium-phosphate-dependent scaffolds that are loaded with bi-
ological compounds such as growth factors or drugs to enhance bone formation. The
main challenge in these scaffolds includes drug/growth factor low solubility, effective
dose assessment, short half-life, and side effects such as ectopic bone formation [
16
]. A
prospective safer approach that has recently been investigated is the addition of natural
bone trace elements such as magnesium, manganese, zinc, and strontium ions (Mg
2+
, Mn
2+
,
Zn
2+
, and Sr
2+
) that can naturally induce growth factor production by cells to promote
osteogenesis [
17
]. Additionally, these elements can improve scaffold biological responses
and physical properties [18].
Zinc (Zn) is the most abundant ion in bones and is widely used to substitute Ca
2+
in HA [
19
]. Zn
2+
is also known to play a key role in the immune system and participate
in numerous anabolic and catabolic activities that help maintain cellular integrity. In
addition, Zn plays an essential role in transcription and gene expression pathways [
20
].
Many studies demonstrated that zinc could promote bone formation and enhance the
expression of osteoblastic gene markers, as well as inhibit osteoclast bone resorption,
although the full pathway remains to be disclosed [
21
]. Yamaguchi et al. proposed that
zinc can potentially act as an NF-
κ
B (nuclear factor kappa light chain enhancer of activated
B cells) pathway antagonist in both osteoblasts and osteoclasts, decreasing both bone
resorption and formation; the negative effect on bone formation will later be modulated
by activating the Smad (Suppressor of Mothers against Decapentaplegic) pathway that
plays a critical role in osteoblastic lineage commitment and proliferation, hence increasing
bone mineralization [
22
]. Furthermore, Grandjean-Laquerriere et al. showed that zinc can
increase the production of anti-inflammatory cytokines such as interleukin (IL) IL-10 and
IL-8, simultaneously decreasing the production of inflammatory cytokines such as TNF-
α
(tumor necrosis factor alpha) [
23
]. In addition, Zn
2+
is also known to have antibacterial
properties that have been reported in many studies [24–27].
Zn’s antibacterial property is very important as bacterial growth at the implantation
site is one of the major failures in the bone grafting and healing process. The infection is
mainly caused by bacterial strains that can form a biofilm and produce an extracellular
polymeric layer that can help bacteria to survive and escape the immune system, as well
as antimicrobial agents [
28
]. A total of 75% of post-implant infections are caused by the
pathogen Staphylococcus aureus (S. aureus) which can colonize asymptomatically, resulting
in a life-threatening disease and implant failure [
29
]. To overcome the post-implantation
Polymers 2023,15, 1370 3 of 18
bacterial infection, recent studies have investigated physical antibacterial mechanisms
using surface coats that can inhibit bacterial adhesion and growth, such as nanopillars
which can stick to the bacterial cell membrane, rupturing it and eventually preventing
biofilm formation [
30
]. However, the use of this method has been limited due to the high
temperature, pressure, and electrical energy techniques that are adopted in the synthesis
of these surfaces which can interfere with polymers’ physical properties [
31
]. In contrast,
several studies investigated combining the scaffold with chemical antimicrobial agents such
as antibiotics and metal ions (e.g., zinc, silver, and copper) [
32
], with zinc ions showing
preferable cell differentiation and antimicrobial properties [33].
Despite all of zinc’sadvantages, many studies showed that higher Zn
2+
concentra-
tions are cytotoxic to cells and can lead to the accumulation of zinc ions in mitochondria
leading to functional impairment and cell apoptosis [
34
]. Guo et al. showed that cells
exposed to
10 µg/mL
of zinc oxide for 4 h can reduce cell viability to less than 30% [
35
]. In
addition, Wang et al. reported that higher Zn
2+
concentrations of more than 5% showed
a cytotoxic effect on human bone marrow mesenchymal stem cell (hBMSCs) cultures,
with 10% Zn showing reduced alkaline phosphatase (ALP) activity compared to lower Zn
concentrations [36].
Strontium (Sr) is a divalent cation that has chemical properties analogous to calcium
and can be processed by the body in a similar way to calcium [
37
]. Sr has a dual effect on
both osteoblasts and osteoclasts. In osteoblasts, Sr
2+
binds to Ca
2+
receptors and enhances
cell proliferation and bone formation. Concurrently, in osteoclasts, strontium ions (Sr
2+)
can induce conformational changes in the Ca
2+
receptors, resulting in cell apoptosis and
inhibited bone resorption [
38
]. Chattopadhyay et al. demonstrated that Sr
2+
could bind
to Ca
2+
-sensing receptors and act as an agonist to activate several cellular responses that
increase the expression of rat osteoblastic genes and induce osteoblast proliferation [
39
].
Furthermore, Sr
2+
has been used as a surrogate for Ca
2+
remedies to treat bone disorders
and prevent bone fractures. Sr-ranelate (SrR) is widely used as an effective treatment for
osteoporosis and found to enhance bone formation and increase overall bone quality and
mineral density [
40
]. Despite all these advantages, prolonged oral administration of SrR
was found to increase the risk of a cardiovascular infarction, and many studies proved
that local administration of Sr
2+
is more beneficial [
41
–
43
]. Recently, with the emergence of
BTE and biomaterials in the treatment of bone diseases, Sr-enriched biomaterials showed
an advantageous effect that allowed local Sr delivery, reducing the side effects [
44
]. Sr
2+
has been introduced with Ca
2+
phosphate biomaterials due to their resemblance to natural
bone and the easiness of Sr incorporation into their structure [
45
]. Additionally, a Sr-HA
scaffold showed higher ALP activity
in vitro
compared to pure HA, while
in vivo
animal
implantation enhanced osteogenesis [46].
Polymeric nanoparticles that possess antimicrobial properties such as silver, zirconia,
zinc oxide, etc., have been introduced to medical devices in the field of dental prosthetics
and periodontal diseases. However, further
in vivo
experiments are needed to assess
their biocompatibility, cytotoxicity, and degradation [
47
]. In addition, polymer matrices
that contain silver nanoparticles proved to be effective in wound dressing and prevented
bacterial growth at the wound site; however, these particles tend to agglomerate with time
which reduces their antimicrobial effect [48].
Poly (lactic-co-glycolic acid) (PLGA) is an FDA-approved polymer that is biocompat-
ible and has controllable mechanical properties and degradation rate. PLGA is widely
used as a delivery vehicle for drugs or bioactive factors [
49
]. However, its use as a scaffold
in BTE has been limited due to its low osteoinductivity. As a result, PLGA is always
used in a composite accompanied by other biomaterials [
50
]. Studies have shown that
the incorporation of HA into a PLGA polymer can enhance osteogenic cell proliferation
and differentiation, while an
in vivo
HA-PLGA scaffold exhibited good structural stability
and mechanical properties [
51
,
52
]. Furthermore, some studies used PLGA to encapsulate
antibacterial agents which significantly reduced bacterial growth [
53
–
55
], but this could
increase the burden of antibiotic-resistant bacterial species, and also raise the concern of
Polymers 2023,15, 1370 4 of 18
a burst release of the encapsulated antibiotic [
56
]. Silver and copper ions (Ag
+
and Cu
2+
)
have also been extensively studied due to their antimicrobial properties [
57
]. Ag
+
was
found to have a high cytotoxic effect compared to Cu
2+
, while Cu
2+
’s antibacterial effect
remained only for a short time frame [58].
This study aimed to fabricate a multipurpose bio-composite bone scaffold of Sr/Zn
doped nHAp-PLGA that has antibacterial activity and is cell friendly. Zn
2+
and Sr
2+
were
chosen to dope nHAp in this study as Zn
2+
has a dual effect of enhancing bone regeneration
and reducing bacterial growth, while Sr
2+
can enhance osteoblastic gene expression and
increase bone density. Meanwhile, PLGA was added to increase the scaffold stability and
mechanical properties.
In our previously published work [
59
], we analyzed the influence of Sr
2+
and Zn
2+
doping on nHAp crystallinity, and we were successfully able to synthesize Sr/Zn-doped
nHAp-PLGA composite scaffolds with adequate porosity, bioactivity, and degradability.
The XRD pattern and FTIR spectra revealed the phase composition and crystal properties
of nHAp in both Sr/Zn-doped powders and composite scaffolds and also confirmed
the incorporation of PLGA in the scaffolds. Crystallinity decreased while the Sr
2+
/Zn
2+
concentration increased, and composite scaffolds with less crystalline nHAp produced a
bioactive layer that was suitable for bone regeneration. The scaffolds were able to form an
orthophosphate layer on the surface when immersed in simulated body fluid, which was
confirmed by SEM and TEM studies [59]. Composite scaffolds with PLGA and 4% Sr/Zn-
nHAp exhibited the most abundant crystal growth after 2 weeks of submersion in SBF [
59
].
The porous structure of the scaffolds was confirmed in the SEM images. The composite
scaffolds showed interconnected, widely distributed pores. The average pore size for PLGA-
nHAp, PLGA-2.5% Sr/Zn-nHAp, and PLGA-4% Sr/Zn-nHAp scaffolds ranged between
189
±
10.26 to 406
±
26.54 (mean
±
SEM). The results suggested a statistically significant
increase (p< 0.0001) in the pore size of the composite scaffolds when we doped them with
strontium and zinc ions [
59
]. In addition, PLGA incorporation proved to reinforce the
mechanical properties and scaffolds were able to release Sr and Zn ions
in vitro
for up to
three weeks [59].
In the present work, we investigated the antimicrobial properties of Zn /Srdoped
nHAp with and without PLGA polymer, and assessed the effect of PLGA incorporation
on Zn
2+
antimicrobial activity. Furthermore, this work examined cytocompatibility and
osteoblastic cell proliferation on Zn/Sr-nHAp-PLGA composite scaffolds. The study also
optimized the concentration of doping elements (Zn/Sr) and measured their release in
simulated body fluid (SBF) using ICP-MS, making sure that the levels of these ions were
within the normal range that could enhance cell viability, hence augmenting bone formation.
2. Methods
2.1. Preparation of Nano-Hydroxyapatite
nHAp was prepared using the chemical precipitation method [
60
,
61
]. Briefly, 0.06 M
ammonium phosphate dibasic [(NH
4
)
2
HPO
4
, 98% Sigma-Aldrich (Saint Louis, MO, USA)]
and 0.1 M calcium nitrate tetrahydrate [Ca(NO
3
)
2
.4H
2
O, 99% Sigma-Aldrich] solutions
were prepared separately and dissolved in deionized water. After that, the phosphate-
containing solution was added dropwise to the calcium-containing solution and the Ca/P
ratio was kept at 1.67. The pH was adjusted to 11–12 using 10 N sodium hydroxide [NaOH,
98% Sigma-Aldrich] and the resulting solutions were kept under stirring conditions for 1 h.
The mixture was then aged overnight, and the white precipitate was filtered and washed
with distilled water 3 times. The final slurry was dried in an oven at 80
◦
C for 24 h and
calcinated at 300 ◦C for 1 h.
2.2. Preparation of Zn/Sr-Substituted Nano-Hydroxyapatite
A phosphate-containing solution and a calcium-containing solution were prepared as
previously mentioned. Zn in the form of zinc nitrate hexahydrate [Zn (NO
3
)
2
.6H
2
O, 98%
Daejung Chemicals & Metals Co., Ltd.] and/or strontium nitrate [Sr (NO
3
)
2
, 99% Merck,
Polymers 2023,15, 1370 5 of 18
Darmstadt, Germany] was added to the Ca-containing solution and the concentration of Zn
and/or Sr was set at 1%, 2.5%, and 4%. Then, the phosphorous (P)-containing solution was
added dropwise to the Ca and Zn/Sr solution, and the pH was adjusted to 11–12. After
that, the mixture was stirred at 100
◦
C for 1 h and aged overnight. The resulting white
precipitate was filtered and washed 3 times with distilled water then dried in an oven at
80 ◦C for 24 h, followed by calcination at 300 ◦Cfor1h[62].
2.3. Preparation of Zn/Sr-nHAp-PLGA
The composite scaffolds were prepared according to [
63
] with some modifications.
Briefly, different ratios of Zn/Sr-nHAp (see Table 1) and PLGA polymer [lactide: glycolide
(75:25), mol wt. 66,000–107,000, Sigma Aldrich] were dissolved separately in organic
solvent dichloromethane (DCM, Merck) and vortexed at 1000 RPM for 15 min. Zn/Sr-
nHAp dispersion was added dropwise to the PLGA/DCM solution and the resulting
mixture was vortexed at 2000 RPM for 30 min, and then kept in the oven at 70
◦
C for 15 min
to evaporate the solvent. The resulting slurry was washed with ethanol 3 times to remove
the DCM residues and left in the oven at 50
◦
C for 48 h to dry, followed by calcination at
150 ◦C for 4 h.
Table 1. Composition of different Sr/Zn-nHAp-PLGA scaffolds.
Scaffolds Sr
(mol%)
Zn
(mol%)
nHAp
(mol%)
PLGA
(mol%)
nHAp 0 0 1 0
PLGA-nHAp 0 0 1 3
1% Sr/Zn-nHAp-PLGA 1 1 1 3
2.5% Sr/Zn-nHAP-PLGA 2.5 2.5 1 3
4% Sr/Zn-nHAp-PLGA 4 4 1 3
2.4. Scaffold Fabrication
From each prepared powder, 500 mg was compressed using a hydraulic pellet press
at 5000–10,000 psi for 1 min to form a disc shape of 13
×
3 (W
×
H) mm (Figure 1). Disc
scaffolds were subjected to supercritical CO
2
, as in our previously published study [
59
].
Before each experiment, discs were placed on 24-well plates and sterilized using a UV light
for 30 min on each side.
Polymers2023,15,xFORPEERREVIEW5of18
2.2.PreparationofZn/Sr‐SubstitutedNano‐Hydroxyapatite
Aphosphate‐containingsolutionandacalcium‐containingsolutionwereprepared
aspreviouslymentioned.Znintheformofzincnitratehexahydrate[Zn(NO3)2.6H2O,98%
DaejungChemicals&MetalsCo.,Ltd.]and/orstrontiumnitrate[Sr(NO3)2,99%Merck,
Darmstadt,Germany]wasaddedtotheCa‐containingsolutionandtheconcentrationof
Znand/orSrwassetat1%,2.5%,and4%.Then,thephosphorous(P)‐containingsolution
wasaddeddropwisetotheCaandZn/Srsolution,andthepHwasadjustedto11–12.
Afterthat,themixturewasstirredat100°Cfor1handagedovernight.Theresulting
whiteprecipitatewasfilteredandwashed3timeswithdistilledwaterthendriedinan
ovenat80°Cfor24h,followedbycalcinationat300°Cfor1h[62].
2.3.PreparationofZn/Sr‐nHAp‐PLGA
Thecompositescaffoldswerepreparedaccordingto[63]withsomemodifications.
Briefly,differentratiosofZn/Sr‐nHAp(seeTable1)andPLGApolymer[lactide:glycolide
(75:25),molwt.66,000–107,000,SigmaAldrich]weredissolvedseparatelyinorganicsol‐
ventdichloromethane(DCM,Merck)andvortexedat1000RPMfor15min.Zn/Sr‐nHAp
dispersionwasaddeddropwisetothePLGA/DCMsolutionandtheresultingmixture
wasvortexedat2000RPMfor30min,andthenkeptintheovenat70°Cfor15minto
evaporatethesolvent.Theresultingslurrywaswashedwithethanol3timestoremove
theDCMresiduesandleftintheovenat50°Cfor48htodry,followedbycalcinationat
150°Cfor4h.
Table1.CompositionofdifferentSr/Zn‐nHAp‐PLGAscaffolds.
ScaffoldsSr
(mol%)
Zn
(mol%)
nHAp
(mol%)
PLGA
(mol%)
nHAp0010
PLGA‐nHAp0013
1%Sr/Zn‐nHAp‐PLGA1113
2.5%Sr/Zn‐nHAP‐PLGA2.52.513
4%Sr/Zn‐nHAp‐PLGA4413
2.4.ScaffoldFabrication
Fromeachpreparedpowder,500mgwascompressedusingahydraulicpelletpress
at5000–10,000psifor1mintoformadiscshapeof13×3(W×H)mm(Figure1).Disc
scaffoldsweresubjectedtosupercriticalCO2,asinourpreviouslypublishedstudy[59].
Beforeeachexperiment,discswereplacedon24‐wellplatesandsterilizedusingaUV
lightfor30minoneachside.
Figure1.Fabricatedscaffolds’(a)sideviewand(b)topview.
Figure 1. Fabricated scaffolds’ (a) side view and (b) top view.
Polymers 2023,15, 1370 6 of 18
2.5. Assessment of Scaffolds’ Antibacterial Activity
Scaffold antibacterial activity was measured against Staphylococcus aureus [ATCC
(25923), Microbiologics, St Cloud, MN, USA] by counting bacterial colonies on tryptone
soya agar (TSA, Mast, Bootle, UK)) after direct contact with the scaffold, as described
by Resmim et al. and Ofudje et al. [
62
,
64
]. Briefly, the bacterial density was adjusted to
5×106CFU/mL
using a McFarland densitometer and tryptone soya broth. A total of 2 mL
of the adjusted bacterial suspension was added into each scaffold in wells; a well without
scaffold was used as a growth-positive control (G.C), and plates were incubated at 37
◦
C for
24 h. After incubation, the bacterial suspension was aspirated, 1 mL of phosphate-buffered
saline (PBS) was added to each scaffold, and the plates were placed in a shaker at 100 RPM
for 15 min to remove scaffold-attached bacteria [
64
]. Thereafter, 100
µ
L of the test solution
(bacteria in PBS) was retrieved and serially diluted to 10
−8
. The agar plates were then
divided into 8 equal square sectors and 5
µ
L of the appropriate dilution was dropped onto
the agar surface of each sector and left upright to spread and dry for 15–20 min. Agar
plates were then inverted and incubated at 37
◦
C for 24 h for colony formation. The final
CFU/mL in the original sample was calculated according to the equation: CFU/mL = the
average number of colonies for a dilution
×
total dilutions of the sample. Furthermore,
the percentage of antibacterial properties of different scaffolds was calculated using the
formula: antibacterial rate % = (CFU
control −
CFU
test
)/CFU
control ×
100% [
65
]. All
trials were performed in triplicate and the results were normalized by calculation of the
arithmetic average.
2.6. Cell Culture
In this study, primary rat osteoblasts’ cell line (ROb) was purchased from (Cell Appli-
cations Inc., San Diego, CA, USA) and all the tissue culture reagents were purchased from
Sigma-Aldrich. Cells were cultured using standard protocols, as per company instructions,
following the standard sterilization technique and safety rules. ROb cells were cultured in a
rat osteoblast growth medium supplemented with 10% FBS and 1% penicillin-streptomycin
antibiotic and incubated in a 5% CO
2
incubator at 37
◦
C. Cells were passaged when reach-
ing 80–90% confluency (Figure 2). The medium was refreshed every 2–3 days and passages
number 4–7 were used for scaffold seeding in this experiment. Cells in the culture flask
were observed using an inverted Olympus microscope IX70 with a digital camera DP70.
Polymers2023,15,xFORPEERREVIEW6of18
2.5.AssessmentofScaffolds’AntibacterialActivity
ScaffoldantibacterialactivitywasmeasuredagainstStaphylococcusaureus[ATCC
(25923),Microbiologics,StCloud,MN,USA]bycountingbacterialcoloniesontryptone
soyaagar(TSA,Mast,Bootle,UK))afterdirectcontactwiththescaffold,asdescribedby
Resmimetal.andOfudjeetal.[62,64].Briefly,thebacterialdensitywasadjustedto5×
10
6
CFU/mLusingaMcFarlanddensitometerandtryptonesoyabroth.Atotalof2mLof
theadjustedbacterialsuspensionwasaddedintoeachscaffoldinwells;awellwithout
scaffoldwasusedasagrowth‐positivecontrol(G.C),andplateswereincubatedat37°C
for24h.Afterincubation,thebacterialsuspensionwasaspirated,1mLofphosphate‐
bufferedsaline(PBS)wasaddedtoeachscaffold,andtheplateswereplacedinashaker
at100RPMfor15mintoremovescaffold‐attachedbacteria[64].Thereafter,100μLofthe
testsolution(bacteriainPBS)wasretrievedandseriallydilutedto10
−8
.Theagarplates
werethendividedinto8equalsquaresectorsand5μLoftheappropriatedilutionwas
droppedontotheagarsurfaceofeachsectorandleftuprighttospreadanddryfor15–20
min.Agarplatesweretheninvertedandincubatedat37°Cfor24hforcolonyformation.
ThefinalCFU/mLintheoriginalsamplewascalculatedaccordingtotheequation:
CFU/mL=theaveragenumberofcoloniesforadilution×totaldilutionsofthesample.
Furthermore,thepercentageofantibacterialpropertiesofdifferentscaffoldswascalcu‐
latedusingtheformula:antibacterialrate%=(CFU
control
−CFU
test
)/CFU
control
×100%[65].
Alltrialswereperformedintriplicateandtheresultswerenormalizedbycalculationof
thearithmeticaverage.
2.6.CellCulture
Inthisstudy,primaryratosteoblasts’cellline(ROb)waspurchasedfrom(CellAp‐
plicationsInc.,
SanDiego
,CA,USA)andallthetissueculturereagentswerepurchased
fromSigma‐Aldrich.Cellswereculturedusingstandardprotocols,aspercompanyin‐
structions,followingthestandardsterilizationtechniqueandsafetyrules.RObcellswere
culturedinaratosteoblastgrowthmediumsupplementedwith10%FBSand1%penicil‐
lin‐streptomycinantibioticandincubatedina5%CO
2
incubatorat37°C.Cellswerepas‐
sagedwhenreaching80–90%confluency(Figure2).Themediumwasrefreshedevery2–
3daysandpassagesnumber4–7wereusedforscaffoldseedinginthisexperiment.Cells
inthecultureflaskwereobservedusinganinvertedOlympusmicroscopeIX70witha
digitalcameraDP70.
Figure2.CulturedRObcellsshowingtheircharacteristicstellate‐to‐spindle‐shapedappearance,in‐
dicatedwitharedarrow(Scalebar=200μm).
Figure 2.
Cultured ROb cells showing their characteristic stellate-to-spindle-shaped appearance,
indicated with a red arrow (Scale bar = 200 µm).
Polymers 2023,15, 1370 7 of 18
2.7. Scaffold Seeding
The fabricated scaffolds used to check cell viability were nHAp, PLGA-nHAp, 1%
Sr/Zn-nHAp-PLGA, 2.5% Sr/Zn-nHAp-PLGA, and 4% Sr/Zn-nHAp-PLGA, and n= 3 for
each scaffold. Prior to cell seeding, the scaffolds were placed aseptically in 24-well plates
and sterilized using a UV light for 30 min on each side, then soaked in 1 mL of complete
culture medium for 1 h. Cells were trypsinized and the seeding density was adjusted to
1×105
cells per scaffold. A total of 35
µ
L of the adjusted cells suspension was added
dropwise to each scaffold, followed by incubation at 37
◦
C in 5% CO
2
for 1 h to allow cell
attachment. After that, 2 mL of complete culture medium was added to each scaffold,
and the plates were returned to the incubator. The culture medium was changed every
2–3 days [66].
2.8. MTT Assay
On day 2 and day 7, osteoblast cell proliferation was assessed using a 3-(4,5-dimethyl
thiazolyl-2)-2,5-diphenyl-tetrazolium bromide (MTT) assay kit (Abcam, ab211091, Boston,
MA, USA). In this assay, living cells cleave MTT-soluble tetrazolium salts and convert them
into insoluble purple formazan, which is then solubilized, and the resulting color intensity
is directly proportional to the number of living cells [
67
]. Briefly, the medium was aspirated
from the scaffolds, 1 mL of MTT reagent was added to each scaffold, and then plates were
incubated at 37
◦
C for 4 h. After incubation, the MTT reagent was removed and 1 mL of
MTT solvent was added to each well. Subsequently, plates were wrapped with aluminum
foil and agitated on an orbital shaker for 15 min. The test solution was transferred into
a new 24-well plate and the optical density (OD) was measured at 590 nm using a Tecan
infinite M200 PRO microplate reader [68].
2.9. Sr and Zn Ion Release Study
The ion release study was performed according to our previously published work [
59
].
Briefly, each scaffold was immersed in SBF at 37
◦
C for 28 days. The scaffolds were removed
at different time intervals and the Sr and Zn ions’ release in SBF fluid was measured using
inductively coupled plasma mass spectrometry (ICP-MS; NexION 300X, PerkinElmer,
Waltham, MA, USA).
2.10. Statistical Analysis
All experiments were measured in triplicate and data were analyzed using SPSS
software (version 28.0) using one-way analysis of variance (ANOVA) and post hoc for
multiple comparisons. Differences were considered statistically significant when the
p-value was <0.05.
3. Results and Discussion
3.1. Scaffolds Antibacterial Activity
Our focus in this study was to assess the antibacterial property of the fabricated
scaffolds [nHAp, PLGA, nHAp-PLGA, Zn-nHAp, Sr-nHAp, Sr/Zn-nHAp, and Sr/Zn-
nHAp-PLGA] with different doping percentages of Sr and Zn ions (1%, 2.5%, and 4%).
Figure 3represents colony forming units (CFU) of S. aureus in TSA after 24 h of direct contact
with the different scaffolds. Figure 3a–d demonstrates bacterial CFU in the growth control
(G.C), i.e., growth without any scaffold, nHAp, PLGA, and PLGA-nHAp, which showed a
relatively similar bacterial number. Moreover, in the Zn-nHAp scaffolds in Figure 3e–g, all
Zn concentrations showed a decrease in CFU numbers, the reduction was dose-dependent,
and the scaffolds with the highest Zn
2+
concentration (4% Zn-nHAp) showed the lowest
CFU numbers; hence, they had the best antibacterial activity. Sr-nHAp scaffolds did not
show any antibacterial activity in Figure 3h–j and the CFU number was similarly close to
G.C and nHAp. Sr/Zn nHAp with and without PLGA in Figure 3k–p showed a reduction
in CFU number as the Zn
2+
concentration increased, and a 4% doping percentage attained
the maximum inhibitory effect for both scaffolds with and without PLGA.
Polymers 2023,15, 1370 8 of 18
Polymers2023,15,xFORPEERREVIEW8of18
reductioninCFUnumberastheZn2+concentrationincreased,anda4%dopingpercent‐
ageattainedthemaximuminhibitoryeffectforbothscaffoldswithandwithoutPLGA.
Figure3.PhotographsofbacterialcoloniesinTSAplatesafter24hofdirectcontactwithdifferent
scaffolds.Eachsquaresectorrepresentsaserialdilutionfrom10−1to10−8,lefttoright.(a)G.C,(b)
purenHAp,(c)PurePLGA,(d)nHAp‐PLGA,(e)1%Zn‐nHAp,(f)2.5%Zn‐nHAp,(g)4%Zn‐nHAp,
(h)1%Sr‐nHAp,(i)2.5%Sr‐nHAp,(j)4%Sr‐nHAp,(k)1%Sr/Zn‐nHAp,(l)2.5%Sr/Zn‐nHAp,(m)
Figure 3.
Photographs of bacterial colonies in TSA plates after 24 h of direct contact with different
scaffolds. Each square sector represents a serial dilution from 10
−1
to 10
−8
, left to right. (
a
) G.C,
(
b
) pure nHAp, (
c
) Pure PLGA, (
d
) nHAp-PLGA, (
e
) 1% Zn-nHAp, (
f
) 2.5% Zn-nHAp, (
g
) 4%
Zn-nHAp, (
h
) 1% Sr-nHAp, (
i
) 2.5% Sr-nHAp, (
j
) 4% Sr-nHAp, (
k
) 1% Sr/Zn-nHAp, (
l
) 2.5%
Sr/Zn-nHAp, (
m
) 4% Sr/Zn-nHAp, (
n
) 1% Sr/Zn-nHAp-PLGA, (
o
) 2.5% Sr/Zn-nHAp-PLGA,
(p) 4% Sr/Zn-nHAp-PLGA.
Polymers 2023,15, 1370 9 of 18
Figure 4a,b shows the exact numbers of bacteria CFU. The results showed that the
bacterial number in G.C was 4
×
10
7
CFU/mL; in nHAp, it was 2
×
10
7
CFU/mL, while
in PLGA, the CFU count was 2.6
×
10
7
CFU/mL. A drop in the CFU number was ob-
served in all Zn
2+
-containing scaffolds, with 4% Zn showing the lowest CFU number
of
28 ×104CFU/mL
in 4% Sr/Zn-nHAp scaffolds, and 6
×
10
4
CFU/mL in 4% Sr/Zn-
nHAp-PLGA, indicating that PLGA incorporation into the scaffolds did not affect Zn
2+
’s
antibacterial action.
Polymers2023,15,xFORPEERREVIEW9of18
4%Sr/Zn‐nHAp,(n)1%Sr/Zn‐nHAp‐PLGA,(o)2.5%Sr/Zn‐nHAp‐PLGA,(p)4%Sr/Zn‐nHAp‐
PLGA.
Figure4a,bshowstheexactnumbersofbacteriaCFU.Theresultsshowedthatthe
bacterialnumberinG.Cwas4×107CFU/mL;innHAp,itwas2×107CFU/mL,whilein
PLGA,theCFUcountwas2.6×107CFU/mL.AdropintheCFUnumberwasobservedin
allZn2+‐containingscaffolds,with4%ZnshowingthelowestCFUnumberof28×104
CFU/mLin4%Sr/Zn‐nHApscaffolds,and6×104CFU/mLin4%Sr/Zn‐nHAp‐PLGA,
indicatingthatPLGAincorporationintothescaffoldsdidnotaffectZn2+’santibacterial
action.
Figure4.BacterialCFUcountsafter24hofincubationwithdifferentscaffolds(a)G.C,nHAp,
PLGA,PLGA‐nHAp,1%Sr‐nHAp,2.5%Sr‐nHAp,4%Sr‐nHAp;(b)1%Zn‐nHAp,2.5%Zn‐nHAp,
4%Zn‐nHAp,1%Sr/Zn‐nHAp,2.5%Sr/Zn‐nHAp,4%Sr/Zn‐nHAp,1%Zn/Sr‐nHAp‐PLGA,2.5%
Zn/Sr‐nHAp‐PLGAand4%Zn/Sr‐nHAp‐PLGA.
Thebacterialgrowthinhibitionpercentageofthedifferentscaffoldswascalculated
fromCFUnumbers,usingthenHApscaffoldasacontrol(Table2).Zn2+‐containingscaf‐
foldsshowedthebestgrowthinhibitionpercentagethatrangedbetween98.6±0.2and
Figure 4.
Bacterial CFU counts after 24 h of incubation with different scaffolds (
a
) G.C, nHAp,
PLGA, PLGA- nHAp, 1% Sr-nHAp, 2.5% Sr-nHAp, 4% Sr-nHAp; (
b
) 1% Zn-nHAp, 2.5% Zn-
nHAp,
4% Zn-nHAp
, 1% Sr/Zn-nHAp, 2.5% Sr/Zn-nHAp,
4% Sr/Zn-nHAp
, 1% Zn/Sr-nHAp-
PLGA, 2.5% Zn/Sr-nHAp-PLGA and 4% Zn/Sr-nHAp-PLGA.
The bacterial growth inhibition percentage of the different scaffolds was calculated
from CFU numbers, using the nHAp scaffold as a control (Table 2). Zn
2+
-containing
scaffolds showed the best growth inhibition percentage that ranged between
98.6 ±0.2
and 99.7
±
0.1% in 4% Zn concentrations. Sr
2+
-containing scaffolds in 1%, 2.5%, and
4% Sr-nHAp did not show any bacterial inhibitory effect unless accompanied with Zn
2+
,
which is also in agreement with other studies where Sr-HA did not reveal any antibacterial
Polymers 2023,15, 1370 10 of 18
properties unless accompanied by other metals such as Ag
+
(silver) and Se
4+
(selenium), as
they are known to exhibit antibacterial effect [69–71].
Table 2. Bacterial growth inhibition percentage for different scaffolds ±SEM.
Composite Scaffolds Growth Inhibition %
Pure nHAp 0%
PLGA 0%
PLGA-nHAp 0%
1%, 2.5%, 4% Sr-nHAp 0%
1% Zn-nHAp 98.9 ±0.5%
2.5% Zn-nHAp 99.7 ±1.65%
4% Zn-nHAp 99.8 ±0.1%
1% Sr/Zn-nHAp 93 ±3.4%
2.5% Sr/Zn-nHAp 98.2 ±0.4%
4% Sr/Zn-nHAp 98.6 ±0.2%
1% Zn/Sr-nHAp-PLGA 10 ±4.5%
2.5% Zn/Sr-nHAp-PLGA 98 ±0.57%
4% Zn/Sr-nHAp-PLGA 99.7 ±0.1%
Several previous studies have reported the antimicrobial properties of Zn-nHAp
against Staphylococcus aureus and Escherichia coli [
25
,
57
,
62
,
72
–
74
]. Multiple factors could
contribute to this bactericidal effect. One of them is the fact that Zn ions can bind to some
structural proteins in the bacterial cell membrane, altering the membrane permeability
and killing the bacteria [62,75]. Additionally, the decrease in Zn-nHAp crystallinity in the
higher Zn
2+
concentrations will enhance the growth of the apatite layer (Figure 5) and,
consequently, increase the surface area which will facilitate the contact between Zn ions
and the bacterial cell membrane, resulting in more bacterial death [
72
]. This explains the
dose-dependent reduction in bacterial CFU numbers as the Zn
2+
concentration increased.
The enhanced antibacterial effect in higher Zn
2+
concentrations was also observed by
Ofudje et al. and Valarmathi et al. [
62
,
76
]. The PLGA polymer has been extensively studied
as a drug carrier due to its many beneficial properties [
77
–
79
]. In many studies, PLGA
was used to encapsulate antibiotic agents, whereas, in some studies, PLGA was used as
a composite in bone scaffolds. In our previous study, PLGA incorporation into nHAp
showed an increase in the scaffold mechanical properties [
59
], while in this study, PLGA
was found to have no impact or a weakening effect on Zn
2+
antibacterial activity, which
makes it a suitable candidate to use in antimicrobial bone scaffolds in combination with
Sr/Zn-doped nHAp.
3.2. Cell Proliferation Using MTT Assay
MTT assay was performed to assess osteoblast cell proliferation in the different fab-
ricated scaffolds to evaluate their cytocompatibility. Many previous studies confirmed
nHAp cytocompatibility and its favorable effect on osteoblast proliferation and differentia-
tion [
80
–
82
]. Therefore, cell proliferation on nHAp was used as a control to evaluate the
Sr/Zn doping effect. MTT assay (Figure 6) showed that the number of metabolically active
cells on day 2 in nHAp and nHAp-PLGA scaffolds was less when compared to Sr and Zn
ion-doped scaffolds; however, the difference was not statistically significant. On day 7,
the optical densities of cells in scaffolds devoid of Sr/Zn ions were slightly decreased and
cells were not able to maintain their numbers, while all scaffolds co-doped with Sr/Zn ions
showed a significant increase in the cell numbers compared to pure nHAp (p< 0.001). The
highest concentration of Sr/Zn ions as in the 4% example were able to maintain the number
of viable cells for 7 days and showed the greatest number of viable cells. These results
suggest that 4% Sr/Zn-nHAp-PLGA scaffolds could be preferable for cell proliferation
and viability. Wang et al. also demonstrated that BMSCs cultured in Sr/Zn co-doped HA
scaffolds for 7 days, showed a stronger proliferative ability and ALP activity compared to
single-ion-doped and pure HA scaffolds [83].
Polymers 2023,15, 1370 11 of 18
Polymers2023,15,xFORPEERREVIEW11of18
Figure5.MechanismofZn2+antibacterialeffect.CreatedwithBioRender.com.*[59]Hassanetal.
3.2.CellProliferationUsingMTTAssay
MTTassaywasperformedtoassessosteoblastcellproliferationinthedifferentfabricatedscaffolds
toevaluatetheircytocompatibility.ManypreviousstudiesconfirmednHApcytocompatibilityand
itsfavorableeffectonosteoblastproliferationanddifferentiation[80–82].Therefore,cellprolifera‐
tiononnHApwasusedasacontroltoevaluatetheSr/Zndopingeffect.MTTassay(Figure6)
showedthatthenumberofmetabolicallyactivecellsonday2innHApandnHAp‐PLGAscaffolds
waslesswhencomparedtoSrandZnion‐dopedscaffolds;however,thedifferencewasnotstatis‐
ticallysignificant.Onday7,theopticaldensitiesofcellsinscaffoldsdevoidofSr/Znionswere
slightlydecreasedandcellswerenotabletomaintaintheirnumbers,whileallscaffoldsco‐doped
withSr/ZnionsshowedasignificantincreaseinthecellnumberscomparedtopurenHAp(p<
0.001).ThehighestconcentrationofSr/Znionsasinthe4%examplewereabletomaintainthenum‐
berofviablecellsfor7daysandshowedthegreatestnumberofviablecells.Theseresultssuggest
that4%Sr/Zn‐nHAp‐PLGAscaffoldscouldbepreferableforcellproliferationandviability.Wang
etal.alsodemonstratedthatBMSCsculturedinSr/Znco‐dopedHAscaffoldsfor7days,showeda
strongerproliferativeabilityandALPactivitycomparedtosingle‐ion‐dopedandpureHAscaffolds
[83].
Figure 5. Mechanism of Zn2+ antibacterial effect. Created with BioRender.com. * [59] Hassan et al.
Polymers2023,15,xFORPEERREVIEW12of18
Figure6.MTTassayforosteoblastsculturedfor2and7daysondifferentscaffolds:(nHAp,nHAp‐
PLGA,1%Zn/Sr‐nHAp‐PLGA,2.5%Zn/Sr‐nHAp‐PLGA,and4%Zn/Sr‐nHAp‐PLGA).***p≤0.001
comparedtonHAp.
ThemechanismbywhichZnandSrionscanaffectosteoblastbehaviorwasinvesti‐
gatedpreviously.ZnandSrionscanstimulateosteogenesisbyinterferingwithseveral
signalingpathwaysthatcanregulategeneexpression,proliferation,differentiation,and
collagenmatrixmineralizationinculturedosteoblasts[84,85].Additionally,anearlier
studyshowedthatzinc‐containingnanoparticlesloadedtocelluloseandchitosan‐based
hydrogelspromotedvascularendothelialgrowthfactor(VEGF)expressionandangiogen‐
esis,whichisusefulforcellproliferationandtissueregeneration[86].
TheoptimaleffectivedosageforZnorSrionsthatcanacceleratecellproliferation
withoutbeingtoxictothecellsisdebatable.Wefoundtheliteraturetobecontradictory
onzincconcentration;somestudiesstatedthat5%andupto6%Znionconcentrationcan
enhancecellproliferationwhileincreasingtheZn2+concentrationto10%and20%resulted
inasignificantdropincellnumbers[87–89].Additionally,Fernandesetal.demonstrated
that5%Zn‐HApowderimprovedtheosteogenesisofratcalvarialbonedefectsalmostas
muchasautografts,anddemonstratedaslowerdegradationratecomparedtopureHA
andautografts[90].HAwith13%Zn2+contentscanleadtoMgiondepletionandreplace‐
mentwithZnions,whichwilljeopardizecellsurvival,asMgionsarenormallyfoundto
bindtosomestructuralproteinsthathelptomaintaincellmembraneintegrityandare
involvedinseveralvitalprocessessuchasDNAsynthesisandpolymeraseactivity[74].
AnincreaseinROSformationwhichmayinduceoxidativestressandleadtocelldamage
isalsoreportedwiththeuseofhigherconcentrationsofZn2+.[74].Ontheotherhand,
Popaetal.andUllahetal.reportednocytotoxiceffectata10%Znconcentration[91,92].
X‐raydiffraction(XRD)andFourier‐transforminfraredspectroscopy(FTIR)studies
provedthatincreasingZn2+concentrationstomorethan12.3wt.%canweakentheHA
structureduetothelossoftheapatitephaseandreplacementwithanon‐apatitephaseof
Zn‐βtricalciumphosphate(β‐TCP),asmoreCaionswillbesubstitutedbyZnions,result‐
ingindifferentsolubilityandionreleaseratesbythescaffold[93].Ontheotherhand,
severalstudiesdidnotshowacytotoxiceffectfor10%Sr‐HAorreportedweakcytotoxi‐
city[94,95],suggestingthatcellscantoleratehighSr2+concentrationsincontradistinction
toZn2+.Thisstudyinvestigated1%,2.5%,and4%ofZn2+andSr2+dopedtonHAp.Ac‐
cordingtoourresults,a4%Zn2+andSr2+dopingpercentagemaximizedthescaffoldanti‐
bacterialeffectandenhancedosteoblasticcellsurvivalandproliferation.Inaddition,
PLGAincorporationintothescaffolddidnotjeopardizeZn2+antibacterialactionanden‐
hancedscaffoldcytocompatibilityandmechanicalproperties.
Figure 6.
MTT assay for osteoblasts cultured for 2 and 7 days on different scaffolds: (nHAp,
nHAp-PLGA, 1% Zn/Sr-nHAp-PLGA, 2.5% Zn/Sr-nHAp-PLGA, and 4% Zn/Sr-nHAp-PLGA).
*** p≤0.001 compared to nHAp.
The mechanism by which Zn and Sr ions can affect osteoblast behavior was investi-
gated previously. Zn and Sr ions can stimulate osteogenesis by interfering with several
signaling pathways that can regulate gene expression, proliferation, differentiation, and
collagen matrix mineralization in cultured osteoblasts [
84
,
85
]. Additionally, an earlier study
Polymers 2023,15, 1370 12 of 18
showed that zinc-containing nanoparticles loaded to cellulose and chitosan-based hydro-
gels promoted vascular endothelial growth factor (VEGF) expression and angiogenesis,
which is useful for cell proliferation and tissue regeneration [86].
The optimal effective dosage for Zn or Sr ions that can accelerate cell proliferation
without being toxic to the cells is debatable. We found the literature to be contradictory
on zinc concentration; some studies stated that 5% and up to 6% Zn ion concentration can
enhance cell proliferation while increasing the Zn
2+
concentration to 10% and 20% resulted
in a significant drop in cell numbers [
87
–
89
]. Additionally, Fernandes et al. demonstrated
that 5% Zn-HA powder improved the osteogenesis of rat calvarial bone defects almost as
much as autografts, and demonstrated a slower degradation rate compared to pure HA and
autografts [
90
]. HA with 13% Zn
2+
contents can lead to Mg ion depletion and replacement
with Zn ions, which will jeopardize cell survival, as Mg ions are normally found to bind to
some structural proteins that help to maintain cell membrane integrity and are involved in
several vital processes such as DNA synthesis and polymerase activity [
74
]. An increase in
ROS formation which may induce oxidative stress and lead to cell damage is also reported
with the use of higher concentrations of Zn
2+
. [
74
]. On the other hand, Popa et al. and Ullah
et al. reported no cytotoxic effect at a 10% Zn concentration [
91
,
92
]. X-ray diffraction (XRD)
and Fourier-transform infrared spectroscopy (FTIR) studies proved that increasing Zn
2+
concentrations to more than 12.3 wt.% can weaken the HA structure due to the loss of the
apatite phase and replacement with a non-apatite phase of Zn-
β
tricalcium phosphate (
β
-
TCP), as more Ca ions will be substituted by Zn ions, resulting in different solubility and ion
release rates by the scaffold [
93
]. On the other hand, several studies did not show a cytotoxic
effect for 10% Sr-HA or reported weak cytotoxicity [
94
,
95
], suggesting that cells can tolerate
high Sr
2+
concentrations in contradistinction to Zn
2+
. This study investigated 1%, 2.5%,
and 4% of Zn
2+
and Sr
2+
doped to nHAp. According to our results, a 4% Zn
2+
and Sr
2+
doping percentage maximized the scaffold antibacterial effect and enhanced osteoblastic
cell survival and proliferation. In addition, PLGA incorporation into the scaffold did
not jeopardize Zn
2+
antibacterial action and enhanced scaffold cytocompatibility and
mechanical properties.
3.3. Sr and Zn Ion Release Using ICP-MS
The ions release profile of Sr/Zn-nHAp-PLGA scaffolds was measured using inductive
coupled plasma-mass spectrometry ICP-MS on days 1, 7, 14, and 28 after immersion in SBF.
The results in Figure 7a,b show that the release profile started on day 1 and was maximized
in 4% Sr/Zn-nHAp-PLGA scaffolds for all days. This will be helpful for bone healing as
ions will be readily available as early as the first week. Zn and Sr ion release peaked at day
14 for Zn ions and day 7 for Sr ions.
A key factor that needs to be under control is the amount of Zn/Sr ions released into
the surrounding medium as the scaffold degrades, which will be taken up by cells and
affect their viability. The reference range for Zn
2+
levels in human serum is
60–120 µg/dL
,
while for Sr
2+
, it is 1.9–9.6
µ
g/dL [
96
,
97
]. The highest release profile for Zn and Sr ions was
6.9 µg/dL
and 9.02
µ
g/dL, respectively. These levels did not exceed the normal concentra-
tion range; hence, we can consider 4% Zn/Sr-nHAp-PLGA as a safe and cytocompatible
scaffold according to
in vitro
studies. In our study, we tried to limit Zn
2+
concentrations to
4%, as higher concentrations may result in a higher release profile that could outrange the
normal levels and may increase the risk of cytotoxicity. Scaffold implantation in an animal
model is needed further to confirm
in vivo
biocompatibility and bone regeneration ability.
Polymers 2023,15, 1370 13 of 18
Polymers2023,15,xFORPEERREVIEW13of18
3.3.SrandZnIonReleaseUsingICP‐MS
TheionsreleaseprofileofSr/Zn‐nHAp‐PLGAscaffoldswasmeasuredusinginductivecoupled
plasma‐massspectrometryICP‐MSondays1,7,14,and28afterimmersioninSBF.Theresultsin
Figure7a,bshowthatthereleaseprofilestartedonday1andwasmaximizedin4%Sr/Zn‐nHAp‐
PLGAscaffoldsforalldays.Thiswillbehelpfulforbonehealingasionswillbereadilyavailableas
earlyasthefirstweek.ZnandSrionreleasepeakedatday14forZnionsandday7forSrions.
Figure7.IonreleaseprofileusingICP‐MSfor1%Zn/Sr‐nHAp‐PLGA,2.5%Zn/Sr‐nHAp‐PLGA,
and4%Zn/Sr‐nHAp‐PLGA.(a)Zincions;(b)strontiumions.
AkeyfactorthatneedstobeundercontrolistheamountofZn/Srionsreleasedinto
thesurroundingmediumasthescaffolddegrades,whichwillbetakenupbycellsand
affecttheirviability.ThereferencerangeforZn2+levelsinhumanserumis60–120μg/dl,
whileforSr2+,itis1.9–9.6μg/dl[96,97].ThehighestreleaseprofileforZnandSrionswas
6.9μg/dland9.02μg/dl,respectively.Theselevelsdidnotexceedthenormalconcentra‐
tionrange;hence,wecanconsider4%Zn/Sr‐nHAp‐PLGAasasafeandcytocompatible
scaffoldaccordingtoinvitrostudies.Inourstudy,wetriedtolimitZn2+concentrationsto
4%,ashigherconcentrationsmayresultinahigherreleaseprofilethatcouldoutrangethe
normallevelsandmayincreasetheriskofcytotoxicity.Scaffoldimplantationinananimal
modelisneededfurthertoconfirminvivobiocompatibilityandboneregenerationability.
Designinganidealbonescaffoldwithappropriateporosity,biocompatibility,and
tunablemechanicalpropertiesisstillachallenge.Naturalpolymerssuchaschitosan,al‐
ginate,silk,andcollagendemonstrategoodbiocompatibility,degradability,andbioactiv‐
ity.However,theyhavepoormechanicalproperties.Asaresult,theyareusuallyusedin
theformofblendsoftwoorthreecomponentstoimprovetheirmechanicalproperties
[98,99].Inaddition,thefunctionalizationofthesepolymersisdifficult,whichreduces
theirboneregenerationability[11].Thecompositescaffoldproposedinthisstudyiscom‐
posedmainlyofanaturalbonecomponent(nHAp)anddopedwithSr2+andZn2+metal
ions,whicharealsoanaturalconstituentofbonethatcanintroduceadualeffectofen‐
hancingboneregenerationandanantibacterialeffecttothescaffold.Moreover,thescaf‐
folds’mechanicalpropertieswereimprovedbytheadditionofthePLGApolymertothe
scaffold,asshowninourpreviouswork[59].
4.Conclusions
Introducinganantibacterialfunctiontothescaffoldwithoutcompromisingscaffold
biocompatibilityissubstantial.Thescaffoldsimpregnatedwithantibioticsmayincrease
theburdenofantibioticresistancespeciesandincreasetheconcernofburstreleaseof
thesedrugs,whichwillleavethescaffoldineffectiveforbacterialkilling.Thesynthesisof
Figure 7.
Ion release profile using ICP-MS for 1% Zn/Sr-nHAp-PLGA, 2.5% Zn/Sr-nHAp-PLGA,
and 4% Zn/Sr-nHAp-PLGA. (a) Zinc ions; (b) strontium ions.
Designing an ideal bone scaffold with appropriate porosity, biocompatibility, and
tunable mechanical properties is still a challenge. Natural polymers such as chitosan, algi-
nate, silk, and collagen demonstrate good biocompatibility, degradability, and bioactivity.
However, they have poor mechanical properties. As a result, they are usually used in the
form of blends of two or three components to improve their mechanical properties [
98
,
99
].
In addition, the functionalization of these polymers is difficult, which reduces their bone
regeneration ability [
11
]. The composite scaffold proposed in this study is composed mainly
of a natural bone component (nHAp) and doped with Sr
2+
and Zn
2+
metal ions, which
are also a natural constituent of bone that can introduce a dual effect of enhancing bone
regeneration and an antibacterial effect to the scaffold. Moreover, the scaffolds’ mechanical
properties were improved by the addition of the PLGA polymer to the scaffold, as shown
in our previous work [59].
4. Conclusions
Introducing an antibacterial function to the scaffold without compromising scaffold
biocompatibility is substantial. The scaffolds impregnated with antibiotics may increase
the burden of antibiotic resistance species and increase the concern of burst release of
these drugs, which will leave the scaffold ineffective for bacterial killing. The synthesis of
scaffolds with surface coats that inhibit bacterial adhesion and growth such as nanopillars
needs an extreme synthesis environment which can affect the polymer’s properties. Scaf-
folds doped with metal ions such as Ag
+
were found to have a high cytotoxic effect, while
Cu
2+
’s antibacterial effect remained only for a short time. In comparison, Zn
2+
has good
antibacterial activity at certain limited concentrations, while it was also found to enhance
osteoblast gene expression and increase bone mineralization [33].
In this study, we presented a novel Sr/Zn co-doped nHAp-PLGA scaffold with an
antibacterial effect against S. aureus that reached 99.7% bacterial growth inhibition in the
4% Sr/Zn-nHAp-PLGA composite scaffold; hence, this was the best dosage required.
This antibacterial activity increased with increasing Zn ion concentrations, while PLGA
polymer incorporation into the scaffold did not affect this antibacterial activity. At the same
time, it enhanced the scaffold’s mechanical property and stability, as shown in our earlier
study [
59
]. MTT assay showed that Zn/Sr co-doped nHAp-PLGA scaffolds provided a
friendly environment for osteoblast cell proliferation compared to pure nHAp. Our results
suggest that 4% Sr/Zn-nHAp-PLGA is a promising candidate for bone tissue engineering
applications with excellent antimicrobial activity and cytocompatibility.
Polymers 2023,15, 1370 14 of 18
Author Contributions:
Conceptualization, S.M.; methodology, S.M., M.H., and A.K.; validation, S.M.,
A.H.A.-M., S.M.K., and A.K.; formal analysis, M.H., S.M., I.u.R., S.M.K., and A.K.; investigation, S.M.,
A.H.A.-M., and A.K.; resources, S.M., A.H.A.-M., S.M.K., and A.K.; data curation, M.H. and S.M.;
writing—original draft preparation, M.H.; writing—review and editing, S.M., A.H.A.-M., S.M.K.,
I.u.R., and A.K.; visualization, M.H., A.K., and S.M.; supervision, S.M., A.H.A.-M., and A.K.; project
administration, S.M.; funding acquisition, S.M., I.u.R., and A.K. All authors have read and agreed to
the published version of the manuscript.
Funding:
This research was funded by United Arab Emirates University, UAEU Program for Ad-
vanced Research (UPAR) grant number G00003460, and “The APC will be funded by the same”.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Acknowledgments:
We would like to express our appreciation to Subi Sugathan from the Department
of Anatomy for his help with the cell culture. We are also grateful to Lana Daoud from the Department
of Microbiology & Immunology for assistance with bacterial culture.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Wippert, P.-M.; Rector, M.; Kuhn, G.; Wuertz-Kozak, K. Stress and Alterations in Bones: An Interdisciplinary Perspective. Front.
Endocrinol. 2017,8, 96. [CrossRef]
2.
Feng, X. Chemical and Biochemical Basis of Cell-Bone Matrix Interaction in Health and Disease. Curr. Chem. Biol.
2009
,3, 189–196.
[CrossRef] [PubMed]
3.
Fuchs, R.K.; Thompson, W.R.; Warden, S.J. 2—Bone Biology. In Bone Repair Biomaterials, Woodhead Publishing Series
in Biomaterials; 2nd ed.; Pawelec, K.M., Planell, J.A., Eds.; Woodhead Publishing: Sawston, UK, 2019; pp. 15–52,
ISBN 978-0-08-102451-5.
4.
Offner, D.; de Grado, G.F.; Meisels, I.; Pijnenburg, L.; Fioretti, F.; Benkirane-Jessel, N.; Musset, A.-M. Bone Grafts, Bone Substitutes
and Regenerative Medicine Acceptance for the Management of Bone Defects Among French Population: Issues about Ethics,
Religion or Fear? Cell Med. 2019,11, 2155179019857661. [CrossRef]
5.
Wang, W.; Yeung, K.W.K. Bone grafts and biomaterials substitutes for bone defect repair: A review. Bioact. Mater.
2017
,2, 224–247.
[CrossRef]
6.
Amini, A.R.; Laurencin, C.T.; Nukavarapu, S.P. Bone tissue engineering: Recent advances and challenges. Crit. Rev. Biomed. Eng.
2012,40, 363–408. [CrossRef]
7.
Elsalanty, M.E.; Genecov, D.G. Bone grafts in craniofacial surgery. Craniomaxillofac. Trauma Reconstr.
2009
,2, 125–134. [CrossRef]
8. Moore, W.R.; Graves, S.E.; Bain, G.I. Synthetic bone graft substitutes. ANZ J. Surg. 2001,71, 354–361. [CrossRef]
9. Bohner, M. Resorbable biomaterials as bone graft substitutes. Mater. Today 2010,13, 24–30. [CrossRef]
10.
Zimmermann, C.E.; Gierloff, M.; Hedderich, J.; Açil, Y.; Wiltfang, J.; Terheyden, H. Biocompatibility of bone graft substitutes:
Effects on survival and proliferation of porcine multilineage stem cells in vitro. Folia Morphol. 2011,70, 154–160.
11.
Guo, L.; Liang, Z.; Yang, L.; Du, W.; Yu, T.; Tang, H.; Li, C.; Qiu, H. The role of natural polymers in bone tissue engineering.
J. Control Release 2021,338, 571–582. [CrossRef]
12.
Donnaloja, F.; Jacchetti, E.; Soncini, M.; Raimondi, M.T. Natural and Synthetic Polymers for Bone Scaffolds Optimization. Polymers
2020,12, 905. [CrossRef]
13.
Baino, F.; Novajra, G.; Vitale-Brovarone, C. Bioceramics and Scaffolds: A Winning Combination for Tissue Engineering. Front.
Bioeng. Biotechnol. 2015,3, 202. [CrossRef]
14.
Kilpadi, K.L.; Chang, P.-L.; Bellis, S.L. Hydroxylapatite binds more serum proteins, purified integrins, and osteoblast precursor
cells than titanium or steel. J. Biomed. Mater. Res. 2001,57, 258–267. [CrossRef]
15.
Family, R.; Solati-Hashjin, M.; Nik, S.N.; Nemati, A. Surface modification for titanium implants by hydroxyapatite nanocomposite.
Casp. J. Intern. Med. 2012,3, 460–465.
16.
Bose, S.; Fielding, G.; Tarafder, S.; Bandyopadhyay, A. Understanding of dopant-induced osteogenesis and angiogenesis in
calcium phosphate ceramics. Trends Biotechnol. 2013,31, 594–605. [CrossRef] [PubMed]
17.
Boanini, E.; Gazzano, M.; Bigi, A. Ionic substitutions in calcium phosphates synthesized at low temperature. Acta Biomater.
2010
,
6, 1882–1894. [CrossRef] [PubMed]
18. Shepherd, J.H.; Best, S.M. Calcium phosphate scaffolds for bone repair. JOM 2011,63, 83–92. [CrossRef]
19.
Miyaji, F.; Kono, Y.; Suyama, Y. Formation and structure of zinc-substituted calcium hydroxyapatite. Mater. Res. Bull.
2005
,40,
209–220. [CrossRef]
20.
Murakami, M.; Hirano, T. Intracellular zinc homeostasis and zinc signaling. Cancer Sci.
2008
,99, 1515–1522. [CrossRef] [PubMed]
Polymers 2023,15, 1370 15 of 18
21.
Mikael, P.E.; Amini, A.R.; Basu, J.; Arellano-Jimenez, M.J.; Laurencin, C.T.; Sanders, M.M.; Carter, C.B.; Nukavarapu, S.P. Func-
tionalized carbon nanotube reinforced scaffolds for bone regenerative engineering: Fabrication,
in vitro
and
in vivo
evaluation.
Biomed. Mater. 2014,9, 035001. [CrossRef]
22.
Yamaguchi, M.; Weitzmann, M.N. Zinc stimulates osteoblastogenesis and suppresses osteoclastogenesis by antagonizing NF-
κ
B
activation. Mol. Cell. Biochem. 2011,355, 179. [CrossRef]
23.
Grandjean-Laquerriere, A.; Laquerriere, P.; Jallot, E.; Nedelec, J.-M.; Guenounou, M.; Laurent-Maquin, D.; Phillips, T.M. Influence
of the zinc concentration of sol–gel derived zinc substituted hydroxyapatite on cytokine production by human monocytes
in vitro
.
Biomaterials 2006,27, 3195–3200. [CrossRef]
24.
Predoi, D.; Iconaru, S.L.; Predoi, M.V.; Motelica-Heino, M.; Guegan, R.; Buton, N. Evaluation of Antibacterial Activity of
Zinc-Doped Hydroxyapatite Colloids and Dispersion Stability Using Ultrasounds. Nanomaterials
2019
,9, 515. [CrossRef]
[PubMed]
25.
Liu, Y.-C.; Lee, Y.-T.; Huang, T.-C.; Lin, G.-S.; Chen, Y.-W.; Lee, B.-S.; Tung, K.-L. In Vitro Bioactivity and Antibacterial Activity of
Strontium-, Magnesium-, and Zinc-Multidoped Hydroxyapatite Porous Coatings Applied via Atmospheric Plasma Spraying.
ACS Appl. Bio Mater. 2021,4, 2523–2533. [CrossRef]
26.
Ohtsu, N.; Kakuchi, Y.; Ohtsuki, T. Antibacterial effect of zinc oxide/hydroxyapatite coatings prepared by chemical solution
deposition. Appl. Surf. Sci. 2018,445, 596–600. [CrossRef]
27.
Charlena; Suparto, I.H.; Kurniawan, E. Synthesis and Characterization of Hydroxyapatite-Zinc Oxide (HAp-ZnO) as Antibacterial
Biomaterial. IOP Conf. Ser. Mater. Sci. Eng. 2019,599, 12011. [CrossRef]
28.
Arciola, C.R.; Campoccia, D.; Montanaro, L. Implant infections: Adhesion, biofilm formation and immune evasion. Nat. Rev.
Microbiol. 2018,16, 397–409. [CrossRef] [PubMed]
29.
Ribeiro, M.; Monteiro, F.J.; Ferraz, M.P. Infection of orthopedic implants with emphasis on bacterial adhesion process and
techniques used in studying bacterial-material interactions. Biomatter 2012,2, 176–194. [CrossRef] [PubMed]
30.
Tripathy, A.; Sen, P.; Su, B.; Briscoe, W.H. Natural and bioinspired nanostructured bactericidal surfaces. Adv. Colloid Interface Sci.
2017,248, 85–104. [CrossRef] [PubMed]
31.
Cho, Y.S.; Kim, H.K.; Ghim, M.S.; Hong, M.W.; Kim, Y.Y.; Cho, Y.S. Evaluation of the antibacterial activity and cell response
for 3D-printed polycaprolactone/ nanohydroxyapatite scaffold with zinc oxide coating. Polymers
2020
,12, 2193. [CrossRef]
[PubMed]
32.
Lemire, J.A.; Harrison, J.J.; Turner, R.J. Antimicrobial activity of metals: Mechanisms, molecular targets and applications. Nat. Rev.
Microbiol. 2013,11, 371–384. [CrossRef]
33.
Saxena, V.; Hasan, A.; Pandey, L.M. Effect of Zn/ZnO integration with hydroxyapatite: A review. Mater. Technol.
2018
,33, 79–92.
[CrossRef]
34.
Kao, Y.-Y.; Chen, Y.-C.; Cheng, T.-J.; Chiung, Y.-M.; Liu, P.-S. Zinc Oxide Nanoparticles Interfere with Zinc Ion Homeostasis to
Cause Cytotoxicity. Toxicol. Sci. 2012,125, 462–472. [CrossRef]
35.
Guo, D.; Bi, H.; Liu, B.; Wu, Q.; Wang, D.; Cui, Y. Reactive oxygen species-induced cytotoxic effects of zinc oxide nanoparticles in
rat retinal ganglion cells. Toxicol. Vitro 2013,27, 731–738. [CrossRef] [PubMed]
36.
Wang, H.; Zhao, S.; Xiao, W.; Cui, X.; Huang, W.; Rahaman, M.N.; Zhang, C.; Wang, D. Three-dimensional zinc incorporated
borosilicate bioactive glass scaffolds for rodent critical-sized calvarial defects repair and regeneration. Colloids Surf. B Biointerfaces
2015,130, 149–156. [CrossRef]
37.
Blaschko, S.D.; Chi, T.; Miller, J.; Flechner, L.; Fakra, S.; Kapahi, P.; Kahn, A.; Stoller, M.L. Strontium substitution for calcium in
lithogenesis. J. Urol. 2013,189, 735–739. [CrossRef]
38.
Cannata-Andía, J.B.; Rodríguez-García, M.; Gómez-Alonso, C. Action mechanism of strontium ranelate. Rev. Osteoporos. Metab.
Miner. 2010,2, 5–9.
39.
Chattopadhyay, N.; Quinn, S.J.; Kifor, O.; Ye, C.; Brown, E.M. The calcium-sensing receptor (CaR) is involved in strontium
ranelate-induced osteoblast proliferation. Biochem. Pharmacol. 2007,74, 438–447. [CrossRef]
40.
Liao, J.; Blake, G.M.; McGregor, A.H.; Patel, R. The effect of bone strontium on BMD is different for different manufacturers’ DXA
Systems. Bone 2010,47, 882–887. [CrossRef] [PubMed]
41.
Querido, W.; Rossi, A.L.; Farina, M. The effects of strontium on bone mineral: A review on current knowledge and microanalytical
approaches. Micron 2016,80, 122–134. [CrossRef] [PubMed]
42.
Durmus, K.; Turgut, N.H.; Dogan, M.; Tuncer, E.; Ozer, H.; Altuntas, E.E.; Akyol, M. Histopathological evaluation of the effect of
locally administered strontium on healing time in mandibular fractures: An experimental study. Adv. Clin. Exp. Med.
2017
,26,
1063–1067. [CrossRef] [PubMed]
43.
Al-Duliamy, M.J.; Ghaib, N.H.; Kader, O.A.; Abdullah, B.H. Enhancement of orthodontic anchorage and retention by the local
injection of strontium: An experimental study in rats. Saudi Dent. J. 2015,27, 22–29. [CrossRef] [PubMed]
44.
Henriques Lourenço, A.; Neves, N.; Ribeiro-Machado, C.; Sousa, S.R.; Lamghari, M.; Barrias, C.C.; Trigo Cabral, A.; Barbosa,
M.A.; Ribeiro, C.C. Injectable hybrid system for strontium local delivery promotes bone regeneration in a rat critical-sized defect
model. Sci. Rep. 2017,7, 5098. [CrossRef]
45.
Kołodziejska, B.; St˛epie ´n, N.; Kolmas, J. The influence of strontium on bone tissue metabolism and its application in osteoporosis
treatment. Int. J. Mol. Sci. 2021,22, 6564. [CrossRef] [PubMed]
Polymers 2023,15, 1370 16 of 18
46.
Chandran, S.; Shenoy, S.J.S.; Nair, R.P.; Varma, H.K.; John, A. Strontium Hydroxyapatite scaffolds engineered with stem cells aid
osteointegration and osteogenesis in osteoporotic sheep model. Colloids Surf. B Biointerfaces 2018,163, 346–354. [CrossRef]
47.
Yudaev, P.; Chuev, V.; Klyukin, B.; Kuskov, A.; Mezhuev, Y.; Chistyakov, E. Polymeric Dental Nanomaterials: Antimicrobial
Action. Polymers 2022,14, 864. [CrossRef]
48.
Yudaev, P.; Mezhuev, Y.; Chistyakov, E. Nanoparticle-Containing Wound Dressing: Antimicrobial and Healing Effects. Gels
2022
,
8, 329. [CrossRef]
49.
Zhao, D.; Zhu, T.; Li, J.; Cui, L.; Zhang, Z.; Zhuang, X.; Ding, J. Poly(lactic-co-glycolic acid)-based composite bone-substitute
materials. Bioact. Mater. 2021,6, 346–360. [CrossRef]
50.
Gentile, P.; Chiono, V.; Carmagnola, I.; Hatton, P.V. An overview of poly(lactic-co-glycolic) Acid (PLGA)-based biomaterials for
bone tissue engineering. Int. J. Mol. Sci. 2014,15, 3640–3659. [CrossRef] [PubMed]
51.
Wei, J.; Yan, Y.; Gao, J.; Li, Y.; Wang, R.; Wang, J.; Zou, Q.; Zuo, Y.; Zhu, M.; Li, J. 3D-printed hydroxyapatite microspheres
reinforced PLGA scaffolds for bone regeneration. Biomater. Adv. 2022,133, 112618. [CrossRef]
52.
Park, J.-W.; Hwang, J.-U.; Back, J.-H.; Jang, S.-W.; Kim, H.-J.; Kim, P.-S.; Shin, S.; Kim, T. High strength PLGA/Hydroxyapatite
composites with tunable surface structure using PLGA direct grafting method for orthopedic implants. Compos. Part B Eng.
2019
,
178, 107449. [CrossRef]
53.
Cheng, Y.; Qin, J.; Huang, Y.; Wang, T. The antimicrobial effects of PLGA microspheres containing the antimicrobial peptide
OP-145 on clinically isolated pathogens in bone infections. Sci. Rep. 2022,12, 14541. [CrossRef]
54.
Chen, L.; Shao, L.; Wang, F.; Huang, Y.; Gao, F. Enhancement in sustained release of antimicrobial peptide and BMP-2 from
degradable three dimensional-printed PLGA scaffold for bone regeneration. RSC Adv. 2019,9, 10494–10507. [CrossRef]
55.
McLaren, J.S.; White, L.J.; Cox, H.C.; Ashraf, W.; Rahman, C.V.; Blunn, G.W.; Goodship, A.E.; Quirk, R.A.; Shakesheff, K.M.;
Bayston, R.; et al. A biodegradable antibiotic-impregnated scaffold to prevent osteomyelitis in a contaminated
in vivo
bone defect
model. Eur. Cells Mater. 2014,27, 332–349. [CrossRef]
56.
Cobb, L.H.; McCabe, E.M.; Priddy, L.B. Therapeutics and delivery vehicles for local treatment of osteomyelitis. J. Orthop. Res.
2020,38, 2091–2103. [CrossRef] [PubMed]
57.
Sinulingga, K.; Sirait, M.; Siregar, N.; Doloksaribu, M.E. Investigation of Antibacterial Activity and Cell Viability of Ag/Mg and
Ag/Zn Co-doped Hydroxyapatite Derived from Natural Limestone. ACS Omega 2021,6, 34185–34191. [CrossRef]
58.
Shimabukuro, M. Antibacterial property and biocompatibility of silver, copper, and zinc in titanium dioxide layers incorporated
by one-step micro-arc oxidation: A review. Antibiotics 2020,9, 716. [CrossRef]
59.
Hassan, M.; Sulaiman, M.; Yuvaraju, P.D.; Galiwango, E.; Rehman, I.U.; Al-Marzouqi, A.H.; Khaleel, A.; Mohsin, S. Biomimetic
PLGA/Strontium-Zinc Nano Hydroxyapatite Composite Scaffolds for Bone Regeneration. J. Funct. Biomater.
2022
,13, 13.
[CrossRef] [PubMed]
60.
Anwar, A.; Kanwal, Q.; Akbar, S.; Munawar, A.; Durrani, A.; Hassan Farooq, M. Synthesis and characterization of pure and
nanosized hydroxyapatite bioceramics. Nanotechnol. Rev. 2017,6, 149–157. [CrossRef]
61.
Dou, L.; Zhang, Y.; Sun, H. Advances in Synthesis and Functional Modification of Nanohydroxyapatite. J. Nanomater.
2018
,2018,
3106214. [CrossRef]
62.
Ofudje, E.A.; Adeogun, A.I.; Idowu, M.A.; Kareem, S.O. Synthesis and characterization of Zn-Doped hydroxyapatite: Scaffold
application, antibacterial and bioactivity studies. Heliyon 2019,5, e01716. [CrossRef] [PubMed]
63.
Liuyun, J.; Chengdong, X.; Lixin, J.; Lijuan, X. Effect of hydroxyapatite with different morphology on the crystallization behavior,
mechanical property and
in vitro
degradation of hydroxyapatite/poly(lactic-co-glycolic) composite. Compos. Sci. Technol.
2014
,
93, 61–67. [CrossRef]
64.
Resmim, C.M.; Dalpasquale, M.; Vielmo, N.I.C.; Mariani, F.Q.; Villalba, J.C.; Anaissi, F.J.; Caetano, M.M.; Tusi, M.M. Study of
physico-chemical properties and
in vitro
antimicrobial activity of hydroxyapatites obtained from bone calcination. Prog. Biomater.
2019,8, 1–9. [CrossRef]
65.
Xu, K.; Yuan, Z.; Ding, Y.; He, Y.; Li, K.; Lin, C.; Tao, B.; Yang, Y.; Li, X.; Liu, P.; et al. Near-infrared light triggered multi-mode
synergetic therapy for improving antibacterial and osteogenic activity of titanium implants. Appl. Mater. Today
2021
,24, 101155.
[CrossRef]
66.
Kurzyk, A.; Ostrowska, B.; ´
Swi˛eszkowski, W.; Pojda, Z. Characterization and Optimization of the Seeding Process of Adipose
Stem Cells on the Polycaprolactone Scaffolds. Stem Cells Int. 2019,2019, 1201927. [CrossRef]
67.
Sylvester, P.W. Optimization of the Tetrazolium Dye (MTT) Colorimetric Assay for Cellular Growth and Viability BT—Drug
Design and Discovery: Methods and Protocols. In Drug Design and Discovery: Methods and Protocols; Satyanarayanajois, S.D., Ed.;
Humana Press: Totowa, NJ, USA, 2011; pp. 157–168. ISBN 978-1-61779-012-6.
68.
Sari, M.; Hening, P.; Chotimah; Ana, I.D.; Yusuf, Y. Bioceramic hydroxyapatite-based scaffold with a porous structure using
honeycomb as a natural polymeric Porogen for bone tissue engineering. Biomater. Res. 2021,25, 2. [CrossRef]
69.
Ressler, A.; Ivankovi´c, T.; Polak, B.; Ivaniševi ´c, I.; Kovaˇci´c, M.; Urli ´c, I.; Hussainova, I.; Ivankovi´c, H. A multifunctional
strontium/silver-co-substituted hydroxyapatite derived from biogenic source as antibacterial biomaterial. Ceram. Int.
2022
,48,
18361–18373. [CrossRef]
70.
Fielding, G.A.; Roy, M.; Bandyopadhyay, A.; Bose, S. Antibacterial and biological characteristics of silver containing and strontium
doped plasma sprayed hydroxyapatite coatings. Acta Biomater. 2012,8, 3144–3152. [CrossRef] [PubMed]
Polymers 2023,15, 1370 17 of 18
71.
Maqbool, M.; Nawaz, Q.; Rehman, M.A.U.; Cresswell, M.; Jackson, P.; Hurle, K.; Detsch, R.; Goldmann, W.H.; Shah, A.T.;
Boccaccini, A.R. Synthesis, characterization, antibacterial properties, and
in vitro
studies of selenium and strontium co-substituted
hydroxyapatite. Int. J. Mol. Sci. 2021,22, 4246. [CrossRef]
72.
Stani´c, V.; Dimitrijevi´c, S.; Anti´c-Stankovi´c, J.; Mitri´c, M.; Joki´c, B.; Ple´caš, I.B.; Raiˇcevi´c, S. Synthesis, characterization and
antimicrobial activity of copper and zinc-doped hydroxyapatite nanopowders. Appl. Surf. Sci. 2010,256, 6083–6089. [CrossRef]
73.
Tank, K.P.; Chudasama, K.S.; Thaker, V.S.; Joshi, M.J. Pure and zinc doped nano-hydroxyapatite: Synthesis, characterization,
antimicrobial and hemolytic studies. J. Cryst. Growth 2014,401, 474–479. [CrossRef]
74.
Oshita, M.; Umeda, K.; Kataoka, M.; Azuma, Y.; Furuzono, T. Continuous antimicrobial mechanism of dispersible hydroxyapatite
nanoparticles doped with zinc ions for percutaneous device coatings. J. Biomater. Appl. 2022,37, 659–667. [CrossRef] [PubMed]
75.
Zare, E.N.; Jamaledin, R.; Naserzadeh, P.; Afjeh-Dana, E.; Ashtari, B.; Hosseinzadeh, M.; Vecchione, R.; Wu, A.; Tay, F.R.;
Borzacchiello, A.; et al. Metal-Based Nanostructures/PLGA Nanocomposites: Antimicrobial Activity, Cytotoxicity, and Their
Biomedical Applications. ACS Appl. Mater. Interfaces 2020,12, 3279–3300. [CrossRef] [PubMed]
76.
Valarmathi, N.; Sabareeswari, K.; Sumathi, S. Antimicrobial and larvicidal activity of zinc-substituted hydroxyapatite. Bull. Mater.
Sci. 2020,43, 218. [CrossRef]
77. Chereddy, K.K.; Vandermeulen, G.; Préat, V. PLGA based drug delivery systems: Promising carriers for wound healing activity.
Wound Repair Regen. 2016,24, 223–236. [CrossRef]
78.
Kapoor, D.N.; Bhatia, A.; Kaur, R.; Sharma, R.; Kaur, G.; Dhawan, S. PLGA: A unique polymer for drug delivery. Ther. Deliv.
2015
,
6, 41–58. [CrossRef]
79.
Ruirui, Z.; He, J.; Xu, X.; Li, S.; Peng, H.; Deng, Z.; Huang, Y. PLGA-based drug delivery system for combined therapy of cancer:
Research progress. Mater. Res. Express 2021,8, 122002. [CrossRef]
80.
Kattimani, V.S.; Kondaka, S.; Lingamaneni, K.P. Hydroxyapatite—Past, Present, and Future in Bone Regeneration. Bone Tissue
Regen. Insights 2016,7, BTRI.S36138. [CrossRef]
81.
Ha, S.-W.; Jang, H.L.; Nam, K.T.; Beck, G.R. Nano-hydroxyapatite modulates osteoblast lineage commitment by stimulation of
DNA methylation and regulation of gene expression. Biomaterials 2015,65, 32–42. [CrossRef]
82.
Abe, Y.; Okazaki, Y.; Hiasa, K.; Yasuda, K.; Nogami, K.; Mizumachi, W.; Hirata, I. Bioactive Surface Modification of Hydroxyapatite.
Biomed Res. Int. 2013,2013, 626452. [CrossRef]
83.
Wang, Q.; Tang, P.; Ge, X.; Li, P.; Lv, C.; Wang, M.; Wang, K.; Fang, L.; Lu, X. Experimental and simulation studies of
strontium/zinc-codoped hydroxyapatite porous scaffolds with excellent osteoinductivity and antibacterial activity. Appl. Surf.
Sci. 2018,462, 118–126. [CrossRef]
84.
Maleki-Ghaleh, H.; Siadati, M.H.; Fallah, A.; Koc, B.; Kavanlouei, M.; Khademi-Azandehi, P.; Moradpur-Tari, E.; Omidi, Y.;
Barar, J.; Beygi-Khosrowshahi, Y.; et al. Antibacterial and cellular behaviors of novel zinc-doped hydroxyapatite/graphene
nanocomposite for bone tissue engineering. Int. J. Mol. Sci. 2021,22, 9564. [CrossRef]
85.
Xue, W.; Moore, J.L.; Hosick, H.L.; Bose, S.; Bandyopadhyay, A.; Lu, W.W.; Cheung, K.M.C.; Luk, K.D.K. Osteoprecursor cell
response to strontium-containing hydroxyapatite ceramics. J. Biomed. Mater. Res. Part A
2006
,79A, 804–814. [CrossRef] [PubMed]
86.
Ahtzaz, S.; Nasir, M.; Shahzadi, L.; Amir, W.; Anjum, A.; Arshad, R.; Iqbal, F.; Chaudhry, A.A.; Yar, M.; ur Rehman, I. A study on
the effect of zinc oxide and zinc peroxide nanoparticles to enhance angiogenesis-pro-angiogenic grafts for tissue regeneration
applications. Mater. Des. 2017,132, 409–418. [CrossRef]
87.
Ito, A.; Kawamura, H.; Otsuka, M.; Ikeuchi, M.; Ohgushi, H.; Ishikawa, K.; Onuma, K.; Kanzaki, N.; Sogo, Y.; Ichinose, N.
Zinc-releasing calcium phosphate for stimulating bone formation. Mater. Sci. Eng. C 2002,22, 21–25. [CrossRef]
88.
Kojima, C.; Watanabe, K.; Murata, H.; Nishio, Y.; Makiura, R.; Matsunaga, K.; Nakahira, A. Controlled release of DNA from zinc
and magnesium ion-doped hydroxyapatites. Res. Chem. Intermed. 2019,45, 23–32. [CrossRef]
89.
Sergi, R.; Bellucci, D.; Candidato, R.T.; Lusvarghi, L.; Bolelli, G.; Pawlowski, L.; Candiani, G.; Altomare, L.; De Nardo, L.; Cannillo,
V. Bioactive Zn-doped hydroxyapatite coatings and their antibacterial efficacy against Escherichia coli and Staphylococcus aureus.
Surf. Coatings Technol. 2018,352, 84–91. [CrossRef]
90.
Fernandes, G.V.O.; Calasans-Maia, M.; Mitri, F.F.; Bernardo, V.G.; Rossi, A.; Almeida, G.D.S.; Granjeiro, J.M. Histomorphometric
analysis of bone repair in critical size defect in rats calvaria treated with hydroxyapatite and zinc-containing hydroxyapatite 5%.
Key Eng. Mater. 2009,396–398, 15–18. [CrossRef]
91.
Popa, C.L.; Deniaud, A.; Michaud-Soret, I.; Guégan, R.; Motelica-Heino, M.; Predoi, D. Structural and Biological Assessment of
Zinc Doped Hydroxyapatite Nanoparticles. J. Nanomater. 2016,2016, 1062878. [CrossRef]
92.
Ullah, I.; Siddiqui, M.A.; Kolawole, S.K.; Liu, H.; Zhang, J.; Ren, L.; Yang, K. Synthesis, characterization and
in vitro
evaluation of
zinc and strontium binary doped hydroxyapatite for biomedical application. Ceram. Int. 2020,46, 14448–14459. [CrossRef]
93.
Chaudhry, A.A.; Khalid, H.; Zahid, M.; Ijaz, K.; Akhtar, H.; Younas, B.; Manzoor, F.; Iqbal, F.; Ur Rehman, I. Zinc containing
calcium phosphates obtained via microwave irradiation of suspensions. Mater. Chem. Phys. 2022,276, 124921. [CrossRef]
94.
Fu, Y.; Chen, D.; Zhang, J.
In vitro
study on the cytotoxicity of strontium substituted hydroxyapatite. Shanghai Kou Qiang Yi Xue
2002,11, 229–232.
95.
Sun, L.; Li, T.; Yu, S.; Mao, M.; Guo, D. A Novel Fast-Setting Strontium-Containing Hydroxyapatite Bone Cement with a Simple
Binary Powder System. Front. Bioeng. Biotechnol. 2021,9, 643557. [CrossRef]
96.
Barman, N.; Salwa, M.; Ghosh, D.; Rahman, M.W.; Uddin, M.N.; Haque, M.A. Reference value for serum zinc level of adult
population in Bangladesh. Electron. J. Int. Fed. Clin. Chem. Lab. Med. 2020,31, 117–124.
Polymers 2023,15, 1370 18 of 18
97.
Somarouthu, S.; Ohh, J.; Shaked, J.; Cunico, R.L.; Yakatan, G.; Corritori, S.; Tami, J.; Foehr, E.D. Quantitative bioanalysis of
strontium in human serum by inductively coupled plasma-mass spectrometry. Futur. Sci. OA
2015
,1, FSO76. [CrossRef]
[PubMed]
98.
Grabska-Zieli ´nska, S.; Sionkowska, A.; Carvalho, Â.; Monteiro, F.J. Biomaterials with Potential Use in Bone Tissue Regeneration—
Collagen/Chitosan/Silk Fibroin Scaffolds Cross-Linked by EDC/NHS. Materials 2021,14, 1105. [CrossRef]
99.
Hatton, J.; Davis, G.R.; Mourad, A.-H.I.; Cherupurakal, N.; Hill, R.G.; Mohsin, S. Fabrication of Porous Bone Scaffolds Using
Alginate and Bioactive Glass. J. Funct. Biomater. 2019,10, 15. [CrossRef] [PubMed]
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