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Use of strontium doping glass-ceramic material for bone regeneration in critical

defect: In vitro and in vivo analyses

Panmella Pereira Maciel, Joyce Andreza Moreira Pessôa, Eudes Leonnan Gomes de
Medeiros, Andre Ulisses Dantas Batista, Lucas Ricardo Fernandes Figueiredo, Eliton
Souto de Medeiros, Dennis França de Oliveira Duarte, Adriano Francisco Alves,
Frederico Barbosa de Sousa, Basilio Rodrigues Vieira, Roberta Ferreti Bonan Dantas
Batista, Danyel Elias Cruz Perez, Romualdo Rodrigues Menezes, Lúcio Roberto
Cançado Castellano, Paulo Rogério Ferreti Bonan

PII: S0272-8842(20)31940-4
DOI: https://doi.org/10.1016/j.ceramint.2020.06.280
Reference: CERI 25707

To appear in: Ceramics International

Received Date: 19 March 2020

Revised Date: 8 June 2020
Accepted Date: 25 June 2020

Please cite this article as: P.P. Maciel, J.A. Moreira Pessôa, E.L. Gomes de Medeiros, A.U. Dantas
Batista, L.R. Fernandes Figueiredo, E. Souto de Medeiros, D. França de Oliveira Duarte, A.F. Alves,
F. Barbosa de Sousa, B.R. Vieira, R.F. Bonan Dantas Batista, D.E. Cruz Perez, R.R. Menezes,
Lú.Roberto. Cançado Castellano, Paulo.Rogé. Ferreti Bonan, Use of strontium doping glass-ceramic
material for bone regeneration in critical defect: In vitro and in vivo analyses, Ceramics International
(2020), doi: https://doi.org/10.1016/j.ceramint.2020.06.280.

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Use of strontium doping glass-ceramic material for bone regeneration in
critical defect: in vitro and in vivo analyses
Panmella Pereira Maciel1, Joyce Andreza Moreira Pessôa1, Eudes Leonnan Gomes
de Medeiros2, Andre Ulisses Dantas Batista3, Lucas Ricardo Fernandes Figueiredo3,
Eliton Souto de Medeiros3, Dennis França de Oliveira Duarte1, Adriano Francisco
Alves1, Frederico Barbosa de Sousa4, Basilio Rodrigues Vieira4, Roberta Ferreti
Bonan Dantas Batista3, Danyel Elias Cruz Perez5, Romualdo Rodrigues Menezes2,
Lúcio Roberto Cançado Castellano1, Paulo Rogério Ferreti Bonan1*

1
Laboratory for Cell Culture and Analysis (LACEC), Dentistry Graduation Program,
Universidade Federal da Paraíba, Paraiba, Brazil.
2
Laboraroty of Materials Technology (LTM), Department of Materials Engineering,
Universidade Federal de Campina Grande, Paraiba, Brazil.
3
LAMAB-Laboratory of Materials and Biosystems, Material Engineering Graduation
Program, Universidade Federal da Paraíba, Paraiba, Brazil.
4
Laboratory of Microscopy and Hard Tissues, Dentistry Graduation Program,
Universidade Federal da Paraíba, Paraiba, Brazil.
5
Laboratory of Oral Pathology, Dentistry Graduation Program, Universidade Federal
de Pernambuco, Pernambuco, Brazil.

*Corresponding author: DSc. Paulo Rogério Ferreti Bonan - Cidade Universitária,

Castelo Branco, Universidade Federal da Paraíba, Zip Code:58000-000, João
Pessoa, Paraíba, bonanpr@gmail.com
Abstract
The in vitro bioactivity and in vivo bone neoformation in critical-size bone
defects of a glass-ceramic material containing strontium ions (Sr2+) were
evaluated in the present study as well as the antimicrobial effect against oral
pathogens. A glass-ceramic bioactive material in powder (CP), based on the
composition of S53P4 bioactive glass, was produced by partially replacing
calcium with SrO using the sol-gel route. The amount of SrO added was
2.2wt.% (CPSr12.5) and 5.0wt.% (CPSr25). The amount of Sr2+ ions released
by the CP increase with time achieving 18 mg/L at 6 h and the release rate
decreased at longer times. pH values higher than 10 were obtained in the first
6 h, in agreement with an inhibitory microbial effect. These materials showed in
vitro bioactivity, with total surface coated by hydroxyapatite (HA) after 7 days of
immersion in simulated body fluid (SBF). The pH of SBF increased rapidly after
immersion of CP, reaching a maximum value of 8.72 after 168 h. HA formation
was observed in vitro for all samples. On microtomography and
histomorphometric analysis, CPSr25 showed higher values than CP without
Sr2+ (CPSr0) for bone volume (p=0.016), density (p=0.016) and neoformation
area (p=0.025) at 28 days. Histological analysis revealed higher degree of
vascularization after 28 days for CPSr25 when compared to CPSr0 (p=0.003).
In addition, CP showed an inhibitory effect on oral pathogens. Substitution of
CaO by SrO (CPSr25) presented the best results on the healing of critical-size
bone defects, as evidenced by microtomographic and histological analyses.
These data confirmed that higher concentrations of Sr2 + doped CP materials are
potential alternatives to improve bone healing and regeneration in critical-size
bone defects.

Keywords: Bioactive glass-ceramic, Sol-gel, Strontium, Bone Regeneration,

Critical Defects
1. INTRODUCTION

Successful reconstruction of critical-size bone defects caused by

traumatic lesions, inflammation, congenital deformity and tumor resection is a
problem which motivates the creation of new biomaterials [1]. The most
commonly used bone grafts for the rehabilitation of affected tissue form and
function could be autogenous, homogeneous, heterogeneous and alloplastic
[2]. Alloplastic grafts have been highlighted because of the limitations and risks
associated with other surrogates, such as limited bone supply, donor bank
morbidity, prolonged hospitalization, potential risks of infection, and rejection of
the immune system [3]. Synthetic materials most used in critical-size bone
defects include hydroxyapatite (HA), beta-phosphate-tricalcium (β-TCP),
metals, polymers and bioactive glasses (BG) [4].
S53P4 (53% silicon dioxide, 23% sodium oxide, 20% calcium oxide and
4% phosphorus pentoxide) is a bioactive glass (BG) generally produced by
melting and used as bone graft substitute. This material is one of the most
recently studied synthetic materials in researches addressing bone regeneration
because of potential osteoconduction [5], osteogenic differentiation of stem cells
[6], angiogenesis [5], and unlike other BGs, it has an antibacterial effect [7,8].
This antibacterial effect especially important for protection of biomaterial
surfaces against colonization of microorganisms. Moreover, BGs chemical
composition is responsible for the bioactive HA layer formation that binds
strongly to bone tissue [9,10], which is an advantage for applications in medical
areas [5,7,11] and dentistry [12,13,14].
Melting process is the standard method for BG production [15]. However,
this method presents drawbacks, resulting in chemically heterogeneous
materials, with possible contaminations [16] and defects in material integration
[17]. On other hand, sol-gel route offers an alternative method to traditional
procedure for preparing glass and glass-ceramic materials [16] and has
advantages of obtaining greater purity, homogeneity, composition control [18],
ease of microstructural alteration [15] and presence of porosity allowing an
increase of surface area [18]. In addition, gels containing sodium, calcium and
silicium stabilized at temperatures around 600 to 800ºC resulting in partially
crystalline materials, composed of highly bioactive crystalline phases, such as
Na2CaSi2O6 and Na2Ca2Si3O9 [15,16].
Different ions can be added to BG, such as zinc, magnesium, zirconia,
titanium, boron, copper, silver and strontium (Sr2+), leading to enhance
functionality and bioactivity [19]. Sr2+ ions are known for therapeutic potential on
bone strengthening via two mechanisms: osteoblastic differentiation and
apoptosis of osteoclasts by calcium-like (Ca2+) pathways [20].
Studies with addition of Sr2+ to BG demonstrate increased bone
formation, chemotaxis of osteoblasts [21], multiplication of stromal
mesenchymal cells [21,22] and stimulation of expression of osteoblast
differentiation markers [9,22]. Other in vitro studies demonstrated Sr2+
replacement of Ca2+ and delayed formation of a HA-like phase in simulated
body fluid (SBF) [10,23]. This behavior has been attributed to the inability to
form a precursor phase of octacalcium phosphate at lower Ca2+ concentrations
[23], indicating that more studies are necessary to define the influence of Sr2+
on bone regeneration, principally when used in critical-size defects.
Sr2+ doped S53P4 formulations have already been performed [10,24].
However, there are no evaluations of its use over glass-ceramics in powder
(CP) based on the composition of S53P4. It includes an animal model with
critical-size defects in rat calvaria. In addition, no studies verified antimicrobial
effect of this Sr2+ doped formulation against oral fungi, which can cause bone
infections [25,26]. Thus, the present work aimed to evaluate the in vitro
bioactivity and in vivo bone neoformation in critical-size bone defects in rat
calvaria of CP based on the composition of S53P4 doped by Sr2+, and its
antimicrobial effect against oral pathogens.

2. MATERIALS AND METHODS

2.1. Preparation of crystalline powders

 Tetraethyl orthosilicate (TEOS, 99%), triethyl phosphate (TEP), calcium
nitrate [Ca(NO3)2·4H2O], sodium nitrate [Na(NO3)] and strontium chloride (SrCl2)
(Sigma-Aldrich, St. Louis, MO, USA) were used for production of the studied
materials. Acetic acid (VWR Chemicals, 99.8%) was used as a catalyst [15].
TEOS was added to an aqueous solution of acetic acid, 0.1mol/L. The
relation between the mols number of water and TEOS was maintained at 35.
After 3h under vigorous magnetic stirring, the remaining reagents were added:
TEP, calcium nitrate and sodium nitrate, with interval between additions of 1h
and under constant agitation. For compositions containing Sr, strontium chloride
was added to replace 12.5 and 25% of the CaO by SrO in the final composition.
The produced materials were named as CPSr0, CPSr12.5 CPSr25 according to
the amount of SrO, 0%, 2.5% and 5wt.%, respectively. Table 1 presents the
compositions of the materials studied in the present work.
The solutions were oven-heated at 110° C for 48 h for gelation and
drying. After these processes, final materials were calcined at 800oC, with a
heating rate of 30oC/min and dwell time of 5 h. Calcination temperatures were
determined based on the thermogravimetric analysis of the gels. Materials were
desagglomerated and sieved using a 325 mesh sieve (45 µm) prior to other
studies.

2.2. Physical-chemical characterization of samples

Thermogravimetric analyses (TG/DTG) were performed (DTG-60H,

Shimadzu) using samples with about 5 mg held in platinum crucibles, from room
temperature to 1000ºC at a heating rate of 15oC/min. Crystallization behavior
was analyzed by X-ray diffraction (XRD) (XRD-6000, Shimadzu) operated with
copper K alpha radiation (Cu Kα, λ = 0.15418Å) and a monochromator. XRD
analyses was performed using fixed time scan mode with angular step size of
0.02o and counting time of 0.6s. The database of the International Center of
Diffraction Data, version 2.4, Software PCPDFWIN 2003, was used to
identifying the crystalline phases. Infrared spectra were obtained using Fourier
Transform Infrared (FTIR) spectrophotometer (IRPrestige-21, Shimadzu), using
KBr pellets, 64 scan, and resolution of 4cm-1. Particle size determination was
performed by analysing scanning electron microscopy (SEM) micrographs with
an image freeware (ImageJ; Version 1.48, NIH), measuring at least 100
individual particles for each sample at two different sites. Materials morphology
was assessed on gold-sputtered samples (layer thickness of 24 nm) using SEM
(Quanta 450, FEI).

Sr2+ release was determined by Flame Atomic Absorption Spectrometry

(FAAS) (Nov AAS3003, Analytik Jena). Briefly, 0.225g of each material doped
with Sr2+ (CPSr12.5 and CPSr25) was immersed in a beaker with 150mL of
deionized water under continuous agitation (mean rate of 75 rpm) at a
temperature of 22°C. Using a syringe, 50mL of the homogeneous solution (60,
360 and 1440 min) were removed. After each withdrawal, the solutions were
filtered with a millipore filter of 0.22μm [28]. The experiments were performed in
triplicate [28].

2.3. In vitro bioactivity test

Bioactivity was investigated by immersing pellets in simulated body fluid

(SBF) (pH 7.4) [29]. Pellets were produced by uniaxial pressing at 9.8 N for 20
min without binder to obtain scaffolds. Each sample was ultrasonically cleaned
for 10s in acetone prior to immersion in polyethylene bottles containing SBF
[15,16]. The specimens were immersed in SBF using a 2mg/mL ratio and kept
in a shaking incubator at 37oC [15] for 24, 72 and 168h [10]. At the end of each
test, samples were washed in acetone to eliminate the SBF and to terminate
any surface reaction [15,30]. After drying, samples were analyzed by XRD,
FTIR and SEM to evaluate the formation of hydroxycarbonate apatite (HCA).
SBF was analyzed after each period to determine variations in pH (pH21,
HANNA) due to the partial dissolution of the materials [10,15].

2.4. Antibacterial and antifungal assays

 Reference strains (ATCC - American Type Culture Collection - and UA -
University of Alabama) were used to evaluate the materials antimicrobial
potential. Staphylococcus aureus (ATCC 15656), Escherichia coli (ATCC
25922), Enterococcus faecalis (ATCC 14506) and Streptococcus mutans
(ATCC 11006), were cultured in Brain Heart Infusion (BHI) broth medium and
seeded on BHI agar (HiMedia Laboratories Pvt Ltd, India). Candida tropicalis
(ATCC 750), Candida glabrata (ATCC 2001), Candida albicans (ATCC 11006)
and Candida krusei (ATCC 34135), were cultured in Sabouraud Dextrose broth
medium and seeded on Sabouraud Dextrose Agar (SDB/SDA, Kasvi).
Antibacterial and antifungal activity was evaluated through microbial
death curve [31,32] and minimum inhibitory concentration (MIC) [33,34] and
minimal bactericidal/fungicide concentration (MBC/MFC) [35]. Growth controls
(BHI or SDB culture medium and inoculum) and sterility (BHI or SDB and
materials culture medium) were performed for study validation. The experiments
were performed in triplicate. Measurements of the pH values of solution
containing the powder material were performed to try to correlate with the
antimicrobial effect [7,36].

2.4.1. Microbial death curve

In 48-well plates containing 800μL of culture medium, 100 mg of the

materials were added to a 200μL aliquot of bacterial or fungal suspension (105
and 103 UFC/mL, respectively) in each well, resulting in a concentration of
100mg/mL [36]. Plates were incubated at 35-37 °C for 24 to 48 hours. Aliquots
of 10μL were seeded by an agar scattering method after serial dilution (10-1 to
10-21) at 1, 6, 12, 24, 48 and 72 h of incubation. Data were collected after 24h of
subcultures by counting of CFUs. Results were expressed in CFU/mL, through
the equation (Eq.1):
CFU/mL = (CFU/10n) x SA (Eq.1)
Where, n is the potency of the dilution performed and SA is an aliquot of
subculture plated on agar medium.
2.4.2. pH analysis

In tubes containing BHI broth medium and the materials (100 mg/mL), pH
values were measured at 0, 6, 12, 24, 48 and 72h to determine the pH changes
by a pHmeter (pH21, HANNA). pH values variation curve was subsequently
correlated with the determined microbial growth [7,36].

2.4.3. Macrodilution

Powders (100mg, 50mg, 25mg, 12.5mg and 6.25mg) were inserted into
48-well plates. Afterwards, 800μL of culture medium (BHI or SDB broth) was
added to each well. Next, the plates were incubated at 37°C for 48h to condition
the powders [7]. 200μL of the strain suspensions, previously standardized
(optical density equal to 0.5 McFarland) [33,34], were added to each well at the
final concentration of 105CFU/mL for bacteria and 103CFU/mL for yeast. Plates
were incubated in a bacteriological oven at 35-37 °C for 24-48 h. After this
period, MIC was determined visually and 10μL of the subcultures were plated
on agar medium and incubated under the above conditions. After the incubation
period, minimum bactericidal concentration (MBC) or minimum fungicidal
concentration (MFC) was defined as the lowest materials concentration, and
99.9% of colonies were eliminated [35].

2.5. In vivo mineralization experiment

2.5.1. Animals and Surgical procedure

Male Wistar rats (Rattus norvegicus albinus) weighing between 300 g

and 400g were submitted to intraperitoneal anesthesia [37]. Trichotomy was
performed on the calvaria region and skin antisepsis was performed with 2%
chlorhexidine digluconate. A 2-cm long bicoronal cutaneous incision was made,
extending from the skin to the fascial and periosteal layers overlapping sagittal
suture of the calvaria. Subcutaneous tissue, temporal muscle and periosteum
were divided to allow the visualization of the bone to be surgically exposed. Two
critical circular defects, each 7 mm in diameter, were performed on the medial
sagittal suture with a 6 mm trephine drill (Harte, Precision Grip, Brazil) coupled
to a portable surgical motor (Smart plus Maillefer, DENTSPLY, Switzerland),
under low turnover and abundant irrigation with 0.9% sterile saline solution [38].
To avoid damaging the underlying tissues, the bone was partially pierced with a
trephine and gently raised with morse tip scythe (Sickle Excavator Tip Morse
0/00, Millennium) to separate from the dura mater [37].
Animals were randomly divided into three groups and evaluated in 2
distinct periods (14 and 28 days), totalizing six groups (n=5). The groups
received different filling materials: blood clot (Clot), CPSr0 and CPSr25 [38]. For
the experimental groups treated with biomaterials, 20 mg of materials was
placed to fully occupy the created bone defect. A polypropylene mesh (Propy-
Mesh Flat Mesh Polypropylene Mesh, Atramat, Mexico) was positioned on top
of the filling material in order to promote a surgical area free from epithelial
invasion [39] and for protection of the dura mater. Skin suture was made with 3-
0 silk thread (TECHNEWl).
During the postoperative period, animals were placed in single-ply
polypropylene cages (Pinus Biotério – QUIMIA), with day/night cycle of 12/12h,
with facilitation of access to water and feed (QUIMIA). After surgery, a single
dose administration of 0.2 mL of the pentabiotic (Pentabiotico Veterinário Porte,
Fort Dodge Saúde Animal Ltda, Brazil) and 50 mg/kg of dipyrone were used,
every 8h for 5 days, both subcutaneously [40].
After 14 and 28 postoperative days, the animals underwent euthanasia in
a chamber by inhaling carbon dioxide (CO2). Skulls were dissected and
representative fragments of calvaria with the defect region were collected and
fixed on 10% formaldehyde for at least 48h [38]. The procedures followed
euthanasia practice guidelines of the National Animal Experimentation Control
Council and every effort was made to minimize suffering. The present research
was approved by the Ethics Committee on the Use of Animals (CEUA) of the
University Center of João Pessoa under the protocol number 002/2019.
2.5.2. Computerized Microtomography (Micro-CT)

To evaluate the mineralization of defects, four samples from each at 14

and 28 days, were analyzed by Micro-CT (SkyScan 1172, Bruker micro CT).
The following protocol was used: pixel size of 13.5μm, 80 kV, 124 μA, with a 0.5
mm aluminum filter. Scanning time was approximately 53 minutes, using 180°
of rotation and four projections at each 0.60 rotation. Samples were positioned
and fixed to a saddle device without movement during the tomographic capture.
At the end of the process, data on two dimensions were processed in the
NRecon software (Micro photonics) and repositioned on a dataviewer to align
the cutting planes. Using CT Analyzer software, the study region (ROI) with 7
mm of diameter and a fixed gray levels threshold of 0 to 57 was delimited.
Specimens were analyzed regarding bone volume (BV), bone volume density
(BVD) on Total Tissue Volume (TTV), Trabecular thickness (TB.TH), space
between the bone trabeculae (BT.SP) and number of bone trabeculae (NTB)
[41].

2.5.3. Histological Analysis

After the microtomographic analysis, all specimens fixed in buffered

formalin solution (10%) were placed in nitric acid (10%) solution for 3 days,
dehydrated and diaphanized. Calvaries were sectioned coronally, separating
two defects, anterior and posterior. Samples were included in paraffin and
coronal sections of 5μm [38] were made from the central area with greater bone
neoformation [42]. Histological slides were stained with Hematoxylin and Eosin
(H & E) and Masson's Trichrome (TM) [42]. Histological images were obtained
in 5X, 10X, 40X increments, later evaluated by two blinded examiners,
calibrated and experienced with the aid of an optical microscope (Carl Zeiss)
[43].
TM stained sections were histomorphometrically assessed on the 5X
objective, with the aid of the Image Analysis Software (ImageJ, National
Institutes of Health, USA), to establish the percentage of calculated bone
neoformation area. First, edges of the defects were delineated, and the
neoformed bone region (NB) was identified and quantified. Total area (TA) was
measured between the edges of defect. NBF (New Bone Formation) was
expressed as a percentage of TA [44], according to equation 2 (Eq.2):
NBF = NB / TA × 100 (Eq. 2)
Aspects verified during qualitative analysis were tabulated in a system of scores
based on degree of inflammation and vascularization. The morphological
analysis verified processes such as leukocyte migration, hemorrhage,
vasodilation and cell death (necrosis and apoptosis) [43], as well as count of
inflammatory cells and blood capillaries, using grades presented in the literature
[45], as described in Table 2.

2.6. Statistical analysis.

Results were analyzed with mean values, standard deviation and

standard error. Differences between groups were verified by analysis of
variance (ANOVA) followed by the Tukey's (p <0.05) or Kruskal-Wallis (p <0.05)
and multiple comparison tests using the GraphPad Prisma software program
(Graphpad Software, USA).

3. RESULTS AND DISCUSSION

3.1. Physical-chemical characterization

Figures 1 and 2 show thermogravimetric (TG) and differential thermal

analysis (DTA) curves of produced gel materials, respectively. For all samples,
loss of mass between 65°C and 155°C and in the range of 600°C to 800°C was
observed (Figure 2). Loss of mass up to 200oC was related to residual water
presented in the material. The loss between 600 and 750oC is related with
decomposition of carbonates formed during the gelation process as a
consequence of calcium presence; and the to decomposition of unreacted
Ca(NO3)2 [46]. The loss of mass at around 775oC (peak at DTG) was attributed
to the decomposition of sodium nitrate [17].
The glass transition temperature, Tg, of CPSr0 (S53P4) obtained by
the first derivative of DTA at the inflection temperature of the first negative peak
was 596oC. Literature [47,48,49] reported Tg values ranging from 547 to 608oC
for the S53P4, corroborating with the determined value. On the other hand,
authors [10] indicated a shift to lower temperatures in Tg of S53P4 glass
containing up to 15 wt.% of Sr, used in substitution to the calcium, due to a
decrease in the field strength of the cations. However, the influence of strontium
amount on the Tg of produced materials could not be performed because Tg
related peaks in derivative DTA were not observed in the CPSr12.5 and
CPSr25 samples.
Endothermic peaks ranging from 600 to 800oC were observed in the
DTA and are probably related to decomposition events, such as those observed
in TG, due to similarities in the events temperatures. The crystallization
temperatures are associated with exothermic events; however, these peaks
were not observed in the DTA curves. These events occurred in parallel with
the endothermic events and were probably masked by them.
The gels partially crystallized (Figure 3) forming sodium-calcium-silicate
phases, Na2CaSi2O6 (JCPDS 77-2189) and Na2Ca2Si3O9 (JCPDS 75-1687).
These phases are bioactive crystalline phases [27], and are also observed in
other studies [10,15,27] on bioactive glass-ceramic materials. This result
indicates that the thermal treatment at 800oC is appropriate for the development
of a bioactive glass-ceramic. Sr2+ doped samples presented Sr-containing
crystalline phases, SrSiO3 (JCPDS 30-1302) and Na2SrSi2O6 (JCPDS 32-
1159). A study [50] on bioactive glass doped with Sr2+ observed the
development of Sr2+ substituted phases when the gel was calcined at
temperatures higher than 600oC, such as Na2SrSi2O6. The present study
observed the presence of SrSiO3 when the heat treatment was at temperatures
above 800oC. Other studies also observed the development of SrSiO3 and
Na2SrSi2O6 when calcium is replaced by Sr in bioactive glass. This indicates
that the phase evolution observed in the present work is characteristic of
bioactive glass containing Sr2+. Moreover, it was observed that diffraction peaks
moved subtly to smaller values of 2θ angle with the increase of Sr2+
substitution, which can be explained by a larger Sr2+ atom size than substituted
Ca2+[22,23].
The FTIR spectra of samples (Figure 4) showing a region from 850 cm-1
to 1200 cm-1 characterizes silicate glasses due to bands associated with
symmetrical and asymmetrical stretching vibrations of the silica network
[27,28,51]. The 930 cm-1 band can be attributed to the binding of SiO2 to Na2+ or
Ca2+. The 1035 cm-1 and 694 cm-1 bands are related to asymmetric Si-O-Si
stretching and P-O-P, respectively [51,52]. The 1070 cm-1 band is associated
with the vibration of phosphate group [53]. The 530 and 625 cm-1 bands may be
related to the bonds representing combeite (Na2Ca2Si3O9 and Na2CaSi3O8)
[51]. The range between 1400 cm-1 and 1515 cm-1 is related to carbonate on a
glass structure [10]. The band centered at 3450 cm-1 is related to Si-OH
corresponding to the stretching vibrations of the hydroxyl groups. The spectra of
samples containing strontium show only subtle variations in the absorption
bands, with a 781 cm-1 band attributed to Sr-O binding.
Micrographs showed that samples were composed of particles that
exhibited irregular shapes, variable sizes and showed conglomeration (Figure
5). There is a broad range of particle sizes, from sub-micro particles to
micrometric particles (with sizes up to 20µm). The particle size analysis
presented mean particle size for the CPSr0, CPSr12.5 and CPSr25 of 2.3 ±
6.6μm, 2.9 ± 6.4μm and 4.2 ± 7.5μm, respectively. However, there was a high
amount of particles with sizes lower than 1μm. CPSr0, CPSr12.5 and CPSr25
had D50 (based on the number of particles) of 0.4μm, 0.5μm and 0.7μm,
respectively; and D90 of 4μm, 6.5μm and 13 μm, respectively. This indicates
high surface area and reactivity for the used bioactive glasses when comparing
with the other 45S5 and S53P4 containing strontium glasses reported in the
literature [10,47,49]. Coarse glasses with particle sizes in the range of 300 to
800μm and fine glasses with particle size < 45μm were reported [10,47,49].
Figure 6 showed Sr2+ released from samples containing Sr in distilled
water through FAAS. An increasing rate of dissolution of Sr2+ ions was observed
up to maximum at 360 min (6 hours) for two materials. During the following
minutes, this tendency started to change with a slight slope for the CPSr25 and
a marked fall for the CPSr12.5. The amount of strontium released in the
medium does not cause toxicity to living organisms [54]. The higher amount of
Sr2+ released by the CPSr25 and the similar microstructures of Sr2+ doped
samples indicate the use of CPSr25 over CPSr12.5 for use in vivo tests due to
possible higher and constant plasma concentrations tending to trigger better
biological responses due to higher availability in the medium.

3.2. In vitro mineralization bioreactivity test.

The morphology of powder surfaces (CPSr0, CPSr12.5 and CPSr25)

after immersion in SBF at 24, 72 and 168h was shown on the SEM micrographs
(Figure 7). The formation of a HA-compatible layer was observed in all samples
and confirmed by XRD and FTIR tests (Figures 8 and 9, respectively). There is
the growth of spherical agglomerates of HA which increases with incubation
time, covering the surfaces of the particles. Apparently, samples without
strontium developed a greater amount of hydroxyapatite on their surfaces.
Reflection peaks related of calcium carbonate (CaCO3, JCPDS 05-0586),
HA (Ca10(PO4)6(OH)2, JCPDS 09-4320) and beta-tricalcium phosphate
(Ca3(PO4)2, JCPDS 09-0169) were visualized in XRD patterns (Figure 8). Peaks
related to the original crystalline phases were also observed. At 168 h, more
expressive like-HA and beta-tricalcium phosphate peaks were observed in the
group without Sr2+, in agreement with that observed in the SEM micrographs.
FTIR spectra of samples after immersion in SBF were presented on
Figure 9. FTIR spectra showed adsorption bands at 565 and 605cm-1 in all
samples, which can be attributed to HA [16,55,56], 958 cm-1 and 575 cm-1 can
be attributed to the Si-O-Ca [53] and phosphate group elongation vibration,
respectively [53,57]. In addition, bands at 1411cm-1 were observed
corresponding to substitution of carbonate group into deposited mineral on
samples, confirming deposition of the carbonate HA [53]. The behavior of the
FTIR appears similar to those of XRD and SEM analyses, indicating a lower
amount of hydroxyapatite with an increase of Sr2+ content in the CP. Moreover,
an absorption band at 620 cm-1 (CPSr0, CPSr12.5 and CPSr25), characteristic
of phosphate in the crystalline phase [16] was observed; and peaks of SiO2
gradually disappeared with the appearance of calcium phosphate phases [55],
as observed in the 930 and 1035 cm-1 peaks obtained for the untreated
samples, which disappeared after 24h of immersion in SBF.
pH values of the SBF after samples immersion increased with time on all
groups (Table 3). pH of the control (SBF) was stable around 7.4 during all study
periods (24, 72 and 168h). pH of systems increased rapidly in the first 24h, and
achieved a higher value in the CPSr25, but at 168h all samples presented
similar pH. The substitution of CaO by SrO in the S53P4 increases the
dissolution rate in the SBF [10]. Studies [58] also observed that substituting Sr
for Ca increases the dissolution rate and ion release in Tris buffer due an
expansion of the glass network caused by the larger ionic radius of Sr ions
compared with the Ca. These higher release and dissolution rates can explain
the higher pH value of the CPSr25. However, similar pH at 168h indicates that
the higher development of HA in the CPSr0 is not only related with the pH of the
medium.
Studies [10,23] on Sr2+ replacement of calcium on bioactive glass
observed a decrease in the amount of produced HA in simulated body fluid
(SBF) for Sr2+ doped samples, such as observed by SEM, FTIR and XRD.
However, lower HA layers found in Sr2+ doped CP can facilitate the dissolution
of biomaterials and improve bioactivity [10], which indicates the necessity of in
vivo studies for a more appropriated evaluation of the influence of Sr2+ on the
performance of bioactive glass.

3.3. Antibacterial and antifungal assays

Figure 10 showed growth/death curves of S. aureus, S. mutans, E. coli,

E. faecalis, C. albicans, C. glabrata, C. krusei and C. tropicalis when exposed to
culture medium (growth control) and to the produced materials. All
microorganisms under ideal conditions (temperature and culture medium)
showed increasing growth in the first 12 hours, and then stabilized between 48-
72 hours, except for C. glabrata and C. tropicalis, which showed increasing
growth. These microorganisms are frequently found in bone infections
[25,26,36,59] and the samples inhibited the growth of all microorganisms,
demonstrating complete inhibition in first the 6h, except for S. mutans when
exposed to the CPSr12.5 (complete inhibition in 24h) and CPSr25 (complete
inhibition at 48h) and E. faecalis when exposed to the CPSr0 (complete
inhibition in 24h), CPSr12.5 and CPSr25 (complete inhibition in 72h).
Studies [7,32,36] evaluating antimicrobial activity of S53P4 glass by
means of death curves demonstrated high bacterial inhibition when using
concentrations four [7,32] and eight times [32] greater than 100 mg/mL. These
used concentrations were indicated to be effective on microbial inhibition [36].
Indeed, CFU was lower for all microorganisms after being exposed to samples
in the first 6h in relation to the growth control. This data indicates high inhibitory
effect and corroborates previous studies with the same formulation [7,8].
pH measurements were shown on Figure 11. pH was 7.42 (± 0.01) in the
BHI medium. A rapid increase in the pH in the first 6h (11.35 ± 0.01, 10.73 ±
0.01 and 10.43 ± 0.02, respectively) was observed when samples were
immersed in the medium. pH rapid increase in the first 6h and reached a
maximum value of 11.94 ± 0.01 (CPSr0), 11.28 ± 0.00 (CPSr12.5) and 11.14 ±
0.00 (CPSr25) after 72h. Mediums with pHs higher than 10 is considered to
present effective conditions to antimicrobial activity [7], and the achieved pHs
may explain part of the antibacterial effect of the studied materials. A
decreasing of pH values with the substitution of CaO by SrO was observed,
which may explain the delay in antimicrobial effect of samples with Sr2+. An
increase in pH values for times longer than 24h, but at lower rates, with
tendency to stabilization, was also observed.
The minimum inhibitory concentration (MIC) and minimal bactericidal
(MBC) or fungicide concentration (MFC) are presented in Tables 4 and 5. The
6.25 mg/mL concentration showed inhibition on all microorganisms except for E.
coli for the CPSr25 and E. faecalis in all groups. Study [31] demonstrated that
the increase of Sr2+ in cements containing bioactive glass increase the
bactericidal action. This data does not corroborate with the present study, since
there was a delay in inhibition for E. faecalis and S. mutans when treated with
CP containing Sr2+ (Figure 10). No inhibitory effect was demonstrated at
concentrations lower than those observed for the CPSr0 (Tables 4 and 5).
However, it was observed that the substitution of Ca2+ by Sr2+ did not interfere
with the antimicrobial effect of the materials since with low concentrations, the
samples were able to inhibit and cause death to all tested microorganisms.
It is worth mentioning the importance of the present study when
evaluating samples with a composition equals to the S53P4 produced by the
sol-gel route on fungi of the genus Candida. These microorganisms can
colonize damaged tissue and cause surgical complications, such as bone
infections (osteomyelitis) [25,26]. In addition, an increase in resistance of oral
fungal infections to antifungal agents has been reported in the literature [60]. It
was observed that lower concentration samples were able to inhibit and cause
yeast death. In addition, since antimicrobial effect of these materials is mainly
due to pH and osmotic pressure variation, microorganisms could not develop
resistance [36].

3.4. In vivo mineralization experiment

3.4.1. Computational Microtomography (Micro-CT)

Figure 12 schematized representative 3D images obtained from critical

defects created in calvaria of animals treated with clot, CPSr0 or CPSr25 and
evaluated 14 and 28 days after surgical procedures. Bone regeneration was
higher in the group implanted with CPSr25, although all groups showed bone
healing capacity, with superior mineralization and bone maturation during the
progression of periods.
Figure 13 presents the microtomographic parameters. CPSr25 was
statistically different from clot in 14 and 28 days for the BV (p = 0.0328, p =
0.0016) and PBV (p = 0.029, p = 0.0016), and in 28 days for TB.N (p = 0.0159).
On the other hand, the Clot group showed a higher TB.SP in relation to the
CPSr0 and CPSr25 (p = 0.029; p = 0.0098) in 14 days. The CPSr25, when
compared to the CPSr0, showed higher values for the BV (p = 0.0166) and PBV
(p = 0.0168) in 28 days. An increased speed and quality of bone regeneration
and remodeling through recovery of trabecular connectivity was noticed.
For the first time, bone regenerative potential of Sr2+ ion substitution in
CP equal to S53P4 was investigated using a critical-size bone defect model in
rat calvaria. Critical-size defects represent validated experimental models since
they do not present spontaneous bone regeneration [61]. However, even in a
critical defect (≥ 5 mm in diameter) [62], there was a discrete bone formation in
the Clot group. Polypropylene mesh may have influenced, due to protecting
bone shop from invasion of connective tissue (guided bone regeneration) [39].
Bone regeneration was higher in the group implanted with CPSr25, although all
groups showed bone healing capacity, with superior mineralization and bone
maturation during the progression of periods. Results of microtomographic
analysis indicated Sr2+ doping has a greater bone regenerative potential. Similar
results were seen in a previous study that demonstrated an increase in the
speed and quality of bone regeneration and remodeling [9].

3.4.2. Histological Analysis

Histological sections of the Clot, CPSr0 and CPSr25 groups were stained
by TM and H&E (Figure 14). TM staining showed a tendency for formation of a
single bone block at the edges of defect and presence of dense connective
tissue connecting calcification centers, with bone neoformation directed to the
center. Neoformed bone tissue during both periods showed many osteocytes
and irregular bone gaps, characterizing an immature pattern. There were no
outbreaks of bone necrosis in the specimens, confirming absence of local
toxicity of samples, doped or not with Sr2+. The biomaterials were completely
reabsorbed at 14 days.
Greater bone neoformation was observed in the groups, mainly for the
CPSr25 at 14 and 28 days (Figure 15). Significant differences between groups
implanted with CP were seen in the 28-day period (p=0.025). These results
confirmed the data obtained in the micro-CT, in which, a higher BV and PBV for
the CP groups, especially on doped products were found. Differently from the
studies which improvements in bone quality after doping were not obtained
[54,63], biocompatibility and bone repair potential is verified in the present
study, and substitutions of 25 mol% Sr2+ are safe for therapeutic applications
[9,21,24].
The good results obtained in the CPSr25 group, verified in the
microtomographic and histometric analyses, can be explained by the higher rate
of initial dissolution in medium and constant formation of a thinner layer of HA
that allows a prolonged bioactivity, as previously seen in the literature [10]. This
finding can be explained due to the ability of Sr2+ doped CP to stimulate the
expression of the genes Alpl, Col1a1, Bglap and Runx2 in the periods of 7 to 14
days [9, 22]. Alpl, Col1a1, Bglap and Runx2 are markers of early osteoblastic
differentiation, presence of pre-osteoblasts and initial deposition of osteoid, late
stage of osteogenic differentiation, and regulation of osteogenesis, respectively
[64]. This cascade of markers directly influences osteogenic proliferation and
differentiation, conditioning better bone repair abilities in vivo [9].
An increase in vascularization at 14 to 28 days and absence of
inflammation during two periods for the Clot group was observed (Figure 16).
CPSr0 showed a discrete periosteal inflammatory exudate predominantly
macrophagic immersed in neoformed connective tissue with abundant presence
of young vessels in 14 days and, in 28 days, abundant mononuclear
inflammation with rare lymphocytes and few blood vessels. In the CPSr25
group, there was abundant mononuclear exudate infiltrated in a connective
tissue rich in blood capillaries on periosteum at 14 and 28 days. We found
higher values for the CPSr25 in relation to the Clot group for the degree of
inflammation in both periods (p=0.0005; p=0.0036) and for the degree of
vascularization at 14 days=0.0036). Degree of vascularization was significantly
higher for the CPSr25 group than the CPSr0 only in 28 days (p=0.0036). The
greater and constant degree of vascularization and inflammation verified in the
CPSr25 group were related to significant bone gains. When in contact with
biomaterials, it is essential for biodegradation process of grafts in the signaling
of growth factors and in bone regeneration [65].

According to this mechanism of biological response, a greater degree of

angiogenesis allows an efficient supply of oxygen and nutrients to the bone
defect through release of angiogenic mediators [66,67]. It is suggested that
bioactive silica-based materials with presence of a Sr2+ ions matrix promote
initial angiogenesis by regulating the phenotype of macrophages that express
high levels of platelet - derived growth factor - BB (PDGF) [67], and production
of vascular endothelial growth factors (VEGF) and angiopoietin 1 (Ang-1) [66],
giving efficient vascularization to new bone formation.

5. CONCLUSIONS

In the present study, we evaluated the effect of the addition of Sr2+ ions in
the CP with the composition of S53P4 on the physicochemical characteristics,
in the in vitro bioactivity tests and on bone regeneration of critical-size defects in
rat calvaria. Samples were successfully doped with Sr2+ and presented a
bioactive crystalline phase, with an irregular porous surface and presented high
antibacterial and antifungal action. Although delayed inhibition of S. mutans and
E. faecalis was observed, this did not interfere with the final antimicrobial effect.
Materials doped with Sr2+ presented bioactivity under in vitro tests with the
development of HA and beta-tricalcium phosphate on powder surface after 168
hours. The substitution of CaO by SrO (CPSr25) presented the best results in
the repair of critical-size bone defects in the in vivo studies, from as evidenced
by the microtomographic and histological results. Therefore, Sr2+ doping CP are
good candidates for a variety of dental applications, with bone and infectious
involvement.

ACKNOWLEDGEMENTS
The authors are grateful to the Brazilian research funding agency CNPq, grant
nos. 308822/2018-8 and 420004/2018-1, for the financial support.
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Table 1. Compositions of the studied materials (wt.%).

Components SiO2 Na2O CaO P2O3 SrO

CPSr0a 53 23 20 4 0

CPSr12.5 53 23 17.5 4 2.5

CPSr25 53 23 15 4 5.0
a
S53P4 bioactive glass composition
Table 2. Grades atributted to the histological evaluation. Adapted from Mendes
et al. (2009).45

Score Histological evaluation

(a) Inflammation index

1 Absence of inflammatory cells

2 Slight (up to 15 inflammatory cells at 10X)

3 Moderate (up to 30 inflammatory cells at 10X)

4 Abundant (more than 30 inflammatory cells at 10X)

(b) Vascularization Grade

1 Absence of capillary vessels

2 Slight (less than capillary vessels at 40X)

3 Moderate (3 to 5 capillary vessels at 40X)

4 Abundant (more than 10 capillary vessels at 40X)

Table 3. pH values of groups (2 mg/mL) in simulated body fluid (SFB / pH = 7.4
- Control).

Samples 24 h 72 h 168 h

CPSr0 8.31 ±0.01 8.37 ±0.01 8.72 ±0.03

CPSr12.5 8.33 ±0.09 8.37 ±0.01 8.70 ±0.02

CPSr25 8.39 ±0.04 8.40 ±0.03 8.70 ±0.03

Control 7.40 ±0.01 7.40 ±0.02 7.40 ±0.01

Table 4. Minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) of the samples (mg/mL) against S. aureus, E. coli, E.
faecalis and S. mutans and controls sterility of samples (GC) and growth of
microorganisms (SC). The signs (-) means negative and (+) positive growth.

Powder S. aureus E. coli E. faecalis S. mutans

MIC MBC MIC MBC MIC MBC MIC MBC

CPSr0 6.25 6.25 6.25 12.50 25.00 100.00 6.25 6.25

CPSr12.5 6.25 25.00 6.25 6.25 25.00 100.00 6.25 6.25

CPSr25 6.25 12.50 12.50 25.00 50.00 100.00 6.25 6.25

GC + + + + + + + +

SC - - - - - - - -
Table 5. Minimum inhibitory concentration (MIC) and minimum fungicidal
concentration (MBC) of the samples (mg/mL) against C. albicans, C. glabrata,
C. krusei and C. tropicalis and controls sterility of samples (SC) and growth of
microorganisms (GC). The signs (-) means negative and (+) positive growth.

Powder C. albicans C. glabrata C. krusei C. tropicalis

MIC MFC MIC MFC MIC MFC MIC MFC

CPSr0 6.25 12.50 6.25 12.50 6.25 12.50 6.25 12.50

CPSr12.5 6.25 12.50 6.25 6.25 6.25 25.00 6.25 6.25

CPSr25 6.25 25.00 6.25 12.50 6.25 12.50 6.25 25.00

GC + + + + + + + +

SC - - - - - - - -
Figure 1. Thermogravimetric (TG/DTG) curves of the studied samples.
Figure 2. DTA thermograms of the studied samples.
Figure 3. XRD patterns of the samples thermal treated at 800 °C (Na2CaSi2O6
(●), Na2Ca2Si3O9(○), SrSiO3 (□), Na2SrSi2O6 (■)).
Figure 4. FTIR spectra of the samples treated at 800 °C.
Figure 5. Micrographs (SEM) of the powder samples.
Figure 6. Release of Sr2+ ion from the CPSr12.5 and CPSr25 in distilled water
over a period of 60 (1h), 360 (6h) and 720 (12h) minutes. The release of Sr2+
ions was reported in mg/ L.
Figure 7. Micrographs (SEM) of the powders samples after immersion in
simulated body fluid (SBF) for 24 (above), 72 (middle) and 168h (below).
Figure 8. XRD patterns of the samples after immersion in simulated body fluid
(SBF) for 24, 72 and 168h (Na2CaSi2O6 (●), Na2Ca2Si3O9(○), SrSiO3 (□),
Hydroxyapatite (Ca10(PO4)6(OH)2) (X), Calcium Carbonate (C), Beta Tricalcium
Phosphate (T).
Figure 9. FTIR spectra of the samples after immersion in simulated body fluid
(SBF).
Figure 10. Death curve of microorganisms exposed to the evaluated samples
(CFU/mL in relation to time (h)).
Figure 11. pH values of the samples (100 mg/mL) in culture medium (BHI broth
– Control).
Figure 12. 3D images representative of Micro-CT analysis of rat calvarial
defects after 14 days (14 d) and 28 days (28 d) in the Blood Clot (Clot), CPSr0
and CPSr25 groups.
Figure 13. Micro-CT anaylsis considering the following parameters: (A) Bone
Volume, (b) Percent Bone Volume, (c) Trabecular Number, (c) Trabecular
Number, (c) Trabecular Number, d) Trabecular Thickness and (e) Trabecular
Separation. Values of the mean and standard error, One-way ANOVA followed
by the Tukey's post-test for comparison between groups, being * p ≤ 0.05 and **
p <0.01 for comparisons between the Clot, CPSr0 and CPSr25 groups.
Figure 14. Histological analysis of skull defects treated with clot, CPSr0 and
CPSr25 in the periods of 14 days and 28 days, postoperatively. Histological
sections were stained with Hematoxylin and Eosin, and Masson's Trichrome.
(Magnification = 5x). Caption: v (new bone formation), CT (connective tissue), --
- (border of the bone defect).
Figure 15. Histomorphometric analysis of NBF (New Bone Formation). Values
of the mean and standard error, One-way ANOVA followed by the Tukey’s post-
test for comparison between groups, being * p ≤ 0.05, ** p <0.01 and *** p
<0.005 for comparisons between the Clot CPSr0 and CPSr25 groups.
Figure 16. Histological analysis through the degree of inflammation and degree
of vascularization. Values of mean and standard error, Kruskal-Wallis followed
by Dunn’s post-test for comparison between groups, being * p <0.05, ** p <0.01
and *** p <0.005 for comparisons between the Clot, CPSr0 and CPSr25 groups.
 Declaration of interests

The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

Paulo Rogério Ferreti Bonan

Universidade Federal da Paraíba