Received: 19 May 2019 Revised: 26 July 2019 Accepted: 17 August 2019

DOI: 10.1002/jbm.b.34484

ORIGINAL RESEARCH REPORT

Doxycycline containing hydroxyapatite ceramic microspheres

as a bone-targeting drug delivery system

Carlos Soriano-Souza1 | Helder Valiense2 | Elena Mavropoulos3 |

Victor Martinez-Zelaya4 | Andrea Machado Costa5 | Adriana T. Alves6 |
Mariana Longuinho1 | Rodrigo Resende7 | Carlos Mourão8 | Jose Granjeiro9 |
Maria H. Rocha-Leao10 | Alexandre Rossi1 | Mônica Calasans-Maia11

1
Department of Applied Physics, Brazilian
Center for Physics Research, Rio de Janeiro, Abstract
Brazil Drug delivery technology is a promising way to enhance the therapeutic efficacy of
2
Dentistry School, Federal Fluminense
drugs. The purpose of this study is to evaluate the physical and chemical properties of
University, Rio de Janeiro, Brazil
3
LABIOMAT, Brazilian Center for Physics hydroxyapatite ceramic microspheres loaded with doxycycline (HADOX), their effects
Research, Rio de Janeiro, Brazil on in vitro osteoblast viability, and their antimicrobial activity, and to determine the
4
Department of Physics, Brazilian Center for
effects of DOX on the healing of rat sockets after tooth extraction. The internal micro-
Physics Research, Rio de Janeiro, Brazil
5
CBPF, COMAN, Rio de Janeiro, Brazil
sphere porosity was sensitive to the treatment used to adsorb DOX onto microsphere
6
Department of Oral Pathology, Federal surface; HA microspheres without DOX presented 26% of pores, whereas HADOX0.15
Fluminense University, Rio de Janeiro, Brazil microspheres presented 52.0%. An initial drug release of 49.15 μg/ml was observed in
7
Oral Surgery Department, Fluminense Federal
the first 24 hr. The minimal inhibitory concentration (MIC) tested against Enterococcus
University, Rio de Janeiro, Brazil
8
Unidade de Pesquisa Clínica, Universidade faecalis demonstrated that bacterial growth was inhibited for up to 7 days. Results of cell
Federal Fluminense, Rio de Janeiro, Brazil viability and cell proliferation did not indicate statistical differences in the metabolic
9
Department of Bioengineering, National
activity of HADOX samples relative to HA without DOX microspheres (p > .05). After
Institute of Metrology, Standardization and
Industrial Quality, Rio de Janeiro, Brazil 1 week, a discreet inflammation reaction was observed in the control group, and after
10
Chemistry School, Federal University of Rio 6 weeks, newly-formed bone was observed in the HADOX0.15 (p < .05). The HADOX
de Janeiro, Rio de Janeiro, Brazil
11
did not interfere in the bone repair and controlled the early inflammatory response.
Oral Surgery Department, Dentistry School,
Fluminense Federal University, Rio de Janeiro, HADOX could be a promising biomaterial to promote bone repair in infected sites.
Brazil
KEYWORDS
Correspondence
biocompatibility, cytotoxicity, doxycycline, drug delivery, hydroxyapatite
Mônica Calasans-Maia, Oral Surgery
Department, Dentistry School, Fluminense
Federal University, Rio de Janeiro, Brazil.
Email: monicacalasansmaia@gmail.com

1 | I N T RO D UC T I O N must be evaluated. The surgeon may decide among a variety of available

Congenital diseases, infections, traumatic, and neoplastic processes repre-
sent the most important challenges in reconstructive treatment of bone
defects (Calasans-Maia et al., 2014). The size of the defect is a critical fac-
tor to consider. A circumferential bone loss of 50% or length of 2 cm2 has
been proposed as the point where reconstruction may be required for
human adult patients, although age, glycemic control, and vascular supply
strategies, such as (a) autologous bone graft (for defects <5 cm); (b) syn-
thetic or xenogeneic bone grafts, (c) induction of tissue membrane forma-
tion containing fibroblastic cells, type-1 collagen and growth factors
(Maquelet technique); and (d) delivery of growth factors or bone morpho-
genetic proteins (Mauffrey, Barlow, & Smith, 2015; Sheikh et al., 2017).
Considering synthetic bone grafts, many biomaterials including
polymers, bioceramics, and composites could be applied as a carrier

J Biomed Mater Res. 2019;1–12. wileyonlinelibrary.com/journal/jbmb © 2019 Wiley Periodicals, Inc. 1

2 SORIANO-SOUZA ET AL.
for drug delivery systems. The physical and chemical properties of
these materials such as porosity, interconnected porous, and high sur-
face to volume ratio lead to their effectiveness as a carrier for delivery
of therapeutic agents such as drugs, genes, antigens, enzymes and
other proteins (Jafari & Adibkia, 2014; Soriano-Souza et al., 2015;
Thomas, Harshita, & Mishra, 2015).
Hydroxyapatite (HA), composed of Ca10(PO4)6(OH)2 mineral
matrix that resembles the crystallographic structure of natural bone
and teeth, is widely used as a synthetic bone graft because of its
excellent biocompatibility, absence of toxicity and immunogenicity,
low biodegradability, bone affinity, and lack of inflammatory behavior
(Wang et al., 2011). Previous studies have demonstrated that HA is
indicated as a delivery system for antimicrobial and antibacterial
agents to simultaneously stimulate bone regeneration and prevent
infection (Bose & Tarafder, 2012; Habraken, Wolke, & Jansen, 2007;
Soriano-Souza et al., 2015; Tautzenberger, Kovtun, & Ignatius, 2012).
Tetracycline is a class of antibiotics with a broad spectrum, where
the antimicrobial activity is caused by the inhibition of the bacterial
protein synthesis in a selective form in eukaryotic cells. Tetracycline,
oxytetracycline, doxycycline (DOX), and minocycline are examples of
drugs belonging to this group (Brion & Berthon, 1981).
DOX is a broad-spectrum antibiotic that is clinically applied as an
adjunct to root planning or surgical debridement in order to treat peri-
odontitis and peri-implantitis (Golub et al., 2016). DOX is used in the
treatment of both Gram-positive and Gram-negative bacteria and
inhibits the bacterial protein biosynthesis (Injac, Djordjevic-Milic, &
Srdjenovic, 2007). In addition to its antibacterial action by inhibiting
microbial protein synthesis, DOX suppresses polymorphonuclear leu-
kocytes and anti-collagenase from the altered regulation of cytoplas-
mic calcium mediated by osteoclasts, further inhibiting bone
resorption (Golub et al., 2001). Furthermore, Kinugawa et al. (2012)
have shown that DOX inhibits osteoclast differentiation. These stud-
ies indicate beneficial effects of DOX on bone regeneration. The
effect of DOX on promoting bone formation after implant placement
is expected to be due to its inhibitory properties on inflammation and
osteoclastogenesis (Ding et al., 2018; Walter et al., 2014).
Many studies have investigated combinations of DOX with different
calcium phosphates such as poly (lactide-co-glycolide) encapsulated
HA microspheres (Wang et al., 2012) polycaprolactone/gelatin/HA
nanofibers (Ramírez-Agudelo et al., 2018), and calcium phosphate
substituted with strontium (Alkhraisat et al., 2010). The purpose of these
studies was to promote the incorporation of the antibiotic into calcium
phosphates, which allows adequate release of the entrapped substance
to the specific location.
Regenerative medicine has focused on developing biomaterials
with multiple purposes in order to promote bone tissue regeneration
and provide infection treatment or prevention at the same time.
Soriano-Souza et al. (2015) showed that the low chlorhexidine
(HACHX) loading (0.12%) dose did not impair the biocompatibility and
osteoconductivity of HA during bone repair. These results also indi-
cate that high antimicrobial doses (2.0%) may activate a strong local
inflammatory response and disrupt the long-term osteoconductive
properties of HACHX delivery systems (Soriano-Souza et al., 2015).
The aims of the present study were to characterize the adsorption
pattern of DOX molecules on the HA surface, to evaluate the release
of the drug, to determine its effects on the cell viability of osteoblasts
in vitro, to estimate antimicrobial activity, and to study the effects of
HA microspheres containing DOX in the healing of the alveolar socket
after dental extraction in rats.
2 | MATERIALS AND METHODS
2.1 | HA preparation
HA powders were synthesized by dropwise addition of aqueous
(NH4)2HPO4 solution to a Ca (NO3)2 solution (Merck KGaA, Darm-
stadt, Germany) at 90 C and pH = 10. X-Ray fluorescence (X-Ray
Fluorescence Spectrometer PW 2400, Philips Analytical X-Ray,
Almelo, Netherlands) was used to determine the HA Ca/P molar ratio.
The structure of the HA was analyzed by Fourier transform infrared
spectroscopy (IR-Prestige-21/AIM-880 spectrometer, Shimadzu
Corp.). FTIR spectroscopy of the powder samples was performed in a
transmittance mode using KBr discs operating in the range of
4,000–400 cm−1, with 128 scans. The crystallinity of the HA was ana-
lyzed by X-ray diffraction with Cu Kα radiation at 40 kV and 40 mA
(X'Pert Pro X-ray diffractometer, PAN analytical). The synthesis and
the chemical and physical characterization of both biomaterials were
performed at Biomaterials Laboratory (LABIOMAT) at the Brazilian
Center for Physics Research (CBPF) in Rio de Janeiro, RJ, Brazil.
2.2 | HA microspheres preparation
HA powder was dispersed in a 10 mg/mL aqueous solution of sodium algi-
nate (Fluka Biochemika, Buchs, Switzerland) to achieve a 1:15 alginate-HA
ratio. The alginate/HA mixture was extruded dropwise at room tempera-
ture into a 0.15 M CaCl2 solution using a needle with a 0.55 mm diameter
(BD Precision Glide, Sao Paulo, SP, Brazil). Spherical particles instanta-
neously formed and were allowed to mature in the CaCl2 solution for
24 hr for complete gelation. The HA-alginate microspheres were dried
overnight in an oven at 30 C and sintered at 1,100 C. After sintering, the
microsphere diameters were reduced to a range of 425–600 μm.
2.3 | DOX adsorption onto HA
Adsorption experiments were performed in a batch system according
to the procedure described by de Souza et al. (2011). Briefly, HA
microspheres were incubated in DOX hydrochloride DOX (Sigma-
Aldrich Corp., St. Louis, MO) phosphate-buffered saline (PBS, pH 7.4)
solutions at 1.5 and 20 mg/ml (HADOX0.15; HADOX2.0 samples
respectively) at a proportion of 50 mg HA/ml and moderately shaken
for 24 hr at 37 C. After incubation in the DOX solutions, micro-
spheres (HADOX) were centrifuged (6,000 RPM for 5 min), washed
slightly for 1 min to remove the physically adsorbed DOX fractions,
and dried by freeze-lyophilization method for 24 hr. The solid part
was centrifuged and analyzed by UV–Vis spectrophotometry (UV–Vis
2550 Spectrophotometer with ISR-2200 Integrating Sphere
SORIANO-SOUZA ET AL.
Accessory, Shimadzu Corp.) in a reflectance mode operating in the
range of 200–600 nm. The amount of DOX adsorbed on HA was
determined by UV spectrophotometry with the assistance of a stan-
dard curve at a wavelength of 275 nm. The UV spectra were collected
by using solid aliquots (400 mg) composed by analytical grade BaSO4
powder (300 mg, Sigma–Aldrich) and HADOX (100 mg) or DOX pow-
der (100 mg). Gaussian functions were used to fit the UV spectra of
DOX adsorbed onto the HA surface. The HA group was obtained by
incubating HA microspheres in PBS solution under the same condi-
tions described previously. For all the biological experiments, the HA
and HADOX microspheres were sterilized by gamma rays (15 kGy).
2.4 | Physical and chemical characterization
The topography of the HA microspheres surfaces before and after the
DOX 0.15% adsorption (HA and HADOX0.15, respectively) was ana-
lyzed using a scanning electron microscope (JEOL JSM-6490LV,
Tokyo, Japan) operated at 15 kV. The structure and crystallinity of HA
and HADOX were analyzed by Fourier transform infrared spectropho-
tometer in transmission mode (FTIR-IRPrestige-21, Shimadzu) from
400 to 4,000 cm−1 and X-ray diffraction-(DRX-X'Pert Pro X-ray dif-
fractometer, PANanalytical) operating with CuKα radiation (1.5418 Å)
at 40 kV and 40 mA. Data from obtained DRX patterns were com-
pared with the standards of file 9-432 from ICDD (International Cen-
tre for Diffraction Data) employing the software PCPDF Win 2.1.
For porosity analysis, the HA or HADOX0.15 microspheres were

embedded in epoxy resin (EPON) and polymerized at 60 C for 48 hr.
Thereafter, the resin was mounted on aluminum stubs and polished to
the maximum diameter of the microsphere. The sample was coated
with a thin gold layer, and a carbon tape was attached to the sample
to avoid charging during the SEM analysis.
Images were acquired using a LYRA-3 dual-beam workstation
(TESCAN - Czech Republic). The working distance between the sample
and the beam was 9 mm. SEM images were acquired with a current
mode at 15 kV and backscattered detector (BSED, Oxford). The dimen-
sions of the recorded images were 1,024 × 1,144 pixels, and the dwell
time was 32 μs/pixel. Microsphere porosity was calculated using
threshold images treated by FIJI software. The porous area was del-
imited from the surface to the middle of the microsphere with a width
of 33 μm and height equal to its radius. Synchrotron radiation-based X-
ray microtomography (SR-CT) was used to characterize the HADOX
microsphere internal structure. The samples were analyzed with a
micro X-ray scanned at the high-resolution imaging beamline (IMX), at
the Brazilian Synchrotron Light Source (LNLS). The experiment was
performed using a polychromatic beam (4-24 keV). A 550 μm Si filter
was positioned before the sample to reduce the beam hardening effect
(Brooks et al., 1976). A total number of 1001 projections were cap-
tured by a CCD camera (PCO.2000) coupled with a 10X lens. The pixel
size was of 0.82 μm (Martinez-Zelaya et al., 2019). The count mode
was used to guarantee the same flux on all projections. A fast back-
projection algorithm (Martinez et al., 2017; Miqueles et al., 2014, 2016)
was used to reconstruct the data, and the software Avizo 9.5, was used
to filter, segment, and analyze the images.
3
2.5 | DOX in vitro release assays
DOX release assays were carried out in vitro by incubating
HADOX0.15 microsphere samples in phosphate-buffered saline (PBS)
for up to 9 days. Briefly, 100 mg of HADOX microspheres were incu-
bated in 10 mL of pH 7.4 PBS at 37 C. At defined intervals of
1–120 min and 1, 3, 6, and 9 days, the PBS was completely removed
for analysis and replaced with an equal volume of fresh PBS buffer up
to 9 days when the final sample was collected. The amount of
released DOX was determined by UV–Vis spectrophotometry at
275 nm (UV–VIS 2550, Shimadzu).
2.6 | Microbiological assays
The antimicrobial activity experiments of HADOX 0.15 microspheres
before and after 1, 3, and 7 days were based on standard dilution
methods published by the National Committee for Clinical Laboratory
Standards Institute (Clinical and Laboratory Standards Institute, 2013).
Briefly, Enterococcus faecalis cultures (ATCC 29212), recognized to be
one of the most commonly isolated or detected species from oral per-
sistent infections (Bouillaguet et al., 2018,) were grown from frozen
stocks in trypticase soy broth (TSB, Plast Labor, Rio de Janeiro, RJ,
Brazil). Cultures were freshly prepared for each experiment and
diluted to 0.5 McFarland turbidity standards, approximately 1.5 × 108
cells/ml. The bacterial inoculums were incubated in TSB containing
HADOX microspheres at 25 mg/ml to 0.012 mg/ml concentrations. In
order to evaluate the ability to inhibit growth, the turbidity of the
broth was measured by using a UV spectrophotometer at 595 nm
(UV–VIS 2550, Shimadzu) after 18 hr. The MIC values were defined
by the lowest dilution required to maintain the culture medium with-
out turbidity. The tests were run in triplicate.
2.7 | Cytocompatibility assay
For the cytocompatibility evaluation, osteoblast cells from
periosteum-free fragments of murine femurs (F-OST; Balduino et al.,
2005) were chosen. These cells are highly differentiated and behave
most like osteoblasts. Cells were routinely cultured with Dulbecco's
modified essential medium (DMEM, Gibco) supplemented with 10%
fetal bovine serum (FBS) and incubated at 37 C at 5% CO2 atmo-
sphere. Confluent low passages were trypsinized, counted in a Neu-
bauer chamber, and used in all experiments.
To determine if some release of DOX can cause cell cytotoxicity, a
cell viability assay was performed after exposing the cells to extracts
of each material (HA, HADOX0.05 and HADOX0.15 groups). To pre-
pare the sample extracts, microspheres sterilized by gamma radiation
were immersed in DMEM free of fetal bovine serum and shaken for
24 hr at 37 C as recommended by (ISO 10993-5, 2009). Then, the
extracts were collected and centrifuged at 3,000 rpm for 10 min, to
ensure that no particles would interfere in the biocompatibility assay.
F-OST cells were seeded in a 96-well cell culture plate (8 × 103
cell/well) and cultured in DMEM supplemented with 10% FBS for
24 hr at 37 C in 5% CO2. After 24 hr, the DMEM was removed, and
4 SORIANO-SOUZA ET AL.

the cells were exposed to the samples extracts for another 24 hr. In
addition, 1% of sodium dodecyl sulfate (SDS) was used as the positive
cytotoxicity (C+) control and cells were used as the negative cytotox-
icity control (C−). The negative control is used to demonstrate the cel-
lular response in the assay and the positive control to prove an
appropriate response of the reagents.
Cell viability assay was performed using PrestoBlue™ Cell Viability
Reagent (Invitrogen, Inc., Carlsbad, CA). PrestoBlue reagent is a solution
based on resazurin (7-hydroxy-3H-phenoxazine-3-one-10-oxide). Living
cells reduce this compound to resafurin, and measurements of fluores-
cence and absorbance of the solution give an indication of the cellular met-
abolic activity and, consequently, the presence of viable cells measured at
590 nm. The crystal violet dye elution (CVDE) assay evaluates cell density
by staining DNA, and the absorbance at 540 nm is proportional to the
number of cells in each well. The colorimetric assays were performed by
Synergy II UV–Vis spectrophotometer (Biotek Instruments, Winooski, VT).
2.8 | In vivo study
2.8.1 | Ethics considerations
starting after the procedure on the day of surgery and for 2 additional
days. The animals were placed into boxes based on their experimental
groups. In these boxes, the rats received food and unlimited water.
2.8.4 | Surgical procedures
The upper right incisors were extracted with forceps (Duflex, Minas
Gerais, Brazil) after disconnection of the surrounding gingiva and luxa-
tion with an enamel hatchet with a cutting edge. HA microspheres with
or without DOX (≈50 mg) were placed into the extraction sites. Immedi-
ately after surgery, the gingival tissues were sutured with Mononylon
Ethilon 5-0 (Ethicon®, Johnson & Johnson, São Paulo, SP, Brazil).
2.8.5 | Histological preparation
The animals were sacrificed by an overdose of general anesthesia 1 and
6 weeks postoperatively (n = 5 per group), their maxillae were removed,
and their heads immersed in 10% formalin for 48 hr. After fixation, the
maxilla was dissected and divided along the median sagittal plane. The
right halves were cut tangentially to the distal surface of the molars,

The animal experiments and breeding were performed in accordance with

conventional guidelines in the NIH Guide for the Care and Use of Labora-
tory Animals (US National Institutes of Health 85-23, revised 1996) and
in accordance to the Brazilian legislation on animal use in experimental
research. This study is reported according to the ARRIVE guidelines
(Animal Research: Reporting of in vivo Experiments; Kilkenny et al., 2010)
complemented by PREPARE (Planning Research and Experimental Proce-
dures on Animals: Recommendations for Excellence; Smith, Clutton, Lilley,
Hansen, & Brattelid, 2018), regarding relevant items. The protocols were
approved by the local Institutional Animal Care and Use Committee of
Federal Fluminense University, Niteroi, Brazil (protocol # 348).

2.8.2 | Animals
Thirty adult male and female Wistar rats, with a mean weight of
300/400 g and an age of 5–6 months were used in this study. The ani-
mals were kept in individual cages (n = 2) and received water and food
ad libitum throughout the study. The animals were provided by Labora-
tory Animals Center from Fluminense Federal University, Niteroi, Rio de
Janeiro, Brazil. A senior veterinarian managed the nutritional recommen-
dations, animal care and preoperative and postoperative fasting of ani-
mals. The animals were divided into two groups (HA and HADOX0.15)
and investigated after two experimental periods (1 and 6 weeks).

2.8.3 | Anesthetics and analgesia procedures

All animals were anesthetized with 3 ml of a solution of ketamine
(1 ml/kg; Virbac, São Paulo, Brazil), xylazine (0.5 ml/kg; FortDodge, Rio
de Janeiro, Brazil), Midazolam (0.6 ml/kg; Roche, Rio de Janeiro, Brazil),
Tramadol (0.2 ml/kg; Sun, Goiânia, Brazil) and saline solution (8.5 ml).
F I G U R E 1 XRD pattern of HADOX0.15 microsphere showing the
During the postoperative period, the rats received analgesia with 5 mg/kg expected peaks of a stoichiometric hydroxyapatite structure (a) and
Meloxicam (Eurofarma, São Paulo, Brazil) subcutaneously every 24 hr, HADOX0.15 microsphere FTIR spectra showing (PO4)3− and OH− bands (b)
SORIANO-SOUZA ET AL. 5

decalcified and processed for paraffin embedding. Longitudinal 4-μm
thick sections were cut and stained with hematoxylin and eosin.
2.8.6 | Histomorphometric evaluation
For histomorphometric analysis, a light microscope (Olympus BX43,
Tokyo, Kanto, Japan) with 10× magnification was used. The micro-
scope was connected to a computer, and each HE-stained histological
slice corresponding to the alveolar region was captured by scanning
by image acquisition software (Cellsens® 1.9 Digital, Tokyo, Kanto,
Japan). An expert observer analyzed 10 nonconsecutive images of
each section. With the Image-Pro Plus® 6.0 (Media Cybernetics, Silver
Spring, MD), a grid of 200 points was superimposed on the captured
field, allowing the determination of newly formed bone and the resid-
ual biomaterial. The bone volume density (BV/TV%) was calculated as
bone volume over total volume, indicating how much of the volume
of interest was occupied by newly formed bone. The areas were
expressed as a percentage.

F I G U R E 2 SR-μCT of
HADO×0.15 microspheres. (a,b),
biomaterial space and (c,d), pore
space. The internal porosity was of
14.2% (± 3.6), with 12.3% (± 3.6) of
interconnected porosity; the mean
internal pore size was 5.3 (± 0.3) μm

F I G U R E 3 SEM images of HA
and HADOX0.15 microspheres. (a,b)
HADOX0.15 microspheres cut in half
showing the interior and exterior of
the microspheres; polished section of
HA microspheres: lower magnification
(c) and higher magnification (d);
polished section of HADOX0.15
microspheres lower magnification
(e) and higher magnification (f). Scale
bars: a, c, and e: 100 μm; b: 2 μm; d
and f: 10 μm
6 SORIANO-SOUZA ET AL.
2.8.7 | Statistical evaluation
For in vivo analysis, after the normality test, a quantitative description
with means and confidence interval (CI) of BV/TV% was performed.
The analysis of variance (ANOVA) and Tukey's post hoc test were
applied, and two assignable sources of variation, groups and periods,
were evaluated. The significance level was set at p = .05. The analyses
were performed using Prism Graph Pad® 6.3 software (GraphPad
Software, Inc. La Jolla, CA). For cytocompatibility evaluation, mean
values and standard deviation for each test were calculated, and the
ANOVA test was performed. Post hoc analysis was carried out with
Dunnett's test. The significance level was set at 99.9% (p < .001).
FIGURE 4 Structure of doxycycline molecule
FIGURE 5 UV–Vis spectra: (a) DOX solution, (b) DOX powder, (c) HADOX0.05, and (d) HADOX2.0
3 | RESULTS
3.1 | Physical and chemical characterization
The microsphere X-ray diffraction pattern was typical of crystalline
and nearly stoichiometric HA phase, as shown in Figure 1a. The FTIR
spectrum showed phosphate ([PO4]3−: 560, 601, 632, 962, and
1,039 cm−1) and hydroxyl (OH−: 632 and 3,572 cm−1) bands of a
well-formed HA structure (Figure 1b) confirming the XRD results. SR-
μCT technique was used to estimate HADOX0.15 microsphere poros-
ity with a resolution of 1.5 μm. As shown in Figure 2, the microsphere
interior was composed of dense HA and micropores with 5.3 (± 0.3)
μm mean size occupying 14.2% (± 3.6) of the microsphere volume.
The effective porosity, also called the interconnected porosity, was
12.3% (± 3.6). SEM analyses with the sub-micron resolution were con-
ducted in HA and HADOX0.15 microspheres, as illustrated in
Figure 3. The microspheres had an irregular surface topography with
micrometer-scale roughness (Figure 3a). The inner portion of the
microsphere was composed of dense areas due to sintering treatment
at 1,100 C (Figure 3b). SEM images treated by FIJI software revealed
that the internal microsphere porosity was sensitive to the treatment
SORIANO-SOUZA ET AL. 7

used to adsorb DOX onto microsphere surface: HA microspheres shown in Figure 5d. Comparison of deconvolution bands from both spec-
without DOX presented 26% (± 5.0) of pores (Figure 3c,d), whereas tra indicate a decrease of the band at 275 nm which could be related to
HADOX0.15 microspheres presented 52.0% (± 2.5; Figure 3e,f). an interaction of acidic A-ring chromophore with a basic species of the
HA compound, such as the OH− group. It is important to note that an
additional band at 394 nm from DOX adsorbed on HA surface was not
3.2 | DOX adsorption by HA
present on the UV–Vis spectrum of DOX in buffer solution and seems to
Tetracycline molecules consist of a linear fused tetracyclic nucleus be related to the BCD interaction with Ca groups of HA.
named from A to D rings with several functional groups, as shown in
Figure 4. According to the literature, UV–Vis bands of tetracyclines
are observed near ~270 nm, which are attributed to the most acidic 3.3 | DOX release and microbiological assays
functional group centered in the A-ring chromophore. A second band DOX adsorption onto the HA microspheres was determined UV–Vis
at ~365 nm is attributed to the b-hydroxyketo system from the BCD spectroscopy. DOX uptake was 28.2 ± 4.5 mgDOX/mgHA for
chromophore, which is responsible for the other wavelength absorp-
HADOX0.15 microspheres samples. The drug release profile for
tion bands (Kilkenny et al., 2010).
HADOX0.15 microspheres is shown in Figure 6. It was observed an initial
Figure 5a,b show the UV–Vis spectra of DOX powder and DOX in
drug release of 49.15 μg/mL (~20%) in the first 24 hr, followed by a con-
aqueous buffer solution, respectively. The UV–Vis spectrum of non-
tinuous drug release throughout the experimental periods. About 60% of
interacting DOX molecules in solution consists of bands at 243, 274,
DOX remained retention into HA microspheres after 9 days. In order to
303, and 351 nm. The bands position change to 237, 271, 308, 359, and
confirm the antibacterial activity of DOX associated with HA, microbio-
a new band appears at 411 nm when the spectrum is collected with
logical assays were performed to evaluate potential bacterial growth
DOX sample in the solid state. This change in the UV–Vis spectrum of
inhibition of the biomaterial. The minimal inhibitory concentration (MIC)
DOX is attributed to a modification at electronic transitions due to the
for growth inhibition of E. faecalis was determined. The samples before
interaction between molecules in the crystalline structure. The UV–Vis
(T = 0) and after the desorption experiments after 1, 3, and 7 days were
spectrum of HA sample loaded with DOX 0.05% (HADOX0.05–10 μg
analyzed. Data for the MIC testing against E. faecalis is also presented in
DOX/mg HA) was also decomposed into five absorption bands centered
at 247, 275, 301, 350, and 394 nm, suggesting an interaction of DOX
molecules with the HA surface (Figure 5c). The complexation of
tetracycline-based compounds with calcium ions was discussed in detail
in the work of Schmitt and Schneider (2000). The bands at 247, 275,
301, 350, and 394 nm were also present in the UV–Vis spectrum of HA
samples loaded with DOX 2.0% (HADOX2.0–300 μg DOX/mgHA) as

F I G U R E 7 Cell viability assay using Prestoblue (a), and cell

F I G U R E 6 Drug cumulative release of DOX (μg/ml) and MIC values
(mg/ml). Initial burst of drug release in the first 24 hr followed by an increasing
drug release until 9 days and progressive increase of the HADOX0.15
microspheres concentration for the inhibition of E. faealis growth
proliferation using CVDE (b). For statistical analysis Dunnett's post-test
were used as representative of two independent experiments performed
in sixtuplicates. The samples were normalized in percentage and
compared to HA. The asterisk indicates that the SDS group presented a
significantly lower absorbance than all other groups (p < .001, ANOVA)
8 SORIANO-SOUZA ET AL.
F I G U R E 1 0 New formed bone volume density for HA and
HADOX0.15. The newly formed bone increased significantly between
1 and 6 weeks after biomaterial implantation (p < .05)
F I G U R E 8 Photomicrographs of the
2 groups after 7 days of alveolar
implantation. Biomaterial (BM);
neutrophilic infiltrate (N); new-formed
bone (NFB) a and c: Magnification of 20×
(Bar: 100 μm); b and d: Magnification of
40× (Bar: 50 μm). Stain: Hematoxylin and
eosin
F I G U R E 9 Photomicrographs of the
two groups after 42 days of alveolar
implantation. Biomaterial (BM);
connective tissue (CT); New formed bone
(NFB). a and c. Magnification of 20× (Bar:
100 μm); b and d: Magnification of 40×
(Bar: 50 μm). Stain: Hematoxylin and
eosin
Figure 6 and demonstrated that bacterial growth was inhibited for up to
7 days although the HADOX MIC increased from 1.25 mg/ml (initial) to
25 mg/ml (7 days), about 20 times.
3.4 | Cytocompatibility assay
The cytotoxicity tests were performed on the samples in order to deter-
mine whether the extracts of the HA samples doped with DOX in differ-
ent concentration generated any observable biological effect on F-OST
cells. Results of cell viability through the PrestoBlue reagent did not indi-
cate differences in the metabolic activity of HADOX0.05 and
SORIANO-SOUZA ET AL.
HADOX0.15 samples relative to the HA. The CVDE assay, which analyzes
cell proliferation, also indicated that the samples did not present statistical
differences in cell proliferation when compared to HA. The positive con-
trol presented low cell viability for both reagents, confirming the inherent
toxicity to the material and the performance of the method (Figure 7).
3.5 | In vivo study of HADOX microspheres
Histological evaluation after 7 days.
3.5.1 | HA (control group)
After 7 days, the presence of biomaterial (microspheres) with limited
connective tissue containing acute inflammatory infiltrate, mainly neu-
trophilic infiltrate (Figure 8a,b), was observed.
3.5.2 | DOXHA 0.15%
A presence of fibrocellular connective tissue with a moderate inflamma-
tory infiltrate, mainly mononuclear, between biomaterial microspheres
was observed. It was possible to observe the presence of osteoblast
pavimentation between microspheres (Figure 8c,d).
3.6 | Histological evaluation after 42 days
3.6.1 | HA (control group)
The presence of biomaterial limited by fibrocellular connective tissue
exhibiting a few multinucleated giant cells and newly formed bone
around the microspheres (Figure 9a,b) was observed.
3.6.2 | HADOX 0.15
After 42 days, the experimental group exhibited dense fibrous con-
nective tissue limiting the biomaterial microspheres, with a few
multinucleated giant cells and a few mononuclear cells. Newly formed
bone was observed between microspheres. The biomaterial
maintained the format of microspheres without apparent absorption.
3.7 | Histomorphometric evaluation
The histomorphometric analysis is shown in Figure 10 and shows an
increase (p < .05) at the newly formed bone volume density for HA
and HADOX0.15 group from 1 week to the end of the experiment.
4 | DISCUSSION
In the present study, DOX was adsorbed on the surface of ceramic
microspheres of HA. Despite the thermal treatment at 1,100 C, the
HADOX0.15 microspheres showed 52% of internal microporosity,
which contributed to increase the DOX adsorption in HA structure.
The UV–Vis spectra of HA loaded with DOX 0.05 and 2.0% showed
significant differences from the spectrum of the unloaded sample. The
9
intensity decrease of the band at 275 nm could be related to an inter-
action of the acidic A-ring chromophore with ions of the HA structure,
such as the OH− group. It is worth noting that an additional band at
394 nm from DOX adsorbed on HA was not present on the UV–Vis
spectrum of DOX in buffer solution. This effect seemed to be due to
the BCD interaction with Ca groups of HA. Perrin (1965) proposed
another configuration for HA tetracycline complex. The author stud-
ied the binding of tetracyclines to the bone and suggested a model for
tetracycline´s anchorage on HA, where tetracycline molecule was in a
perpendicular position related to the ab plane of HA. Furthermore,
the Ca ions of HA would be coordinated with oxygen atoms of tetra-
cycline molecule in C2, C10, and C12 positions. Schmitt and
Schneider (2000) demonstrated the differences in the complexation
of distinct tetracycline molecules with Ca and Mg ions. UV–Vis and
fluorescence spectra of tetracycline and oxytetracycline molecules in
a solution containing Ca2+ or Mg2+ indicated two successive complex-
ation equilibria. The first complexation occurred between a metal ion
with tetracycline's BCD chromophore and the second complexation
between a metal ion and the A chromophore. With the DOX mole-
cule, the result was different because metal ion complexations
occurred with the BCD chromophore. This fact corroborates with the
results found in this work and could explain the changes of UV–Vis
band related to the acidic A chromophore at 270 nm, with the com-
plexation by OH− groups of HAs instead of calcium ions.
Regarding release kinetics, an initial drug release of approximately
20% in the first 24 hr was observed, followed by a continuous drug
release throughout the 9 days. About 60% of DOX remained on the
HA microspheres after the experimental period. The retention of this
amount of DOX can be explained by high chemical affinity between
the families of tetracycline antibiotics as described by Perrin (1965)
Concerning the antimicrobial activity, MICs values for E. faecalis
demonstrated that bacterial growth was inhibited for up to 7 days,
although the HADOX MIC increased from 1.25 mg/ml (initial) to
25 mg/ml (7 days), about 20 times. However, taking into account that
after 7 days, the remaining DOX concentration was about 60% of the
initial, in a dynamic system as the infectious-inflammatory sites
in vivo, a different release kinetics could occur due to the solubility of
the HADOX biomaterial, and consequently, the antimicrobial activity
would remain at therapeutic levels for a longer time. This result sug-
gests that an adjustment of the initial concentration of the antibiotic
could increase the antimicrobial efficiency of the biomaterial.
In vitro cytocompatibility assays were carried out to estimate the
effects of DOX association to HA microspheres on bone cells. The cell
viability through the PrestoBlue reagent and CVDE assay for cell den-
sity did not indicate differences in the metabolic activity of
HADOX0.05 and HADOX0.15 samples relative to the HA, despite a
3X concentration difference between the HADOX groups (10 μg
DOX/mg HA for HADOX0.05; 30 μg DOX/mg HA for HADOX0.15).
It was concluded that the analyzed samples were cytocompatible and
that the extracts of the materials analyzed in this study did not show
a toxic effect by the method employed. These results were in accor-
dance with previous studies that demonstrated that DOX did not
10
affect osteoblastic cell survival and proliferation (Perrin, 1965;
Schmitt & Schneider, 2000).
The histological observation clearly demonstrated new bone for-
mation around the microspheres in both groups. However, the group
HADOX0.15 showed an increase in new-formed bone, when com-
pared with group HA. The use of HADOX0.15 could be significant for
many surgical bone procedures, for example, in bone regeneration or
the treatment of local bone disease, when there is insufficient bone
vascularization quality to send the drug (antibiotic) by first-pass
metabolism or intravascular delivery. Therefore, HADOX will probably
increase the success rate in this type of local treatment after surgical
bone procedures. Moreover, the local “drug delivery” that was used in
this study (HADOX) is an excellent surgical option. The advantages of
local delivery include lower dosage, greater control over drug bioavail-
ability and release profile, maintenance of therapeutic concentration
at the infection site, less severe side effects, and reduced cost
(Habraken et al., 2007).
Understanding the role of HA as an osteoconductive substitute
brings this material to a significant level as a vehicle for drug transport
and/or association with molecules and chemical elements that will
modify its geometry, crystallinity, and morphology thus enhancing its
osteoconductive activity. Its chemical composition, consisting of a cat-
ionic radical, an anion and a hydroxyl, allows its connection with
almost all the chemical elements of the periodic table (Narasaraju &
Phebe, 1996), as well as drugs like chlorhexidine (Soriano-Souza et al.,
2015), tetracycline (Narasaraju & Phebe, 1996; Trajano, Costa, Lanza,
Sinisterra, & Cortés, 2016), minocycline (Martin et al., 2019), and DOX
(Chang & Yamada, 2000; Ramírez-Agudelo et al., 2018). The latter has
a high affinity for this compound.
The changes in properties, production technologies and interac-
tion of the material to the tissues leads to more efficient bioactivity,
resulting in an optimization of the bone repair. It is evident that the HA
microspheres through its chemical composition, nano/microstructured
surface and the presence of micropores (3D architecture) are consid-
ered essential parameters that can determine the delivery capacity of
therapeutic molecules (El-Fiqi, Buitrago, Yang, & Kim, 2017). In this
study, it was verified by the porosity evaluation that there was a signifi-
cant increase for HADOX 0.15 concerning HA without DOXs, 52.30%
(±2.52) and 15.11% (±1.69), respectively. The increase in the solubility
of some hydrophobic drugs is directly related to the surface area of
the nanoparticles, as well as the presence of micropores, which is a
fundamental characteristic for the biodegradation of biomaterials
(Granjeiro & Soares, 2012).
Among the advantages cited by Gu, Wu, Chen, and Xiao (2013)
for the use of nanoparticles as a delivery system vehicle for the treat-
ment of bone diseases are the protection of the drug by preventing its
dispersion or degradation by body fluids and by increasing the time of
circulation or retention time in the body. In this way, the medicine is
used in the local site, keeping it concentrated.
An important factor when considering the association of drugs
with HA is the concentration. At a concentration of 0.5 mg/ml chlor-
hexidine and in a lower concentration, the minocycline, both had a
cytotoxic effect on the proliferation of osteoblasts. On the other
SORIANO-SOUZA ET AL.
hand, DOX showed improvement in the maturation and differentia-
tion of osteoblasts by increased alkaline phosphatase enzyme activity
(Almazin, Dziak, Andreana, & Ciancio, 2009). This author attributed to
DOX the property of being anti-inflammatory, an osteoclast inhibitor,
a fibroblast stimulator, anti-collagenolytic and clearly antimicrobial.
The results regarding cell viability and cell proliferation in this study
did not indicate a difference in metabolic activity between the control
group and the test. It was concluded that the samples are cyto-
compatible and do not have a toxic effect in the concentrations used.
A previous in vivo study, Soriano-Souza et al. (2015) evaluated the
biocompatibility of HA microspheres carried with chlorhexidine and
observed that the osteoconductive properties of HA associated with
an antimicrobial agent might be useful in the treatment of bone infec-
tions. However, the concentration of the agent must be controlled
because it observed a statistically significant difference when com-
pared to a concentration of 0.12 and 2%, the latter presented a delay
in the process of bone repair.
The combined treatment of nHA-DOX was performed by
Ramírez-Agudelo, Scheuermann, Gala-García, et al. (2018) and
showed that cytotoxicity is directly dependent on concentration and
the low concentration of DOX (5 μg/ml) exhibited a less significant
inhibitory effect on cancer cells when compared to 30 μg/ml.
HA as a drug carrier is limited and needs to be modified on its sur-
face because small, water-soluble molecules, such as DOX, can experi-
ence a very rapid release of this drug when absorbed in this
phosphate (Wang et al., 2010). The El-Fiqi, Buitrago, Yang, and Kim
(2017) (Chang & Yamada, 2000) study also observed a drop in the sur-
rounding nanoparticle aggregates, which were substantially reduced
from the third and seventh days. This can be explained by the fact
that HA as a drug carrier presents an initial abrupt release which may
limit its application according to Boonsongrit et al. (2008) However,
the retention time in the body seems to be an interesting observation,
since almost 60% of the drug remained adsorbed to the biomaterial
after the ninth day of the experiment. It is essential to point out that
there is a greater liberation of DOX in the environment in the first
4 hr compared to tetracycline.
5 | C O N CL U S I O N
The tested materials were cytocompatible, biocompatible and
osteoconductive after alveolar implantation in rats (7 and 42 days).
HADOX0.15 showed inhibition of bacterial growth for up to 7 days
and presented a neoformed bone (p < .05) after 42 days of implanta-
tion and could be a promising biomaterial to promote bone repair in
infected sites.
ACKNOWLEDG MENTS
This research used resources of the Brazilian Synchrotron Light Labo-
ratory (LNLS), an open national facility operated by the Brazilian Cen-
tre for Research in Energy and Materials (CNPEM) for the Brazilian
Ministry for Science, Technology, Innovations and Communications
SORIANO-SOUZA ET AL.
(MCTIC). The IMX beamline staff is acknowledged for the assistance
during the experiments (Proposal 20160692).
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