International Journal of

Molecular Sciences

Article
A Study of Zn-Ca Nanocomposites and Their
Antibacterial Properties
M. I. Torres-Ramos 1 , U. J. Martín-Camacho 1 , J. L. González 2 , M. F. Yañez-Acosta 2, *, L. Becerra-Solano 3 ,
Y. K. Gutiérrez-Mercado 3 , M. Macias-Carballo 3 , Claudia M. Gómez 4 , O. A. González-Vargas 5 ,
J. A. Rivera-Mayorga 6 and Alejandro Pérez-Larios 1, *

1 Laboratorio de Investigación en Nanomateriales, Agua y Energía, Departamento de Ingeniería, Centro

Universitario de los Altos, Universidad de Guadalajara, Av. Rafael Casillas Aceves 1200,
Tepatitlán de Morelos 47600, Mexico; [email protected] (M.I.T.-R.);
[email protected] (U.J.M.-C.)
2 Especialidad en Odontopediatría, Centro Universitario de los Altos, Universidad de Guadalajara, Av. Rafael
Casillas Aceves 1200, Tepatitlán de Morelos 47600, Mexico; [email protected]
3 Laboratorio Biotecnológico de Investigación y Diagnostico, Departamento de Clínicas, División de Ciencias
Biomedicas, Centro Universitario de los Altos, Universidad de Guadalajara, Av. Rafael Casillas Aceves 1200,
Tepatitlán de Morelos 47600, Mexico; [email protected] (L.B.-S.);
[email protected] (Y.K.G.-M.); [email protected] (M.M.-C.)
4 Departamento de Química, División de Ciencias Naturales y Exactas, Campus Guanajuato de la Universidad
de Guanajuato Noria Alta s/n, Col., Noria Alta, Guanajuato 36050, Mexico; [email protected]
5 Departamento de Ingeniería en Control y Automatización, ESIME-Zacatenco, Instituto Politécnica Nacional,
UPALM, Av. Politécnico s/n, Col., Zacatenco, Alcadía Gustavo A. Madero, Ciudad de México 07738, Mexico;
[email protected]
6 Departamento de Química, Centro Universitario de Ciencias Exactas e Ingenierías, Universidad de
Citation: Torres-Ramos, M.I.;
Martín-Camacho, U.J.; González, J.L.; Guadalajara, Boulevard Marcelino García Barragán, Calzada Olímpica, Guadalajara 44430, Mexico;
Yañez-Acosta, M.F.; Becerra-Solano, [email protected]
* Correspondence: [email protected] (M.F.Y.-A.); [email protected] (A.P.-L.)
L.; Gutiérrez-Mercado, Y.K.;
Macias-Carballo, M.; Gómez, C.M.;
González-Vargas, O.A.; Abstract: This study aimed to develop Ca2+ doped ZnO nanoparticles (NPs) and investigate their
Rivera-Mayorga, J.A.; et al. A Study antibacterial properties against microorganisms of dental interest. Zn-Ca NPs were synthesized
of Zn-Ca Nanocomposites and Their by the sol-gel method with different concentrations of Ca2+ (1, 3, and 5 wt. %) and subsequently
Antibacterial Properties. Int. J. Mol. characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), UV-vis spectroscopy
Sci. 2022, 23, 7258. https://doi.org/ and Fourier transform infrared spectroscopy (FT-IR). The Kirby–Bauer method was used to measure
10.3390/ijms23137258 antibacterial effects. NPs showed the wurzite phase of ZnO and bandgap energies (Eg) from 2.99 to
Academic Editor: Oxana 3.04 eV. SEM analysis showed an average particle size of 80 to 160 nm. The treatments that presented
V. Galzitskaya the best antibacterial activity were Zn-Ca 3% and Zn-Ca 5%. ZnO NPs represent an alternative to
generate and improve materials with antibacterial capacity for dental applications.
Received: 30 April 2022
Accepted: 26 June 2022
Keywords: nanoparticles; ZnO; antibacterial activity; nanocomposites; dental materials
Published: 29 June 2022

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil- 1. Introduction
iations.
Nanotechnology is the most important dynamic exploration region in current of
material science [1]. The significant growth in nanotechnology is best evidenced by the
number of scientific articles and its many applications [2,3]. Recently, the scientific re-
Copyright: © 2022 by the authors.
search community worldwide expressed interest in synthesizing metal and metal oxide
Licensee MDPI, Basel, Switzerland. nanoparticles (NPs) [4]. ZnO-NPs are of great importance due to their wide variety of
This article is an open access article applications in photocatalysis, water purification, and antibacterial disinfection. ZnO-NPs
distributed under the terms and display properties that are distinct from those of typical NPs [5]. The biological activities
conditions of the Creative Commons of ZnO-NPs are size and morphology dependent, and are the subject of investigation by
Attribution (CC BY) license (https:// many researchers [3,6–8]. ZnO-NPs are considered a multi-purpose option and in recent
creativecommons.org/licenses/by/ years, research has focused on these metallic NPs due to their remarkable antimicrobial
4.0/). properties [9,10]. The antibacterial effects of these nanostructured agents is attributed to

Int. J. Mol. Sci. 2022, 23, 7258. https://doi.org/10.3390/ijms23137258 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022, 23, 7258 2 of 13

the high surface/volume ratio since it provides a greater contact area with agents in the
environment. The ability to easily penetrate cell membranes disrupts various intracellular
processes, resulting in high reactivity and antibacterial activity [11,12]. The incorporation
of ZnO into dental components has received special attention, representing an effective
alternative for biomedicine, specifically in oral health [13]. The antibacterial properties of
ZnO as a nanocomposite can be used against Gram-negative bacteria, such as Escherichia
coli (E. coli) [13,14], as well as Gram-positive bacteria, such as Enterococcus faecalis (E. faecalis)
and Staphyococcus aureus (S. aureus), and it has gained interest for the elimination of these
bacteria from the oral cavity [3,6,7].
Although antimicrobial compounds have been reported to decrease the occurrence
of dental disease, the use of antibiotics and chemical bactericides can have a negative
impact on the bacterial flora of the oral cavity and intestinal tract [15,16]. Since pathogens
can acquire resistance against different antibiotics, agents that are characterized for hav-
ing remarkable antibacterial activity and do not develop resistance are now in high de-
mand [17,18]. Bacterial growth in the oral cavity is the main cause of secondary caries [19].
Improvement in the antibacterial properties of dental composites can effectively decrease
the occurrence of secondary caries [20]. Presently, two methods are usually used to improve
the conditions of the oral cavity. The first method involves the addition of antibacterial
agents such as chlorhexidine (CHX), fluoride or silver ions into the resin composite whereas
the second method involves the use of nanotechnology (NP’s, like ZnO) that provided
an effective method for delivering a payload with antibacterial effects [2,21]. Several re-
searchers have tried to improve the antibacterial properties of ZnO by doping it with other
materials that have improved the inhibition of bacteria present in the oral cavity [22–24].
The use of Ca2+ has mainly been seen in bioactive glasses that have been synthesized by
different methods [25,26] due to the need to improve the biocompatibility of materials.
Based on the above, this work aims to present the first report of a Zn-Ca nanocomposite
applied in the dental field that was synthesized with different Ca2+ molar ratios. The NPs
were labeled as Zn-CaX, where X represents the molar ratio of Ca+ , and the antibacterial
properties of each formulation were evaluated against strains of oral interest, such as
Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis),
Streptococcus mutans (S. mutans), Veillonella parvula (V. parvula), Fusobacterium nucleatum (F.
nucleatum), Actinomyces odontolyticus (A. odontolyticus).

2. Results
The images obtained by Scanning Electron Microscopy (SEM) of the Zn-Ca nanocom-
posites (Figure 1) show spherical and conical morphologies with a hexagonal base, and
in some cases, rod-like morphologies were observed (Figure 1b–d), this was confirmed
with Transmission Electron Microscopy (TEM). Table 1 shows the average particle sizes
of each nanomaterial, as measured by ImageJ analysis software. XRD diffraction patterns
of the Zn-Ca and ZnO nanocomposites showed characteristic peaks with an index Miller
at (100), (002), (101), (102), (110), (103) y (112) (Figure 3). FT-IR spectroscopy (Figure 4)
showed bands at 548, 692, 879, 2334, y 2366 cm−1. The UV-vis study (Figure 9) showed
an optical absorption at 400 nm. Antibiograms (Figure 10) for Gram-negative bacteria
(E. coli, V. párvula y F. nucleatum) showed inhibition halos less than 15 mm, with the best
material being Zn-Ca5%, whereas the inhibition halos for Gram-positive bacteria (E. faecalis,
S. Aureus, S. mutans, y A. odontolyticus) had an average size of 20 mm, and Zn-Ca 3% was
the best material.
Int. J. Mol. Sci. 2022, 23, 7258 3 of 13

Figure 1. Scanning electron microscopy images of Zn-Ca nanocomposites. (a) ZnO, (b) Zn-Ca 1,
(c) Zn-Ca 3, (d) Zn-Ca 5.

Table 1. Surface analysis of nanocomposites.

Lattice Parameters
Material Eg (eV) Crystallite Size (nm)
a = b (Å) c (Å)
ZnO 3.03 3.2477 5.2035 42.13
Zn-Ca 1 2.99 3.2476 5.2024 47.93
Zn-Ca 3 3.04 3.2511 5.2059 52.82
Zn-Ca 5 3.01 3.2482 5.2041 34.61

3. Discussion
The presented study was based on the implementation of a nanocomposite (Zn-Ca) as
an antibacterial agent. The use of nanomaterials in the dental field has been reported [13–17]
as an alternative to currently used treatments [4]. The implementation of calcium ions in
nanocomposites provided the material with the specific characteristics demonstrated in this
study. It is known that the structural morphology of zinc oxide nanoparticles encompasses
spherical and hexagonal configurations [27], however, SEM microscopy allowed us to
observe an increase in size as the percentage of doping increased [28]. This behavior is
due to the ionic size of the Ca2+ ion, which is larger than the guest cation Zn2+ [29]. The
substitution of Ca2+ with a larger radius in the Zn2+ sites resulted in an increase in the size
of the nanoparticle, which agrees with other investigations where the concentration of the
dopant directly influenced the morphology and size of the nanoparticles [30–34].
Int. J. Mol. Sci. 2022, 23, 7258 4 of 13

In Figure 2, TEM images of ZnO shows spindle shaped nanoparticles [35]; however,
as the Ca content increases, the size of Zn particles increases and the appearance of small
particles corresponding to Ca was observed. To determine the size distribution of the
nanoparticles, software was used measuring nanoparticle size at 50 nm [34].

Figure 2. Transmission electron microscopy of Zn-Ca nanocomposites. (a) ZnO, (b) Zn-Ca 1, (c) Zn-Ca
3, (d) Zn-Ca 5.

Regarding the crystallinity of the material, the XRD diffraction patterns (Figure 3)
were found to be in agreement with the standard diffraction patterns for the wurtzite
hexagonal phase for ZnO according to a crystallographic chart (JCPDS 01-079-0206) [36].
The diffraction peaks reveal the presence of ZnO but not Ca2+ . These results are in agree-
ment with reported studies that used the sol-gel method to synthesize the material, and
the dopant was not detectable by XRD [37–40]. The average sizes of the ZnO and the
Zn-Ca nanocomposites were calculated by the Scherrer equation (Table 1) and agree with
those reported in ZnO studies [30,41]. The crystal size increased in proportion with the
increase in the concentration of the dopant, except for the Zn-Ca5 material. This was due
to saturation by the dopant, which generates a decrease in crystal size [31]. The lattice
parameters were calculated using the Bragg’s law equation, obtaining similar values for the
parameters between the nanocomposites, which indicates that the incorporation of calcium
ions does not modify the morphology and maintains the phase of the material [42].
Int. J. Mol. Sci. 2022, 23, 7258 5 of 13

Figure 3. X-ray diffraction patterns of Zn-Ca nanocomposites. (a) ZnO, (b) Zn-Ca 1, (c) Zn-Ca 3,
(d) Zn-Ca 5.

FT-IR spectroscopy (Figure 4) confirmed the wurzite phase of the material, identifying
the presence of tetrahedral groups of ZnO and the Zn-O-Ca bond that make up this
structure [43]. Characteristic bands associated with functional groups present in ZnO were
also detected, which correspond to the metal-oxygen vibration modes, shows bands at 548
and 692 cm−1 corresponding to vibrational modes of Zn-O and Zn-OH [44]. The absorption
peaks at 2334 and 2366 cm−1 were assigned to absorption levels that reveal the presence
of C-H stretching vibrations due to the precursor used in the synthesis [36]. This analysis,
together with the XPS (Figure 5) confirm the presence of Zn in the nanocomposite.

Figure 4. FT−IR spectra of Zn-Ca nanocomposites.

Int. J. Mol. Sci. 2022, 23, 7258 6 of 13

Figure 5. XPS spectra for zinc. (a) ZnO, (b) Zn-Ca 1, (c) Zn-Ca 3, (d) Zn-Ca 5.

An XPS analysis was performed for the samples, obtaining signals corresponding to
zinc (Zn 2p), calcium (Ca 2p), oxygen (O 1s) and carbon (C 1s). In Figure 5, we observed
the characteristic peaks for Zn 2p1/2 and Zn 2p3/2 [33]. The signals observed around 1047
and 1024 eV were assigned to Zn in octahedral sites, and the increase in the intensity of
these signals with the increase in doping is appreciable, which is related to the increase in
the concentration of antisite defects [45,46].
The calcium spectra (Figure 6) showed peaks belonging to Ca 2p3/2 at 351 eV and Ca
2p1/2 at 347 eV, and the deconvolution of the spectrum in Figure 5c suggests interactions
between COO- and Ca2+ in accordance with reports by other authors [47,48].

Figure 6. XPS spectra for calcium. (a) Zn-Ca 1, (b) Zn-Ca 3, (c) Zn-Ca 5.

Figure 7 shows the XPS analysis corresponding to oxygen. The peak corresponding to
O 1s can be broken down into various Gaussian components, where the band present at
Int. J. Mol. Sci. 2022, 23, 7258 7 of 13

approximately 533 eV belonged to chemisorbed oxygen (OC ), the band at 531 eV belonged
to lattice oxygen (OL ) species, whereas the signals present at approximately 534 eV can
be assigned to the C = O bond in the nanocomposites, where increases in intensity were
proportional to the percentage of the dopant [33,49].

Figure 7. XPS spectra for oxygen. (a) ZnO, (b) Zn-Ca 1, (c) Zn-Ca 3, (d) Zn-Ca 5.

The spectra for carbon (C 1s) is shown in Figure 8, where the peak deconvolutions at
289, 287, 285, and 284 correspond to O-C = C, C = O, C-O and C = C, respectively [48]. The
spectra indicates a shift towards higher binding energies when calcium ions are used as a
dopant [33,49].

Figure 8. XPS spectra for carbon. (a) ZnO, (b) Zn-Ca 1, (c) Zn-Ca 3 and (d) Zn-Ca 5.

The optical absorbances obtained in the UV-vis analysis (Figure 9) can be attributed to
the Zn-O electron transitions of ZnO. The results show a small shift in the red region (3.3
a 2.99 eV) [50]. Thus, the incorporation of CaO into ZnO produces only small variations
in the band gap energy, Eg. Adding donor or acceptor impurities to a semiconductor
creates energy levels near the conduction or valence band edges, as seen for the Zn-Ca
1 and Zn-Ca 5 nanocomposites. This behavior is in agreement with that reported by
various authors [31,32,42], and indicates that the incorporation of calcium ions to the
Int. J. Mol. Sci. 2022, 23, 7258 8 of 13

material presents small variations in the energy of the forbidden band without generating
modifications in the stability of ZnO.

Figure 9. UV-Vis spectra and determination of the bandgap energies for ZnO, Zn-Ca 1, Zn-Ca 3, and
Zn-Ca 5.

The antibacterial properties of the evaluated nanocomposite can be attributed to

reactive oxygen species (ROS) [51,52] that inhibit bacterial growth because ZnO NPs release
Zn2+ ions that cross the cell wall and react with cytoplasmic content. It is also known that
ZnO NPs produce H2 O2 , which is a strong oxidant capable of causing great damage to the
cell membranes of bacteria [50,53]. These results are in accordance with Kim et al. who
evaluated the antimicrobial activity of films containing ZnO and determined that as the
concentration increased, the antimicrobial activity of the film produced a better inhibition
halo (Figure 10) [54]. In addition, the antimicrobial activity of ZnO and its nanocomposites
synthesized in this work showed better activity than previous reports where the inhibition
halos ranged from 12–15 mm [55,56] Table 2.

Figure 10. Antibiograms of the evaluated treatments, (a) E. coli, (b) E. faecalis, (c) S. Aureus, (d) S.
mutans, (e) A. odontolyticus, (f) V. parvula, (g) F. nucleatum.
Int. J. Mol. Sci. 2022, 23, 7258 9 of 13

Table 2. Antimicrobial activity of ZnO-CaO samples.

Treatment Concentration (µg/mL)

Control
100 200 300 400 500
(+)
E. coli ATCC 8739
ZnO 19.3 6.33 7.33 7.33 7.67 8.67
Zn-Ca 1% 18.3 6.63 6.67 6.67 7.33 8.33
Zn-Ca 3% 18.6 8.33 8.67 11.33 12 13.67
Zn-Ca 5% 17.5 10 10 13 13.33 14.67
E. faecalis ATCC 19433
ZnO 23.6 8.33 10 10.67 14.33 15.33
Zn-Ca 1% 22.6 6.33 9 9 9.33 10.67
Zn-Ca 3% 23.5 11 12.33 13.7 16.33 17.67
Zn-Ca 5% 22 11.33 14 15.67 16.33 18.67
S. Aureus ATCC 33862
ZnO 23.8 13.5 14.3 15 16.3 17.6
Zn-Ca 1% 25 15 15.3 17 19.5 20
Zn-Ca 3% 25.3 19 19.6 23 24.5 31.5
Zn-Ca 5% 22.4 20 20.6 22.6 25 25.6
S. mutans ATCC 25175
ZnO 25.5 14.5 17 18 19.5 20.5
Zn-Ca 1% 25 15.5 16 18 19.5 20
Zn-Ca 3% 25.5 19 21 22.5 23.5 24.5
Zn-Ca 5% 26.5 16.5 17 19.5 21 23.5
A. odontolyticus ATCC 17929
ZnO 26.4 14.5 17.5 20 22 24.5
Zn-Ca 1% 25.6 15 18 24 25 27
Zn-Ca 3% 26 25 28 30 31 31.3
Zn-Ca 5% 27.3 20 27 30 32 35
V. párvula ATCC 10790
ZnO 17.3 9.2 9.6 10.3 10.9 11.4
Zn-Ca 1% 17.9 10.3 10.6 10.8 11.3 11.6
Zn-Ca 3% 17 11.4 11.9 12.5 12.7 12.9
Zn-Ca 5% 18.2 12.3 12.8 13.0 13.4 13.7
F. nucleatum ATCC 25586
ZnO 17.6 10.3 10.7 11.2 11.8 12.3
Zn-Ca 1% 18.4 10.5 10.9 11.6 12.0 12.7
Zn-Ca 3% 18 12.6 12.95 13.4 13.7 14.1
Zn-Ca 5% 17.3 13.3 13.8 14.2 14.6 14.9

4. Materials and Methods

4.1. Chemical Reagents
Zinc oxide (ZnO) was obtained from zinc acetate dihydrate (C4 H6 O4 Zn * 2H2 O, Sigma
Aldrich, St. Louis, MO, EE. UU.), calcium ions were obtained from calcium nitrate A.C.S.
(CaN2 O6 * 4H2 O, MEYER, CDMX, MX.).

4.2. Nanomaterial Synthesis

Zn-CaX nanoparticles were synthesized by the sol-gel method with some modifications
by using zinc acetate as a precursor [38] and Ca2+ as the dopant. For this purpose, 14 g of
Zn acetate was dissolved in 140 mL of ethanol (CTR, VA, EE. UU.) in a three-mouth flask
with the following amounts of Ca2+ : 1% (Zn-Ca 1), 3% (Zn-Ca 3), and 5% (Zn-Ca 5). Then,
a few drops of HNO3 (1 M, Sigma Aldrich) were added to adjust the pH of the solutions to
3. Each solution was heated under reflux at 80 ◦ C for four hours with magnetic stirring.
The solution was then cooled down to 0 ◦ C for 18 h. The resulting gel was dried at 100 ◦ C
and annealed at 500 ◦ C for 4 h in a static air atmosphere (heating rate of 2 ◦ C min−1 ). A
similar procedure was followed for the synthesis of ZnO nanoparticles.
Int. J. Mol. Sci. 2022, 23, 7258 10 of 13

4.3. Sample Characterization

The morphology of the materials was observed by scanning electron microscopy
(Tescan, MIRA 3LMU, LDN, UK) operated at 20 kV. High-resolution images were acquired
using a high-resolution transmission electron microscope (Jeol microscope, JEM-ARM200F,
Boston, MA, USA.) operated at 200 kV. The resulting images were analyzed using Gatan
Micrograph software v. 3.7.0 (Pleasanton, CA, USA).
The absorption spectra of the materials were acquired by a UV-Vis DRS (Shimadzu
UV-2600, Tokyo, Japan) provided with an integration sphere suitable for diffuse reflectance
studies. The UV-Vis DRS spectra were obtained from 190 to 900 nm wavelength. From the
plot, the bandgap energy was calculated using Plank’s equation [32] as follows:

1239.8
Eg = (1)
λ
where energy (Eg ) = band gap energy (eV), and wavelength (λ) = absorption peak value.
The X-ray powder diffraction patterns were acquired using an XRD Panalytical diffrac-
tometer (Empyrean, Almelo, Netherland) equipped with Cu Kα radiation (λ = 0.154 nm).
Data were collected from 10◦ to 90◦ (2θ) with a scan rate of 0.02◦ /0.2 s. The average crystal
size was determined using the Scherrer equation as follows:

kλ
D= (2)
β cos θ

where D is the crystal size, k is the form factor (0.89), λ is the wavelength of Cu Kα radiation,
β is the width evaluated at mid-high of the most intense diffraction peak and θ is the Bragg
angle. The inter-planar distance (d) can also be evaluated from Bragg’s law as follows:

2d sin θ = nλ (3)

The FT-IR spectra for the material were recorded with an FT-IR (Shimadzu, IRTracer-
100, Tokyo, Japan) spectrophotometer using attenuated total reflectance (ATR) with a
diamond waveguide (XR model). A detector with fast recovery deuterated triglycine
sulfate (DTGS) (standard) was used for the analysis. The spectra were recorded at room
temperature with 24 scans and 4 cm−1 of resolution from 4000 cm−1 to 400 cm−1 .
The interactions between chitosan and magnetite in the ChM composite and ChM-
arsenic were characterized by X-ray photoelectron spectroscopy (XPS) using an XPS SPECS
system (Berlin, Germany), which contains a Phoibos 150 analyzer and a 1D DLD detector.
The XPS spectra were obtained with a monochromatic Al Kα source (1486.7 eV) working at
250 W (12.5 kV and 20 mA) and a base pressure of 3 × 10−9 mbar in the analytical chamber.
The high-resolution scans were conducted with a pass energy of 15 eV and step sizes of
0.1 eV using a flood gun source with 20 µA of emission and 2 eV energy to compensate.
Data were analyzed with Analyzer 2.21 software using Lorentzian–Gaussian curves after
background subtraction [57].

4.4. Antibacterial Activity

Zn-Ca NPs were evaluated against S. aureus (ATCC 33862), E. coli (ATCC 8739), E.
Faecalis (ATCC 19433), S. mutans (ATCC 25175), V. parvula (ATCC 10790), F. nucleatum (ATCC
25586) and A. odontolyticus (ATCC 17929) by using the disk diffusion method. Bacteria were
inoculated 108 CFU/mL onto Muller Hinton agar medium in petri dishes. Consequently a
6 mm diameter paper disc was placed on the test organism impregnated with nanoparticles
(100, 200, 300, 400, and 500 µg/mL), prepared in sterile bi-distilled water. The plates were
incubated at 37 ◦ C for 24 h. Discs with ampicillin (10 µg/mL) were used as the positive
control (C+), and discs impregnated with sterile bi-distilled water served as the negative
control (C−). Antimicrobial activity was determined by measuring the zone of inhibition
(mm) around the disc [41].
Int. J. Mol. Sci. 2022, 23, 7258 11 of 13

5. Conclusions
The results of this study show that ZnO synthesized with the sol-gel method does not
present changes in its textural and surface properties, maintaining the characteristic wurzite
phase of ZnO, as well as its morphology. The Zn-Ca nanocomposite had better antibacterial
activity against Gram-negative bacteria, with an increase in efficacy proportional to the
percentage of Ca. Zn-Ca nanocomposites represents an active line of research for the
dental field, and the evidence suggests continuing the evaluation of the applicability of this
nanomaterial and its interaction with other microorganisms of dental interest.
Researchers who are interested in carrying out similar research work are recommended
to carry out a nitrogen physisorption analysis to determine the surface area of the mate-
rial, cytotoxicity and cell viability studies, in addition to carrying out release profiles to
determine the optimal doses of use.

Author Contributions: Writing—original draft preparation, M.I.T.-R., U.J.M.-C. and J.L.G.; writing—
review and editing, M.F.Y.-A., L.B.-S., Y.K.G.-M., M.M.-C., C.M.G., J.A.R.-M., O.A.G.-V. and A.P.-L.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: Torres-Ramos MI and JL Gonzalez thank Conacyt for the scholarships received
(778183 and 926097) and Sergio Oliva and Martin Flores from the UDG for the use of XRD and
SEM equipment at the Centro Universitario de Ciencias Exactas e Ingenierias of the University of
Guadalajara, Jalisco, Mexico.
Conflicts of Interest: The authors declare no conflict of interest.

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