UNIVERSIDADE ESTADUAL PAULISTA

“JÚLIO DE MESQUITA FILHO”

Instituto de Ciência e Tecnologia

Campus de São José dos Campos

ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2025.e4595

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated with

antibiotic/chitosan solution

Propriedades físico-químicas e antibacterianas de agregado trióxido mineral modiﬁcado com nanopartículas de ZnO

hidratado com solução de antibiótico/quitosana

Ismi KHUZAIMAH1 , Sri Juari SANTOSA1 , Fajar Inggit PAMBUDI1 , Dyah IRNAWATI2 , NURYONO1 

1 - Universitas Gadjah Mada, Faculty of Mathematics and Natural Sciences, Department of Chemistry. Yogyakarta, Indonesia.

2 - Universitas Gadjah Mada, Faculty of Dentistry, Department of Dental Biomaterial. Yogyakarta, Indonesia.

How to cite: Khuzaimah I, Santosa SJ, Pambudi FI, Irnawati D, Nuryono. Physicochemical and antibacterial properties of ZnO

nanoparticles-modied mineral trioxide aggregate hydrated with antibiotic/chitosan solution. Braz Dent Sci. 2025;28(2):e4595.

https://doi.org/10.4322/bds.2025.e4595

ABSTRACT

Mineral trioxide aggregate (MTA) modied with ZnO nanoparticles (ZnONP) and hydrated with chitosan

solution denoted MTA-ZnO/Ch is a strategy to solve the MTA limitations as a pulp capping material in root canal

therapy. MTA materials with high antibacterial activity and antibiotic delivery are being developed. Objective:

This study evaluated the effect of various antibiotic additions to MTA-ZnO/Ch on mechanical and antibacterial

activities. Material and Methods: The ZnONP, 5% (w/w), was added during preparing MTA, and the resulting

gel was calcined at 1000 °C to obtain MTA-ZnO. The MTA-ZnO was hydrated with 4% chitosan in 1% acetic acid

solution containing antibiotics (tetracycline, metronidazole, amoxicillin, and ampicillin) at a 1:1 ratio to produce

MTA-ZnO/Ch-AB. The properties studied included compressive strength, diametral tensile strength, radiopacity,

mass loss percentage, pH change, and antibacterial activity against

Pseudomonas aeruginosa

(

P. aeruginosa

) and

Enterococcus faecalis (E. faecalis)

. Results: The synthesized MTA-ZnO/Ch has a compact structure, large surface

area, and mesopore distribution with an average particle size of 747.04 nm. Among antibiotics investigated, only

tetracycline improved the compressive and diametral tensile strengths of MTA-ZnO/Ch. Adding all investigated

antibiotics did not affect the mass loss percentage (except for tetracycline reducing the mass loss), radiopacity,

and the pH of the MTA-ZnO/Ch immersed saliva. Except for ampicillin addition against

E. faecalis,

which did

not show antibacterial activity, antibiotics added to MTA-ZnO/Ch improved antibacterial activities against

aeruginosa

and

E. faecalis

, Conclusion: Adding tetracycline in MTA-ZnO/Ch enhanced physicochemical and

antibacterial properties, and it can potentially be a conservative pulp therapy material.

KEYWORDS

Antibacterial; Antibiotics; Chitosan; Mineral trioxide aggregate; ZnO nanoparticles.

RESUMO

O agregado trióxido mineral (MTA), modicado com nanopartículas de óxido de zinco (ZnONP) e hidratado com

solução de quitosana (MTA-ZnO/Qu), tem sido proposto como alternativa para superar as limitações do MTA

convencional como material de capeamento pulpar em terapias endodônticas. Materiais à base de MTA com maior

atividade antibacteriana e capacidade de liberação de antibióticos vêm sendo desenvolvidos. Objetivo: Avaliar

o efeito da adição de diferentes antibióticos ao MTA-ZnO/Qu sobre suas propriedades mecânicas e atividade

antibacteriana. Material e Métodos: As ZnONP (5% p/p) foram incorporadas ao MTA durante o preparo, e o

gel obtido foi calcinado a 1000 °C para obtenção do MTA-ZnO. Este foi então hidratado com quitosana a 4%

em solução de ácido acético a 1% contendo antibióticos (tetraciclina, metronidazol, amoxicilina ou ampicilina),

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

na proporção 1:1, resultando no composto MTA-ZnO/Qu-AB. As propriedades avaliadas incluíram resistência

à compressão, resistência à tração diametral, radiopacidade, perda de massa, variação de pH e atividade

antibacteriana frente a

Pseudomonas aeruginosa

(

P. aeruginosa

) e

Enterococcus faecalis

(

E. faecalis

). Resultados:

O MTA-ZnO/Qu sintetizado apresentou estrutura compacta, ampla área supercial e distribuição mesoporosa,

com tamanho médio de partícula de 747,04 nm. Dentre os antibióticos testados, apenas a tetraciclina aumentou

signicativamente as resistências à compressão e à tração diametral. A adição dos antibióticos não alterou a

perda de massa (exceto pela tetraciclina, que reduziu esse parâmetro), radiopacidade e pH da saliva em que o

material foi imerso. Todos os antibióticos melhoraram a atividade antibacteriana contra as bactérias testadas,

exceto a ampicilina frente a E. faecalis, que não demonstrou atividade antibacteriana, os antibióticos adicionados

ao MTA-ZnO/Qu melhoraram as atividades antibacterianas contra

P. aeruginosa

E. faecalis

. Conclusão: A

incorporação de tetraciclina ao MTA-ZnO/Qu aprimorou suas propriedades físico-químicas e antibacterianas,

destacando seu potencial como material conservador para terapias pulpares.

PALAVRAS-CHAVE

Antibacterianos; Antibióticos; Quitosana; Agregado trióxido mineral; Nanopartículas de ZnO.

fulll several requirements as a capping agent,

namely having antibacterial properties, adhering

to dentin and restoration materials, resisting

pressure when applying restoration materials,

and looking radiopaque on radiographs after

application [5]. In addition, pulp capping

materials must have good physical properties,

one of which is tensile strength, representing

good adhesion ability with the tooth [6]. Efforts

to overcome the limitations have been reported

in previous research. Our last study proved

that adding 4% chitosan to MTA provides the

highest intimate contact between dentin and

material interface and enhanced antibacterial

activities compared to commercial MTA [7].

However, chitosan addition to MTA lowers

mechanical strength at the early stages of cement

hydration [8]. As the main component of MTA,

compounds have been widely used to repair

hard tissues such as bones and teeth due to their

ability to induce mineralization [9]. Furthermore,

chitosan can deposit on the surface of the dentinal

tubules, resulting in more adhesion between the

dentin and MTA interface [7].

ZnO nanoparticles are widely used in various

biomaterials to improve their mechanical and

biological properties [10]. The release of zinc ions

at low concentrations can increase antibacterial

activity, stimulate cell proliferation in vitro, and

stimulate osteoblast cells in hard tissues such as

bones and teeth. On the other hand, the presence

of ZnO in MTA does not signicantly change its

radiopaque property; hence, the composition

ratio of ZnO and Bi2O3 can be applied to

achieve the qualified radiopacity level. The

promising characteristics of ZnO and chitosan

have made them suitable for modifying MTA.

INTRODUCTION

Oral and dental diseases are significant

global health concerns, affecting millions of

people. The community’s typical dental disease

is caries, which can progress to pulp disease. In

conservative pulp therapy, calcium hydroxide or

mineral trioxide aggregate (MTA) is needed to

maintain pulp vitality in cases of conservative

pulp treatment. Calcium hydroxide is cheaper

and more accessible, but the results are not as

optimal as when using MTA. An in-vivo study

by Cervino et al. [1] showed that MTA can

stimulate the proliferation of pulp cells much

higher than calcium hydroxide. MTA is similar

to Portland cement in that the main ingredient

is ne hydrophilic particles and has the main

components of tricalcium silicate, dicalcium

silicate, and tricalcium aluminate, but with the

addition of bismuth oxide as a radiopaque agent.

This material takes 3-4 hours to harden and

has a pH of 12.5 [1]. The use of MTA in health

applications includes pulp capping treatment,

perforation repair, root tip closure, and spurring

root development in pulpotomy treatment [2].

Even though showing excellent sealing

ability and biocompatibility in pulp capping

applications, MTA has weaknesses, including

bacterial penetration in clinical applications,

which subsequently induces inflammatory

responses [3]. Thus, an ideal dental material

should exhibit antibacterial efficacy without

detrimentally affecting its biological and physical

properties. Another limitation of MTA is poor

mechanical properties in the early stages of

cement hydration, which causes it to wash out

quickly in a liquid environment [4]. MTA must

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

Combining MTA with ZnO and chitosan improves

its mechanical and antibacterial properties [10].

During the past few decades, the most effective

method of infection removal caused by numerous

microbes, particularly aerobes and anaerobes

bacteria, has been using antibiotics or other

antimicrobials incorporated in some antibiotic

delivery systems. Several antibiotic-releasing

systems for dental drug delivery have been

reported, including chitosan lm for metronidazole

and levooxacin delivery [11], chitosan-pectin and

chitosan-alginate polyelectrolyte complex (PEC)

lms for tetracycline [12], and hydroxyapatite/

chitosan composite for minocycline [13]. It is

necessary to develop the pulp capping material,

MTA modied with ZnONP and chitosan (MTA-

ZnO/Ch), to be used as an antibiotic delivery

system. This research aims to evaluate the effect of

adding various antibiotics on the physicochemical

and antibacterial properties of MTA-ZnO/Ch. It is

hypothesized that adding antibiotics increases the

antibacterial activity of MTA without reducing the

essential properties of MTA/ZnO/Ch for a pulp

capping agent. In this research, four antibiotics

(tetracycline, metronidazole, amoxicillin, and

ampicillin) were chosen, and two bacteria,

Pseudomonas aeruginosa

(

P. aeruginosa

) and

Enterococcus faecalis (E. faecalis)

, were tested.

MATERIAL AND METHODS

Materials

The materials used for preparing MTA were

bismuth (III) oxide (Bi2O3) powder, zinc oxide

nanoparticles (ZnONP) powder, 25% ammonia

(NH3) solution, tetraethyl orthosilicate (TEOS),

calcium carbonate (CaCO3) powder, and aluminum

nitrate nonahydrate (Al2(NO3)3.9H2O). Hydration

of MTA material used chitosan (C6H11O4 99% purity

Sigma Aldrich, USA) 4% solution in acetic acid

(CH3COOH 99% purity Merck, USA) 1% solution.

Three antibiotic powders in pharma-grade were

ampicillin, amoxicillin, and tetracycline, while

metronidazole was used in an aqueous solution

with a concentration of 5000 mg/L. Chemicals

used to prepare the artificial saliva included

potassium chloride (KCl) powder, sodium chloride

(NaCl) powder, sodium bicarbonate (NaHCO3)

powder, anhydrous sodium hydrogen phosphate

(Na2HPO4) powder, potassium dihydrogen

phosphate (KH2PO4) powder, urea (CH4N2O)

powder, potassium thiocyanate (KSCN) powder,

and 1 M hydrochloric acid (HCl) solution. OXOID

ciprooxacin test disc, bacteria of

Pseudomonas

aeruginosa

(

P. aeruginosa

) (ATCC 10145) and

Enterococcus faecalis (E. faecalis)

(ATCC 29212),

70% ethanol solution, 0.9% sodium chloride

(NaCl) infusion, and OXOID Muller Hinton Agar

media were used to test the antibacterial activity

of the MTA materials.

Instrumentation

The instruments needed in this research

were an attenuated total reflectance-infrared

spectroscopy (ATR-IR) 8201 PC Shimadzu, X-ray

diffractometer (XRD) Bruker AXS D8 Advance

ECO, scanning electron microscope-energy

dispersive X-ray (SEM-EDX) Jeol JSM-6510LA,

and Surface Area Analyser (SAA) Quantachrome

Novatouch Lx4.

Preparation of MTA modied with ZnO

nanoparticles

ZnONP-modified MTA (MTA-ZnO)

containing CaO, SiO2, Al2O3, Bi2O3, and ZnONP of

60%, 20%, 2%, 13% and 5%, respectively, were

synthesized using a sol-gel method. A mixture

of 200 mL of distilled water and 200 µL of 25%

ammonia solution was stirred for 10 minutes, then

6.93 mL of TEOS was added and stirred for 10

minutes. CaCO3 powder (10.71 g) was added to

the solution and allowed to stand for 30 minutes

while stirring at 80 °C. The white suspension was

then mixed with 0.70 g of Al2(NO3)3.9H2O and

0.50 g (5%) of ZnO and stirred for 1 hour at 80 °C.

The suspension was evaporated while stirring at

120 °C for 90 minutes. Then, the gel was heated

at 100 °C for 2 hours. The resulting powder

was then calcinated at 1000 °C for 3 hours. The

powder was pulverized, and 13% Bi2O3 (1.3 g)

was added and sieved with a 200 mesh sieve [7].

Hydration of MTA-ZnO with chitosan and

antibiotic solution

MTA-ZnO samples were hydrated using a

liquid phase prepared by mixing a 4% (w/v)

chitosan (Ch) in 1% acetic acid solution and a

1500 ppm antibiotic solution with a volume ratio

of 1:1. Antibiotic types were tetracycline (TC),

metronidazole (MET), amoxicillin (AMX), and

ampicillin (AMP). The ratio of MTA material mass

(g) to liquid volume (mL) was 2:1. The hydrated

MTA samples denoted MTA-ZnO/Ch, MTA-ZnO/

Ch-TC, MTA-ZnO/Ch-MET, MTA-ZnO/Ch-AMX,

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

and MTA-ZnO/Ch-AMP were then molded in

acrylic cylinder molds according to ISO 9917-1

standard with 4 mm diameter (d) and 6 mm high

(t) for compressive strength test; d=4 mm and

t=3 mm for diametral tensile strength test, pH

change, mass loss percentage and antibacterial

activity test. For the radiopacity test, the sample

size is d=5 mm and t=1 mm. In addition, the

materials were characterized by XRD, SEM-EDX,

ATR-IR, and SAA, as well.

Measurement of compressive strength and

diametral tensile strength

MTA samples that had been hydrated for

24 hours were tested with a Universal Testing

Machine (UTM) in triplicate (n=3) following ISO

9917 to produce a value in MPa. Each sample

was tested with a pressure and force of 0.01 N,

pre-load speed of 300 mm/min, and test speed

of 10 mm/min. The maximum load required to

break the sample was recorded as the mechanical

test value for compressive strength and diametral

tensile strength.

Radiopacity measurement

The radiopacity of the MTA samples was

measured following ISO 6876; 9971-1 and

ANSI/ADA in triplicate using ImageJ software

for calculating Al distance (mm). The samples

were hydrated and then printed with a size of

d = 5 mm and t = 1 mm. The molded hydrated

samples were left at room temperature (29 oC)

for 24 hours. Next, the sample was placed next

to the aluminum step wedge and irradiated with

7 mA X-rays at 70 kV. The distance between

the specimen and the X-ray source was 30 cm

with an exposure time of 0.2 seconds. The pellet

sample was placed next to the aluminum step

wedge with a standard 1-13 mm thickness. The

radiopacity was determined based on the value

of X-ray intensity that can penetrate the MTA

pellet material and aluminum step wedge. The

radiopacity was calculated by comparing the

thickness of the aluminum step wedge, which

was calculated using Equation 1.

A log 1

255



=−−





(1)

where A is the absorption and G is the grey scale

value of the digital image of the aluminum step

wedge.

Measurement of pH and mass loss percentage

The mass loss of hydrated samples was

determined according to the ISO 6876 standard in

an articial saliva media prepared with an Afnor

method [14]. KCl powder (1.2 g), NaCl (0.7 g),

NaHCO3 (1.5 g), Na2HPO4 (0.26 g), KH2PO4 (0.2 g),

urea (0.13 g), and KSCN (0.33 g) were mixed in

a 1 L glass beaker containing 500 mL of distilled

water. The mixture was stirred with a magnetic

stirrer until homogeneous, and the pH was

adjusted to 6.8 by adding 1 M HCl. The volume was

adjusted to 1 L by adding distilled water, and the

solution temperature was maintained at 37 °C. In

the rst step in measuring pH and determining the

mass loss percentage, each sample was weighed

for mass and recorded as the initial mass. Each

sample was immersed in 2.5 mL articial saliva

solution, and the pH of the solution was measured

at intervals of 1, 3, 7, and 14 days. During the same

periods, the mass was weighed, and the samples

were heated at 150 °C for 7 hours. The mass of the

sample before and after being immersed in saliva

was used to determine the percentage of mass loss.

Antibacterial activity testing

Antibacterial activity was tested for all

samples of MTA-ZnO/Ch, MTA-ZnO/Ch-TC,

MTA-ZnO/Ch-MET, MTA-ZnO/Ch-AMX, MTA-

ZnO/Ch-AMP. This study used ciprofloxacin

(CIP) as a positive control for antibacterial

testing in triplicate on each sample against

aeruginosa

and

E. faecalis

. At rst, all apparatus

and glassware used were sterilized by rinsing

with 70% alcohol and then kept in an autoclave

for 2 hours at 121 °C. Next, Muller Hinton Agar

(MHA) media was made by weighing 19 g of

MHA and dissolved with distilled water until it

reached a volume of 500 mL, then heated until

homogeneous. The media was sterilized using

an autoclave at 120 °C for 2 hours. MHA was

poured into Petri dishes about 25 mL and allowed

to solidify for test treatment. Suspensions of

aeruginosa

and

E. faecalis

test colonies were

made by transferring one bacterial colony from

solid MHA media into a test tube containing

5 mL of 0.9% NaCl. Then, the suspension of

test bacteria was inoculated on 0.1 mL of MHA

media [15].

Antibacterial activity was tested using a well-

diffusion method by embedding the sample on MHA

media. The test bacterial suspension was attened

with a cotton swab on MHA media, allowed to

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

dry, and then incubated for 24 hours at 37 °C. The

clear zone formed around the wells was observed

as the zone of inhibition of antibacterial activity.

The inhibition zone, representing the antibacterial

activity, was measured using a caliper three times

at different positions, and the values were averaged.

Statistical analysis

To evaluate the signicance of the difference

between tested MTA-ZnO/Ch materials groups

for the parameters being assessed, we used

a multiple comparison method (one-way

ANOVA followed by the Tukey HSD test). Each

parameter was analyzed separately, including

diametral tensile, compressive strength, pH

change, mass loss, radiopacity, and antibacterial

activity data. P values less than 0.05 were

considered statistically signicant. All statistical

data were calculated using SPSS (IBM Statistic

25.0) software.

Grouping the MTA-ZnO samples for

properties testing, including compressive strength,

diametral tensile strength, radiopacity, pH

change, mass loss percentage, and antibacterial

activity, are presented in Table I.

RESULTS

Characteristics of MTA-ZnO and MTA-ZnO/Ch

Crystallinity

The crystal phase of ZnO-modified MTA

(MTA-ZnO) and MTA-ZnO hydrated with 4%

chitosan in 1% acetic acid solution (MTA-ZnO/

Ch) was identied based on the XRD pattern as

presented in Figure 1. It is observed that almost all

d-spacing indicating the crystallinity of MTA-ZnO

and MTA-ZnO/Ch are not signicant differences.

Dicalcium silicate (C2S), tricalcium silicate

(C3S), and tricalcium aluminate (C3A) components

are formed during calcination at 1000 °C from the

precursors, with C2S and C3S of about 75%, as

the most signicant components in determining

the MTA properties. The formation of C2S can

be seen at 2θ = 18.04°, 41.19°, 54.29° (ICDD

00-024-0037), C3S at 2θ = 28.67°, 29.36°, and

32.08° (ICDD 00-014-0693), C3A at 2θ = 39.41°,

42.34°, and 50.84° (ICDD 00-033-0251). Calcium

silicate hydrate (CSH) is observed at 2θ = 29.36°

(ICDD 00-033-0306), Zn2SiO4 at 2θ = 47.24°

(ICDD 00-024-1468), Ca(OH)2 at 2θ = 34.10°,

71.55° (ICDD 00-004-0733), Zn(OH)2 at 2θ =

36.28° and 67.92° (ICDD 00-048-1066). Bi2O3

peaks are detected at 2θ = 26.89°, 27.38°, and

47.24° (ICDD 00-014-0659). The characteristic

intensity of C2S and C3S peaks decreases in MTA-

ZnO/Ch (Figure 1b), and the increase in CSH

peak indicates that hydration occurs.

Morphology

The SEM image is expressed in Figure 2a,

and based on the SEM image, the particle size

distribution was determined with ImageJ and

Origin on MTA-ZnO/Ch, resulting in an average

size of 747.04 nm (Figure 2b). ZnO is indicated

by the presence of ne globules scattered around

the MTA. SEM images of MTA-ZnO/Ch show a

dense structure with tiny pores following the

porosity analysis.

Surface area, porosity, and pore size distribution

MTA-ZnO/Ch was characterized with an SAA

for surface area analysis and porosity based on

N2 gas adsorption-desorption via the Brunauer-

Emmett-Teller (BET) method. At the same time,

pore size distribution was analyzed using the

Table I - Grouping the tested MTA-ZnO/Ch samples

No. Group Antibiotics added Material Code (n=3)

1 No addition (control) MTA-ZnO/Ch

2 Tetracycline MTA-ZnO/Ch-TC

3 Metronidazole MTA-ZnO/Ch-MET

4 Amoxicillin MTA-ZnO/Ch-AMX

5 Ampicillin MTA-ZnO/Ch-AMP

Figure 1 - XRD patterns of (a) MTA-ZnO (before hydration) and (b)

MTA-ZnO hydrated with chitosan solution (MTA-ZnO/Ch).

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

Barrett-Joyner Halenda (BJH) method. The test

results are presented in Table II.

Based on the data in Table II, modication

of MTA with ZnO nanoparticles and chitosan

increases the surface area and decreases the

total pore volume and average pore diameter

compared to MTA without modication.

Effect of antibiotics on MTA-ZnO/Ch

properties

Functional groups

FTIR spectra of MTA-ZnO (before hydration),

MTA-ZnO/Ch (after hydration with chitosan

solution), and MTA-ZnO/Ch with antibiotic

addition can be seen in Figure 3. In all FTIR

spectra, the wavenumber of 505 cm-1 shows

the Bi-O vibration of the Bi2O3 and the Zn-O

stretching vibration [16]. The C2S and C3S are

characterized by absorption at 1000-800 cm-1.

At 994 cm-1, corresponding to Si–O stretching

indicates stable γ-C2S phases. Another calcium

silicate phase (Si–O stretching) at 879 cm−1,

indicating the presence of Si(OSi)3O–Ca (C3S).

Adsorption at 1600-1350 cm-1 corresponds to

-O-H bending from Ca(OH)2 in un-hydrated MTA/

ZnO (Figure 2a). The O-H stretching vibration of

Ca(OH)2 was observed at 3740-3640 cm-1, and

the O-H bending was observed at 1500 cm-1. CSH

formed during the hydration is indicated by the

vibration of the O-Si-O chain at 710 cm-1 [10].

Figures 3c-f show the spectra of MTA-ZnO/

Ch with antibiotic addition. It can be seen that the

antibiotics do not give a signicant difference in

the FTIR spectra from MTA-ZnO/Ch. It probably

overlaps with the typical absorption of -CH

vibration at 1600-1396 cm-1. The board peak is

observed with the antibiotic addition, and a new

weak peak (due to low concentration) appears at

2900 cm-1, corresponding to the -C-H vibration from

chitosan and antibiotics. Those results are similar

to that of -NO2 from metronidazole [17], beta-

lactam ring from amoxicillin and ampicillin [18],

and -CH bending vibration from tetracycline [19].

The low concentration of antibiotics leads to weak

absorption bands.

Compressive strength

The effect of antibiotics on the compressive

strength of MTA-ZnO/Ch is presented in Figure 4a.

It can be seen that MTA-ZnO/Ch-TC has a

compressive strength of 8.34±0.47 MPa, which

Table II - BET area and porosity analysis of hydrated samples

Type of Analysis MTA MTA-ZnO/Ch

BET surface area (m2/g) 5.15 11.40

Total pore volume (cm3/g) 0.17 0.03

Average pore diameter (nm) 6.43 5.17

Figure 2 - (a) SEM image (2000x magniﬁcation), (b) particle

size distribution histogram of MTA-ZnO/Ch, and (c) adsorption-

desorption isotherm of MTA-ZnO/Ch.

is higher than that of MTA-ZnO/Ch. In contrast,

the compressive strength MTA-ZnO/Ch with the

addition of other antibiotics (MTA-ZnO/Ch-MET,

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

MTA-ZnO/Ch-AMX, and MTA-ZnO/Ch-AMP) is

smaller than MTA-ZnO/Ch (without antibiotics).

The value of p<0.05 was obtained through the

one-way ANOVA test, meaning that the antibiotics

affected the compressive strength of MTA-ZnO/

Ch. However, based on the Tukey HSD test, the

difference was not signicant.

Diametral tensile strength

The diametral tensile strength of MTA-ZnO/

Ch in the presence of antibiotics is expressed

in Figure 4b. It is observed that similar to the

tensile strength, MTA-ZnO/Ch-TC has the highest

diametral tensile strength compared to MTA-

ZnO/Ch. However, the compressive strength of

MTA-ZnO/Ch-MET, MTA-ZnO/Ch-AMX, MTA-

ZnO/Ch-AMP are lower than MTA-ZnO/Ch. It

can be concluded that the modication of MTA

with ZnO nanoparticles and chitosan and the

addition of antibiotics, except tetracycline, reduce

the diametral tensile strength.

Radiopacity

The calculated radiopacity is presented in

Figure 4c, showing that the radiopacity of all

MTA-ZnO/Ch samples, without antibiotics and

with antibiotic variations, is higher than 3 mmAl.

MTA-ZnO/Ch-AMX has a higher radiopacity than

other samples, namely 9.63±1.16 mmAl. The

magnitude of this value does not indicate that the

quality of radiopacity is always better than other

variations of MTA. Figure 4c shows a difference

in radiopacity between MTA-ZnO/Ch and MTA-

ZnO/Ch antibiotic addition. This is evidenced

by the one-way ANOVA test showing p=0.034,

but this difference is insignicant based on the

Tukey HSD test.

Mass loss percentage

Articial saliva was chosen as the sample’s

immersion medium due to its characteristics

being similar to those of the human biological

system. The results of the mass loss test on MTA-

ZnO/Ch with antibiotic addition can be seen in

Figure 5a.

Figure 3 - IR spectra of (a) MTA-ZnO, (b) MTA-ZnO/Ch, (c) MTA-

ZnO/Ch-TC, (d) MTA-ZnO/Ch-MET, (e) MTA-ZnO/Ch-AMX, (f) MTA-

ZnO/Ch-AMP.

Figure 4 - (a) Compressive strength, (b) diametral tensile strength,

and (c) radiopacity of MTA-ZnO/Ch variation of antibiotics.

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

It is evident that the mass loss percentage of

each sample increases from day 1 to day 14. The

antibiotics do not affect the mass loss percentage,

except tetracycline, which consistently tends to

be lower. However, as evidenced with the Tukey

HSD test, the decrease is not signicantly different.

pH testing

In the results of pH measurement (Figure 5b),

the saliva immersed with MTA-ZnO/Ch-ABs has

a pH of ±12.5, which is relatively constant on

days 1, 3, and 7. On day 14, the pH value drops

drastically to 9-10. The saliva immersed with

MTA-ZnO/Ch-TC has the highest pH among

the saliva immersed with other MTA materials.

However, the difference is insignicant, so it can

be concluded that adding antibiotics does not

affect the pH of MTA-ZnO/Ch.

Antibacterial activity of MTA-ZnO/Ch with anti-

biotic addition

The inhibition zones, illustrating the

antibacterial activity of MTA-ZnO/Ch-Abs, are

presented in Figure 6.

Ciprooxacin (10 µL) used as a positive control

(the data is not included in Figure 6) was found

to have an inhibition zone of 30.78±1.22 mm

against

P. aeruginosa

and 21.97±2.07 mm against

E. faecalis

. As can be observed, MTA-ZnO/Ch used

as a comparison sample showed no antibacterial

property on

E. faecalis

, as indicated by the absence

of an inhibition zone. However,

P. aeruginosa

shows

an inhibition zone of 7.97±0.49 mm, smaller than

the inhibition zone of MTA-ZnO/Ch-ABs. Almost

all MTA-ZnO/Ch samples with various antibiotics

show inhibition zones of 8.76-11.27 mm against

aeruginosa

and 2.00-7.50 mm against

E. faecalis,

with higher inhibition zone than MTA-ZnO/Ch

(without antibiotics). MTA-ZnO/Ch-AMX has the

largest inhibition zone compared to other antibiotic

variation modications, namely 11.27±0.31 mm.

Figure 6 shows that adding antibiotics signicantly

increases the antibacterial inhibition zone (p<0.05)

of MTA-ZnO/Ch based on the one-way ANOVA test,

except for ampicillin addition.

DISCUSSION

Characterization results indicated the

presence of C2S, C3S, C3A, Ca(OH)2, chitosan,

ZnO, and Bi2O3 in the synthesized MTA-ZnO/

Ch. The high peaks of Bi2O3 indicate the natural

Figure 5 - (a) Mass loss percentage and (b) saliva pH after various-

day immersion of MTA-ZnO/Ch-ABs.

Figure 6 - Diagram of antibacterial activity presented with an

inhibition zone of MTA-ZnO/Ch-AB.

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

crystal phase due to being added after the

solid reaction [20]. C2S and C3S are signicant

components of about 75%, essential in determining

the mechanical properties of hydrated MTA. In

contrast, C3A, the most reactive component, reacts

quickly with water, even though it contributes

less to the MTA strength [21]. Pure MTA has

limitations in properties, including slow to

set, creating porosity, and low antibacterial

properties; hence, modification with typical

components needs to be studied.

Hydration with chitosan solution does not

affect the MTA phase (XRD pattern in Figure 1),

except leading to decreased ZnO peak intensity.

This is probably due to the formation of Zn(OH)2

bonded to the CSH chain, which increases the

hydration rate of C3S [22]. Figure 2b shows that

the average size of MTA-ZnO/Ch is 747.04 nm.

Meanwhile, the average particle size of MTA

without modication is currently 1.5-40 µm. This

shows that modication with ZnONP decreases

the particle size due to the formation of zinc

silicate. The smaller radius of zinc ions than

calcium ions gives a shorter bond length between

Zn-Si compared to Ca-Si, resulting in denser

cement grains and increasing the physical

strength of MTA [10]. The added ZnONP partially

reacts with SiO2 by replacing Ca2+ to form zinc

silicate (Zn2SiO4). This substitution can occur due

to their similar ionic charges [23]. In addition, the

large surface area allows good cell adhesion to

interact with cells within the root canal, thereby

increasing contact with dentin [24].

Based on the data in Table II, chitosan

reduces porosity. It strengthens the interaction

between aggregates in the composite due to

hydrogen bonding between the active group -OH

on the chitosan molecule and the silanol group

(-Si-OH) on the silicate chain. This interaction

reduces the void space and thickness zone of

the adjacent CSH interlayer, thus increasing the

physical strength of the MTA [7]. The chitosan in

the hydrated MTA improves the physicochemical

properties because chitosan acts as a reinforcing

polymer to reduce porosity and strengthen the

interaction between aggregates in the composite.

In addition, chitosan enhances the adhesion

between the cement matrix and chitosan,

increasing the cement plasticity. According to

Mariyam et al. [7], chitosan polymers are assumed

to intercalate in the CSH interlayer and further

form hydrogen bonds between the active -OH

group on the chitosan molecule and the silanol

group (-Si-OH) on the CSH silicate chain. These

intercalations and interactions reduce space,

make the CSH interlayer closer, and increase

MTA’s physicochemical strength. Adding chitosan

to MTA neutralizes the acidic environment

through the protonation of amino groups and

increases the pH of the immersion solution.

Therefore, the corrosion, pore formation, and

micro-leakage can be minimized as the acidity

is reduced [7].

Antibiotics added to MTA-ZnO/Ch reduce

mechanical properties, including compressive

and shear tensile strength (Figure 4). This

is following the research of Hegde and

Arora [25], which states that the addition of

DAP (double antibiotic paste) has a minimal

effect on the compressive strength of ProRoot

MTA® compared to the addition of TAP

(triple antibiotic paste) and the compressive

strength value decrease as the concentration

of antibiotics added increases. Antibiotics can

penetrate the root canal wall and bind with

dentin and calcium ions to form insoluble

complexes through chelation. Antibiotics can

create an acidic environment in the root canal

that can affect the binding of calcium silicate-

based materials in the dentinal tubules. Unlike

other antibiotics, the data shows that adding

tetracycline on MTA-ZnO/Ch increases the

compressive strength and diametral tensile

strength. It is probably because the rich number

of hydroxyl groups in tetracycline molecules

interact with hydroxyl groups in chitosan

to form glycoside bonds by releasing H2O

molecules, which makes the distance between

chitosan chains shorter [26], leading to lower

solubility of the material.

The result aligns with data in Figure 5a,

indicating that the MTA-ZnO/Ch-TC sample

shows a minimal mass loss percentage compared

to other samples. According to the American

Dental Association (ADA), the solubility

used as a root canal sealer must have a value

smaller than 3%. From the results of this

study, only MTA-ZnO/Ch-TC is close to the

allowed standard, which is 3.71% on day 1.

Fridland and Rosado [27] obtained data that

the solubility of MTA was 13-14% when soaked

in distilled water for 14 days with a water/MTA

powder ratio of 0.33%. With the same ratio,

Gandol et al. [28] reported the solubility of

MTA Plus, MTA Plus gel, and ProRoot MTA

samples after soaking in distilled water for 14

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

days, namely 18.51%, 14.62%, and 10.79%,

respectively. Therefore, due to the enhanced

physicochemical properties and low mass loss

percentage, adding tetracycline positively affects

MTA-ZnO/Ch and its potential as pulp capping

material in clinical applications.

Even though in this study, all MTA samples

experienced mass loss, the solubility of components

does not always have a negative effect. Fridland

and Rosado [27] found that calcium hydroxide

was the main compound released by MTA upon

soaking. These hydroxides may be responsible

for MTA’s benecial properties with dental and

periapical tissues. Zbańska et al. [29] reported the

regeneration of new cementum from MTA as a

unique phenomenon that may be due to its sealing

ability, biocompatibility, high pH, or the release of

calcium hydroxide that can activate cement oblasts

to lay down the matrix for cement genesis.

The radiopacity of MTA-ZnO/Ch materials,

without and with antibiotic additions, is higher than

3 mmAl. This value follows the standard values

of ISO 6876: 2012 and ANSI/ADA number 57.

Kang et al. [30] stated that root-end lling materials

should be distinguishable from bone and root

adjacent to dentin for easy differentiation during

treatment. In contrast, materials with radiopacity

values smaller than 3 mmAl cannot be distinguished.

Adding bismuth oxide to MTA increases the

radiopacity without damaging physical properties.

The combination of Portland cement with bismuth

oxide positively correlates with radiopacity and

the added bismuth oxide concentration. ZnO is a

radiopaque agent commonly used in endodontic

materials [31]. The radiopacity value of Bi2O3 is

higher than ZnO due to its atomic density, but

when ZnO is added to MTA, it significantly

improves its physicochemical properties without

losing its radiopacity. Adding chitosan with higher

concentration reduces the radiopacity due to the

absence of radiopacication properties in chitosan

material [10]. In other words, the radiopacity of

MTA-ZnO/Ch-ABs does not change and meets the

pulp capping material standard.

MTA-ZnO/Ch, with the addition of

antibiotics, has a pH of ±12.5, which is relatively

constant on days 1, 3, and 7, then drops to

9-10 on day 14. Theoretically, if the pH is high,

the concentration of Ca2+ in water is high. Ca2+

ions bind OH- from H2O so that OH- species in

water are also higher, and the pH increases [32].

Zn(OH)2 and calcium hydroxyzincate solubility

rise in a solution with higher alkalinity, leading

to an elevated pH level. Therefore, a large

amount of Zn(OH)2 and calcium hydroxyzincate

species can increase the pH value in hydrated

products. Even in lower Ca2+ concentrations,

zinc ions can dissolve and form zincate ions. In

addition, a high pH environment causes apatite

precipitation at the material-dentin interface,

resulting in excellent sealing and occlusion

ability of dentinal tubules [10].

Adding antibiotics causes different effects on

the antibacterial activity. The MTA-ZnO/Ch was

used as a comparison sample, and MTA-ZnO/

Ch-AMP showed no antibacterial activity against

E. faecalis

. Adding other antibiotics (TC, MET,

and AMX) led to antibacterial activity in a 2.00-

7.50 mm range.

E. faecalis

bacteria can survive

in very extreme environments, including very

alkaline pH and high salt concentrations [33].

Ampicillin antibiotics have a beta-lactam ring

structure that binds to penicillin-binding protein

(PBP) on the bacterial cell wall. This bond

inhibits cell wall formation and causes damage

to the bacterial cell wall structure. However,

this method is less effective when used on

microorganisms that have beta-lactam enzymes

that work by breaking the beta-lactam ring in

the penicillin group. As a result, the damaged

beta-lactam ring cannot bind to PBP, so its

function as an inhibitor of cell wall formation is

inefcient [34]. According to Moon et al. [35],

reduced ampicillin susceptibility in enterococci

is due to the expression of a low-afnity class B

PBP called PBP4 in

E. faecalis

. Although increased

resistance in

E. faecalis

strains is less common, it

may arise after prolonged beta-lactam treatment,

leading to mutations in PBP4.

In contrast, MTA-ZnO/Ch had an inhibition

zone of 7.97±0.49 mm, lower than the inhibition

zone of MTA-ZnO/Ch with various antibiotics

(8.76-11.27 mm). These results agree with

previously reported research. Zbańska et al. [29]

reported that pure MTA found lower antibacterial

properties than antibacterial material-modied

MTA. Pure MTA is only effective against facultative

bacteria and less effective against anaerobic

bacteria. Adding ZnONP and chitosan to MTA

is an alternative to improve its antibacterial

properties as a pulp capping material [10]. The

antibacterial mechanism of ZNONP is similar to

other nanoparticles, which causes an increase

in cell wall membrane permeability, release

of cytoplasmic content, and cell death [36].

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

In comparison, the antibacterial mechanism

of chitosan is caused by its ability to bind to

negatively charged bacterial cell membranes,

resulting in increased permeability and,

ultimately, cytoplasmic leakage and bacterial

cell death [37].

MTA-ZnO/Ch-TC and MTA-ZnO/Ch-AMX

had higher inhibition zones than MTA-ZnO/Ch

(without antibiotics) for both tested bacteria. By

combining the mechanical properties, in which

adding tetracycline increases the compressive

strength and diametral tensile strength, it

can be concluded that MTA combined with

ZnO, chitosan, and tetracycline is a potential

biomaterial applied for pulp capping treatment.

This research is a primary step to nding applicable

clinical biomaterial; further investigation is still

necessary, including biocompatibility, antibiotic

release kinetics, antibacterial activity against

anaerobic bacteria, and in-vivo tests.

CONCLUSIONS

ZnO nanoparticle-modied mineral trioxide

aggregate (MTA-ZnO) hydrated using a solution

of chitosan in 4%(w/v) acetic acid solvent

indicated the formation of CSH, C2S, C3S,

C3A, ZnSiO4, and Bi2O3 components, a white

solid with a diameter of 747.04 nm. Among

antibiotics investigated, only adding tetracycline

(MTA-ZnO/Ch-TC) reduced the mass loss and

increased mechanical properties (compressive

strength and diametral tensile strength). Other

antibiotics tended to reduce the mechanical

properties and increase the loss mass, although

statistically, the reduction was insignificant.

Adding antibiotics did not affect the radiopacity

and pH of the solution. Meanwhile, the addition

of antibiotics increased the antibacterial activity

against both

P. aeruginosa

and

E. faecalis

except for the MTA-ZnO/Ch-AMP against

faecalis,

which showed no antibacterial activity.

MTA modified with ZnONP/Ch-TC shows

potential material for pulp capping treatment

incorporated with antibiotic delivery. Further

investigation is ongoing by testing the slow-

release kinetics of antibiotics, biocompatibility,

and in-vivo experiments.

Acknowledgements

The authors thank the Directorate of Research,

Technology, and Community Service, Directorate

General of Higher Education, Research, and

Technology, Ministry of Education, Culture,

Research, and Technology, Republic of Indonesia

through the Regular Fundamental Research (PFR)

Scheme with the contract number: 2660/UN1/

DITLIT/PT.01.03/2024 for the nancial support.

Data availability

All the data are included in the manuscript.

Author’s Contributions

IK, DI: Writing – Original Draft Preparation,

Methodology, Formal Analysis, Data Curation.

SJS, FIP: Writing – Review & Editing, Data

Curation. N: Conceptualization, Writing – Review

& Editing, Supervision, Methodology, Funding

Acquisition.

Conict of Interest

No conicts of interest declared concerning

the publication of this article.

Funding

The authors thank the Directorate of

Research, Technology, and Community Service,

Directorate General of Higher Education,

Research, and Technology, Ministry of Education,

Culture, Research, and Technology, Republic of

Indonesia for the nancial support through the

Regular Fundamental Research (PFR) Scheme

with the contract number: 2660/UN1/ DITLIT/

PT.01.03/2024.

Regulatory Statement

This study followed all the provisions of the

Ethical Commission of the Faculty of Dentistry,

Universitas Gadjah Mada, under letter number

8/UN1/KEP/ FKG-RSGM/EC/2024.

Disclosure

The research study was performed as part of

Universitas Gadjah Mada.

REFERENCES

1. Cervino G, Laino L, D’Amico C, Russo D, Nucci L, Amoroso

G,etal. Mineral trioxide aggregate applications in endodontics:

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

a review. Eur J Dent. 2020;14(4):683-91. http://doi.

org/10.1055/s-0040-1713073. PMid:32726858.

2. Gürcan AT, Şişmanoğlu S, Sengez G. Effect of different adhesive

strategies on the microshear bond strength of calcium-silicate-

based materials. J Adv Oral Res. 2022;13(2):191-9. http://doi.

org/10.1177/23202068221118979.

3. Tsesis I, Elbahary S, Venezia NB, Rosen E. Bacterial colonization

in the apical part of extracted human teeth following root-end

resection and filling: a confocal laser scanning microscopy

study. Clin Oral Investig. 2018;22(1):267-74. http://doi.

org/10.1007/s00784-017-2107-1. PMid:28349219.

4. Kim H-J, Lee D, Cho S, Jang J-H, Kim SG, Kim S-Y. Improvement of

the bonding properties of mineral trioxide aggregate by elastin-

like polypeptide supplementation. Scanning. 2019;2019:3484396.

http://doi.org/10.1155/2019/3484396. PMid:31531154.

5. Pushpalatha C, Dhareshwar V, Sowmya SV, Augustine D,

Vinothkumar TS, Renugalakshmi A, et al. Modified mineral

trioxide aggregate - a versatile dental material: an insight

on applications and newer advancements. Front Bioeng

Biotechnol. 2022;10:941826. http://doi.org/10.3389/

fbioe.2022.941826. PMid:36017346.

6. Jang Y, Song M, Yoo IS, Song Y, Roh BD, Kim E. A randomized

controlled study of the use of proroot mineral trioxide

aggregate and endocem as direct pulp capping materials:

3-month versus 1-year outcomes. J Endod. 2015;41(8):1201-6.

http://doi.org/10.1016/j.joen.2015.03.015. PMid:25933707.

7. Mariyam M, Sunarintyas S, Nuryono N. Improving mechanical,

biological, and adhesive properties of synthesized mineral

trioxide aggregate by adding chitosan. Inorg Chem Commun.

2023;149:110446. http://doi.org/10.1016/j.inoche.2023.110446.

8. Subhi H, Husein A, Mohamad D, Nurul AA. Physicochemical,

mechanical and cytotoxicity evaluation of chitosan-

based accelerated portland cement. J Mater Res Technol.

2020;9(5):11574-86. http://doi.org/10.1016/j.jmrt.2020.07.108.

9. Hu D, Ren Q, Li Z, Zhang L. Chitosan-based biomimetically

mineralized composite materials in human hard tissue Repair.

Molecules. 2020;25(20):4785. http://doi.org/10.3390/

molecules25204785. PMid:33086470.

10. Mariyam M, Sunarintyas S, Yuliatun L, Irnawati D, Hatmanto

AD, Nuryono N. Physicochemical and antibacterial properties of

Zno/chitosan-modified mineral trioxide aggregate composites.

Case Stud Chem Environ Eng. 2024;9:100749. http://doi.

org/10.1016/j.cscee.2024.100749.

11. Khan G, Yadav SK, Patel RR, Nath G, Bansal M, Mishra B.

Development and evaluation of biodegradable chitosan films

of metronidazole and levofloxacin for the management of

periodontitis. AAPS PharmSciTech. 2016;17(6):1312-25. http://

doi.org/10.1208/s12249-015-0466-y. PMid:26689408.

12. Kassem AA, Ismail FA, Naggar VF, Aboulmagd E. Preparation

and evaluation of periodontal films based on polyelectrolyte

complex formation. Pharm Dev Technol. 2015;20(3):297-

305. http://doi.org/10.3109/10837450.2013.862262.

PMid:24438021.

13. Gao H, Ge K, Xu Y, Wang Y, Lu M, Wei Y,etal. Controlled release

of minocycline in hydroxyapatite/chitosan composite for

periodontal bone defect repair. Dent Mater J. 2022;41(3):346-

52. http://doi.org/10.4012/dmj.2021-217. PMid:35321974.

14. Sandra Devi L, Prijatmoko D, Joelijanto R, Prasetyarini S,

Herniyati, Soesetijo FXA,etal. The potential of avocado seed

extract (

Persea

americana

) in inhibiting the release of metal ions

in cuniti and stainless steel based orthodont wire. Int J Heal

Pharm. 2024;4(2):405-14. http://doi.org/10.51601/ijhp.v4i2.322.

15. Uwizeyimana JD, Kim D, Lee H, Byun J-H, Yong D. Determination

of colistin resistance by simple disk diffusion test using

modified Mueller-Hinton agar. Ann Lab Med. 2020;40(4):306-11.

http://doi.org/10.3343/alm.2020.40.4.306. PMid:32067429.

16. Indurkar AR, Sangoi VD, Patil PB, Nimbalkar MS. Rapid synthesis

of Bi2O3 nano-needles via ‘green route’ and evaluation of its

anti-fungal activity. IET Nanobiotechnol. 2018;12(4):496-9.

http://doi.org/10.1049/iet-nbt.2017.0070. PMid:29768236.

17. Al-Khodir FAI, Refat MS. Investigation of coordination ability

of Mn(II), Fe(III), Co(II), Ni(II), and Cu(II) with metronidazole,

the antiprotozoal drug, in alkaline media: synthesis and

spectroscopic studies. Russ J Gen Chem. 2017;87(4):873-9.

http://doi.org/10.1134/S107036321704034X.

18. Wojnárovits L, Tóth T, Takács E. Critical evaluation of rate

coefficients for hydroxyl radical reactions with antibiotics:

a review. Crit Rev Environ Sci Technol. 2018;48(6):575-613.

http://doi.org/10.1080/10643389.2018.1463066.

19. Meretoudi A, Banti CN, Siafarika P, Kalampounias AG,

Hadjikakou SK. Tetracycline water soluble formulations with

enhanced antimicrobial activity. Antibiotics. 2020;9(12):845.

http://doi.org/10.3390/antibiotics9120845. PMid:33256054.

20. Li Q, Coleman NJ. Impact of Bi2O3 and ZrO2 radiopacifiers

on the early hydration and C–S–H gel structure of white

Portland cement. J Funct Biomater. 2019;10(4):46. http://doi.

org/10.3390/jfb10040046. PMid:31635346.

21. Kong H, Kwon KB, Park SJ, Noh WS, Lee SJ. Synthesis of C3S, C2S,

C3A powders using ultra-fine calcium oxide powder synthesized

from Eggshell and effect of C3A content on hardened mixed

aggregates. J Korean Powder Metall Inst. 2019;26(6):493-501.

http://doi.org/10.4150/KPMI.2019.26.6.493.

22. Morales-Melgares A, Casar Z, Moutzouri P, Venkatesh A,

Cordova M, Kunhi Mohamed A,etal. Atomic-level structure

of zinc-modified cementitious calcium silicate hydrate. J Am

Chem Soc. 2022;144(50):22915-24. http://doi.org/10.1021/

jacs.2c06749. PMid:36508687.

23. Eltohamy M, Kundu B, Moon J, Lee H-Y, Kim H-W.

Antibacterial zinc-doped calcium silicate cements: bone

filler. Ceram Int. 2018;44(11):13031. http://doi.org/10.1016/j.

ceramint.2018.04.122.

24. Chang SW. Chemical composition and porosity characteristics

of various calcium silicate-based endodontic cements.

Bioinorg Chem Appl. 2018;2018:2784632. http://doi.

org/10.1155/2018/2784632. PMid:29487618.

25. Hegde V, Arora S. Effect of intracanal medicaments on push-

out bond strength of smart-seal system. J Conserv Dent.

2015;18(5):414-8. http://doi.org/10.4103/0972-0707.164059.

PMid:26430308.

26. Bayer IS. Controlled drug release from nanoengineered

polysaccharides. Pharmaceutics. 2023;15(5):1364. http://doi.

org/10.3390/pharmaceutics15051364. PMid:37242606.

27. Fridland M, Rosado R. Mineral Trioxide Aggregate (MTA)

solubility and porosity with different water-to-powder ratios. J

Endod. 2003;29(12):814-7. http://doi.org/10.1097/00004770-

200312000-00007. PMid:14686812.

28. Gandolfi MG, Siboni F, Primus CM, Prati C. Ion release,

porosity, solubility, and bioactivity of MTA plus tricalcium

silicate. J Endod. 2014;40(10):1632. http://doi.org/10.1016/j.

joen.2014.03.025. PMid:25260736.

29. Zbańska J, Herman K, Kuropka P, Dobrzyński M. Regenerative

endodontics as the future treatment of immature permanent

teeth. Appl Sci. 2021;11(13):6211. http://doi.org/10.3390/

app11136211.

30. Kang TY, Choi JW, Seo KJ, Kim KM, Kwon JS. Four different

commercial root-end filling materials: a comparitive study.

Materials. 2021;14(7):1693. http://doi.org/10.3390/ma14071693.

Braz Dent Sci 2025 Apr/Jun;28 (2): e4595

Khuzaimah I et al.

is properly citePhysicochemical and antibacterial properties of ZnO nanoparticles-modified mineral trioxide aggregate hydrated with antibiotic/chitosan solution

Khuzaimah I et al. Physicochemical and antibacterial properties of ZnO

nanoparticles-modiﬁed mineral trioxide aggregate hydrated

with antibiotic/chitosan solution

Date submitted: 2024 Nov 24

Accept submission: 2025 May 02

Nuryono

(Corresponding address)

Universitas Gadjah Mada, Faculty of Mathematics and Natural Sciences,

Department of Chemistry, Yogyakarta, Indonesia.

Email: nuryono_mipa@ugm.ac.id

31. Khan S, Fareed M, Kaleem M, Uddin S, Iqbal K. An updated

review of mineral trioxide aggregate part-1 : compositional

analysis, setting reaction and physical properties. J Pak Dent

Assoc. 2014;23(4):140-7.

32. Ashraf W, Olek J. Carbonation behavior of hydraulic and

non-hydraulic calcium silicates: potential of utilizing low-lime

calcium silicates in cement-based materials. J Mater Sci.

2016;51(13):6173-91. http://doi.org/10.1007/s10853-016-

9909-4.

33. Evans M, Davies JK, Sundqvist G, Figdor D. Mechanisms

involved in the resistance of

Enterococcus

faecalis

calcium hydroxide. Int Endod J. 2002;35(3):221-8. http://doi.

org/10.1046/j.1365-2591.2002.00504.x. PMid:11985673.

34. Rice LB, Desbonnet C, Tait-Kamradt A, Garcia-Solache M, Lonks

J, Moon TM,etal. Structural and regulatory changes in PBP4

trigger decreased β-lactam susceptibility in

Enterococcus

faecalis

. Am Soc Microbiol. 2018;9(2):e00361-18. http://doi.

org/10.1128/mBio.00361-18.

35. Moon TM, D’Andréa ÉD, Lee CW, Soares A, Jakoncic J,

Desbonnet C,etal. The structures of penicillin-binding protein

4 (PBP4) and PBP5 from

Enterococci

provide structural insights

into β-lactam resistance. J Biol Chem. 2018;293(48):18574-84.

http://doi.org/10.1074/jbc.RA118.006052. PMid:30355734.

36. Ibrahim AIO, Moodley DS, Petrik L, Patel N. Use of antibacterial

nanoparticles in endodontics. SADJ. 2017;72(3):105-12.

37. Li J, Zhuang S. Antibacterial activity of chitosan and its

derivatives and their interaction mechanism with bacteria:

current state and perspectives. Eur Polym J. 2020;138:109984.

http://doi.org/10.1016/j.eurpolymj.2020.109984.