ORIGINAL RESEARCH

https://doi.org/10.37819/nanofab.010.2038 Nanofabrication (2025) 10 | 1

Fabrication of Calcium Hydroxide/Manganese Oxide

Nanocomposite as a Novel Antibacterial Agent

Mostafa Goodinia, Sabah Etemadib, Reza Hatamc,

Atefeh Khavidd, Atena Karimie, Mohsen Safaeif

ticles, and their stirring time were investigated. These agents

had three different levels and nally led to the identication of

fect. The nanocomposites made using test conditions 6 (calcium

hydroxide 150 mg/mL, manganese oxide 9 mg/mL, and stirring

time 60 minutes) showed the greatest effect in growth inhi-

composite and its components were evaluated through materi-

In root treatments, the main goal is to prevent or treat apical periodon-

titis. Complete cleaning of root canals is difcult due to complications

(Roghanizad et al., 2019; Youse et al., 2021). The residual rough-

ness in the root canal along with bacteria and their products can cause

problems, although the use of washing solutions can play an effective

role, it is difcult to completely clean these areas (Rodig et al., 2019).

Bacteria play an important role in root canal treatment failure, and one

of the most important bacteria is E. faecalis (Gulabivala et al., 2005).

Biolms protect bacteria, enabling growth and potentially causing

fection treatments are hyaluronidase, gelatinase, toxin, adhesion to

dentin tubules alone through mechanical and chem-

ical preparation using tools and cleaning solutions

(Kishen et al., 2008). The presence of bacteria in

the root canal reduces the long-term effectiveness

of the treatment (Kayaoglu and Orstavik, 2008).

of antibiotics when dealing with infections in the

bacteria that are resistant to these antibiotics, there

are cases where this method of treatment is ineffec-

tive. As a result, alternative treatments with higher

success rates become necessary. With the expansion

of nanoscience and the use of nanoparticles with

antibacterial properties, such as manganese oxide

nanoparticles, attention has increased to their use in

the treatment of resistant infections. Nanotechnol-

overcome biological barriers in the body (Sabouri

et al., 2023; Narm et al., 2024).

Calcium hydroxide is used as a common com-

pound due to its appropriate biological effects inside

the root canal of the tooth (Afkhami et al., 2015).

This compound has antimicrobial effects due to the

release of hydroxyl ions and the creation of an al-

kaline environment (Dianat et al., 2015). Hydroxyl

ions cause inhibition in replication through the gap

in the DNA molecule. It also causes disturbance in

the enzymatic and metabolic activity of the cell and

destroys the cytoplasmic membrane of microbes

(Aguiar et al., 2015).

um hardness and strength and at the same time high

tions such as increasing the physical and mechan-

ical properties of the matrix (Sabouri et al., 2023).

nanocomposite can have an effective antibacterial

effect in the treatment of tooth root canal infections,

timize the synthesis conditions of the nanocompos-

tibacterial properties of a nanocomposite consisting

The production of manganese oxide nanoparticles

in this study was done using the bacterial synthe-

sis method. For this purpose, the bacterial strain

(11083 IBRC-M) Bacillus sp obtained from the Na-

tional Center of Genetic and Biological Resources

of Iran was used. These bacteria can produce en-

zymes and metabolites that reduce and convert

manganese ions into manganese oxide nanopar-

ticles. In other words, bacteria metabolites act as

a reducing agent and facilitate the biosynthesis

next step, the centrifugation process was performed

of 160 rpm, for 72 hours at 30 °C. Subsequently, the

https://doi.org/10.37819/nanofab.010.2038 Nanofabrication (2025), 10 | 3

ing a microbial lter (Safaei and Moghadam, 2022).

2.2. Synthesis of Manganese

In this study, calcium hydroxide nanoparticles were

obtained from Merck. To make calcium hydroxide/

were designed and three factors of calcium hydrox-

ide (mg/mL) 150, 100, and 200, manganese oxide

(mg/mL) 3, 6, and 9, and stirring time 60, 90, and

120 minutes were considered according to Table 1.

In all 9 experiments, nanocomposite was prepared

by the in situ synthesis method. In this method,

different concentrations of calcium hydroxide and

manganese oxide nanoparticles were prepared and

sonicated for 10 minutes. Then, solutions contain-

ing calcium hydroxide were placed on a magnetic

et al., 2019).

2.3. Antibacterial activity

In this study, the antibacterial properties of nano-

composite consisting of calcium hydroxide and

manganese oxide were investigated on E. faecalis

(IBRC-M10740). This bacterium was obtained from

the Center of Genetic and Biological Resources of

Iran. After a 24-hour incubation period on the BHI

culture medium, a suspension with a concentration

equal to half McFarland was prepared from the

mentioned bacteria. To investigate the antibacteri-

were used. Then, for each test, 1 mL of the culture

mixture was added to the BHI medium using the

pour-plate method. After incubation for 24 hours,

the number of colonies was determined. This pro-

cess was repeated for each nanocomposite and al-

lowed a comprehensive assessment of the number

of bacteria (Wang et al., 2020).

ergy diffraction spectroscopy (EDX), element dis-

tribution map (Map) was investigated with SAMX

detector, transmission electron microscope (TEM)

and ultraviolet-visible spectroscopy (UV-Vis).

These investigations were conducted to investi-

gate the morphology and structural properties of

calcium hydroxide, manganese oxide, and calcium

hydroxide/manganese oxide nanocomposite sam-

ples. To perform the tests, Thermo infrared Fourier

transform spectrometer, Philips X ‘Pert X-ray dif-

scope and Shimadzu UV-160 A visible-ultraviolet

spectrometer were used.

3. RESULTS AND DISCUSSION

3.1. Antibacterial activity

production of nanocomposite consisting of calcium

hydroxide and manganese oxide with the strongest

were designed and carried out using the Taguchi

method. After that, the effect of these synthesized

number 6 shows the highest level of antibacterial

and a mixing time of 60 minutes. In this condition,

the synthesized nanocomposite showed the stron-

gest inhibitory effect on the growth of Enterococcus

faecalis bacteria. The exact mechanisms by which

currently under extensive investigation. Several

mechanisms have been widely investigated, such

oxygen species are a set of molecules or reactive

intermediates that have signicant oxidative po-

tential despite their short existence in nature, and

these species can potentially cause toxicity in mi-

croorganisms. Nanoscale materials are attached to

the microorganism cell surface through active parts

and then spread inside the cell and interact with dif-

https://doi.org/10.37819/nanofab.010.2038 Nanofabrication (2025), 10 | 5

of the membrane (proteins, enzymes, and DNA).

Nanoparticles produce ROS inside the cell, and

these ROS induce oxidative stress, resulting in dis-

turbed metabolic pathways and cell death (Safaei

and Taran, 2022; Sarabikia et al., 2023).

hydroxide and manganese oxide nanoparticles can

ronment. This inhibits enzymatic activities at the

bacterial cell membrane, disrupting metabolism,

tach to bacterial cell membranes, causing physical

hydroxide and manganese oxide nanoparticles and

their nanocomposite (Stankic et al., 2016).

manganese oxide, and mixing time on the surviv-

al rate of E. faecalis bacteria. The ndings indicate

that the calcium hydroxide factor in the second lev-

showed a signicant mutual effect on each other

and also on the survival rate of E. faecalis with a

value of 26.78.

mum value of the mutual inuence intensity index for

calcium hydroxide in the rst level and manganese

oxide in the second level was observed as 4.79%.

viability of E. faecalis is presented in Table 1. The

greatest effect on the survival of E. faecalis was

attributed to manganese oxide, whose effect was

44.53%, followed by calcium hydroxide with an ef-

nanocomposite with the strongest antibacterial ac-

ed in Table 2.

greatest effect on the survival rate of E. faecalis,

was close to manganese oxide. The most appropri-

ate level for the factors of calcium hydroxide and

composite under ideal conditions is determined to

be –0.46. As a result, the nanocomposite consisting

of calcium hydroxide and manganese oxide effec-

tively prevents the activity of E. faecalis bacteria.

activity of various nanocomposites, including cal-

Current grand average of performance 1.29

Bacterial survival at optimum condition –0.46

3.2. FTIR analysis

The FTIR spectrum of the nanocomposite com-

posed of calcium hydroxide and manganese oxide,

as well as its components, is presented in Fig. 4 in

shows the FTIR spectrum of calcium hydroxide,

were the result of the out-of-plane bending of the

is the result of O-H bond vibration, which indicates

the absorption of moisture and the presence of a wa-

ter molecule inside these nanoparticles (Karthik et

FTIR analysis of manganese oxide nanopar-

bacteria in the process of reducing and creating

manganese oxide nanoparticles. The signicant ab-

sorption detected in FTIR wavelengths, especially

the peak intensity in FTIR spectra.

https://doi.org/10.37819/nanofab.010.2038 Nanofabrication (2025), 10 | 7

oxide nanoparticles and the spectrum of the man-

ufactured nanocomposite showed that the spectrum

of the nanocomposite is the result of the interaction

between its components. In addition, the peaks due

served in the nanocomposite spectrum.

3.3. XRD analysis

Investigation of phase formation and crystal struc-

ture of nanocomposite and its components was done

using X-ray diffraction (Fig. 5). The X-ray diffrac-

adam, 2022).

observed that the 2θ angles are equal 22.45 cor-

ing to the (131) plane, 42.60 corresponding to the

related to plane (421) (Phuakkhaw et al., 2016;