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.2024.e4552

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in

any medium, provided the original work is properly cited.

Antibacterial and remineralizing eﬀects of orthodontic adhesive

modiﬁed by nano-chitosan loaded with calcium phosphate

Efeitos antibacteriano e remineralizante de adesivo ortodôntico modiﬁcado com nanopartículas de quitosana carregadas

com fosfato de cálcio

Lara Riyadh AL-BANAA1 , Ali R. AL-KHATIB1 , Fawzi Habeeb JABRAIL1 

1 - Mosul University, College of Dentistry, Department of Pedodontics, Orthodontic and Preventive Dentistry. Erbil, Iraq.

How to cite: Al-Banaa LR, Al-Khatib AR, Jabrail FH. Antibacterial and remineralizing effects of orthodontic adhesive modied by nano-

chitosan loaded with calcium phosphate. Braz Dent Sci. 2024;27(4):e4552. https://doi.org/10.4322/bds.2024.e4552

ABSTRACT

Objective: The objective of this study was to evaluate the remineralizing and antibacterial effects of orthodontic

adhesive modied with nano-chitosan loaded with calcium phosphate, and to investigate the adhesive’s physical

and chemical properties. Material and Methods: Transbond™ XT adhesive primer was modied by nano-chitosan

loaded with calcium phosphate, this study compared three groups: Control primer without any additive, 5%

and 10% nano-chitosan/ calcium phosphate primer in terms of Fourier Transform Infrared Spectrometer, Shear

Bond Strength, Degree of monomer Conversion, Contact Angle measurement, Antibacterial properties against

Streptococcus mutans

and

Lactobacillus acidophilus

, and Field Emission Scanning Electron Microscopy with

Energy Dispersive X-ray Spectroscopy to examine remineralization of the demineralized enamel. Statistical Analysis

used to compare the results using descriptive statistics, one-way ANOVA, and Tukey’s post hoc test. Statistical

signicance was set at P<0.05%. Results: Both 5% and 10% nano-chitosan/ calcium phosphate primer showed

signicant increases in the SBS, DC, antibacterial, and remineralization of demineralized enamel (higher mineral

contents and greater Ca/P ratio) when compared to the control group. Contact angle values showed non-signicant

differences among groups. Conclusion: The orthodontic adhesive primer modied with nano-chitosan/ calcium

phosphate showed improved physical, chemical, and biological properties including enhanced antibacterial and

remineralization compared to the commercial non modied adhesive primer. The 10% nano-chitosan/ calcium

phosphate primer group displayed superior improvements across all the tested adhesive properties compared to

the control and 5% nano-chitosan/ calcium phosphate primer groups.

KEYWORDS

Calcium phosphate; Chitosan; Degree of conversion; Orthodontic adhesive; Remineralization.

RESUMO

Objetivo: O objetivo deste estudo foi avaliar o efeito remineralizante e antibacteriano de um adesivo ortodôntico

modicado com nanopartículas de quitosana carregadas com fosfato de cálcio, e investigar as propriedades

físicas e químicas do adesivo. Material e Métodos: O primer do adesivo Transbond™ XT foi modicado com

nanopartículas de quitosana carregadas com fosfato de cálcio, comparando três grupos: Primer controle sem

aditivos; Primer contendo 5% e 10% de nanopartículas de quitosana/fosfato de cálcio; em relação a espectroscopia

no infravermelho por transformada de Fourier, resistência ao cisalhamento, grau de conversão dos monômeros,

mensuração do ângulo de contato, propriedades antibacterianas contra Streptococcus mutans e Lactobacillus

acidophilus, e microscopia eletrônica de varredura com emissão por campo para examinar a remineralização

do esmalte desmineralizado. A análise estatística foi usada para comparar os valores obtidos na estatística

descritiva, com ANOVA um fator e teste de Tukey. O nível de signicância adotado foi p<0,05%. Resultados:

Ambos os primers contendo 5% e 10% de nanopartículas de quitosana/fosfato de cálcio mostraram aumento

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed with nano-chitosan loaded with calcium

phosphate

INTRODUCTION

White spot lesions (WSLs) are the initial

clinical signs of enamel demineralization that

can progress into cavitated lesions with gradual

dissolution of the enamel hydroxyapatite crystals

and loss of tooth tissue [1,2]. WSLs are classied

according to Nyvad et al. [3] criteria in to: Active

lesion, characterized by a whitish or yellowish

rough chalky surface with loss of luster; feels

rough when the tip of the probe is moved gently

across the surface; or inactive lesion, if they have

a smooth shiny surface and feels hard and smooth

when the tip of the probe is moved gently across

the surface.

Two types of bacteria are involved in tooth

decay;

Streptococcus mutans

which is primarily

responsible for initiating dental caries, and

Lactobacillus acidophilius

, which contributes

to the development and progression of caries

pathogenesis [4]. Both bacteria can produce acid

during the metabolism of sugar fermentation.

The acid produced in the dental plaque on

tooth surface readily diffuse in all directions

in the mouth, it also diffuses into the pores of

enamel and begins to dissolve it. In the mouth,

this process progresses long enough; the result

is a cavity. This process usually takes several

months to years for progression to cavitation,

the end‐point of the disease process known as

dental caries [5].

The remineralization mechanism is a natural

repairing process that occurs under a neutral

physiological pH condition, it restores the ionic

forms of the minerals to the hydroxyapatite crystal

lattice (HAP). Plaque and saliva are sources of

calcium and phosphate ions which precipitate

into the demineralized enamel lesion forming

larger HAP crystals with greater resistance to acid

dissolution [6]. Various types of remineralizing

agents are used, they are classied into uorides

and non-fluoride agents. Calcium phosphate

materials are non-uoride remineralizing agents

and the main components of HAP crystals [7].

Calcium phosphate, considered a source

of Ca+2 and PO4

-3 ions, is used to improve the

saturation of HAP in early carious lesions.

In nature, there are various types of calcium

phosphate compounds which include: Tri

Calcium Phosphate, Tetra Calcium Phosphate,

Amorphous Calcium Phosphate (ACP), Octa

Calcium Phosphate, Mono Calcium Phosphate

Monohydrate, and HAP [8].

Combining remineralizing agents with

antibacterial strategies can enhance their

effectiveness in treatment of WSL. It might

be beneficial to combine remineralization

systems such as calcium phosphate with natural

antibacterial agents, like chitosan.

Chitosan is a natural carbohydrate polymer

prepared by the de-N-acetylation of chitin,

which is the main component of the exoskeleton

of insects, shrimps, and crustaceans [9]. It has

been incorporated in to various dental products,

including composite resin, glass ionomer cement,

and many adhesive materials, for improving

mechanical and antibacterial properties because

it is biocompatible, has antioxidant activity and

exhibits no toxicity [10].

Chitosan is useful in preventing dental

caries for several reasons: (i) it has adhesive

property so in acidic media, chitosan’s amino

groups become protonated, resulting in positively

charged molecules that adhere to negatively

charged surfaces such as cell membranes and

tooth enamel [11]; (ii) it possesses antibacterial

activity. Its mechanism involves interaction with

bacterial cell wall, promoting the displacement

of Ca++ from anionic membrane sites, leading

to cellular destruction [12]; (ii) It inhibits

demineralization by acting as a barrier against

signicante em relação à resistência ao cisalhamento, grau de conversão, ação antibacteriana e remineralização

do esmalte (alto conteúdo mineral e maior relação Ca/P) quando comparado ao grupo controle. Os valores do

ângulo de contato mostraram-se sem diferenças signicantes entre os grupos. Conclusão: O primer do adesivo

ortodôntico modicado com nanopartículas de quitosana carregadas com fosfato de cálcio mostrou melhores

propriedades físicas, químicas e biológicas, incluindo melhora antibacteriana comparada ao primer comercial

não modicado. O primer contendo 10% de nanopartículas de quitosana/fosfato de cálcio mostrou melhores

resultados comparado ao controle e o primer contendo 5%.

PALAVRAS-CHAVE

Fosfato de cálcio; Quitosana; Grau de conversão; Adesivo ortodôntico; Remineralização.

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

acid penetration (iii) it prevents phosphorus

release from the enamel [13]; (iiii) The structure

of chitosan acts as a drug delivery system as it

has specialized active regions that combine with

various bioactive materials to release ions required

for remineralization [9,14]. Chitosan has been

applied as a composite agent in the formation

of hybrid materials with calcium phosphates,

which are widely used for dental restorations

and bone tissue regeneration [15]. Several

hybrid remineralizing agents combining chitosan

and calcium phosphates have been evaluated,

including: hybrid Chitosan-Hydroxyapatite [16],

Carboxymethyl chitosan- Amorphous Calcium

Phosphate nanocomplexes [17], Chitosan/

Calcium Pyrophosphate microowers [18], etc.

Previous studies analyses showed that

no studies have investigated the biological

properties of adding nano chitosan loaded with

calcium phosphate to an orthodontic primer.

Therefore, the current study aimed to evaluate

the effect of adding nano- chitosan loaded

with calcium phosphate as an antibacterial

and remineralizing agent while maintaining

acceptable physical and chemical properties of

the orthodontic adhesive.

The null hypothesis assumed that modication

of the orthodontic adhesive primer has no effect

on the remineralizing, antimicrobial, physical,

and chemical properties of the orthodontic

adhesive.

MATERIAL AND METHODS

The University of Mosul, College of Dentistry’s

ethics committee approved this study on February

5, 2023, with the reference number (UoM.Dent.

23/24).

The materials used in this study included:

1. Nano-chitosan powder (Nanochemazone

Company, Canada) with particles size range

between 30-50 nm, molecular formula

(C6H11NO4), purity 99.9%, and molecular

weight of 161 g/mol;

2. Tricalcium Phosphate (Sigma-Aldrich

Germany) with the chemical formula

Ca3(PO4)2, molecular weight: 310.18, and

assay analysis ≥98% β-phase basis (sintered

Powder);

3. Transbond XT primer (3M Unitek, Monrovia,

USA) was used in the study.

Preparation of nano-chitosan solution

loaded with calcium phosphate

1.0 g of nano-chitosan powder was dissolved

in 100 mL of deionized distilled water containing

1 mL of 2% (v/v) acetic acid solution (Merck

KGaA, Darmstadt, Germany), The mixture was

stirred using magnetic stirring for approximately

three hours at room temperature to achieve a

homogenous nano-chitosan solution. The pH

of the dissolved chitosan solution was adjusted

to 7.0 by adding a 0.05 M dropwise of NaOH

solution [19].

Nano-chitosan loaded with calcium

phosphate solution was prepared by mixing

chitosan with calcium phosphate in a 5:1 weight

ratio. Specically, 200mg of calcium phosphate

powder was added to the chitosan solution

under magnetic stirring for 3 hours at room

temperature.

Preparation of the modied orthodontic

primer

This study compared three groups, as shown

in Table I. To prepare 0%, 5%, and 10% nano-

chitosan/calcium phosphate modified primer

(ChCPP) in weight-to-weight ratios, one drop

(50 µl in size) of primer was measured using a

micropipette (10-100 µm) for standardization.

A digital scale was used to weigh one drop

which was equivalent to 0.05 g, for each group,

20 drops of modied primer were prepared by

mixing 1 g of primer with 0.05, and 0.1 g of

nano-chitosan/calcium phosphate to prepare

5% ChCPP, and 10% ChCPP groups respectively,

along with a control group. Mixing was done in

a semi-dark environment inside microtubes by

Table I - Groups of the study

No. Name Description

1 Control No additive

2 5% ChCPP 5% Chitosan loaded with Calcium Phosphate modiﬁed Primer

3 10% ChCPP 10% Chitosan loaded with Calcium Phosphate modiﬁed Primer

ChCPP: Chitosan/Calcium Phosphate Primer.

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

using the straight head of a dental probe until a

uniform consistency was achieved. Furthermore,

an ultrasonic bath was used to enhance the

distribution of the material into the primer for

30 min. The microtubes were wrapped in dark

tape to prevent light exposure, the method of

modied primer preparation followed that of

Nader et al. [20].

Testing procedures

Field Emission Scanning Electron Microscopy and

Energy Dispersive X-ray spectroscopy (FESEM-

EDX) characterization of nano-chitosan/calcium

phosphate

The morphology and distribution of calcium

phosphate particles within the nano-chitosan

solution were frozen by liquid nitrogen and then

analyzed using Field Emission Scanning Electron

Microscopy(FESEM) at 20 kV accelerating voltage

and 10 mA. Energy Dispersive X-ray Spectroscopy

(EDX) was employed to determine the elemental

composition of the prepared material.

Fourier Transform Infrared Spectrometry (FTIR)

The chemical characteristics of the primer

before and after modication with nano-chitosan

loaded with calcium phosphate were analyzed

using Fourier Transform Infra-Red spectroscopy

(FTIR/ATR Alpha II, Platinum, Bruker Optic,

Germany) at a wavelength range of 400-4000 cm-1,

24 scans at 4 cm-1 resolution. The FTIR spectrum

of unmodied primer was used as a reference for

the changes detected [21,22].

Degree of Monomer Conversion (DC)

Using FTIR Spectroscopy, the DC

assessment was comparable to that reported

by Gutiérrez et al. [23], 5 specimens for each

group were prepared by applying a drop of

primer (10µL) on a celluloid strip fixed over

a glass slide and gently pressed by another

strip. Then the specimen was light cured at

1500W/cm 2 for 10s (5 seconds from each side),

with consideration for a standard distance of

1 mm between the tip of the light unit and the

sample. The upper celluloid strips were removed,

and the cured specimen (4*6 mm in dimensions

and thickness of approximately 0.1 mm), was

carefully removed with a narrow surgical blade

and stored for 24 hours in a dry dark container

until analysis. After that the specimen was

placed on the diamond of the metal circlip of a

“Fourier-transform infrared spectroscope (FTIR)”

equipped with attenuated total reection, the

spectrum was carried out in the absorbance

mode with a wavelength range 400-4000 cm-1,

24 scans at 4 cm−1 resolution. Non-cured drops

of control and modied primer specimens were

also subjected to FTIR spectroscopy [24].

The DC% was measured by a relative

percentage basis (the tangent baseline and the

two-frequency method) employing the C═C

aliphatic bond stretching vibrations (analytical

bond at 1638 cm-1) and the C..C aromatic

bond stretching vibrations (reference bond at

1608 cm-1) which were not inuenced by the

setting reaction. The following Equation 1 was

used to determine the DC:

(C C) (C C)

% 100 1 (C C) (C C)

AP AM



=∗−

= ×−



−∗ =



(1)

where: A (C=C), A(C..C): the net peak absorbance

areas of the set (P) and unset (M) material at the

specic bands, respectively.

Shear bond strength (SBS)

The sample size was calculated using the

following Formula 2:

()

( )

4 2N ZZ E

αβ







= ÷

+

(2)

where: N: The number of experimental

samples; σ: The assumed standard deviation,

it was =2.31 [25]; Zα =1.96 for a=0.05

(two-tailed), Zβ = 0.80 for the 80% power; E:

The detectable difference between treatment

means = 4.

The sample size estimation involved 10 teeth

for each study group. In accordance with the

above formula, the study samples consisted of

30 human premolar teeth, free of enamel cracks,

filling, and caries which were extracted for

orthodontic purposes. The teeth were cleaned

from debris and stored in a 0.1% thymol solution

to inhibit bacterial growth [26,27]. The roots

of all teeth were embedded in blocks of acrylic

resin for stability during testing with only the

crown surfaces exposed. After polishing the

buccal surfaces of the teeth with non-uoridated

pumice, they were rinsed with water and dried.

For brackets bonding, (37%) phosphoric acid was

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

used to etch the teeth for (20) sec, rinsed for (10)

sec, and air dried.

The unmodied and modied primer was

then painted on the premolar buccal surfaces,

and a gentle airow was applied to disperse any

excess primer. Orthodontic brackets (Stainless

Steel Metallic Brackets, Standard Edgewise

type, Dentaurum, Germany) were positioned

with a Boone gauge approximately 4 mm from

the tip of the buccal cusp [28] and pressed by a

load of 200 g, monitored by computer software

connected to the universal testing machine

(Gester instrument Co, Fujian, PR China). after

removing the excess adhesive, the brackets

were cured from both the mesial and distal

sides for 20 secs, using LED light curing device

(1500 mW/cm2), keeping a distance of 2 mm

away from the bracket. The SBS test

was conducted using a universal testing

machine [29-31]. A knife-edge chisel was positioned

at the tooth-bracket interface (Figure 1) and the

crosshead speed was set to 0.5mm/min. The results

of the fracture force were electronically recorded

in Newton and transformed into Megapascals by

dividing the force by the surface area of the bracket

base (10 mm2).

Testing the wettability

Wettability was assessed by measuring the

contact angle of the adhesive primer [32] by a

sessile drop method at room temperature (25 ±

0.5 °C) using a contact angle goniometer a drop

(5 µl) of each group (control primer, 5%, and

10% ChCPP) was dispensed using a micropipette

onto a smooth, at aluminum plate covered with

polytetrauoroethylene tape [33] The plate was

then positioned on the sample table of a contact

angle goniometer. This procedure was repeated

ve times for each group, and a total of 15 images

were captured. The contact angles were measured

using drop shape analysis in the Image J software

program (Figure 2).

Disc diffusion method for antibacterial analysis:

(Kirby-Bauer method)

Plastic molds (5 mm in diameter and

1 mm in thickness) were used to fabricate discs

for the control, 5% ChCPP, and 10% ChCPP

groups (total discs =15, n=5 for each group).

The primer was poured into the molds and

light-cured for 20 sec, the discs were removed

from the molds and sterilized with 70% alcohol

for 30 minutes at room temperature and placed

inside a sterilization pouch. A sterilization check

was performed by incubating one disc in broth

media for 24 hours, showing no growth.

The antibacterial activity of the modied

adhesive discs was tested against

Streptococcus

mutans

(gram positive) and

Lactobacillus

acidophilus

(gram negative) bacteria.

Brain heart infusion (BHI) broth was used to

culture

S. mutans

and

Lactobacillus

individually;

with turbidity equal to 0.5 standard (about

150 million cells per mL), the tubes were

incubated for 18 hours at 37 °C.

The Mueller-Hinton Agar plate was smeared

with bacterial culture and allowed to dry for

about (5) min. A sterilized forceps was used to

press the discs onto the agar surface [34]. Plates

incubations were done in anaerobic conditions at

37 °C for 48 hours. The diameter of the bacterial

inhibition zone surrounding the discs was

measured in millimeters using Image J software.

An inter-examiner calibration was carried

out by repeating the measurements of all groups

by a second experienced operator (both of them

were blinded to the experimental groups),

The results revealed no signicant differences

between the two sets of readings.

Figure 1 - Bracket under constant pressure during the shear bond

strength testing.

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

Five Petri dishes were used for each type

of bacteria with each dish containing 3 discs:

control, 5% ChCPP, and 10% ChCPP [35].

FESEM-EDX analysis of remineralization

Before assessing the remineralization effect of

modied orthodontic primer, a demineralization

process was conducted to eliminate false positive

results. The demineralizing solution contained

(2.2) mmol/L NaH2PO4, (2.2) mmol/L CaCl2,

and (50) mmol/L acetic acid, with NaOH added

dropwise to adjust pH to 4.5 [36]. Fifteen maxillary

premolar crowns of all groups (n=5) were painted

with an acid-resistant varnish, leaving a window

of enamel about 3 × 4 mm in the middle third of

the buccal surface exposed to the acid attack, while

the rest of the crown was protected by the varnish.

Each group was immersed in 40 mL of

acidified buffered demineralizing solution at

room temperature for 96 hours to develop initial

artificial carious lesions. After removal from

the demineralization solution, the specimens

were washed and dried. The exposed enamel

window on the demineralized specimen was

then etched and bonded with control primer,

5% ChCPP, and 10% ChCPP. Each group was

placed in a separate container with 40 mL

articial saliva for four months. Articial saliva

composition included: (0.4g/L) KCl, (0.4g/L)

NaCl, (0.906 g/L) CaCL2.2H2O, (0.906 g/L)

NaH2PO4.2H2O, (0.005 g/L) Na2S.9H2O and

(1 g/L) Urea, dissolved in 1000 mL of distilled

water [37].

For FESEM-EDX analysis, the crowns were

separated from the roots using a low-speed

water-cooled diamond cut-off disc, followed

by a longitudinal section at the buccolingual

direction in the mid of the occlusal surface of

the premolars. The cut surfaces were stored in

distilled water until measurements.

All specimens were thoroughly dried and

gold-coated to prepare the surface suitable for

examination by the FESEM.

The EDX analysis unit attached to the

FESEM apparatus was used to measure elemental

precipitation (calcium and phosphate weight

%). The cut surfaces of the teeth were examined

carefully at a depth of 100 µm from the bonding

surface to obtain representative photographs and

measurements.

RESULTS

Characterization of nano-chitosan/calcium

phosphate by FESEM-EDX

The FESEM image (Figure 3A) of the

prepared chitosan/ calcium phosphate shows

calcium phosphate particles as pale spots, forming

clusters within the chitosan matrix. In Figure 3B

a homogeneous distribution of the calcium

phosphate particles in the chitosan matrix is

observed with a nano-size scale range of 30-60nm.

The homogenous distribution and cluster

formation of the prepared solution indicates a

Figure 2 - Contact angle measurement of droplet by Image J software.

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

successful loading of the calcium phosphate in

to the chitosan solution.

The EDX of chitosan/ calcium phosphate

sample represented a higher proportion of Carbon

nitrogen (N), calcium (Ca), and phosphate (P)

(Figure 3C).

FTIR Characterization of modied primer by

nano-chitosan loaded with calcium phosphate:

FTIR spectra of all groups are shown in

Figures 4A, B and C indicating that no chemical

reaction occurred between the primer and the

calcium phosphate-loaded chitosan at 5% and

10%. This is evidenced by the absence of additional

new bands or disappearing bands in the spectra.

Chitosan displays a distinct peak at

3436 cm-1 belongs to the hydroxyl stretch(O-H),

and a peak at 1636 belongs to the (C = O) stretch

of the amide I band.

The FTIR spectrum of the primer displays

distinct peaks at 3436 cm-1, 2959, 1714 cm-1,

and 1636 cm-1, corresponding to hydroxyl(O-H),

aliphatic(C-H), carbonyl (C=O), and alkene

(C=C) stretching, respectively. these peaks

overlap with those of chitosan indicating that no

additional peaks are present between the FTIR

spectra of the unmodied and modied primer.

Shear bond strength and degree of

monomer conversion

The 10% ChCPP group exhibited the greatest

significant mean value for both SBS and DC,

followed by the 5% ChCPP group. The control

group had the lowest mean values. No signicant

differences in SBS and DC were observed between

5% and 10% ChCPP groups as shown in Table II.

Figure 3 - (A) FESEM image of nano-chitosan loaded with calcium phosphate at 200 kx magniﬁcation showing a cluster formation of calcium

phosphate within chitosan matrix; (B) Size of calcium phosphate particles; (C) EDX spectrum with elements percentage in weight of the

prepared solution.

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

Wettability and contact angle measurement

Table III shows that the control group had a

higher mean value of contact angle measurement

compared to the modied groups, although the

differences were not statistically significant.

This indicates that 5% and 10% ChCPP groups

improved the wettability of the orthodontic resin.

Antibacterial Action on

S. mutans

and

Lacto-

bacillus acidophilus

The antimicrobial test results displayed

that both the 5% and 10% ChCPP experimental

discs exhibited antibacterial activity against

mutans

and

Lactobacillus

as indicated by the clear

inhibition zones around the discs on the agar

plates, as shown in Figure 5A and B. In contrast,

no clear zone was observed around the discs

in the control group indicating no antibacterial

effect. The antibacterial effect significantly

increased from 5% to 10% ChCPP (Table IV).

FESEM-EDX analysis of remineralized enamel

The morphology of hydroxy appetite crystals,

inter-rod spaces, and mineral depositions on the

surface of treated groups is demonstrated in the

FESEM images (Figure 6 from A1 to C2).

After 4 months of remineralization with

ChCPP, grain-like new materials modified

the nanocrystalline structures of the enamel

(prisms and inter-prismatic areas). Filling the

acid-demineralized lesions with enamel-like

structures. These changes suggest that non-

classical crystallization mechanisms likely

contributed to the formation of the new structures.

Additionally, the treated groups displayed

mineral deposition and the formation of

hydroxyapatite layers on the demineralized enamel

beyond the adhesive layer (Figure 6: B1, B2, C1, C2).

The 10% ChCPP group showed a greater

reduction in inter-rod spaces and more mineral

deposition than 5% ChCPP group, while no such

changes were observed in the control group

In the EDX analysis (Figure 6: A3, B3, C3),

the primary elements identied in the enamel

were oxygen (O), calcium (Ca), and phosphorus

(P) with small amounts of carbon (C), magnesium

(Mg), sodium (Na), and chlorine (Cl). Table V

summarizes the means and standard deviation of

the weight percentages of mineral concentrations.

Figure 4 - FTIR spectra of (A) control; (B) 5% ChCPP; (C) 10%.

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

Figure 5 - Bacterial inhibition zone against: (A)

S. mutans

and (B)

Lactobacillus.

Disc A for control group, Disc B for 5% ChCPP and Disc C for

10% ChCPP.

Table II - Descriptive statistics and multiple comparisons Post-hoc Tukey test of shear bond strength and degree of conversion of the control,

5% ChCPP, 10% ChCPP, signiﬁcant diﬀerence at (P ≤ 0.05)

Shear Bond Strength Degree of Conversion

Groups n Mean (MPa) SD p - value Post-hoc test* N Mean (%) SD p - value Post-hoc test*

Control 10 15.2 2.4

0.01

A 5 60.26 1.25

0.000

5% ChCPP 10 18.52 2.2 B 5 67 1.45 B

10% ChCPP 10 20.85 3.7 B 5 68.2 1.42 B

ChCPP: Chitosan/Calcium Phosphate Primer; SD: Standard deviation. *Diﬀerent letters mean signiﬁcant diﬀerence (p ≤ 0.05).

Table III - Descriptive statistics and multiple comparisons Post-hoc Tukey test of contact angle measurements of the control, 5% ChCPP, 10%

ChCPP, signiﬁcant diﬀerence at (P ≤ 0.05)

Groups N Mean SD Min. Max. p - value Post-hoc test*

Control 5 68.1 3.1 65 72.3

0.06

5% ChCPP 5 63.4 3.5 58 68.4 A

10% ChPP 5 63.6 1.865 60.5 65.3 A

ChCPP: Chitosan/Calcium Phosphate Primer; SD: Standard deviation. *Diﬀerent letters mean signiﬁcant diﬀerence (p ≤ 0.05).

Table IV - Descriptive statistics and multiple comparisons Post-hoc Tukey test of

S. mutans

and

Lactobacillus growth

inhibition of the control,

5% ChCPP, 10% ChCPP, signiﬁcant diﬀerence at (P ≤ 0.05).

Streptococcus mutans Lactobacillus

Groups n Mean

(mm) SD p- value Post-hoc

test* nMean

(mm) SD p- value Post-hoc

test*

Control 5 0 0

0.000

A 5 0 0

0.000

5% ChCPP 5 7.42 0.88 B 5 5.64 .75 B

10% ChCPP 5 12.26 0.93 C 5 11.72 .74 C

ChCPP: Chitosan/Calcium Phosphate Primer; SD: Standard deviation. *Diﬀerent letters mean signiﬁcant diﬀerence (p ≤ 0.05).

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

Table V - Descriptive statistics and multiple comparisons Post-hoc Tukey test of EDX of calcium wt %, phosphate wt % and Ca/P ratio of the

control, 5% ChCPP, 10% ChCPP, signiﬁcant diﬀerence at (P ≤ 0.05)

Elements Control

(n=5)

5% ChCPP

(n=5)

10% ChCPP

(n=5) p-value Tukey test*

control

Tukey test*

5% ChCPP

Tukey test*

10% ChCPP

Ca wt % (mean ± SD) 26.42 ± 6.46 32.92 ± 3.08 36.04 ± 3.21 0.017 A AB B

P wt % (mean ± SD) 13.98 ± 2.18 15.83 ± 1.37 17.30 ± 1.9 0.046 A AB B

Ca/P (mean ± SD) 1.75 ± 0.19 2.07 ± 0.79 2.08 ± 1.57 0.007 A B B

ChCPP: Chitosan/Calcium Phosphate Primer; SD: Standard deviation. *Diﬀerent letters mean signiﬁcant diﬀerence (p ≤ 0.05).

Figure 6 - SEM images of the teeth cross section after remineralization at 100kx and 200kx magniﬁcations: A1, A2 for the control group; B1, B2

for 5%ChCPP, C1, C2 for 10% ChCPP. Yellow arrow showed the inter-rods spaces of control group. Red arrows showed decreased spaces. The

green arrows represent the calcium phosphate precipitation. A3, B3, and C3 showed the EDX analysis for each group.

ANOVA analysis revealed significant

differences in calcium and phosphorus

concentration across the three groups following

remineralization (p < 0.05) (Table V). Both 5%

and 10% ChCPP groups had higher mean calcium

and phosphorus levels than the control group.

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

The10% ChCPP showed a signicant difference

from the control group, while the 5% ChCPP

group did not show signicant differences from

both the control and 10% ChCPP groups.

On comparing the Ca/P ratio, ANOVA showed

signicant differences in the Ca/P ratio among the

three groups after remineralization (p < 0.05)

(Table V). The Ca/P ratios of the 5% and 10%

ChCPP groups were signicantly higher than those

of the control group. Additionally, the 10% ChCPP

group had a nonsignicant higher mean value of

Ca/P ratio compared to 5% ChCPP group.

DISCUSSION

In this study, the addition of nano-chitosan

loaded with tricalcium phosphate to the orthodontic

adhesive showed antimicrobial activity against

dental caries-producing bacteria and showed

clear mineral precipitation indicating its potential

for remineralizing of the demineralized enamel.

Additionally, it enhanced the physical and

chemical properties of the primer. Therefore, the

null hypothesis was rejected.

The modication of the orthodontic adhesive

primer with antibacterial and remineralizing

agents requires careful testing of bond strength

because the treatment is negatively affected by

both high and low SBS. Excessive SBS values

above 60 MPa can lead to enamel fractures during

brackets debonding, while lower SBS values

below 6-8 MPa may result in brackets debonding

and prolong orthodontic treatment [38,39].

The current study showed a signicant increase in

SBS by incorporating 5% and 10% nano-chitosan

loaded with calcium phosphate into orthodontic

primer within the acceptable range compared

to the control group. This enhancement can be

attributed to the nanoparticles acting as stress-

absorbing elements, which provide structural

reinforcement and reduce the concentration

of interfacial stress [40]. These results are

comparable to other studies [40,41] that

found the addition of chitosan to orthodontic

primer increased the SBS. However, other

researches [39,42], using different molecular

weights and concentrations of chitosan

nanoparticles in orthodontic adhesives found no

signicant differences in SBS, suggesting that the

impact of chitosan addition on the mechanical

properties may vary based on formulation.

Degree of conversion (DC) was assessed in

this study to evaluate the proportion of monomers

that react to form polymers or the proportion of

C=C double bonds that convert into C-C single

bonds [43]. The DC% plays a crucial role in

determining the physical properties of cross-

linked polymers. A high DC typically correlates

with improved mechanical properties, less

susceptibility to degradation, and lower levels of

residual monomers in the organic matrix leading to

reduced monomer leaching and cytotoxicity [44].

Notably, residual monomers can still be detected

after prolonged immersion of up to 52 weeks [45].

Our study showed a significant increase

in the degree of monomer conversion and

improved adhesive polymerization in both 5%

and 10% ChCPP groups compared to the control

group. This result aligns with the findings of

Tanaka et al. [46], who observed an increase

in DC in dental composites with the addition

of chitosan-loaded dibasic calcium phosphate

llers. Similarly, Chanachai et al. [47] modied

orthodontic adhesives with calcium phosphate

and nisin, achieving a comparable degree of

conversion to commercial adhesives, although

with decreased mechanical properties. In contrast,

Mahapoka et al. [48] reported a non-signicant

decrease in the DC of Chitosan whiskers

incorporated into dental resin compared to the

control group. Moreover, Machado et al. [44]

found that the adding 2 or 5 wt.% chitosan or

triclosan-loaded chitosan showed non-signicant

differences in DC from the control group.

The discrepancies between these ndings

and our results may be attributed to variations

in methodology. Both Mahapoka’s and

Machado’s studies used solid chitosan, which

may have hindered light transmission through

the adhesive film, thereby slowing down the

photopolymerization process [49]; In contrast,

our study used chitosan loaded with calcium

phosphate in liquid form, which did not affect

the translucency of the primer and thus did not

interfere with the polymerization process.

The mean DC values across all groups in

our study were within the clinically acceptable

range of 55% to 75%, as suggested by Kauppi

and Combe [50]. The improvement in monomer

conversion observed in our study may be due

to the photooxidative degradation of chitosan

during light curing, which leads to polymer

chain breakage. This process generates free

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

radicals, reducing the molecular weight and

viscosity of the polymer, ultimately enhancing the

polymerization process and increasing DC [51].

Regarding the contact angle measurements

of the primer, the angle is formed at a three-

phase junction where solid, liquid, and gas

intersect [52]. When the CA is less than 90°, it

indicates that the liquid (primer) wets the etched

enamel surface, promoting better adhesion.

Conversely, if the CA is greater than 90°, it

suggests non-wetting of the substrate, which may

negatively affect adhesion. Complete wetting is

achieved when the contact angle is zero degrees,

signifying ideal adhesion conditions [32].

The bonding efciency of the bracket base is

primarily inuenced by the ability of the primer

to wet and adapt to the etched enamel surface.

The wettability of a liquid primer is closely

related to its contact angle (CA), with an inverse

relationship between them, meaning better

wettability is associated with a lower CA [32].

In this study, the CA of the modied adhesive

with 5% and 10% chitosan/calcium phosphate

was lower compared to the control group,

although no statistically signicant differences

were found between the modied groups.

The reduction in CA might be attributed to

the dilution effect caused by the incorporation

of the chitosan solution, which likely led to a

decrease in the primer’s viscosity, enhancing

its ability to spread across the enamel surface.

Improved wettability at a lower contact angle

and contributes to better adaptation of the primer

to the surface, enhancing the bonding strength.

These findings align with the study by

Katyal et al. [41], which also reported a decrease

in CA for the nano-chitosan modied orthodontic

primer compared to non-modied primer. This

suggests that the incorporation of chitosan in

primer formulations can improve surface wetting

and potentially improve bond strength without

negatively affecting the adhesive’s performance.

A study of Niu et al. [53] reported decreased

values of CA by increasing the concentration of

phosphorylated chitosan loaded with amorphous

calcium phosphate (Pchi/ACP) added into

a composite resin. This reduction in CA was

attributed to the hydrophilic components of

HEMA (hydroxyethyl methacrylate) and Pchi.

which promote better surface wetting and

interaction with the substrate [53].

Chitosan also possesses notable antibacterial

activity [54,55], as it acts as an adhesive agent

that binds to the negatively charged cell wall. This

interaction disrupts the bacterial cell’s functions

by interfering with DNA replication leading to

cell destruction [41].

Two types of bacteria were investigated

in this study:

Streptococcus mutans

and

Lactobacillus acidophilus

. The results showed

that 5% and 10% ChCPP groups had antibacterial

effects against both

S. mutans

and

Lactobacillus

acidophilus

with a significant increase in its

activity by increasing the concentration, these

results agree with Yao et al. [56] who concluded

that 20 mg/mL carboxymethyl chitosan (CMC)

modied adhesive system, revealed antibacterial

action against

S. mutans

. Kikuchi et al. [57]

showed that reduction of biolm formation can

be achieved by 16 hours crosslinking of chitosan

particles loaded with dibasic calcium phosphate

(DCPA). Another study by Harini et al. [58]

claimed that a maximum inhibition zone against

S. mutans

was achieved by chitosan-based dental

varnish. Also, Mirhashemi et al. [59] suggested

that orthodontic composite modied by chitosan

had antibacterial activity against

S. sangius, S.

mutans

, and

Lactobacillus acidophilus

Overall, the data supports the use of

chitosan and its derivatives in enhancing the

antibacterial properties of orthodontic materials,

potentially improving oral health outcomes

during orthodontic treatment [60].

The FESEM images in Figure 6 showed

mineral precipitation indicating that both 5 and

10% ChCPP groups were capable of remineralizing

the demineralized enamel and this nding aligns

with the study by Simeonov et al. [61], which

reported that chitosan/calcium phosphate

microgels effectively remineralize demineralized

enamel surfaces by promoting calcium phosphate

nucleation and development. Chitosan served

as a scaffold for calcium phosphate deposition,

facilitating the in-situ formation of amorphous to

weakly crystalline hydroxyapatite (HAP) crystals.

Furthermore, Song et al. [62] loaded ACP

by carboxy-methyl chitosan/lysozyme nano

gel and evaluated remineralization ability by

forming a prismatic enamel-like structure over

the demineralized surface. Wahied et al. [63]

reported that nanohydroxyapatite and nano

β-TCP loaded by nano-chitosan acted as active

remineralizing agents with promising results.

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

In this study, EDX analysis revealed

that greater calcium and phosphate were

precipitated in the area beyond the adhesive

layer. These findings are consistent with the

study by Neu et al. [53], which suggested

that incorporating phosphorylated chitosan/

amorphous calcium phosphate (Pchi/ACP) into

composite resin led to higher mineral deposits

of calcium and phosphorus weight fractions on

dentin surfaces of treated dentin slabs.

It has been found from previous studies that

the Ca/P ratio is a key element in the nucleation

of calcium phosphate [64]. In our study, the

Ca/P increased in the 5% and 10% ChCPP

groups compared to the control group. This

observation aligns with the research conducted by

Paik et al. [65] who added a calcium phosphate

ion cluster solution to demineralized enamel and

reported enhancements in the Ca/P ratio and

remineralization of early tooth lesions.

The advantages of chitosan loaded with

calcium phosphate in enamel remineralization

over calcium phosphate alone can be explained

by the fact that chitosan acts as a template for

molecular interaction with the minerals to control

HAP growth in the mineralization process.

In acidic conditions, the protonation of the amino

groups in the chitosan molecule results in a

positive charge. The positively charged chitosan

calcium phosphates attract to the negatively

charged enamel surfaces and promote the

adhesion between them, furthermore, chitosan

functions as a reservoir for Ca and P ions, giving

the demineralized enamel with the mineral ions

essential for the biomimetic mineralization [63].

CONCLUSIONS

Under the limitations of this study, the

following conclusions can be drawn based on the

results and discussion:

Improved Adhesive Properties

: The modied

orthodontic primer containing 5% and

10% chitosan/calcium phosphate (ChCPP)

demonstrated signicant improvements in

shear bond strength (SBS), contact angle

(CA), and degree of conversion (DC);

Antibacterial Activity

: Both the 5% and

10% ChCPP formulations exhibited

effective antibacterial properties against

Streptococcus mutans

and

Lactobacillus

acidophilus

, suggesting their potential for

caries prevention;

Remineralization of Enamel

: The ChCPP

formulations were shown to promote

remineralization of demineralized enamel

after four months of immersion in articial

saliva, resulting in mineral deposition and

an increased calcium-to-phosphate (Ca/P)

ratio compared to the control group;

Enhanced Performance of 10% ChCPP:

The 10% ChCPP group displayed superior

improvements across all adhesive properties

compared to the 5% ChCPP group, making it

a more promising candidate for orthodontic

adhesive applications.

These findings support the potential of

incorporating ChCPP into orthodontic adhesives

to enhance their performance and contribute to

dental health.

Limitation

1. This in vitro study was conducted under

controlled laboratory conditions, which may

not accurately replicate the complex oral

environment, including variations in saliva

composition, oral hygiene practices, and

dietary factors.

2. The remineralization assessment was

conducted over a xed period (4 months).

Longer-term studies may provide a more

comprehensive understanding of the

durability and effectiveness of the modied

adhesives.

3. Antibacterial efcacy was evaluated against

specic strains (

S. mutans

and

Lactobacillus

acidophilus

). Future studies could assess

the effectiveness against a broader range of

bacteria, including oral biolm and other

cariogenic pathogens.

Suggestions

• Evaluation of the remineralizing properties

of chitosan loaded with calcium phosphate

in vivo conditions;

• Assess the calcium and phosphate ion

release;

• The effects of adding chitosan/ calcium

phosphate to other dental products like

toothpaste, varnishes, and other preventive

measures.

Braz Dent Sci 2024 Oct/Dec;27 (4): e4552

Al-Banaa LR et al.

Antibacterial and remineralizing effects of orthodontic adhesive modified with nano-chitosan loaded with calcium phosphate

Al-Banaa LR et al. Antibacterial and remineralizing eﬀects of orthodontic

adhesive modiﬁed by nano-chitosan loaded with calcium

phosphate

Acknowledgements

The authors wish to express their gratitude

to College of Dentistry, University of Mosul for

providing all necessary assistance that helped

them to conduct this research.

Author’s Contributions

LRAB: Methodology, Investigation, Resources,

Data Curation, Writing – Original Draft Preparation,

Writing – Review & Editing, Visualization. ARAK:

Supervision. FHJ: Supervision.

Conict of Interest

No conicts of interest declared concerning

the publication of this article.

Funding

The authors declare that no nancial support

was received.

Regulatory Statement

The present study did not include the use of

hazardous materials, or any procedures that could

potentially harm the environment. It was conducted

in accordance with all the provisions of University

of Mosul Ethical Committee Agency. The approval

code for this study is: UoM.Dent. 23/24.

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Lara Riyadh Al-Banaa

(Corresponding address)

Mosul University, College of Dentistry, Department of Pedodontics,

Orthodontic and Preventive Dentistry, Erbil, Iraq.

Email: dr-lara-ra@uomosul.edu.iq

Date submitted: 2024 Oct 19

Accept submission: 2024 Dec 26