Photodiagnosis and Photodynamic Therapy 42 (2023) 103141

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Photodiagnosis and Photodynamic Therapy

journal homepage: www.elsevier.com/locate/pdpdt

Effects of photodynamic therapy on Streptococcus mutans and enamel

remineralization of multifunctional TiO2-HAP composite nanomaterials
Ranxu Wang a, 1, Conghui Jia a, 1, Nannan Zheng b, Shujuan Liu c, Zhilin Qi b, Ruiwen Wang d,
Lu Zhang b, Yumei Niu a, *, Shuang Pan a, *
a
The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China
b
School of Life Science and Technology, Key Laboratory of Micro-systems and Micro-structures Manufacturing Ministry of Education, Micro/Nano Technology Research
Center, Harbin Institute of Technology, Harbin 150080, China
c
School of Materials Science and Engineering, Harbin Institute of Technology, Harbin 150001, PR China
d
Material Science and Engineering college, Northeast Forestry University, Harbin, Heilongjiang 150080, China

A R T I C L E I N F O A B S T R A C T

Keywords: Background: As photosensitizer and photocatalyst, titanium dioxide (TiO2) can produce a photodynamic reaction
Anti-biofilm for antibacterial treatment. This study aims to explore a Titanium dioxide/nano-hydroxyapatite (TiO2-HAP)
Dental caries composite combined with the dental curing lamp (385–515 nm) in clinical which could inhibit the dental plaque
Remineralization
biofilm formed by Streptococcus mutans (S. mutans) and promote the enamel surface remineralization
Dental materials
Photodynamic Therapy
simultaneously.
Methods: X-ray Diffraction (XRD) and high resolution transmission electron microscope (HRTEM) were used to
detect the characterization of TiO2-HAP composite nanomaterials. Photodynamic properties of TiO2-HAP were
detected by Diffuse reflectance spectrum (DRS) and fluorescence spectroscopy. Bacterial growth was measured
by reading the absorbance of bacterial cultures and confocal microscope was used to observe the biofilm removal
ability of nanomaterials. The ability of TiO2-HAP to promote enamel remineralization was measured by Scanning
electron microscope (SEM).
Results: The OD 600 of S. mutans was 0.76 in the control group and 0.13 in group of TiO2-HAP with exposure to
light-emitting diode (LED) (150 mW/cm2) for 5 min, suggesting its sustained antibacterial potency and inhibition
of the metabolic activity of dental plaque microcosm biofilm. Also, the release of calcium and phosphorus ions in
TiO2-HAP can promote enamel mineralization simultaneously. After 15 days of remineralization, the Ca/P ratio
of demineralized enamel surface increased from 1.28 to 1.67, which was similar to that of normal enamel.
Conclusions: The TiO2-HAP exhibit a promising anti-bacterial activity and remineralization capacity which can
prevent the occurrence of caries to the greatest extent and promote the biomimetic mineralization of dental
tissues.

1. Introduction formation of dental plaques that are difficult to remove, the acid pro­
duced by dental plaques further causes the demineralization of dental
Caries is one of the most prevalent chronic diseases across the globe, hard tissue [7]. The single component antibacterial material or
which has seriously threatened human oral health and even caused mineralization-promoting material could not fully prevent the occur­
systemic diseases such as dentally associated endocarditis [1] and can­ rence and development of caries. Thus, it is in urgent need to develop
cer [2,3]. It is also the leading cause of missing teeth [4]. The occurrence bifunctional materials that can efficiently destroy dental plaques and
of caries is attributed to the ecological imbalance between inorganic improve remineralization of enamel synchronously.
components of hard dental tissues and dental plaques [5,6]. Oral bac­ Removal of dental plaque to achieve continuous antibacterial effect
teria such as S.mutans colonizing on the surface of tooth leads to the is the first step to achieve synergistic anti-caries and dental repairment.

* Corresponding authors.
E-mail addresses: [email protected] (Y. Niu), [email protected] (S. Pan).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.pdpdt.2022.103141
Received 20 July 2022; Received in revised form 5 September 2022; Accepted 29 September 2022
Available online 3 October 2022
1572-1000/© 2022 Elsevier B.V. All rights reserved.
R. Wang et al. Photodiagnosis and Photodynamic Therapy 42 (2023) 103141

The formation processes of dental plaque (biofilm) consist of three realize synergistic therapeutic effects including antibacterial and remi­
stages: the formation of an acquired salivary pellicle, aggregation of neralization in dental caries treatment. Herein, we have reported (TiO2-
bacteria, and maturation of dental plaque [8]. The extracellular poly­ HAP) nanocomposites to disrupt dental plaque and promote enamel
meric substances (EPS) interconnects bacterial cells and apatitic surfaces surface remineralization based on its photocatalytic properties com­
and then forms a stable and cohesive biofilm that is difficult to remove bined photocatalyst with dental curing lamp. HAP in TiO2-HAP com­
[9–11]. Dental brushing and flossing are routine therapeutic approaches posite is observed to red-shift the absorption wavelength of the TiO2 and
to remove dental plaque. However, they are not able to fully clear away further improve the performance of TiO2 catalytic material responding
the bacteria on adjacent surfaces of teeth [12]. The use of mouthwash is to visible light [38–40]. To investigate the ability of TiO2-HAP in pro­
another routine therapeutic approach to remove dental plaque, but most moting the remineralization of teeth provides a theoretical basis for the
antimicrobials are unable to penetrate biofilm due to its EPS [13]. In prevention of caries and the treatment of early enamel caries.
recent years, using photodynamic technology to mediate bacterial
deactivation has been extensively studied. TiO2, a classical photosensi­ 2. Materials and methods
tizer, photocatalyst [14] and antibacterial material [15,16], which has
excellent biocompatibility [17,18], economic availability, and minimal 2.1. Materials
risk of dental staining, has been widely used to promote osseointegration
of dental implant surface in clinical [19]. TiO2 irradiated by ultraviolet Unless specified, all the chemicals were analytical grade and used
(UV) light will lead to the formation of electron-hole pairs, by combining without further purification. TiO2 and HAP were purchased from Hope
with O2 and OH− to produce superoxide and free hydroxyl radicals Bio-Technology (Qingdao, China). S. mutans strain UA159
(⋅OH), which could attack the microbial surface of the bacterial cells and (ATCC700610) was purchased from BeNa Culture Collection (BNCC,
cause the death of bacterial cell [20]. However, TiO2 can only be acti­ China). Propidium iodide (PI) was obtained from Sigma-Aldrich. Arti­
vated by UV light but not the visible light, which limits its application in ficial saliva and demineralizing solution were purchased from Yuanye
daily clinical practice. It is extremely important to develop TiO2-based Bio-Technology (Shanghai, China).
materials that can make full use of visible light and have a good
bactericidal effect, especially can eradicate bacterial film and achieve a 2.2. Characterization
continuous bactericidal effect.
As the principal acidogenic component in dental plaques, S.mutans XRD was used to assess the structural properties of the samples and
can cause demineralization and eventually lead to dental decay [21]. identify the crystalline phases. The samples were scanned in the range
Sterilization can only slow down the development of caries but cannot 2θ =20◦ ~80◦ . The surface morphology of the samples was characterized
repair the demineralized enamel. Antibacterial and restoration of by SEM and HRTEM. The elemental composition was measured by EDS.
demineralized teeth must be carried out simultaneously. Enamel The optical properties of the samples were determined by UV-vis spec­
demineralization as the precursor of caries was caused by exposure to trophotometer and fluorescence spectrophotometer.
acid environment [22]. Normally, the calcium and phosphate ions in
human saliva can remineralize the demineralized enamel. However, 2.3. Synthesis of TiO2-HAP nanocomposites
when demineralization outpaces remineralization, dental diseases result
[23]. NaF is widely used to prevent tooth demineralization in clinical. Briefly, hydroxyapatite nanoparticles were dissolved in HCl (0.1
While excess exposure to toothpaste and other fluorine-containing mol/L) and added nano-TiO2 suspension. the molar ratio of HAP and
components increases the risks of fluorosis [24–26]. Hydroxyapatite TiO2 is 2:1. The ammonia solution was added to the suspension to adjust
(HAP) and amorphous calcium phosphate showed a good performance the pH to 11-12. The solutions were heated to 90 ◦ C under stirring for
on repairment of tooth enamel and biomimetic mineralization [27]. 6 h. The precipitate was filtered and subsequently washed three times
Remineralization after the removal of biofilms on the tooth surface will be with distilled water. The samples were dried at 80 ◦ C under vacuum to
more effective because HAP could interact with tooth surfaces directly. obtain the powders of TiO2-HAP.
In actual practice, Ca, P ions, which can promote tooth remineralization,
need to pass through the bacterial biofilm before tooth repairment, 2.4. Antibacterial effect of TiO2-HAP on bacterial suspension
which inhibits its ability to mineralize teeth. Therefore, it is more useful
to combine plaque removal material directly with in-situ mineralization Bacteria were cultured in Brain Heart Infusion (BHI) media and
material. Till now, there have been no reports of coordination of incubated overnight at 37 ◦ C in 5% CO2 conditions. Bacteria were
removing biofilms and promoting remineralization at the same time. diluted to 1 × 105 CFU/ml and added into 96-well plates, followed
Previous studies have shown that photocatalyst instability under by the addition of serial dilutions of TiO2 and TiO2-HAP (0.2, 0.4, 0.6,
light conditions promotes ion release due to self-oxidation/reduction 0.8, 1.0, 1.5 mg/mL) with or without dental curing lamp irradiation.
caused by accumulated holes/electrons [28–30]. The release of photo­ Dental curing lamp (385 nm-515 nm, WOODPECKER, X-cure, Soft
catalysts promoted under the irradiation of light can also be taken as an mode) was used as the light source with 150 mW/cm2 and yielded a total
advantage to enhance enamel remineralization. According to the liter­ energy density of 45 J/cm2. The distance between the lamp and the
ature, hydroxyapatite shows superior mechanical properties to remi­ liquid surface was 0.5 cm. The samples were irradiated with LED for 5
neralize initial enamel lesions and form a protective layer [[31],[32]]. min. The cultures were grown in an anaerobic atmosphere at 37 ◦ C for
Also, HAP can form nanocomposites with other photocatalytic materials 24 h, and OD 600 values were measured using a microplate reader. Also,
such as Ag3PO4/HAP [33] and Au-HAP [34] and improve photocatalytic two hundred microlitres of each sample were pipetted out and evenly
performance. According to literature, TiO2-HAP nanocomposites can swabbed on solid medium. After 24 h of incubation, the colonies were
improve the low efficiency [35] and narrow light response range prop­ observed and recorded.
erties of pure TiO2 in the antimicrobial area according to the following
Equation [36,37]: 2.5. Cell culture

Ca10(PO4)6(OH)2⇋10Ca 2+
+6PO3− −
+2OH (1)
4
Murine L929 fibroblasts were purchased from the cell bank of the
Degree of saturation (DS) = [(Ca2+)5(PO3− −
4 )3(OH )/ Ksp]
1/9
(2) Chinese Academy of Sciences, Shanghai, China. L929 cells were
routinely cultured in a DMEM cell culture medium containing 10% fetal
HAP based photocatalyst is expected to take advantage of the slow bovine serum (FBS) and antibiotics (100 mg mL− 1 of streptomycin and
release of Ca and P ions during the photocatalytic sterilization process to 100 U mL− 1 of penicillin) at 37 ◦ C with 5% CO2.

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R. Wang et al. Photodiagnosis and Photodynamic Therapy 42 (2023) 103141

2.6. Cytotoxicity assays with P < 0.05 were considered statistically significant.

The cytotoxicity of TiO2-HAP on L929 cells was evaluated by CCK8 3. Results

assay. Briefly, 5 × 103 per well L929 cells were seeded in 96-well cell
culture plates for 24 h attachment. The medium was then discarded and 3.1. Characterization of the as-synthesized TiO2-HAP nanocomposites
cells were rinsed with PBS twice, followed by co-incubation with TiO2-
HAP nanospheres with various concentrations for 24 h. The medium was The TiO2-HAP nanocomposite was fabricated according to previous
then removed and the cells were washed with PBS. 100 mL of media and method [41]. The XRD pattern of TiO2-HAP nanorods are represented in
10 μL of CCK-8 solution were added to each well and all together Fig. 1a. The synthesized nanorods shows diffraction peak values that are
incubated for 2 h. The absorbance of each well was read on a microplate matched with (1 0 1), (0 0 4), (2 0 0) and (1 0 5) crystal planes of anatase
reader (Infinite M200, Tecan) at a test wavelength of 450 nm. TiO2 (JCPDS card 75-1537) and (1 1 2), (1 3 0), (1 3 2) and (5 0 2) crystal
planes of HAP (JCPDS No-74-0565). According to the SEM images in
2.7. Live/dead bacterial staining assay Fig. 1b, the TiO2 were homogeneously located on the HAP surface, the
as-prepared TiO2-HAP nanorods display a uniform size with a mean
To analyze the bacterial viability of S. mutans in mature biofilm, a diameter of 200 ± 20 nm. The chemical composition and distribution of
biofilm preformed on HAP disc surfaces for 24 h was exposed to TiO2- the TiO2-HAP are examined by energy-dispersive X-ray (EDX) spec­
HAP suspension and LED light, then the HAP disc surfaces were washed troscopy and elemental mappings (Fig. c and d). Element mapping im­
with PBS and stained by a LIVE/DEAD BacLight Bacterial Viability Kit ages of TiO2-HAP reveal the even distribution of Ti, Ca, and P (Fig. 1c).
(Molecular Probes, USA) containing SYTO 9 and propidium iodide (PI) Fig. 1d indicates that the surface of nanorods was composed of 39.03%
for 30 min at room temperature. The final concentrations of SYTO 9 and titanium, 23.72% phosphorus, and 37.25% calcium by atomic compo­
PI were 6 μmol/L and 30 μmol/L, respectively. The fluorescence sition. In addition, the HRTEM image revealed a clear lattice fringe and
photograph was taken using a CLSM (Olympus Fluoview FV1000, interfacial contact of TiO2 and HAP(Fig. 1e). These results suggest the
Japan). The excitation and emission wavelengths were 488 and 532 nm, successful synthesis of the TiO2-HAP.
respectively, and the fluorescence was detected in the ranges 500-530
nm and 630-700 nm, respectively. 3.2. Hydroxyl radical detection

2.8. Demineralization of teeth The catalytic activity of photocatalytic semiconductors mainly de­
pends on the absorption and bandgap of the photocatalyst. Fig. 2a shows
Human mandibular premolars were extracted due to orthodontic and UV-vis diffuse reflectance spectroscopy (UV-vis DRS) of TiO2 and TiO2-
periodontal reasons and teeth with single straight roots were selected HAP. The TiO2-HAP shows higher absorption efficiency than TiO2
and stored in normal saline till use. Firstly, the cylinder-like enamel within the wavelength range from 400 nm to 700 nm due to the
samples (approximately 5 mm in diameter) were cut from the normal loading of HAP granules. Compared with TiO2, the nanocomposite
teeth, then the enamels samples were polished gradually using a pol­ showed a red shift in absorbtion (λ > 390 nm), which improved the weak
ishing unit with silicon carbide disks of 600, 1200, and 2500 grits to photocatalytic ability of TiO2 in the visible light region. TiO2-HAP
remove all dental plaque from the tooth surfaces. The enamel slices were activated by visible light could generate more hydroxyl radicals
immersed in the demineralizing solution for 48 h and then placed in enhancing the antimicrobial efficacy. Fig. 2b shows that the capacity to
artificial saliva, and stored at a 37 ◦ C constant temperature water bath. generate free radicals of TiO2-HAP under LED. Terephthalic acid (TA)
can react with the ⋅OH radicals to generate 2-hydroxyterephthalic acid
2.9. Bacterial morphology on enamel (TAOH) with a strong fluorescence. TiO2-HAP under LED shows high
fluorescence intensity at 425 nm, which are expressed as 17.07-fold of
SEM was used to observe the changes in cell morphologies. A biofilm controls, 4.79-fold, and 1.25-fold of TiO2 and TiO2+LED. As shown in
preformed on enamel surfaces for 24 h was exposed to TiO2-HAP sus­ Fig. 2c, the concentration of ⋅OH in solution increased with increasing
pension and LED light. Cells dehydrated with a graded ethanol series LED irradiation time. The above results indicate that TiO2-HAP nano­
(50, 75, 85, 95, and 100%) followed by gold spraying and imaged by composites showed higher ⋅OH formation ability and photocatalytic
SEM (HITACHI, SU5000) at an extra high tension of 15.0 kV. Cell treated activity than pure TiO2. ⋅OH can damage bacterial proteins, DNA and
with LED only were served as control. membranes to produce antibacterial effects, and increased hydroxyl
radicals can improve antibacterial ability. Also, the wavelength of dental
2.10. Studies on TiO2-HAP promoted remineralization curing lamp used in clinical caries treatment is mostly in the visible
light, the photocatalytic materials responding to the visible light are
The crown portion of the tooth was sectioned and preserved in more consistent with the clinical characteristics of oral treatment.
artificial saliva, and the ions in artificial saliva were cleaned with
distilled water before use. To mimic the symptoms of dental caries, the 3.3. Sustained antimicrobial properties of TiO2-HAP nanocomposites
tooth is demineralized by immersion in a demineralized solution, fol­
lowed by a quick rinse of water to remove the acid from the crown slice. Under UV irradiation, TiO2 has emerged as an outstanding photo­
The specimens of demineralized enamels were randomly divided into 6 sensitizer and photocatalytic material due to its unique structure and
groups (n = 3), to be treated with artificial saliva, 1.5 mg/mL TiO2, 1.5 characteristics. Based on the previous experimental results, the photo­
mg/mL TiO2-HAP, 20 mg/ml NaF, with or without LED illumination. catalytic activity of TiO2 nanomaterials under visible light illumination
Each agent was applied onto the demineralized surface of enamel is enhanced after HAP is introduced, and the formation of hydroxyl
covering the 5 × 5 mm2 area and exposed to LED illumination for 5 min radical is boosted. S. mutans is an important etiological factor for dental
before immersion in freshly artificial saliva. The remineralized agents caries, therefore, the antibacterial activity of TiO2-HAP against plank­
were applied twice a day, with a 12 h interval in between, for 15 days. tonic S. mutans was investigated by absorbance at 600 nm (OD 600) and
the agar plate test [42]. As shown in Fig. 3b and c, The OD 600 of
2.11. Statistical analysis S. mutans was 0.76 of the control group and 0.73 for LED (150 mW/cm2)
for 5 min. The OD 600 of S. mutans treated by TiO2 +LED (0.12) and
All data are presented as the mean ± standard deviation and assessed TiO2-HAP+LED (0.13) were significantly enhanced the bacteriostatic
for significance by a two-tailed independent Student’s t-test. Differences efficiency of TiO2 (0.33) and TiO2-HAP (0.40) in the suspension. The

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R. Wang et al. Photodiagnosis and Photodynamic Therapy 42 (2023) 103141

Fig. 1. Characterization of TiO2-HAP nanorods. (a) XRD pattern. (b) SEM image. (c) Element mapping images for Ca, P and Ti. (d). EDS profile. (e) HRTEM image.

Fig. 2. Photocatalytic Properties of TiO2-HAP nanorods. (a) DRS spectra of the TiO2-HAP. (b) The fluorescence emission spectra in the presence of TiO2, TiO2-HAP,
and TA with and without LED. (c). The fluorescence emission spectra in the presence of TiO2-HAP with LED for a different time. This effect may be due to the specific
texture of the composite which provides a higher specific surface area.

Fig. 3. Antibacterial activities of TiO2 and TiO2-HAP nanocomposite with and without visible light. (a) Antibacterial activity by the agar plate test. (b) The
macroscopic turbidity observation of S. mutans.(c) Effects of TiO2 and TiO2-HAP on bacterial growth measured as OD 600. (***) Denotes a significant difference (p <
0.05, Student’s t-test).

bacteriostatic effect of TiO2-HAP against S. mutans exhibited a which diffuse into the tooth matrix and dissolve calcium phosphate at
concentration-dependent manner (Fig. S1). Therefore, the TiO2-HAP has vulnerable areas. Frequent exposure to fermentable carbohydrates
a stronger bactericidal effect on the planktonic bacteria after exposure to causes demineralization of dental hard tissues as well as changes in the
LED. Caries-related biofilms promote calcium and phosphate loss from nature of the oral biofilm’s microbial population [43]. The existence of
tooth surfaces by converting dietary carbohydrates into organic acids, EPS in biofilm promote selective adhesion and accumulation of

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R. Wang et al. Photodiagnosis and Photodynamic Therapy 42 (2023) 103141

pathogenic streptococcus in caries lesions and allow more fermentable

carbohydrate substrates to permeate to the tooth hard tissue surface and
promoting the progression of dental caries [10,44–46–46]. Thus, we
further analyzed the inhibition of biofilm and EPS formation on HAP
disc surfaces. SYTO 9 (green) and PI (red) were used to stain live bac­
teria with intact cell membranes and dead bacteria with compromised
membranes, respectively. As depicted in Fig. 4, No dead cells could be
detected in the biofilms of the LED group compared with the control
group. Compared with the biofilm from TiO2 and TiO2-HAP group, the
S. mutans biofilm treated with TiO2+LED and TiO2-HAP+LED dramat­
ically destroyed S. mutans and EPS in biofilm. The confocal laser scan­
ning microscope of S. mutans biofilms is also consistent with the
above-mentioned experiments.
The oxidative stress caused by ROS including ⋅OH oxidized intra­
cellular protein, changed the membrane potential, and altered cellular Fig. 5. SEM micrograph of the morphological changes of Streptococcus mutans
respiration of bacteria, eventually resulting in the death of bateria biofilm (Scale bars: 10 μm (lower magnification); 5 μm (higher magnification);
[47–49–49]. Encouraged by the prominent bactericidal effect of TiO2-­ The red arrows indicate dead bacteria.
HAP nanorods against S. mutans on HA disc surfaces, the assessment of
bacterial viability on the surface of ex vivo teeth was investigated by 3.4. Effect of TiO2-HAP by enhancing remineralization of enamel carious
SEM. Numerous S. mutans bacteria attached to and colonized the tooth lesions
surface, forming a dense layer of biofilm. In the control group, a large
number of bacteria formed a dense structure with an adhesive extra­ Caries is caused by bacterial fermentation and acid production,
cellular matrix-like structure between the bacteria. The bacterial which causes demineralization and loss of the tooth’s hard structures
membranes and cell borders were intact and clear. For the [50]. Therefore, in addition to antibacterial and inhibiting plaque for­
TiO2-HAP+LED group, the biofilm of S. mutans cells was almost mation, repairing demineralized enamel is also the key to the treatment
removed, the bacterial cell membrane was damaged and morphology of caries. The results above confirmed that TiO2-HAP combined with
had become rough. Based on the above results, the TiO2-HAP combined curing lamp can significantly inhibit the activity of planktonic bacterial
with LED irradiation demonstrated the excellent antibacterial effect and destroy dental plaques. Next, the ability of TiO2-HAP for enamel
against S. mutans on the isolated tooth. In the oral cavity, the formation surface remineralization is investigated. We further performed the
of dental plaque is a continuous and repetitive process since planktonic remineralization effect of TiO2-HAP nanorods with LED irradiation on
S. mutans could adhere to the surface of the tooth in a short time after the isolated tooth. The concentration of Ca/P ratio and Ca was used as
they are removed, so the sustained antibacterial ability of TiO2-HAP is markers of the mineralization development [51]. Fig. S4 showed a
also important (Fig. 5). smooth enamel surface with calcium-to-phosphorus ratios of 1.63 [52,
To investigate the sustainable antibacterial properties of TiO2-HAP, 53]. All groups were immersed in a demineralization solution to
the sustainable antibacterial experiments were tested. After being construct a demineralized dental enamel model before therapy as indi­
treated by TiO2-HAP, the antibacterial photocatalytic effect of TiO2-HAP cated in Fig. 6. Demineralized enamel showed an irregular surface of the
was maintained for at least 24 h (Fig. S2), The result indicates that TiO2- enamel with the Ca/P ratios of 1.28, which represented the successful
HAP can attach to the surface of enamel and provide long-term pro­ establishment of the demineralization model. The Ca/P ratios of TiO2
tection against caries to all tooth samples. Good biocompatibilityis the and TiO2+LED are 1.21 and 1.24-fold of demineralized enamel,
prerequisite for application of biomaterials. Here, biocompatibility and respectively (Fig. 6b and c). The titanium dioxide nanoparticles showed
cell proliferation studies were performed on the mouse fibroblast L929 effectiveness in remineralization because of their negative charge,
cells. When L929 cells were treated with TiO2-HAP nanorods (maximal attracting Ca ions to induce the growth of new hydroxyapatite on
concentration: 2 mg mL− 1) respectively for 24 h, no obvious cytotoxicity enamel [54]. The white particles of the mineral accumulation area are
was observed (Fig. S3). TiO2-HAP nanorods can even promote the pro­ hydroxyapatite crystals on TiO2, TiO2+LED, TiO2-HAP, and
liferation of L929 cells when its concentration is lower than 1.5 mg TiO2-HAP+LED groups. The Ca/P ratio of TiO2-HAP+LED reached 1.67
mL− 1, indicating good biocompatibility of TiO2-HAP nanorods. which was higher than the TiO2-HAP group and was attributed to the
photochemical dissolution of TiO2-HAP caused by LED [55]. Sodium

Fig. 4. Confocal laser scanning microscope (CLSM) of S. mutans biofilms treated by different methods. Live cells were stained green by Syto 9, and dead cells were
stained red by PI.

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R. Wang et al. Photodiagnosis and Photodynamic Therapy 42 (2023) 103141

Fig. 6. SEM images and EDS photograph of demineralized and remineralized enamel. (a.) Demineralized enamel. (b) Enamel surface remineralized with TiO2. (c)
Enamel surface remineralized with TiO2+LED. (d) Enamel surface remineralized with TiO2-HAP. (e) Enamel surface remineralized with TiO2-HAP +LED. (f) Enamel
surface remineralized with NaF. Red arrows depict the area of remineralization.

fluoride (NaF, 20 mg/mL) was used as positive control could increase but cannot repair the tooth damage. Similarly, the bacteria can continue
the Ca/P ratio to 2.42. The concentration of Ca2+ in TiO2-HAP with and to demineralize the enamel after the tooth is remineralized without
without LED irradiation was detected by ICP-OES. Without UV light inhibiting the bacteria. In this experiment, nano-TiO2 was combined
irradiation, the concentration of Ca2+ in the DI water after 300 min with HAP to prepare nano-TiO2-HAP composite, so as to achieve the
reached only 2.55 mg/L, by comparison, a much higher dual effects of inhibiting bacteria and remineralization of enamel. The
Ca2+concentration of 4.86 mg/L was observed after 300 min under LED simultaneous effect of TiO2-HAP on anti-plaque and remineralization of
light, approximately 1.9-fold the concentration in the dark (Figure S5). by comparing the viability of planktonic S. mutans and dental plaque
LED may promote the release of insoluble TiO2-HAP, which is caused by biofilm, surface changes, and Ca/P ratio with and without irradiation by
the direct oxidation function of h+ [30]. The enamel surface of the dental curing lamp. The results of this study showed that the influence of
TiO2-HAP+LED group is smooth and the micropores are reduced TiO2-HAP on the growth of S. mutans combined with the light curing
compared with demineralized enamel. The supersaturated levels of ionic lamp. Compared with the control group, the TiO2 group and the TiO2-
calcium and phosphate released by TiO2-HAP increased the efficacy of HAP group both had significant growth inhibition, but there was no
teeth remineralization. significant difference between the two groups. It indicates that the
introduction of HAP as a mineralization promoting material has a minor
4. Discussion negative influence on the photocatalytic activity of TiO2.
Dental plaque biofilm is a three-dimensional microbial colony
Dental caries is a multifaceted, chronic, and complicated disease. formed by bacteria co-aggregation and embedded in EPS. Dental plaque
The main causes of dental caries include cariogenic bacteria, frequent is closely related to caries and periodontal disease. On one hand, the
consumption of fermentable carbohydrates, abnormalities in salivary bacteria in dental plaque metabolize carbohydrates to produce acid, and
production and composition, and demineralize the tooth surfaces. A the presence of extracellular matrix changes the local barrier and
large number of bacteria naturally exist in the human mouth, forming microenvironment of plaque, the acid is not easy to diffuse out of plaque,
oral microbiota. Under normal circumstances, the bacteria in the oral resulting in a decrease in local pH, which will lead to the demineral­
flora cooperate and antagonize each other, and will not cause oral dis­ ization of enamel surface and ultimately lead to the occurrence of caries.
eases. However, excessive sugar intake and residual sugar in the mouth On the other hand, EPS reduces the amount of antimicrobial agents
can lead to the proliferation of caries bacteria such as S. mutans, which entering into the dental plaque, and enhances the mechanical stability of
ferments sugars into tooth-destroying lactic acid [56]. The demineral­ dental plaque, making it difficult to remove [10,46,58]. The use of
ization and organic matrix degradation of teeth can lead to changes in traditional antibacterial agents such as chlorhexidine and H2O2 can not
their structure and morphology over time, eventually lead to caries [57]. degrade EPS matrix and reduce acid degradation of tooth enamel and
Thus the process of antibacterial therapy alone is inadequate, methods can lead to tooth staining, taste disorders and bacterial community
provide both antimicrobial and remineralization properties are disorders, etc [59–61–61]. As a photosensitizer, TiO2 is only active
required. under UV light, which limits its application in biomedicine. Fortunately,
As a classical photodynamic antibacterial material, TiO2 has been TiO2-HAP nanocomposites showed good photocatalytic activity under
widely concerned in recent years because of its strong antibacterial ac­ blue light, and the antibacterial effect was good at the wavelength of
tivity, high biocompatibility and good economic applicability. Also, dental curing lamp (385-515 nm), the antibacterial efficiency was up to
HAP is the main component of human natural tooth enamel. A large 82.8%. TiO2-HAP, with a particle size of about 200 nm, can penetrate
number of studies have shown that HAP has a significant effect on the outer matrix of the biofilm, thus generating hydroxyl radicals and
enamel remineralization. At present, there have been many studies on effectively eliminating the dental plaque. However, the toxicity of
the use of photocatalysis technology to achieve tooth antibacterial, but nanoparticles limited their clinical application. The potential cytotoxic
the use of photocatalysis to achieve both antibacterial and mineralizing response of nano-HAP and TiO2 NPs were widely investigated because
research is less. antibacterial can only delay the development of caries, they are attractive materials to promote the repair and reconstruction of

6
R. Wang et al. Photodiagnosis and Photodynamic Therapy 42 (2023) 103141

bone [62]. The toxicity degree of TiO2 has been reported to be lower Supplementary materials
than that of other inorganic nanoparticles (Ag > CuO > ZnO > Au >
TiO2 > SiO2) [63]. In numerous products for the treatment of dental Supplementary material associated with this article can be found, in
hypersensitivity and enamel remineralization, nano-hydroxyapatite has the online version, at doi:10.1016/j.pdpdt.2022.103141.
been employed as an oral care component. The use of hydroxyapatite
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