Remineralization effectiveness of the PAMAM dendrimer with different terminal groups on artificial initial enamel caries in vitro

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Remineralization effectiveness of the PAMAM dendrimer with different terminal groups on artificial initial enamel caries in vitro Menglin Fan a,1 , Min Zhang a,1 , Hockin H.K. Xu b,c,d , Siying Tao a , Zhaohan Yu a , Jiaojiao Yang a , He Yuan a , Xuedong Zhou a , Kunneng Liang a,b,∗∗ , Jiyao Li a,∗ a

State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Cariology and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China b Department of Advanced Oral Sciences and Therapeutics, University of Maryland School of Dentistry, Baltimore, MD 21201, USA c Center for Stem Cell Biology & Regenerative Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA d Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, USA

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objective. Disruption of the demineralization–remineralization balance could trigger the

Received 21 July 2019

development of dental caries, making it challenging for enamel to “self-heal”. Thus, extrinsic

Received in revised form

assistance is needed to restore enamel lesions and stop undermining progression. The aim

9 November 2019

of this study was to investigate enamel remineralization in a simulated oral environment

Accepted 15 November 2019

via poly (amino amine) (PAMAM) dendrimers quantitatively.

Available online xxx

Methods. Bovine enamel specimens were shaken in demineralization solution (pH 4.5, 37 ◦ C,

Keywords:

specimens were then divided into four groups: enamel treated with PAMAM-NH2 , enamel

50 rpm/min) for 72 h to create initial enamel carious lesions. The subsurface-demineralized Enamel

treated with PAMAM−COOH, enamel treated with PAMAM−OH, and enamel treated with

Subsurface demineralization

deionized water. The treated specimens underwent subsequent 12-day pH cycling. Enamel

Remineralization

blocks were analyzed by transverse microradiography (TMR), surface microhardness testing

PAMAM dendrimers

and scanning electron microscopy (SEM) before and after demineralization and pH cycling.

TMR

Results. Groups treated with PAMAM dendrimers showed lower lesion depth and less min-

Quantitative measurement

eral loss, attained more vertical-section surface microhardness recovery, and adsorbed more mineral deposits (p < 0.05). The enamel lesion remineralization values of PAMAM-NH2 , PAMAM-COOH, and PAMAM-OH groups were 76.42 ± 3.32%, 60.07 ± 5.92% and 54.52 ± 7.81%, respectively.

∗

Corresponding author. Corresponding author at: State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Cariology and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China. E-mail addresses: [email protected] (K. Liang), [email protected] (J. Li). https://doi.org/10.1016/j.dental.2019.11.015 0109-5641/© 2019 Published by Elsevier Inc. on behalf of The Academy of Dental Materials. ∗∗

Please cite this article in press as: M. Fan, M. Zhang, H.H.K. Xu et al.. Remineralization effectiveness of the PAMAM dendrimer with different terminal groups on artificial initial enamel caries in vitro. Dent Mater (2019), https://doi.org/10.1016/j.dental.2019.11.015

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Significance. In conclusion, PAMAM with different terminal groups could induce enamel remineralization, among which PAMAM-NH2 showed the most prominent competence, followed by PAMAM-COOH and PAMAM-OH, in that order. © 2019 Published by Elsevier Inc. on behalf of The Academy of Dental Materials.

1.

Introduction

Caries, cancers and cardiovascular disease rank as the top 3 noncommunicable diseases (NCDs) [1]. The prevalence of dental caries is high, with untreated dental caries being the most common disease affecting humans worldwide [2]. There are approximately 2.3 billion people (32% of the population) with dental caries in their permanent teeth, comprising approximately 9% of infants and nearly all adults [3]. Except for local infections and inflammation, such as pulpitis and periapical periodontitis [4], the pathogenic bacteria and their toxins serve as a reservoir, leading to infectious and inflammatory diseases of distant body sites, especially in immunocompromised hosts [5,6]. Unfortunately, the prevention and treatment of caries remain unsolvable. Enamel consists of the outermost layer of the dental crown, the first to be attacked by injurious factors. As the hardest tissue of human body, enamel serves for mastication, cutting, tearing, crushing and grinding food. Additionally, the highly mineralized structure offers protection for dentin and pulp, slowing down the progress of dental and endodontic diseases [7]. Generally, the demineralization and remineralization of enamel coexist and maintain the balance, and disruption of the de/remineralization balance triggers the development of dental caries [8–10]. If threatened by acids or caries, the reacquisition of mineral crystals from saliva calcium (Ca) and phosphate (P) reservoir can reconstruct the surface layer of enamel to some extent. When destruction goes beyond reconstruction, subsurface demineralization and hard tissue disintegration would be too extensive for enamel to “self-heal”. Therefore, extrinsic assistance is needed to restore enamel lesion and stop undermining progression [11–13]. PAMAM dendrimers, known as “artificial proteins” for its protein-like structure, are highly branched polymers comprising internal cavities and external terminal groups, internal cavities for drug or ion delivery and external terminal groups for specific functions or connections [14]. It was reported that PAMAM dendrimers are prone to self-assemble into hierarchical structures in aqueous solution: first nanospheres, then nanochains and microfibers, and finally macroscopic aggregates [15,16]. The self-assembly behavior and similar structure make it possible for PAMAM dendrimers to mimic the function of amelogenin, which plays an essential role in the crystallization process of hydroxyapatite [17–19]. As the most abundant protein in forming enamel, amelogenin constitutes more than 90% of the extracellular organic matrix,

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These authors contributed equally to this work.

modulating enamel formation [20,21]. Thus, PAMAM dendrimers act as nuclear templates, forming new crystals with the same structure, orientation and mineral phase as intact enamel [15,22–24]. Therefore, PAMAM dendrimers are recognized as promising restorative materials for hard tissues [25,26]. PAMAM dendrimers with different terminal groups have shown remarkable remineralization ability. For example, PAMAM-COOH was applied to the phosphoric acid-etched and nitric acid-etched enamel surface, inducing mineral crystal deposition [27–29]. Additionally, phosphoric acid-etched enamel coated with phosphorylated PAMAM realized remineralization in vitro [30,31]. However, PAMAM-NH2 with the best ability of dentin remineralization and PAMAM-OH with excellent biocompatibility have never been utilized for enamel remineralization [32–34]. Additionally, the enamel lesion model in previous studies mentioned above was surface demineralization produced by strong acids such as phosphoric acid, inconsistent with the subsurface demineralization in actual initial enamel caries [27–30]. Additionally, the remineralization process was conducted in artificial saliva or other remineralization solutions rather than pH cycling conditions similar to the oral cavity. Furthermore, most previous studies measured the remineralization capability of PAMAM dendrimers qualitatively such as scanning electron microscope (SEM) and X-ray diffraction analysis, and no comparison in their enamel-repairing capacities has been reported thus far. Thus, it would be beneficial for the clinical application of PAMAM dendrimers if quantitative measurement in a more biomimetic environment was carried out. The present study was the first to quantify the remineralization effectiveness of PAMAM dendrimers on the subsurface-demineralization enamel, first to examine the remineralization capacity in demineralization–remineralization cycling, which is more similar to the oral cavity, first to specify the comparison of PAMAM with different terminal groups, and first to apply PAMAM-NH2 and PAMAM-OH to enamel remineralization. Accordingly, the objectives of this study were: (1) to measure the remineralization competence quantitatively when PAMAM is coated onto subsurface-demineralized enamel in a simulated oral environment and (2) to compare the enamel remineralization effectiveness of PAMAM-NH2 , PAMAM-COOH and PAMAM-OH by quantitative measurements for the first time. It was hypothesized that: (1) PAMAM with three different terminal groups would be capable of in vitro remineralization on artificial enamel initial caries in a pH cycling environment and (2) PAMAM with a certain type of terminal group would prove to be the most effective for enamel restoration by quantitative measurements.

Please cite this article in press as: M. Fan, M. Zhang, H.H.K. Xu et al.. Remineralization effectiveness of the PAMAM dendrimer with different terminal groups on artificial initial enamel caries in vitro. Dent Mater (2019), https://doi.org/10.1016/j.dental.2019.11.015

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2.

Materials and methods

2.1.

Enamel sample preparation

Bovine incisors free of lesions and cracks were collected. The incisors were cut at the cement-enamel junction by a low-speed, water-cooled diamond saw (Minitom, Struers, Copenhagen, Denmark) to separate the crown part. Enamel blocks were then embedded in epoxy resin (the labial side up). The labial surfaces were polished with 800, 1500, 2000, and 2400-grit carbide-polishing papers under running water. The surface was subsequently covered with acid-resistant nail varnish, leaving a 3 × 4-mm window. The polished specimens were ultrasonicated for 10 min in an ultrasonic cleaner (FS20; Fisher Scientific Co., Pittsburgh, PA, USA) to remove the smear layer [33]. The initial hardness of the superficial surface and cross section was measured using a Vickers hardness tester (MMT-X7A, Matsuzawa, Japan) with a diamond indenter under a 50 gf (for superficial surface)/5 gf (for cross section) load for 10 s [35,36]. Each sample was given five indentations, and the average value was calculated. Enamel samples with microhardness between 3.3 and 4.0 Gpa were included in the following steps to eliminate demineralized specimens following a previous study [37]. The selected samples were stored in 0.5% thymol solution at 4 ◦ C before use [28,30].

2.2.

Enamel carious lesion formation

Initial enamel caries was created as described in previous studies [38,39]. The prepared enamel blocks were immersed in demineralization solution (50 mM acetic acid, 2.2 mM Ca(NO3 )2 , 2.2 mM KH2 PO4 , 5.0 mM NaN3 , and 0.5 ppm NaF, pH 4.5) and were incubated in a shaker (37 ◦ C, 50 rpm/min) for 72 h. The demineralization process produced significant subsurface demineralization-like enamel caries [37,38]. After 3-day demineralization, eight samples were left for surface microhardness measurement and transverse microradiography analysis to identify the formed subsurface demineralization. Additionally, the results showed that the subsurface lesion was well formed. Next, the exposed surfaces of remanent samples were half-covered with acid-resistant nail varnish, leaving a 3 × 2-mm window, the same in all groups, for following treatments.

2.3.

pH cycling

The subsurface-demineralized specimens were divided into four groups—treated with PAMAM-NH2 , PAMAM−COOH, PAMAM−OH and deionized water, respectively—and then were subjected to the designated treatments and pH cycling as described previously [37]. pH cycling models were widely used to estimate the potential of anti-caries agents, as they mimicked the caries process well [40–43]. The pH-cycling scheme included 2-h demineralization and 22-h remineralization incubating in a shaker (37 ◦ C, 50 rpm/min). The demineralization solution contained 50 mM acetic acid, 2.2 mM Ca(NO3 )2 , 2.2 mM KH2 PO4 , and 1.0 mM NaN3 , with the pH adjusted to 4.5. The remineralization solution contained 20 mM HEPES, 0.9 mM KH2 PO4 , 1.5 mM CaCl2 , 130 mM KCl, and 1.0 mM NaN3 ,

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with the pH adjusted to 7.0 [37]. The treatments were carried out at 9:00 am and 17:00 am, before and after 1-h demineralization, respectively. PAMAM dendrimers used in the present study were 4th generation PAMAM-NH2 , PAMAM-COOH and PAMAM-OH, obtained commercially (Chenyuan Dendrimer Tech., Weihai, China). PAMAM dendrimers were dissolved in distilled water to prepare PAMAM solution (1 mg/mL). It was confirmed that PAMAM dendrimers of this concentration performed well in remineralization effectiveness and biocompatibility in previous studies [28,44]. A drop of 100 ␮L of PAMAM solution was applied on the exposed surfaces of demineralized enamel for 15 min each time to adsorb PAMAM dendrimers onto the samples. The whole pH cycling lasted for 12 days, as shown in Fig. 1 [38,45]. All solutions were replaced fresh daily.

2.4. Microhardness measurement of the superficial surface and cross section The surface microhardness of superficial surface and cross section of enamel blocks was measured after pH cycling as described previously. The hardness was tested using a Vickers hardness tester (MMT-X7A, Matsuzawa, Japan) with a diamond indenter. The superficial surface microhardness was tested under a 50-gf load for 10 s [36]. For the cross section, the microhardness of the enamel samples at depth (“depth” means the distance from the indentations to the superficial surface of enamel blocks) of 5 ␮m, 15 ␮m, 25 ␮m, 35 ␮m, 45 ␮m, 55 ␮m, 75 ␮m, 95 ␮m, 115 ␮m, and 135 ␮m was tested under a 5-gf load for 10 s [35]. Five indentations were made for each sample and each depth (n = 9). The microhardness of the demineralized enamel without any treatments was also measured to serve as controls.

2.5.

Transverse microradiography (TMR)

Six enamel blocks of each group were prepared for transverse microradiography. The selected samples were cut perpendicularly to the exposed surfaces by the low-speed, water-cooled diamond saw (Minitom, Struers, Copenhagen, Denmark) to obtain 1-mm slices. The tooth slices were then polished with 800- and 1500-grit carbide-polishing papers under running water to obtain approximately 100- to 120-␮m slices for TMR detection [46]. Each slice was fixed to a transparent plastic flake by invisible tape, and the plastic flake was clipped previously into a particular shape to match the sample container. The prepared plastic flake with fixed slices was placed on a Plexiglass negative in the custom-made TMR sample holder (Inspektor Research Systems BV, Amsterdam, Netherlands). Slices were then microradiographed alongside an aluminum calibration step-wedge with a monochromatic CuK X-ray source (Philips, Eindhoven, Netherlands) operated at 20 kV and 20 mA and an exposure time of 30 min [47,48]. Imaging software (Transversal Microradiography Software 2006, Inspektor Research Systems BV) was used to analyze the lesion depth, mineral loss and mineral content at selected depths. Three slices were analyzed from each enamel block. Six TMR traces were measured on each slice: three traces from areas without exposure to pH cycling and the other three from exposed areas [49].

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Fig. 1 – Flowchart of enamel carious lesion creation and 12-day pH cycling (“Re” stands for remineralization solution; “De” stands for demineralization solution). The pH cycling was carried out following the number noted in the flowchart.

Remineralization of enamel samples after 12-day pH cycling is calculated as: remineralization value (R) = (Mbefore Mafter )/Mbefore , where Mbefore is mineral loss of enamel blocks before pH cycling, and Mafter is mineral loss of the same specimen after pH cycling [48].

2.6.

Scanning electron microscopy (SEM)

To observe the mineral deposits of subsurface remineralization, each enamel block before and after pH cycling was separated into half perpendicularly to the exposed surfaces by the low-speed, water-cooled diamond saw (Minitom, Struers, Copenhagen, Denmark). The cross sections were sputtercoated with gold and examined via SEM (Inspect F50; FEI, USA).

2.7.

no significant hardness recovery was observed in the groups with or without treatments. The microhardness of cross sections (Fig. 2B) showed similar results to the mineral content (Fig. 5B) of the four groups. For sound enamel, the hardness increased with the depth and then levelled off. For demineralized enamel, the hardness decreased in a particular range of depth and then increased with depth. The remineralized enamel also showed an upand-down tendency, and the concave part of the curves was related to the mineral content: the PAMAM-NH2 group possessed the most mineral content (Fig. 5B), while the concave part of the curve was the smallest and the minimum microhardness of cross sections was highest in all experimental groups, followed by that in the PAMAM-COOH and PAMAM-OH groups (Fig. 2B); similar relationships of the mineral content and curve shape were observed in the four groups.

Statistical analysis

Statistical analyses were performed using SPSS software version 24.0 (SPSS, Inc., an IBM Company, Chicago, IL, USA). All the data were checked for normal distribution using the Kolmogorov–Smirnov test. One-way analyses of variance (ANOVA) were performed to detect the significant effects of the variables. Student–Newman–Keuls multiple comparison tests were used at a p value of 0.05.

3.

Results

3.1.

Surface microhardness measurement

Fig. 2 shows the results of surface microhardness measurement. The microhardness of superficial sections (Fig. 2A) decreased was significantly after 3-day demineralization, and

3.2.

Transverse microradiography (TMR)

Fig. 3 shows the microradiographs of artificial enamel carious lesions before and after pH cycling. After 3-day demineralization, significant subsurface demineralization was found. Although the demineralization zone of the control group was less translucent after 12-day pH cycling, only a little noticeable remineralization was observed. The enamel blocks of the PAMAM-COOH and PAMAM-OH groups showed better effects of demineralization zone narrowing than the control group, certifying their remineralization effectiveness. The demineralization zone of the PAMAM-NH2 group was the tiniest and least translucent, showing the most significant remineralization. Fig. 4 shows the lesion depth (Fig. 4A) and mineral loss (Fig. 4B) of enamel blocks in four groups. The lesion depth

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group retained more mineral content than the demineralized group, less than the PAMAM-COOH and PAMAM-OH groups. The enamel of the PAMAM-NH2 group retained the most mineral content volume.

3.3.

Scanning electron microscopy (SEM)

Figs. 6 and 7 and show representative SEM images of enamel block sections perpendicular to exposed surfaces, within a 100-␮m depth from the superficial surface, with a magnification of 5000 and 20,000, respectively. For sound enamel, the rod-like structure could be seen clearly in cross-section images. After 3-day demineralization, the space between the rod-like structure was broader and more noticeable due to the loss of mineral crystals. Specimens in the control group attained a few mineral deposits. Abundant mineral deposits were observed in groups treated with PAMAM dendrimers, filling the space exposed through 3-day demineralization.

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Fig. 2 – (A) Microhardness of the superficial surface of groups with or without treatments: sound enamel, after 3-day demineralization, PAMAM-NH2 group, PAMAM-COOH group, PAMAM-OH group and control group (mean ± SD, n = 9). The value decreased significantly after 3-day demineralization and no significant recovery was observed after 12-day pH cycling. (B) Microhardness of the cross sections in the six groups above (mean ± SD, n = 9): the microhardness of cross sections in the four experimental groups showed a similar tendency to the mineral content (Fig. 5B). Different letters indicate significantly different values (P < 0.05).

(Fig. 4A) of PAMAM-COOH, PAMAM-NH2 , PAMAM-OH and control groups resulted in significant differences between each pair. The PAMAM-OH group showed the highest lesion depth, and the PAMAM-NH2 group showed the lowest lesion depth. Similar results were observed in mineral loss (Fig. 4B): the mineral loss of PAMAM-NH2 was the least, followed by that of the PAMAM-COOH and PAMAM-OH groups. Although attaining some remineralization, the control group had the most mineral loss. Fig. 5 shows the remineralization value (Fig. 5A) and relationship of the lesion depth and mineral content volume (Fig. 5B) of enamel blocks in four groups. The enamel lesion remineralization value (Fig. 5A) showed that the PAMAM-NH2 group had the highest remineralization value (mean ± SD) of 76.42 ± 3.32%, higher than 60.07 ± 5.92% in the PAMAM−COOH group and 54.52 ± 7.81% in the PAMAM-OH group. Fig. 5B showed that specimens treated with deionized water had the least mineral content volume. PAMAM-NH2 , PAMAM-COOH and PAMAM-OH groups resulted in significantly higher mineral content than the control group. Samples of the control

Discussion

The present study quantitatively examined the remineralization capability of the PAMAM dendrimer with different terminal groups in a subsurface-demineralized enamel model and demineralization–remineralization cycling for the first time. PAMAM with amino groups, carboxyl groups and hydroxyl groups all displayed capabilities of inducing enamel remineralization, in which PAMAM-NH2 showed the strongest remineralization ability, followed by PAMAM-COOH and PAMAM-OH, in that order. As a common disease that attacks nearly all the adults at some point in time, dental caries leads to white-spot lesions of enamel at the beginning, followed by cavities, and finally infection and death of the pulp tissue. Enamel comprises approximately 96% of inorganic substances. Basically, mineral crystals taken from saliva can remedy the damage caused by acids or caries, and the disruption of the deremineralization balance initiates dental caries [10]. On the one hand, it is impossible for acellular dental enamel to generate new crystals such as cellular tissue; on the other hand, mineral precipitation from saliva is insufficient. Hence, extrinsic assistance with remineralization efficacy is needed to restore enamel lesion and stop undermining progression [13]. Amelogenin is essential for the organization of the prismatic pattern, control of crystal size and regulation of elongated crystal growth and enamel thickness [50,51]. Similar to amelogenin, PAMAM dendrimers can self-assemble and act as nuclear templates, inducing the growth and organization of enamel crystals [15–17]. PAMAM dendrimers and PAMAM modified with HAP-targeting peptide-dendron, laspartic acids, alendronate, tri-phosphate or bis-phosphate peripheral groups have been used to adsorb to hydroxyapatite or regulate the formation of hydroxyapatite [15,27,31]. However, there were some limitations in previous studies mentioned above. First, the artificial enamel caries created by strong acids, leading to surface demineralization, was inconsistent with the subsurface demineralization in actual enamel caries. There is a much highly calcified region located on the

Please cite this article in press as: M. Fan, M. Zhang, H.H.K. Xu et al.. Remineralization effectiveness of the PAMAM dendrimer with different terminal groups on artificial initial enamel caries in vitro. Dent Mater (2019), https://doi.org/10.1016/j.dental.2019.11.015

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Fig. 3 – Representative microradiographs of enamel lesions (white arrows). (A) Healthy enamel: no demineralization lesion was observed. (B) After 3-day demineralization: a significant demineralization zone was observed. (C) Control group: a small amount of remineralization was obtained. (D) PAMAM-NH2 group: showing the best level of remineralization. (E) The PAMAM-COOH group and (F) PAMAM-OH group achieved remarkable diminishment of the translucent district.

Fig. 4 – (A) Lesion depth of enamel blocks in four groups (mean ± SD, n = 9): The lesion depth of PAMAM-NH2 , PAMAM-COOH, PAMAM-OH and control groups resulted in significant differences between each pair; the PAMAM-OH group showed the highest lesion depth, and the PAMAM-NH2 group showed the lowest lesion depth. (B) Mineral loss of enamel blocks in the four groups (mean ± SD, n = 9): Similar results to the lesion depth. Different letters indicate significantly different values (P < 0.05). Please cite this article in press as: M. Fan, M. Zhang, H.H.K. Xu et al.. Remineralization effectiveness of the PAMAM dendrimer with different terminal groups on artificial initial enamel caries in vitro. Dent Mater (2019), https://doi.org/10.1016/j.dental.2019.11.015

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Fig. 5 – (A) Enamel lesion remineralization value after 12-day pH cycling: the R value of enamel lesions in the PAMAM-NH2 , PAMAM-COOH and PAMAM-OH groups were 76.42 ± 3.32%, 60.07 ± 5.92%, and 54.52 ± 7.81%, respectively. Different letters indicate significantly different values (P < 0.05). (B) Average mineral content profiles after 12-day pH cycling: PAMAM with three different terminal groups all contributed to a higher mineral content than the control group, in which PAMAM-NH2 group owned the highest mineral content.

outermost layer of enamel, approximately 30 ␮m, appearing more radiopaque in X-ray radiographs, endowing the outer layer with stronger acid resistance than the inner part [54]. Consequently, enamel caries manifests as subsurface demineralization (an X-ray translucent district with an intact upper surface according to the TMR micrographs and data analysis) incipiently, mainly created by demineralization solution of pH 4.5 [38,39]. Second, the artificial remineralization condition included artificial saliva only, regardless of the acid challenge during meals in the oral cavity [27–30]. Third, the remineralization effectiveness of PAMAM dendrimers have been proved qualitatively, while no quantitive measurements was found and no comparison was made among different PAMAM dendrimers [27–31]. To imitate the physiological environment better, the present study created the subsurface demineralization and applied demineralization–remineralization cycling so that the results may predict the actual effectiveness of PAMAM dendrimers better. To compare the remineralization effectiveness of different PAMAM dendrimers quantitively, the present study chose the most commercially used PAMAM-

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NH2 , PAMAM-COOH, and PAMAM-OH and evaluated their remineralization effectiveness by TMR and microhardness of cross sections. Furthermore, the quantified comparisons of other modified PAMAM dendrimers are needed to draw a more comprehensive conclusion. Transverse microradiography has been considered the gold-standard technique to evaluate enamel lesions and is appropriate to quantitatively measure mineral loss or gain in de- and remineralization of dental hard tissue [52,53]. A monochromatic X-ray beam is used in TMR examination to evaluate the volume percent of mineral (vol% min) by mass attenuation. Surface microhardness is sensitive to the mineral volume of dental enamel and dentin, which is widely used in previous studies [39,54]. The morphology variation and newly formed mineral crystals can be presented vividly by scanning electron microscopy, which acts as supplementary evidence [27,29,39]. For TMR examination, the control group realized a small amount of remineralization compared with the baseline after 3-day demineralization, and enamel of the PAMAM-COOH and PAMAM-OH group showed less lesion depth and mineral loss, while the carious lesions of the PAMAM-NH2 group was the smallest and least translucent. The diminishment of the translucent region indicated the subsurface remineralization by attracting mineral crystal deposits. The enamel itself could restore the mineral loss to some extent as can be seen in control group, while treatments with PAMAM dendrimers provided better remineralization effectiveness. Similar results were shown in the microhardness of the superficial surface and cross section. It was observed that the microhardness of the superficial surface showed little hardness recovery in the four groups. One reason was that PAMAM dendrimers, inducing subsurface remineralization (as shown in TMR data and photographs), were aimed at minimizing the carious lesion instead of eliminating the lesion completely. Thus the existing tiny demineralization zone after treatments may affect superficial microhardness recovery negatively. Another reason was that the surface microhardness was competent to distinguish the mechanical resilience differences of dental hard tissue within the depth of 10 ␮m accurately [55]. However, the mineral content of four groups showed significant differences at the depth range of 20–100 ␮m as shown in Fig. 5B, beyond the range of surface microhardness mentioned above. That may explain the lack of significant differences in the surface microhardness among four groups after being given four treatments. The microhardness of the cross section deserves more attention, showing a similar tendency to the TMR results. As the depth increased, the hardness first decreased and then increased, finally leveling off in the three experimental groups and control group. The more the lesion depth and mineral loss was, the lower the microhardness values of the cross section tended to be. The latter and former findings were in agreement with previous findings [55]. In summary, demineralization and remineralization affected the microhardness of cross sections rather than to that of the superficial surface. SEM revealed the regenerated rod-like mineral crystals directly. The space between column-like structures was exposed after 3-day demineralization, which was filled with rod-like mineral crystals after treatments. The enamel of the

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Fig. 6 – SEM micrographs (×5000) of enamel cross sections parallel to the long axis of enamel rods: (A) sound enamel; (B) after 3-day demineralization; (C) control group; (D) PAMAM-NH2 group; (E) PAMAM-COOH group; and (F) PAMAM-OH group. The space between rod-like structures (white arrows) was broader after 3-day demineralization (Fig. 6B). The control group (Fig. 6C) showed a few mineral deposits. Groups with PAMAM treatments (Fig. 6D-F) showed abundant mineral precipitation on the surface of demineralized enamel, filling the space between rod-like structures.

Fig. 7 – SEM micrographs (×20,000) of enamel cross sections parallel to the long axis of enamel rods: (A) sound enamel; (B) after 3-day demineralization; (C) control group; (D) PAMAM-NH2 group; (E) PAMAM-COOH group; and (F) PAMAM-OH group.

Please cite this article in press as: M. Fan, M. Zhang, H.H.K. Xu et al.. Remineralization effectiveness of the PAMAM dendrimer with different terminal groups on artificial initial enamel caries in vitro. Dent Mater (2019), https://doi.org/10.1016/j.dental.2019.11.015

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control group showed significantly fewer mineral deposits, while the regenerated crystals of the experimental groups were arranged more regularly. Conclusively, PAMAM dendrimers could adsorb abundant Ca and P ions for mineral deposits and crystal regeneration. Additionally, SEM did not discriminate between treatments compared to TMR analysis. It was concluded that PAMAM-OH showed the least remineralization ability, followed by PAMAM-COOH, with PAMAM-NH2 showing the best remineralization ability. The reason might be explained as follows. It is well known that the enamel surface is negatively charged [56]. Regarding PAMAM dendrimers, PAMAM-NH2 is positively charged, PAMAM-COOH is negatively charged, and PAMAM-OH is electrically neutral [57–59]. Thus, PAMAM with amino groups can adsorb to the enamel surface through electrostatic forces between the positive charge and negative charge, providing a firm combination for PAMAM-NH2 and enamel. With regard to PAMAM-COOH, the carboxyl groups bind to Ca2+ on the surface of hydroxyapatite via coordination bond [60]. Accordingly, both PAMAM-NH2 and PAMAM-COOH performed well in adsorbing to enamel, acting as a nuclear template, grasping Ca and P ions to regenerate new mineral crystals, and realizing the prevention and treatment of enamel caries. By contrast, the electrically neutral hydroxyl groups were not as strong in enamel adsorption so the remineralization effectiveness was inferior. The results agreed with the study results by Clarkson that concluded that the binding capacity of a dendrimer to hydroxyapatite crystals decreased in the following order: positively charged –NH2 , negatively charged −COOH, and neutral charged –NHC(O)CH3 [32]. Moreover, further studies are needed to explore the relevant mechanism. It was demonstrated that the PAMAM dendrimers could attract mineral crystal deposits, narrowing the demineralization zone and realizing subsurface remineralization. Considering more applications, PAMAM dendrimers are promising to be ingredients of enamel-protective paint, mouthwash or therapeutic remineralization solution. However, this study possesses some limitations. More observations and comparison about the microstructures of regenerated crystals are needed to display the features of subsurface reconstruction by PAMAM dendrimers. Furthermore, this is an in vitro study and behavior in vivo may be different. Therefore, in vivo experiments are needed to certify the intracorporal effectiveness.

5.

Conclusion

PAMAM dendrimers functioned well in the remineralization of subsurface-demineralized enamel. PAMAM-NH2 exhibited the best performance, resulting in the most hardness recovery of cross sections and the least lesion depth and mineral loss. PAMAM−COOH was also able to induce enamel subsurfaceremineralization, while PAMAM-OH was not so effective in repairing demineralized enamel. Since subsurface demineralization is the common characteristics of enamel white-spot lesions and initial enamel caries, PAMAM-NH2 has great potential for enamel protection and remineralization.

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Acknowledgements This work was supported by the National Natural Science Foundation of China81670977 (L.J.Y.) and 81800965 (L.K.N), Sichuan Science and Technology2017SZ0030 (L.J.Y), Fundamental Research Funds2018SCU12016 (L.K.N), China Postdoctoral Foundation2018M643507 (L.K.N), Research Fund of West China Hospital WCHS-201705 (L.K.N), Research Fund of Chinese Stomatological AssociationCSA-R2018-06 (L.K.N), Research Fund of Chinese Stomatological Association CSAB2018-05 (Z.M), University of Maryland School of Dentistry bridging fund (HX), and University of Maryland Baltimore seed grant (HX).

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