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Efficacy of amelogenin-chitosan hydrogel in biomimetic repair of human enamel in pH-cycling systems

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Abstract

Amelogenin-chitosan (CS-AMEL) hydrogel has shown great potential for the prevention, restoration, and treatment of defective enamel. As a step prior to clinical trials, this study aimed to examine the efficacy of CS-AMEL hydrogel in biomimetic repair of human enamel with erosive or caries-like lesions in pH-cycling systems. Two models for enamel defects, erosion and early caries, were addressed in this study. Two pH-cycling systems were designed to simulate the daily cariogenic challenge as well as the nocturnal pH conditions in the oral cavity. After pH cycling and treatment with CS-AMEL hydrogel, a synthetic layer composed of oriented apatite crystals was formed on the eroded enamel surface. CS-AMEL repaired the artificial incipient caries by re-growing oriented crystals and reducing the depth of the lesions by up to 70% in the pH-cycling systems. The results clearly demonstrate that the CS-AMEL hydrogel is effective at the restoration of erosive and carious lesions under pH-cycling conditions.
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119
ORIGINAL ARTICLES
Efficacy of amelogenin-chitosan hydrogel
in biomimetic repair of human enamel in
pH-cycling systems
Qichao Ruan1, David Liberman1, Rucha Bapat1, Karthik Balakrishna Chandrababu1, Jin-Ho Phark2,
Janet Moradian-Oldak1
1. Center for Craniofacial Molecular Biology, Division of Biomedical Sciences, Herman Ostrow School of Dentistry,
University of Southern California, Los Angeles, USA. 2. Division of Restorative Sciences, Herman Ostrow School of
Dentistry, University of Southern California, Los Angeles, USA.
Correspondence: Janet Moradian-Oldak. Address: Center for Craniofacial Molecular Biology, Herman Ostrow School of
Dentistry, University of Southern California, Los Angeles, CA 90033, USA. Email: joldak@usc.edu
Received: August 17, 2015 Accepted: October 19, 2015 Online Published: November 2, 2015
DOI: 10.5430/jbei.v2n1p119 URL: http://dx.doi.org/10.5430/jbei.v2n1p119
Abstract
Amelogenin-chitosan (CS-AMEL) hydrogel has shown great potential for the prevention, restoration, and treatment of
defective enamel. As a step prior to clinical trials, this study aimed to examine the efficacy of CS-AMEL hydrogel in
biomimetic repair of human enamel with erosive or caries-like lesions in pH-cycling systems. Two models for enamel
defects, erosion and early caries, were addressed in this study. Two pH-cycling systems were designed to simulate the
daily cariogenic challenge as well as the nocturnal pH conditions in the oral cavity. After pH cycling and treatment with
CS-AMEL hydrogel, a synthetic layer composed of oriented apatite crystals was formed on the eroded enamel surface.
CS-AMEL repaired the artificial incipient caries by re-growing oriented crystals and reducing the depth of the lesions by
up to 70% in the pH-cycling systems. The results clearly demonstrate that the CS-AMEL hydrogel is effective at the
restoration of erosive and carious lesions under pH-cycling conditions.
Key words
Amelogenin-chitosan hydrogel, Enamel biomimetic, pH-cycling, Enamel erosion, Early carious lesion
1 Introduction
Dental enamel is the hardest tissue in human body and forms the outer layer of the tooth, providing protection against
physical and chemical damage during dental function. Unlike other mineralized tissues, mature enamel is a non-living
tissue and cannot regenerate after the substantial mineral loss that often occurs due to dental caries or erosion. Dental
erosion is one of the most common human diseases and affects the vast majority of individuals. It can be defined as an
irreversible loss of dental hard tissue due to a chemical process without the involvement of microorganisms [1]. The
causative agents are usually demineralizing acidic substances, such as foods, beverages, and gastroesophageal reflux [2].
Another worldwide chronic oral disease is dental caries, which is caused by acid-producing bacteria on the teeth. Caries
presents as progressive subsurface demineralization and ultimately results in mechanical failure and cavitation [3, 4]. In
contrast to an erosive lesion, an incipient carious lesion in enamel possesses a typical micromorphology with a
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ISSN 2377-9381 E-ISSN 2377-939X
120
pseudo-intact surface layer on top of the subsurface body of the lesion as a result of re-precipitating minerals [5]. Despite
efforts to remineralize enamel using agents containing fluoride or casein phosphopeptide-amorphous calcium phosphate
(CPP-ACP) [6, 7], the prevention and treatment of incipient carious lesions and submicrometer erosion are still major
clinical challenges. Currently, advanced lesions are treated by removing the compromised tissue and filling the resulting
cavity with restorative materials, such as amalgam, composites, or ceramics. These restorations fail over time due to the
more or less weak adhesion at the interface between the original enamel and the artificial materials, with secondary caries
often forming at this interface.
As a potential alternative to conventional treatment, biomimetic reconstruction of tooth enamel may provide an ideal
solution that regrows organized enamel-mimicking apatite crystals with robust attachment to the natural enamel surface [8].
Such an approach will lead to a strong tooth surface and will eliminate the problem of secondary caries. Biomimetic
strategies for enamel repair have therefore attracted increasing interest in materials science and dentistry, and are widely
considered to be promising approaches to the prevention, restoration, and treatment of defective enamel. In our recent
studies, we developed a biomimetic amelogenin-containing chitosan (CS-AMEL) hydrogel for superficial enamel
reconstruction [9-11]. Amelogenin is the most abundant protein in forming enamel and is essential for organization of its
characteristic prismatic pattern, control of crystal size and regulation of oriented and elongated crystal growth [12]. We
found that amelogenin assemblies carried in chitosan hydrogel could stabilize Ca-P clusters and arrange them into linear
chains, which could fuse with enamel crystals and then develop into enamel-like co-aligned crystals. The chitosan used as
a carrier did not affect the crystal orientation but demonstrated the potential to protect repaired enamel from secondary
caries and erosion due to its apparent antimicrobial and pH-responsive properties. After treatment with CS-AMEL
hydrogel, an organized enamel-like layer formed on the etched enamel surface, significantly improving its hardness and
elastic modulus [9]. Most importantly, this biomimetic in situ regrowth of apatite crystals generated a robust enamel–
restoration interface, which is important for ensuring the efficacy and durability of restorations.
It should be noted that, in order to produce formation of apatite on enamel in vitro, artificial saliva was used in our previous
studies to provide an ion concentration similar to that of human saliva, with the pH remaining at a constant 7.0. However,
natural saliva is a more complicated environment for enamel remineralization, due in part to regular variations in pH
conditions. Normal salivary pH is from 6 to 7 but varies over a wider range according to fluctuations in salivary flow, from
5.3 (low flow) to 7.8 (peak flow) [13]. A clinical study has shown that the oral pH could change immediately to 3.8 ~ 5.4
after consuming a variety of beverages [14]. As a result, to generate reliable data promoting an appropriate design for
clinical trials, it is important to evaluate the effectiveness of the CS-AMEL hydrogel at promoting enamel regrowth under
more realistic model conditions in vitro.
Among in vitro protocols, pH-cycling models have become the preferred method to evaluate the anti-caries efficacy of
developing and recently marketed products [15, 16]. In the typical pH-cycling models, dental substrates (enamel or dentin)
are subjected to a scheme in which a pH-neutral environment is periodically interrupted by acid challenges, simulating
what occurs in the mouth when sugars are metabolized [17, 18]. In this way, they mimic the dynamics of mineral loss and
gain involved in caries formation, which is an important advantage of pH-cycling models [19]. Other advantages include the
high level of scientific control and the resulting lower variability intrinsic to in vitro models, as well as the smaller
necessary sample size [19, 20]. These key advantages have made pH-cycling models a superior tool for improving
understanding of the caries process and evaluating developing materials in vitro.
As a necessary step prior to clinical trails, the present study aimed to investigate the efficacy of CS-AMEL hydrogel for
biomimetic repair of human enamel under pH-cycling conditions. We addressed models of two types of enamel defects:
erosion and early carious lesions. After treatment with CS-AMEL hydrogel under pH-cycling conditions, the morphology
and composition of the repaired enamel were characterized by scanning electron microscopy (SEM) and X-ray diffraction
(XRD), and the depths of the caries-like lesions were observed by fluorescent microscopy.
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To further evaluate the efficacy of CS-AMEL hydrogel in the repair of enamel with caries-like lesions, a modified
pH-cycling model (cycle II) was applied to mimic the oral environment during the nighttime. Based on the finding that the
salivary pH during the nighttime is around 6.5 in the healthy oral cavity [25], a modified remineralization solution with a pH
of 6.5 was utilized in cycle II instead of the demineralization solution (pH 4.6) in cycle I. In addition, the duration of
treatment in the modified remineralization solution was set to 8 hours to simulate the typical duration of nighttime sleep.
To obtain a better comparison, part of the white spot lesion was covered with nail varnish (white arrows in Figure 6A)
before subjecting it to pH cycle II. Because of the protection of nail vanish, the progression of the carious lesion was
stopped in the control. No obvious changes in lesion depth could be observed in the region covered by nail vanish after 7
days of cycle II. In contrast, the carious lesion decreased from ~100 μm to ~30 μm with the CS-AMEL hydrogel treatment
after 7 days of cycle II. Interestingly, some parts of lesions were almost recovered by remineralized crystals. The
fluorescent images and XRD analysis clearly revealed that CS-AMEL hydrogel is effective at repairing early carious
lesions of the enamel by regrowing enamel-like crystals.
4 Discussion
In our previous in vitro studies, we have demonstrated the potential of CS-AMEL hydrogel in biomimetic reconstruction
of dental enamel. Chitosan is a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine
(deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). In the CS-AMEL hydrogel, the potential advantage of
chitosan is to provide effective protection from enamel erosion and by interacting with amelogenin to avoid its loss into the
saliva, at pH values below the pKa of chitosan (6.5) [9]. On the other hand, amelogenin assemblies carried in chitosan
hydrogel could stabilize Ca-P clusters and guide arrangement of the clusters into linear chains that eventually evolve into
enamel-like co-aligned crystals anchored to the natural enamel substrate [9]. This in situ regrowth promoted a dense
interface and a strong bonding between the newly grown layer and the tooth surface. Our previous in vitro study has shown
that the newly grown layer formed in the CS-AMEL hydrogel was tightly bound to the enamel surface and the organized
structure was unaffected even after an ultrasonic washing process [9]. In addition, the hardness and elastic modulus of
etched enamel were increased significantly after treatment with CS-AMEL hydrogel. Furthermore, CS-AMEL hydrogel is
easy to handle under clinical conditions. We have designed a user-friendly dental tray that will be custom-made for the
patients and can be easily used for the application of CS-AMEL hydrogel. However, further testing is still necessary to
establish whether it is effective in the real oral cavity, which has a more complex environment for crystal growth. The
present experiments under pH-cycling conditions described here constitute one step towards that goal.
One major difference between typical in vitro studies and the natural oral cavity is the fluctuating pH of the saliva,
especially the acidic environment after consuming food. We sought to determine here whether those conditions affect the
efficacy of CS-AMEL hydrogel during treatment for erosive or carious lesions. To tackle this challenge, tooth samples
modeling erosive and early carious lesions were subjected to a pH-cycling model involving an acidic challenge (pH 4.6) of
demineralization solution in the present study. After 5 days of pH cycling with CS-AMEL treatment, an organized layer of
enamel-like crystals was regrown on the surface of erosive lesions, indicating that both chitosan and amelogenin were still
stable under the acidic challenge, even though chitosan is considered to be soluble at pH < 6.5 [26, 27]. However, in this case
it is still able to re-grow the erosive enamel during the pH cycling due to its unique adhesive property. In the
demineralization solution, the amino groups of chitosan capture hydrogen ions, resulting in an overall positive charge that
gives a bioadhesive property to negatively charged surfaces of erosive enamel. It has been reported that this positively
charged chitosan layer might act as a barrier against acid penetration, inhibiting the demineralization process [28, 29].
Moreover, the pH-sensitivity of chitosan may provide protection for amelogenin under acidic conditions [9]. At pH values
below the pKa of chitosan (6.5), it can interact with amelogenin through electrostatic interaction to avoid protein loss into
the saliva. When the normal pH of the saliva is restored (to the range 6.3-7.0), the weakly interacting amelogenin is
released from the chitosan to regulate the remineralization of enamel. Together with this evidence from previous studies,
our present results clearly validate the effectiveness of CS-AMEL hydrogel at repairing erosive enamel lesions in a
pH-cycling system.
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Another concern is whether the CS-AMEL hydrogel can repair early carious lesion. Unlike superficial erosive lesions, in
early caries the enamel has an intact mineral layer on top of the lesion. We therefore sought to test whether CS-AMEL
hydrogel could penetrate through this mineral layer to reach the subsurface lesion. Both in vivo and ex vivo studies have
observed that the surface morphology of an initial carious lesion is different from that of sound enamel [30, 31]. In initial
carious lesions, the relatively intact surface layer generally exhibits a clearer perikymata pattern and in numerous studies
so-called focal holes have been seen [32]. Using scanning electromicroscopy, de Marsillac et al. found that micro-sized
diffusion pathways through intercrystalline and interprismatic spaces could be detected on the surface of carious
enamel [33]. On the other hand, our previous investigation has revealed that CS-AMEL hydrogel works through the
nano-sized amelogenin-Ca-P clusters that eventually evolve into enamel-like co-aligned crystals [9]. Based on previous
observations, it is reasonable to believe that this pseudo-intact surface layer is permeable by the active ingredient in
CS-AMEL hydrogel. Indeed, in the present study, the artificial carious lesions were successfully repaired by the
CS-AMEL hydrogel in the pH-cycling systems. In cycle I, the depth of the artificial incipient carious lesions was reduced
by up to 50%. After 7 days of cycle II with the CS-AMEL treatment, the depth of the artificial caries was significantly
reduced by up to ~ 70%. Some portions of the carious lesions were almost refilled by remineralized crystals in the
CS-AMEL-treated samples. In comparison, in a previous study it was reported that only ~ 24% of caries was recovered
after 12 days of pH cycling with NaF treatment [34]. The CS-AMEL-treated sample in this study exhibited a superior degree
of recovery in terms of depth compared to other treatments reported in the literature.
In addition, the results from this study may provide guidance for the design of future clinical study protocols for testing
CS-AMEL. Considering the successful repair of carious lesions in cycle II, patients could be instructed to apply the
hydrogel before and after sleep. Nevertheless, it should be noted that this study did not take into account the effect of the
saliva proteins, which is very important for the clinical application of CS-AMEL hydrogel. Among the non-immunologic
salivary protein components, there are enzymes (lysozyme, lactoferrin, and peroxidase), mucin glycoproteins, agglutinins,
histatins, proline-rich proteins, statherins, and cystatins [35]. Lysozyme can degrade the chitosan molecule [36], and it may
also perturb and interfere with the native intermolecular amelogenin interactions because of its positive charge at neutral
pH [37]. It has also been reported that proline-rich proteins and statherins inhibit the spontaneous precipitation of calcium
phosphate salts and the growth of hydroxyapatite crystals on the tooth surface [35]. Examining the effect of saliva on the
hydrogel is a subject for future investigation.
In conclusion, the efficacy of amelogenin-chitosan (CS-AMEL) hydrogel for the biomimetic repair of human enamel with
erosive or carious lesions was investigated in two pH-cycling systems. The results showed that CS-AMEL hydrogel is
effective at pH 4.6, which is similar to the pH of the mouth after consumption of food, as well as at pH 6.5, which is the
average pH during the nighttime. CS-AMEL hydrogel was effective in forming a new organized layer of enamel-like
crystals on the surface of erosive lesions. In addition, CS-AMEL could repair artificial incipient caries by regrowing
oriented crystals and reducing the depth of the lesions by up to 50%-70% under pH-cycling conditions. This study clearly
demonstrates the potential of CS-AMEL hydrogel for the prevention, restoration, and treatment of defective dental enamel.
Furthermore, the results presented here may promote the appropriate design for clinical trials; however, the effect of
salivary proteins should also been considered in human studies.
Acknowledgements
This work was supported by NIH-NIDCR grants DE-13414 and DE-020099 to J.M.O and the USC Coulter Translational
Partnership Program. The authors would like to thank the Center for Electron Microscopy and Microanalysis (CEMMA)
at USC for electron microscopy.
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... Medium (3-5) Ruan, 2013 35 3 Medium (3-5) Ruan, 2014 36 3 Medium (3-5) Ruan, 2016 37 3 Medium (3-5) ...
... 34 Amelogenin-containing chitosan hydrogels can induce the apatite crystals to form on the damaged human enamel. [35][36][37] Amelogenin derivative is a small domain derived from native amelogenin. It can construct a mineral layer on the demineralized enamel. ...
... Chitin is a hydrogel scaffold that is extensively employed for upgrading the performance of tissue-engineered constructs (Jayakumar et al., 2011). Another example of modified hydrogels is amelogeninchitosan, designed and used for the prevention, restoration, and treatment of defective dental enamel (Ruan et al., 2016). ...
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Direct utility of molecular drugs has been hindered in dental and bone engineering because of their short-term biological effects in vivo, fast metabolism or difficulties in achieving drug delivery targetability. To solve these problems, drug delivery systems (DDSs) have been studied extensively in recent years. As a natural polysaccharide, chitosan (CS) has been widely studied as a bioactive carrier for DDSs due to its excellent biocompatibility, biodegradability, polycation characteristic, bioadhesion, bacteriostasis and simplicity of modification. Numerous new designs of CS-based DDSs combining the chitosan carrier with natural or synthetic polymers and bioactive molecules have been fabricated to promote remineralization and osteogenesis. This paper presents the main properties of CS as a carrier for human hard tissue repair and has tried to review the current CS-based applications for bone regeneration and dental repair.
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Mature tooth enamel is acellular and does not regenerate itself. Developing technologies that rebuild tooth enamel and preserve tooth structure is therefore of great interest. Considering the importance of amelogenin protein in dental enamel formation, its ability to control apatite mineralization in vitro, and its potential to be applied in fabrication of future bio-inspired dental material this review focuses on two major subjects: amelogenin and enamel biomimetics. We review the most recent findings on amelogenin secondary and tertiary structural properties with a focus on its interactions with different targets including other enamel proteins, apatite mineral, and phospholipids. Following a brief overview of enamel hierarchical structure and its mechanical properties we will present the state-of-the-art strategies in the biomimetic reconstruction of human enamel.
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Abstract We recently reported an amelogenin-chitosan (CS-AMEL) hydrogel as a promising biomimetic material for future in situ human enamel regrowth. To further optimize the necessary conditions for clinical applicability of CS-AMEL hydrogel, herein we studied the effects of viscosity and supersaturation degree on the size and orientation of synthetic crystals by means of scanning electron microscopy (SEM) and X-ray diffraction (XRD). Raising the hydrogel viscosity by increasing chitosan concentration from 1% to 2% (w/v) improved the orientation of the crystals, while a higher supersaturation (σ(HAp) >10.06, [Ca(2+)] >5 mM) resulted in the formation of random crystals with larger sizes and irregular structures. We conclude that optimal conditions to produce organized enamel-like crystals in a CS-AMEL hydrogel are: 2% (w/v) chitosan, 2.5 mM calcium, and 1.5 mM phosphate (degree of supersaturation = 8.23) and 200 µg/ml of amelogenin.
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Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.
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The earliest evidence of demineralization on the smooth enamel surface of a crown is a white spot lesion. The conventional treatment of these white spot lesions includes topical fluoride application, iamproving the oral hygiene, and use of remineralizing agents. The following article illustrates the use of a novel approach to treat smooth surface noncavitated white spot lesions microinvasively based on infiltration of enamel caries with low-viscosity light curing resins called infiltrants. This treatment aims upon both the prevention of caries progression and improving esthetics, by diminishing the opacity.
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Developing experimental models to understand dental caries has been the theme in our research group. Our first, the pH-cycling model, was developed to investigate the chemical reactions in enamel or dentine, which lead to dental caries. It aimed to leverage our understanding of the fluoride mode of action and was also utilized for the formulation of oral care products. In addition, we made use of intra-oral (in situ) models to study other features of the oral environment that drive the de/remineralization balance in individual patients. This model addressed basic questions, such as how enamel and dentine are affected by challenges in the oral cavity, as well as practical issues related to fluoride toothpaste efficacy. The observation that perhaps fluoride is not sufficiently potent to reduce dental caries in the present-day society triggered us to expand our knowledge in the bacterial aetiology of dental caries. For this we developed the Amsterdam Active Attachment biofilm model. Different from studies on planktonic ('single') bacteria, this biofilm model captures bacteria in a habitat similar to dental plaque. With data from the combination of these models, it should be possible to study separate processes which together may lead to dental caries. Also products and novel agents could be evaluated that interfere with either of the processes. Having these separate models in place, a suggestion is made to design computer models to encompass the available information. Models but also role models are of the utmost importance in bringing and guiding research and researchers.
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To assess the long-term (>3 months) remineralizing effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on early caries lesions in vivo. PubMed, Web of Science, Embase, Cochrane-Central, Science Direct, CBM, and CNKI were searched up to April 2013. Only articles in English and Chinese were included. Grey literature was also searched. Randomized or quasi-randomized clinical trials in which CPP-ACP was delivered by any method were considered. All relevant studies underwent two independent reviews. Of the 738 studies screened, 83 studies were reviewed and eight selected for inclusion in the final sample. The follow-up period of the studies included varied from 3 to 24 months. The long-term remineralizing effect of CPP-ACP in vivo was demonstrated in comparison with placebo in randomized controlled trial. However, there is conflicting evidence regarding the clinical efficacy of CPP-ACP when used in conjunction with fluoride toothpastes. No specific side effect related to CPP-ACP usage was found. CPP-ACP has a long-term remineralizing effect on early caries lesions in comparison with placebo, although this does not appear to be significantly different from that of fluorides. The advantage of using CPP-ACP as a supplement to fluoride-containing products is still unclear. High-quality, well-designed clinical studies in this area are still required before definitive recommendations can be made. Clinical significance: CPP-ACP is a promising remineralizing agent with a significant remineralizing effect that has been demonstrated in both in vivo and in vitro studies. The evidence to support its synergistic effect with fluoride is insufficient based on the current existing long-term human randomized controlled trials.
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Peptides containing 8 repeats of aspartate-serine-serine (8DSS) have been shown to promote the nucleation of calcium phosphate from solution into human enamel. Here we tested the ability of 8DSS to promote the remineralization of demineralized enamel in an in vitro model of artificial early enamel caries. Initial caries lesions were created in bovine enamel blocks, which were then subjected to 12 d of pH cycling in the presence of 25 µM 8DSS, 1 g/L NaF (positive control) or buffer alone (negative control). Absorption of 8DSS was verified by X-ray photoelectron spectroscopy. Mineral loss, lesion depth, and mineral content at the surface layer and at different depths of the lesion body were analyzed before and after pH cycling by polarized light microscopy and transverse microradiography. Mineral loss after pH cycling was significantly lower in the 8DSS samples than in the buffer-only samples, and lesions in the 8DSS samples were significantly less deep. Samples treated with 8DSS showed significantly higher mineral content than buffer-only samples in the region extending from the surface layer (30 µm) to the average lesion depth (110 µm). No significant differences were found between the samples treated with 8DSS and those treated with NaF. These findings suggest that 8DSS has the potential to promote remineralization of demineralized enamel.
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Aims: To assess through scanning electron microscopy (SEM) and cross-sectional microhardness (CSMH) test whether the methodology exposed in this experiment can be used to produce artificial active white spot lesions (AAWSLs) on smooth unabraded human dental enamel. Materials and methods: Ten human permanent molars were used in this experiment. One section of each tooth was double coated with nail varnish except for a limited central area sized 2.5 mm × 1 mm (2.5 mm 2 ). Each specimen was individually exposed to 10.4 ml of a demineralizing solution at pH 5.0, during 42 days (37°C) without agitation. Samples were sectioned in the center of the AAWSL and one half was analyzed in SEM and the other half was subjected to CSMH. Descriptive statistics was performed to determine mean depth of the lesion. Results: The mean depth of AAWSL was 100 μm (s.d. =12.1) and a white dull rough surface could be detected by the unaided eye. SEM images demonstrated that although some surface areas of the lesion appeared to be relatively intact, erosion was present. A prismatic pattern of dissolution was observed in all samples with an enlargement of the prism sheaths and some samples had also sites of destruction of prism cores. Conclusion: This methodology can be used to induce AAWSLs in human dental enamel but surface erosion has to be taken into account when performing CSMH test.
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Biomimetic reconstruction of tooth enamel is a significant topic of study in material science and dentistry as a novel approach for prevention, restoration, and treatment of defective enamel. We developed a new amelogenin-containing chitosan hydrogel for enamel reconstruction that works through amelogenin supramolecular assembly, stabilizing Ca-P clusters and guiding their arrangement into linear chains. These amelogenin Ca-P composite chains further fuse with enamel crystals and eventually evolve into enamel-like co-aligned crystals, anchoring to the natural enamel substrate through a cluster growth process. A dense interface between the newly-grown layer and natural enamel was formed and the enamel-like layer had improved hardness and elastic modulus compared to etched enamel. We anticipate that chitosan hydrogel will provide effective protection against secondary caries because of its pH-responsive and antimicrobial properties. Our studies introduce amelogenin-containing chitosan hydrogel as a promising biomaterial for enamel repair and demonstrate the potential of applying protein-directed assembly to biomimetic reconstruction of complex biomaterials.