Preventing and Arresting Primary Tooth Enamel Lesions Using Self- Assembling Peptide P11-4 In Vitro

At a Glance

Metadata	Details
Publication Date	2022-01-01
Journal	Journal of International Society of Preventive and Community Dentistry
Authors	Nour Wahba, Falk Schwendicke, Mohamed A Kamel, Gehan Allam, Noha Kabil
Institutions	Ain Shams University, British University in Egypt
Citations	9

Abstract

INTRODUCTION Caries in primary dentition is one of the most prevalent conditions of humankind,[1] with more than 500 million untreated cases and more than 120 million incident cases each year. Conventional treatment of caries lesions in primary teeth using restorative approaches is challenging due to a combination of behavioral and micro- and macro-anatomic factors, and failure rates of plastic restorations in the primary dentition being high specifically due to secondary caries.[2] Caries in the primary dentition is a major reason for hospitalization for both routine treatments and emergencies.[3] Hence, there is a great need for both preventing and arresting carious lesions in the primary dentition. The most accepted strategies for prevention are the delivery of fluoride, mainly via toothpaste or, in high-risk individuals, varnishes, gels, or mouthwashes, as well as routine oral hygiene care and dietary control. The use of fluoridated toothpaste for caries prevention is supported by a large body of evidence.[4] Similarly, fluoride varnish (FV) and fluoride mouthwash (FMW) have been found to be highly efficacious for caries prevention,[56] are if applied risk-adjusted are cost-effective.[7] Fluoride has been well known for its inhibitory effect on demineralization and enhancing effect of remineralization. During demineralization phases in vivo, sugars are converted into acids and once the critical pH of enamel (pH = 5.5) is reached, mineral dissolution occurs. The presence of fluoride in the dental biofilm can inhibit the demineralization process by the formation of fluoroapatite because of hydroxyapatite dissolution. Since the critical pH of fluoroapatite is pH = 4.5, it allows the precipitation of minerals back to the tooth structure and prevents the net demineralization. In addition, fluoride can enhance the remineralization process. When sugar ingestion has stopped, the pH rises above 5.5, and the salivary remineralizing effect is enhanced by the presence of fluoride in the biofilm. At high pH levels, biofilm fluid is supersaturated with respect to hydroxyapatite as well as fluoroapatite and hence the lost ions are efficiently recovered by the tooth.[8] Alternative interventions to control the balance between de- and remineralization on the surface of primary teeth, specifically enamel, have been sought; among them are (a) casein-phosphopeptide amorphous calcium phosphate (CPP-ACP) with or without the addition of fluoride for home and in-office use, (b) nanohydroxyapatite (nHA), or (c) self-assembling peptides P11-4 (SAP).[9] After its application, CPP-ACP accumulates in the supragingival plaque and, under low pH conditions, releases Ca2+ and PO43- ions, which precipitate and are thought to prevent net demineralization and enhance remineralization of incipient lesions.[10] Synthetic nHA is known for being a bioactive and biocompatible material that resembles enamel apatite crystals in their morphology, structure, and crystallinity. The nano-sized nHA particles are believed to fill enamel defects on the enamel surface and create a new layer of synthetic surface enamel, preventing demineralization and enhancing remineralization.[111213] SAP are oligomer β-sheet-forming peptides (Ace-Gln-Gln-Arg-Phe-Glu-Trp-Glu-Phe-Glu-Gln-Gln-Nh2), which when subjected to specific environmental conditions have the ability to self-assemble into fibrillar scaffolds, thereby creating a β-sheet called “nanotapes.” The process of self-assembly continues while the nanotapes connect by pairing and transform into ribbons, which further self-assemble to form fibrils and fibers,[14] leading to scaffold-like structures attracting calcium and phosphate deposition.[141516] SAP is thereby supposed to facilitate biomimetic remineralization of hard dental tissue[15] and has been found to be efficacious clinically as well.[17] Similarly, for lesion arrest, the in-office application of FV or CPP-ACP, for example in higher concentrations,[18] or SAP has been suggested.[19] Alternatively, resin infiltration (RI), where the lesion is infiltrated with lowly filled resins, which are light-cured and subsequently block any acid diffusion into the lesion body and hence mineral loss from it, can be applied to inhibit caries lesion progression.[20] There is a robust body of clinical data supporting RI, for example to arrest proximal carious lesions, mainly in the permanent dentition.[21] Overall, most studies on preventing and/or arresting carious lesions using the described measures were conducted in the permanent, not the primary dentition. The body of evidence comparing fluoride applications, other mineral suppliers, RI or SAP is extremely limited. Therefore, we aimed at comparing caries prevention and inhibition of lesion progression using SAP against those of other established measures in vitro, hypothesizing SAP to have no superior caries-preventive and arresting properties compared with the other materials under investigation. MATERIALS AND METHODS STUDY DESIGN This study followed the CRIS (Checklist for Reporting In vitro Studies) guidelines[22] and was based on the fundamentals of ethical research practice. Informed consent was obtained from all patients’ legal guardians to include children’s teeth in the experiments. This study assessed the caries-preventive and -arresting effect of SAP and various alternatives, namely FV, FMW, CPP-ACP, CPP-ACP with fluoride (CPP-ACPF), and nHA for prevention, and FV, CPP-ACPF, and RI for arrest in primary tooth enamel in vitro [Table 1].Table 1: The caries-preventive and caries-arresting products usedA combined study design was chosen, with samples being either prepared, the preventive strategies applied (Experiment 1, Preventive Study), and then challenged with demineralization (using pH cycling), or with the samples being prepared, pre-demineralized using acetic acid for 21 days (to induce an artificial carious lesion), the application of arresting interventions (Experiment 2, Arrest Study), and then challenged with demineralization (using pH cycling). For both study parts, mineral loss and lesion depth were assessed using transverse microradiography (TMR). The study flow is summarized in Figure 1.Figure 1: Study flow. Sound enamel was ground, polished, and sectioned to enamel-dentin samples. Parts of the sound polished enamel were protected against demineralization using nail varnish to serve as sound controls. The remaining exposed enamel was either submitted to preventive strategy application (left side) or pre-demineralized to induce artificial caries lesions (right side), to be arrested using arresting strategies. The treated surfaces were exposed to pH cycling for 14 days. Samples of 100 ± 10 µm thickness were prepared for transverse microradiography and microradiographically analyzed (left side: the lesion induced by pH cycling, sound enamel, and dentin beneath it; right side: I sound enamel and dentin, II pre-demineralized enamel, sound enamel, and dentin beneath it, III the lesion after further demineralization using pH cycling and sound enamel and dentin beneath it)SAMPLE SIZE ESTIMATION A sample size estimation was performed to have adequate power to apply a two-sided statistical test of the research hypothesis (null hypothesis) that there is no difference SAP and FV in preventing and inhibiting carious lesions progression of primary teeth. The estimation was built on the results of Sindhura, Vemulapalli et al., in which the mean difference was 0.26 and the standard deviation was 0.21.[23] Assuming an alpha (α) level of 0.05 (5%), a beta (β) level of 0.20 (20%), that is, a power = 80%, and an effect size (d) of (1.24), the required sample size (n) was 12 samples per group. Sample size calculation was performed using G*Power version 3.1.9.2.[24] Several studies done on remineralization comparing SAP with other remineralizing agents used a range of 10-22 samples per group, with an average of 16 samples/group. Therefore, in Experiment 1 (Prevention Study), a sample size of n = 20 and in Experiment 2 (Arrest Study), n = 15 was chosen, to have a minimum of 25% extra samples to compensate for any loss of samples during preparation expected due to the limited thickness of enamel in primary anterior teeth. SPECIMEN PREPARATION One hundred eighty sound primary anterior teeth (incisors and canines) obtained from Egyptian patients after exfoliation under an ethically approved protocol (ethical committee of the Ain Shams University, FDASURecIR022024) were collected and stored in 0.5% Chloramine T solution for a maximum of two months. There are more than 15 different storage solutions suggested in the literature for the storage of extracted teeth or enamel specimens, Chloramine T was chosen for being a well-known storage medium with antibacterial properties that can prevent bacterial growth during the storage period.[25] Teeth were cleaned and those with stains, cracks, and carious or developmental defects were excluded. The root was separated from the crowns at the cemento-enamel junction using a water-cooled diamond coated band saw (Band Saw Exakt 300cl; Exakt Apparatebau, Norderstedt, Germany). The samples were embedded in epoxy resin (Technovit 4071, Heraeus Kulzer, Hanau, Germany), with the labial surfaces of incisors and the lingual surfaces of canines facing upward, ground flat, and polished sequentially (Mikroschleifsystem; Abrasive Paper WS flex 18C, SiC 1200-4000, Exakt Apparatebau, Norderstedt, Germany) until a surface of approximately 2 mm × 2 mm enamel was exposed. All samples were checked under light microscopy (Durchlichtmikroskop “Axioskop 2,” Fa. Zeiss, Oberkochen, Deutschland) to make sure that an enamel surface was still present, and that dentine was not exposed. INTERVENTIONS The 180 samples were divided into the two experimental arms: In Experiment 1 (Prevention Study), 120 samples were used to assess the caries-preventive effect, whereas in Experiment 2 (Arrest Study), 60 samples were used to assess the lesion arrest [Figure 2]. One-third of the exposed surfaces of all samples was protected against the subsequent demineralization challenge using a nail varnish (Maybelline New York Express Finish 40, New York, USA), serving as baseline (sound control).Figure 2: Flow chart, showing the sample sizes for all groups in both Experiments 1 and 2. Experiment 1: SAPP (self-assembling peptide for prevention); FV (fluoride varnish); CPP-ACP/CPP-ACPF (casein-phosphopeptide amorphous-calcium-phosphate without/with fluoride); FMW (fluoride mouthwash); nHA (nanohydroxyapatite). Experiment 2: SAPR (self-assembling peptide for repair); FV (fluoride varnish); CPP-APF (casein-phosphopeptide amorphous-calcium-phosphate fluoride); RI (resin infiltration)Experiment 1 (Prevention Study) To test the caries-preventive effect, the remaining two-thirds of the exposed surfaces of the 120 samples were treated using one of six interventions (n = 20/group, Table 1) before being challenged for demineralization:[1] SAPP (Curodont Protect, Credentis, Windisch, Switzerland),[2] FV (5% NaF Profluorid, Voco, Cuxhaven, Germany),[3] CPP-ACP (Tooth Mousse, GC, Tokyo, Japan),[4] CPP-ACP plus fluoride (CPP-ACPF, MI Paste Plus, GC, Tokyo, Japan),[5] 500 ppm sodium FMW (pharmacy of the Charité - Universitätsmedizin Berlin),[6] and nHA mouthwash (Biorepair Mouth Wash, Dr. Kurt Wolff, Bielefeld, Germany). Curodont Protect gel was applied on a semidry surface with a microbrush, rubbed in, and left for a couple of minutes to dry. It was then washed away as instructed by the manufacturer. FV, CPP-ACP, and CPP-ACPF were applied on a dry surface with a microbrush and left 30 min to set; then, they were rinsed off with water to mimic the conditions of the oral cavity. Both mouthwashes were utilized once daily after the demineralization cycle for 15 min by storing the samples in them, whereas the other samples were stored in distilled water during that time. Experiment 2 (Arrest Study) To test lesion arrest, the remaining two-thirds of the exposed surfaces of the 60 samples were pre-demineralized using 3 mM CaCl2, 3 mM KH2PO4, 0,006 mM methylhydroxydiphosphanate (MHDP), 50 mM CH3COOH, and 10 M KOH. The pH was adjusted to 4.95 using KOH for 21 days (Carl Roth, Karlsruhe, Germany).[26] Half of the demineralized surface was covered with a nail varnish to allow the assessment of the mineral loss of the lesions after the first demineralization challenge and prior to the second demineralization challenge. The remaining one-third of the exposed surface of the sample received one of four interventions (n = 15/group, Table 1) prior to being challenged again for demineralization:[1] SAPR (Curodont Repair, Credentis, Windisch, Switzerland),[2] 5% FV (5% NaF Profluorid, Voco, Cuxhaven, Germany),[3] CPP-ACPF (MI Paste Plus, GC),[4] and RI (Icon DMG, Hamburg, Germany). For SAPR, samples were etched with 37% phosphoric acid (Fine Etch 37, Spident, Korea) for 5 s and rinsed with tap water. After drying the surface, Curodont Repair (In Vitro Vial, Credentis, Windisch, Switzerland) was dissolved without any further purification in 50 µL distilled water applied on each sample and left 5 min for setting. FV and CPP-ACPF were applied as described in experiment 1. Before RI, samples were etched using 37% phosphoric acid (FineEtch 37) for 5 s. The specimens were thereafter washed and dried using Icon Dry for 30 s and infiltrated using Icon Infiltrant for 3 min. After removing the excess material, light-curing was performed using an LED curing light (Valo, Ultradent, Salt Lake City, USA) with an intensity of 1400 mW/cm2 for 40 s from < 1 mm distance. The procedure was repeated, with the infiltrant being applied for only 1 min, as recommended by the manufacturer. DEMINERALIZATION CHALLENGE USING pH CYCLING All samples were subsequently subjected to a demineralizing pH cycling using a demineralization solution containing 2.2 mM CaCl2, 2.2 mM NaH2PO4, and 50 mM acetic acid adjusted to a pH of 4.8 by NaOH (Carl Roth, Karlsruhe, Germany). The remineralizing solution contained 1.5 mM CaCl2, 0.9 mM NaH2PO4, and 0.15 M KCl adjusted to a pH of 7.0 by KOH (Carl Roth, Karlsruhe, Germany). Each group was cycled separately for 8 h in 100 mL demineralizing solution and 16 h in 100 mL remineralizing solution for 14 days at room temperature without agitation. Between the de- and remineralizing cycles, the samples were washed with distilled water. The mouthwashes in the prevention groups were renewed daily.[27] TRANSVERSAL MICRORADIOGRAPHY Samples were cut along their longitudinal axes (Band Saw Exakt, Exakt Apparatebau, Norderstedt, Germany) and thereafter, thin plano-parallel slices with a thickness of 100 ± 10 µm were prepared (Mikroschleifsystem, Exakt Apparatebau, Norderstedt, Germany). In Experiment 1, a total of five samples were lost during their preparation for the TMR analysis, since the enamel of human primary anterior teeth was very thin. Among the five samples, two were from FV group, one from CPP-ACP, one from FMV, and one from nHA. Despite this loss, the estimated sample size was reached. The samples were placed on film holders and exposed to a nickel-filtered copper radiation source operating at 20 kV and 20 mA with an exposure time of 10 s. Films (Fine 71337, Fujifilm, Tokyo, Japan) were developed according to the manufacturer’s instructions under standardized conditions. The microradiographs were analyzed with a digital image-analyzing system (XC 77 CE, Sony, Tokyo, Japan) interfaced with a universal microscope (Axioskop 60318, Zeiss, Oberkochen, Germany) and a personal computer (TMR for Windows 2.0.27.2, Inspector, Research, Amsterdam, Netherlands). Calibration standardization was done using an aluminum step-wedge with different aluminum thicknesses, and a calibration curve between aluminum thickness and gray levels was constructed. STATISTICAL ANALYSIS Our primary outcome was mineral loss (vol%/µm), and the secondary outcome was the lesion depth (µm). Numerical data were tested for normality by checking their distribution and by using Shapiro-Wilk’s test. Data were found to be nonparametric, so they were presented as median and interquartile range values and were analyzed for intergroup comparisons using Kruskal-Wallis test followed by pairwise comparisons utilizing multiple Mann-Whitney U tests with bonferroni correction. The significance level was set at P < 0.05 within all tests. Statistical analysis was performed with R statistical analysis software version 4.1.0 for Windows.[28] RESULTS EXPERIMENT 1 (PREVENTION STUDY) Descriptive statistics for mineral loss and lesion depth change are presented in Tables 2 and 3, respectively.Table 2: Experiment 1 (Prevention Study) descriptive statistics for mineral loss difference (baseline-intervention)Table 3: Experiment 1 (Prevention Study) descriptive statistics for lesion depth difference (baseline-intervention)Results of intergroup comparisons for mineral loss change presented in Table 4 showed that there was a difference between different groups < The median was found in CPP-ACP followed by SAPP and then CPP-ACPF nHA and FMW whereas the was found in FV pairwise comparisons showed values of CPP-ACP, and CPP-ACPF to be higher than values of FMW and FV < In addition, they showed the of nHA to be higher than FV < values for change in mineral loss in different groups are presented in Figure Experiment 1 (Prevention Study) intergroup comparisons for mineral loss difference and lesion depth difference 3: Experiment 1 (Prevention Study) median values for mineral loss of intergroup comparisons for lesion depth change presented in Table 4 showed that there was a difference between different groups < The median was found in CPP-ACPF followed by SAPP and then CPP-ACPF nHA and FMW whereas the was found in FV pairwise comparisons showed values of CPP-ACP, CPP-ACPF, and nHA to be higher than values of FMW and FV < values for change in lesion depth in different groups are presented in Figure Experiment 1 (Prevention Study) median values for lesion depth 2 STUDY) Descriptive statistics for mineral loss and lesion depth change are presented in Tables 5 and of intergroup comparisons for mineral loss change presented in Table showed that there was no difference between different groups the change of mineral loss values from baseline to application = and from demineralized samples to application = For the median was found in SAPR followed by CPP-ACPF and then FV whereas the was found in RI For the median was found in SAPR followed by CPP-ACPF and then RI whereas the was found in FV values for change in mineral loss in different groups are presented in Figure Experiment 2 (Arrest Study) descriptive statistics for mineral loss Experiment 2 (Arrest Study) descriptive statistics for lesion depth Experiment 2 (Arrest Study) intergroup comparisons for mineral loss difference and lesion depth Experiment 2 (Arrest Study) showing median values for mineral loss of intergroup comparisons for lesion depth change presented in Table showed that there was no difference between different groups the change of lesion depth values from baseline to application = and from demineralized samples to application = For the median was found in CPP-ACPF followed by FV and then SAPR whereas the was found in RI For the median was found in SAPR followed by CPP-ACPF and then FV whereas the was found in RI values for change in lesion depth in different groups are presented in Figure Experiment 2 (Arrest Study) median values for lesion depth A range of caries-preventive and -arresting strategies based on both inhibition of demineralization and of remineralization are in and primary teeth, the of efficacious to fluoride are of of and of fluoride and the SAP have been as one In the a range of strategies that are suggested to prevent caries and inhibit lesion progression against each other were compared with primary tooth enamel in In Experiment 1 (Prevention Study), six different agents were compared for their caries-preventive effect, namely FV, CPP-ACP, CPP-ACPF, FMW, and nHA. the prevention of caries lesions, established as FV and FMW were found to mineral loss, the mineral loss whereas strategies as CPP-ACP, CPP-ACPF, and nHA not have any preventive effect in depth were to those of mineral loss, where FV and FMW the lesion depth difference compared with CPP-ACP, CPP-ACPF, and nHA. In Experiment 2 (Arrest Study), four agents were tested for their remineralizing effect, namely SAPR, FV, CPP-ACPF, and SAPR was the material under that is, to remineralization compared with FV, the standard remineralizing compared against CPP-ACPF and There was no difference the four groups in their ability to inhibit lesion when comparing the four RI was found to inhibit the lesions most from followed by FV, whereas again strategies as CPP-ACPF and SAPR not have any inhibition of lesion progression effect in accepted This study has and data on are and so no study has tested SAP to prevent caries or inhibit lesion progression in primary teeth. studies on SAP on human enamel from permanent teeth or Both different and than human primary tooth SAP have been tested for lesion arrest of not and this study is one of the this strategy for preventive as well as caries the assessment of mineral loss using TMR is highly and a that was not in studies on microscopy surface or been the chosen assessment has not the specific mineral based on the thickness of each of on the mean ground sound enamel has in this in a thickness along specimens, which have this be groups with the of and as a the in vitro protocol have to and the enamel surface has the enamel surface which is to be required for SAP and SAPR enamel, calcium of the hydroxyapatite is not any it is that peptide be to in compared with the described was used as only then mineral loss in TMR are and the layer of enamel is off in a clinical at and any clinical of self-assembling peptides be if on this enamel being In addition, studies by polished specimens, which is it was aimed at this for of SAP was applied only that is, before the pH cycling for for standardization FV, were only applied The it once or per the fluoride products tested have a high 100 of and the formation of is their of the calcium source for formation is whereas in study human artificial was for SAP calcium and phosphate from the formation of hydroxyapatite crystals within the peptide and the of the between and those in and in the of to all and the of FV, for not be with an was when lesion arrest using SAP to mimic its clinical of polished samples have the remaining surface layer of the lesion to be for SAP to the The was suggested in to the surface of enamel from and mineral both are not in induced enamel A range of to be The SAP were not in preventing caries or inhibiting lesion A limited body of evidence on SAP is as and with not all of the This be due to the time and of enamel, of and for there a of its preventive SAP are to in the of an caries lesion and then minerals in human It is this apply to prevent In it be that the material was washed away during the first demineralization cycles, or that only very thin peptide on the sound enamel to the enamel from subsequent demineralization. fluoride is the standard for caries prevention and lesion This was by There is evidence that SAP be combined with fluoride to their and of when it to inhibiting lesion progression (fluoride mainly on the surface of whereas SAP is suggested to into the body of the In a of the used FV (5% was its and It is that FV not only a effect by the surface, thereby it from demineralization. a effect has been described for FV in studies on root caries that we found FMW 500 ppm to be this not FMW was daily in to most other RI is well known for its lesion progression inhibition in lesions by carious enamel and thereby the diffusion leading to caries RI has been to be superior to for lesion arrest by a range of clinical in the primary and data in this the application of RI is that be in the primary dentition and in we the excess infiltrant material, a effect of RI of the can be excluded. CPP-ACP and CPP-ACPF not any caries-preventive or lesion progression inhibiting effect in which be to the that their effect is believed to be enhanced by the presence of a which as a for the calcium and phosphate ions and hence prevents mineral loss in of demineralization. and as for the application of CPP-ACP and CPP-ACPF have been on a range of can be in vitro studies SAP at the enamel layer when using ground the application of prior to remineralization be recommended in an in vitro to allow formation and clinical conditions as as in be mineral loss not on as or as they to the for example using transverse or clinical studies be before into any clinical so clinical data have on lesion as In and within the described FV (5% and FMW ppm showed and caries-preventive on human primary teeth enamel in vitro, whereas RI and FV were to be to inhibit caries lesion progression in this CPP-ACP, CPP-ACPF, and nHA not any caries-preventive or progression inhibition AND This research not any specific from in the or The no of and the study has received no of analysis and of of AND This study followed the CRIS (Checklist for Reporting Studies) and was based on the fundamentals of ethical research practice. Informed consent was obtained from all patients’ legal guardians to include children’s teeth in the experiments. Data are on from the

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DOI: https://doi.org/10.4103/jispcd.jispcd_257_21

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