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BDJOpen www.nature.com/bdjopen ARTICLE OPEN Comparative efﬁcacy of a hydroxyapatite and a ﬂuoride toothpaste for prevention and remineralization of dental caries in children 1 1 1 1 Bennett T. Amaechi , Parveez Ahamed AbdulAzees , Dina Ossama Alshareif , Marina Adel Shehata , 1 1 1 1 Patrícia Paula de Carvalho Sampaio Lima , Azadeh Abdollahi , Parisa Samadi Kalkhorani and Veronica Evans OBJECTIVE: This in situ study compared the effectiveness of two toothpastes containing hydroxyapatite or 500 ppm ﬂuoride in promoting remineralization and inhibiting caries development. MATERIALS AND METHODS: Two enamel blocks (human primary teeth), one sound and one with artiﬁcially-produced caries lesion, were exposed to toothpaste containing either 10% hydroxyapatite or 500 ppm F (amine ﬂuoride) via intra-oral appliance worn by 30 adults in two-arm double blind randomized crossover study lasting 14 days per arm (ClinicalTrials.gov: NCT03681340). Baseline and post-test mineral loss and lesion depth (LD) were quantiﬁed using microradiography. One-sided t-test of one group mean was used for intragroup comparison (baseline vs. post-test), while two-sided t-test of two independent means was used to compare the two toothpaste groups. RESULTS: Pairwise comparison (baseline vs. test) indicated signiﬁcant (p < 0.0001) remineralization and LD reduction by either toothpaste; however, when compared against each other, there was no statistically signiﬁcant difference in remineralization or LD reduction between the two toothpastes. No demineralization could be observed in sound enamel blocks exposed to either toothpaste. While F induced lesion surface lamination, HAP produced a more homogenous lesion remineralization. CONCLUSIONS: 10% hydroxyapatite achieved comparable efﬁcacy with 500 ppm F in remineralizing initial caries and preventing demineralization. Thus the HAP toothpaste is conﬁrmed to be equal to the ﬂuoride toothpaste in this study. BDJ Open (2019) 5:18 ; https://doi.org/10.1038/s41405-019-0026-8 INTRODUCTION to what application of ﬂuoride alone can achieve in relation to Although preventable, dental caries continues to be one of the caries prevention and remineralization. These limitations may be most prevalent chronic diseases among children in the U.S. and associated with facts that ﬂuoride becomes less effective below a 12 2+ 3– the world, and one of the most common unmet healthcare needs pH of about 4.5; ﬂuoride still needs Ca and PO ions in a of poor children. As much as 80% of caries incidence is bioavailable form in saliva and other sources to be effective; and experienced by only 20–25% of children, with 10% having ﬂuoride remineralization of initial lesions is most effective at the 13,14 untreated cavities, and those from low socioeconomic and outer 30 μm of the lesion, thus leading to surface-zone minority groups experiencing signiﬁcantly higher rates and at remineralization at the expense of the lesion body, making full 2,3 15,16 younger ages. remineralization difﬁcult to achieve. Furthermore, although It is well documented that saliva has a caries protective effects the efﬁcacy of ﬂuoride is dose-dependent and increases with 2+ 3– 7 due to its supersaturation with Ca and PO ions in a increase dose, there is a limit to which you can increase the bioavailable form and enrichment with various proteins playing dosage of ﬂuoride to avoid the risk of ﬂuorosis in children and 18,19 multiple roles in maintenance of hard tissue integrity throughout toxicity in all ages. The effect of dose limitation on ﬂuoride 4 2+ 3– life. Furthermore, the supersaturation of saliva with Ca and PO effectiveness may be more pronounced in children below 6 years, ions, at physiological pH, ensures that these ions are bioavailable to since the ﬂuoride dose recommended for this group is even lower diffuse into mineral deﬁcient lesions to induce remineralization. than the regulatory 1000–1500 ppm ﬂuoride concentration in However, the natural caries protective and remineralizing effects of non-prescription toothpastes, and as such probably suboptimal saliva is not only a slow process but obviously insufﬁcient to protect for effective remineralization of initial lesions. individuals against caries and remineralize existing lesions without The above mentioned limitations of the saliva homeostatic additional agents to enhance its effects. mechanisms and ﬂuoride-based strategies in caries prevention Although ﬂuoride interventions seem to have the most and remineralization, especially in highly cariogenic oral environ- consistent beneﬁt in preventing caries development and reminer- ments justify the need for new-age strategies that could work alizing initial lesions with the highest level of supporting either better than or as effective as ﬂuoride but can permit 6–8 evidence, caries still develop in high risk individuals of all ages, increasing dosage for increase effectiveness without safety 9,10 irrespective of the dose of ﬂuoride used. There are limitations concerns. It is envisaged that the presence of additional extrinsic Department of Comprehensive Dentistry, School of Dentistry, University of Texas Health San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA Correspondence: Bennett T. Amaechi (firstname.lastname@example.org) Received: 28 June 2019 Revised: 12 October 2019 Accepted: 21 October 2019 © The Author(s) 2019 1234567890();,: Comparative efﬁcacy of a hydroxyapatite and a ﬂuoride toothpaste for. . . B.T. Amaechi et al. 2+ 3– sources of stabilized Ca and PO ions could augment the to wear the in situ appliance and use only assigned products for natural caries preventive and remineralization potential of saliva oral hygiene throughout the duration of the study; the ability to by increasing diffusion gradients favoring faster and deeper read and understand English; ability to provide informed consent; subsurface remineralization. One of the new caries remineraliz- and no self-reported history of allergy to personal care/consumer ing technologies is the biomimetic systems, among which are the products or any ingredient in the test products. Exclusion criteria synthetic hydroxyapatite (HAP; Ca (PO ) (OH) applied in micro- were the presence of advanced periodontal disease or other oral 5 4 3 21,22 cluster or nanocrystalline forms in oral care products. HAP is a pathology; medical condition that requires premedication prior to bioactive and biocompatible material with similar chemical dental procedures; use of antibiotics one month prior to or during 21–24 composition to the apatite crystals of human enamel. Several this study; self-reported pregnancy or breastfeeding; and use of in vitro and in situ studies have provided evidences supporting tobacco products. the caries remineralization and prevention potential of HAP in oral care products based on its demonstrated ability to strongly adsorb Creation of artiﬁcial initial caries and fabrication of the in situ 24–29 to tooth surfaces, plaque components and bacteria. Rando- appliance mized controlled clinical trials, some of which have led to the Following consent from the donors, freshly extracted primary approval of HAP as an anti-caries agent in Japan in 1993 and in teeth were collected from the pediatric clinics of the UTHSA Canada in 2015, have demonstrated its non-inferiority and School of dentistry and stored in 0.1% thymol solution at 4 °C prior 30–32 equivalence to ﬂuoride. Therefore, the objective of this to use. The teeth were examined with a transilluminator, and thirty in situ study was to determine whether Karex Kid’s toothpaste two teeth without caries, cracks, or enamel malformations were containing 10% HAP microclusters (Kinder Karex Zahnpasta, Dr. selected and cleaned with pumice using electronic toothbrush. Kurt Wolff GmbH and Co. KG, Bielefeld, Germany) is as effective as Using a water-cooled diamond wire saw, 4 tooth blocks were Elmex Kid’s toothpaste containing 500 ppm ﬂuoride as amine produced from buccal and lingual surfaces of each of the selected ﬂuoride (Elmex Kinder Zahnpasta, CP GABA GmbH, Hamburg, teeth, with each block measuring ~2 mm length × 2 mm width × Germany) in promoting the remineralization and inhibiting the 1.5 mm thickness. Two of the 4 blocks were retained as sound development of carious lesions. We hypothesized that (1) each of enamel blocks for demineralization inhibition assessment while the two toothpaste formulations promotes remineralization and the other two blocks targeted for remineralization assessment had lesion depth reduction that is signiﬁcantly greater than zero, and artiﬁcial initial caries produced in them as follows. All surfaces of (2) neither toothpaste is inferior to the other with respect to each block were painted with two coats of acid resistant nail promoting the remineralization and inhibiting the development of varnish except buccal or lingual on which an initial caries lesion carious lesions. was created by subjecting this exposed surface to 7 days demineralization in an acidiﬁed gel system (0.10 M lactic acid, 0.10 M sodium hydroxide, 6% / hydroxyethyl cellulose, pH 4.5). MATERIALS AND METHODS Following lesion formation, the nail varnish was carefully removed This is a double-blind, randomized, crossover, single center, with acetone. A tooth section (~150 µm thick) was cut from each controlled in situ study to establish the equivalence of two tooth block for the measurement of the baseline mineral loss (Δz ) children toothpaste formulations, containing either 10% HAP and lesion depth (LD ) of each produced initial caries lesion, and microclusters (crystallite size: length ≈ 80 nm (median) × width ≈ for selection of the suitable lesions for the remineralization 30 nm (median)) or 500 ppm ﬂuoride provided as amine ﬂuoride assessment. The sections were processed for transverse micro- (AMF), in inducing the remineralization and inhibiting the radiography (TMR) as follows. Both sides of the sections were development of initial caries lesions. The primary outcomes to polished using adhesive back lapping ﬁlm in a MultiPrep™ be examined were (1) the percentage remineralization and lesion Precision Polishing machine (Allied High Tech, USA) to achieve depth reduction measured relative to the baseline mineral loss planoparallel surfaces, as well as reduce the thickness of the slice and lesion depth for initial caries, and (2) the amount of mineral to 100 µm (the appropriate thickness for TMR). Following this, the loss and lesion depth for the sound enamel. The study was sections were microradiographed on a type lA high resolution conducted at the clinical research facility of the University of Texas glass X-ray plate (Microchrome Technology, CA, USA) using a Health San Antonio (UTHSA) School of Dentistry. The UTHSA Phillips X-ray generator system set up for this purpose. The plates Institutional Review Board (IRB) approved the study (protocol #: were exposed for 10 min at an anode voltage of 20 kV and a tube HSC20180416H), and the study was registered with ClinicalTrials. current of 10 mA, and then processed. Processing consisted of a 5 gov (NCT03681340). The study was conducted in accordance with min development in Kodak HR developer and 15 min ﬁxation in the ethical standards outlined in the 1964 Declaration of Helsinki Kodak Rapid-ﬁxer before a ﬁnal 30 min wash period. After drying, and its later amendments, and in compliance with the Interna- the microradiographs were examined under a Leica DMR optical tional Conference on Harmonization (ICH) Good Clinical Practice microscope linked via a Sony model XC-75CE CCTV camera to a Guidelines. The participants were recruited from among different personal computer. Using TMR2006 version 126.96.36.199 image ethnic origins and varied socio-economic status in the local San analysis software (Inspektor Research Systems, Amsterdam, Antonio area, written informed consent was obtained from all Netherlands), the enhanced image of the microradiographs were participants prior to their participation in the study. analyzed under standard conditions of light intensity and magniﬁcation along with the image of a step wedge as described Participant recruitment by de Josselin de Jong et al. At this point, the images were used Fifty subjects aged from 18 to 60 years were given screening only for selection of the suitable lesions for the study. Only the examination that included sialometry, medical/dental history, and controls that showed caries-like lesions with subsurface lesions, oral examination (Fig. 1). Thirty two subjects qualiﬁed and were which display a fairly uniform width throughout their length, were enrolled in the study. Inclusion criteria were age of 18 through 60 selected for the remineralization process, and their test blocks years; normal salivary function with unstimulated and stimulated were used for construction of the in situ appliance. It is pertinent salivary ﬂow rates ≥0.2 ml/min and ≥0.7 ml/min, respectively, to mention that the baseline measurements were not conducted measured according to the Sreebny and Valdini procedure. for the sound tooth blocks to be used for demineralization- Other inclusion criteria were not taking any antibiotics or prevention study because the TMR does not measure the mineral medications which could affect saliva ﬂow rate; the presence of density of sound tooth tissue rather the software uses the known at least 20 natural uncrowned teeth (excluding third molars); a mineral density of sound enamel or dentin to determine the past history of dental caries but no clinically active caries; willing amount of mineral loss in a demineralized tissue. BDJ Open (2019) 5:18 Comparative efﬁcacy of a hydroxyapatite and a ﬂuoride toothpaste for. . . B.T. Amaechi et al. Fig. 1 Flow Diagram detailing the stepwise methodology. This is a crossover study so the 30 completers received the two intervention in a crossover design as phase I and II. As stated above, the four tooth blocks from each tooth were started with one week of washout period and then 4 weeks of distributed as follows: two lesion-bearing blocks for remineraliza- treatment, consisting of two 2-week periods during which each tion assessment and two sound blocks for demineralization subject used his/her assigned treatment under the following inhibition assessment. These four blocks were used to fabricate conditions. First 2-week period, with the subject wearing an in situ the in situ appliances as follows. Each block was covered with appliance with sound enamel block, and the second 2-week polyester gauze (Bard Peripheral Vascular, Inc., Tempe, AZ, USA) period, with the subject wearing an in situ appliance with lesion- and mounted within an in situ appliance, a customized bearing tooth block. orthodontic bracket. The polyester gauze facilitated plaque Subjects who satisﬁed enrollment criteria were given a specially retention on the surface of the tooth blocks on intra-oral exposure. manufactured washout toothpaste with neither ﬂuoride nor HAP The appliance consists of an orthodontic molar pad with retentive (Dr. Kurt Wolff GmbH and Co. KG, Bielefeld, Germany) and an adult mesh backing (American Orthodontics Corp., Sheboygan, US), soft-bristled toothbrush to use for a washout period of one week. which has a ring of 0.7 mm orthodontic wire welded to it so that The washout period allows for attenuation of any residual effect of the ring closely encircles each test-block. The block was retained the subject's previously used toothpaste. There was no washout within the bracket with ﬂuoride-free intermediate restorative period between individual 2-week treatment periods within each material (IRM). All appliances were sterilized with gamma phase since the subjects used the same product for the 4 weeks. irradiation prior to delivery to the subject. During the washout period, subjects were instructed to use the toothpaste and toothbrush for 2 min twice a day (morning after Study treatment breakfast and night last thing before bed) in place of their The study was performed in two distinct treatment phases during normally used toothpaste and toothbrush, and as their only oral which subjects were exposed to one of the following two hygiene product. Subjects were given no restriction on treatments in a randomized crossover design; (A) Karex Kid’s dietary habit. toothpaste containing 10% HAP microclusters (Kinder Karex, Dr. At the end of the washout period, patients returned to the Kurt Wolff GmbH and Co. KG, Bielefeld, Germany), and (B) Elmex clinic, and were assigned to a group to use either HAP or AMF by Kid’s toothpaste containing 500 ppm ﬂuoride as AMF (Elmex the Study Coordinator using randomization numbers generated Kinder zahnpasta, GABA GmbH, Hamburg, Germany). Each phase by a computer program designed and operated by our BDJ Open (2019) 5:18 Comparative efﬁcacy of a hydroxyapatite and a ﬂuoride toothpaste for. . . B.T. Amaechi et al. biostatistics team. However, to ensure that both the operators and soft and hard palate, gingival mucosa, buccal mucosa, mucogingival the subjects were blinded as to product assignment, all toothpaste fold areas, tongue, sublingual and submandibular areas, salivary tubes were packaged identically and coded (A, B, or washout) by glands, and the tonsillar and pharyngeal areas. In addition to oral the manufacturing/packaging company, who retained the code examination, subjects were screened for adverse events using a until the completion of the study and data interpretation. questionnaire. Following randomization, the 4 block-bearing in situ appliances originating from one tooth were assigned to one subject. Then the Post-treatment processing and study outcomes ﬁrst of the four assigned appliances was bonded, in accordance Following intra-oral exposure, a tooth section (~150 µm thick) was with current principles of orthodontic practice, on the buccal cut from each tooth block, both sound and lesion-bearing blocks. surface of the chosen lower molar tooth. The appliance was The sections were processed for microradiography as described bonded by a qualiﬁed dentist licensed in the state of Texas, who above for the control sections used for baseline data. Although the was different from the laboratory technician that process and lesion-bearing control sections have been microradiographed and analyze the samples to produce the ﬁnal data. To bond the analyzed for selection of the appropriate lesions, they were appliance, the buccal surface of the tooth chosen was carefully microradiographed again together with the post-test sections and etched for 30 s, washed with water spray and dried for a further both analyzed together for quantiﬁcation of the Δzand LD of the 30 s, and isolated using cotton rolls. The bottom of the lesions as described for baseline sections. This step enabled both appliance was loaded with Transbond™ XT light-cure adhesive control and test sections from same block to be microradiographed paste (3M Unitek, Monrovia, CA, USA), and carefully positioned to and analyzed under the same conditions. For the lesion-bearing avoid causing occlusal interference and to avoid soft tissue sections, this process yielded the pre-test mineral loss (Δz )and irritation. The excess composite material that spilled out from lesion depth (LD ), the post-test mineral loss (Δz ), and lesion depth 1 2 the sides of the appliance was used to cover the sides, beveling (LD ), and the pre-test and post-test microradiograms of the lesions. it to present a comfortable streamlined (non-catching) surface For the sections from sound tooth blocks, the process yielded the when the slab comes in contact with a soft tissue surface (e.g., post-test mineral loss (Δz) and lesion depth (LD) if any lesion tongue). The adhesive paste was cured using an Ortholux XT developed, and the microradiograms. Using the microradiograms, visible Light Curing Unit (3M Unitek, Monrovia, Ca, USA) applied the pattern and the extent of remineralization produced within each for 20 s. lesion by each treatment arm was examined by comparing the pre- Following bonding of the appliance, each subject was given his/ test and post-test images side-by-side. For each participant the post- her respective test toothpaste and a soft-bristled toothbrush treatment mineral loss was subtracted from the pre-treatment designed for use with orthodontic brackets with shorter bristles at mineral loss, and then standardized across participants by dividing the center to accommodate the bracket. Subjects were instructed to that difference by the pre-treatment mineral loss to obtain the % continue with the routine of brushing two times daily, morning after remineralization. The lesion depth pre-treatment and post-treatment breakfast and last thing before bed, for 2 min before rinsing with was handled the same way to obtain the % lesion depth reduction. only 10 ml of water. Subjects were also given special instruction on The two products were compared using the % remineralization and dispensing of the toothpaste, and were advised not to brush directly the % lesion depth reduction. on the appliance but rather to brush around it to prevent disruption of the dental plaque on the surface of the tooth block. Subjects were Power analysis and sample size calculation restricted from eating nor drinking for at least 30 min after brushing. The sample size calculations, which were based on a power A timer and measuring cup were provided to each subject. As a analysis, were performed using nQuery Advisor software (Statis- method of monitoring compliance, a diary was provided to each tical Solutions, Cork, Ireland). Based on previous studies in which subject for recording the time of each brushing episode, and in the mean % remineralization was equal to 30.3 with a standard 27,35–37 addition, toothpaste tubes were weighed at the time of randomiza- deviation equal to 16.3, and for the hypothesis that each of tion and at each study visit. Subjects were instructed to the two toothpaste formulations promotes remineralization and maintain their normal dietary habits and were prohibited lesion depth reduction that is signiﬁcantly greater than zero, an from using any other oral hygiene product (e.g., mouthwash, effective sample size of 30 subjects will have power greater than chewing gum) or tooth-whitening product for the duration of the 0.95 with a 0.05 one-sided signiﬁcance level to detect a difference study. Immediately after bonding of the ﬁrst appliance, each subject between a hypothesis mean of zero and a sample mean % used the test product under the supervision of the Study remineralization equal to or greater than 10% using a two-sided t- Coordinator, and for the remainder of each treatment phase, test of two independent means. However, 32 subjects were subjects completed the procedure at home and as instructed by the enrolled to make provision for 5% dropout. Study Coordinator Subjects returned to the clinical research facility after 2 weeks Statistical analysis without using the product that morning, and the ﬁrst appliance was For measurements by both mineral loss and lesion depth, three detached and sent to the laboratory for analysis. The appliance for endpoints were analyzed. (1) The mean amount of remineraliza- the second 2-week treatment period of the phase was bonded, the tion and mean amount of lesion depth reduction were dairy checked, the toothpaste weighed, and safety evaluation determined for Karex toothpaste as a percentage of pre- performed. Upon completion of the second 2-week treatment treatment mineral loss and pre-treatment lesion depth respec- period, the subject again returned to the clinic for detachment of tively, and these percentages were compared to a value of 0%, the second appliance, and was given washout toothpaste and a soft- which is what would be expected if the toothpaste had no effect. bristle toothbrush to undergo another 7-day washout period The statistical test used was a one-sided t-test of one group mean. without an appliance in preparation for his/her phase 2 of the (2) In the same way, the mean amount of remineralization and study. After completion of the washout period without an appliance, mean amount of lesion depth reduction was determined for subjects return to the clinic, and the procedure of phase 1 was Elmex toothpaste, and also compared to 0%. (3) Finally, the repeated until the second 2-week treatment period was completed, primary endpoint was to compare the means of Karex toothpaste and each subject has gone through the two arms of the study. to Elmex toothpaste to check for non-inferiority/equivalence of the HAP toothpaste to the ﬂuoride toothpaste, using the two- Safety monitoring sided t-test of two independent means. Non-inferiority/equiva- At all visits, the dental examiner visually examines the oral cavity and lence was established if the difference between the two tooth- peri-oral area, and this examination included an evaluation of the paste formulations for any one measurement method was not BDJ Open (2019) 5:18 Comparative efﬁcacy of a hydroxyapatite and a ﬂuoride toothpaste for. . . B.T. Amaechi et al. Table 1. Mean rates of remineralization (%) and lesion depth reduction (%) for each toothpaste. Measurement Karex Elmex p-value, two means % Remineralization 55.8 (s.d. 13.8) 56.9 (s.d. 14.9) 0.81 p-value, One group: <0.0001 <0.0001 % Lesion depth reduction 27.1 (s.d. 10.6) 28.4 (s.d. 9.8) 0.68 p-value, One group: <0.0001 <0.0001 Table 2. Mean (Standard deviation) values of mineral loss (vol%µm) and lesion depth (µm) in the two study groups before and after treatment and their differences (with conﬁdence intervals). Treatment Before treatment After treatment Difference (95% CI) P value ΔZ [Mean (SD)] Karex (10%HAP) 2357.5 (454.63) 1013.5 (273.59) 1344 (1119.93–1568.06) <0.0001 Elmex (500 ppm) 2378.5 (593.16) 1009 (392.48) 1369.5 (1117.40–1621.59) <0.0001 LD [Mean (SD)] Karex (10%HAP) 92.89 (17.15) 67.07 (11.79) 25.82 (19.79–31.84) <0.0001 Elmex (500 ppm) 91.91 (17.94) 65.46 (13.64) 26.44 (21.15–31.74) <0.0001 Paired t-test (n = 30, α = 0.05) regarded to be clinically relevant and was set to Δ ≤ 20%. The (surface zone) of the lesion (lesion surface lamination) i.e., two statistical package R, version 3.5.0, was used for analysis. zones of contrasting density can clearly be observed (Fig. 5b). These remineralization patterns in Fig. 4b and 5b were consistent in all specimens exposed to HAP and AMF respectively. There RESULTS were no incidences of adverse effects reported by subjects or Of the 32 subjects recruited into this study, 1 subject declined to ascertained clinically. participate further during the washout period, and one other subject declined to participate midway into the ﬁrst 2-week treatment while wearing his ﬁrst appliance that bears sound tooth DISCUSSION block (Fig. 1). Thirty subjects (19 females, 11 males) with a mean Despite being a preventable disease and amid the presence of (SD) age of 39.5 (15.0) years completed the study. The uoride in oral dentifrices, dental caries prevalence in children unstimulated and stimulated saliva ﬂow rates of the subjects continues to increase globally, and at a faster rate among ranged from 0.2 to 1.5 ml/min and 0.9 to 3.5 ml/min respectively. children from low socioeconomic backgrounds. This indicates The ethnic distribution of the subjects was as follows: Hispanic 17 that although ﬂuoride interventions have the highest level of (57%), Black (not Hispanic) 3 (10%), White (not Hispanic) 7 (23%), supporting evidence as having the most consistent beneﬁtin 6–8 Asian 1 (3%), and others 2 (7%). preventing caries and remineralizing initial caries lesions; The mean rates of remineralization and lesion depth reduction additional remineralizing and preventive agents are often needed 9,10 are shown in Tables 1 and 2. Each toothpaste had a mean percent to enhance ﬂuoride effects in high caries risk individuals. One remineralization in excess of 50%, and a mean percent lesion may suggest increasing the dose of ﬂuoride since its effectiveness depth reduction better than 25%. For both toothpastes, the mean is dose-dependent, unfortunately, there is a limit to ﬂuoride dose % remineralization and mean % lesion depth reduction were allowed in oral care products to avoid the risk of ﬂuorosis in 17 18,19 statistically signiﬁcantly greater than 0% (p < 0.0001). When children and toxicity in all ages. The ﬂuoride dose compared against each other, there was no statistically signiﬁcant recommended for toddlers and children is even lower than the difference in remineralization (p = 0.81), or in lesion depth regulatory 1000–1500 ppm ﬂuoride concentration in non- reduction (p = 0.68). The 95% conﬁdence interval of the difference prescription toothpastes, which is probably suboptimal for between Karex™ (HAP) and Elmex (AMF) for remineralization was effective remineralization of initial lesions; thus the effect of dose −8.8% to +6.5%, and the 95% conﬁdence interval of the limitation on ﬂuoride effectiveness may be more pronounced in difference for lesion depth reduction was −6.8% to +4.1%. toddlers and children below 6 years. Besides safety concerns, Therefore, in this study the HAP toothpaste is conﬁrmed to be higher ﬂuoride dose in remineralization materials results to non-inferior to the ﬂuoride toothpaste in effectiveness. surface-zone remineralization at the expense of the lesion body, On analysis of the sound tooth blocks that examined the ability thus preventing fuller and homogenous remineralization of the 15,16 of the two toothpastes to inhibit demineralization of sound tooth lesion. It is envisaged that an agent as effective as ﬂuoride, but surface, there was no evidence of demineralization in any of the can permit increasing dosage for increase effectiveness without tooth blocks following intra-oral exposure to either toothpaste safety concerns, may offer a more effective choice, especially for (Fig. 2a, b) and (Fig. 3a, b). Critical examination of the children. Hydroxyapatite, a bioactive and biocompatible material microradiograms from the lesion-bearing samples exposed to with wide applications in both medicine (e.g., bone substitute) HAP (Fig. 4a, b) and AMF (Fig. 5a, b) toothpastes in comparison and dentistry, is currently used in nanocrystaline or microcluster with their respective control microradiograms, shows that while forms in toothpaste and mouthrinses in varying concentrations for 23–25 HAP induced a more homogenous remineralization distributed caries prevention and remineralization. The equivalence (non- throughout the entire thickness of the subsurface lesion (Fig. 4b), inferiority) of 10% HAP in microcluster forms to 500 ppm ﬂuoride the remineralization induced by AMF was denser in the out half provided as AMF in remineralization of initial caries lesion and BDJ Open (2019) 5:18 Comparative efﬁcacy of a hydroxyapatite and a ﬂuoride toothpaste for. . . B.T. Amaechi et al. Fig. 2 Representative microradiographic images of sound tooth tissue before (a) and after (b) intra-oral exposure for demineralization while the research subject is using Karex Kid’s toothpaste (10% HAP microclusters). Fig. 3 Representative microradiographic images of sound tooth tissue before (a) and after (b) intra-oral exposure for demineralization while the research subject is using Elmex Kid’s toothpaste (500 ppm ﬂuoride as AMF). Fig. 4 Representative microradiographic images of enamel subsurface lesions (Initial caries lesions), before (a) and after (b) in situ remineralization via treatment with Karex Kid’s toothpaste (10% HAP microclusters). inhibition of sound enamel demineralization was investigated in in children and adolescents at high caries risk undergoing this study. The ﬁndings of the present in situ study accepted the orthodontic treatment also reported similar results and non- two hypotheses that each of the two toothpaste formulations inferiority of microcrystalline HAP to 1400 ppm ﬂuoride provided promotes remineralization and lesion depth reduction that as AMF and stannous ﬂuoride. Effectiveness of AMF in these is signiﬁcantly greater than zero, and that neither toothpaste is reports and this study is in agreement with the long-established inferior to the other with respect to promoting the remineraliza- fact that ﬂuoride in varying concentrations are effective in tion and inhibiting the development of initial caries lesions. preventing caries development and remineralizing initial car- 9,39,40 Although this study was conducted with human primary teeth, ies, and that the various ﬂuoride salts were equally 41 42 similar result was observed in a previous in situ study that used effective. Furthermore, Hellwig et al. in an in situ study human permanent teeth. A randomized controlled clinical trial demonstrated the remineralization of initial caries lesions of BDJ Open (2019) 5:18 Comparative efﬁcacy of a hydroxyapatite and a ﬂuoride toothpaste for. . . B.T. Amaechi et al. Fig. 5 Representative microradiographic images of enamel subsurface lesions (Initial caries lesions), before (a) and after (b) in situ remineralization via treatment with Elmex Kid’s toothpaste (500 ppm ﬂuoride as AMF). permanent teeth, and concluded that remineralization of primary laminations (surface zone remineralization) were apparent in − 55 teeth with ﬂuorides may be possible in the same way as the lesions subjected to the 250-ppm and 500-ppm F solutions. permanent teeth. The effectiveness of the HAP toothpaste in this Another in situ study demonstrated no signiﬁcant difference in study is in agreement with previous clinical and in vitro studies, the effectiveness of 500, 1000, and 1500 ppm ﬂuoride in and it is not surprising considering the various mechanisms remineralizing initial caries in primary teeth, further conﬁrming through which HAP has been demonstrated to effect reminer- the effect of lesion lamination. The ﬁndings of these previous 25,27,28,32,43–47 alization of initial caries. Based on its strong afﬁnity studies and the lesion lamination effect demonstrate that the 24,26,43 and adsorption to tooth surfaces, HAP has been shown to dose-dependent effect of ﬂuoride effectiveness that reﬂects as induce remineralization of initial caries lesions by directly ﬁlling increased effect of high ﬂuoride toothpaste or addition of 26,48 56 micropores in demineralized tooth surfaces, where it acts as a further ﬂuoride sources, has limit at which it plateaus and further crystal nucleus, and promotes crystal deposition and growth by increase may not improve the effectiveness. In contrast to the continuously attracting large amounts of calcium and phosphate effect of AMF, Fig. 4b shows that HAP induced a more 48,49 ions from the surrounding remineralization solution. homogenous remineralization distributed throughout the entire The absence of any evidence of demineralization in all the thickness of the subsurface lesion, and this may indicate that sound tooth blocks following intra-oral exposure to either HAP or increasing the dose of HAP or continued usage of the toothpaste AMF toothpaste further demonstrated the inhibition of deminer- may result to increased remineralization of the lesion, and alization by both toothpastes. The caries prevention potential of ultimately lead to complete or fuller remineralization of the initial 31,32 HAP, which has been established in previous studies, has been lesion. shown to be based on multiple mechanisms. HAP in toothpaste Based on above discussions one may suggest that HAP- has been reported to elevate calcium and phosphate ions containing toothpaste may be a better choice for children and concentrations in saliva, plaque and tooth surfaces; thus acting individuals at high caries risk since the dosage can be increased to as a calcium and phosphate reservoir, helping to maintain a obtain higher efﬁcacy without any safety issue such as the risk of topical state of supersaturation of these ions with respect to tooth ﬂuorosis in children associated with high ﬂuoride dose. Further- 43,51 minerals. The discussed mechanism must have applied in this more, the use of HAP in oral care products may eliminate the need study considering that the surface of the tooth blocks was covered of combining ﬂuoride and antimicrobials in a dentifrice, as well as with polyesther gauze, which encouraged and maintained plaque having different dosages for infants, children and adults. It is accumulation over the tooth surface, providing a nest for the logical but scientiﬁc that since the remineralizing efﬁcacy of accumulations of the mineral ions. The high potential of HAP to topical ﬂuorides is strictly dependent on the availability of calcium adsorb to bacterial cell wall has been shown to facilitate an and phosphate ions, HAP dentifrices may be a more effective for antibioﬁlm effect by inducing coaggregation of bacteria within the xerostomic patients with diminished amounts of saliva. This may HAP particles, thus aiding bioﬁlm removal from the tooth need to be conﬁrm through a clinical trial on patients suffering 52,53 29,46,47,54 surfaces, and hindering oral bioﬁlm formation. Again, from xerostomia. this mechanism may have contributed the present ﬁndings by In this study, there was no incidences of adverse effects limiting the virulence of the bioﬁlm on the surface of the tooth reported by subjects or ascertained clinically. Previous clinical blocks. studies observed similar ﬁndings, and reported there is no safety 27,32,57 The ﬁndings in this study further conﬁrmed the surface zone issue with HAP in oral care products. The fact that these 13,14,55 remineralization by the ﬂuoride agents. Two zones of studies investigated varying doses of HAP in toothpastes, and contrasting density can clearly be seen in Fig. 5b, showing the none reported any safety issue, conﬁrms that the dose of HAP in remineralization induced by AMF to be denser in the out half of oral care dentifrices can be safely increased for an increase the lesion (surface zone). Observation of such surface zone effectiveness when necessary. remineralization with only 500 ppm ﬂuoride present in the toothpaste used in this study actually demonstrated that this ‘lesion lamination’ effect is not limited to materials with high CONCLUSION ﬂuoride concentration, rather it is based on the established fact that ﬂuoride remineralization of initial lesions, irrespective of the concentration, is most effective at the outer 30 μm of the This study conﬁrmed hydroxyapatite toothpaste is equivalent 13,14,55 lesion. This is supported by the ﬁndings of a previous study, or non-inferior to the ﬂuoride toothpaste with respect to which reported that higher ﬂuoride concentrations did not remineralization of initial caries lesions and prevention of produce any further signiﬁcant increase in remineralization, rather carious lesion development. BDJ Open (2019) 5:18 Comparative efﬁcacy of a hydroxyapatite and a ﬂuoride toothpaste for. . . B.T. Amaechi et al. Future research should be large multicenter clinical trials to 25. Tschoppe, P., Zandim, D. L., Martus, P. & Kielbassa, A. M. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. J. Dent. 39, 430–437 further establish the effectiveness of HAP dentifrices and its (2011). equivalence to ﬂuoride. 26. Huang, S. B., Gao, S. S. & Yu, H. Y. Effect of nano-hydroxyapatite concentration on remineralization of initial enamel lesion in vitro. Biomed. Mater. 4, 034104 (2009). 27. Najibfard, K., Ramalingam, K., Chedjieu, I. & Amaechi, B. T. Remineralization of ACKNOWLEDGEMENTS early caries by nano-hydroxyapatite dentifrice. J. Clin. Dent. 22, 139–143 (2011). The authors would like to thank Michael Mader for his assistance with the statistical 28. Juntavee, N., Juntavee, A. & Plongniras, P. Remineralization potential of nano- analysis. hydroxyapatite on enamel and cementum surrounding margin of computer- aided design and computer-aided manufacturing ceramic restoration. Int J. Nanomed. 8, 2755–2765 (2018). ADDITIONAL INFORMATION 29. Kensche, A. et al. Efﬁcacy of a mouthrinse based on hydroxyapatite to reduce Competing interests: The authors declare no competing interests. initial bacterial colonisation in situ. Arch. Oral. Biol. 80,18–26 (2017). 30. Shimura, N. et al. Field study on the anticaries effect of toothpaste containing Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims hydroxyapatite (First Report). Shika Janaru 15, 213–218 (1982). in published maps and institutional afﬁliations. 31. Kani, T. et al. Effect of apatite-containing dentifrices on dental caries in school children. J. Dent. Hlth. 39, 104–109 (1989). 32. Schlagenhauf, U. et al. Impact of a non-ﬂuoridated microcrystalline hydro- xyapatite dentifrice on enamel caries progression in highly caries-susceptible REFERENCES orthodontic patients: a randomized, controlled 6-month trial. J. Investig. Clin. 1. Kassebaum, N. J. et al. 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