The Open Dentistry Journal




ISSN: 1874-2106 ― Volume 13, 2019
REVIEW ARTICLE

Modes of Action and Clinical Efficacy of Particulate Hydroxyapatite in Preventive Oral Health Care − State of the Art



Joachim Enax1, Helge-Otto Fabritius2, 3, Kathia Fabritius-Vilpoux3, Bennett T. Amaechi4, Frederic Meyer1, *
1 Department of Research, Dr. Kurt Wolff GmbH & Co. KG, Johanneswerkstr. 34-36, 33611 Bielefeld, Germany
2 Bionics and Materials Development, Hamm-Lippstadt University of Applied Sciences, Marker Allee 76-78, 59063 Hamm, Germany
3 Microstructure Physics and Alloy Design, Max-Planck-Institut für Eisenforschung GmbH, Max-Planck-Str. 1, 40237 Duesseldorf, Germany
4 Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, 703 Floyd Curl Drive, San Antonio, Texas 78229-3900, USA

Abstract

Background:

Particulate Hydroxyapatite (HAP; Ca5(PO4)3(OH)) is being increasingly used as multifunctional active ingredient in oral care. Due to its high similarity to human enamel crystallites, it is considered as a biomimetic agent.

Objective:

The aim of this narrative review is to identify the modes of action of HAP in preventive oral health care based on published studies. The outcomes are expected to improve the understanding of the effects of HAP in the oral cavity and to provide a knowledge base for future research in the field of biomimetic oral care.

Methods:

The data analyzed and discussed are primarily based on selected published scientific studies and reviews from in vivo, in situ, and in vitro studies on HAP in the field of preventive oral health care. The databases Cochrane Library, EBSCO, PubMed and SciFinder were used for literature search.

Results:

We identified different modes of action of HAP in the oral cavity. They are mainly based on (I) Physical principles (e.g. attachment of HAP-particles to the tooth surface and cleaning properties), (II) Bio-chemical principles (e.g. source of calcium and phosphate ions under acidic conditions and formation of an interface between HAP-particles and the enamel), and (III) Biological principles (e.g. HAP-particles interacting with microorganisms).

Conclusion:

Although more mechanistic studies are needed, published data show that HAP has multiple modes of action in the oral cavity. Since the effects address a wide range of oral health problems, HAP is a biomimetic agent with a broad range of applications in preventive oral health care.

Keywords: Teeth, Hydroxyapatite, Biomimetics, Caries, Periodontitis, Toothpaste.


Article Information


Identifiers and Pagination:

Year: 2019
Volume: 13
First Page: 274
Last Page: 287
Publisher Id: TODENTJ-13-274
DOI: 10.2174/1874210601913010274

Article History:

Received Date: 18/04/2019
Revision Received Date: 03/06/2019
Acceptance Date: 11/07/2019
Electronic publication date: 31/07/2019
Collection year: 2019

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© 2019 Enax et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Research, Dr. Kurt Wolff GmbH & Co. KG, Johanneswerkstr. 34-36, 33611 Bielefeld, Germany; Tel: +49 521 8808-6061; Fax: +49 521 8808-626064;
E-mail: frederic.meyer@drwolffgroup.com






1. BACKGROUND

1.1. Teeth

The mineral phase of human tooth enamel and dentin consists of Hydroxyapatite (HAP; Ca5(PO4)3(OH)), a crystal line calcium phosphate (Fig. 1). Enamel has an average HAP content of 97% while dentin is far less mineralized with an average HAP content of 70% [1Dorozhkin SV, Epple M. Biological and medical significance of calcium phosphates. Angew Chem Int Ed Engl 2002; 41(17): 3130-46.
[http://dx.doi.org/10.1002/1521-3773(20020902)41:17<3130::AID-ANIE3130>3.0.CO;2-1] [PMID: 12207375]
-6Meyer F, Amaechi BT, Fabritius HO, Enax J. Overview of calcium phosphates used in biomimetic oral care. Open Dent J 2018; 12: 406-23.
[http://dx.doi.org/10.2174/1874210601812010406] [PMID: 29988215]
]. From an evolutionary point of view, tooth enamel consisting of HAP is an ancestral character present in the large majority of vertebrate groups. The only notable exception are sharks and a small number of bony fishes whose teeth are covered with enamel-like enameloid consisting of fluoroapatite (Ca5(PO4)3F) [4Lowenstam HA, Weiner S. On Biomineralization 1989., 7Enax J, Prymak O, Raabe D, Epple M. Structure, composition, and mechanical properties of shark teeth. J Struct Biol 2012; 178(3): 290-9.
[http://dx.doi.org/10.1016/j.jsb.2012.03.012] [PMID: 22503701]
, 8Enax J, Janus AM, Raabe D, Epple M, Fabritius HO. Ultrastructural organization and micromechanical properties of shark tooth enameloid. Acta Biomater 2014; 10(9): 3959-68.
[http://dx.doi.org/10.1016/j.actbio.2014.04.028] [PMID: 24797528]
]. Under healthy conditions, human tooth enamel is already fully mineralized when a tooth erupts [9Moll K-J, Moll M. Kurzlehrbuch Anatomie 2000.]. The mineralization of enamel is a highly complex process, where ameloblasts, the enamel-building cells, interact with calcium and phosphate ions building the HAP-crystals and finally the enamel prisms [9Moll K-J, Moll M. Kurzlehrbuch Anatomie 2000.-11Moradian-Oldak J. The regeneration of tooth enamel. Dimens Dent Hyg 2009; 7(8): 12-5.
[PMID: 20651953]
]. Enamel is a tissue that cannot be rebuilt naturally [11Moradian-Oldak J. The regeneration of tooth enamel. Dimens Dent Hyg 2009; 7(8): 12-5.
[PMID: 20651953]
]. Teeth are subjected to constant mechanical and chemical wear often accompanied by various clinical conditions during their service. Both phenomena are promoted to different extents by locally varying dietary habits [12Fejerskov O, Kidd E. Dental caries: The disease and its clinical management 2009., 13Petersen PE, Bourgeois D, Ogawa H, Estupinan-Day S, Ndiaye C. The global burden of oral diseases and risks to oral health. Bull World Health Organ 2005; 83(9): 661-9.
[PMID: 16211157]
]. In consequence, almost the whole world population is affected by negative enamel and dentin conditions with the major factors being excessive erosion and caries [12Fejerskov O, Kidd E. Dental caries: The disease and its clinical management 2009., 14Lussi A, Ganss C. Erosive Tooth Wear: From Diagnosis to Therapy 2014.
[http://dx.doi.org/10.1159/isbn.978-3-318-02553-8]
].


Fig. (1)
Schematic depiction of the hexagonal HAP crystal structure (Ca5(PO4)3(OH)). Legend: Yellow=OH-; red=Ca2+, blue/white=PO43-; modified after Lu et al. [37Lu X, Zhang H, Guo Y, Wang Y, Ge X, Leng Y, et al. Hexagonal hydroxyapatite formation on TiO2 nanotubes under urea modulation. CrystEngComm 2011; 13: 3741-9.
[http://dx.doi.org/10.1039/c0ce00971g]
].


1.2. Hydroxyapatite in Oral Care

Contemporary everyday oral care strategies focus on the protection and preservation of enamel and dentin, mainly by using fluorides [15Lussi A, Hellwig E, Klimek J. Fluorides - mode of action and recommendations for use. Schweiz Monatsschr Zahnmed 2012; 122(11): 1030-42.
[PMID: 23192605]
-17Walsh T, Worthington HV, Glenny AM, Appelbe P, Marinho VC, Shi X. Fluoride toothpastes of different concentrations for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2010; (1): CD007868
[http://dx.doi.org/10.1002/14651858.CD007868.pub2] [PMID: 20091655]
]. In the field of clinical dentistry, tooth repair and replacement are relying on ceramics, composites, polymers, and other materials [18Craig RG, Welker D, Rothaut J, Krumbholz KG, Stefan K, Dermann K, et al. Dental materials 2006.
[http://dx.doi.org/10.1002/14356007.a08_251.pub2]
, 19Lübke A, Enax J, Wey K, Fabritius HO, Raabe D, Epple M. Composites of fluoroapatite and methylmethacrylate-based polymers (PMMA) for biomimetic tooth replacement. Bioinspir Biomim 2016; 11(3)035001
[http://dx.doi.org/10.1088/1748-3190/11/3/035001] [PMID: 27159 921]
]. Despite a constant increase both in the treatment as well as financial efforts, the problems with dental health still prevail [20Kassebaum NJ, Bernabé E, Dahiya M, Bhandari B, Murray CJ, Marcenes W. Global burden of untreated caries: A systematic review and metaregression. J Dent Res 2015; 94(5): 650-8.
[http://dx.doi.org/10.1177/0022034515573272] [PMID: 25740856]
, 21Kassebaum NJ, Smith AGC, Bernabé E, et al. Global, regional, and national prevalence, incidence, and disability-adjusted life years for oral conditions for 195 countries, 1990-2015: A systematic analysis for the global burden of diseases, injuries, and risk factors. J Dent Res 2017; 96(4): 380-7.
[http://dx.doi.org/10.1177/0022034517693566] [PMID: 28792274]
]. Therefore, the demand for alternative and complementary treatment strategies is on the rise and researchers are increasingly investigating new approaches [3Enax J, Epple M. Synthetic hydroxyapatite as a biomimetic oral care agent. Oral Health Prev Dent 2018; 16(1): 7-19.
[PMID: 29335686]
, 6Meyer F, Amaechi BT, Fabritius HO, Enax J. Overview of calcium phosphates used in biomimetic oral care. Open Dent J 2018; 12: 406-23.
[http://dx.doi.org/10.2174/1874210601812010406] [PMID: 29988215]
, 22Hannig M, Hannig C. Nanomaterials in preventive dentistry. Nat Nanotechnol 2010; 5(8): 565-9.
[http://dx.doi.org/10.1038/nnano.2010.83] [PMID: 20581832]
-30Meyer F, Enax J. Hydroxyapatite in oral biofilm management. Eur J Dent (in press)]. One strategy focuses on the analysis of bioinspired concepts that rely on mimicking the natural constituents of human teeth like crystalline calcium phosphate minerals [3Enax J, Epple M. Synthetic hydroxyapatite as a biomimetic oral care agent. Oral Health Prev Dent 2018; 16(1): 7-19.
[PMID: 29335686]
, 6Meyer F, Amaechi BT, Fabritius HO, Enax J. Overview of calcium phosphates used in biomimetic oral care. Open Dent J 2018; 12: 406-23.
[http://dx.doi.org/10.2174/1874210601812010406] [PMID: 29988215]
, 22Hannig M, Hannig C. Nanomaterials in preventive dentistry. Nat Nanotechnol 2010; 5(8): 565-9.
[http://dx.doi.org/10.1038/nnano.2010.83] [PMID: 20581832]
-26Schlagenhauf U, Kunzelmann KH, Hannig C, et al. Impact of a non-fluoridated microcrystalline hydroxyapatite dentifrice on enamel caries progression in highly caries-susceptible orthodontic patients: A randomized, controlled 6-month trial. J Investig Clin Dent 2019; 10(2)e12399
[http://dx.doi.org/10.1111/jicd.12399] [PMID: 30701704]
, 31Meyer F, Enax J. Early childhood caries: Epidemiology, aetiology, and prevention. Int J Dent 2018; 20181415873
[http://dx.doi.org/10.1155/2018/1415873] [PMID: 29951094]
]. Among the group of these calcium phosphates, HAP is the most extensively investigated in the field of oral care e.g. [1Dorozhkin SV, Epple M. Biological and medical significance of calcium phosphates. Angew Chem Int Ed Engl 2002; 41(17): 3130-46.
[http://dx.doi.org/10.1002/1521-3773(20020902)41:17<3130::AID-ANIE3130>3.0.CO;2-1] [PMID: 12207375]
, 3Enax J, Epple M. Synthetic hydroxyapatite as a biomimetic oral care agent. Oral Health Prev Dent 2018; 16(1): 7-19.
[PMID: 29335686]
, 6Meyer F, Amaechi BT, Fabritius HO, Enax J. Overview of calcium phosphates used in biomimetic oral care. Open Dent J 2018; 12: 406-23.
[http://dx.doi.org/10.2174/1874210601812010406] [PMID: 29988215]
, 26Schlagenhauf U, Kunzelmann KH, Hannig C, et al. Impact of a non-fluoridated microcrystalline hydroxyapatite dentifrice on enamel caries progression in highly caries-susceptible orthodontic patients: A randomized, controlled 6-month trial. J Investig Clin Dent 2019; 10(2)e12399
[http://dx.doi.org/10.1111/jicd.12399] [PMID: 30701704]
, 27Hu M-L, Zheng G, Zhang Y-D, Yan X, Li X-C, Lin H. Effect of desensitizing toothpastes on dentine hypersensitivity: A systematic review and meta-analysis. J Dent 2018; 75: 12-21.
[http://dx.doi.org/10.1016/j.jdent.2018.05.012] [PMID: 29787782]
, 32Loveren Cv. Toothpastes 2013.
[http://dx.doi.org/10.1159/isbn.978-3-318-02207-0]
]. Dorozhkin & Epple [1Dorozhkin SV, Epple M. Biological and medical significance of calcium phosphates. Angew Chem Int Ed Engl 2002; 41(17): 3130-46.
[http://dx.doi.org/10.1002/1521-3773(20020902)41:17<3130::AID-ANIE3130>3.0.CO;2-1] [PMID: 12207375]
] and Enax & Epple [3Enax J, Epple M. Synthetic hydroxyapatite as a biomimetic oral care agent. Oral Health Prev Dent 2018; 16(1): 7-19.
[PMID: 29335686]
], for example, describe the synthesis routes and characterization of various non-biogenic apatites. In addition to synthetic HAP, HAP from natural sources (e.g. bovine bone as well as fish bone and scales) can be used for medical applications [33Granito RN, Muniz Renno AC, Yamamura H, de Almeida MC, Menin Ruiz PL, Ribeiro DA. Hydroxyapatite from fish for bone tissue engineering: A promising approach. Int J Mol Cell Med 2018; 7(2): 80-90.
[PMID: 30276163]
].

Several studies show the abilities of HAP to remineralize early caries lesions [3Enax J, Epple M. Synthetic hydroxyapatite as a biomimetic oral care agent. Oral Health Prev Dent 2018; 16(1): 7-19.
[PMID: 29335686]
, 6Meyer F, Amaechi BT, Fabritius HO, Enax J. Overview of calcium phosphates used in biomimetic oral care. Open Dent J 2018; 12: 406-23.
[http://dx.doi.org/10.2174/1874210601812010406] [PMID: 29988215]
, 24Fabritius-Vilpoux K, Enax J, Herbig M, Raabe D, Fabritius H-O. Quantitative affinity parameters of synthetic hydroxyapatite and enamel surfaces in vitro. Bioinspir Biomim Nan 2019; 0(0): 1-40.
[http://dx.doi.org/10.1680/jbibn.18.00035]
, 26Schlagenhauf U, Kunzelmann KH, Hannig C, et al. Impact of a non-fluoridated microcrystalline hydroxyapatite dentifrice on enamel caries progression in highly caries-susceptible orthodontic patients: A randomized, controlled 6-month trial. J Investig Clin Dent 2019; 10(2)e12399
[http://dx.doi.org/10.1111/jicd.12399] [PMID: 30701704]
, 34Kani K, Kani M, Isozaki A, Shintani H, Ohashi T, Tokumoto T. Effect of apatite-containing dentifrices on dental caries in school children. J Dent Health 1989; 19: 104-9.
[http://dx.doi.org/10.5834/jdh.39.104]
, 35Najibfard K, Ramalingam K, Chedjieu I, Amaechi BT. Remineralization of early caries by a nano-hydroxyapatite dentifrice. J Clin Dent 2011; 22(5): 139-43.
[PMID: 22403978]
]. The effectiveness of HAP as active ingredient is not limited by the quantity of calcium and phosphate present in saliva, since HAP can intrinsically act as source for these ions. This as well as its high chemical and structural similarity to natural enamel crystallites [1Dorozhkin SV, Epple M. Biological and medical significance of calcium phosphates. Angew Chem Int Ed Engl 2002; 41(17): 3130-46.
[http://dx.doi.org/10.1002/1521-3773(20020902)41:17<3130::AID-ANIE3130>3.0.CO;2-1] [PMID: 12207375]
, 3Enax J, Epple M. Synthetic hydroxyapatite as a biomimetic oral care agent. Oral Health Prev Dent 2018; 16(1): 7-19.
[PMID: 29335686]
, 24Fabritius-Vilpoux K, Enax J, Herbig M, Raabe D, Fabritius H-O. Quantitative affinity parameters of synthetic hydroxyapatite and enamel surfaces in vitro. Bioinspir Biomim Nan 2019; 0(0): 1-40.
[http://dx.doi.org/10.1680/jbibn.18.00035]
, 36Roveri N, Battistella E, Foltran I, Foresti E, Iafisco M, Lelli M, et al. Synthetic biomimetic carbonate-hydroxyapatite nanocrystals for enamel remineralization. Adv Mat Res 2008; 47-50: 821-4.
[http://dx.doi.org/10.4028/www.scientific.net/AMR.47-50.821]
, 37Lu X, Zhang H, Guo Y, Wang Y, Ge X, Leng Y, et al. Hexagonal hydroxyapatite formation on TiO2 nanotubes under urea modulation. CrystEngComm 2011; 13: 3741-9.
[http://dx.doi.org/10.1039/c0ce00971g]
] qualifies HAP as a promising preventive oral health care agent.

The similarity to human enamel and dentin crystallites is the reason why particulate HAP is already in use as a biomimetic agent in oral care formulations [3Enax J, Epple M. Synthetic hydroxyapatite as a biomimetic oral care agent. Oral Health Prev Dent 2018; 16(1): 7-19.
[PMID: 29335686]
, 6Meyer F, Amaechi BT, Fabritius HO, Enax J. Overview of calcium phosphates used in biomimetic oral care. Open Dent J 2018; 12: 406-23.
[http://dx.doi.org/10.2174/1874210601812010406] [PMID: 29988215]
, 22Hannig M, Hannig C. Nanomaterials in preventive dentistry. Nat Nanotechnol 2010; 5(8): 565-9.
[http://dx.doi.org/10.1038/nnano.2010.83] [PMID: 20581832]
, 31Meyer F, Enax J. Early childhood caries: Epidemiology, aetiology, and prevention. Int J Dent 2018; 20181415873
[http://dx.doi.org/10.1155/2018/1415873] [PMID: 29951094]
, 32Loveren Cv. Toothpastes 2013.
[http://dx.doi.org/10.1159/isbn.978-3-318-02207-0]
, 38Gillam DG. Dentine hypersensitivity: Advances in diagnosis, management, and treatment 2015., 39Daculsi G, Kerebel B. High-resolution electron microscope study of human enamel crystallites: size, shape, and growth. J Ultrastruct Res 1978; 65(2): 163-72.
[http://dx.doi.org/10.1016/S0022-5320(78)90053-9] [PMID: 731784]
]. Several clinical trials have demonstrated the effectiveness of HAP-toothpastes regarding plaque reduction, improvement of periodontal health, and caries prophylaxis among caries-risk individuals [25Harks I, Jockel-Schneider Y, Schlagenhauf U, et al. Impact of the daily use of a microcrystal hydroxyapatite dentifrice on de novo plaque formation and clinical/microbiological parameters of periodontal health. A randomized trial. PLoS One 2016; 11(7)e0160142
[http://dx.doi.org/10.1371/journal.pone.0160142] [PMID: 27467683]
, 26Schlagenhauf U, Kunzelmann KH, Hannig C, et al. Impact of a non-fluoridated microcrystalline hydroxyapatite dentifrice on enamel caries progression in highly caries-susceptible orthodontic patients: A randomized, controlled 6-month trial. J Investig Clin Dent 2019; 10(2)e12399
[http://dx.doi.org/10.1111/jicd.12399] [PMID: 30701704]
, 34Kani K, Kani M, Isozaki A, Shintani H, Ohashi T, Tokumoto T. Effect of apatite-containing dentifrices on dental caries in school children. J Dent Health 1989; 19: 104-9.
[http://dx.doi.org/10.5834/jdh.39.104]
, 40Cosola S, Marconcini S, Giammarinaro E, Marchisio O, Lelli M, Roveri N, et al. Antimicrobial efficacy of mouthwashes containing zinc-substituted nanohydroxyapatite and zinc L-pyrrolidone carboxylate on suture threads after surgical procedures. J Oral Sci Rehabil 2017; 3(4): 24-30., 41Hegazy SA, Salama IR. Antiplaque and remineralizing effects of Biorepair mouthwash: A comparative clinical trial. Pediatr Dent J 2016; 26: 89-94.
[http://dx.doi.org/10.1016/j.pdj.2016.05.002]
]. Hu et al. recently showed in a meta-analysis the clinical evidence of HAP with reducing dentin hypersensitivity [27Hu M-L, Zheng G, Zhang Y-D, Yan X, Li X-C, Lin H. Effect of desensitizing toothpastes on dentine hypersensitivity: A systematic review and meta-analysis. J Dent 2018; 75: 12-21.
[http://dx.doi.org/10.1016/j.jdent.2018.05.012] [PMID: 29787782]
]. Compositional and structural accordance with tooth minerals confers an excellent biocompatibility to HAP [1Dorozhkin SV, Epple M. Biological and medical significance of calcium phosphates. Angew Chem Int Ed Engl 2002; 41(17): 3130-46.
[http://dx.doi.org/10.1002/1521-3773(20020902)41:17<3130::AID-ANIE3130>3.0.CO;2-1] [PMID: 12207375]
, 24Fabritius-Vilpoux K, Enax J, Herbig M, Raabe D, Fabritius H-O. Quantitative affinity parameters of synthetic hydroxyapatite and enamel surfaces in vitro. Bioinspir Biomim Nan 2019; 0(0): 1-40.
[http://dx.doi.org/10.1680/jbibn.18.00035]
, 42Epple M. Review of potential health risks associated with nanoscopic calcium phosphate. Acta Biomater 2018; 77: 1-14.
[http://dx.doi.org/10.1016/j.actbio.2018.07.036] [PMID: 30031162]
, 43Ramis J, Coelho C, Córdoba A, Quadros P, Monjo M. Safety assessment of nano-hydroxyapatite as an oral care ingredient according to the EU cosmetics regulation. Cosmetics 2018; 5(3): 53.
[http://dx.doi.org/10.3390/cosmetics5030053]
]. Consequently, the applicable dosage of HAP is not quantitatively restricted, and no fluorosis-risk is present [31Meyer F, Enax J. Early childhood caries: Epidemiology, aetiology, and prevention. Int J Dent 2018; 20181415873
[http://dx.doi.org/10.1155/2018/1415873] [PMID: 29951094]
, 44Limeback H, Robinson C. Fluoride therapy Comprehensive preventive dentistry 2012; 251-82.
[http://dx.doi.org/10.1002/9781118703762]
, 45Limeback H. Comprehensive preventive dentistry 2012.
[http://dx.doi.org/10.1002/9781118703762]
]. Therefore, HAP as active ingredient may be especially suitable for individuals where high fluoride exposure is not recommended such as children (risk of swallowing), pregnant women, and individuals suf fering from hyposalivation (i.e. lack of calcium and phosphate ions for remineralization) [31Meyer F, Enax J. Early childhood caries: Epidemiology, aetiology, and prevention. Int J Dent 2018; 20181415873
[http://dx.doi.org/10.1155/2018/1415873] [PMID: 29951094]
, 35Najibfard K, Ramalingam K, Chedjieu I, Amaechi BT. Remineralization of early caries by a nano-hydroxyapatite dentifrice. J Clin Dent 2011; 22(5): 139-43.
[PMID: 22403978]
, 46Bashash M, Thomas D, Hu H, et al. Prenatal fluoride exposure and cognitive outcomes in children at 4 and 6-12 years of age in Mexico. Environ Health Perspect 2017; 125(9)097017
[http://dx.doi.org/10.1289/EHP655] [PMID: 28937959]
]. Another important factor is the ability of HAP-particles to interact with enamel and dentin surfaces (Fig. 2) [23Kensche A, Holder C, Basche S, Tahan N, Hannig C, Hannig M. Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ. Arch Oral Biol 2017; 80: 18-26.
[http://dx.doi.org/10.1016/j.archoralbio.2017.03.013] [PMID: 2836 4672]
, 24Fabritius-Vilpoux K, Enax J, Herbig M, Raabe D, Fabritius H-O. Quantitative affinity parameters of synthetic hydroxyapatite and enamel surfaces in vitro. Bioinspir Biomim Nan 2019; 0(0): 1-40.
[http://dx.doi.org/10.1680/jbibn.18.00035]
, 47Hannig C, Basche S, Burghardt T, Al-Ahmad A, Hannig M. Influence of a mouthwash containing hydroxyapatite microclusters on bacterial adherence in situ. Clin Oral Investig 2013; 17(3): 805-14.
[http://dx.doi.org/10.1007/s00784-012-0781-6] [PMID: 22782257]
, 48Lelli M, Putignano A, Marchetti M, et al. Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: A comparative in vivo study. Front Physiol 2014; 5: 333.
[http://dx.doi.org/10.3389/fphys.2014.00333] [PMID: 25249980]
].

Despite this wide range of applications in preventive oral health care, the exact modes of action of HAP in some areas remain quite elusive. References indicate that key effects are related to the ability of HAP to bio-chemically interact with tooth tissue as well as intrinsic and extrinsic substances present in the oral cavity, such as salivary components or microorganisms. Other effects seem to be more related to the particulate delivery form. In either case, understanding HAP’s mode of action is very important to exploit the full potential of HAP to improve its efficacy in biomimetic oral care formulations and to enable effective evidence-based oral hygiene recommendations for patients.

1.3. Aims and Literature Search Strategy

To date, a review that correlates the various described effects of HAP on different oral/tooth tissues (enamel, dentin, and gingiva) and oral diseases with the underlying modes of action has, to our knowledge, not been presented yet. Therefore, the aim of this narrative review is to analyze published scientific studies related to the effects of HAP in the clinical management of different oral and dental diseases. Based on the numerous studies recently reviewed by Meyer et al. and Enax & Epple [3Enax J, Epple M. Synthetic hydroxyapatite as a biomimetic oral care agent. Oral Health Prev Dent 2018; 16(1): 7-19.
[PMID: 29335686]
, 6Meyer F, Amaechi BT, Fabritius HO, Enax J. Overview of calcium phosphates used in biomimetic oral care. Open Dent J 2018; 12: 406-23.
[http://dx.doi.org/10.2174/1874210601812010406] [PMID: 29988215]
] as well as a further comprehensive literature search, the authors develop hypotheses about the modes of action of HAP used in oral care formulations with a focus on the prevention of caries, periodontitis, dental erosion, and dentin hypersensitivity. Cochrane Library, EBSCO, PubMed, and SciFinder were screened for relevant studies. Full text search terms were: hydroxyapatite AND (in vitro study OR in situ study OR in vivo study OR clinical study AND bacteria OR plaque OR biofilm OR caries OR periodontitis OR remineralization) AND (toothpaste OR dentifrice OR mouth rinse OR mouthwash) (Fig. 3).

Fig. (2)
Scanning electron micrographs showing the attachment of synthetic HAP particles to the tooth surface in vitro (the hydroxyapatite phase of the particles was confirmed by X-ray powder diffraction). (A) Overview of attached synthetic HAP particles (arrows) as used in various oral care applications on clean bovine enamel substrate. (B) Synthetic HAP particles (arrows) attached to the surface of bovine teeth including enamel lesions and pellicle. (C) High magnification at the dentin-enamel junction shows a mineral–mineral interphase between HAP crystallites from synthetic particles and enamel. (D) Synthetic HAP particles attached to polished dentin surface including open tubules where particles (arrows) can be observed inside the open tubules. For more details see Fabritius-Vilpoux et al. [24Fabritius-Vilpoux K, Enax J, Herbig M, Raabe D, Fabritius H-O. Quantitative affinity parameters of synthetic hydroxyapatite and enamel surfaces in vitro. Bioinspir Biomim Nan 2019; 0(0): 1-40.
[http://dx.doi.org/10.1680/jbibn.18.00035]
].


Fig. (3)
Literature search strategy for this review was adopted from two recent papers by Enax and Epple [3Enax J, Epple M. Synthetic hydroxyapatite as a biomimetic oral care agent. Oral Health Prev Dent 2018; 16(1): 7-19.
[PMID: 29335686]
] and Meyer et al. [6Meyer F, Amaechi BT, Fabritius HO, Enax J. Overview of calcium phosphates used in biomimetic oral care. Open Dent J 2018; 12: 406-23.
[http://dx.doi.org/10.2174/1874210601812010406] [PMID: 29988215]
]. Additionally, Cochrane Library, EBSCO, PubMed, and SciFinder were screened for further relevant studies using the search terms shown above.


We correlate the objectives of the studies with proposed mechanisms of HAP in preventive oral health care (e.g. prevention of caries, periodontitis, dental erosion, dentin hypersensitivity) and conclude presumable modes of action based on physical, bio-chemical, and biological principles as well as combinations of these.

2. SHORT OVERVIEW OF PREVALENT ORAL DISEASES THAT ARE AFFECTED BY ORAL HYGIENE HABITS

The oral cavity is a highly complex environment that can be affected by a variety of diseases, some of which are highly prevalent among our society. The most predominant of which are dental caries, periodontitis, erosion, and dentin hypersensitivity. All of these have in common that they can be prevented up to a certain level without clinical intervention, namely by oral hygiene habits, reduced sugar (and acid) diet, smoking cessation, and stress-free life style.

2.1. Caries

Caries is a disease that affects nearly every individual at least once in their life time and has the highest prevalence among all diseases that are known worldwide [49Vos T, Abajobir AA, Abate KH, Abbafati C, Abbas KM, Abd-Allah F, et al. GBD 2016 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: A systematic analysis for the Global Burden of Disease Study 2016. Lancet 2017; 390(10100): 1211-59.
[http://dx.doi.org/10.1016/S0140-6736(17)32154-2] [PMID: 2891 9117]
]. Even though its prevalence has declined in some parts of the world over the last years, it is still prevalent in all age groups and its treatment remains the highest unmet need in the world [20Kassebaum NJ, Bernabé E, Dahiya M, Bhandari B, Murray CJ, Marcenes W. Global burden of untreated caries: A systematic review and metaregression. J Dent Res 2015; 94(5): 650-8.
[http://dx.doi.org/10.1177/0022034515573272] [PMID: 25740856]
, 31Meyer F, Enax J. Early childhood caries: Epidemiology, aetiology, and prevention. Int J Dent 2018; 20181415873
[http://dx.doi.org/10.1155/2018/1415873] [PMID: 29951094]
, 50Frencken JE, Sharma P, Stenhouse L, Green D, Laverty D, Dietrich T. Global epidemiology of dental caries and severe periodontitis - A comprehensive review. J Clin Periodontol 2017; 44(Suppl. 18): S94-S105.
[http://dx.doi.org/10.1111/jcpe.12677] [PMID: 28266116]
, 51Meyer F, Karch A, Schlinkmann KM, et al. Sociodemographic determinants of spatial disparities in early childhood caries: An ecological analysis in Braunschweig, Germany. Community Dent Oral Epidemiol 2017; 45(5): 442-8.
[http://dx.doi.org/10.1111/cdoe.12308] [PMID: 28547864]
]. Untreated caries affects more than 2.4 billion people around the world [20Kassebaum NJ, Bernabé E, Dahiya M, Bhandari B, Murray CJ, Marcenes W. Global burden of untreated caries: A systematic review and metaregression. J Dent Res 2015; 94(5): 650-8.
[http://dx.doi.org/10.1177/0022034515573272] [PMID: 25740856]
]. Caries is a biofilm-driven disease where the biofilm switches from homeostasis to dysbiosis [52Kilian M, Chapple ILC, Hannig M, et al. The oral microbiome - An update for oral healthcare professionals. Br Dent J 2016; 221(10): 657-66.
[http://dx.doi.org/10.1038/sj.bdj.2016.865] [PMID: 27857087]
]. Microorganisms that form biofilms on tooth surfaces metabolize sugars as energy-source, producing organic acids, mostly lactic acid, as end-products [53Takahashi N, Nyvad B. The role of bacteria in the caries process: ecological perspectives. J Dent Res 2011; 90(3): 294-303.
[http://dx.doi.org/10.1177/0022034510379602] [PMID: 20924061]
, 54Sanz M, Beighton D, Curtis MA, et al. Role of microbial biofilms in the maintenance of oral health and in the development of dental caries and periodontal diseases. Consensus report of group 1 of the Joint EFP/ORCA workshop on the boundaries between caries and periodontal disease. J Clin Periodontol 2017; 44(Suppl. 18): S5-S11.
[http://dx.doi.org/10.1111/jcpe.12682] [PMID: 28266109]
]. By locally lowering the pH within the biofilm on the tooth surface, these acids are able to demineralize the underlying tooth tissue [1Dorozhkin SV, Epple M. Biological and medical significance of calcium phosphates. Angew Chem Int Ed Engl 2002; 41(17): 3130-46.
[http://dx.doi.org/10.1002/1521-3773(20020902)41:17<3130::AID-ANIE3130>3.0.CO;2-1] [PMID: 12207375]
, 55Lingström P, van Ruyven FO, van Houte J, Kent R. The pH of dental plaque in its relation to early enamel caries and dental plaque flora in humans. J Dent Res 2000; 79(2): 770-7.
[http://dx.doi.org/10.1177/00220345000790021101] [PMID: 10728 979]
]. This process is slowed down to individually different extents by the natural remineralization process, the rate and the extent of which depend on the availability of free calcium and phosphates from saliva and extrinsic sources. Local dissolution/recrystallization processes of the enamel surface on the microscopic scale caused by local concentration gradients may be an additional reason for superficial remineralization phenomena.

2.2. Periodontitis

Periodontitis is an inflammatory disease affecting the whole periodontium. In most cases periodontitis will start with gum inflammation and bleeding, also known as gingivitis [56Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. Lancet 2005; 366(9499): 1809-20.
[http://dx.doi.org/10.1016/S0140-6736(05)67728-8] [PMID: 16298 220]
]. Periodontitis can affect up to 90% of the population, while the severe form of this disease can be detected within up to 20% of some populations [50Frencken JE, Sharma P, Stenhouse L, Green D, Laverty D, Dietrich T. Global epidemiology of dental caries and severe periodontitis - A comprehensive review. J Clin Periodontol 2017; 44(Suppl. 18): S94-S105.
[http://dx.doi.org/10.1111/jcpe.12677] [PMID: 28266116]
, 56Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. Lancet 2005; 366(9499): 1809-20.
[http://dx.doi.org/10.1016/S0140-6736(05)67728-8] [PMID: 16298 220]
]. Periodontal disease is one of the most prevalent diseases in the world (rank 11 in 2016) [21Kassebaum NJ, Smith AGC, Bernabé E, et al. Global, regional, and national prevalence, incidence, and disability-adjusted life years for oral conditions for 195 countries, 1990-2015: A systematic analysis for the global burden of diseases, injuries, and risk factors. J Dent Res 2017; 96(4): 380-7.
[http://dx.doi.org/10.1177/0022034517693566] [PMID: 28792274]
, 49Vos T, Abajobir AA, Abate KH, Abbafati C, Abbas KM, Abd-Allah F, et al. GBD 2016 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: A systematic analysis for the Global Burden of Disease Study 2016. Lancet 2017; 390(10100): 1211-59.
[http://dx.doi.org/10.1016/S0140-6736(17)32154-2] [PMID: 2891 9117]
, 57Tonetti MS, Jepsen S, Jin L, Otomo-Corgel J. Impact of the global burden of periodontal diseases on health, nutrition and wellbeing of mankind: A call for global action. J Clin Periodontol 2017; 44(5): 456-62.
[http://dx.doi.org/10.1111/jcpe.12732] [PMID: 28419559]
]. The incidence of periodontitis increases with the age, as teeth nowadays remain longer in the oral cavity [21Kassebaum NJ, Smith AGC, Bernabé E, et al. Global, regional, and national prevalence, incidence, and disability-adjusted life years for oral conditions for 195 countries, 1990-2015: A systematic analysis for the global burden of diseases, injuries, and risk factors. J Dent Res 2017; 96(4): 380-7.
[http://dx.doi.org/10.1177/0022034517693566] [PMID: 28792274]
]. Periodontal disease is caused by a dysbiotic state of the dental plaque and can be triggered by a suppressed immune response or genetic factors [58Meuric V, Le Gall-David S, Boyer E, et al. Signature of microbial dysbiosis in periodontitis. Appl Environ Microbiol 2017; 83(14): e00462-17.
[http://dx.doi.org/10.1128/AEM.00462-17] [PMID: 28476771]
-60Cardoso EM, Reis C, Manzanares-Céspedes MC. Chronic periodontitis, inflammatory cytokines, and interrelationship with other chronic diseases. Postgrad Med 2018; 130(1): 98-104.
[http://dx.doi.org/10.1080/00325481.2018.1396876] [PMID: 2906 5749]
]. Traditional agents for prevention of this inflammatory disease focus on antimicrobial effects [61Marsh PD, Head DA, Devine DA. Ecological approaches to oral biofilms: Control without killing. Caries Res 2015; 49 (Suppl.1): 46-54.
[http://dx.doi.org/10.1159/000377732] [PMID: 25871418]
, 62Meyer F, Enax J. Die Mundhöhle als Ökosystem. Biol Unserer Zeit 2018; 48(1): 62-8.
[http://dx.doi.org/10.1002/biuz.201810641]
]. Frequently used oral care products are, for example, based on chlorhexidine as well as stannous and zinc salts [30Meyer F, Enax J. Hydroxyapatite in oral biofilm management. Eur J Dent (in press), 62Meyer F, Enax J. Die Mundhöhle als Ökosystem. Biol Unserer Zeit 2018; 48(1): 62-8.
[http://dx.doi.org/10.1002/biuz.201810641]
, 63Marsh PD. Contemporary perspective on plaque control. Br Dent J 2012; 212(12): 601-6.
[http://dx.doi.org/10.1038/sj.bdj.2012.524] [PMID: 22722123]
]. All of these antimicrobial agents might lead to a dysbiosis of the oral microbiota. In contrast to that, the modern approach in preventing periodontitis is focused on keeping the ecological balance of the microbiota: Not killing but controlling the harmful microorganisms [61Marsh PD, Head DA, Devine DA. Ecological approaches to oral biofilms: Control without killing. Caries Res 2015; 49 (Suppl.1): 46-54.
[http://dx.doi.org/10.1159/000377732] [PMID: 25871418]
, 64Marsh PD. In sickness and in health-what does the oral microbiome mean to us? An ecological perspective. Adv Dent Res 2018; 29(1): 60-5.
[http://dx.doi.org/10.1177/0022034517735295] [PMID: 29355410]
].

2.3. Erosion

Dental erosion, otherwise known as erosive tooth wear, is a dissolution primarily of the enamel caused by acids of non-bacterial origin, both from extrinsic or intrinsic sources such as seen with patients suffering from gastroesophageal disease or bulimia [14Lussi A, Ganss C. Erosive Tooth Wear: From Diagnosis to Therapy 2014.
[http://dx.doi.org/10.1159/isbn.978-3-318-02553-8]
, 65Amaechi BT. Dental Erosion and Its Clinical Management 2015.
[http://dx.doi.org/10.1007/978-3-319-13993-7]
-67Bretz WA. Oral profiles of bulimic women: Diagnosis and management. What is the evidence? J Evid Based Dent Pract 2002; 2(4): 267-72.
[http://dx.doi.org/10.1016/S1532-3382(02)70078-X] [PMID: 2228 7937]
]. Erosion is an increasing challenge for dentists, oral care practitioners and manufacturers of oral care products [14Lussi A, Ganss C. Erosive Tooth Wear: From Diagnosis to Therapy 2014.
[http://dx.doi.org/10.1159/isbn.978-3-318-02553-8]
, 65Amaechi BT. Dental Erosion and Its Clinical Management 2015.
[http://dx.doi.org/10.1007/978-3-319-13993-7]
]. The prevalence known from studies varies between 6 and 100% depending on the age group and the geographical region [65Amaechi BT. Dental Erosion and Its Clinical Management 2015.
[http://dx.doi.org/10.1007/978-3-319-13993-7]
]. The highest prevalence was found with children aged 9 to 17 years [65Amaechi BT. Dental Erosion and Its Clinical Management 2015.
[http://dx.doi.org/10.1007/978-3-319-13993-7]
]. Within adults, the prevalence ranges between 4 and 83% [65Amaechi BT. Dental Erosion and Its Clinical Management 2015.
[http://dx.doi.org/10.1007/978-3-319-13993-7]
]. Erosive tooth wear is found to be mainly a consequence of modern dietary habits with fruits, juices, lemonades, and other acidic aliments [14Lussi A, Ganss C. Erosive Tooth Wear: From Diagnosis to Therapy 2014.
[http://dx.doi.org/10.1159/isbn.978-3-318-02553-8]
].

2.4. Dentin Hypersensitivity

Patients often report dentin hypersensitivity to their dentists [38Gillam DG. Dentine hypersensitivity: Advances in diagnosis, management, and treatment 2015.]. Prevalence ranges from 3 to 98% within several studies [68Splieth CH, Tachou A. Epidemiology of dentin hypersensitivity. Clin Oral Investig 2013; 17(Suppl. 1): S3-8.
[http://dx.doi.org/10.1007/s00784-012-0889-8] [PMID: 23224064]
]. Depending on the age group, women are slightly more affected than men and most of the people at the age between 30 and 40 years suffer from dentin hypersensitivity [68Splieth CH, Tachou A. Epidemiology of dentin hypersensitivity. Clin Oral Investig 2013; 17(Suppl. 1): S3-8.
[http://dx.doi.org/10.1007/s00784-012-0889-8] [PMID: 23224064]
]. Buccal surfaces are mostly affected. Reasons might be gingival recession or abrasion at the cemento-enamel junction exposing the dentin into the oral cavity [69Addy M, West NX. The role of toothpaste in the aetiology and treatment of dentine hypersensitivity. Monogr Oral Sci 2013; 23: 75-87.
[http://dx.doi.org/10.1159/000350477] [PMID: 23817061]
]. Patients describe dentin hypersensitivity as a short, sharp pain [38Gillam DG. Dentine hypersensitivity: Advances in diagnosis, management, and treatment 2015.]. The pain will occur when the nerves in the pulp are stimulated via the liquid-filled tubules in exposed dentin when triggered by stimuli, typically thermal, evaporative, tactile, osmotic or chemical that cannot be ascribed to another defect or disease [38Gillam DG. Dentine hypersensitivity: Advances in diagnosis, management, and treatment 2015.]. In general, two different concepts can be described to treat dentin hypersensitivity with oral care agents: Desensitization of the pulp / nerve and occlusion of dentinal tubules [32Loveren Cv. Toothpastes 2013.
[http://dx.doi.org/10.1159/isbn.978-3-318-02207-0]
, 38Gillam DG. Dentine hypersensitivity: Advances in diagnosis, management, and treatment 2015., 70van Loveren C, Schmidlin PR, Martens LC, Amaechi BT. Dentin hypersensitivity management. Clin Dent Rev 2018; 2(1): 6.
[http://dx.doi.org/10.1007/s41894-017-0019-8]
]. Occlusion might last longer due to physical mechanisms, as ions for desensitization (i.e. potassium) are only momentary pain-relieving.

3. HAP IN ORAL CARE

Studies show the efficacy of particulate synthetic HAP in prevention of caries [26Schlagenhauf U, Kunzelmann KH, Hannig C, et al. Impact of a non-fluoridated microcrystalline hydroxyapatite dentifrice on enamel caries progression in highly caries-susceptible orthodontic patients: A randomized, controlled 6-month trial. J Investig Clin Dent 2019; 10(2)e12399
[http://dx.doi.org/10.1111/jicd.12399] [PMID: 30701704]
, 34Kani K, Kani M, Isozaki A, Shintani H, Ohashi T, Tokumoto T. Effect of apatite-containing dentifrices on dental caries in school children. J Dent Health 1989; 19: 104-9.
[http://dx.doi.org/10.5834/jdh.39.104]
], remineralization of early stages of dentin and enamel caries [35Najibfard K, Ramalingam K, Chedjieu I, Amaechi BT. Remineralization of early caries by a nano-hydroxyapatite dentifrice. J Clin Dent 2011; 22(5): 139-43.
[PMID: 22403978]
, 71Tschoppe P, Zandim DL, Martus P, Kielbassa AM. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. J Dent 2011; 39(6): 430-7.
[http://dx.doi.org/10.1016/j.jdent.2011.03.008] [PMID: 21504777]
, 72de Carvalho FG, Vieira BR, Santos RL, Carlo HL, Lopes PQ, de Lima BA. In vitro effects of nano-hydroxyapatite paste on initial enamel carious lesions Pediatr Dent 2014; 36(3): 85-9.], prevention of periodontitis [23Kensche A, Holder C, Basche S, Tahan N, Hannig C, Hannig M. Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ. Arch Oral Biol 2017; 80: 18-26.
[http://dx.doi.org/10.1016/j.archoralbio.2017.03.013] [PMID: 2836 4672]
, 25Harks I, Jockel-Schneider Y, Schlagenhauf U, et al. Impact of the daily use of a microcrystal hydroxyapatite dentifrice on de novo plaque formation and clinical/microbiological parameters of periodontal health. A randomized trial. PLoS One 2016; 11(7)e0160142
[http://dx.doi.org/10.1371/journal.pone.0160142] [PMID: 27467683]
, 47Hannig C, Basche S, Burghardt T, Al-Ahmad A, Hannig M. Influence of a mouthwash containing hydroxyapatite microclusters on bacterial adherence in situ. Clin Oral Investig 2013; 17(3): 805-14.
[http://dx.doi.org/10.1007/s00784-012-0781-6] [PMID: 22782257]
], reduction of gingival bleeding [25Harks I, Jockel-Schneider Y, Schlagenhauf U, et al. Impact of the daily use of a microcrystal hydroxyapatite dentifrice on de novo plaque formation and clinical/microbiological parameters of periodontal health. A randomized trial. PLoS One 2016; 11(7)e0160142
[http://dx.doi.org/10.1371/journal.pone.0160142] [PMID: 27467683]
, 41Hegazy SA, Salama IR. Antiplaque and remineralizing effects of Biorepair mouthwash: A comparative clinical trial. Pediatr Dent J 2016; 26: 89-94.
[http://dx.doi.org/10.1016/j.pdj.2016.05.002]
], prevention of acid erosion [48Lelli M, Putignano A, Marchetti M, et al. Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: A comparative in vivo study. Front Physiol 2014; 5: 333.
[http://dx.doi.org/10.3389/fphys.2014.00333] [PMID: 25249980]
, 73Colombo M, Beltrami R, Rattalino D, Mirando M, Chiesa M, Poggio C. Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study. Ann Stomatol (Roma) 2017; 7(3): 38-45.
[PMID: 28149449]
, 74Poggio C, Gulino C, Mirando M, Colombo M, Pietrocola G. Protective effect of zinc-hydroxyapatite toothpastes on enamel erosion: An in vitro study. J Clin Exp Dent 2017; 9(1): e118-22.
[PMID: 28149475]
], and reduction of dentin hypersensitivity [27Hu M-L, Zheng G, Zhang Y-D, Yan X, Li X-C, Lin H. Effect of desensitizing toothpastes on dentine hypersensitivity: A systematic review and meta-analysis. J Dent 2018; 75: 12-21.
[http://dx.doi.org/10.1016/j.jdent.2018.05.012] [PMID: 29787782]
, 75Orsini G, Procaccini M, Manzoli L, Giuliodori F, Lorenzini A, Putignano A. A double-blind randomized-controlled trial comparing the desensitizing efficacy of a new dentifrice containing carbonate /hydroxyapatite nanocrystals and a sodium fluoride /potassium nitrate dentifrice. J Clin Periodontol 2010; 37(6): 510-7.
[http://dx.doi.org/10.1111/j.1600-051X.2010.01558.x] [PMID: 20507 374]
-78Vano M, Derch G, Barone A, Covani U. Effectiveness of nano-hydroxyapatite toothpaste in reducing dentin hypersensitivity: A double-blind randomized controlled trial. Quint int 2014; 45: 703-11.]. The fact that HAP is effective within multiple clinical indications in a number of different preventive oral care applications strongly indicates that this biomimetic calcium phosphate mineral has different relevant modes of action. These are either related to inherent physico-chemical properties of HAP or the chosen administration form. To develop formulations that are even more efficacious in combating caries and other oral diseases, the understanding of these modes of action is a very important key. Following this, the effects as demonstrated by the results of the scientific studies are discussed with respect to presumable modes of action of HAP.

3.1. Studies and Mechanisms of HAP in Preventing Caries

3.1.1. Studies

(1) The first clinical trial using HAP in the field of caries prophylaxis was published in 1989 [34Kani K, Kani M, Isozaki A, Shintani H, Ohashi T, Tokumoto T. Effect of apatite-containing dentifrices on dental caries in school children. J Dent Health 1989; 19: 104-9.
[http://dx.doi.org/10.5834/jdh.39.104]
]. Two different schools with at least 200 fourth-graders were included in the study. The study duration was three years. One group used a (fluoride-free) toothpaste containing 5% HAP, and the other group used a placebo-toothpaste without any active ingredients. Every year, the dmft/DMFT was acquired. While on the level of the primary dentition only minor effects could be observed, the caries reduction on the level of newly-erupted teeth was statistically significant in the HAP-group compared to placebo (mean caries inhibition: about 45%) [34Kani K, Kani M, Isozaki A, Shintani H, Ohashi T, Tokumoto T. Effect of apatite-containing dentifrices on dental caries in school children. J Dent Health 1989; 19: 104-9.
[http://dx.doi.org/10.5834/jdh.39.104]
].

(2) These above results were confirmed by a randomized clinical trial (non-inferiority-trial) from Schlagenhauf et al. [79Schlagenhauf U, Kunzelmann K-H, Hannig C, et al. Impact of a non-fluoridated microcrystalline hydroxyapatite dentifrice on enamel caries progression in highly caries-susceptible orthodontic patients: A randomized, controlled 6-month trial. J Investig Clin Dent 2019; 10(2)e12399
[http://dx.doi.org/10.1111/jicd.12399] [PMID: 30701704]
]. This study was performed at five German university-hospitals using high-caries risk-patients undergoing orthodontic treatment. Since it is well known that these patients develop caries within four weeks [80Enaia M, Bock N, Ruf S. White-spot lesions during multibracket appliance treatment: A challenge for clinical excellence. Am J Orthod Dentofacial Orthop 2011; 140(1): e17-24.
[http://dx.doi.org/10.1016/j.ajodo.2010.12.016] [PMID: 21724067]
, 81O'Reilly MM, Featherstone JDB. Demineralization and remineralization around orthodontic appliances: An in vivo study American journal of orthodontics and dentofacial orthopedics: Official publication of the American Association of Orthodontists, its constituent societies, and the American Board of Orthodontics 1987; 92(1): 33-40.], the study was conducted over a period of six months and the 147 patients were randomly distributed using either a (fluoride-free) toothpaste containing 10% microcrystalline HAP, and a toothpaste containing amine fluoride/stannous(II)fluoride (1400 ppm fluoride). Caries incidence was measured using ICDAS-scores 1 and 2. Out of the 133 participants overall who finished the study per protocol, the HAP-group was not inferior to the fluoride-group [79Schlagenhauf U, Kunzelmann K-H, Hannig C, et al. Impact of a non-fluoridated microcrystalline hydroxyapatite dentifrice on enamel caries progression in highly caries-susceptible orthodontic patients: A randomized, controlled 6-month trial. J Investig Clin Dent 2019; 10(2)e12399
[http://dx.doi.org/10.1111/jicd.12399] [PMID: 30701704]
].

(3) Lelli et al. used extracted teeth from an in vivo study (so called ex-in vivo study) to investigate whether a calcium-layer can be observed after using a HAP-toothpaste. Due to orthodontic or prosthetic reasons, teeth had to be extracted. Participants had either brushed their teeth with a fluoride-toothpaste or with a HAP-toothpaste for at least 8 weeks. The extracted teeth were analyzed using scanning electron microscopy (SEM) to visualize a possibly formed superficial layer and energy dispersive X-ray spectroscopy (EDX) to analyze its elemental composition. While in the fluoride-group no additional layer could be observed, the use of HAP-toothpaste leads to the formation of a calcium phosphate-containing layer on top of the natural enamel [48Lelli M, Putignano A, Marchetti M, et al. Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: A comparative in vivo study. Front Physiol 2014; 5: 333.
[http://dx.doi.org/10.3389/fphys.2014.00333] [PMID: 25249980]
].

(4) Najibfard et al. conducted a double-blind in situ study with thirty participants using enamel slabs with and without artificial carious lesions [35Najibfard K, Ramalingam K, Chedjieu I, Amaechi BT. Remineralization of early caries by a nano-hydroxyapatite dentifrice. J Clin Dent 2011; 22(5): 139-43.
[PMID: 22403978]
]. Using a crossover-design (washout-phase in between the phases was seven days), participants brushed for at least 28 days with a fluoride-toothpaste (1100 ppm fluoride) or a HAP-toothpaste (5% HAP and 10% HAP, respectively). The slabs were analyzed using microradiography to quantify mineral loss and lesion depth. The results showed that HAP-toothpaste prevented the enamel from demineralization. Additionally, remineralization of enamel was not different between the toothpastes containing fluoride (1100 ppm fluoride), and HAP-toothpaste (5% and 10% HAP) [35Najibfard K, Ramalingam K, Chedjieu I, Amaechi BT. Remineralization of early caries by a nano-hydroxyapatite dentifrice. J Clin Dent 2011; 22(5): 139-43.
[PMID: 22403978]
].

(5) A clinical trial compared three different mouth rinses containing either HAP, CHX or fluoride with respect to plaque accumulation, gingival indices, and remineralization properties [41Hegazy SA, Salama IR. Antiplaque and remineralizing effects of Biorepair mouthwash: A comparative clinical trial. Pediatr Dent J 2016; 26: 89-94.
[http://dx.doi.org/10.1016/j.pdj.2016.05.002]
]. 81 children used the respective mouth rinse twice a day under parental supervision for two weeks. The children were examined after 1, 2, 4, and 6 weeks. With respect to caries (remineralization), HAP performed equally compared to fluoride and was also effective in reducing dental plaque and improving gingival health [41Hegazy SA, Salama IR. Antiplaque and remineralizing effects of Biorepair mouthwash: A comparative clinical trial. Pediatr Dent J 2016; 26: 89-94.
[http://dx.doi.org/10.1016/j.pdj.2016.05.002]
].

(6), (7) Two in situ studies investigated the bacterial adhesion to enamel surfaces after rinsing with an (6) aqueous dispersion containing HAP-particles [23Kensche A, Holder C, Basche S, Tahan N, Hannig C, Hannig M. Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ. Arch Oral Biol 2017; 80: 18-26.
[http://dx.doi.org/10.1016/j.archoralbio.2017.03.013] [PMID: 2836 4672]
] and (7) a mouth rinse containing HAP-particles [47Hannig C, Basche S, Burghardt T, Al-Ahmad A, Hannig M. Influence of a mouthwash containing hydroxyapatite microclusters on bacterial adherence in situ. Clin Oral Investig 2013; 17(3): 805-14.
[http://dx.doi.org/10.1007/s00784-012-0781-6] [PMID: 22782257]
]. Participants wore enamel slabs and rinsed with the respective dispersions. As positive control, CHX-containing mouth rinse was used, and negative control was also studied. Enamel slabs were carried in the oral cavity for six to twelve hours and then analyzed using bacterial staining and microscopy-techniques. Additionally, in vitro experiments regarding antimicrobial effects were also carried out. HAP does not kill the bacteria but leads to a decrease of bacterial attachment on enamel surfaces. The reduction of initial bacterial colonization to enamel surfaces in situ was comparable with the reduction of the antimicrobial agent chlorhexidine [23Kensche A, Holder C, Basche S, Tahan N, Hannig C, Hannig M. Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ. Arch Oral Biol 2017; 80: 18-26.
[http://dx.doi.org/10.1016/j.archoralbio.2017.03.013] [PMID: 2836 4672]
, 47Hannig C, Basche S, Burghardt T, Al-Ahmad A, Hannig M. Influence of a mouthwash containing hydroxyapatite microclusters on bacterial adherence in situ. Clin Oral Investig 2013; 17(3): 805-14.
[http://dx.doi.org/10.1007/s00784-012-0781-6] [PMID: 22782257]
].

Several in vitro studies have investigated the remineralization-potential of particulate HAP [71Tschoppe P, Zandim DL, Martus P, Kielbassa AM. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. J Dent 2011; 39(6): 430-7.
[http://dx.doi.org/10.1016/j.jdent.2011.03.008] [PMID: 21504777]
, 72de Carvalho FG, Vieira BR, Santos RL, Carlo HL, Lopes PQ, de Lima BA. In vitro effects of nano-hydroxyapatite paste on initial enamel carious lesions Pediatr Dent 2014; 36(3): 85-9., 82Esteves-Oliveira M, Meyer-Lueckel H, Wierichs RJ, Santos NM, Rodrigues JA. Caries-preventive effect of anti-erosive and nano-hydroxyapatite-containing toothpastes in vitro Clin Oral Investig 2016.
[PMID: 26993660]
-84Gjorgievska ES, Nicholson JW, Slipper IJ, Stevanovic MM. Remineralization of demineralized enamel by toothpastes: A scanning electron microscopy, energy dispersive X-ray analysis, and three-dimensional stereo-micrographic study. Microsc Microanal 2013; 19(3): 587-95.
[http://dx.doi.org/10.1017/S1431927613000391] [PMID: 23659606]
]. Most of the evaluated studies used demineralized enamel or dentin, and applied HAP-particles on these slabs [71Tschoppe P, Zandim DL, Martus P, Kielbassa AM. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. J Dent 2011; 39(6): 430-7.
[http://dx.doi.org/10.1016/j.jdent.2011.03.008] [PMID: 21504777]
, 83Huang SB, Gao SS, Yu HY. Effect of nano-hydroxyapatite concentration on remineralization of initial enamel lesion in vitro Biomed Mater 2009; 4: 034104/1-/6., 84Gjorgievska ES, Nicholson JW, Slipper IJ, Stevanovic MM. Remineralization of demineralized enamel by toothpastes: A scanning electron microscopy, energy dispersive X-ray analysis, and three-dimensional stereo-micrographic study. Microsc Microanal 2013; 19(3): 587-95.
[http://dx.doi.org/10.1017/S1431927613000391] [PMID: 23659606]
]. Two studies, however, used pH-cycling models [72de Carvalho FG, Vieira BR, Santos RL, Carlo HL, Lopes PQ, de Lima BA. In vitro effects of nano-hydroxyapatite paste on initial enamel carious lesions Pediatr Dent 2014; 36(3): 85-9., 82Esteves-Oliveira M, Meyer-Lueckel H, Wierichs RJ, Santos NM, Rodrigues JA. Caries-preventive effect of anti-erosive and nano-hydroxyapatite-containing toothpastes in vitro Clin Oral Investig 2016.
[PMID: 26993660]
]. Out of these, Esteves-Oliveira et al. did not detect a caries-preventive effect of HAP in vitro [82Esteves-Oliveira M, Meyer-Lueckel H, Wierichs RJ, Santos NM, Rodrigues JA. Caries-preventive effect of anti-erosive and nano-hydroxyapatite-containing toothpastes in vitro Clin Oral Investig 2016.
[PMID: 26993660]
], while de Carvalho et al. described caries-inhibiting effects of HAP [72de Carvalho FG, Vieira BR, Santos RL, Carlo HL, Lopes PQ, de Lima BA. In vitro effects of nano-hydroxyapatite paste on initial enamel carious lesions Pediatr Dent 2014; 36(3): 85-9.].

3.1.2. Mechanisms

The effects described in the results of the analyzed studies imply a number of different mechanisms where HAP plays a major role. As caries is a biofilm-associated disease, this biofilm should consequentially be reduced. HAP has already been shown to reduce the bacterial biofilm on the teeth [23Kensche A, Holder C, Basche S, Tahan N, Hannig C, Hannig M. Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ. Arch Oral Biol 2017; 80: 18-26.
[http://dx.doi.org/10.1016/j.archoralbio.2017.03.013] [PMID: 2836 4672]
]. Bacteria need a rough surface for attachment [85Hannig C, Hannig M. The oral cavity-A key system to understand substratum-dependent bioadhesion on solid surfaces in man. Clin Oral Investig 2009; 13(2): 123-39.
[http://dx.doi.org/10.1007/s00784-008-0243-3] [PMID: 19137331]
].

HAP in oral care products might help smoothen the surface through two different effects: Firstly, particulate HAP attaching to the enamel surface may preferentially deposit in small depressions such as scratches and superficial defects because it can more firmly attach and may be harder to remove by either abrasive food particles or brushing than on the smooth enamel surface areas, thus leveling the surface on the microscopic scale. Secondly, the remineralization effect and the resulting bio-chemically induced formation of a protective layer may also be more pronounced in surface depressions of the outer enamel. Both effects lead to a smoother surface that consequently decelerates the attachment of potentially harmful bacteria.

Furthermore, bacteria can erratically attach to individual HAP particles that are either loosely attached to the enamel surface or float in the oral cavity, thus inducing bacteria co-aggregation. In consequence, bacterial load will be reduced by spitting out the residual HAP after thorough tooth brushing. Despite all routine daily oral hygiene, bacteria can attach on the teeth after a certain time. If a protective layer of HAP particles is present and regularly sustained, this biofilm is formed on its surface instead of the calcium phosphate of the original enamel. When acids are produced within this biofilm, this protective layer might be dissolved, and calcium and phosphate ions will be released. This can have two main effects: On the one hand, the biofilm formation might be disrupted by influencing the energy metabolism of oral bacteria [86Astasov-Frauenhoffer M, Varenganayil MM, Decho AW, Waltimo T, Braissant O. Exopolysaccharides regulate calcium flow in cariogenic biofilms. PLoS One 2017; 12(10)e0186256
[http://dx.doi.org/10.1371/journal.pone.0186256] [PMID: 29023506]
]. On the other hand, the excess of released calcium and phosphate ions become available for other, different effects. They might for instance help and promote remineralization of demineralized tooth surfaces and support the natural remineralization-mechanisms of saliva, i.e. shifting the equilibrium from de- to remineralization. If residual plaque is present (i.e. plaque that has not been removed by toothbrushing), HAP may also be incorporated into the biofilm as demonstrated for other calcium phosphates before [6Meyer F, Amaechi BT, Fabritius HO, Enax J. Overview of calcium phosphates used in biomimetic oral care. Open Dent J 2018; 12: 406-23.
[http://dx.doi.org/10.2174/1874210601812010406] [PMID: 29988215]
, 87Vogel GL, Zhang Z, Carey CM, Ly A, Chow LC, Proskin HM. Composition of plaque and saliva following a sucrose challenge and use of an alpha-tricalcium-phosphate-containing chewing gum. J Dent Res 1998; 77(3): 518-24.
[http://dx.doi.org/10.1177/00220345980770031101] [PMID: 9496925]
].

When acids are produced by bacteria, the dissociation products of HAP may act as buffering-solution. Particularly the phosphate ions are able to neutralize acids to a certain level, likewise the salivary phosphate-buffer. If present in excess, the released calcium and phosphate ions can also switch the solubility product balance back to remineralization.

In addition to the mechanisms described above, HAP can remineralize early caries-lesions directly which was shown under in situ conditions (Fig. 4) [35Najibfard K, Ramalingam K, Chedjieu I, Amaechi BT. Remineralization of early caries by a nano-hydroxyapatite dentifrice. J Clin Dent 2011; 22(5): 139-43.
[PMID: 22403978]
]. From a histological point of view, initial caries-lesions show larger distances between the rod-shaped HAP crystallites of the enamel than healthy portions. Small HAP particles from oral-care products might be able to penetrate through these gaps between enamel-rods and thus fill these initial lesions.

The HAP particles might bind to enamel/pellicle due to electrochemical forces, because of partial electronegativity: Zeta-potential of enamel and HAP-particles show a negative net surface potential of -15 to -30 mV, and basic proteins (i.e. from saliva/pellicle) a positive net surface potential of +20 to + 30 mV [88Young A, Smistad G, Karlsen J, Rölla G, Rykke M. Zeta potentials of human enamel and hydroxyapatite as measured by the Coulter DELSA 440. Adv Dent Res 1997; 11(4): 560-5.
[http://dx.doi.org/10.1177/08959374970110042501] [PMID: 9470517]
, 89Reynolds EC, Wong A. Effect of adsorbed protein on hydroxyapatite zeta potential and Streptococcus mutans adherence. Infect Immun 1983; 39(3): 1285-90.
[PMID: 6301991]
]. HAP particles might also act as crystal nuclei by attracting calcium and phosphate from saliva [32Loveren Cv. Toothpastes 2013.
[http://dx.doi.org/10.1159/isbn.978-3-318-02207-0]
]. Local concentration gradients can induce the formation of calcium phosphate through crystal growth on the enamel surface.

Fig. (4)
Microradiographic images of early caries (A) before and (B) after in situ remineralization by use of a 5% HAP toothpaste. A decrease in surface roughness is accompanied by a decrease of the demineralized region below the surface indicated by the smaller dark grey area. The images are taken with publisher’s permission from Najibfard et al. [35Najibfard K, Ramalingam K, Chedjieu I, Amaechi BT. Remineralization of early caries by a nano-hydroxyapatite dentifrice. J Clin Dent 2011; 22(5): 139-43.
[PMID: 22403978]
].


Since HAP is a calcium phosphate source itself, the described mechanisms do not only rely on ions provided with the salivary flow. As soon as bacteria are able to attach to a HAP-layer on tooth surfaces and acids are produced, calcium and phosphate ions will be released which may have a positive influence on the remineralization process (Fig. 5A-C).

HAP has a wide range of actions with respect to caries prevention. The main advantage of HAP compared to other oral care active ingredients is its high biocompatibility, i.e. no potentially toxically side effects like fluorosis. In consequence to this, there is no regulation in dosage [42Epple M. Review of potential health risks associated with nanoscopic calcium phosphate. Acta Biomater 2018; 77: 1-14.
[http://dx.doi.org/10.1016/j.actbio.2018.07.036] [PMID: 30031162]
, 43Ramis J, Coelho C, Córdoba A, Quadros P, Monjo M. Safety assessment of nano-hydroxyapatite as an oral care ingredient according to the EU cosmetics regulation. Cosmetics 2018; 5(3): 53.
[http://dx.doi.org/10.3390/cosmetics5030053]
].

Fig. (5)
Schematic overview showing the modes of action of HAP in the remineralization and protection of enamel. (A) HAP-particles from oral care products are introduced into the oral cavity. (B) HAP particles from oral care formulations adhere to enamel surfaces as a protective layer [23Kensche A, Holder C, Basche S, Tahan N, Hannig C, Hannig M. Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ. Arch Oral Biol 2017; 80: 18-26.
[http://dx.doi.org/10.1016/j.archoralbio.2017.03.013] [PMID: 2836 4672]
, 48Lelli M, Putignano A, Marchetti M, et al. Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: A comparative in vivo study. Front Physiol 2014; 5: 333.
[http://dx.doi.org/10.3389/fphys.2014.00333] [PMID: 25249980]
]. Bacterial adhesion is reduced. Free HAP-particles act as “magnet” for oral bacteria and are spit out. (C) Bacteria forming a biofilm on top of the HAP-layer after a certain time will produce acids when sugar/starch is provided, and acids are produced. These acids might lead to dissolution of the protective HAP-layer. The tooth will be protected, because of free calcium ions, and phosphate buffer mechanisms from the HAP-layer.


3.2. Studies and Mechanisms of HAP in Preventing Periodontitis

3.2.1. Studies

Several clinical trials report an improvement of gingival health after having used HAP-containing oral care products. (8) Harks et al. tested in a randomized clinical trial the differences in the plaque-formation-rate between a HAP-toothpaste and an antibacterial amine fluoride/stannous (II) fluoride-toothpaste. Sixty-seven patients with mild periodontitis completed the whole double-blind 12 weeks-trial. Besides plaque-formation-rate, several other clinical parameters were tested (i.e. bleeding on probing). The two groups used their respective toothpaste for 4 weeks without any therapy at home. After these 4 weeks supragingival cleaning was performed and the toothpastes were used for another 8 weeks. Plaque-formation rate did not change within that time. However, gingival health improved within both groups after 4 and 12 weeks. There were no differences between the antibacterial toothpaste and the HAP-toothpaste [25Harks I, Jockel-Schneider Y, Schlagenhauf U, et al. Impact of the daily use of a microcrystal hydroxyapatite dentifrice on de novo plaque formation and clinical/microbiological parameters of periodontal health. A randomized trial. PLoS One 2016; 11(7)e0160142
[http://dx.doi.org/10.1371/journal.pone.0160142] [PMID: 27467683]
].

(9) Plaque samples from this study were also investigated using 16S rRNA amplicons for next generation sequencing. No differences of the plaque-composition could be observed between these two groups [90Hagenfeld D, Prior K, Harks I, et al. No differences in microbiome changes between anti-adhesive and antibacterial ingredients in toothpastes during periodontal therapy. J Periodontal Res 2019; 54(4): 435-43.
[http://dx.doi.org/10.1111/jre.12645] [PMID: 30851050]
]. While mainly Fusobacterium and Prevotella species were observed in the interproximal and subgingival sites, buccal and lingual surfaces were dominated by Streptococcus and Veillonella species. However, the use of either antibacterial or anti-adhesive toothpastes did not change the composition noticeably indicating the stability of the ecological niche. Interestingly, gingival bleeding was reduced in both groups showing an effect on periodontal health of both treatments [25Harks I, Jockel-Schneider Y, Schlagenhauf U, et al. Impact of the daily use of a microcrystal hydroxyapatite dentifrice on de novo plaque formation and clinical/microbiological parameters of periodontal health. A randomized trial. PLoS One 2016; 11(7)e0160142
[http://dx.doi.org/10.1371/journal.pone.0160142] [PMID: 27467683]
].

(10) Additionally, Cosola et al. tested a mouth rinse containing HAP (and zinc-PCA) with patients after surgical procedures [40Cosola S, Marconcini S, Giammarinaro E, Marchisio O, Lelli M, Roveri N, et al. Antimicrobial efficacy of mouthwashes containing zinc-substituted nanohydroxyapatite and zinc L-pyrrolidone carboxylate on suture threads after surgical procedures. J Oral Sci Rehabil 2017; 3(4): 24-30.]. Twenty-six patients were randomly allocated to either CHX-group or HAP-group. All patients received oral surgery. As postoperative treatment, patients rinsed with the mouth rinse. After their removal the suture threads from both groups, CHX and HAP (and zinc-PCA) were tested in vitro for colony-forming units. Despite different treatment, threads from both groups were found to have the same effect on bacterial growth. Since prevention of periodontitis relies on controlling bacterial growth, it is important to note that this study shows HAP (with addition of zinc-PCA) to reduce bacterial growth on suture threads. This goes in line with the in vivo (5) and in situ (6), (7) studies presented before [23Kensche A, Holder C, Basche S, Tahan N, Hannig C, Hannig M. Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ. Arch Oral Biol 2017; 80: 18-26.
[http://dx.doi.org/10.1016/j.archoralbio.2017.03.013] [PMID: 2836 4672]
, 25Harks I, Jockel-Schneider Y, Schlagenhauf U, et al. Impact of the daily use of a microcrystal hydroxyapatite dentifrice on de novo plaque formation and clinical/microbiological parameters of periodontal health. A randomized trial. PLoS One 2016; 11(7)e0160142
[http://dx.doi.org/10.1371/journal.pone.0160142] [PMID: 27467683]
, 41Hegazy SA, Salama IR. Antiplaque and remineralizing effects of Biorepair mouthwash: A comparative clinical trial. Pediatr Dent J 2016; 26: 89-94.
[http://dx.doi.org/10.1016/j.pdj.2016.05.002]
].

3.2.2. Mechanisms

The results of these studies provide a number of useful hints with regard to HAP’s possible involvement in the prevention of periodontitis.

Dental plaque bacteria can interact with extrinsic HAP when present in the oral cavity. Thus, early colonizers, such as streptococci bind to free HAP derived from oral-care products instead of the tooth surface. The HAP can be either free particles in the oral cavity or particles bound to the tooth surfaces thereby creating a protective film of alternative substrate. As the early colonizers are needed for further biofilm-formation, the biofilm will be consequentially reduced either by directly spitting out of the free particles or by abrasion of the HAP layer through oral hygiene related cleaning activity.

It is known that gingivitis and periodontitis are promoted by a dysbiotic dental plaque if it is not removed. Hindering the settlement of early colonizers and reducing growth of the dental plaque can slow and potentially even prevent reaching a dysbiotic state.

The mode of action of HAP with respect to periodontitis is indirect. HAP is not able to directly interact with the immune system. Nevertheless, by reducing bacterial load and controlling the biofilm-formation, periodontal pathogens (i.e. Porphyromonas gingivalis and Tannerella forsythia) will not be able to lead to a shift in the taxonomic composition of the biofilm, as they are late colonizers and with that they are dependent of the early colonizers and bridge microorganisms (i.e. Fusobacterium nucleatum) [52Kilian M, Chapple ILC, Hannig M, et al. The oral microbiome - An update for oral healthcare professionals. Br Dent J 2016; 221(10): 657-66.
[http://dx.doi.org/10.1038/sj.bdj.2016.865] [PMID: 27857087]
, 62Meyer F, Enax J. Die Mundhöhle als Ökosystem. Biol Unserer Zeit 2018; 48(1): 62-8.
[http://dx.doi.org/10.1002/biuz.201810641]
].

HAP also acts as an biomimetic cleaning agent (same hardness as the tooth) in toothpaste formulations, directly reducing dental plaque [32Loveren Cv. Toothpastes 2013.
[http://dx.doi.org/10.1159/isbn.978-3-318-02207-0]
, 91Enax J, Epple M. Die Charakterisierung von Putzkörpern in Zahnpasten. Dtsch Zahnarztl Z 2018; 73: 116-24.].

The authors hypothesize that a HAP-layer applied to a tooth surface directly after mechanical dental plaque removal might lead to an aggravated biofilm formation [48Lelli M, Putignano A, Marchetti M, et al. Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: A comparative in vivo study. Front Physiol 2014; 5: 333.
[http://dx.doi.org/10.3389/fphys.2014.00333] [PMID: 25249980]
]. Formulations with the addition of zinc salts or lactoferrin may boost the effect of HAP, especially against already existing or developing biofilms [30Meyer F, Enax J. Hydroxyapatite in oral biofilm management. Eur J Dent (in press), 40Cosola S, Marconcini S, Giammarinaro E, Marchisio O, Lelli M, Roveri N, et al. Antimicrobial efficacy of mouthwashes containing zinc-substituted nanohydroxyapatite and zinc L-pyrrolidone carboxylate on suture threads after surgical procedures. J Oral Sci Rehabil 2017; 3(4): 24-30., 92Nocerino N, Fulgione A, Iannaccone M, et al. Biological activity of lactoferrin-functionalized biomimetic hydroxyapatite nanocrystals. Int J Nanomedicine 2014; 9: 1175-84.
[PMID: 24623976]
]. Zinc-substituted HAP, where Zn2+ions replace a part of the Ca2+ ions [5Brown PW, Constantz B. Hydroxyapatite and related materials 1994., 48Lelli M, Putignano A, Marchetti M, et al. Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: A comparative in vivo study. Front Physiol 2014; 5: 333.
[http://dx.doi.org/10.3389/fphys.2014.00333] [PMID: 25249980]
], might also be dissolved in acidic environments, and the released zinc might act as an antimicrobial agent. This might promote the positive effects of HAP with respect to gingival health [25Harks I, Jockel-Schneider Y, Schlagenhauf U, et al. Impact of the daily use of a microcrystal hydroxyapatite dentifrice on de novo plaque formation and clinical/microbiological parameters of periodontal health. A randomized trial. PLoS One 2016; 11(7)e0160142
[http://dx.doi.org/10.1371/journal.pone.0160142] [PMID: 27467683]
, 40Cosola S, Marconcini S, Giammarinaro E, Marchisio O, Lelli M, Roveri N, et al. Antimicrobial efficacy of mouthwashes containing zinc-substituted nanohydroxyapatite and zinc L-pyrrolidone carboxylate on suture threads after surgical procedures. J Oral Sci Rehabil 2017; 3(4): 24-30., 47Hannig C, Basche S, Burghardt T, Al-Ahmad A, Hannig M. Influence of a mouthwash containing hydroxyapatite microclusters on bacterial adherence in situ. Clin Oral Investig 2013; 17(3): 805-14.
[http://dx.doi.org/10.1007/s00784-012-0781-6] [PMID: 22782257]
, 92Nocerino N, Fulgione A, Iannaccone M, et al. Biological activity of lactoferrin-functionalized biomimetic hydroxyapatite nanocrystals. Int J Nanomedicine 2014; 9: 1175-84.
[PMID: 24623976]
].

In addition to that, HAP might also interact with proteins derived from microorganisms. Proteins are known to act as virulence factors, i.e. arginine deiminase arc A from P. gingivalis [59Deng Z-L, Szafrański SP, Jarek M, Bhuju S, Wagner-Döbler I. Dysbiosis in chronic periodontitis: Key microbial players and interactions with the human host. Sci Rep 2017; 7(1): 3703.
[http://dx.doi.org/10.1038/s41598-017-03804-8] [PMID: 28623321]
]. HAP particles present in the vicinity of the bacteria might bind these proteins [93Gorbunoff MJ, Timasheff SN. The interaction of proteins with hydroxyapatite. III. Mechanism. Anal Biochem 1984; 136(2): 440-5.
[http://dx.doi.org/10.1016/0003-2697(84)90241-0] [PMID: 6721144]
]. Being immobilized, the virulence factors will not be able to interact with the human immune system.

Finally, HAP does not show unwanted side effects like discoloration of teeth or irritation of taste and can be used on a daily basis, in contrast to substances like chlorhexidine [23Kensche A, Holder C, Basche S, Tahan N, Hannig C, Hannig M. Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ. Arch Oral Biol 2017; 80: 18-26.
[http://dx.doi.org/10.1016/j.archoralbio.2017.03.013] [PMID: 2836 4672]
, 30Meyer F, Enax J. Hydroxyapatite in oral biofilm management. Eur J Dent (in press), 47Hannig C, Basche S, Burghardt T, Al-Ahmad A, Hannig M. Influence of a mouthwash containing hydroxyapatite microclusters on bacterial adherence in situ. Clin Oral Investig 2013; 17(3): 805-14.
[http://dx.doi.org/10.1007/s00784-012-0781-6] [PMID: 22782257]
].

Furthermore, HAP does not kill bacteria (in contrast to e.g. stannous ions and chlorhexidine) but controls them by its physical presence or by passively withdrawing potentially harmful metabolic products from the oral cavity environment. Modern approaches focus on controlling the oral bacteria rather than killing them [94Adams SE, Arnold D, Murphy B, et al. A randomised clinical study to determine the effect of a toothpaste containing enzymes and proteins on plaque oral microbiome ecology. Sci Rep 2017; 7: 43344.
[http://dx.doi.org/10.1038/srep43344] [PMID: 28240240]
], since it is known that the use of antibacterial agents might lead to resistance or tolerance of bacteria against these agents. Biomimetic concepts have been shown to be promising alternatives or supplements in promoting periodontal health.


3.3. Studies and Mechanisms of HAP in Preventing Dental Erosion

3.3.1. Studies

Clinical studies in the field of erosion are rare due to a feasible study population and an acceptable study duration for the participants and ethical issues [95Huysmans MCDNJM, Chew HP, Ellwood RP. Clinical Studies of Dental Erosion and Erosive Wear. Caries Res 2011; 45 (suppl1): 60-8.
[http://dx.doi.org/10.1159/000325947]
]. Therefore, in situ and in vitro studies using a sophisticated study design are much more important. (3) An ex-in vivo study by Lelli et al. has shown the formation of a (protective) calcium phosphate layer on top of the teeth in the HAP-group [48Lelli M, Putignano A, Marchetti M, et al. Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: A comparative in vivo study. Front Physiol 2014; 5: 333.
[http://dx.doi.org/10.3389/fphys.2014.00333] [PMID: 25249980]
]. While some in vitro studies were not able to show a positive effect of HAP-toothpastes on erosion [96Aykut-Yetkiner A, Attin T, Wiegand A. Prevention of dentine erosion by brushing with anti-erosive toothpastes. J Dent 2014; 42(7): 856-61.
[http://dx.doi.org/10.1016/j.jdent.2014.03.011] [PMID: 24704085]
, 97Ganss C, Lussi A, Grunau O, Klimek J, Schlueter N. Conventional and anti-erosion fluoride toothpastes: Effect on enamel erosion and erosion-abrasion. Caries Res 2011; 45(6): 581-9.
[http://dx.doi.org/10.1159/000334318] [PMID: 22156703]
], other studies were able to show protective effects of HAP-toothpastes [24Fabritius-Vilpoux K, Enax J, Herbig M, Raabe D, Fabritius H-O. Quantitative affinity parameters of synthetic hydroxyapatite and enamel surfaces in vitro. Bioinspir Biomim Nan 2019; 0(0): 1-40.
[http://dx.doi.org/10.1680/jbibn.18.00035]
, 71Tschoppe P, Zandim DL, Martus P, Kielbassa AM. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. J Dent 2011; 39(6): 430-7.
[http://dx.doi.org/10.1016/j.jdent.2011.03.008] [PMID: 21504777]
, 73Colombo M, Beltrami R, Rattalino D, Mirando M, Chiesa M, Poggio C. Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study. Ann Stomatol (Roma) 2017; 7(3): 38-45.
[PMID: 28149449]
, 74Poggio C, Gulino C, Mirando M, Colombo M, Pietrocola G. Protective effect of zinc-hydroxyapatite toothpastes on enamel erosion: An in vitro study. J Clin Exp Dent 2017; 9(1): e118-22.
[PMID: 28149475]
, 98Poggio C, Lombardini M, Vigorelli P, Colombo M, Chiesa M. The role of different toothpastes on preventing dentin erosion: An SEM and AFM study®. Scanning 2014; 36(3): 301-10.
[http://dx.doi.org/10.1002/sca.21105] [PMID: 23784952]
, 99Poggio C, Gulino C, Mirando M, Colombo M, Pietrocola G. Preventive effects of different protective agents on dentin erosion: An in vitro investigation. J Clin Exp Dent 2017; 9(1): e7-e12.
[PMID: 28149456]
].

(11) Fabritius-Vilpoux et al. tested in vitro, if HAP-particles from an aqueous dispersion can attach to (mechanically and chemically eroded) enamel-surfaces [24Fabritius-Vilpoux K, Enax J, Herbig M, Raabe D, Fabritius H-O. Quantitative affinity parameters of synthetic hydroxyapatite and enamel surfaces in vitro. Bioinspir Biomim Nan 2019; 0(0): 1-40.
[http://dx.doi.org/10.1680/jbibn.18.00035]
]. Bovine enamel slabs were pre-treated with phosphoric acid. Afterwards, these slabs were dipped into agitated aqueous dispersions containing different HAP-concentrations. HAP particle attachment and surface-coverage was determined using SEM. Even a dispersion containing only 1% of HAP resulted in an area coverage of 10% of the eroded enamel surface. A dispersion with 10% HAP covered more than 30% of the enamel after one-time application [24Fabritius-Vilpoux K, Enax J, Herbig M, Raabe D, Fabritius H-O. Quantitative affinity parameters of synthetic hydroxyapatite and enamel surfaces in vitro. Bioinspir Biomim Nan 2019; 0(0): 1-40.
[http://dx.doi.org/10.1680/jbibn.18.00035]
].

(12) Another in vitro study used also enamel slabs and tested Vickers-hardness after erosive challenges and application of toothpastes [74Poggio C, Gulino C, Mirando M, Colombo M, Pietrocola G. Protective effect of zinc-hydroxyapatite toothpastes on enamel erosion: An in vitro study. J Clin Exp Dent 2017; 9(1): e118-22.
[PMID: 28149475]
]. Enamel slabs were erosively challenged four consecutive times (0, 8, 24, and 32 hrs.) for two minutes. After each challenge, toothpaste was applied on the surface. Vickers-hardness measurements revealed that acidic attacks significantly reduce enamel surface hardness. However, HAP-containing toothpastes caused significant re-hardening of the surface, indicating the occurrence of remineralization [74Poggio C, Gulino C, Mirando M, Colombo M, Pietrocola G. Protective effect of zinc-hydroxyapatite toothpastes on enamel erosion: An in vitro study. J Clin Exp Dent 2017; 9(1): e118-22.
[PMID: 28149475]
].

(13) The same group also used human enamel slabs in another study [99Poggio C, Gulino C, Mirando M, Colombo M, Pietrocola G. Preventive effects of different protective agents on dentin erosion: An in vitro investigation. J Clin Exp Dent 2017; 9(1): e7-e12.
[PMID: 28149456]
]. Here, the slabs were pre-treated with the respective toothpastes. Several fluoride-containing and calcium-containing toothpastes were tested besides HAP-toothpaste. These pre-treated slabs were then dipped into an acidic soft-drink for a maximum of 32 min. As measurement for erosive challenge, loss of weight of the slabs was determined. The HAP-toothpaste showed significantly less loss of mineral compared to the other products. This indicates a protective effect of HAP against erosive challenges [99Poggio C, Gulino C, Mirando M, Colombo M, Pietrocola G. Preventive effects of different protective agents on dentin erosion: An in vitro investigation. J Clin Exp Dent 2017; 9(1): e7-e12.
[PMID: 28149456]
].

(14) Colombo et al. used a visual rating system imaged by SEM to evaluate the enamel surface after application of a HAP-toothpaste [73Colombo M, Beltrami R, Rattalino D, Mirando M, Chiesa M, Poggio C. Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study. Ann Stomatol (Roma) 2017; 7(3): 38-45.
[PMID: 28149449]
]. Human incisor specimens were prepared and underwent an erosive challenge. In four different groups (positive control with no erosive treatment, negative control without toothpaste-application, fluoride toothpaste, HAP-toothpaste), different toothpastes were applied for 3 min. and rinsed off with distilled water at 0, 8, 24, and 36 hrs time intervals. In between the applications, specimens were stored in artificial saliva. Using a systematic assessment-method by SEM, enamel damage was recorded by four observers. The HAP-toothpaste was the only tested toothpaste where mineral deposition was observed after the erosive challenges. The grade of damage was consistently lowest in the HAP-group [73Colombo M, Beltrami R, Rattalino D, Mirando M, Chiesa M, Poggio C. Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study. Ann Stomatol (Roma) 2017; 7(3): 38-45.
[PMID: 28149449]
].

3.3.2. Mechanisms

From to above studies, one can deduce the following mechanisms: HAP forms, after application, a protective layer on the tooth surface. The protective layer might act as expendable shield that protects the tooth material from acidic attacks. Consequently, the protective HAP layer will be dissolved, releasing calcium- and phosphate ions that can also act as buffer system:

Ca5(PO4)3(OH) + 4 H+ → 5 Ca2+ + 3 HPO42- + H2O

Furthermore, it is known that the content of calcium within an erosive surrounding might lead to a shift of the equilibrium from dissolution to homeostasis. When parts of the teeth are eroded by acids, HAP is potentially able to remineralize attacked surfaces. Dental erosion and dental caries are both diseases caused by acids. However, the amount of acids and the origin of the acids is different. Consequently, pathogenesis of these two acid-driven diseases differs. Erosion is a fast process compared to caries: Acids produced by bacteria slowly penetrate through the outer layers of enamel to create a subsurface demineralization (caries), while extrinsic acids lead to a dissolution of the enamel’s outer layers (erosion). Enamel crystallites and prisms can be analyzed, when using SEM [73Colombo M, Beltrami R, Rattalino D, Mirando M, Chiesa M, Poggio C. Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study. Ann Stomatol (Roma) 2017; 7(3): 38-45.
[PMID: 28149449]
]. In erosion, HAP can cover the demineralized surfaces and might also directly remineralize the underlying demineralized tissue. In vitro experiments using clean enamel surfaces indicate that HAP particles attach to the surface of eroded teeth not only by electrochemical forces, but directly form solid interfaces between HAP crystallites from the particles and the enamel [24Fabritius-Vilpoux K, Enax J, Herbig M, Raabe D, Fabritius H-O. Quantitative affinity parameters of synthetic hydroxyapatite and enamel surfaces in vitro. Bioinspir Biomim Nan 2019; 0(0): 1-40.
[http://dx.doi.org/10.1680/jbibn.18.00035]
]. Hornby et al. performed several experiments where they used HAP in combination with fluoride treatment [100Hornby K, Evans M, Long M, Joiner A, Laucello M, Salvaderi A. Enamel benefits of a new hydroxyapatite containing fluoride toothpaste Int Dent J 2009; 59(6S1): 325-1.
[http://dx.doi.org/10.1002/idj.2009.59.6s1.325]
]. However, one experiment was performed using 45Ca-labelled HAP to determine Ca-uptake from a HAP slurry after an erosive attack. While the Ca2+-uptake from HAP with sound surfaces was 1.05 mg/mm2, Ca2+-uptake with demineralized enamel was three times higher (3.16 mg/mm2). The authors concluded that this indicates an availability of Ca2+-ions from HAP for remineralization [100Hornby K, Evans M, Long M, Joiner A, Laucello M, Salvaderi A. Enamel benefits of a new hydroxyapatite containing fluoride toothpaste Int Dent J 2009; 59(6S1): 325-1.
[http://dx.doi.org/10.1002/idj.2009.59.6s1.325]
].

In conclusion, HAP can form a protective layer on tooth surfaces and remineralize eroded enamel and dentin [48Lelli M, Putignano A, Marchetti M, et al. Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: A comparative in vivo study. Front Physiol 2014; 5: 333.
[http://dx.doi.org/10.3389/fphys.2014.00333] [PMID: 25249980]
, 71Tschoppe P, Zandim DL, Martus P, Kielbassa AM. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. J Dent 2011; 39(6): 430-7.
[http://dx.doi.org/10.1016/j.jdent.2011.03.008] [PMID: 21504777]
, 73Colombo M, Beltrami R, Rattalino D, Mirando M, Chiesa M, Poggio C. Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study. Ann Stomatol (Roma) 2017; 7(3): 38-45.
[PMID: 28149449]
, 74Poggio C, Gulino C, Mirando M, Colombo M, Pietrocola G. Protective effect of zinc-hydroxyapatite toothpastes on enamel erosion: An in vitro study. J Clin Exp Dent 2017; 9(1): e118-22.
[PMID: 28149475]
, 99Poggio C, Gulino C, Mirando M, Colombo M, Pietrocola G. Preventive effects of different protective agents on dentin erosion: An in vitro investigation. J Clin Exp Dent 2017; 9(1): e7-e12.
[PMID: 28149456]
]. By forming a “sacrificial layer”, acidic attacks will not directly demineralize the teeth. Additionally, HAP leads to a shift of the solubility equilibrium (Fig. 6). Regular use of HAP is needed to cope with regular acidic challenges.

Fig. (6)
(A) shows a caries attack in the absence of HAP. The natural tooth will get demineralized at a pH around < 5.5. After a certain time, saliva will clear the acids and salivary ions will lead to a (partial) remineralization of the enamel. (B) shows the same conditions, but in the presence of extrinsic HAP (from toothpaste or mouth rinse), the dissolution of the natural enamel is decreased. Due to the presence of HAP, and a change of the solubility equilibrium, the natural enamel will be protected [115Min JH, Kwon HK, Kim BI. The addition of nano-sized hydroxyapatite to a sports drink to inhibit dental erosion: In vitro study using bovine enamel J Dent 2011; 39(9): 629-35.
[http://dx.doi.org/10.1016/j.jdent.2011.07.001] [PMID: 21763390]
, 116Min JH, Kwon HK, Kim BI. Prevention of dental erosion of a sports drink by nano-sized hydroxyapatite in situ study Int J Paediatr Dent 2015; 25(1): 61-9.
[http://dx.doi.org/10.1111/ipd.12101] [PMID: 24628844]
].


3.4. Studies and Mechanisms of HAP in Preventing Dentin Hypersensitivity

3.4.1. Studies

(15) Hu et al. conducted a systematic review and a meta-analysis that proved the effect of HAP in relieving dentin hypersensitivity [27Hu M-L, Zheng G, Zhang Y-D, Yan X, Li X-C, Lin H. Effect of desensitizing toothpastes on dentine hypersensitivity: A systematic review and meta-analysis. J Dent 2018; 75: 12-21.
[http://dx.doi.org/10.1016/j.jdent.2018.05.012] [PMID: 29787782]
]. The authors searched five databases for randomized controlled trials investigating dentin hypersensitivity pain relief. Risk of bias was assessed following the Cochrane guidelines. Confidence intervals and evidence were also calculated in this study. Particulate HAP was evidentially proven in relieving dentin hypersensitivity [27Hu M-L, Zheng G, Zhang Y-D, Yan X, Li X-C, Lin H. Effect of desensitizing toothpastes on dentine hypersensitivity: A systematic review and meta-analysis. J Dent 2018; 75: 12-21.
[http://dx.doi.org/10.1016/j.jdent.2018.05.012] [PMID: 29787782]
].

Amaechi et al. performed an (16)in situ study [28Amaechi BT, Mathews SM, Ramalingam K, Mensinkai PK. Evaluation of nanohydroxyapatite-containing toothpaste for occluding dentin tubules. Am J Dent 2015; 28(1): 33-9.
[PMID: 25864240]
] and an (17) in vivo study [29Amaechi BT, Lemke KC, Saha S, Gelfond J. Clinical efficacy in relieving dentin hypersensitivity of nanohydroxyapatite-containing cream: A randomized controlled trial. Open Dent J 2018; 12: 572-85.
[http://dx.doi.org/10.2174/1874210601812010572] [PMID: 30288181]
] in the field of dentin hypersensitivity using HAP. (16) The in situ study tested the occlusion of dentin tubules after application of either 10% or 15% HAP [28Amaechi BT, Mathews SM, Ramalingam K, Mensinkai PK. Evaluation of nanohydroxyapatite-containing toothpaste for occluding dentin tubules. Am J Dent 2015; 28(1): 33-9.
[PMID: 25864240]
]. Overall, 20 participants per group were recruited to wear human dentine blocks for at least 14 days. Additionally, a toothpaste containing fluoride and another containing NovaMin were also tested. Untreated blocks were used as control. After 7 and 14 days, respectively, dentine occlusion was visualized using SEM. Both tested concentrations showed after 7 and 14 days a higher degree (up to 50%) of completely occluded dentin tubules than the non-HAP toothpastes. In the 15% HAP-group, the test surfaces were 100% covered with a precipitate layer [28Amaechi BT, Mathews SM, Ramalingam K, Mensinkai PK. Evaluation of nanohydroxyapatite-containing toothpaste for occluding dentin tubules. Am J Dent 2015; 28(1): 33-9.
[PMID: 25864240]
]. (17) These results were confirmed by the clinical trial from the same group where reduction of dentin hypersensitivity was investigated [29Amaechi BT, Lemke KC, Saha S, Gelfond J. Clinical efficacy in relieving dentin hypersensitivity of nanohydroxyapatite-containing cream: A randomized controlled trial. Open Dent J 2018; 12: 572-85.
[http://dx.doi.org/10.2174/1874210601812010572] [PMID: 30288181]
].

(18) Huettemann and Doenges published already in the year 1987 a double blind clinical trial using different toothpastes with HAP particles of different diameters [101Huettemann RW, Doenges H. Untersuchungen zur Therapie überempfindlicher Zahnhälse mit Hydroxylapatit. Dtsch Zahnarztl Z 1987; 42: 486-8.]. 140 patients with dentine hypersensitivity were recruited. Study duration was four weeks and sensitivity was clinically tested using standardized tests (i.e. cold stimulus). The test pastes containing HAP (diameter 2 µm and 6 µm) were compared to a placebo. While in the placebo-group no improvement could be measured, 90% of the HAP-group reported an improvement of dentin hypersensitivity already after 3-5 days. 50% of the HAP-group were pain-free within the study-period (four weeks) [101Huettemann RW, Doenges H. Untersuchungen zur Therapie überempfindlicher Zahnhälse mit Hydroxylapatit. Dtsch Zahnarztl Z 1987; 42: 486-8.]. These results were confirmed by several other clinical trials using HAP-containing formulations for relieving dentin hypersensitivity [75Orsini G, Procaccini M, Manzoli L, Giuliodori F, Lorenzini A, Putignano A. A double-blind randomized-controlled trial comparing the desensitizing efficacy of a new dentifrice containing carbonate /hydroxyapatite nanocrystals and a sodium fluoride /potassium nitrate dentifrice. J Clin Periodontol 2010; 37(6): 510-7.
[http://dx.doi.org/10.1111/j.1600-051X.2010.01558.x] [PMID: 20507 374]
, 76Orsini G, Procaccini M, Manzoli L, et al. A 3-day randomized clinical trial to investigate the desensitizing properties of three dentifrices. J Periodontol 2013; 84(11): e65-73.
[http://dx.doi.org/10.1902/jop.2013.120697] [PMID: 23489232]
, 78Vano M, Derch G, Barone A, Covani U. Effectiveness of nano-hydroxyapatite toothpaste in reducing dentin hypersensitivity: A double-blind randomized controlled trial. Quint int 2014; 45: 703-11., 102Vano M, Derchi G, Barone A, Pinna R, Usai P, Covani U. Reducing dentine hypersensitivity with nano-hydroxyapatite toothpaste: A double-blind randomized controlled trial. Clin Oral Investig 2017.
[PMID: 28361171]
].

(19) Hiller et al. tested the in vitro permeability of dentin after application of particulate HAP [77Hiller K-A, Buchalla W, Grillmeier I, Neubauer C, Schmalz G. In vitro effects of hydroxyapatite containing toothpastes on dentin permeability after multiple applications and ageing Sci Rep 2018; 8(1): 4888.
[http://dx.doi.org/10.1038/s41598-018-22764-1] [PMID: 29559639]
]. For this, they used bovine dentin slabs where a HAP-toothpaste was applied. Subsequently, the hydraulic conductance was tested and used as a measure for the degree of occlusion of the dentin tubules. Permeability was significantly reduced after application of all tested toothpastes, including the HAP-application [77Hiller K-A, Buchalla W, Grillmeier I, Neubauer C, Schmalz G. In vitro effects of hydroxyapatite containing toothpastes on dentin permeability after multiple applications and ageing Sci Rep 2018; 8(1): 4888.
[http://dx.doi.org/10.1038/s41598-018-22764-1] [PMID: 29559639]
].

3.4.2. Mechanisms

The mechanisms of HAP in preventing dentin hypersensitivity can be summarized as follows: In exposed dentin the dentinal tubules are open towards the oral cavity and to the pulp. Thus, external stimuli can propagate through the dentinal fluid directly to the nerve tissue in the pulp via the odontoblastic processes located in dentinal tubules. Since the applied HAP-particles have a high polarity, they are able to bind both to collagen and hydroxyapatite from dentin. Thus, these particles will attach to dentinal surfaces and eventually occlude exposed dentinal tubule openings if they are smaller than the tubule diameter. The size of tubules/diameter close to the tooth surface or the DEJ, respectively, is about 3.5 µm [103Lenzi TL, Guglielmi CdeA, Arana-Chavez VE, Raggio DP. Tubule density and diameter in coronal dentin from primary and permanent human teeth. Microsc Microanal 2013; 19(6): 1445-9.
[http://dx.doi.org/10.1017/S1431927613012725] [PMID: 23947480]
]. Most oral care products contain HAP-particles of sizes between 0.1 and 10 µm [38Gillam DG. Dentine hypersensitivity: Advances in diagnosis, management, and treatment 2015.]. Particles need to have diameters <5 µm to reliably occlude dentinal tubules [38Gillam DG. Dentine hypersensitivity: Advances in diagnosis, management, and treatment 2015.]. HAP particles close the tubules by being pressed into the tubule openings during for instance brushing. Within a certain time, HAP-particles that have occluded tubules will bio-chemically bind to the collagen-rich dentin and bio-chemically fuse with the inner mineral lining of the tubule. Deposited HAP soon gets mineralized by attracting calcium and phosphate ions from saliva. Mineralization will lead to fewer open tubules, and consequently less possibilities for external stimuli to induce pain. When using HAP containing oral care products regularly, dentinal tubules can be completely occluded due to the filling effect building up over time and the concurrent formation of a protective layer on the dentin. As positive side effect, dentin might also be protected from acidic attacks by the mineralized deposited HAP that can act as sacrificial layer.

In conclusion, HAP is known for decades to reduce dentin hypersensitivity. Several studies have shown a significant improvement of clinical parameters when using HAP-based oral care products, which is confirmed by a recently published meta-analysis [27Hu M-L, Zheng G, Zhang Y-D, Yan X, Li X-C, Lin H. Effect of desensitizing toothpastes on dentine hypersensitivity: A systematic review and meta-analysis. J Dent 2018; 75: 12-21.
[http://dx.doi.org/10.1016/j.jdent.2018.05.012] [PMID: 29787782]
].

3.5. Studies and Mechanisms of HAP in Promoting Tooth Whitening

3.5.1. Studies

Tooth whitening becomes more and more popular, because the social demand for whiter and brighter smiles is increasing [104Christensen GJ. Are snow-white teeth really so desirable? J Am Dent Assoc 2005; 136(7): 933-5.
[http://dx.doi.org/10.14219/jada.archive.2005.0295] [PMID: 16060 475]
]. Beside in-office bleaching, several oral care products for home use show whitening properties [105Carey CM. Tooth whitening: What we now know. J Evid Based Dent Pract 2014; 14(Suppl.): 70-6.
[http://dx.doi.org/10.1016/j.jebdp.2014.02.006] [PMID: 24929591]
-108Joiner A. Whitening toothpastes: A review of the literature. J Dent 2010; 38(Suppl. 2): e17-24.
[http://dx.doi.org/10.1016/j.jdent.2010.05.017] [PMID: 20562012]
]. Most whitening toothpastes on the market are characterized by high abrasiveness. Their effect relies on removing the outermost stained layer of enamel which may lead to increased roughness or other side effects, such as dentin hypersensitivity [91Enax J, Epple M. Die Charakterisierung von Putzkörpern in Zahnpasten. Dtsch Zahnarztl Z 2018; 73: 116-24., 104Christensen GJ. Are snow-white teeth really so desirable? J Am Dent Assoc 2005; 136(7): 933-5.
[http://dx.doi.org/10.14219/jada.archive.2005.0295] [PMID: 16060 475]
, 105Carey CM. Tooth whitening: What we now know. J Evid Based Dent Pract 2014; 14(Suppl.): 70-6.
[http://dx.doi.org/10.1016/j.jebdp.2014.02.006] [PMID: 24929591]
, 109da Silva EM. Maia JNdSMD, Mitraud CG, Russo JdES, Poskus LT, Guimarães JGA. Can whitening toothpastes maintain the optical stability of enamel over time? J Appl Oral Sci 2018; 26e20160460
[http://dx.doi.org/10.1590/1678-7757-2016-0460]
]. In addition to an increased amount of abrasives, toothpastes often use phosphate-systems for stain removal [106Soeteman GD, Valkenburg C, Van der Weijden GA, Van Loveren C, Bakker E, Slot DE. Whitening dentifrice and tooth surface discoloration-A systematic review and meta-analysis. Int J Dent Hyg 2018; 16(1): 24-35.
[http://dx.doi.org/10.1111/idh.12289] [PMID: 28573755]
, 107Schemehorn BR, Moore MH, Putt MS. Abrasion, polishing, and stain removal characteristics of various commercial dentifrices in vitro J Clin Dent 2011; 22(1): 11-8.
[PMID: 21290981]
]. However, only extrinsic stain can be removed when using whitening toothpastes. (20), (21), (22), (23), (24) There are studies showing an effect of HAP with respect to tooth whitening [110Dabanoglu A, Wood C, García-Godoy F, Kunzelmann KH. Whitening effect and morphological evaluation of hydroxyapatite materials. Am J Dent 2009; 22(1): 23-9.
[PMID: 19281109]
-114Bommer C, Flessa HP, Xu X, Kunzelmann KH. Hydroxyapatite and self-assembling peptide matrix for non-oxidizing tooth whitening. J Clin Dent 2018; 29(2): 57-63.
[PMID: 30211992]
] and their results are promising. Basically, HAP acts as cleaning agent, but in contrast to other abrasives used for tooth whitening products (i.e. perlite and alumina) it has the same hardness as enamel [91Enax J, Epple M. Die Charakterisierung von Putzkörpern in Zahnpasten. Dtsch Zahnarztl Z 2018; 73: 116-24.]. Consequently, HAP does not lead to excessive enamel and dentin abrasion. (20) Dabanoglu et al. showed whitening properties of HAP. HAP-particles remained stable on the tooth surface after application of hydrodynamic shear force [110Dabanoglu A, Wood C, García-Godoy F, Kunzelmann KH. Whitening effect and morphological evaluation of hydroxyapatite materials. Am J Dent 2009; 22(1): 23-9.
[PMID: 19281109]
]. (21), (22), (23) The whitening-effect can be attributed to an attachment of HAP to the tooth surface, rather than a polishing process [111Jin J, Xu X, Lai G, Kunzelmann KH. Efficacy of tooth whitening with different calcium phosphate-based formulations. Eur J Oral Sci 2013; 121(4): 382-8.
[http://dx.doi.org/10.1111/eos.12063] [PMID: 23841792]
-113Niwa M, Sato T, Li W, Aoki H, Aoki H, Daisaku T. Polishing and whitening properties of toothpaste containing hydroxyapatite. J Mater Sci Mater Med 2001; 12(3): 277-81.
[http://dx.doi.org/10.1023/A:1008927502523] [PMID: 15348313]
]. (23) The whitening effect of HAP could also originate from physical properties related to its particulate structure [112Kim BI, Jeong SH, Jang SO, Kim KN, Kwon HK, Park YD. Tooth whitening effect of toothpastes containing nano-hydroxyapatite. Key Eng Mater 2006; 309-311: 541-4.
[http://dx.doi.org/10.4028/www.scientific.net/KEM.309-311.541]
]. (24) In vivo results showed whitening effects of HAP-particles [114Bommer C, Flessa HP, Xu X, Kunzelmann KH. Hydroxyapatite and self-assembling peptide matrix for non-oxidizing tooth whitening. J Clin Dent 2018; 29(2): 57-63.
[PMID: 30211992]
]. These results were verified with in vitro testing: Whitening properties of HAP are mainly based on diffuse reflection leading to optical whitening effects [114Bommer C, Flessa HP, Xu X, Kunzelmann KH. Hydroxyapatite and self-assembling peptide matrix for non-oxidizing tooth whitening. J Clin Dent 2018; 29(2): 57-63.
[PMID: 30211992]
].

3.5.2. Mechanisms

Besides removing stain mechanically from tooth surfaces during routine oral hygiene, HAP particles might also bind proteins which often lead to discolorations. These would then be removed together with the particles during the cleaning process. The protective layer formed by HAP particles on tooth surfaces appears inherently white in colour as long as it remains firmly attached to the tooth surface. The size and irregular orientation of the HAP crystallites constituting the particles makes them ideal scatterers within the wavelength range of visible light. The resulting reflection of light makes the tooth surface appear even brighter white than natural, untreated tooth surfaces, especially if they are naturally darker tainted or contain incorporated amounts of extrinsic stains.

In conclusion, HAP particles are promising agents for tooth whitening, as they do not lead to tooth abrasion. HAP particles might remove staining proteins, cover tooth surfaces and enhance the perceived whiteness by scattering and reflection of light.

4. MODES OF ACTION OF HAP IN PREVENTIVE ORAL HEALTH CARE: CONCLUSIONS AND OUTLOOK

In contrast to other active ingredients in oral care, HAP represents a multifunctional biomimetic agent whose different effects in preventive oral health care are based both on its bio-chemical activity and the ability to physically interact with the oral cavity environment due to its particulate nature. The modes of action of HAP-particles in preventive oral care derived from data of the analyzed publications are summarized in Table 1. They are mainly based on the following principles:

Table 1
Multifunctionality of HAP and correlation of the proposed modes of action with the described preventive properties of HAP. The numbers indicated in the table refer to the respective sections mentioned in the text.


(I) Physical principles (e.g. attachment of HAP-particles to the tooth surface and cleaning properties) [23Kensche A, Holder C, Basche S, Tahan N, Hannig C, Hannig M. Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ. Arch Oral Biol 2017; 80: 18-26.
[http://dx.doi.org/10.1016/j.archoralbio.2017.03.013] [PMID: 2836 4672]
, 24Fabritius-Vilpoux K, Enax J, Herbig M, Raabe D, Fabritius H-O. Quantitative affinity parameters of synthetic hydroxyapatite and enamel surfaces in vitro. Bioinspir Biomim Nan 2019; 0(0): 1-40.
[http://dx.doi.org/10.1680/jbibn.18.00035]
, 34Kani K, Kani M, Isozaki A, Shintani H, Ohashi T, Tokumoto T. Effect of apatite-containing dentifrices on dental caries in school children. J Dent Health 1989; 19: 104-9.
[http://dx.doi.org/10.5834/jdh.39.104]
, 48Lelli M, Putignano A, Marchetti M, et al. Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: A comparative in vivo study. Front Physiol 2014; 5: 333.
[http://dx.doi.org/10.3389/fphys.2014.00333] [PMID: 25249980]
, 91Enax J, Epple M. Die Charakterisierung von Putzkörpern in Zahnpasten. Dtsch Zahnarztl Z 2018; 73: 116-24.].

(II) Bio-chemical principles (e.g. source of calcium and phosphate ions [100Hornby K, Evans M, Long M, Joiner A, Laucello M, Salvaderi A. Enamel benefits of a new hydroxyapatite containing fluoride toothpaste Int Dent J 2009; 59(6S1): 325-1.
[http://dx.doi.org/10.1002/idj.2009.59.6s1.325]
] and formation of an interface between HAP-particles and the enamel surface) [24Fabritius-Vilpoux K, Enax J, Herbig M, Raabe D, Fabritius H-O. Quantitative affinity parameters of synthetic hydroxyapatite and enamel surfaces in vitro. Bioinspir Biomim Nan 2019; 0(0): 1-40.
[http://dx.doi.org/10.1680/jbibn.18.00035]
].

(III) Biological principles (e.g. HAP-particles interact with microorganisms [reduction of bacterial colonization to tooth surfaces]) [23Kensche A, Holder C, Basche S, Tahan N, Hannig C, Hannig M. Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ. Arch Oral Biol 2017; 80: 18-26.
[http://dx.doi.org/10.1016/j.archoralbio.2017.03.013] [PMID: 2836 4672]
].

Several studies show the efficacy of HAP with respect to prevention and remineralization of caries, biofilm control, protection against erosion, and relief of dentin hypersensitivity [3Enax J, Epple M. Synthetic hydroxyapatite as a biomimetic oral care agent. Oral Health Prev Dent 2018; 16(1): 7-19.
[PMID: 29335686]
, 6Meyer F, Amaechi BT, Fabritius HO, Enax J. Overview of calcium phosphates used in biomimetic oral care. Open Dent J 2018; 12: 406-23.
[http://dx.doi.org/10.2174/1874210601812010406] [PMID: 29988215]
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[http://dx.doi.org/10.1016/j.archoralbio.2017.03.013] [PMID: 2836 4672]
, 25Harks I, Jockel-Schneider Y, Schlagenhauf U, et al. Impact of the daily use of a microcrystal hydroxyapatite dentifrice on de novo plaque formation and clinical/microbiological parameters of periodontal health. A randomized trial. PLoS One 2016; 11(7)e0160142
[http://dx.doi.org/10.1371/journal.pone.0160142] [PMID: 27467683]
, 26Schlagenhauf U, Kunzelmann KH, Hannig C, et al. Impact of a non-fluoridated microcrystalline hydroxyapatite dentifrice on enamel caries progression in highly caries-susceptible orthodontic patients: A randomized, controlled 6-month trial. J Investig Clin Dent 2019; 10(2)e12399
[http://dx.doi.org/10.1111/jicd.12399] [PMID: 30701704]
, 35Najibfard K, Ramalingam K, Chedjieu I, Amaechi BT. Remineralization of early caries by a nano-hydroxyapatite dentifrice. J Clin Dent 2011; 22(5): 139-43.
[PMID: 22403978]
, 36Roveri N, Battistella E, Foltran I, Foresti E, Iafisco M, Lelli M, et al. Synthetic biomimetic carbonate-hydroxyapatite nanocrystals for enamel remineralization. Adv Mat Res 2008; 47-50: 821-4.
[http://dx.doi.org/10.4028/www.scientific.net/AMR.47-50.821]
, 41Hegazy SA, Salama IR. Antiplaque and remineralizing effects of Biorepair mouthwash: A comparative clinical trial. Pediatr Dent J 2016; 26: 89-94.
[http://dx.doi.org/10.1016/j.pdj.2016.05.002]
, 48Lelli M, Putignano A, Marchetti M, et al. Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: A comparative in vivo study. Front Physiol 2014; 5: 333.
[http://dx.doi.org/10.3389/fphys.2014.00333] [PMID: 25249980]
, 71Tschoppe P, Zandim DL, Martus P, Kielbassa AM. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. J Dent 2011; 39(6): 430-7.
[http://dx.doi.org/10.1016/j.jdent.2011.03.008] [PMID: 21504777]
, 73Colombo M, Beltrami R, Rattalino D, Mirando M, Chiesa M, Poggio C. Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study. Ann Stomatol (Roma) 2017; 7(3): 38-45.
[PMID: 28149449]
-75Orsini G, Procaccini M, Manzoli L, Giuliodori F, Lorenzini A, Putignano A. A double-blind randomized-controlled trial comparing the desensitizing efficacy of a new dentifrice containing carbonate /hydroxyapatite nanocrystals and a sodium fluoride /potassium nitrate dentifrice. J Clin Periodontol 2010; 37(6): 510-7.
[http://dx.doi.org/10.1111/j.1600-051X.2010.01558.x] [PMID: 20507 374]
, 77Hiller K-A, Buchalla W, Grillmeier I, Neubauer C, Schmalz G. In vitro effects of hydroxyapatite containing toothpastes on dentin permeability after multiple applications and ageing Sci Rep 2018; 8(1): 4888.
[http://dx.doi.org/10.1038/s41598-018-22764-1] [PMID: 29559639]
, 102Vano M, Derchi G, Barone A, Pinna R, Usai P, Covani U. Reducing dentine hypersensitivity with nano-hydroxyapatite toothpaste: A double-blind randomized controlled trial. Clin Oral Investig 2017.
[PMID: 28361171]
]. Furthermore, HAP particles within oral care products can be used for tooth whitening [110Dabanoglu A, Wood C, García-Godoy F, Kunzelmann KH. Whitening effect and morphological evaluation of hydroxyapatite materials. Am J Dent 2009; 22(1): 23-9.
[PMID: 19281109]
, 114Bommer C, Flessa HP, Xu X, Kunzelmann KH. Hydroxyapatite and self-assembling peptide matrix for non-oxidizing tooth whitening. J Clin Dent 2018; 29(2): 57-63.
[PMID: 30211992]
]. Due to its similarity to the tooth mineral phase, HAP is considered a biocompatible and biomimetic agent, and as such the applicable dosage is not limited by health concerns as it is the case with fluoride in fluorosis [42Epple M. Review of potential health risks associated with nanoscopic calcium phosphate. Acta Biomater 2018; 77: 1-14.
[http://dx.doi.org/10.1016/j.actbio.2018.07.036] [PMID: 30031162]
, 44Limeback H, Robinson C. Fluoride therapy Comprehensive preventive dentistry 2012; 251-82.
[http://dx.doi.org/10.1002/9781118703762]
]. Thus, oral care products with HAP can be used for all age-groups, including children. From the studies investigated and discussed it became evident that the effect of HAP on each of the clinical conditions relies on more than one mode of action. Therefore, HAP may experience future uses in other fields too. Based on the current state of the art, it can be concluded that HAP can be used as active biomimetic ingredient in preventive oral care for several indications. For future research, the physical, bio-chemical, and biological mechanisms of action need to be investigated in much greater detail using suitable in vitro and in vivo approaches, especially the interactions of HAP particles with teeth under conditions that mimic the situation in the oral cavity including pellicle, biofilms, and the entire spectrum of clinical conditions. This will enable to optimize the intrinsic modes of action and thus the efficiency of HAP by tailoring formulations of applications and the physical and chemical properties of the particles themselves.

In summary, HAP is a promising multifunctional biomimetic active ingredient with verified efficacy for different oral health concerns.

CONSENT FOR PUBLICATION

Not applicable.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

The authors thank Dr. Med. Dent. Barbara Simader for helpful discussions.

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