Rastrelli Gianbattista, Rastrelli Francesco, Bifulco Guglielmo , Kalichem Srl04.01.20
With an emphasis on tooth health, oral care active ingredients have evolved significantly in the past decade. Traditional benchmark actives are fluoride-based salts,1 but today, remineralizers are based on hydroxyapatite (HAP),2,3 the mineral physiologically found in enamel and dentin, and its derivatives. This article details the use of biomimetic materials based on HAP and its derivatives, commercialized by Kalichem srl, under the brand Kalident. Tooth remineralization usually relies on two strategies: the first refers to fluoride-based actives, the latter to Ca++ and PO4—sources.4 In both cases, the functional target is to promote the in vivo formation of physiological HAP and fluorohydroxyapatite (F-HAP).
The tooth is composed of a rich and constantly evolving mineral phase, characterized by the presence of HAP in enamel and dentin, where it represents, respectively, 97% and 70% of total composition. The remineralization is the process by which the tooth mineral phase is constantly subject to a restoring phase, as a mechanism of physiological homeostasis and balance against daily demineralization.5,6,7
This article details three different remineralizing solutions (not based on nano material) with complementary mechanisms inspired by the biomimetic philosophy.8 “Biomimetic” is a term pointing out the study of the formation, structure or function of biologically produced substances and biological mechanisms and processes, for the purpose of synthesizing similar products which mimic natural ones.9 The following proposals meet the biomimetic concept through different modalities and efficiently respond to the functional requirements to counteract the onset of events like caries, hypersensitivity and whitening boosting.
Materials and Methods
The biomimetic HAP compositional analysis was carried through X-ray diffraction. Tests were carried out to evaluate its hypersensitivity reduction action through subjective evaluation, when used in a toothpaste formulation at 5% on 50 subjects, two applications a day, for 28 days.
Subjective evaluation on a toothpaste containing biomimetic HAP at 4% were assessed to quantify its whitening action. In vivo tests were made on a toothpaste containing biomimetic HAP and fluorhydroxyapatite at 6%, against a control reference containing a benchmark remineralizing active + 1,500ppm of fluoride. After a three-day treatment on 45 people with positive anamnesis for hypersensitivity, a tactile stimulus was applied on subjects’ teeth through a blunt probe. The hypersensitivity score was reported according to VAS (visual analogic scale): the score was given through ratings from 0 to 1 no pain, 2–3 for mild, 4–6 for moderate, and 7–10 for severe pain. SEM images were taken out of ex vivo tests on bovine teeth to observe the functionalized amorphous calcium phosphate (F-ACP) complex behavior. The images are observable with different resolution (5.0 and 15.0K), the differences between teeth demineralized after acid treatment and after 1–2 week treatments with a toothpaste including 10% of F-ACP solution, to highlight the new mineral phase deposition on the tooth.
Measurements of Vickers microhardness were made comparing F-ACP against a benchmark reference by a prior demineralizing treatment followed by a hardness assessment, carried through a diamond indenter and a light load to produce tooth indentation. The depth of indentation was converted into the hardness value.
The tooth fluoride delivery was analyzed by Energy Dispersive X-ray Analysis, comparing the results of the complex against a blend of the single actives (ACP and sodium fluoride).
Biomimetic HAP
The biomimetic HAP differentiates from the stoichiometric HAP (which has the defined chemical formula [Ca5(PO4)3(OH)], and is found in nature in mineral form or can be industrially produced) for its different composition and behavior.
Biomimetic HAP shows variable stoichiometry, related to its Ca++ and OH– sites functionalization; its surface shows a rough layer resulting from the vacancies in the Ca++ and OH– sites, replaced by other ions such as CO3– and Mg++; such a roughness comprises surface irregularities in the order of single unit cells size,10 and corresponds to the tendency to increase protein binding in the bioremineralization.
Previous studies confirmed that rough surfaces improve the biocompatibility and adhesion of the material,10 confirming the HAP capability to easily get linked to substrates like tooth biofilm glycoproteins11 and microlesions. The different surface nature differentiates stoichiometric and biomimetic HAP for their crystalline index, too.
The crystalline phase is more accentuated in the stoichiometric HAP, determining different reactivity:10 substances with inferior crystalline nature, such as Biomimetic HAP, increase the release of Ca++ and PO4– ions available for the remineralization cycle, intending for remineralization the capability of releasing such elements in the saliva on a balanced, pH dependent and time-prolonged basis. The Biomimetic HAP reactivity is a key element exploited for bone prosthesis design,11 underscoring its biocompatibility and safety.
Previous studies suggest HAP more efficiently promotes mineral deposition on the tooth in comparison to benchmark Ca++ and PO4– based materials.13 The Biomimetic HAP presented here was subject to several tests to identify its chemical nature and functional features when included in toothpaste.
Figure 1 shows x-ray diffraction analysis comparison between biomimetic, physiological and stoichiometric HAP. Biomimetic HAP gives peaks comparable to the HAP expressed in the human body, confirming its mimic nature, particularly in relationship to the detection of CO3– ions in the OH– vacant sites. This pattern is in contrast to the significantly different peaks observed with the stoichiometric form, suggesting significant differences in the compositional background of the product.
The reactive nature of biomimetic HAP explains its tendency to release in the saliva-remineralizing ions with a pH dependent mechanism: solubility tests highlighted very low reactivity at pH values around 7.0, and a slow and steady release at values below 5.5, which are critical for demineralization.
Biomimetic HAP promotes tooth remineralizing action,5,14 through a multi-step mechanism implying its high compatibility tooth adhesion, the formation of a mineral phase “pool” deposited on the tooth, and a time prolonged and pH dependent ions release to the saliva to boost the remineralization process.
In vivo tests using a toothpaste containing biomimetic HAP show consistent reduction of dentin hypersensitivity (Figure 2); further evaluations highlighted a perceived high efficacy in 80% of the panel, with no cases of inflammation or low tolerance in gums and oral mouth reported. Additional studies also prove increased efficacy in tooth whitening, confirming previous studies results.15,16 Further data obtained from the same panel prove hypersensitivity reduction after drinking hot beverages in 46.6% of the panelists and in 50% in the case of cold beverages.
Fluorohydroxyapatite (F-HAP)
F-HAP is a naturally occurring mineral found in various rocks, laterilic soils17 and shark tooth enamel, where it represents most of the mineral phase.18 F-HAP provides more resistance against acid attack, given its low solubility.19 It boasts Ksp value of 2.34 x 10–59, implying a higher resistance to acid erosion and low ions dissolution,20 which only becomes tangible below pH 4.0. The F-HAP deposition21 on the tooth mineral phase, promoted by its biomimetic nature and optimized particle size, improves the tooth resistance against acid erosion, enamel strengthening,22 avoiding excessive mineral loss during the daily oral pH swings, ranging between pH 4.0–7.0.23 Regarding F-HAP biomimetic aspects, its use implies the deposition of a thick layer firmly adhered on the tooth, even when mechanical stress for separation is applied or tooth fracture is performed, suggesting a strong chemical interaction at the interface; dentistry radiographic images24 revealed no significant difference on radiopacity between the F-HAP layer formed on the tooth and the enamel, suggesting similar density on the enamel surface, backing up its biomimetic nature. Moreover, the F-HAP mineral phase presence on the tooth represents a hostile environment for the bacterial biofilm formation, given the recognized fluoride features in inhibiting microbes glycolisis.25,26 For these reasons, F-HAP is a candidate for inclusion in toothpaste with specific action on hyper-sensitivity reduction and contextual multi-level supporting action against caries.
In vivo tests27 were carried out through VAS (Figure 3), comparing a toothpaste with biomimetic HAP and F-HAP at 6%, against a control toothpaste (containing a remineralizing benchmark active ingredient plus 1500 ppm fluoride), and a placebo. The tests evaluated the anti-hypersensitivity action after 3 days, and demonstrated the higher efficiency of the biomimetic HAP/F-HAP toothpaste and the benchmark over the placebo. The highest hypersensitivity reduction was observed with the HAP/F-HAP paste.
F-ACP Complex
Functionalized Amorphous Calcium Phosphate (F-ACP) complex is based on amorphous calcium phosphate functionalized with citrate, fluoride and carbonate ions through a patented technology28 that foresees the citrates coating of the complex and the inclusion of fluoride and carbonates inside the ACP phase, aimed to increase actives delivery on the tooth.
Citrates are physiologically occurring on tooth organic matrix with a content up to 5% and play a key role in mineral tissues.29,30 Their presence is a key biomimetic factor, optimizing the complex link to the tooth surface and minimizing the active ingredient loss with rinsing water during toothbrushing. The complex shows an amorphous status that, in presence of water, is broken31 with massive ions in the saliva in short term: at this level, the ions crystallize in fluorohydroxyapatite and carbonate hydroxyapatite. This mechanism represents a smart solution for professional and daily treatments where intense remineralization is required.32
Ex vivo tests using a paste with F-ACP complex at 10%, showed the remineralizing action of the complex after one and two weeks compared to the tooth demineralized with phosphoric acid at the beginning of the assessment (Figure 4). The results show significant deposition of the mineral phase after one week, and more evident results after two weeks.
Comparative analysis against a control anhydrous toothpaste were carried out measuring the Vickers microhardness. The analysis of the depth indentation showed microhardness values of the F-ACP complex of 110hv against the 87hv value of the benchmark.
EDX analysis was carried out to detect the biomimetic character of the F-ACP through the measurement of the fluoride content found in the tooth, comparing it to a blend of ACP and sodium fluoride. The fluoride amount used in the analyzed systems was the same. The results show a four-fold increase of fluoride deposited after the treatment with the F-ACP complex, with 1.5% w/w detected in the tooth. The results confirm the optimized ions delivery accomplished through the patented biomimetic technology.
Conclusion
Three different approaches in remineralization were taken under evaluation.
The biomimetic HAP with vacancies in OH– sites showed suitability for daily oral care routine to promote a smart, balanced and long-lasting remineralization, aimed to prevent dental hypersensitivity, caries onset and promote tooth whitening effect boosting.
Fluorohydroxyapatite, through biomimetic behavior given by its compatibility to the tooth surface in terms of adhesivity, shows great potential for applications on severe tooth microlesions, due to its resistance to acid attack and its enamel strengthening features, representing a state of the art treatment approach against caries and acid attack.
The F-ACP complex, through a patented technology aimed to optimize the actives delivery on the tooth, allows to formulate anhydrous toothpaste specific for immediate tooth remineralization, and is indicated for professional and daily treatments following dental cleaning, or severe hypersensitivity or anti-caries treatments.
REFERENCES
The tooth is composed of a rich and constantly evolving mineral phase, characterized by the presence of HAP in enamel and dentin, where it represents, respectively, 97% and 70% of total composition. The remineralization is the process by which the tooth mineral phase is constantly subject to a restoring phase, as a mechanism of physiological homeostasis and balance against daily demineralization.5,6,7
This article details three different remineralizing solutions (not based on nano material) with complementary mechanisms inspired by the biomimetic philosophy.8 “Biomimetic” is a term pointing out the study of the formation, structure or function of biologically produced substances and biological mechanisms and processes, for the purpose of synthesizing similar products which mimic natural ones.9 The following proposals meet the biomimetic concept through different modalities and efficiently respond to the functional requirements to counteract the onset of events like caries, hypersensitivity and whitening boosting.
- Biomimetic hydroxyapatite;
- Fluorohydroxyapatite; and
- Amorphous calcium phosphate functionalized with citrates, fluoride and carbonate ions.
Materials and Methods
The biomimetic HAP compositional analysis was carried through X-ray diffraction. Tests were carried out to evaluate its hypersensitivity reduction action through subjective evaluation, when used in a toothpaste formulation at 5% on 50 subjects, two applications a day, for 28 days.
Subjective evaluation on a toothpaste containing biomimetic HAP at 4% were assessed to quantify its whitening action. In vivo tests were made on a toothpaste containing biomimetic HAP and fluorhydroxyapatite at 6%, against a control reference containing a benchmark remineralizing active + 1,500ppm of fluoride. After a three-day treatment on 45 people with positive anamnesis for hypersensitivity, a tactile stimulus was applied on subjects’ teeth through a blunt probe. The hypersensitivity score was reported according to VAS (visual analogic scale): the score was given through ratings from 0 to 1 no pain, 2–3 for mild, 4–6 for moderate, and 7–10 for severe pain. SEM images were taken out of ex vivo tests on bovine teeth to observe the functionalized amorphous calcium phosphate (F-ACP) complex behavior. The images are observable with different resolution (5.0 and 15.0K), the differences between teeth demineralized after acid treatment and after 1–2 week treatments with a toothpaste including 10% of F-ACP solution, to highlight the new mineral phase deposition on the tooth.
Measurements of Vickers microhardness were made comparing F-ACP against a benchmark reference by a prior demineralizing treatment followed by a hardness assessment, carried through a diamond indenter and a light load to produce tooth indentation. The depth of indentation was converted into the hardness value.
The tooth fluoride delivery was analyzed by Energy Dispersive X-ray Analysis, comparing the results of the complex against a blend of the single actives (ACP and sodium fluoride).
Biomimetic HAP
The biomimetic HAP differentiates from the stoichiometric HAP (which has the defined chemical formula [Ca5(PO4)3(OH)], and is found in nature in mineral form or can be industrially produced) for its different composition and behavior.
Biomimetic HAP shows variable stoichiometry, related to its Ca++ and OH– sites functionalization; its surface shows a rough layer resulting from the vacancies in the Ca++ and OH– sites, replaced by other ions such as CO3– and Mg++; such a roughness comprises surface irregularities in the order of single unit cells size,10 and corresponds to the tendency to increase protein binding in the bioremineralization.
Previous studies confirmed that rough surfaces improve the biocompatibility and adhesion of the material,10 confirming the HAP capability to easily get linked to substrates like tooth biofilm glycoproteins11 and microlesions. The different surface nature differentiates stoichiometric and biomimetic HAP for their crystalline index, too.
The crystalline phase is more accentuated in the stoichiometric HAP, determining different reactivity:10 substances with inferior crystalline nature, such as Biomimetic HAP, increase the release of Ca++ and PO4– ions available for the remineralization cycle, intending for remineralization the capability of releasing such elements in the saliva on a balanced, pH dependent and time-prolonged basis. The Biomimetic HAP reactivity is a key element exploited for bone prosthesis design,11 underscoring its biocompatibility and safety.
Previous studies suggest HAP more efficiently promotes mineral deposition on the tooth in comparison to benchmark Ca++ and PO4– based materials.13 The Biomimetic HAP presented here was subject to several tests to identify its chemical nature and functional features when included in toothpaste.
Figure 1 shows x-ray diffraction analysis comparison between biomimetic, physiological and stoichiometric HAP. Biomimetic HAP gives peaks comparable to the HAP expressed in the human body, confirming its mimic nature, particularly in relationship to the detection of CO3– ions in the OH– vacant sites. This pattern is in contrast to the significantly different peaks observed with the stoichiometric form, suggesting significant differences in the compositional background of the product.
The reactive nature of biomimetic HAP explains its tendency to release in the saliva-remineralizing ions with a pH dependent mechanism: solubility tests highlighted very low reactivity at pH values around 7.0, and a slow and steady release at values below 5.5, which are critical for demineralization.
Biomimetic HAP promotes tooth remineralizing action,5,14 through a multi-step mechanism implying its high compatibility tooth adhesion, the formation of a mineral phase “pool” deposited on the tooth, and a time prolonged and pH dependent ions release to the saliva to boost the remineralization process.
In vivo tests using a toothpaste containing biomimetic HAP show consistent reduction of dentin hypersensitivity (Figure 2); further evaluations highlighted a perceived high efficacy in 80% of the panel, with no cases of inflammation or low tolerance in gums and oral mouth reported. Additional studies also prove increased efficacy in tooth whitening, confirming previous studies results.15,16 Further data obtained from the same panel prove hypersensitivity reduction after drinking hot beverages in 46.6% of the panelists and in 50% in the case of cold beverages.
Fluorohydroxyapatite (F-HAP)
F-HAP is a naturally occurring mineral found in various rocks, laterilic soils17 and shark tooth enamel, where it represents most of the mineral phase.18 F-HAP provides more resistance against acid attack, given its low solubility.19 It boasts Ksp value of 2.34 x 10–59, implying a higher resistance to acid erosion and low ions dissolution,20 which only becomes tangible below pH 4.0. The F-HAP deposition21 on the tooth mineral phase, promoted by its biomimetic nature and optimized particle size, improves the tooth resistance against acid erosion, enamel strengthening,22 avoiding excessive mineral loss during the daily oral pH swings, ranging between pH 4.0–7.0.23 Regarding F-HAP biomimetic aspects, its use implies the deposition of a thick layer firmly adhered on the tooth, even when mechanical stress for separation is applied or tooth fracture is performed, suggesting a strong chemical interaction at the interface; dentistry radiographic images24 revealed no significant difference on radiopacity between the F-HAP layer formed on the tooth and the enamel, suggesting similar density on the enamel surface, backing up its biomimetic nature. Moreover, the F-HAP mineral phase presence on the tooth represents a hostile environment for the bacterial biofilm formation, given the recognized fluoride features in inhibiting microbes glycolisis.25,26 For these reasons, F-HAP is a candidate for inclusion in toothpaste with specific action on hyper-sensitivity reduction and contextual multi-level supporting action against caries.
In vivo tests27 were carried out through VAS (Figure 3), comparing a toothpaste with biomimetic HAP and F-HAP at 6%, against a control toothpaste (containing a remineralizing benchmark active ingredient plus 1500 ppm fluoride), and a placebo. The tests evaluated the anti-hypersensitivity action after 3 days, and demonstrated the higher efficiency of the biomimetic HAP/F-HAP toothpaste and the benchmark over the placebo. The highest hypersensitivity reduction was observed with the HAP/F-HAP paste.
F-ACP Complex
Functionalized Amorphous Calcium Phosphate (F-ACP) complex is based on amorphous calcium phosphate functionalized with citrate, fluoride and carbonate ions through a patented technology28 that foresees the citrates coating of the complex and the inclusion of fluoride and carbonates inside the ACP phase, aimed to increase actives delivery on the tooth.
Citrates are physiologically occurring on tooth organic matrix with a content up to 5% and play a key role in mineral tissues.29,30 Their presence is a key biomimetic factor, optimizing the complex link to the tooth surface and minimizing the active ingredient loss with rinsing water during toothbrushing. The complex shows an amorphous status that, in presence of water, is broken31 with massive ions in the saliva in short term: at this level, the ions crystallize in fluorohydroxyapatite and carbonate hydroxyapatite. This mechanism represents a smart solution for professional and daily treatments where intense remineralization is required.32
Ex vivo tests using a paste with F-ACP complex at 10%, showed the remineralizing action of the complex after one and two weeks compared to the tooth demineralized with phosphoric acid at the beginning of the assessment (Figure 4). The results show significant deposition of the mineral phase after one week, and more evident results after two weeks.
Comparative analysis against a control anhydrous toothpaste were carried out measuring the Vickers microhardness. The analysis of the depth indentation showed microhardness values of the F-ACP complex of 110hv against the 87hv value of the benchmark.
EDX analysis was carried out to detect the biomimetic character of the F-ACP through the measurement of the fluoride content found in the tooth, comparing it to a blend of ACP and sodium fluoride. The fluoride amount used in the analyzed systems was the same. The results show a four-fold increase of fluoride deposited after the treatment with the F-ACP complex, with 1.5% w/w detected in the tooth. The results confirm the optimized ions delivery accomplished through the patented biomimetic technology.
Conclusion
Three different approaches in remineralization were taken under evaluation.
The biomimetic HAP with vacancies in OH– sites showed suitability for daily oral care routine to promote a smart, balanced and long-lasting remineralization, aimed to prevent dental hypersensitivity, caries onset and promote tooth whitening effect boosting.
Fluorohydroxyapatite, through biomimetic behavior given by its compatibility to the tooth surface in terms of adhesivity, shows great potential for applications on severe tooth microlesions, due to its resistance to acid attack and its enamel strengthening features, representing a state of the art treatment approach against caries and acid attack.
The F-ACP complex, through a patented technology aimed to optimize the actives delivery on the tooth, allows to formulate anhydrous toothpaste specific for immediate tooth remineralization, and is indicated for professional and daily treatments following dental cleaning, or severe hypersensitivity or anti-caries treatments.
REFERENCES
- ten Cate JM, Contemporary perspective on the use of fluoride products in caries prevention, Br Dent J. 2013 Feb;214(4):161-7. doi: 10.1038/sj.bdj.2013.162
- Tokoko Kani, Mizuo Kani, Effect to Apatite containing dentifrices on dental caries in school children, Journal of dental health- 39, 104-109 (1988)
- Shimura N., Yonemitu O., Yamada et al., Caries prevention and clinical trial of hydroxyapatite containing dentifrices, Dental Journal, 15; 213, 1982
- Cochrane, N. J., Cai, F., Huq, N. L., Burrow, M. F. & Reynolds, E. C., New approaches to enhanced remineralization of tooth enamel, Journal of Dental Research 89, 1187–1197 (2010)
- Neel, E. A. A. et al., Demineralization–remineralization dynamics in teeth and bone, International Journal of Nanomedicine 11, 4743–4763 (2016)
- Koulourides T, Feagin F, Pigman W. - Remineralization of dental enamel by saliva in vitro Ann N Y Acad Sci. 1965 Sep 30;131(2):751-7. PMID: 5214661 DOI: 10.1111/j.1749-6632.1965.tb34839.x
- Arends, J. et al., Rate and mechanism of enamel demineralization in situ, Caries research 26, 18–21 (1992)
- Ruan, Q. & Moradian-Oldak, J. Amelogenin and enamel biomimetics, Journal of Materials Chemistry B 3, 3112–3129 (2015)
- Nathan F Lepora, Paul Verschure, Tony J Prescott, The state of the art in biomimetics, Bioinspir. Biomim. 8 (2013) 013001
- Jaime Gómez-Morales, Michele Iafisco, José Manuel Delgado-López, Stéphanie Sarda, Christophe Drouet, Progress on the preparation of nanocrystalline apatites and surface characterization: Overview of fundamental and applied aspects, Chem. Mater. 2010, 22, 12, 3653-3663
- Souza, Comar et al, Effect of an experimental paste with hydroxyapatite nanoparticles and fluoride on dental demineralization and remineralization in situ. Caries Research 2015;49:499-507
- X. Lin K. de Groot, D. Wang, Q. Hu, D. Wismeijer, and Y. Liu, A review paper on biomimetic calcium phosphate coatings, Open Biomed Eng J. 2015; 9: 56–64. Published online 2015 Feb 27. doi: 10.2174/1874120701509010056
- M.Bajaj, Poornima P., Praveen S et al., Comparison of CPP-ACP, tri-calcium phosphate and hydroxyapatite remineralization of artificial caries like lesions on primary enamel- an in vitro study, The Journ. Of Clinical Pediatric Dentistry Vol.40, Nr 5/2016
- Roveri, N. et al., Surface enamel remineralization: biomimetic apatite nanocrystals and fluoride ions different effects, Journal of Nanomaterials (2009)
- C.Guo, H.Liu, I. Katayama, Effect of hydroxyapatite toothpaste on vital color, Journal of Dental Reearch, 81 Special Issue 2002 A-254
- Niwa M1, Sato T, Li W, Aoki H, Aoki H, Daisaku T., Polishing and whitening properties of toothpaste containing hydroxyapatite, J Mater Sci Mater Med. 2001 Mar;12(3):277-81
- Handbook of mineralogy- 2001-2005 Mineral Data Publishing, version 1
- Adrian Lussi, Elmar Hellwig, Joachim Klimek, Fluorides – mode of action and recommendations for use, Research and Science, October 2011
- HB Pan, BW Darvell, Solubility of calcium fluoride and fluorapatite by solid titration, Arch. Oral. Biol. 52, 861-8
- Nora H. de Leeuw, Resisting the onset of hydroxyapatite dissolution through the incorporation of fluoride, J. Phys. Chem. B 2004, 108, 6, 1809-1811
- H Yamazaki, A Litman, HC Margolis, Effect of fluoride in fluoride on artificial caries lesion progression and repair in human enamel-Regulation of mineral deposition and dissolution under in vivo like conditions. Arch. Oral. Biol., 52(22):110-20
- Featherstone JD, Prevention and reversal of dental caries: role of low level fluoride, Community Dent Oral Epidemiol, 1999 Feb;27(1):31-40
- Bowen WH, The Stephan Curve revisited, Odontology 2013 Jan;101(1):2-8. doi: 10.1007/s10266-012-0092-z. Epub 2012 Dec 6
- Oliveira M, Sander H, Synthetic tooth enamel: SEM characterization of a fluoride hydroxyapatite coating for dentistry applications, Mat. Res. vol.10 no.2 São Carlos Apr./June 2007 http://dx.doi.org/10.1590/S1516-14392007000200004
- Marquis RE, Antimicrobial actions of fluoride for oral bacteria. Can J Microbiol. 1995 Nov;41(11):955-64
- Loskill P, Zeitz C, Grandthyll S, Thewes N, Müller F, Bischoff M, Herrmann M, Jacobs K . Reduced adhesion of oral bacteria on hydroxyapatite by fluoride treatment. Langmuir 2013 May 7;29(18):5528-33. doi: 10.1021/la4008558. Epub 2013 Apr 23
- Weinstein T., Basso M, Tassera C, Ionescu EC et al- 3 Day clinical study on three different toothpastes on dentin hypersensitivity. International Association for Dental Research IADR-PEF Congress, Jerusalem 2016- Pos Ref 2530694
- Iafisco, Degli Esposti, Rodríguez, Carella, Morales, Ionescu, Brambilla et al., Fluoride-doped amorphous calcium phosphate nanoparticles as a promising biomimetic material for dental remineralization- Scientific reports, 2018) 8:17016 | DOI:10.1038/s41598-018-35258-x 1
- Zipkin, I. & Piez, K. A. The Citric Acid Content of Human Teeth, J. Dent. Res. 29, 498–505 (1950).
- Hu, Y. Y., Rawal, A. & Schmidt-Rohr, K., Strongly bound citrate stabilizes the apatite nanocrystals in bone, Proc. Natl. Acad. Sci. USA 107, 22425–22429 (2010).
- Boskey, A. L. & Posner, A. S., Conversion of amorphous calcium phosphate to microcrystalline hydroxyapatite, a pH-dependent, solution-mediated, solid-solid conversion. J. Phys. Chem. 77, 2313–2317 (1973).
- Zhao, J., Liu, Y., Sun, W.-b. & Zhang, H., Amorphous calcium phosphate and its application in dentistry, Chemistry Central Journal, 1–7 (2011).