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Table of Contents   
ORIGINAL RESEARCH  
Year : 2021  |  Volume : 32  |  Issue : 1  |  Page : 92-97
Development of strontium-doped nano hydroxyapatite dentifrice and compare its remineralising potential with a topical cream containing casein phosphopeptide- amorphous calcium phosphate – An In Vitro study


1 Department of Conservative Dentistry and Endodontics, Saveetha Dental College, Saveetha Institute of Medical and Technical Science, Saveetha University, Chennai, Tamil Nadu, India
2 Department of Conservative Dentistry and Endodontics, Azeeziza Dental College and Research, Kollam, Kerala, India
3 Department of Public Health Dentistry, Azeeziza Dental College and Research, Kollam, Kerala, India

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Date of Submission11-Mar-2019
Date of Decision01-Feb-2020
Date of Acceptance08-Dec-2020
Date of Web Publication13-Jul-2021
 

   Abstract 


Aim: To develop and evaluate the efficacy of synthesised strontium-doped nano hydroxyapatite dentifrice and compare its remineralizing potential with a topical cream containing Casein Phospho Peptide – Amorphous Calcium Phosphate, in remineralizing artificial carious lesion on enamel. Materials and Methods: Enamel specimens of 4 x 4 x 1 mm were prepared from 90 freshly extracted teeth. Specimens were divided into 3 groups of 30 samples each, based on the type of dentifrice applied that is a control group (Group I) and two experimental groups (Groups II, III). Surface topography and the calcium/phosphorous ratio of all sound specimen were evaluated using Scanning electron microscope and Energy Dispersive X-ray Analysis (SEM-EDAX). The samples in group I and each of the experimental groups were subjected to demineralisation and the calcium/phosphorous ratio of the demineralized specimen were analysed. The samples were then subjected to remineralisation using different agents in each group. Samples in the control group (Group I) were brushed with a conventional dentifrice. In the experimental groups, Group II topical cream with Casein Phosphopeptide and Amorphous Calcium phosphate (CPP-ACP) was used and in Group III laboratory synthesized Strontium-doped nanohydroxyapatite paste (Sr-nHAP), respectively for 28 consecutive days. The samples in the both the control and the two experimental groups were again subjected to SEM-EDAX analysis to analyse the calcium phosphorus ratio following remineralisation cycle. Groupwise comparison of the data was done with one way ANOVA followed by Tukeys Post hoc Test. Results: Both experimental groups (II, III) showed statistically significant remineralisation potential after demineralisation, compared to the control group I. Intergroup comparison showed that the samples in Group III showed the higher remineralisation potential than Group II and was statistically significant. Conclusion: Both CPP- ACP containing tooth cream as well as Sr doped nHAp showed remineralisation potential. Sr doped nanohydroxyapatite showed better remineralisation than CPP ACP and can be considered for enamel repair in incipient carious lesions.

Keywords: CPP-ACP, remineralisation, SEM-EDAX, Strontium-doped nanohydroxyapatite

How to cite this article:
Rajendran R, Nair K R, Sandhya R, Krishnan AV, Anilkumar A, Rakhi P V. Development of strontium-doped nano hydroxyapatite dentifrice and compare its remineralising potential with a topical cream containing casein phosphopeptide- amorphous calcium phosphate – An In Vitro study. Indian J Dent Res 2021;32:92-7

How to cite this URL:
Rajendran R, Nair K R, Sandhya R, Krishnan AV, Anilkumar A, Rakhi P V. Development of strontium-doped nano hydroxyapatite dentifrice and compare its remineralising potential with a topical cream containing casein phosphopeptide- amorphous calcium phosphate – An In Vitro study. Indian J Dent Res [serial online] 2021 [cited 2021 Aug 3];32:92-7. Available from: https://www.ijdr.in/text.asp?2021/32/1/92/321368



   Introduction Top


Recently, dentistry aims at minimal intervention with the least invasive treatment options such that tissue loss and patient discomfort is minimised. One such area of focus of minimally invasive dentistry is the early diagnosis of incipient carious lesions and remineralisation of such lesions. White spot lesions are considered the earliest phase of the caries process and they are reversible.

Biological approaches focus on application of remineralising agents to incipient carious lesions and are aimed at controlling demineralisation and promoting remineralisation. Remineralising agents can also promote the deposition of mineral aggregates in the openings of exposed dentinal tubules thereby reducing dentine hypersensitivity.[1] A variety of remineralising agents like fluorides, casein calcium phosphopeptides, bioactive glass like NovaMin®, are available commercially. These remineralising agents control the demineralization, depending upon the microenvironment around the tooth.

Casein phosphopeptide (CPP) is a phosphopeptide obtained from milk protein casein that contains phosphoseryl sequences and stabilises the calcium phosphate in nanocomplexes. CPP prevents dissolution of calcium and phosphate ions by binding with amorphous calcium phosphate (ACP).[2] The CPP-ACP solution is supersaturated and provides Ca2+ and PO42− ions for remineralisation.

Nanohydroxyapatite (nHAp) has been used recently as an active ingredient in some remineralizing toothpastes for repair of demineralising lesions in enamel, and tooth sensitivity. It has the ability to induce mineralisation from within the teeth, which can be further enriched by natural therapy of the saliva, as opposed to fluoride which has been known to cause hypermineralization of the surface layers and thus failing to strengthen the teeth from within.[3] However, these particles have a larger molecular size and penetration into the minute pores of enamel surface is prevented and it fails to produce the desired effect. Moreover, the strength of the commercially available nanohydroxyapatite (nHAp) is not enough to provide adequate protection for the enamel surface leaving it in the same way as before and further amenable to plaque accumulation and acid attack from microbials in the oral cavity.[4] Carbonated hydroxyapatite and strontium-substituted hydroxyapatite (above 10 mol% substitution), are more soluble than nHAp and brings in alterations in the degree of crystallinity and change some of the material properties, including phase stability, and reactivity.[4]

In a study by Vinod Krishnan et al, strontium-doped nanohydroxyapatite in solution was found to be superior to ACP-CPP cream and nanohydroxyapatite toothpaste for repair of demineralized enamel surface.[4] However, preparation of a thicker material like tooth paste whilst incorporating this particle in its formulation will be beneficial because of its greater retention on tooth surface and it can also be used like a normal toothpaste. Hence, in this study, laboratory synthesised paste with Strontium-doped nanohydroxyapatite crystals having smaller particle size was used.

The purpose of the study was to evaluate and compare the remineralising potential of a laboratory synthesised paste containing Strontium-doped nanohydroxyapatite to a topical cream containing CPP-ACP.


   Materials and Methods Top


Tooth samples

Ninety intact premolars extracted for orthodontic reasons were collected from healthy young adult patients after obtaining written consent. The study was approved by the institutional Ethical Committee.

Preparation of enamel specimens

The samples were cleaned of calculus and soft tissues and stored in normal saline. The teeth were sectioned horizontally using a diamond disc, with a slow speed straight hand piece at 15,000 rpm at the level of CEJ, separating the coronal part of the tooth. Ninety enamel specimens of 4 X 4 X 1 mm in size, were prepared from the buccal surfaces of the premolars. Following this, all samples were coated with nail varnish on the palatal side to make them resistant to the subsequent acid treatment on the same side. The samples were divided into three groups i.e., Groups I, II and III. Group I was taken as the control group in which the samples were treated with conventional toothpaste. Group II specimen treated with commercially available CPP-ACP tooth paste and Group III specimen treated with custom made tooth paste with strontium-doped nanohydroxyapatite.

Preparation of strontium-doped nanohydroxyapatite paste

Nanohydroxyapatite particles of smaller size (2-7 um) were synthesized in the laboratory. Strontium was doped into the nanohydroxyapatite crystals. The paste was prepared by mixing the powder of lab synthesised strontium-doped nanohydroxyapatite of smaller particle size (2-7 μm), with sodium alginate, sorbitol, sodium lauryl sulfate, glycerine and water respectively in adequate proportion.

All enamel samples were subject to scanning electron microscopy to assess the surface topography of sound enamel specimen and energy dispersing X-ray analysis to assess the mean calcium and phosphorus values of sound enamel.

Demineralisation of the samples

The samples in Groups I, II and III were subjected to demineralisation with McInne's demineralising solution consisting of l ml of 36% hydrochloric acid, 1 ml of 30% hydrogen peroxide and 0.2 ml of anaesthetic ether which was freshly mixed in the ratio of 5:5:1 in a dappen dish before each application.[5]

All enamel specimen were subject to two cycles of demineralisation. The demineralising agent so prepared was applied to the surface of 90 enamel samples using a cotton applicator for five minutes. It was then washed under running tap water, damped dry with absorbent paper and then stored in artificial saliva. After 24 hours the second application of demineralising agent was carried out as described earlier for all the 90 enamel samples.

The demineralized specimen were subjected to Scanning Electron Microscope (SEM) and Energy Dispersive X-ray Analysis (EDX) and mean calcium and phosphorus content of demineralized specimen were recorded.
Figure 1: SEM and EDAX images of normal tooth samples

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Figure 2: SEM and EDAX images of demineralized samples

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Figure 3: SEM and EDAX images of group 1

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Figure 4: SEM and EDAX images of group 2

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Figure 5: SEM and EDAX images of group 3

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Remineralisation of the samples

The demineralised specimens in group I, II and III were now subjected to remineralisation with the corresponding remineralising agents. Group I conventional tooth paste without specific remineralising agent, Group II subjected to remineralisation with Casein Phosphopeptide - Amorphous Calcium Phosphate containing toothpaste.

Group III remineralisation with laboratory synthesised Strontium-doped nanohydroxyapatite toothpaste.

All the specimens were brushed with the specific remineralising agent using a motorized toothbrush for three minutes twice daily (12 hour interval) for 28 consecutive days. After each application of remineralising agent the samples were gently rinsed with water and stored in artificial saliva for 28 days. The samples were then subjected to SEM-EDX analysis and mean calcium and phosphorus values of the remineralized specimen of Group I, II and III were analysed.

Statistical analyses

Data was analysed statistically using Statistical Package for Social Sciences. (SPSS) version - 22. Data were expressed in its mean and standard deviation and were analysed using one way ANOVA and Tukey post hoc test.


   Results Top


Mean calcium and phosphorus levels of sound enamel, demineralised enamel and remineralized enamel across all groups were calculated. Sound enamel specimen of Group I showed a mean calcium and phosphorus value of 65.32 ± 0.53 and 21.05 ± 0.76, respectively. After demineralisation, mean calcium and phosphorus values obtained were 55.15 ± 0.47 and 15.77 ± 0.57, respectively. Following remineralisation with regular toothpaste the mean calcium and phosphorus values obtained were 55.14 ± 0.47 and 15.77 ± 0.57 respectively which is lesser than that of sound enamel and was statistically significant. The values obtained were similar to the mean calcium and phosphorus content of demineralised samples and was not statistically significant [Table 1] and [Graph 1].
Table 1: Mean calcium and phosphorous values obtained after treating with conventional tooth paste

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Group II showed a mean calcium and phosphorus value of 65.32 ± 0.65 and 20.80 ± 0.75 respectively for sound enamel. Demineralisation showed mean calcium and phosphorus values of 55.23 ± 0.45 and 15.69 ± 0.53, respectively. After remineralisation of the demineralised specimen with toothpaste containing CPP-ACP, the mean calcium and phosphorus values obtained were 63.0 ± 0.37 and 18.51 ± 0.37, respectively. Mean calcium and phosphorus values of the group II specimen after remineralisation with CPP-ACP was higher than the demineralised samples and was statistically significant (p = 0.000) [Table 2] and [Graph 2].
Table 2: Mean calcium and phosphorous values obtained after treating with casein phosphopeptide-amorphous calcium phosphate

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Group III showed a mean calcium and phosphorus value of 65.25 ± 0.59 and 20.76 ± 0.81 respectively for sound enamel. Demineralisation showed mean calcium and phosphorus values of 55.03 ± 0.59 and 15.59 ± 0.48. After remineralisation with laboratory synthesised strontium-doped nanohydroxyapatite containing tooth paste the values obtained were 65.4 ± 0.86 and 20.5 ± 0.76 respectively, which is higher than the values obtained for demineralized samples and was statistically significant (p = 0.000) [Table 3] and [Graph 3].
Table 3: Mean calcium and phosphorous values obtained after treating with laboratory synthesised strontium-doped nanohydroxyapatite

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Intergroup comparison between groups I, II and III using Tukey post hoc test showed statistically significant differences in the mean calcium and phosphorus levels, with group III yielding higher mean calcium and phosphorus levels and group I showing least value. [Table 4] and [Graph 4].
Table 4: Intergroup comparison of mean calcium and phosphorous of group 1 group 2 and group 3

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SEM Images of the sound enamel specimens show smooth surfaces. After demineralisation, specimen showed an uneven and irregular surface with porosities. After remineralisation, group II and group III specimens show mineral deposition, pores covered partially with crystals. Group III showed smoother surface after remineralisation than Group II specimen.


   Discussion Top


The removal of the caries affected tissues and replacement with a restorative material is the conventional treatment concept for all caries affected teeth. Recently, focus on caries is directed to the development of methodologies for the detection of caries lesions in the early stages and the use of non-invasive treatment for these lesion. Prevention of initiation and interruption in progression of early lesions are the desirable modes of caries management. Non-cavitated lesions as well as caries extending up to the dentinoenamel junction can be arrested if the cariogenic challenges of the specific micro environment are sufficiently controlled or/and if therapeutic agents are applied for tissue healing.[6]

Early diagnosis with newer caries detection aids enables small lesions to be identified so that remineralisation of lesions by preventive measures can be attempted. Timely intervention can convert a lesion from an active to an inactive state.

Demineralisation is basically the loss of mineral apatite from the enamel.[7],[8] When organic acids, such as lactic and formic acids are produced by acidogenic bacteria, they diffuse in various directions through the enamel and dentin organic matrix into underlying tissues.[9],[10] The organic matrix accelerates the demineralisation process by providing permeable channel networks for acid invasion. When the acid reaches a susceptible site on the crystal surface, minerals dissolve into the surrounding aqueous phase.[8] This is the first step of demineralisation, which takes place at the atomic level before any clinically visible sign is observed. Critical pH, which is about 5.5 for enamel, is the pH level at which demineralisation occurs.

In this study, in order to mimic the caries demineralisation process, the surface layer of the specimens in the experimental groups were placed in the Mc Innes solution as it was an accepted protocol.[5] Demineralisation with McInne's bleaching solution was done in two cycles as it has been proved in the previous studies that only after completing the second cycle of demineralisation after 24 hours, was there a significant reduction in microhardness of enamel. All the specimens were kept in artificial saliva to simulate oral environment.

There are various remineralising agents available today such as fluorides, tricalciumphosphate, calcium sodium-phosphosilicate (novamin), Casein Phosphopeptide – Amorphous Calcium Phosphate which are incorporated either into a tooth paste or in a topical cream. In this study, Group II was treated with topical cream containing Casein Phosphopeptide – Amorphous Calcium Phosphate. CPP - ACP nanocomplexes have been proven to be efficacious in both the prevention and reversal of enamel lesions in caries models.[11],[12] It has been shown that CPP - ACP can be used to prevent demineralisation and promote remineralisation of early enamel lesions and it has a short-term remineralising effect in clinical in situ trials and long term caries-preventing effect in, in-vivo randomised control trial.[13],[14] The CPP has been shown not only to stabilise ACP, but also to deliver and localise ACP at the tooth surface.[11] The proposed anticariogenic mechanism for CPP – ACP is by the localisation of amorphous calcium phosphate on the tooth surface, which buffers the free calcium and phosphate ion activities, thereby helping to maintain a state of supersaturation with respect to the tooth enamel and thus preventing demineralisation and enhancing remineralisation.[15] It has also been stated that CPP - ACP is a valid preventive system against demineralisation of early enamel lesions.

Synthetic hydroxyapatite crystals have also been used for remineralisation. However, synthetic hydroxyapatite has a large crystal size. Limitations of nHAp are lackof strength, brittleness, high degree of crystallinity and low solubility at neutral pH requiring an acidic pH to dissolve.[16] The present study evaluated the remineralizing potential of a newly developed strontium-doped nanohydroxyapatite incorporated tooth paste with less particle size which can overcome the above mentioned problems. It is reported that the incorporation of strontium (Sr) for calcium (Ca) in hydroxyapatite (HA) allows the formation of a pure but non-stoichiometric hydroxyapatite with low (Sr + Ca)/P ratio. This partial replacement of Ca+2ions by Sr+2ions in HA matrix is responsible for the increase in solubility of HA.[17] 25% SrnHAp and 50% SrnHAp used in previous studies had a particle size in the range of 2-7 um and 4-9 um, respectively. Even though the agglomerating capacity of 50% SrnHAp is on the higher side, the increase in particle as well as crystal size makes it a poor choice to form a paste system to be used over dental enamel. Hence, 25% Sr-nHAp is the material of choice due its increased diffusibility through small incipient caries lesions.[4]

Mean calcium and phosphorus values have been determined using energy dispersive X-ray analysis. EDX is considered as the 'gold standard' for the determination of mineral loss or gain in experimentally induced initial carious lesions. The EDX provides a very precise quantitative measurement of the mineral content.[18],[19] EDX has been used for elemental analysis at the ultrastructural level. It is a micro analytical technique used in conjunction with SEM wherein SEM does the structural analysis and the elemental analysis is done by EDX.[20] The topographic changes of the enamel layer that is in sound enamel, demineralised and remineralised specimen can be assessed with scanning electron microscopy. SEM evaluation of the specimens after remineralisation showed favourable surface changes in both the experimental groups (Group II and Group III). The SEM images of group III treated with Sr doped nanohydroxyapatite showed smoother surface compared to Group I with regular dentifrice.

In this study Group I (control group treated with regular toothpaste) showed mean calcium and phosphorous levels of 55.14 ± 0.47 and 15.77 ± 0.57, respectively which was lesser than that of sound enamel. Group II treated with CPP ACP containing tooth cream showed a mean calcium and phosphorus value of 63.0 ± 0.37 and 18.51 ± 0.37, respectively. Group III in which samples were treated with strontium doped nanohydroxyapatite toothpaste showed mean calcium and phosphorous levels of 65.4 ± 0.86 and 20.5 ± 0.76, respectively. The samples in Group III and group II showed a net increase in calcium and phosphorous content after remineralisation compared to the corresponding demineralised values and was statistically significant. Intergroup comparison of Group I (control regular toothpaste), Group II (CPP-ACP) and Group III (Sr doped nHAp) showed that Group III gave higher mean calcium and phosphorous values after remineralisation and the difference was statistically significant. Similar findings was also obtained in a study done by Vinod Krishna et al. where strontium-doped nanohydroxyapatite had better remineralisation potential to CPP-ACP and nanohydroxyapatite.[4] The study concluded that the presence of strontium indicates increased dissolving capacity of the material and improved retention on the tooth surface making it a better choice than CPP-ACP and Novamin for remineralisation or repair of enamel. Furthermore, there is evidence of complete obliteration of the tubules/surface pores in comparison with other treatment modalities. In this study a, laboratory synthesised paste with Strontium-doped nanohydroxyapatite crystals having smaller particle size was used unlike the solution form used in previous study. A thicker material like a paste provides greater retention on tooth surface and could be responsible for the improved results of this group. However, further evaluation with different concentrations of strontium-doped nanohydroxyapatite and toxicology assessment is required before application clinically.


   Conclusion Top


Within the limitations of the present study it can be concluded that both CPP- ACP containing tooth cream as well as Sr-doped nHAp showed significant remineralisation potential compared to regular dentifrice. SEM-EDAX evaluation revealed favourable surface changes in enamel with both CPP- ACP as well as Sr-doped nanohydroxyapatite. The novel laboratory synthesized Sr-doped nanohydroxyapatite showed better remineralisation than CPP-ACP and can be considered for enamel repair in incipient carious lesions. Further evaluation of remineralisation potential of Sr-nHAP will provide a basis for support of the outcome obtained in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Ratheesh Rajendran
Department of Conservative Dentistry and Endodontics, Saveetha Dental College, Saveetha Institute of Medical and Technical Science, Saveetha University, Chennai
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_238_19

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