| Abstract|| |
Background: Research on dentin remineralisation protocols in particular 'biomimetic remineralisation' has gained huge momentum. Aim of this study was to evaluate if biomimetic analogs, incorporated in n-HAp, as an experimental formulation could aid in remineralization of artiﬁcial caries-like dentin and have anti-microbial effect on cariogenic bacteria, S mutans. Materials and Methodology: An experimental paste was formulated using nano-hydroxyapatite (nHAp) with Non-Collagenous Protein analogs- polyacrylic acid (PAA), sodium tri-poly phosphate (STPP) with Simulated Body Fluid. Partially demineralised dentin specimens were divided into three groups (n=10) based on the remineralisation treatment as, Group A- n-HAp paste, Group B- n-HAp and NCP analogues and Group C (Control) - no treatment. At the end of the experimental period, the specimens were assessed using SEM-EDS analysis and Vickers microhardness testing. Further, the antimicrobial efficacy of the paste was assessed. Statistical Analysis: The results were statistically analyzed using ANOVA with post-hoc Bonferroni test. Results: Dentin specimens treated with the experimental paste revealed greater tubular occlusion, with intra tubular deposits and increased mineral content. Specimens treated with n-HAp alone had higher microhardness values and inhibitory effect on the cariogenic bacteria. Conclusion: Non-Collagenous Protein analogs incorporated in n-HAp could remineralize the demineralised dentin and had antibacterial efficacy against S mutans.
Keywords: Biomimetic remineralisation, dentin remineralisation, nano-hydroxyapatite, non -collagenous protein analogues
|How to cite this article:|
Nambiar S, Kumari M, Mathew S, Hegde S, Ramesh P, Shetty N. Effect of nano-hydroxyapatite with biomimetic analogues on the characteristics of partially demineralised dentin: An in-vitro study. Indian J Dent Res 2021;32:385-9
|How to cite this URL:|
Nambiar S, Kumari M, Mathew S, Hegde S, Ramesh P, Shetty N. Effect of nano-hydroxyapatite with biomimetic analogues on the characteristics of partially demineralised dentin: An in-vitro study. Indian J Dent Res [serial online] 2021 [cited 2022 May 26];32:385-9. Available from: https://www.ijdr.in/text.asp?2021/32/3/385/338129
| Introduction|| |
Dental caries is caused by an imbalance between demineralisation and remineralisation process. Demineralisation of dentin exposes the collagen fibres, creating an ideal environment for the rapid destruction of the entire dentin network. Excavating the infected dentin often weakens the tooth structure and may result in accidental pulpal exposure. In this era of minimal invasive dentistry, a more conservative approach would be to facilitate remineralisation of affected dentin. Fluoride, casein phosphopeptide in amorphous calcium phosphate, calcium hydroxide-based materials, mineral trioxide aggregate (MTA), biodentine, calcium-enriched mixtures, hydroxyapatite, β-tricalcium phosphate, HA/β-TCP mixture and α-tricalcium phosphate have been researched in an attempt to facilitate remineralisation.
Dentin remineralisation is more challenging due to the absence of seed crystallites. Bio-mineralisation, is a non-classical crystallisation pathway in which the particle-mediated remineralisation strategy independent of ion transport and seed crystallites have been applied to remineralise dentin. This bottom-up remineralisation strategy may be considered as a feasible method for remineralisation of demineralised dentin. Intrafibrillar mineralisation occurs within the pore spaces and gap zones of collagen fibrils, progressively replacing loosely bound water, improving the mechanical properties of demineralised dentin thereby contributing to enhanced resin-dentin bonds.,
Nano-hydroxyapatite (n-HAp) similar to the apatite crystals of enamel is biocompatible, bioactive and is increasingly used in dental cements and fillings., One of the primary features of hydroxyapatite is its capacity for ion substitution and induction of mineralisation from within the teeth as opposed to fluoride which have been known to cause only hypermineralisation of the surface layers failing to strengthen the teeth from within. It was observed that use of hydroxyapatite containing toothpastes resulted in higher remineralising effects on bovine dentine compared to amine fluoride-containing toothpastes. However, n-HAp lacks strength, is brittle and has a high degree of crystallinity.
Noncollagenous protein (NCP) analogues such as polyacrylic acid (PAA), polyvinyl phosphonic acid (PVPA), agarose gel, phosphorylated chitosan and sodium trimetaphosphate (STMP) have been explored to promote intrafibrillar dentin remineralisation.,,, Addition of dual biomimetic analogues to nano-hydroxyapatite (n-HAp) could enhance its clinical efficacy.
There are limited studies evaluating the remineralising efficacy of dual NCP analogues, PAA and sodium tripolyphosphate (STPP) in n-HAp. Therefore, the aim of this study was to evaluate if biomimetic analogues, incorporated in n-HAp as an experimental formulation, could aid in remineralisation of artiﬁcial caries-like dentin and have anti-microbial effect on cariogenic bacteria, S. mutans.
| Materials and Methodology|| |
Intact maxillary premolars extracted for orthodontic reasons were mounted in die stone and the occlusal enamel was removed using a diamond disc. It was further sectioned horizontally to obtain 2.5 ± 0.5 mm thick sections using low speed saw (Minitom, Struers, Copenhagen, Denmark) under water cooling and further polished using 120 grit Si-C paper. One side of these sections was coated with nail varnish. The specimens were ultrasonicated in distilled water for 10 min (FS20, Fisher Scientific Co., Pittsburgh, PA, USA) and placed in 15 mL freshly prepared demineralising solution (1.5 mM of CaCl2, 0.9 mM of KH2PO4, 50 mM of acetic acid and 0.02% of NaN3, pH ~4.5) for 7 days at 37°C. Following demineralisation, samples were rinsed with deionised water. Confirmation of demineralisation was done using scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM-EDS) analysis. The current study was approved by the University Ethics Committee for Human trials, MSRUAS, UECHT/2016-18/PGDT/CD04 on15/09/2018.
Specimens were divided into three groups (n = 10) based on the remineralisation protocol as Group A- n-HAp paste, Group B- n-HAp and NCP analogues, 3 wt% PAA and 8 wt% STPP and Group C (Control)- no treatment. 10 wt% n-HAp (Merck, India), 3 wt% PAA and 8 wt% STPP (Merck, India) powders were weighed and blended using a mortar and pestle to form a homogeneous powder., The powder and freshly prepared simulated body fluid (SBF) were mixed on a glass slab in a ratio of 3:1 and applied on the dentin samples using microtip brushes for 2 to 5 min and subjected to a 10-day pH cycling regimen. Each cycle consisted of individual immersion of the dentin sections in demineralising solution for 3 h (30 mL per group) followed by a 2-h immersion in a remineralisation solution of SBF and pH was adjusted to 7.0 (30 mL per specimen group) at 37°C without agitation. A 2 to 5 min treatment with the formulated pastes was carried out before and after the demineralising process. The experimental specimens were placed in the remineralising solution overnight. The excess paste was removed, and the dentin sections were washed in deionised water for 5 s before being immersed in either of the solutions. The solutions were renewed daily. At the end of 10 days, three specimens from each group were sputter-coated with gold and examined under SEM (EDAX, Ametek Inc. USA).
Microhardness of seven samples of each group was tested using microhardness tester (Clemex Technologies Inc. Canada) applying a load of 25 gf for 10s. Hardness was calculated using the formula-
Dentin sections 4 mm × 4 mm and 200 μm +/− 0.05 thick were obtained as described previously and polished using 120 grit Si-C paper. Following ultrasonication in distilled water for 10 min, it was autoclaved at 121°C for 30 min. The sections were then placed in 15 mL demineralising solution for 7 days at 37°C and rinsed with deionised water.
Stock cultures of Streptococcus mutans (MTCC 497) were obtained from the lyophilised form by serial dilution, stored in 50% glycerol at −20°C and cultivated in brain-heart infusion broth (BHI, HiMedia, India) over 24h at 37°C.
Samples were divided into four groups (n = 10) based on the treatment protocol:
- Group 1: n-HAp paste
- Group 2: n-HAp and NCP Analogues-STPP and PAA
- Group 3: Negative Control -no treatment group
- Group 4: Positive Control-Calcium hydroxide.
Agar well diffusion test
A loopful of the inoculum from the revived stock culture was spread on the surface of Muller Hilton Agar plate with the help of a swab. Four holes with a diameter of 6 mm were punched representing the 4 groups. A volume (20–100 mL) of each tested paste was introduced into the wells; Group C received no treatment. Agar plates were then incubated aerobically for 48 h after which the zone of inhibition was measured.
Assessment of bactericidal effects on dentin
The pastes were applied to the demineralised discs and left in place for 20 s (Groups 1, 2 & 4), no treatment was done in Group 3. The discs were homogenised in phosphate-buffered saline (PBS). The homogenised solution was diluted immediately with BHI broth, and a 100 μl aliquot was inoculated on Mitis salivarius agar (Becton Dickinson) plates and incubated aerobically for 48 h, and the number of viable bacteria (CFU) was assessed by counting the colonies formed.
The data was analysed using SPSS version 19 (IBM® SPSS® Statistics, United States). ANOVA with post-hoc test (Bonferroni) was used to compare among the groups. The significance level was set at 0.05 (95% confidence interval).
| Results|| |
Scanning electron microscopy and energy-dispersive X-ray analysis
In Group A, few open dentinal tubules were observed [Figure 1]a and in Group B, complete obliteration of dentinal tubules was observed [Figure 1]b. In Group C, the demineralised specimens showed dentinal tubule widening [Figure 1]c. EDS analysis revealed higher mineral content in both Group A (Calcium-13.86 wt% and phosphorus at 8.14 wt%) and Group B (Ca-22.13 wt% and P-18.45 wt%) as compared to Group C [Figure 1].
Group A specimens had the highest hardness value of 51.32 HV, followed by Group B (43.73 HV) and least for Group C (33.74 HV) [Figure 2]. There was a significant difference in the pairwise comparisons between the three groups (Post-hoc Bonferroni analysis) (P ≤ 0.05).
Agar well diffusion test
Group 1 exhibited the highest zone of inhibition of 4.10 mm followed by Group 2, 1.50 mm and Group 4 with 2.00 mm and no inhibition zone observed in Group 3. Comparison using ANOVA and Post-hoc Bonferroni revealed that there was a significant difference in the pairwise comparisons between Group 1 and Group 2. However, no significant difference was observed between Group 2 and Group 4 (CH) (P ≤ 0.05) [Table 1].
|Table 1: Mean growth inhibition diameters against tested microorganisms in mm|
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Assessment of bactericidal effects on dentin
Therefore, if the dilution factor is 103 and 100 μl was aliquot then
E.g.: CFU/ml = (480 × 103)/0.1 = 4.8 × 106
Group 1 displayed the least CFU/ml value of 0.73 × 106 CFU/ml, followed by Group 4 (2.09 × 106 CFU/ml) and Group 2 (2.67 × 106 CFU/ml); maximum CFU/ml was noted for Group 3 (6.55 × 106 CFU/ml) [Table 2]. ANOVA and post-hoc Bonferroni revealed a significant difference in the pairwise comparisons between n-HAp and n-HAp with NCP analogues and no significant difference between Groups 2 and 4 (P ≤ 0.05).
|Table 2: Mean distribution of the microbial count (*106) in all the groups|
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| Discussion|| |
An experimental paste containing nano-hydroxyapatite with NCP analogues was evaluated for its antimicrobial activity as well as remineralisation efficacy on partially demineralised dentin. Dentin specimens in the study were subjected to pH cycling to mimic the dynamic remineralisation-demineralisation cycle that occurs in the oral cavity., The remineralisation efficacy was evaluated using SEM-EDS analysis and microhardness testing [Figure 1]. SEM-EDS analysis provides information about the surface topography and elemental composition of the demineralised specimens prior to and post treatment with the experimental paste.
In the present study, the demineralised specimens (Group C) which underwent only pH cycling revealed numerous open tubules as well as an increase in tubule diameter. The EDS analysis confirmed noticeable reduction in mineral content, with only 9.99 wt% Ca and 5.79 wt% P. SEM micrographs of dentin specimens in Group A (n-HAp) revealed fewer open tubules with spherical deposits within them. At higher magnification, narrowing of the tubular lumen could also be appreciated. EDS analysis revealed increased Ca (13.86 wt%) and P (8.14 wt%) with C and O2 present in increased amounts [Figure 1].
SEM micrographs in Group B (n-HAp with NCP analogues) revealed complete tubular obliteration with no patent dentinal tubules and a protective layer covering the surface. At 10,000 × magnification, agglomerated, spherical precipitates within the tubules were observed. The EDS analysis revealed higher amounts of Ca (22.13 wt%) and P (18.45 wt%) [Figure 2]. This is in accordance to a previous study where incorporation of biomimetic analogues, PAA and STPP to MTA, increased Ca and P content of demineralised dentin specimens as well as facilitated biomimetic remineralisation [Figure 1].
Mechanical properties of dentin can also indirectly reflect the degree of demineralisation/remineralisation. Vickers microhardness test revealed an increase in hardness values for both groups A (51.32 HV) and B (43.73 HV) as compared to the control Group C (33.74 HV) which was significant and p studies have reported that compressive strength increases proportionately with increasing precipitation of HA crystals in the calcium phosphate cement., The increase in microhardness values proves that there was a considerable improvement in mechanical properties of demineralised dentin in both the tested groups [Figure 2].
Furthermore, in this study, the antibacterial activity of the experimental paste against the most predominant microorganism implicated with the process of dental caries, i.e., S. mutans was assessed. Demineralisation of the specimens was done prior to inoculation of S. mutans, in order to simulate artificial carious lesions. Thin dentin specimens were used to eliminate any negative influence due to the inherent buffering capacity of dentin. One of the primary features of hydroxyapatite contributing to anti-bacterial property is its capacity for ion substitution.
Antibacterial activity was evaluated by the Agar well diffusion method, which could not distinguish whether the materials exhibit bactericidal or bacteriostatic effects, as the production of inhibition zones on the agar plate only indicate that the bacterial growth was hindered. Here, group 1 (n-HAp paste) exhibited the maximum zone of inhibition (4.1 mm) as compared to groups 2 (1.5 mm) and 4 (2.00 mm). When the bactericidal effect on infected demineralised dentin was assessed, group 1 (n-HAp) recorded the least mean value of 0.73 × 106 CFU/ml exhibiting better antimicrobial efficacy as compared to the other groups. Group 2, containing NCP analogues (STPP and PAA), also exhibited antimicrobial activity with mean zone of inhibition of 1.5 mm and 2.67 × 106 CFU/ml. This observation could be explained by the mechanism of ion exchange and pH change. PAA has antibacterial action, best at an acidic pH (range of 4.5–6) which impairs pH homeostasis and eventually leads to damage of proteins, membranes and DNA.
Literature review supports the in situ applied delivery system with anti-bacterial property for ACP-stabilisation analogues. Biomimetic remineralisation of dentin can be accomplished through bio-agents incorporated into restorative materials. Therefore, the present study with the aim of developing a biomimetic formulation with bactericidal properties might create a new generation of materials which reinforces the dentin and reduces the incidence of failure by secondary caries. However, despite the results achieved in this study, there are certain limitations which need to be addressed. Different proportions and combinations of the NCP analogues can be compared to attain maximum benefit. Advanced techniques to assess remineralisation like x-ray diffraction, Raman spectroscopy, atomic force microscopy or transmission electron microscopy, can be performed to further validate the findings of this study.
| Conclusion|| |
Within the limitations of this in vitro study, it can be concluded that the NCP analogues—PAA and STPP incorporated in n-Hap—were able to remineralise the demineralised dentin sections as observed by the occluded dentinal tubules, increased calcium/phosphorus content, improved microhardness and also exhibited antimicrobial activity against the tested cariogenic bacteria, S. mutans. Thus, this experimental paste has potential application as a remineralising paste to treat dentinal hypersensitivity and also as an antibacterial pulp capping agent.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Sharanya Nambiar
Department of Conservative Dentistry and Endodontics, Faculty of Dental Sciences, Ramaiah University of Applied Sciences, Bangalore - 560 054, Karnataka
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2]