Comparative Evaluation of Influence of Self-assembling Peptide P11-4 Based Remineralization Agent on Incipient Dental Caries Lesions of Primary Teeth: A Polarized Light Microscopic Study
Corresponding Author: Zaina Gayas, Department of Paedodontics and Preventive Dentistry, Sri Rajiv Gandhi College of Dental Sciences & Hospital, Bengaluru, Karnataka, India, Phone: +91 9845398333, e-mail: email@example.com
Received on: 12 December 2022; Accepted on: 04 January 2023; Published on: 22 April 2023
Background: Interception of the progression of incipient dental caries lesions with remineralization agents can facilitate their regression noninvasively.
Aim: To evaluate the influence of self-assembling peptide P11-4 (oligopeptide 104) based remineralization agent in comparison to fluoride-enhanced hydroxyapatite gel on incipient caries lesions in primary teeth.
Materials and methods: A total of 36 sound deciduous molars were selected and coated with two layers of acid-resistant varnish, leaving two windows (2 × 2 mm) on the buccal surface. The teeth were immersed in the demineralizing solution and incubated at 37°C for 4 days to produce artificial enamel caries lesions. One of the windows was assigned as a baseline lesion, while the other was designated as an experimental lesion and exposed to the test product and pH cycling. The samples were divided into three groups (n = 12). Group I—self-assembling peptide P11-4 gel, group II—fluoride-enhanced hydroxyapatite gel, and group III—control. The self-assembling peptide P11-4 and fluoride-enhanced hydroxyapatite were applied to the specimens for 5–3 minutes, respectively. The specimens were subjected to a 7-day pH-cycling regimen, followed by sectioning and examination under a polarized light microscope.
Results: The remineralization agents demonstrated a decrease in mean lesion depth in comparison to baseline, with the group I demonstrating greater reduction (165.235 ± 7.569–107.704 ± 6.735 μm) in comparison to group II (175.365 ± 12.238–127.462 ± 7.111 μm).
Conclusion: The self-assembling peptide P11-4 and fluoride-enhanced hydroxyapatite gel exhibited significant potential in the remineralization of artificial enamel caries lesions in primary teeth. However, the self-assembling peptide P11-4 demonstrated greater remineralization potential.
How to cite this article: Gayas Z, Azher U, Paul ST, et al. Comparative Evaluation of Influence of Self-assembling Peptide P11-4 Based Remineralization Agent on Incipient Dental Caries Lesions Of Primary Teeth: A Polarized Light Microscopic Study. J South Asian Assoc Pediatr Dent 2023;6(1):3-8.
Source of support: Nil
Conflict of interest: None
Keywords: Dental caries, Fluoride, Remineralization, Self-assembling peptide, White spot lesion.
Early childhood caries is a prevalent oral health problem in preschool children, characterized by a multifactorial etiology and rapid progression leading to an enduring deleterious impression on a child’s general, dental and emotional health, and also the overall quality of life. The white spot lesion represents the first clinical evidence of the inception of dental caries and is characterized by subsurface demineralization of the enamel that results in porous mineral surface covering the body of the lesion. Failure to intercept at the initial reversible stage leads to collapse and irreversible breakage of tooth structure, leading to cavitation after approximately 30% of demineralization.1 The demineralization and remineralization processes are dynamic in the initiation, progression, and reversal of dental caries. Hence, maintenance of the equipoise between these processes is pivotal in the avoidance of dental caries. A wide range of remineralization agents has been developed that aim to treat initial caries lesions noninvasively.2 The decline in dental caries is attributed to the cariostatic potential of fluorides.3 However, despite the profound influence of fluoride in the inhibition of dental caries progression, its usage is associated with limitations. One of the major drawbacks is that fluoride (in high concentrations) has shown to predominantly lead to surface remineralization at the expense of the body of the caries lesion, making full remineralization difficult to achieve.4 Moreover, fluoride does not inhibit the cascade of events that lead to reactivation and progression of caries lesions. Also, as the formation of 1-unit cell of fluorapatite requires 10 calcium (Ca2+) and six phosphate (PO43–) ions for every two fluoride ions, the unavailability of adequate amounts of Ca2+ and PO43– ions following topical application of fluorides can limit the net enamel remineralization.5 Hence, a quest for new and highly efficacious tooth remineralization technologies paved the path for the development of various contemporary remineralizing agents. The current tooth remineralization approaches reduce the solubility of enamel by inhibiting the demineralization of hydroxyapatites. However, they fail to achieve matrix-mediated mineralization similar to the natural process.6 A regenerative approach that enables the regeneration of hydroxyapatite crystals using the natural remineralization capability of saliva has been suggested. The self-assembling peptide P11-4 forms a three-dimensional (3D) matrix that contributes to de novo hydroxyapatite formation and remineralization of subsurface lesions, thus mimicking enamel matrix proteins.7
Remin Pro®, a remineralization agent comprising hydroxyapatite and fluoride, has shown to decrease tooth hypersensitivity, prevent enamel demineralization, and enhance remineralization of enamel lesions.8 Although a myriad range of remineralization agents are commercially available, a paucity of evidence on the remineralization efficacy of self-assembling peptide P11-4 impelled the designing of the present in vitro study with an aim to evaluate and compare the remineralization potential of novel biomimetic self-assembling peptide P11-4 and fluoride-enhanced hydroxyapatite gel in primary teeth using polarized light microscopy.
MATERIALS AND METHODS
An experimental in vitro study was conducted in the Department of Pediatric and Preventive Dentistry after obtaining approval from the Institutional Review Board and Ethical Committee. A total of 36 sound deciduous second molars, which exfoliated naturally or were extracted for reasons of over-retention or orthodontic purposes were collected for the study after obtaining written consent from parents. Surface debridement of all the teeth was done and stored in distilled water containing 0.1% thymol for 1 week and then transferred to normal saline at room temperature until use. The specimens were blot dried and coated with two layers of acid-resistant nail varnish, leaving two windows measuring 2 × 2 mm on the buccal surface (Fig. 1). The samples were immersed in demineralizing solution (10 mL/tooth) and incubated at 37°C for 4 days (96 hours) to produce artificial carious lesions, following which they were stored in artificial saliva until use. One of the windows in each tooth was assigned as a baseline lesion and coated with two layers of acid-resistant nail varnish, while the other was designated as experimental lesion and exposed to the test product and pH cycling process. The samples were divided into three groups (n = 12). Group I—self-assembling peptide P11-4 gel (CurodontTM Protect; Credentis, Switzerland), group II—fluoride-enhanced hydroxyapatite gel (Remin Pro®, VOCO America Inc), and group III—control (distilled water). The self-assembling peptide P11-4 gel (CurodontTM Protect) and fluoride-enhanced hydroxyapatite gel (Remin Pro) was applied to the tooth samples according to the manufacturer’s instructions for 5–3 minutes, respectively (Fig. 2). The specimens were subjected to pH cycling for a period of 7 days. Each cycle comprised 3 hours of demineralization twice daily followed by 2 hours of remineralization in between, and then the specimens were stored in remineralizing solution overnight at 37°C in an incubator. After completion of the pH cycling, the acid-resistant nail varnish was removed from the specimens using acetone solvent. Longitudinal transection of the lesions along the occluso-gingival axis was done using a slow-speed diamond saw under water spray to obtain 600 μm thick sections and then ground to 400–450 μm thickness. The depth of the caries-like lesion was analyzed using a polarized light microscope at 10× magnification. Photomicrographs were taken and analyzed using a computerized calculation with IS Capture software (Figs 3 to 5).
A one-way analysis of variance test was used to compare the mean lesion depth in the study groups at baseline and postintervention. The intragroup comparison of the mean lesion depth at baseline and postintervention in each group was done using a student-paired t-test. Multiple pairwise comparisons of mean difference in lesion depth between groups postintervention were done using Tukey’s post hoc analysis.
At baseline in group I (Curodont™ Protect), the mean lesion depth was 165.235 ± 7.569 μm, with a range between 153.81 and 176.62 μm. In group II (Remin Pro®), the mean lesion depth was 175.365 ± 12.238 μm with a range between 160.23 and 196.23 μm. In group III (control), the mean lesion depth was 170.192 ± 15.743 μm with a range between 136.26 and 198.47 μm. The difference in the mean lesion depth (in μm) among the three groups at the baseline period was not statistically significant (p = 0.92).
Following pH cycling, in group I, the mean lesion depth was 107.704 ± 6.735 μm, with a range between 96.30 and 116.86 μm. In group II, the mean lesion depth was observed to be 127.462 ± 7.111 μm with a range between 116.67 and 141.75 μm. In group III (control), the mean lesion depth was 151.083 ± 20.644 μm with a range between 92.41 and 169.25 μm. The difference in the mean lesion depth (in μm) among the three groups postintervention was statistically significant (p < 0.001).
On intragroup comparison using the student-paired t-test, it was observed that all the groups showed a statistically significant reduction in lesion depth between the baseline and postintervention values (Table 1 and Fig. 6). The mean lesion depth (in μm) in group I, at postintervention period, significantly reduced to 107.704 ± 6.735 μm as compared to its baseline depth of 165.235 ± 7.569 μm; the difference was statistically significant at p < 0.001. Similarly, the postintervention mean lesion depth in group II significantly reduced to 127.462 ± 7.111 μm as compared to its baseline depth of 175.365 ± 12.238 μm, and the difference was statistically significant at p < 0.001. The mean lesion depth in group III (control) also showed a significant reduction in postintervention period to 151.083 ± 20.644 μm as compared to its baseline depth of 170.192 ± 15.743 μm. The difference was statistically significant at p < 0.001. Multiple pairwise comparisons of mean difference in lesion depth (in μm) between study groups postintervention using Tukey’s post hoc analysis exhibited a statistically significant difference between the groups (Table 2).
|Group||Time||N||Mean||Standard deviation||Mean difference||p-value|
|Post 7 days||12||107.704||6.735|
|Group II (Remin Pro®)||Baseline||12||175.365||12.238||47.903||<0.001*|
|Post 7 days||12||127.462||7.111|
|Group III (Control)||Baseline||12||170.192||15.743||19.108||<0.001*|
|Post 7 days||12||151.083||20.644|
|(I) Groups||(J) Groups||Mean difference (I−J)||95% confidence interval for the difference||p-value|
|Group I (Curodont™ Protect)||Group II (Remin Pro®)||−19.758||−32.973||−6.542||0.002*|
|Group III (Control)||−43.379||−56.594||−30.164||<0.001*|
|Group II||Group III|
The onset of dental caries is distinguished by the dissolution of subsurface minerals of the tooth enamel, thus leading to a subsurface demineralized lesion body, that is, white spots9 followed by advancement to irreversible cavitation of the tooth surface. The white spot lesions, however, are clinically detectable and can be remineralized noninvasively. Hence, the early identification and interception of these incipient lesions are recommended. Several contemporary tooth remineralization agents have been developed for the noninvasive management of incipient lesions. The Curolox technology developed by scientists from the University of Leeds for regenerating enamel comprises P11-4, a rationally-designed peptide. Studies have shown that in response to specific environmental triggers, the monomers of P11-4 peptide undergo well-characterized self-assembly into a biocompatible fibrillar scaffold mimics the enamel matrix. Around this matrix, enamel crystals are formed from salivary calcium phosphate [Ca3(PO4)2].10,11
The remineralization of carious lesions has been studied using a wide array of methods that include microradiography,12 polarized light microscopy,13 microhardness test,14 mineral analysis of Ca3(PO4)2 and fluoride phases,15 and transmission, and electron microscopy.16 Polarized light microscopy is one of the most commonly used qualitative methods in the depth-related analysis of artificial caries lesions.13
Polarized light microscopic examination of the lesion depth postintervention demonstrated a decrease in lesion depth in both the test groups. It was observed that both CurodontTM Protect and Remin Pro® facilitated a decrease in the depth of the artificial caries lesions when compared to the control group. The possible hypothesis explained for matrix-mediated mineralization by self-assembling peptide P11-4 is that bioactive peptide synthesized from amino acids diffuses into the porosities and assembles within the subsurface lesions into a 3D fibrillar scaffold resembling extracellular matrix.1 This 3D fibrillar scaffold acts as a nucleus for hydroxyapatites, attracting Ca2+, and PO43– ions from saliva, thus increasing the Ca2+:PO43– ratio and initiating tissue regeneration. Furthermore, when the peptide assembles into the fiber, it contains clusters of negative charges made up of four glu residues providing potential Ca2+ binding sites. These binding sites are approximately 9.4 Å apart, the distance normally present in the natural hydroxyapatite crystal.17 These peptides structurally resemble biological macromolecules in the mammalian skeleton and provide matrix-mediated mineralization.11 Furthermore, as CurodontTM Protect also comprises sodium monofluorophosphate (900 μm), the fluoride may exert a synergistic influence on remineralization potential.
Ultrasonic assessment and scanning electron microscope studies have shown that the application of P11-4 to demineralized enamel surfaces could inhibit demineralization and promote hydroxyapatite crystal formation.18 According to Silvertown et al.,19 a single treatment with P11-4 enabled enamel regeneration through the promotion of de novo remineralization of the early caries lesion. Schmidlin et al.20 observed that CurodontTM Repair exhibited high mechanical properties even at a depth of 125 μm and emphasized that the mechanical properties of Curodont Repair were superior to the fluoride-containing agent at a depth of 200 μm. Alkilzy et al.,7 in a randomized controlled clinical trial, reported that the combination of P11-4 and fluoride-containing agents improved remineralization. The treatment of early caries lesions with P11-4 is safe, and with a single application, significant enamel regeneration is observed, presumably by promoting mineral deposition within the subsurface.10 According to Jablonski-Momeni et al.,21 treatments with self-assembling peptide P11-4 revealed large areas of the remineralized enamel surface, thereby proving to be efficacious. However, Wierichs et al.,22 observed that self-assembling peptides could neither mask the lesions nor inhibit lesion progression considerably.
The samples in group III (control) were not treated with any remineralization agent and were subjected to a 7-day pH-cycling regimen. However, the mean lesion depth in group III (control) was also significantly reduced during the postintervention period. The reduction in the lesion depth of the control group can be attributed to the protective role of ions in the remineralizing solution mimicking the mineral composition and pH of saliva.
Inability to simulate the intraoral conditions and biological aspects involved in the initiation and progression of dental caries lesions.
Absence of the effect of salivary proteins, pellicle, and plaque on the de/remineralization processes.
Inadvertent experimental errors.
Variation in the microstructure of the enamel between the specimens.
SCOPE FOR FUTURE STUDIES
The results of the present study provide useful information on the remineralization potential of novel biomimetic self-assembling peptide P11-4 and fluoride-enhanced hydroxyapatite gel-based remineralization agents. Future comparative clinical studies/trials would be beneficial to verify the remineralization efficacy of the test materials.
The self-assembling peptide P11-4 and fluoride-enhanced hydroxyapatite gel-based remineralization agents both exhibited significant potential in the remineralization of artificial caries lesions in primary tooth enamel. However, the novel biomimetic self-assembling peptide P11-4-based remineralization agents demonstrated greater remineralization potential compared to fluoride-enhanced hydroxyapatite gel.
3. Prabhakar AR, Arali V. Comparison of the remineralizing effects of sodium fluoride and bioactive glass using bioerodible gel systems. J Dent Res Dent Clin Dent Prospects 2009;3(4):117–121. DOI: 10.5681/joddd.2009.029
4. Kamal D, Hassanein H, Elkassas D, et al. Comparative evaluation of remineralizing efficacy of biomimetic self-assembling peptide on artificially induced enamel lesions: an in vitro study. J Conserv Dent 2018;21(5):536–541. DOI: 10.4103/JCD.JCD_123_18
5. Soares R, Fernandes M, Lambor R. Assessment of enamel remineralization after treatment with four different remineralising agents: a scanning electron microscopy (SEM) study. J Clin Diagn Res 2017;11(4):ZC136–ZC141. DOI: 10.7860/JCDR/2017/23594.97882017
6. Sindhura V, Uloopi KS, Vinay C, et al. Evaluation of enamel remineralizing potential of self-assembling peptide P11-4 on artificially induced enamel lesions in vitro. J Indian Soc Pedod Prev Dent 2018;36(4):352–356. DOI: 10.4103/JISPPD.JISPPD_255_18
12. Featherstone JD, Shariati M, Arends J, et al. Comparison of artificial caries-like lesions by quantitative microradiography and microhardness profiles. Caries Res 1983;17(5):385–391. DOI: 10.1159/000260692
13. Hicks MJ, Flaitz CM. Enamel caries formation and lesion progression with a fluoride dentifrice and a calcium-phosphate containing fluoride dentifrice: a polarized light microscopic study. ASDC J Dent Child 2000;67(1):21–28.
14. Magalhães AC, Moron BM, Comar LP, et al. Comparison of cross-sectional hardness and transverse microradiography of artificial carious enamel lesions induced by different demineralising solutions and gels. Caries Res 2009;43(6):474–483. DOI: 10.1159/000264685
15. Buzalaf MAR, Hannas AR, Magalhães AC, et al. pH-cycling models for in vitro evaluation of the efficacy of fluoridated dentifrices for caries control: strengths and limitations. J Appl Oral Sci 2010;18(4):316–334. DOI: 10.1590/s1678-77572010000400002
17. Yetkiner E, Eden E, Attin R, et al. Comparative evaluation of fluoride varnishes, self-assembling peptide-based remineralization agent, and enamel matrix protein derivative on artificial enamel remineralization in vitro. Prog Orthod 2021;22(1):4. DOI: 10.1186/s40510-020-00345
18. Takahashi F, Kurokawa H, Shibasaki S, et al. Ultrasonic assessment of the effects of self-assembling peptide scaffolds on preventing enamel demineralization. Acta Odontol Scand 2016;74(2):142–147. DOI: 10.3109/00016357.2015.1066850
19. Silvertown JD, Wong BPY, Sivagurunathan KS, et al. Remineralization of natural early caries lesions in vitro by P11-4 monitored with photothermal radiometry and luminescence. J Investig Clin Dent 2017;8(4):e12257. DOI: 10.1111/jicd.12257
20. Schmidlin P, Zobrist K, Attin T, et al. In vitro re-hardening of artificial enamel caries lesions using enamel matrix proteins or self-assembling peptides. J Appl Oral Sci 2016;24(1):31–36. DOI: 10.1590/1678-775720150352
21. Jablonski-Momeni A, Heinzel-Gutenbrunner M. Efficacy of the self-assembling peptide P11-4 in constructing a remineralization scaffold on artificially-induced enamel lesions on smooth surfaces. J Orofac Orthop 2014;75(3):175–190. DOI: 10.1007/s00056-014-0211-2
22. Wierichs RJ, Kogel J, Lausch J, et al. Effects of self-assembling peptide P11-4, fluorides and caries infiltration on artificial enamel caries lesions in vitro. Caries Res 2017;51(5):451–459. DOI: 10.1159/000477215
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