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Year : 2018  |  Volume : 36  |  Issue : 4  |  Page : 352-356

Evaluation of enamel remineralizing potential of self-assembling peptide P11-4 on artificially induced enamel lesions in vitro

Department of Pediatric Dentistry, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India

Date of Web Publication16-Oct-2018

Correspondence Address:
Dr. Vemulapalli Sindhura
Department of Pediatric Dentistry, Vishnu Dental College, Vishnupur, Bhimavaram - 534 202, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JISPPD.JISPPD_255_18

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Purpose: The purpose of this study was to evaluate the remineralizing efficacy of self-assembling peptide (SAP) P11-4 qualitatively and quantitatively using scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX), respectively. Methodology: Enamel samples (n = 24) were prepared by sectioning extracted premolars and subjected for demineralization to create artificial enamel lesions. The structural and elemental concentrations (calcium and phosphate weight %) were assessed to obtain baseline data using SEM and EDX spectroscopy, respectively. The samples were randomly allocated into two groups and were treated with SAP P11-4 (test group) and casein phosphopeptides-amorphous calcium phosphate (CPP-ACP) (control group) followed by storage in artificial saliva to evaluate the remineralizing efficacy at 1-week, 1-month, and 3-month intervals. Results: Data were analyzed using the ANOVA and unpaired t-test. From the observed results, CPP-ACP showed a significant increase in Ca: P ratio (2.04 ± 0.2) with irregular surface calcific deposition at 1-week interval and this reduced with time (1.87 ± 0.11 at 3-month interval). Whereas P11-4 showed a significant increase in Ca: P ratio (1.95 ± 0.10) with uniform ion deposition suggestive of hydroxyapatite nucleation over a 3-month period. Conclusion: SAP P11-4 exhibited superior remineralization with uniform mineral deposition compared to CPP-ACP at 3-month interval.

Keywords: Casein phosphopeptides-amorphous calcium phosphate, enamel remineralization, self-assembling peptide P11-4

How to cite this article:
Sindhura V, Uloopi K S, Vinay C, Chandrasekhar R. 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;36:352-6

How to cite this URL:
Sindhura V, Uloopi K S, Vinay C, Chandrasekhar R. Evaluation of enamel remineralizing potential of self-assembling peptide P11-4 on artificially induced enamel lesions in vitro. J Indian Soc Pedod Prev Dent [serial online] 2018 [cited 2022 Nov 27];36:352-6. Available from: http://www.jisppd.com/text.asp?2018/36/4/352/243456

   Introduction Top

Enamel is the uniquely organized outermost covering of the tooth and is predisposed to numerous challenges within the oral environment. However, mature enamel cannot be regenerated due to lack of functional capacity of ameloblasts.[1] Owing to this inherent limitation and any drop in pH below the critical value begins the demineralization process. Once a certain degree of demineralization has occurred the lesion will appear as white spot. This marks the process of caries initiation with most of the mineral loss from the subsurface area and a relatively intact superficial layer. These lesions can be clinically detectable as well as remineralized noninvasively.[2] After approximately 30% of demineralization, the mineralized surface collapses and breaks irreversibly.[3] Hence, early detection and management of these incipient lesions with remineralizing agents are recommended.

Various remineralizing agents are available for noninvasive management of incipient lesions. However, these agents fall short of achieving matrix-mediated mineralization similar to the natural process. To overcome this, self-assembling peptide (SAP) P11-4 was developed to regenerate enamel by providing matrix-mediated mineralization. These short-chain peptides have the property of assembling into a three-dimensional structure resembling the extracellular matrix, thereby promote regeneration.[4] As there is little information available on remineralizing efficacy of SAPs, a study was planned with an aim to assess and to compare the remineralizing efficacy of novel biomimetic SAP P11-4, with casein phosphopeptides-amorphous calcium phosphate (CPP-ACP) on artificially induced enamel lesions.

   Methodology Top

Enamel samples were prepared from 12 recently extracted human premolars in children requiring orthodontic treatment. The samples were stored in 10% formalin solution until use. The root portions were dissected at the level of cementoenamel junction [Table 1], and crowns were made into two halves by sectioning them in mesiodistal plane with intact buccal and lingual surfaces using a low-speed diamond disk.[5] Thus, the obtained samples (n = 24) were allocated randomly into two groups with 12 samples in each group [Table 2]. Artificial enamel lesions were created by placing a 4 mm × 4 mm wax sheet on the tooth surface, and the remaining surface was coated with acid-resistant nail varnish, later the wax sheet was removed, and the samples were stored in demineralizing solution[6] for 4 days [Table 3]. The samples were then subjected for scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) to evaluate the structural appearance and elemental concentration of artificially created enamel lesions which served as a baseline data. According to manufacturer's instructions, the samples of the test group (P11-4) were etched with 37% phosphoric acid for 20 s and rinsed thoroughly with water, the surfaces were dried; later SAP P11-4 was applied on the tooth surface with a brush and allowed to diffuse for 5 min.[7] In Group 2, the sample surface was dried, and a sufficient amount of CPP-ACP was applied on the tooth surface with gloved finger and left undisturbed for 4 min.[8] Artificial saliva was used for the storage of samples at room temperature.[9] The solution was changed for every 14 days to prevent bacterial contamination.[10] SEM and EDX evaluation was done to assess the amount of remineralization at 1-week, 1-month, and 3-month intervals.
Table 1: Comparison of calcium, phosphate weight percentages, and calcium: phosphorus ratio at different time intervals in P11-4 group

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Table 2: Comparison of calcium, phosphate weight percentages, and calcium: phosphorus ratio at different time intervals in casein phosphopeptides-amorphous calcium phosphate group

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Table 3: Comparison of calcium phosphate ratio between P11-4 and casein phosphopeptides-amorphous calcium phosphate groups

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   Results Top

Quantitative evaluation of remineralization

EDX spectrometry was used to determine calcium and phosphorus weight % of the samples. The calcium and phosphorus content was then converted into Ca/P ratios for each group.

Data were statistically analyzed using ANOVA and unpaired t-test. In test group (P11-4), calcium phosphate ratio decreased at 1-week (1.71 ± 0.13) interval compared to baseline (1.73 ± 0.15). The ratio gradually increased at 1-month (1.74 ± 0.11) and 3-month (1.95 ± 0.10) evaluation; however, at 3-month observation, the difference was found to be statistically significant compared to baseline. On the contrary, with CPP-ACP group, a significant increase in Ca: P ratio was observed at 1 week (2.04 ± 0.2) itself, and then gradually the ratio reduced at 1-month (1.86 ± 0.14) and 3-month (1.87 ± 0.11) intervals. However, the increase in Ca: P ratio at all the intervals is significantly higher when compared to baseline.

On comparing Ca: P ratios with P11-4 and CPP-ACP groups at three intervals, a higher Ca: P ratio was found with CPP-ACP compared to P11-4 at 1-week and 1-month intervals which are highly significant (P = 0.000). However, at 3-month intervals, it is noticed that a higher Ca: P ratio was seen in P11-4 group compared to CPP-ACP which is statistically significant (P = 0.027).

Qualitative analysis of remineralization

Demineralized enamel samples [Figure 1]a and [Figure 2]a: in both the groups, after demineralization evidence of porosities with increase in pore volume were seen suggestive of demineralization.
Figure 1: Scanning electron microscopic images of enamel surface treated with P11-4; (a) demineralized enamel surface with loss of interprismatic substance showing evidence of porosities; (b) enamel surface treated with P11-4 at 1week interval showing evidence of reduction in pore volume; (c) 1month evaluation of enamel surface showed evidence of mineral deposition around the prism cores; (d) 3month evaluation of enamel surface depicts evidence of mineral deposition resembling natural enamel surface

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Figure 2: Scanning electron microscopic images of enamel surface treated with CPP-ACP; (a) demineralized enamel surface with evidence of porosities suggestive of demineralisation; (b) 1week evaluation of enamel surface treated with CPP-ACP showed evidence of Mineral deposition around the prism cores; (c) enamel surface treated with CPP- ACP observed at 1month interval showed reduction in pore volume; (d) 3month evaluation of enamel surface represents globular deposition of ions around prism cores suggestive of remineralisation

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SAP P11-4 [Figure 1]b, [Figure 1]c, [Figure 1]d: remineralization was noticed at 1-month interval with evidence of ion deposition around the prism, and a reduction in pore volume was noticed. However, at 3-month interval, uniform ion deposition was found resembling natural enamel surface suggestive of hydroxyapatite nucleation.

CPP-ACP group [Figure 2]b, [Figure 2]c, [Figure 2]d: mineral deposition was noticed around the demineralized enamel prisms with reduced pore volume at 1-week interval. On 1-month and 3-month observation, although ion deposition was seen, irregular deposition with globular arrangement of ions was observed.

   Discussion Top

Incipient lesions manifest as early signs of caries activity. These lesions clinically appear as a white opaque spot and are softer than the adjacent sound enamel. They are capable of being reversible.[10] Remineralizing agents being marketed fail to regenerate enamel by simulating the natural mineralization process. Understanding the basic principles of matrix-mediated mineralization paved a way for the formulation of biomimetic enamel remineralizing agents such as SAP P11-4. In the present study, the remineralizing efficacy of SAP P11-4 was compared with CPP-ACP both qualitatively and quantitatively using SEM and EDX, respectively.

EDX is a spectroscopic technique which determines the presence and relative abundance of the elements that are present in the specimen whereas SEM produces images of sample by scanning the surface with a focused beam of electrons.[11] Although atomic force microscopy (AFM), scanning tunneling microscopy, and profilometry can be used to determine enamel surface characteristics, SEM and EDX were used in the present study as they determine the ion concentration and surface morphology in noncontact method. Studies conducted to evaluate the remineralizing efficacy of P11-4 using AFM studies and by clinical evaluation showed effective remineralization at 3 months.[7] We preferred three-time intervals to understand the pattern of remineralization and analyze the mineral content (Ca: P ratio).

Initially (at 1-week interval), SAP P11-4 showed a decrease in Ca: P ratio compared to baseline value. This could be attributed to additional acid etching before the application of P11-4 as recommended by the manufacturer which could have caused further loss of minerals from the tooth surface. In contrary, CPP-ACP initially had shown an increase in Ca: P ratio with evidence of mineral deposition and reduction in pore volume on SEM evaluation at 1 week. Similar observations were noticed when the remineralizing efficacy of CPP-ACP was evaluated at different time intervals with EDX and SEM, it was found that CPP-ACP showed increase in Ca: P ratio at 1-week interval itself.[8] However, contrary findings were reported in a study done by Lata et al., where surface microhardness evaluation of CPP-ACP following single application did not show effective remineralization after 5 days.[12]

Interestingly, SAP P11-4 showed a gradual increase in the Ca: P ratio at 1-month and 3-month intervals. Evidence suggests that remineralization in 93% of samples was confirmed by SEM examination after 3 months.[7] Similar findings were reported after ultrasonic assessment of SAP P11-4 with the formation of needle-shaped crystals.[13] The possible hypothesis explained for matrix-mediated mineralization by SAP P11-4 is that bioactive peptide synthesized from amino acids diffused into the porosities and assembled within the subsurface lesion into a three-dimensional fibrillar scaffold resembling extracellular matrix.[3] This acts as a nucleus for hydroxyapatite, which initiates tissue regeneration by attracting calcium and phosphate ions from saliva resulting in increased Ca: P ratio. Furthermore, when peptides assemble into fibers, it contains clusters of negative charges made up of four Glu residues providing a potential Ca2+-binding site. These binding sites are approximately 9.4 A apart, the distance normally present in the natural hydroxyapatite crystal.[14] The structural resemblance of these peptides to the biological macromolecules in mammalian skeleton accounts for the peptides to provide matrix-mediated mineralization.[15],[16]

CPP-ACP, on the other hand, showed a decrease in the Ca: P ratio at 1-month and 3-month intervals. A single application of CPP-ACP could be the possible reason for decrease in the ratio with time. CPP has been shown to stabilize calcium and phosphate, preserving the ions in an amorphous or soluble form termed as ACP. CPP-bound ACP acts as a reservoir of calcium and phosphate ions. The calcium and phosphate ion diffuses through the pores of carious surface into the body of enamel lesion. In the body of the enamel lesion, dissociation of ions increases the activities of Ca2+ and PO43−, thereby increasing the degree of saturation with respect to hydroxyapatite.[17] The formation of hydroxyapatite in the lesion would lead to the generation of acid and phosphate, including the neutral phosphoric acid, which would diffuse out of the lesion down a concentration gradient.[18],[19]

Although both remineralizing agents had shown comparable evidence of remineralization at 3 months, SAP P11-4 demonstrated improved mineral content with surface morphology resembling natural enamel on quantitative and qualitative evaluation. The findings of the present study promptly reveal the quality of remineralization produced with SAP P11-4 and give further importance to carry out clinical studies and to authenticate its efficacy over other remineralizing agents in a large sample group with long-term evaluation.

   Conclusion Top

SAP P11-4 showed superior remineralization with higher Ca: P ratio and uniform mineral deposition on incipient lesions compared to CPP-ACP.

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Conflicts of interest

There are no conflicts of interest.

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Kyle S, Aggeli A, Ingham E, McPherson MJ Production of self-assembling biomaterials for tissue engineering. Trends Biotechnol 2009;27:423-33.  Back to cited text no. 4
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  [Table 1], [Table 2], [Table 3]

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