Physico-chemical Characterisation of the Processes Involved in Enamel Remineralisation by CPP-ACP
Casein phosphopeptides derived from tryptic digests of milk caseins spontaneously assemble with calcium and phosphate ions at high pH to form casein phosphopeptide-amorphous calcium phosphate complexes (CPP-ACP). These complexes have been shown to be able to repair lesions in tooth enamel (biohydroxyapatite – HA) both in vitro and in vivo (specifically white spot lesions in the early stages of tooth decay). In order to better understand the processes involved in enamel remineralisation, the chemical equilibria between the CPP and calcium and phosphate ions as a function of pH were investigated. Furthermore, a thin-enamel slab technique was developed with enhanced sensitivity to monitor the diffusion of radio-opaque ions into individual lesions over a period of days to weeks.
KeywordsEnamel CPP-ACP Remineralisation Hydroxyapatite Diffusion NMR Model
The aim of this study was to investigate the interactions between the peptides and crystalline and non-crystalline mineral components during the remineralisation process.
23.2 Materials and Methods
Extracted human third molars were obtained from patients attending the Melbourne Dental School with ethics approval (1340048). Enamel slices (~300 μ thick) were prepared from these teeth for examination.
23.2.2 Ion-Binding Studies
To evaluate the influence of pH on the calcium and phosphate ion equilibria, solutions of CPP-ACP, αS1-CN(59-79)-ACP, and β-CN(1-25) were subjected to pH titrations. Calcium and phosphate ion concentrations were determined using microfiltration using previously described protocols, (Cross et al. 2005) modified to use a Dionex ion analyser.
23.2.3 Nuclear Magnetic Resonance Studies
NMR spectra were acquired at 599.741 MHz on a Varian Unity Inova spectrometer as described previously (Cross et al. 2016). Solution-phase diffusion measurements were performed using the sLED experiment as described previously (Altieri et al. 1995).The amplitude of the NMR signal is a function of the applied magnetic field gradient and the Stokes-Einstein radius of the diffusing species (S ∝ exp (−αDG2),where D is the diffusion coefficient, G is the magnetic field gradient, and α depends on experimental values. The diffusion coefficient is related to the hydrodynamic radius by D = kBT/6πρR where kB is the Boltzmann constant, T the absolute temperature, ρ the solution viscosity, and R the hydrodynamic radius of the spherical particles.
23.2.4 Remineralisation Studies
The novel technique utilised thin slabs of enamel (~300 μ thick) cut from human third molars that allowed sound mineral portions of the slabs to be used as controls in measuring the time course of remineralisation of artificial lesions. The technique is an extension of that previously described (Cochrane et al. 2008), with remineralisation of individual slabs being assessed after 0, 2, 3, 6, 12, 15, and 20 days immersion in a remineralisation solution at either pH 5·5 or pH 7·0. Diffusion of radio-opaque ions into the artificially prepared lesions was monitored by TWIM (Thomas et al. 2006). Acid-resistant nail polish was used to define the remineralisation zone and applied three times to prevent leakage during the soaking in the remineralisation solutions. Remineralising solutions consisted of 1% solutions of either CPP-ACP or β-CN(1-25)-ACP prepared at either pH 7.0 or 5.5 to compare the effects of neutral and acidic pH.
Lesion-sections were subjected to transversal wavelength-independent microradiography (TWIM) at days 0, 2, 3, 6, 12, 15, and 20 to visualise the time dependence of mineral ion uptake during enamel remineralisation. Microradiographs acquired on day 0 were used as control images for each lesion-section. The lesion-sections remained soaked in the remineralisation solutions except when being X-rayed.
DOSY experiments were performed using the sLED technique to determine the relative rates of diffusion of the complexes using either the integrated aromatic or aliphatic signals of a β-CN(1-25)-ACP sample.
SDS-PAGE of CPP cross-linked using glutaraldehyde was conducted to analyse the multimerisation of CPP as described previously (Cross et al. 2016).
23.3.1 The CPP-ACP Complexes Exist in Equilibria with Both Bound and Free Calcium and Phosphate Ions
23.3.2 The CPP-ACP Complexes Are Small Readily Diffusible Species
SDS-PAGE of CPP cross-linked using glutaraldehyde, in the presence of either calcium ions (Fig. 23.4b) or calcium and phosphate ions (Cross et al. 2016), suggests that the complexes contain up to six CPP peptides. Figure 23.4c shows a model of the CPP-ACP complex consistent with the results of these experiments.
23.3.3 Both CPP-ACP and β-CN(1-25)-ACP Complexes Release Mineral Ions that Remineralise Demineralised Enamel Lesions
Figure 23.5c shows a plot of the time-dependent, X-ray opacity of a specific enamel slab after demineralisation at day 0 and following remineralisation until day 20. The X-ray data showed a diffusion-dependent increase in electron density, interpreted as mineralisation.
A time-dependent uptake of mineral was observed in the presence of both CPP-ACP and β-CN(1-25)-ACP at both pH values. The calculated data from each sample was fitted to the time-dependent part of a diffusion equation of the form
In this study, a variety of methods have been used to characterise the complexes formed by the casein phosphopeptides with calcium and phosphate ions. CPP bind calcium and phosphate to form stable CPP-ACP complexes in alkaline solution. These studies show that the ratio of bound calcium to bound phosphate is constant and independent of pH in the range of pH 7–9. Furthermore, the ion activity product fits a single curve for a calcium-rich, non-stoichiometric calcium phosphate phase. DOSY experiments using the sLED sequence demonstrated that the complex has a small but significant variation in size with pH. These experiments further revealed the ability of the small complexes to aggregate as concentrations were increased. The model of the CPP-ACP complex with all amino acids in the peptides interacting with the ACP surface is consistent with earlier findings that the peptide length influences the extent of binding to calcium and phosphate ions (Cross et al. 2005).
The importance of small readily diffusible CPP-ACP complexes is confirmed by the experiments using thin slabs of human enamel with artificial lesions that mimic early carious lesions. To enable the time-dependent monitoring, the current remineralisation procedures (Shen et al. 2011) required extensive improvements. The use of the single section for all-time points required a thicker section to withstand repeated handling. To accommodate the increased thickness, TWIM was used instead of the commonly used transverse microradiography (TMR) technique, both methods being validated techniques for monitoring carious lesions (Thomas et al. 2006). In addition, the acid-resistant nail varnish was reapplied three times to prevent leakage during the soaking in remineralisation solutions. To improve the signal-to-noise ratio, an additional 500-μ-thick aluminium filter was used with an optimal X-ray tube voltage of 30 kV.
We observed statistically significant differences in the extent of remineralisation within the multiple lesion-sections derived from the same individual tooth. These differences were attributed to varying microporosities of the demineralised lesion-sections of each individual tooth.
In conclusion, the CPP-ACP complexes were characterised to be a small readily diffusible species that can release the mineral ions on contact with demineralised enamel lesions. Furthermore both CPP-ACP and β-CN(1-25) complexes were able to remineralise within a few days in in vitro experiments.
This study was funded by the Oral Health Cooperative Research Centre and NHMRC. Extracted human third molars were obtained from patients attending the Melbourne Dental School with ethics approval (1340048).
- Huq NL, Cross KJ, Myroforidis H, Stanton DP, Chen YY, Ward BR, Reynolds EC (2018) Molecular interactions of peptide encapsulated calcium phosphate delivery vehicle at enamel surfaces. Proc BIOMIN XIVGoogle Scholar
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