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Journal of Dental Research
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Biomaterials & Bioengineering

Self-assembling Peptide Scaffolds Promote Enamel Remineralization

J. Kirkham1,*, A. Firth1,2, D. Vernals2, N. Boden2, C. Robinson1, R.C. Shore1, S.J. Brookes1 and A. Aggeli2,{dagger}

1 Department of Oral Biology, Leeds Dental Institute, University of Leeds, Clarendon Way, Leeds LS2 9LU, UK; and
2 Centre for Self Organising Molecular Systems, School of Chemistry, University of Leeds, UK

Correspondence: * corresponding author, J.Kirkham{at}leeds.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationally designed β-sheet-forming peptides that spontaneously form three-dimensional fibrillar scaffolds in response to specific environmental triggers may potentially be used in skeletal tissue engineering, including the treatment/prevention of dental caries, via bioactive surface groups. We hypothesized that infiltration of caries lesions with monomeric low-viscosity peptide solutions would be followed by in situ polymerization triggered by conditions of pH and ionic strength, providing a biomimetic scaffold capable of hydroxyapatite nucleation, promoting repair. Our aim was to determine the effect of an anionic peptide applied to caries-like lesions in human dental enamel under simulated intra-oral conditions of pH cycling. Peptide treatment significantly increased net mineral gain by the lesions, due to both increased remineralization and inhibition of demineralization over a five-day period. The assembled peptide was also capable of inducing hydroxyapatite nucleation de novo. The results suggest that self-assembling peptides may be useful in the modulation of mineral behavior during in situ dental tissue engineering.

Key Words: biomimetic • self-assembly • peptides • scaffolds • hydroxyapatite • remineralization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oligomeric β-sheet-forming peptides that spontaneously undergo hierarchical self-assembly into fibrillar scaffolds in response to specific environmental triggers have provided a new generation of well-defined biopolymers with potentially important roles across a range of applications (Aggeli et al., 1997a,b). Under specific conditions, these peptides undergo one-dimensional self-assembly, forming micrometer-long, β-sheet "nanotapes". Further assembly can then be induced, such that the nanotapes stack in pairs to form ribbons (Nyrkova et al., 2000b; Aggeli et al., 2001b), which in turn can further assemble to form fibrils (Fig. 1Go), and pairs of fibrils entwine edge-to-edge to form fibers. This assembly process has been well-characterised (Nyrkova et al., 2000a; Aggeli et al., 2001a) and is principally driven by intermolecular H bonding arising from the peptide backbone, together with additional interactions between specific side-chains.


Figure 1
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  		Figure 1. Schematic representation of a self-assembled fibrillar network and its relationship with individual self-assembled fibrils and peptide primary structure. (a) Fibrillar network of assembled peptide. (b) Each fibril is comprised of 4 ribbons. The thin lines on the ribbons represent individual oligopeptides, each in β-strand conformation. (c) Dimeric antiparallel β-sheet tape of P11-4, the self-assembling peptide used in the present study. The labels correspond to the side-chains of the peptide at the back only.

 
Rational peptide design is central to potential applications for self-assembling peptide fibrillar scaffolds. The type of structure formed (tape, ribbon, fibril, etc.), its surface properties, and its responsiveness to external chemical triggers can be controlled (Aggeli et al., 2001b, 2003). In addition, control of the distances between specific functional groups on the surfaces of the polymers can be achieved through the properties of the underlying β-sheet lattice, imposing fixed distances between specific side-chains on adjacent strands. This offers exciting prospects for the use of self-assembling peptide fibrillar scaffolds as a route to novel nanostructured biomimetic biopolymers with applications in materials science (Meegan et al., 2004), medicine, and nanotechnology.

Once assembled, these fibrillar networks can form scaffold-like structures that mirror the biological macromolecules found in extracellular matrices, including those of the mammalian skeleton, where (predominantly anionic) matrix proteins are known to control the deposition and growth of hydroxyapatite crystals (Boskey, 2003). Dental enamel is a biological ceramic and the most highly mineralized of the skeletal tissues. In contrast to bone, its extracellular organic matrix is degraded and removed from the tissue prior to tooth eruption. During enamel development, enamel matrix proteins—themselves known to form self-assembling supramolecular structures—are believed to control the disposition and morphology of the hydroxyapatite crystals, ultimately determining the physicomechanical properties of the mature tissue (Simmer and Fincham, 1995; Wen et al., 1999; Kirkham et al., 2002).

Enamel caries is probably the most common of all skeletal tissue pathologies, presenting as progressive subsurface demineralization (mineral loss) and ultimately resulting in mechanical failure and cavitation (Robinson et al., 2000). The fundamentally invasive treatment strategy for enamel restoration has changed little over the years, despite advances in dental materials themselves. The long-term goal of this work is to offer a new generation of bioactive materials which recapitulate normal histogenesis by providing biomimetic scaffolds capable of inducing mineral deposition in situ.

The specific aim of this study was to determine the effects of a rationally designed self-assembling anionic peptide, P11-4 (Fig. 1cGo), on the de- and remineralization behavior of caries-like lesions of enamel under simulated intra-oral conditions.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis of P11-4
P11-4 (Ace-Gln-Gln-Arg-Phe-Glu-Trp-Glu-Phe-Glu-Gln-Gln-NH2; Fig. 1cGo) was synthesized by standard solid-phase peptide synthesis (Aggeli et al., 1997a). The HPLC-purified peptide was checked by analytical HPLC, mass spectrometry, and amino acid analysis. The mass spectrum of the purified peptide showed a single peak corresponding to a molecular weight of 1596 Da, and amino acid analysis confirmed the expected peptide composition.

Transmission Electron Microscopy (TEM) of Peptide Gels
P11-4 samples (15 mg/mL in phosphate buffer, pH 7.4) were diluted with water to a peptide concentration of 20 µM immediately before direct application to carbon-coated, 400-hexagonal-mesh copper grids. The grids were stained with uranyl acetate solution (4% w/v in water) for 20 sec and examined under a Phillips CM10 TEM. Gels prepared and incubated in "mineralizing" solution as described below, for the investigation of hydroxyapatite nucleation, were removed from the mineralizing solution, washed briefly in deionized water, fixed in 3% phosphate-buffered glutaraldehyde solution (pH 7.3), and embedded in Lowicryl K4M resin according to the manufacturers’ instructions. Ultrathin sections were deposited on 300-mesh carbon/formvar copper grids, stained with 3% methanolic uranyl acetate, and viewed in the Philips 400T TEM at an accelerating voltage of 80 KV.

Preparation of Lesions and pH Cycling
To test whether application of monomeric peptide affected remineralization and demineralization behavior of caries-like lesions, we used a previously published oscillating pH model (Robinson et al., 1992). Caries-like lesions were prepared in human permanent premolar teeth extracted for orthodontic purposes at Leeds Dental Institute. All teeth were used with the individuals’ written consent in fulfillment of the requirements of the Leeds Teaching Hospitals NHS Trust, and were used in accordance with standard protocols. Teeth were brushed clean under running water, but the natural surfaces were left intact. Following removal of the roots, a window of sound enamel, approximately 0.75 cm2, was delineated by the application of 2 coats of acid-resistant nail varnish on the buccal surfaces. The crowns were then immersed in acidified gelatin gel, pH 4.8, for 6 wks (Silverstone, 1966), and then washed in hot water to remove the gelatin for immediate use. This resulted in the production of subsurface lesions of approximately 100-µm depth. We treated the caries-like lesions by applying 10 µL of either (a) a solution of 5 mg/mL monomeric P11-4 in distilled water, titrated to pH 8 using dilute NaOH, or (b) the same solution without P11-4, directly to the surface using a paintbrush. The crowns were then left for 30 min at room temperature to allow the solution to soak into the lesion. From 4–6 treated crowns were then sequentially cycled through chemically defined solutions for 5 days in a pH-cycling model at 35°C (Robinson et al., 1992). Each 24-hour period included 3 x 20 min exposures to acid [demineralizing solution: 1.5 mM Ca(NO3)2, 0.9 mM KH2PO4, 50 mM acetic acid, pH 4.8], with incubation in remineralizing solution [1.5 mM Ca(NO3)2, 0.9 mM KH2PO4, 130 mM KCl, 60 mM Tris, pH 7.4) for the intervening periods. The degree of saturation (DS) with respect to hydroxyapatite for each solution (calculated according to a previously published algorithm (Shellis, 1988) ) was 0.29 and 14.06, respectively. After cycling, mineral loss or gain by the lesions was calculated following spectrophotometric determination of P in the incubation solutions (Chen et al., 1956). Exposed areas were calculated by image analysis, and results expressed as µg P/mm2 of exposed enamel. Control and experimental data were then compared by analysis of variance, based on the results obtained from 8 separate pH-cycling runs for both control and experimental lesions.

De novo Precipitation of Hydroxyapatite in vitro
To determine whether the peptide in its assembled form was capable of nucleating mineral crystals de novo, we prepared a birefringent P11-4 gel by mixing 15 mg P11-4 in 1 mL of 20 mM phosphate buffer, pH 7.0. We prepared gelatin gels of the same concentration by mixing gelatin (SIGMA, Poole, UK) with the same buffer, heating to 60°C, and cooling to room temperature. The gels were then incubated in 10 mL of "mineralizing" solution (120 mM NaCl, 22 mM NaHCO3, 3.75 mM CaCl2, 1.67 mM Na2HPO4, pH 7.4, DS with respect to hydroxyapatite = 26.86) for 7 days at 35°C. Gels were then removed from the incubation solutions, washed in distilled water (pH 7.4), and embedded for TEM. Thin sections were viewed, unstained, in a Philips 400T TEM fitted with an EDAX 9100 EDX spectrometer. Electron diffraction and elemental analysis of electron-dense deposits were then carried out.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A single application with a monomeric solution of P11-4 significantly increased net mineral gain by the caries-like lesions, compared with controls, after 5 days of pH cycling (Fig. 2aGo). This net effect was due to significantly decreased demineralization during exposure to acid, together with a strong trend toward increased remineralization at neutral pH. Treatment with P11-4 also resulted in significant net mineral gain on a day-by-day basis throughout the five-day cycling period, compared with controls (Fig. 2bGo). This effect diminished with increasing cycling time, but P11-4-treated lesions still showed significant net mineral gain, even during the fifth and final day of the cycling period.


Figure 2
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  		Figure 2. Effect of P11-4 on remineralization and demineralization behavior of caries-like lesions in human enamel under simulated intra-oral conditions. (a) Effect of treatment with P11-4 on mineral loss/gain by caries-like lesions of human enamel following 5 days of cycling in an oscillating pH model (5 x 24-hour periods, each comprised of 3 x 20-minute acid challenges [pH 4.8], intervening time at neutral pH; solutions supersaturated with respect to hydroxyapatite at pH 7.4, undersaturated at pH 4.8). Histograms show net mineral gain by lesions treated with the self-assembling peptides after 5 days of cycling, compared with untreated controls, and mineral loss/gain during periods at low pH compared with intervening periods at neutral pH. Results show mean values of 8 separate experiments ± SD. (b) Effect of treatment with a self-assembling peptide on mineral loss/gain by caries-like lesions of human enamel during each 24-hour period of 5 days’ cycling in an oscillating pH model (5 x 24-hour periods, each comprised of 3 x 20-minute acid challenges [pH 4.8], intervening time at neutral pH; solutions supersaturated with respect to hydroxyapatite at pH 7.4, undersaturated at pH 4.8). Histograms show net mineral gain by lesions treated with the self-assembling peptides compared with untreated controls. Results show mean values of 8 separate experiments ± SD.

 
When P11-4 gels were incubated for 7 days at pH 7.4 in physiologically relevant "mineralizing" solutions supersaturated with respect to hydroxyapatite, electron-dense deposits were observed throughout the body of the gel (Figs. 3a, 3cGo, 4aGo). Gelatin gels incubated under the same conditions contained no electron-dense material. Elemental analysis of the deposits within the P11-4 gel revealed a Ca:P molar ratio of 1.67 (Fig. 4cGo). Electron diffraction suggested the presence of a poorly crystalline hydroxyapatite (Fig. 4bGo).


Figure 3
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  		Figure 3. TEM of P11-4 gels and fibrils. TEM micrographs show: (a) section through an unstained, undiluted P11-4 gel following 7 days’ incubation at 35°C in "mineralizing" solution (22 mM NaHCO3, 3.75 mM CaCl2, 1.67 mM Na2HPO4, pH 7.4). Electron-dense deposits, some apparently aligned, may be seen (arrows). Scale bar corresponds to 250 nm. (b) P11-4 fibrils from gels that were not incubated in "mineralizing solution", stained with uranyl acetate and prepared by dilution of the gels immediately before application to the TEM grid. Individual fibrils with clear helical twist can be seen. Scale bar corresponds to 100 nm. (c) Section through an undiluted P11-4 gel stained with uranyl acetate after 7 days’ incubation at 35°C in "mineralizing" solution (22 mM NaHCO3, 3.75 mM CaCl2, 1.67 mM Na2HPO4, pH 7.4, supersaturated with respect to hydroxyapatite). Fibrils appear to be arranged in twisted bundles with a width of hundreds of nm to µm and lengths of several µm, reminiscent of collagen fibers. Electron-dense deposits associated with the bundles following in vitro mineralization may reflect sites where hydroxyapatite nucleation has taken place. Arrows show bundles of fibrils in cross-section (white arrow) and running longitudinally (black arrow). Scale bar corresponds to 1 µm.

 

Figure 4
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  		Figure 4. De novo nucleation of hydroxyapatite by P11-4 in vitro. (a) Transmission electron micrographs (unstained) showing needle-like electron-dense deposits in the body of a self-assembled P11-4 gel following 7 days’ incubation at 35°C in "mineralizing" solution (22 mM NaHCO3, 3.75 mM CaCl2, 1.67 mM Na2HPO4, pH 7.4). (b) Electron diffraction patterns of needle-like deposits. Lattice planes 110 (A); 002 (B); 211 (C), camera focal length 400 mm; 100 (D); 200 (E); and 102 (F), camera focal length 575 mm, are indicated. (c) EDX spectrum of needle-like deposits.

 
The self-assembled state of P11-4 under physiological-like conditions was examined in the TEM. Diluted preparations of P11-4 nematic liquid crystalline gels in phosphate buffer at neutral pH revealed well-defined, µm-long, semi-rigid fibrils with a typical width of approximately 12 nm and a full-twist pitch of approximately 240 nm (Fig. 3bGo). Electron micrographs of the undiluted, P11-4 gels used in the de novo precipitation experiments described above were obtained after some of the gels were fixed and stained at the end of the experiment. The results showed fibrils within the gel arranged in twisted bundles of 20–100 aligned fibrils, hundreds of nm to µm wide and several µm in length, reminiscent of collagen fibers. Electron-dense deposits were observed in association with the bundles following in vitro mineralization (Figs. 3a, 3cGo), possibly reflecting sites where hydroxyapatite nucleation had taken place.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The study and control of organic-inorganic interactions to create hybrid materials is an emerging field in materials science. Here we describe the use of rationally designed β-sheet-forming peptides that spontaneously assemble in response to environmental triggers to form 3D biomimetic scaffolds capable of nucleating hydroxyapatite de novo. Many existing scaffolds are constrained by the need for surface modification via the attachment of bioactive motifs (such as RGD sequences) to biocompatible polymers (Sofia et al., 2001; Yang et al., 2001), and/or the need for pre-fabrication of the scaffolds. The introduction of injectable bioactive materials such as chemically modified hydrogels and polyester scaffolds represents an important advance in tissue engineering (Jo and Park, 2000; Behravesh et al., 2003). Synthetic amphipathic self-assembling polymers and a biomimetic peptide based upon human phosphophoryn capable of nucleating hydroxyapatite de novo and in vitro have been reported (Hartgerink et al., 2001; Chang et al., 2006), and nucleation of hydroxyapatite crystals by self-assembled β-sheet cationic domains of DMP-1, an extracellular matrix protein of dentin and bone, has been described (He et al., 2003). We have advanced this concept further by exploring the use of a simple, rationally designed, self-assembling, biomimetic peptide scaffold, and have illustrated its potential application to enamel repair.

The responsiveness of the peptide to external triggers is a key property to potential applications, offering the possibility for application as a monomeric fluid and subsequent in situ-triggered assembly/gelation inside areas of tissue porosity, including caries lesions and exposed dentin. A further opportunity is for precise control of the surface properties of the peptide aggregates by appropriate peptide design, resulting in not only optimal interactions with the enamel surface, but also spatially determined de novo nucleation of hydroxyapatite crystals on the scaffold surface itself.

Previous work has systematically characterized P11-4 assembly under a range of different environmental conditions. At pH > 8.0 and low ionic strength, P11-4 forms a Newtonian fluid comprised of monomeric, random coil peptides over a wide range of peptide concentrations, due to intermolecular electrostatic repulsions among the 3 negatively charged side-chains at Glu5, Glu7, and Glu9 (Aggeli et al., 2003). When the pH of the solution is decreased below 8.0, P11-4 spontaneously self-assembles to produce 3D gels comprising β-sheet aggregates, due to the partial neutralization of the negative charges. Solution salt concentration (including Ca++ ions) also promotes self-assembly and gelation of P11-4, due to screening of the electrostatic repulsion between negatively charged Glu side-chains. We therefore predict that in the present study, the peptide would be in its monomeric form when originally applied to the lesions’ surfaces in the pH-cycling experiments, but would be rapidly driven to self-assembly following exposure to the tissue and experimental conditions.

Our results clearly demonstrate an effect for P11-4 on the de- and remineralization behavior of caries-like lesions under conditions of oscillating pH in vitro. Treatment with monomeric solutions of P11-4 resulted in a significant net mineral gain by the lesions after 5 days of cycling, compared with untreated controls. A single application of P11-4 to the lesion surface resulted in significant net mineral gain on each of the 5 days of pH cycling, suggesting a sustained or incremental effect on tissue repair under these conditions.

The mechanism(s) underpinning these effects is not yet known. Two broad mechanisms are possible and are not necessarily mutually exclusive. If the predicted transition from low-viscosity isotropic liquid to elastomeric nematic gel is triggered in situ within the lesion pores, the peptide would be expected to form a fibrillar gel within the pores of the lesion. The anionic groups of the side-chains would attract Ca++ ions, potentially inducing de novo precipitation of calcium phosphate salts from the supersaturated supernatant solutions in a regular array on the fibrils’ surface. TEM of the pre-assembled gels revealed fibrils arranged in bundles, thus producing periodic surfaces of aligned fibrils that may further facilitate hydroxyapatite precipitation over and above that of an isotropic gel network containing randomly positioned fibrils. The results obtained in vitro following incubation of the assembled P11-4 in "mineralizing" solutions indicated the presence of needle-like electron-dense deposits, which electron diffraction and EDX suggested to be poorly crystalline hydroxyapatite. It is therefore possible that the observed increase in mineral gain by the lesions was due to precipitation of mineral within an assembled scaffold in situ. This remains to be confirmed, since no analysis of mineral within the lesions themselves has yet been carried out.

However, our results also demonstrated that the increase in net mineral gain by the treated lesions following pH cycling was due to a significant decrease in demineralization during exposure to acid. Increase in remineralization at neutral pH was also apparent, but this was not statistically significant, due to the greater variability. The structure of P11-4 suggests that it would bind to mineral. Our results could therefore be reflecting a stabilization of the mineral surfaces, due to the presence of the peptide. Both this and the above effects could contribute toward the observed net increase in mineral gain by the treated lesions after cycling.

Taken together, our results suggest that self-assembling peptides offer a potentially exciting route to "smart" dental biomaterials, though much work remains to be carried out. We do not yet know, for example, whether the peptides are susceptible to proteolytic degradation, which might limit their use, especially as a surface treatment. Further work is also clearly required to clarify the precise mechanism(s) of their observed actions, in longer-term in situ and in vivo studies.


   ACKNOWLEDGMENTS
 
This work was supported by the Special Trustees of the Leeds NHS Teaching Hospitals Trust and the Royal Society of Great Britain (A.A. is a Royal Society University Research Fellow). The authors thank Mr. Lee Graham and Mrs. Jackie Hudson for their excellent technical assistance and Dr. M. Bell, Dr. I. Nyrkova, and Dr. L.M. Carrick for many helpful discussions. A.F. is supported by an EPSRC/GlaxoSmithKline CASE studentship.


   FOOTNOTES
 
{dagger} queries re self-assembling peptides, a.aggeli{at}leeds.ac.uk Back

Received for publication August 7, 2006. Revision received November 25, 2006. Accepted for publication December 31, 2006.


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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
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Journal of Dental Research, Vol. 86, No. 5, 426-430 (2007)
DOI: 10.1177/154405910708600507
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