RGUHS Nat. J. Pub. Heal. Sci Vol No: 16 Issue No: 1 eISSN: pISSN:
Aparna B K* , Yashoda R, Manjunath P Puranik
Department of Public Health Dentistry, Government Dental College and Research Institute, Bangalore, Karnataka, India
*Corresponding author:
Aparna B K, Postgraduate student, Department of Public Health Dentistry, Government Dental College and Research Institute, Bangalore, Karnataka, India. E-mail: aparnabk17@gmail.com
Received date: 11/02/22; Accepted date: 5/05/22; Published date: 30/09/2022
Abstract
Dental enamel has poor regeneration capability due to the lack of regenerative cells and vascularization. However, under extreme laboratory circumstances, certain procedures can make enamel-like hydroxyapatite nanorods. Self-assembling peptide P11-4 has been introduced as a biomimetic approach for ‘guided enamel regeneration’. It is a rationally designed peptide, the monomers of which self-assemble into a biocompatible fibrillar scaffold that mimics the enamel matrix in response to particular environmental cues. Information on self-assembling peptide P11-4 was gathered using PubMed. A boolean search of PubMed data was conducted using the keywords: (self-assembling peptide) OR (P11) OR (P11-4 OR P11-4 peptide) AND (self-assembling peptide P11-4) AND (dental caries OR white spot OR incipient caries OR early enamel caries) AND (tooth demineralization OR tooth remineralization OR tooth regeneration). The last search was done on September 2021. Using a similar search method, studies were also obtained from the Cochrane Library and Google Scholar. 10687 articles were retrieved using the search method. More articles were gathered by manually searching the reference lists of articles. In this review, the most relevant papers were picked and used. Available evidence shows that self-assembling peptide P11-4 fibers could bind to calcium ions and template hydroxyapatite formation for the treatment of initial carious lesions, supporting remineralization in the same way that amelogenin stimulates enamel formation.
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Introduction
Peptide molecules self-assemble due to their common peptide backbone. An array of nanostructured materials and devices that are appropriate for specific applications can be artificially created by accurately using the property of self-assembly in peptides.1,2 Artificial oligomeric peptides that self-assemble into hierarchical structures in solutions in response to defined physicochemical environmental triggers can be generated in controlled conditions, allowing them to be used as scaffolds in tissue engineering.2
The concept of minimal intervention dentistry advocates a non-invasive approach to managing non-cavitated subsurface demineralized dental lesions via remineralization.3 The routine restorative materials used in dentistry cannot replicate or induce enamel to facilitate repair or remineralization. Various research efforts have been reported to synthesize a humanlike enamel to mimic the biomineralization process to recreate the enamel layer and repair it.4
Peptide monomers can self-assemble into scaffolds with external stimuli, which, along with their great affinity for calcium ions in saliva, can result in the formation of enamel crystals around the enamel matrix.5 This property can be made use of initiating crystal deposition in areas of enamel demineralization. This review presents a narrative overview of the application of self-assembling peptide P11-4 (SAP P11- 4) in therapeutics and hard tissue engineering, including enamel regeneration via self-assembly of organized nanostructures. It is based on a search for articles focusing on SAP P11-4 and its enamel regeneration ability.
Materials and Methods
PubMed search engine was used to gather information relevant to self-assembling peptide P11-4 and its role in biomimetics and enamel regeneration. A boolean search of PubMed data was conducted using the keywords: (self-assembling peptide) OR (P11) OR (P11-4) OR (P11-4 peptide) AND (self-assembling peptide P11-4) AND (dental caries OR white spot OR incipient caries OR early enamel caries) AND (tooth demineralization OR tooth remineralization OR tooth regeneration). The last search was done on September 2021. Using a similar search method, studies were also selected from the Cochrane Library and Google Scholar. Using this search strategy 10687 articles were retrieved. More articles were included by manually examining the reference lists of articles. The most relevant papers were chosen and used in this review.
Self-assembling peptides
1.1.1. Discovery
Zhang S found a protein Zuotin that could bind to left-handed Z-DNA when he was working on yeast genetics to explore the left-handed Z-DNA structure. He discovered a repeated segment with the sequence n-AEAEAKAKAEAEAKAK-c in Zuotin and named it EAK16 indicating its amino acid composition and peptide length. The EAK16 peptide showed a property of self-assembly and took a stable β-sheet structure by itself to form a well-ordered nano-fiber scaffold.6
Later this concept of self-assembly aided in the development of various kinds of biomaterials. Fiberforming peptide, amphiphile molecules’ biological potential to self-assemble was identified in 2001. The peptide amphiphile molecules were created by joining a hydrophobic tail to a hydrophilic amino acid sequence. Similar peptide amphiphiles were later developed employing effective sequences chosen to promote β-sheet configurations, cell adhesive characteristics (e.g., including epitope Arg-Gly-Asp-Ser, RGDS, ligands), and mineralization by favoring hydroxyapatite (HAp) growth.7
1.1.1. Structure
The intrinsic chirality of the 20 naturally occurring α-amino acids that constitute the proteins and peptides can result in a similar formation of the hierarchy of supramolecular structures. By self-assembly, they may form “helical tapes (single molecule thick)”, “twisted ribbons (double tapes)”, “fibrils (twisted stacks of ribbons)”, and fibers with increasing concentration.8 Because they contain both hydrophilic and hydrophobic components, most self-assembling molecules are amphiphilic. The main driving force for the selfassembly in peptides is their amphiphilicity which enables the functional groups to be present on the surface of the structure. Due to structural folding, giving rise to ‘faces’, different folded surfaces are exposed to different environments. These peptides are more complex in their amphiphilicity suiting them better for self-assembly.9
2.1.2.1 Building blocks of self-assembling peptides
In self-assembled peptide structures, different constituent amino acids and various bound chains or motifs can be observed. Some of these motifs called “peptide building blocks” are dipeptides, surfactant-like peptides, peptide amphiphiles with an alkyl group, bola-amphiphilic peptides, and ionic-complementary self-assembling peptides.10
In peptide nanotechnology, dipeptides are the most fundamental ‘building block.’ In dipeptide self-assembly, β-amino acids, the simplest amino acids with an extra C–C link are used.10 The hydrophobic tail of a typical surfactant-like peptide has numerous hydrophobic amino acids, while the hydrophilic head contains hydrophilic amino acids.11
The most prevalent peptide building blocks are peptide amphiphiles with alkyl groups connected with hydrophobic alkyl chains.10 The alkyl tail, β-sheet section, and glycine linker are typically conserved in a designer peptide amphiphile to ensure the self-assembling process, whereas the hydrophilic end has numerous epitopes with distinct functionalities to ensure the self-assembling process.11 These peptide amphiphiles make excellent templates for HAp crystal formation.7
Two hydrophilic heads are linked by a hydrophobic part in bola-amphiphiles. Because of their double-headed form, bola-amphiphilic molecules have unique characteristics and an extremely complex assembly phenomenon.10 Ionic-complementary peptides (ICP) have both positive and negative residues with a unique alternating charge distribution, which when they combine to form membrane-like structures.10 These self-assembling structures based on ICP predominantly assume a β-sheet structure that then can further self-assemble spontaneously into fibrils and hydrogels.12
2.1.2.2. Designing of P11-4 Self-Assembling Peptide
Aggeli et al created a de novo 11-residue peptide CH3 CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH2 that forms β-sheet polymer tapes in water based on criteria for the design of gel generating peptides obtained from their observations and existing literature.13
By incorporating Glu (-CH2 CH2 COOH) or Orn (-CH2 CH2 CH2 NH2 ) into the main structure of the 11 amino acid peptides, Aggeli et al showed that self-assembly may be regulated quickly (in seconds) and reversibly by adjusting the pH.14 P11-4 (CH3 CO-Gln-Gln-Arg-Phe-Glu-Trp-Glu-Phe-Glu-Phe-Glu-Phe-GluGln-Gln-NH2 ) is a synthetic peptide that hierarchically self-assembles into scaffolds in response to specific environmental stimuli and as peptide concentrations rise.15 Self-assembled P11-4 with negative charge domains has scaffold-like structures that are similar to biological macromolecules found in the extracellular matrix of mineralized tissues.16 (Fig. 1).
1.1.2. Peptide matrix formation
The SAP P11-4 has been shown to support enamel formation on its surface by supporting hydroxyapatite nucleation via the resulting fibers. Two in vitro studies successfully depicted the secondary conformation of the fibrils formed over the lesion surface, predominantly the β pleated sheets using Transmission Electron Microscope (TEM) and Congo red stain after the surface treatment with SAP P11-4.15,17
2.1.3.1. TEM examination
Kirkham J et al used TEM to demonstrate the self-assembled form of P11-4 in physiological condition revealing nematic gels with well-defined, micrometer-long, semi-rigid fibrils with a typical width of roughly 12 nm and a full-twist pitch of approximately 240 nm.17 Kind L et al also depicted self-assembled P11-4 forming nematic gels in acidic conditions.15
2.1.3.2. Fourier-transform infrared spectroscopy (FTIR) and Congo Red Stain
Kind et al employed FTIR to examine the absorption spectra of the monomeric and fibrillary forms of P11-4, as well as Congo red stain to show the fibrils’ secondary shape as a primarily pleated sheet.15
1.1. Self-Assembling Peptides as Biomaterial Candidates
The ability of molecular self-assembly ushered in the creation of new biomaterials. By providing a microenvironment with certain features, self-assembling peptides favor the formation of mature or primitive cell types with diverse possibilities.1
• In Magnetic Resonance Imaging (MRIs) contrast agents containing self-assembling peptides increase the sensitivity in the area of interest.
• Targeted fluorescence imaging can be done in vivo using self-assembled peptide complexes.
• Drugs, genes, and other therapeutic substances are carried by self-assembling peptides in therapeutics.18
• Using the hydrophobic core, hydrophobic medicines can be integrated into micellar structures created by amphiphilic peptides.11
• In cancer therapy, the self-assembly of oligomeric peptides around the cancer cell membrane could lead to the death of the cancer cells.12
• The self-assembling properties of Lanreotide in water (a cyclic octapeptide), which aid in the formation of nanotubes, highlights the potential applications such as a growth hormone inhibitor in acromegaly treatment.19
• Nanocomposites can be created using procedures that are based on biological systems and follow a hierarchical self-assembly process. Electrostatic interactions between calcium and phosphate ions in developing HAp and functional groups outside the collagen molecules can cause self-organization.20
1.1.1.Self-assembling Peptides in Enamel
Regeneration Dental enamel is known to be the highly mineralized biological ceramic of skeletal tissues. The enamel matrix proteins control the deposition and morphology of the hydroxyapatite crystals during enamel development, which resembles the self-assembly of supramolecular structures, determining the physicomechanical properties of the mature enamel tissue.17
Extracellular macromolecules in enamel undergo natural supramolecular self-assembly during enamel mineralization. At the time of enamel growth, the amelogenin-rich extracellular organic matrix is constantly secreted, assembled, and mineralized.21 Self-assembling peptide P11-4 was designed in a way to form fibrils at low pH and can act as a scaffold for hydroxyapatite crystals formation in response to external triggers. It has specific molecular features of the extracellular matrix (ECM) of tissues, such as the RGD cell-binding peptide sequence, a domain present in fibronectin protein of basement membrane of enamel during the early phase of enamel formation. The dental epithelium is in continual contact with the proteinaceous matrix of the basement membrane during tooth development, which provides important signals for enamel deposition. The selfassembling peptide P11-4 filaments have been designed to attract calcium ions and act as a template for the synthesis of hydroxyapatite, aiding remineralization in the same way that the proteinaceous matrix aids enamel development.22,23
The P11 family of self-assembling peptides can self-assemble under controlled conditions, resulting in a single molecule thick, micrometer-long β-sheet nanotapes.24 SAP P11-4 is a practically excellent scaffold for enamel remineralization because the repeat unit in β-sheet structures and the cell dimensions of the hexagonal phase of HAp have a clear correlation between them.7
Because of the acidic pH, calcium phosphate minerals dissolve and form pores between crystallites during the demineralization phase, whereas saliva becomes supersaturated with calcium phosphate and redeposits minerals either on existing crystallites or triggers de novo formation of crystallites during the remineralization process. This represents the natural regeneration process of the enamel tissue.15 However, mature enamel cannot be regenerated due to the lack of functional capacity of ameloblasts. Also, any drop in pH below the critical value begins the demineralization process.25 An imbalance between re- and de-mineralization takes place at the site of loss of minerals in hard dental tissue that can result in dental caries, enamel erosion, or dentine sensitivity.26
When an enamel lesion undergoes remineralization, the equilibrium toward remineralization is shifted by reducing the solubility of the dental tissue or increasing the surface area for mineral redeposition.27 During this process, partially demineralized tooth structures can absorb minerals from the environment, such as saliva or biofilm. In demineralized enamel and dentin, remineralization can restore minerals and cause amorphous material to precipitate in the inter-crystal and inter-rod gaps.28,29 Synthetic peptide amphiphiles can be created to self-assemble into nanofibers that aid in the mineralization of HAp.7
P11-4 was found to induce hydroxyapatite nucleation, which results in the remineralization of early carious lesions and enamel erosions by diffusing into mineral loss sites and increasing hydroxyapatite precipitation.30 P11-4 converts from a low viscosity isotropic liquid to an elastomeric nematic gel at pH 7.4 and in the presence of cations. These conditions are known to exist within a carious lesion.16 This property can be made use of initiating crystal deposition in areas of enamel demineralization.
1.1.1.1. Early Enamel Caries
Enamel caries is the most common of all the skeletal tissue problems that produce subsurface demineralization (mineral loss) and eventually cavitation.17 A biological compartment lies under the subsurface body of an initial carious lesion, similar to a void created during guided tissue regeneration. A biomimetic scaffold can help hard tissue remineralization via saliva in this location, resulting in guided enamel regeneration, similar to guided tissue and guided bone regeneration.31
In vitro studies have demonstrated that the P11-4 matrix has an affinity for Ca2+, acting as a nucleator for de novo hydroxyapatite formation, implying that the P11-4 matrix aids the body’s natural enamel remineralization process. This allows more severe enamel lesions to be remineralized, but not cavitated lesions.17,31
A synthetic amelogenin protein trapped between two layers of the membrane could mimic the ameloblast cell membrane with an electrolytic deposition system. It allows only calcium ions to enter the system one-directionally which can act as an organic matrix to induce mineralization. Within the enamel prisms, such a device has been successfully employed to generate apatite crystals with an organization comparable to that of enamel. This could also explain why P11-4 was used to design and manufacture enamel-like materials.21
These self-assembling peptide networks can build scaffold-like structures, comparable to biological macromolecules found in extracellular matrices such as dental enamel, where matrix proteins have been demonstrated to impact hydroxyapatite crystal deposition and growth.17
The surface of the P11-4 fibers may aid in the spontaneous remineralization caused by saliva by increasing the surface area available for calcium phosphate deposition.32 Due to its low viscosity, P11-4 penetrates the pores of the white spot lesion, causing self-assembly and the formation of negatively charged fibers that attract calcium ions thereby resulting in the formation of hydroxyapatite mineral.33
By stabilizing critical nuclei to allow crystal development in demineralized tissue voids, these nucleators function to bring constituent mineral ions from the surrounding disorganized ionic milieu into a highly ordered crystal lattice structure. To govern the formation of hydroxyapatite crystals during biomineralization, critical ionic nuclei must be produced by the collision of important ions. If such nuclei are stable, further crystal development will occur.16
Fully self-assembled fibrillar P11-4 can inhibit demineralization of the enamel if applied on nondemineralized enamel surfaces, by forming a layer on the tooth that buffers the acids and retains calcium phosphate during the demineralization phase.34 The SAP P11-4 fibers were constructed in a way that they promote the binding of calcium ions and the creation of template hydroxyapatite, aiding remineralization in the same way as amelogenin aids enamel development.23
Curodont™ Repair is a commercially available material that contains P11-4 developed for enamel regeneration by CUROLOX® technology at the University of Leeds. Before application, the tooth is polished (with standard prophy paste), wiped with diluted NaOCl (on a cotton swab), etched (20 seconds; 35% phosphoric acid), rinsed, and dried. This preparation can take anywhere from 3 to 5 minutes depending on the degree of experience. CurodontTM Repair is normally only used once, however, it can be repeated for a greater cosmetic effect after a few months if needed. Therapy can be provided by both a dentist and a dental hygienist, although a dentist will confirm the diagnosis and advise treatment first.35
1.1.1.2. Erosion
An early erosive lesion is one in which the enamel has not lost volume but has begun to demineralize, causing the enamel to become softer. Peptide P11-4 can self-assemble into a three-dimensional (3D) scaffold on the surface of the tooth, increasing hydroxyapatite precipitation while decreasing diffusive mineral loss. Because the majority of intraoral erosive lesions are caused by an acidic environment, the SAP P11-4, which at low pH can form fibrils and be monomeric at higher pH, could aid in improved remineralization of such surfaces. The dissolved Ca2+ is attracted by negatively charged surfaces and serine residues that have been phosphorylated in the peptide could further augment the remineralization ability.36
CurodontTM Protect is a commercial product that uses CuroloxTM technology, along with fluoride and calcium phosphate, to produce a gel or film over the tooth to prevent acid erosion.35
1.1.1.3. Dentin hypersensitivity
The dentinal tubuli are thought to be occluded by depositing a peptide matrix (SAPM) layer over the teeth in a gel. The SAPM is composed of hydrophilic fibers, formed by the self-assembling peptides P11-4, that carry hydroxyapatite binding sites on its surface. The fibers show a high affinity to the dentin surface because these binding sites keep the matrix electrostatically bound to the surface of the tooth. By placing SAPM (i.e.hydrogel) on the exposed root surface the dentinal tubuli are not blocked from within. As the occlusion does not depend on an additional chemical reaction, tubuli occlusion can occur immediately.37 Curodont™ Protect can be used for the treatment of hypersensitive teeth.
Various in vivo and in vitro studies had shown remineralization of early enamel lesions and the clinical beneficial effect after SAP P11-4 application in various age groups. Added benefits of accelerating and improving the remineralization process were obtained via formulations of the self-assembling peptide with other remineralizing agents allowing much faster and enhanced regenerative repair for non-cavitated caries lesions. This approach may present a safe, preventive, and minimally invasive treatment for initial enamel lesions as superior remineralization potential had been found in treating early white spot lesions, enamel erosion, and dentin hypersensitivity in various studies.
1.2. Clinical performance
1.2.1. Comparison with Fluoride Varnish
2.3.1.1. Clinical studies
The remineralization potential of SAP and fluoride varnish was compared in five clinical trials and found that SAP was a superior treatment for early caries lesions compared to fluoride varnish alone23,31,34,38,39. Alkilzy M et al and Doberdoli D et al applied SAP P11-4 along with fluoride varnish and found P11-4 and fluoride varnish combination had a superior effect.31,39 The SAP P11-4 groups showed the same relative decreases in laser fluorescence in both trials. The P11-4 and fluoride varnish considerably increased the remineralization and arresting of carious lesions compared to the control.31 The magnitude of the laser fluorescence signal decreased for both test groups in the study done by Doberdoli D et al 39 which differed from Alkilzy M et al. 31 The relative decrease in laser fluorescence among the SAP P11-4 groups in both the studies were identical. The international caries detection and assessment system (ICDAS-II) code data verifying the findings were also similar, with 6.7%–20 % regression into lower ICDAS-II codes in the current.39
In vitro studies
Four in vitro studies were done to compare the treatment effect of SAP P11-4 with fluoride varnish3,5,13,40. Kamal D et al showed that SAP treated sites had higher surface hardness when compared to fluoride varnish treated sites which was similar to a study reported by Schmdlin P et al.13 wherein he compared SAP with amine fluoride. Soares R et al when compared the microhardness of SAP with fluoride-enhanced hydroxyapatite gel, found that the least amount of surface remineralization was exhibited by fluoride-enhanced HA gel 40. Ustun N et al used micro-CT to examine the treated surfaces and discovered that P11-4 had a considerably larger change in mineral density and lesion depth than NaF.21
1.2.2. Comparison with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP)/casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF)
Five in vitro studies compared the relative efficacy of SAP with CPP-ACP and found that SAP had superior remineralization potential in artificially induced early enamel lesions 3,5,25,29,40. Soare R et al found a superior remineralization for the SAP group followed by CPPACP.40 Similar results were obtained by in vitro studies conducted by Kamal D et al and Kamal D et al.3,29. It is demonstrated that SAP+CPP-ACPF combination treatment had a superior surface microhardness compared to SAP+fluoride.29
Ustun N et al discovered that the fluorescence values of P11-4 and the amount of change in mineral density and lesion depth were higher in the SAP group than in the CPP-ACP group. There was no difference in surface area and volume between SAP and CPP-ACP.5 Sindura V et al found similar results in their research where the qualitative examination of the SAP P11-4 treated lesion surfaces using Scanning Electron Microscop (SEM) was done. A decrease in pore volume approximating natural enamel surface and consistent ion deposition around the prism were discovered, indicating hydroxyapatite nucleation. The mineral deposition was seen surrounding the demineralized enamel prisms in the CPP-ACP group with decreased pore volume, although the deposition was uneven with globular ion organisation.25
1.2.3. Safety
The self-assembled P11-4 fibers were found to be biocompatible and immunogenic in various in vitro and in vivo tests. According to Brunton PA et al, clinical use of P11-4 is safe, non-invasive, and patient-acceptable based on clinical judgment, and treated early lesions demonstrated considerable improvement in clinical appearance. Even though all of the measures revealed that enamel regeneration was favorable, the study was a non-controlled safety clinical trial, making the data difficult to interpret. 31
2.3.4. Efficacy
The clinical efficacy of SAP P11-4 in successful remineralization of early lesions has been reported in three studies 27,38,41.
1.1.5. Adverse effects
During the study period, Brunton PA et al reported 11 adverse events, two of which were deemed by the investigator to be likely attributable to the trial procedure. The first was a brief dental hypersensitivity, and the second was a sensitivity to the corsodyl mouthwash that was supplied as part of the trial.16
1.3. Future perspectives
According to current clinical trials, SAP P11-4 may change the routine management of early carious lesions from a wait-and-see approach to early, non-invasive interventions to regenerate enamel tissue. It provides clinicians with a new, effective, non-aerosol generating, and non-invasive therapeutic alternative by stimulating the de novo hydroxyapatite crystals formation deep within and throughout the carious lesion body.
Therefore, as a modern approach to caries treatment without tissue removal, self-assembling peptide P11-4 can be used as a non-invasive scaffold to induce natural enamel remineralization in early enamel lesion surfaces. This novel method presents as a minimally invasive preventive technique that is safe and acceptable as evidenced by various types of research.
Summary and conclusion
The enamel, as the teeth’s outermost layer, must survive a variety of physical and chemical stressors. Caries and dental erosion are two prevalent oral health disorders that manifest as gradual demineralization, eventually leading to cavitation and tooth loss. Caries is routinely treated by cutting and filling, which involves the replacement of injured tissue with foreign material. Such invasive therapy, as well as the removal of major portions of the tooth, weakens the tooth, eventually leading to tooth failure. This vicious cycle eventually leads to tooth loss. Monomeric low-viscosity peptide solutions have been shown to create scaffolding spontaneously in enamel lesions, which may help with hydroxyapatite nucleation and remineralization. SAP P11-4 may infiltrate into early lesions and cause the formation of new hydroxyapatite crystals.
Conflicts of interest
The authors declare that they have no conflict of interest.
Funding
This research did not receive any specific grants from funding agencies in the public, commercial or not-forprofit sectors.
Supporting Files
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