Bioengineered α-Hairpin peptide TKH with GKG linker: a LLPS-mediated biomimetic mineralization system for intrafibrillar remineralization and deep dentin tubule occlusion

Abstract

Dentin hypersensitivity (DH) originates from collagen exposure and dentinal tubules (DTs) patency caused by mechanical abrasion or demineralization. For an effective long-term therapeutic desensitizing agent, the ability to achieve deep tubular occlusion, stable remineralization, and satisfactory biocompatibility simultaneously is essential. In this study, we designed a novel α-hairpin peptide, TKH, engineered by incorporating a flexible GKG linker into an α-helical template, and provide an analysis of its liquid–liquid phase separation (LLPS) and self-assembly properties. TKH effectively stabilized amorphous calcium phosphate and facilitated intrafibrillar mineralization within collagen fibrils. In vitro and in vivo remineralization studies combined with molecular dynamics (MD) simulations demonstrated that TKH promoted the peptide self-assembly through LLPS, hydroxyapatite (HA) adsorption, and amorphous calcium phosphate (ACP) stabilization, ultimately achieving intrafibrillar and extrafibrillar mineralization to seal DTs, alongside excellent biosafety. These findings highlight the potential of TKH as a promising peptide-based biomaterial for dentin remineralization and the treatment of dentin hypersensitivity.

References

[1] Pitts NBet al. . Dental caries. Nat. Rev. Dis. Primers. 2017, 3: 17030.

[2] Mantzourani M, Sharma D. Dentine sensitivity: Past, present and future. J. Dent.. 2013, 41S3-S17.

[3] Tan S, Chen S, Lei Q, Ma D. A novel rapidly mineralized biphasic calcium phosphate with high acid-resistance stability for long-term treatment of dentin hypersensitivity. Dent. Mater.. 2023, 39: 260-274.

[4] Reis BOet al. . Analysis of permeability and biological properties of dentin treated with experimental bioactive glasses. J. Dent.. 2021, 111: 103719.

[5] Alawad FIet al. . Effect of desensitizing agents on the resin bond strength to sound dentin. Odontology. 2024, 112435-443.

[6] Sauro S, Osorio R, Watson TF, Toledano M. Therapeutic effects of novel resin bonding systems containing bioactive glasses on mineral-depleted areas within the bonded-dentine interface. J. Mater. Sci.: Mater. Med.. 2012, 23: 1521-1532

[7] Kim JW, Park J-C. Dentin hypersensitivity and emerging concepts for treatments. J. Oral. Biosci.. 2017, 59: 211-217.

[8] Srisomboon Set al. . Effects of Silver Diamine Nitrate and Silver Diamine Fluoride on Dentin Remineralization and Cytotoxicity to Dental Pulp Cells: An In Vitro Study. J. Funct. Biomater.. 2022, 13: 16.

[9] Gao Wet al. . A Drop-By-Drop Self-Assembled All-Natural Hydrogel as a Desensitizer for Rapid and Enduring Management of Dentin Hypersensitivity. Adv. Healthc. Mater.. 2024, 13. 2303153

[10] Tang S, Dong Z, Ke X, Luo J, Li J. Advances in biomineralization-inspired materials for hard tissue repair. Int. J. Oral Sci.. 2021, 1342.

[11] Moradian-Oldak J, George A. Biomineralization of Enamel and Dentin Mediated by Matrix Proteins. J. Dent. Res.. 2021, 1001020-1029.

[12] Yu L, Wei M. Biomineralization of Collagen-Based Materials for Hard Tissue Repair. Int. J. Mol. Sci.. 2021, 22: 944.

[13] Nudelman F, Lausch AJ, Sommerdijk NA, Sone ED. In vitro models of collagen biomineralization. J. Struct. Biol.. 2013, 183258-269.

[14] Olszta MJ, Douglas EP, Gower LB. Scanning Electron Microscopic Analysis of the Mineralization of Type I Collagen via a Polymer-Induced Liquid-Precursor (PILP) Process. Calcif. Tissue Int.. 2003, 72: 583-591.

[15] Xu Yet al. . Microscopic structure of the polymer-induced liquid precursor for calcium carbonate. Nat. Commun.. 2018, 9. 2582

[16] Niu LNet al. . Collagen intrafibrillar mineralization as a result of the balance between osmotic equilibrium and electroneutrality. Nat. Mater.. 2016, 16: 370-378.

[17] Ma YXet al. . Involvement of prenucleation clusters in calcium phosphate mineralization of collagen. Acta Biomaterialia. 2021, 120: 213-223.

[18] Alberti S, Gladfelter A, Mittag T. Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell. 2019, 176: 419-434.

[19] Yuan C, Li Q, Xing R, Li J, Yan X. Peptide self-assembly through liquid-liquid phase separation. Chem. 2023, 9: 2425-2445.

[20] Song Set al. . Peptide-Based Biomimetic Condensates via Liquid–Liquid Phase Separation as Biomedical Delivery Vehicles. Biomacromolecules. 2024, 25: 5468-5488.

[21] Olszta MJ, Douglas EP, Gower LB. Intrafibrillar Mineralization of Collagen using a Liquid-Phase Mineral Precursor. MRS Online Proc. Libr.. 2004, 774: 710.

[22] Zhou Yet al. . Biochemistry-Enabled 3D Foams for Ultrafast Battery Cathodes. ACS Nano. 2015, 94628-4635.

[23] Peng Xet al. . The Amelogenin-Derived Peptide TVH-19 Promotes Dentinal Tubule Occlusion and Mineralization. Polymers (Basel). 2021, 13: 2473.

[24] Chang R, Yuan C, Zhou P, Xing R, Yan X. Peptide Self-assembly: From Ordered to Disordered. Acc. Chem. Res. 2024, 57289-301.

[25] Karthika S, Radhakrishnan TK, Kalaichelvi P. A Review of Classical and Nonclassical Nucleation Theories. Cryst. Growth Des.. 2016, 16: 6663-6681.

[26] George A, Veis A. Phosphorylated Proteins and Control over Apatite Nucleation, Crystal Growth, and Inhibition. Chem. Rev.. 2008, 108: 4670-4693.

[27] Padovano JDet al. . DMP1-derived peptides promote remineralization of human dentin. J. Dent. Res. 2015, 94: 608-614.

[28] Bewernitz MA, Gebauer D, Long J, Cölfen H, Gower LB. A metastable liquid precursor phase of calcium carbonate and its interactions with polyaspartate. Faraday Discuss.. 2012, 159: 291-312.

[29] Jee SS, Kasinath RK, DiMasi E, Kim Y-Y, Gower L. Oriented hydroxyapatite in turkey tendon mineralized via the polymer-induced liquid-precursor (PILP) process. CrystEngComm. 2011, 13: 2077-2083.

[30] Schenk ASet al. . Polymer-induced liquid precursor (PILP) phases of calcium carbonate formed in the presence of synthetic acidic polypeptides—relevance to biomineralization. Faraday Discuss.. 2012, 159: 327-344.

[31] Saxena Net al. . Influence of fluoride on the mineralization of collagen via the polymer-induced liquid-precursor (PILP) process. Dent. Mater.. 2018, 34: 1378-1390.

[32] Yuan Cet al. . Nucleation and Growth of Amino Acid and Peptide Supramolecular Polymers through Liquid–Liquid Phase Separation. Angew. Chem. Int. Ed.. 2019, 58: 18116-18123.

[33] Narang N, Sato T. Unique phase behaviour and self-assembly of a lysine derivative, Fmoc-homoarginine, in water–DMSO mixtures. Soft Matter. 2022, 18: 7968-7974.

[34] De Santis E, Ryadnov MG. Peptide self-assembly for nanomaterials: the old new kid on the block. Chem. Soc. Rev.. 2015, 44: 8288-8300.

[35] Fu K, Wu H, Su Z. Self-assembling peptide-based hydrogels: Fabrication, properties, and applications. Biotechnol. Adv.. 2021, 49: 107752.

[36] Nam Jet al. . Structural and Functional Assessment of mBjAMP1, an Antimicrobial Peptide from Branchiostoma japonicum, Revealed a Novel alpha-Hairpinin-like Scaffold with Membrane Permeable and DNA Binding Activity. J. Med Chem.. 2018, 6111101-11113.

[37] Han Set al. . Amelogenin-Inspired Autoadaptive Peptide in a Caries Microenvironment Facilitates Long-Term Protection of the Dentin–Pulp Complex. ACS Appl. Mater. Interfaces. 2024, 16: 41518-41533.

[38] Levin Aet al. . Biomimetic peptide self-assembly for functional materials. Nat. Rev. Chem.. 2020, 4: 615-634.

[39] Jin Set al. . Plant-Derived Polyphenol and LL-37 Peptide-Modified Nanofibrous Scaffolds for Promotion of Antibacterial Activity, Anti-Inflammation, and Type-H Vascularized Bone Regeneration. ACS Appl. Mater. Interfaces. 2023, 15: 7804-7820.

[40] Qiao ZYet al. . Polymer–KLAK Peptide Conjugates Induce Cancer Cell Death through Synergistic Effects of Mitochondria Damage and Autophagy Blockage. Bioconjugate Chem.. 2017, 28: 1709-1721.

[41] Gelain F, Luo Z, Zhang S. Self-Assembling Peptide EAK16 and RADA16 Nanofiber Scaffold Hydrogel. Chem. Rev.. 2020, 120: 13434-13460.

[42] Zhang Wet al. . Supramolecular Self-Assembly Bioinspired Synthesis of Luminescent Gold Nanocluster-Embedded Peptide Nanofibers for Temperature Sensing and Cellular Imaging. Bioconjugate Chem.. 2017, 28: 2224-2229.

[43] Abramson Jet al. . Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024, 630: 493-500.

[44] Pettersen EFet al. . UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci.. 2021, 30: 70-82.

[45] Baruch Leshem Aet al. . Biomolecular condensates formed by designer minimalistic peptides. Nat. Commun.. 2023, 14. 421

[46] Sarker P, Jani PK, Hsiao LC, Rojas OJ, Khan SA. Interacting collagen and tannic acid Particles: Uncovering pH-dependent rheological and thermodynamic behaviors. J. Colloid Interface Sci.. 2023, 650: 541-552.

[47] Li Met al. . An engineered dual-functional peptide with high affinity to demineralized dentin enhanced remineralization efficacy in vitro and in vivo. J. Mater. Chem. B. 2023, 11: 5170-5184.

[48] Li Zet al. . Promoting effect of a calcium-responsive self-assembly β-sheet peptide on collagen intrafibrillar mineralization. Regen. Biomater.. 2022, 9. rbac059

[49] Hu Det al. . Unveiling the mechanism of an amelogenin-derived peptide in promoting enamel biomimetic remineralization. Int. J. Biol. Macromol.. 2023, 253: 127322.

[50] Sun Bet al. . A bifunctional lactoferrin-derived amyloid coating prevents bacterial adhesion and occludes dentinal tubules via deep remineralization. Acta Biomaterialia. 2024, 188393-405.

[51] Lin TJ, Heinz H. Accurate Force Field Parameters and pH Resolved Surface Models for Hydroxyapatite to Understand Structure, Mechanics, Hydration, and Biological Interfaces. J. Phys. Chem. C.. 2016, 120: 4975-4992.

[52] Zhou L, Li QL, Wong HM. A Novel Strategy for Caries Management: Constructing an Antibiofouling and Mineralizing Dual-Bioactive Tooth Surface. ACS Appl. Mater. Interfaces. 2021, 13: 31140-31152.

Funding

National Natural Science Foundation of China (National Science Foundation of China)(82370951)

Sichuan Science and Technology Program(2023ZYD0105)

RIGHTS & PERMISSIONS

The Author(s)