Skip to main content Accessibility help

Repairing human tooth enamel with leucine-rich amelogenin peptide–chitosan hydrogel

  • Kaushik Mukherjee (a1), Qichao Ruan (a1), David Liberman (a1), Shane N. White (a2) and Janet Moradian-Oldak (a3)...

We have recently reported the repair of carious enamel using a full-length amelogenin–chitosan hydrogel through guided stabilization and growth of mineral clusters. The objective of this study was to further evaluate the enamel repair potential of smaller amelogenin peptides like LRAP (leucine-rich amelogenin peptide) and compare their efficiency with their full-length counterpart. The demineralized tooth slices treated with a single application of LRAP–chitosan hydrogel for 3 days showed a dense mineralized layer consisting of highly organized enamel-like apatite crystals. Focus-ion beam technique showed a seamless growth at the interface between the repaired layer and native enamel. There was a marked improvement in the surface hardness after treatment of the demineralized sample with almost 87% recovery of the hardness value to that of sound enamel sections. This current approach can inspire the design of smaller peptide analogues based on naturally occurring amelogenin as a competent, low-cost, and safe strategy for enamel biomimetics to curb the high prevalence of incipient dental caries.

Corresponding author
a) Address all correspondence to this author. e-mail:
Hide All
1. Chai, H., Lee, J.J.W., Constantino, P.J., Lucas, P.W., and Lawn, B.R.: Remarkable resilience of teeth. Proc. Natl. Acad. Sci. U. S. A. 106, 72897293 (2009).
2. Moradian-Oldak, J.: Protein-mediated enamel mineralization. Front. Biosci. 17, 19962023 (2012).
3. Cate, A.R.T.: Tooth enamel—Its composition properties and fundamental structure. J. Anat. 100, 416 (1966).
4. Fincham, A.G., Moradian-Oldak, J., and Simmer, J.P.: The structural biology of the developing dental enamel matrix. J. Struct. Biol. 126, 270299 (1999).
5. Moradian-Oldak, J.: Amelogenins: Assembly, processing and control of crystal morphology. Matrix Biol. 20, 293305 (2001).
6. Deyhle, H., White, S.N., Bunk, O., and Beckmann, F., Muller, B.: Nanostructure of carious tooth enamel lesion. Acta Biomater. 10, 355364 (2014).
7. Ruan, Q. and Moradian-Oldak, J.: Amelogenin and enamel biomimetics. J. Mater. Chem. B 3, 31123129 (2015).
8. Kirkham, J., Firth, A., Vernals, D., Boden, N., Robinson, C., Shore, R.C., Brookes, S.J., and Aggeli, A.: Self-assembling peptide scaffolds promote enamel remineralization. J. Dent. Res. 86, 426430 (2007).
9. Mann, S., Fletcher, J., Walsh, D., and Fowler, C.E.: Electrospun mats of PVP/ACP nanofibres for remineralization of enamel tooth surfaces. CrystEngComm 13, 36923697 (2011).
10. Fan, Y., Xu, X., Zhang, J.F., Twomley, J.T., Wen, Z.T., Liao, S., Lallier, T., Hagan, J.L., and Sun, Z.: Novel amelogenin-releasing hydrogel for remineralization of enamel artificial caries. J. Bioact. Compat. Polym. 27(6), 585603 (2012).
11. Reynolds, E.C., Cochrane, N.J., Saranathan, S., Cai, F., and Cross, K.J.: Enamel subsurface lesion remineralisation with casein phosphopeptide stabilised solutions of calcium, phosphate and fluoride. Caries Res. 42, 8897 (2008).
12. Yamagishi, K., Onuma, K., Suzuki, T., Okada, F., Tagami, J., Otsuki, M., and Senawangse, P.: Materials chemistry: A synthetic enamel for rapid tooth repair. Nature 433(7028), 819 (2005).
13. Ruan, Q.C., Zhang, Y.Z., Yang, X.D., Nutt, S., and Moradian-Oldak, J.: An amelogenin-chitosan matrix promotes assembly of an enamel-like layer with a dense interface. Acta Biomater. 9, 72897297 (2013).
14. Ruan, Q.C. and Moradian-Oldak, J.: Development of amelogenin-chitosan hydrogel for in vitro enamel regrowth with a dense interface. J. Visualized Exp. (89), e51606 (2014).
15. Habelitz, S., DenBesten, P.K., Marshall, S.J., Marshall, G.W., and Li, W.: Self-assembly and effect on crystal growth of the leucine-rich amelogenin peptide. Eur. J. Oral Sci. 114, 315319 (2006).
16. Tarasevich, B.J., Perez-Salas, U., Masica, D.L., Philo, J., Kienzle, P., Krueger, S., Majkrzak, C.F., Gray, J.L., and Shaw, W.J.: Neutron reflectometry studies of the adsorbed structure of the amelogenin, LRAP. J. Phys. Chem. B 117, 30983109 (2013).
17. Shaw, W.J., Ferris, K., Tarasevich, B., and Larson, J.L.: The structure and orientation of the C-terminus of LRAP. Biophys. J. 94, 32473257 (2008).
18. Addadi, L., Moradianoldak, J., Furedimilhofer, H., Weiner, S., and Veis, A.: Stereochemical aspects of crystal regulation in calcium phosphate-associated mineralized tissues. Int. Congr. Ser. 1002, 153162 (1992).
19. Addadi, L. and Weiner, S.: Interactions between acidic proteins and crystals—Stereochemical requirements in biomineralization. Proc. Natl. Acad. Sci. U. S. A. 82, 41104114 (1985).
20. DeOliveira, D.B. and Laursen, R.A.: Control of calcite crystal morphology by a peptide designed to bind to a specific surface. J. Am. Chem. Soc. 119, 1062710631 (1997).
21. Tarasevich, B.J., Philo, J.S., Maluf, N.K., Krueger, S., Buchko, G.W., Lin, G.Y., and Shaw, W.J.: The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution. J. Struct. Biol. 190, 8191 (2015).
22. Tarasevich, B.J., Lea, S., and Shaw, W.J.: The leucine rich amelogenin protein (LRAP) adsorbs as monomers or dimers onto surfaces. J. Struct. Biol. 169, 266276 (2010).
23. Le Norcy, E., Kwak, S.Y., Wiedemann-Bidlack, F.B., Beniash, E., Yamakoshi, Y., Simmer, J.P., and Margolis, H.C.: Leucine-rich amelogenin peptides regulate mineralization in vitro. J. Dent. Res. 90, 10911097 (2011).
24. Shafiei, F., Hossein, B.G., Farajollahi, M.M., Fathollah, M., Marjan, B., and Tahereh, J.K.: Leucine-rich amelogenin peptide (LRAP) as a surface primer for biomimetic remineralization of superficial enamel defects: An in vitro study. Scanning 37, 179185 (2015).
25. Boabaid, F., Gibson, C.W., Kuehl, M.A., Berry, J.E., Snead, M.L., Nociti, F.H., Katchburian, E., and Somerman, M.J.: Leucine-rich amelogenin peptide: A candidate signaling molecule during cementogenesis. J. Periodontol. 75, 11261136 (2004).
26. Warotayanont, R., Zhu, D.H., Snead, M.L., and Zhou, Y.: Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells. Biochem. Biophys. Res. Commun. 367, 16 (2008).
27. Robinson, C., Shore, R.C., Brookes, S.J., Strafford, S., Wood, S.R., and Kirkham, J.: The chemistry of enamel caries. Crit. Rev. Oral Biol. Med. 11, 481495 (2000).
28. Busch, S., Schwarz, U., and Kniep, R.: Chemical and structural investigations of biomimetically grown fluorapatite–gelatin composite aggregates. Adv. Funct. Mater. 13, 189198 (2003).
29. Prymak, O., Sokolova, V., Peitsch, T., and Epple, M.: The crystallization of fluoroapatite dumbbells from supersaturated aqueous solution. Cryst. Growth Des. 6, 498506 (2006).
30. Al Sagheer, F.A., Al-Sughayer, M.A., Muslim, S., and Elsabee, M.Z.: Extraction and characterization of chitin and chitosan from marine sources in Arabian Gulf. Carbohydr. Polym. 77, 410419 (2009).
31. Lagarto, A., Merino, N., Valdes, O., Dominguez, J., Spencer, E., de la Paz, N., and Aparicio, G.: Safety evaluation of chitosan and chitosan acid salts from Panurilus argus lobster. Int. J. Biol. Macromol. 72, 13431350 (2015).
32. Tarasevich, B.J., Lea, S., Bernt, W., Engelhard, M., and Shaw, W.J.: Adsorption of amelogenin onto self-assembled and fluoroapatite surfaces. J. Phys. Chem. B 113, 18331842 (2009).
33. Tarasevich, B.J., Lea, S., Bernt, W., Engelhard, M.H., and Shaw, W.J.: Rapid communication changes in the quaternary structure of amelogenin when adsorbed onto surfaces. Biopolymers 91, 103107 (2009).
34. Chen, C.L., Bromley, K.M., Moradian-Oldak, J., and DeYoreo, J.J.: In situ AFM study of amelogenin assembly and disassembly dynamics on charged surfaces provides insights on matrix protein self-assembly. J. Am. Chem. Soc. 133, 1740617413 (2011).
35. Elhadj, S., De Yoreo, J.J., Hoyer, J.R., and Dove, P.M.: Role of molecular charge and hydrophilicity in regulating the kinetics of crystal growth. Proc. Natl. Acad. Sci. U. S. A. 103, 1923719242 (2006).
36. Piana, S., Jones, F., Taylor, Z., Raiteri, P., and Gale, J.D.: Exploring the role of ions and amino acids in directing the growth of minerals from solution. Mineral. Mag. 72, 273276 (2008).
37. Yang, X.D., Xie, B.Q., Wang, L.J., Qin, Y.L., Henneman, Z.J., and Nancollas, G.H.: Influence of magnesium ions and amino acids on the nucleation and growth of hydroxyapatite. CrystEngComm 13, 11531158 (2011).
38. Bowman, K. and Leong, K.W.: Chitosan nanoparticles for oral drug and gene delivery. Int. J. Nanomed. 1(2), 117128 (2006).
39. Stephan, R.M.: pH and dental caries. J. Dent. Res. 26, 340 (1947).
40. Masica, D.L., Gray, J.J., and Shaw, W.J.: Partial high-resolution structure of phosphorylated and non-phosphorylated leucine-rich amelogenin protein adsorbed to hydroxyapatite. J. Phys. Chem. C 115, 1377513785 (2011).
41. Kwak, S-Y., Wiedemann-Bidlack, R.B., Beniash, E., Yamakoshi, Y., Simmer, J.P., Litman, A., and Margolis, H.C.: Role of 20-kDa amelogenin (P148) phosphorylation in calcium phosphate formation in vitro. J. Biol. Chem. 284, 1897218979 (2009).
42. Lu, J-X., Xu, S.Y., and Shaw, W.J.: Phosphorylation and ionic strength alter the LRAP–HAP interface in the N-terminus. Biochemistry 52, 21962205 (2013).
43. Gkioni, K., Leeuwenburgh, S.C.G., Douglas, T.E.L., Mikos, A.G., and Jansen, J.A.: Mineralization of hydrogels for bone regeneration. Tissue Eng. 16, 577585 (2010).
44. Ruan, Q., Liberman, D., Bapat', R., Balakrishna, K.C., Phark, J-H., and Moradian-Oldak, J.: Efficacy of amelogenin-chitosan hydrogel in biomimetic repair of human enamel in pH-cycling systems. J. Biomed. Eng. Health Inform 2(1), 123124 (2016).
45. Prajapati, S., Tao, J., Ruan, Q., De Yoreo, J.J., and Moradian-oldak, J.: Matrix Metalloproteinase-20 mediates dental enamel biomineralization by preventing protein occlusion inside apatite crystals. Biomaterials 75, 260270 (2016).
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Journal of Materials Research
  • ISSN: 0884-2914
  • EISSN: 2044-5326
  • URL: /core/journals/journal-of-materials-research
Please enter your name
Please enter a valid email address
Who would you like to send this to? *


Type Description Title
Supplementary materials

Mukherjee supplementary material
Figure S2

 Unknown (4.8 MB)
4.8 MB
Supplementary materials

Mukherjee supplementary material
Figure S1

 Unknown (4.9 MB)
4.9 MB


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed