Hydroxyapatite Nucleation using Biomimetic Peptides

Objectives: Formation of calcified tissues is a cellular process. Several macromolecules orchestrate this event, however, type I collagen and noncollagenous proteins (NCP) are the key players. Dentin matrix protein-1 (DMP1) is an acidic, NCP associated with the collagen matrix of mineralized tissues such as dentin and bone. DMP1 has been shown to have collagen binding and HA nucleating domains that allow the protein to adhere to the collagen matrix and subsequently nucleate HA.

Methods: In this study, we have engineered 2 designer nanopeptides containing the collagen binding and apatite nucleating domains of DMP1.   The two peptides that were synthesized had molecular weights of 1726 (pA) and 2815 (pB) daltons, each containing both a single collagen binding and HA nucleating domain. Freshly extracted human third molars were sectioned to provide 3mm x 1.4mm x 250µm coronal dentin wafers. The wafers were then demineralized in 0.5M neutral buffered EDTA for 10 days, trypsinized twice with 1.0% trypsin for 2 days, and finally treated with 1M NaCl for 2 hours. Complete demineralization was verified by X-ray analysis for radio opacity. Removal of NCPs was verified using Stains-all. The wafers were independently incubated over night in varying ratios of pA and pB. Recombinant DMP1 (rDMP1) and BSA coated wafers served as controls. Nucleation was carried out in a specialized nucleation chamber under physioilogical concentrations of calcium and phosphate ions for 2 weeks.

Results: SEM showed calcium phosphate HA crystals associated with the collagen matrix of wafers incubated in both peptides pA and pB along with rDMP1. Dentin wafers were also characterized by energy-dispersive X-ray spectroscopy (EDX), and X-ray diffraction.

Conclusions: These data suggest that our designer nanopeptides were able to bind the native 3-dimentional collagen scaffolds and nucleate HA crystals. Thus, biomimetic based nanopeptides could be utilized as a clinically relevant tool for remineralization.