Bio-inspired Synthesis of Mineralized Collagen Fibrils

Abstract

Mineralized collagen fibrils constitute a basic structural unit of collagenous mineralized tissues such as dentin and bone. Understanding of the mechanisms of collagen mineralization is vital for development of new materials for the hard tissue repair. We carried out bio-inspired mineralization of reconstituted collagen fibrils using poly-l-aspartic acid, as an analog of non-collagenous acidic proteins. Transmission electron microscopy and electron diffraction studies of the reaction products revealed stacks of ribbon-shaped apatitic crystals, deposited within the fibrils with their c-axes co-aligned with the fibril axes. Such structural organization closely resembles mineralized collagen of bone and dentin. Initial mineral deposits formed in the fibrils lacked a long range crystallographic order and transformed into crystals with time. Interestingly, the shape and organization of these amorphous deposits was similar to the crystals found in the mature mineralized fibrils. We demonstrate that the interactions between collagen and poly-l-aspartic acid are essential for the mineralized collagen fibrils formation, while collagen alone does not affect mineral formation and poly-l-aspartic acid inhibits mineralization in a concentration dependant manner. These results provide new insights into basic mechanisms of collagen mineralization and can lead to the development of novel bio-inspired nanostructured materials.

Figure 1

TEM images of positively stained self-assembled collagen fibril in PBS after incubation at 37°C for 3 hours at low (A) and intermediate magnification (B). The collagen fibrils show typical banding pattern. Based on the Fourier transform data (Figure 1B, inset) the D-spacing of the fibrils is ~67 nm.

Figure 2

TEM images of mineral particles obtained by mineralization carried out at 37 °C for 16 hours without polyAsp on (A) bare TEM grid (control) and (B) on TEM grid coated with single layer of collagen fibrils. Corresponding electron diffreaction patterns (insets) show distinct reflection rings with the d-spacings corresponding to 002 and 211 planes of hydroxyapatite.

Figure 3

TEM images from mineralization experiments carried out on bare grids at 37 °C for 6 hours in presence of polyAsp at concentrations of 62.5µg/ml (A), 15.62 µg/ml (B), 7.81 µg/ml (C) and 3.90 µg/ml (D). Corresponding electron diffraction pattern (insets) show reflections corresponding to hydroxyapatite phase. Note the increase in intensity of 002 and 211 reflections with decrease in polyAsp concentration from 15.62 to 7.81 µg/ml.

Figure 4

TEM images and corresponding SAED patterns (insets) of mineralized collagen fibrils incubated for 2 hours. Note the arrays of the ribbon-like mineral particles oriented along the collagen fibril axes. Corresponding diffraction patterns indicate a presence of both amorphous (B) and crystalline mineral phases (C) in the sample.

Figure 5

TEM images of mineralized collagen fibrils for 16 hours at low (A) and intermediate (B) magnifications and (C) the diffraction pattern taken from area in (B).

Figure 6