Bioinspired 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 coaligned 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.

Techniques to assess bone ultrastructure organization: orientation and arrangement of mineralized collagen fibrils

“…(g) Single mineralized collagen fibril and diffraction pattern (inset) showing the orientation of unit crystal cells imaged using TEM and electron diffraction, respectively. (Image from [7] with kind permission of ACS Publications.) (Online version in colour.…”

“…It is used to provide information on the orientation of mineral crystals [23,24,174], derived from the diffraction pattern of the electrons that interact with the crystal lattice planes (figure 7). However, extensive preparation protocols for TEM that typically include fixation, dehydration, drying, enhancing feature contrast, preparing small samples for subsequent cutting with an ultramicrotome and the following handling of very small specimens have restricted the use of electron diffraction for quantifying bone ultrastructure organization to a handful of studies over the past decades [6,7,[175][176][177][178] (figure 7), examining either the crystal arrangement in single platelets or fibrils or a limited number of points within a TEM section. TEM gives access to very small features at the nanometre scale (e.g.…”

“…TEM has also allowed local qualitative assessment of the ultrastructure arrangement of healthy and osteoporotic bone [233,234], but in two dimensions only ( figure 13). Furthermore, TEM has been applied to make important contributions to what we know about bone structure at very small scales, through investigations of the shape and size of bone crystals [14,235], their spatial relationship and arrangement in relation to the collagen fibrils [175,236] or features of the collagen fibrils such as the approximately 67 nm D-spacing [7] (figure 13). Finally, TEM is also playing an important role in studying crystal arrangement during the bone mineralization process [237].…”

“…More specifically, regarding imaging techniques, AFM and electron microscopy techniques can directly image mineralized collagen fibrils, with a FOV covering areas at a scale of tens of nanometres to a few tens of micrometres [9]. Imaging bone at this scale provides useful information on other structural features, such as the collagen -mineral interface [21], mineral platelet sizes and shapes [13,14] or collagen D-period [7,263] for very high-resolution techniques such as TEM and AFM, and offers data revealing structural details of the lacuno-canalicular network [245] and bone remodelling sites [270] for high-resolution techniques such as SEM. X-ray phase-contrast techniques such as ptychographic CT [225] or X-ray phase nanotomography [226] operate at similar scales to volume SEM, and in addition offer the important advantage of tomographic (i.e.…”

Energetic basis for the molecular-scale organization of bone

“…Within this highly organized scaffold, apatite crystallites form nanometer-scale platelets (6-9) with their [001] axes preferentially aligned parallel to the fibril axis (10)(11)(12)(13)(14)(15) and the platelet faces defined by {100} crystal planes (16). Recent in vitro investigations of hydroxyapatite (HAP) formation within collagen fibrils revealed a multistage process in which amorphous calcium phosphate (ACP) first infiltrated through the hole zones and then converted into HAP platelets with initial mineral deposition occurring near the hole zones (11,13).…”