ACOM‐TEM
analysis of the effect of heating on the mineral nanocrystals in bone
Authors: Verezhak Mariana , Rauch Edgar F. , Veron Muriel , Bordet Pierre , Plazanet Marie , Gourrier Aurélien 
Published: December 2016

The evolution of the crystalline structure of bone mineral nanoparticles upon heating is a topic of interest in archeology, paleo‐anthropology and forensic science. Archaeological bone remains contain a considerable amount of information which can be altered by heating (e.g. radiocarbon dating). Traces of heating are often observed on bone fragments, but it becomes quite difficult to distinguish its' effect, particularly at temperatures below 600°C. Below this temperature, macroscopic structural parameters become inadequate to characterize the effect of heating and the nanoscale composite structure needs to be considered [1]. At the nanoscale bone consist of two principal components: collagen fibrils of ~ 100 nm in diameter and platelet‐shaped calcium phosphate mineral crystals of about 5 x 50 x 100 nm
3
dimensions.

The closest crystallographic structure that describes the bone mineral phase is hydroxyapatite, as determined by x‐ray diffraction. However, significant differences in crystal structure with respect to the ideal hydroxyapatite are generally observed. Those have been shown to be related to modifications in crystalline chemistry by spectroscopy, as well as nanocrystal size and strain by X‐ray scattering.

Therefore, the main difficulty stands in the necessity of analyzing both the nanocrystals morphology and organization as well as crystal structure which, generally, requires using different methods. Electron microscopy is the most widely used technique to visualize the nanocrystals in real space, while X‐ray diffraction is generally used to analyze the crystal structure in reciprocal space, thus giving information on the crystal‐chemistry disorder. However, the ideal method should provide both insights. Automated Crystal Orientation Mapping at TEM (ACOM‐TEM, also known as ASTAR
TM
tool from NanoMEGAS) [2] allows such complex study.

The ACOM‐TEM method operates in scanning mode and relies on the comparison between the electron diffraction patterns collected at every scan position and the simulated patterns calculated for a given crystal structure in all possible orientations. Maps of the structural parameters can therefore be reconstructed which allow analyzing the crystal size and shape distribution in real space as well as the crystalline orientation. Different model crystal structures can also be tested to access the crystal chemistry fluctuations.

Bovine bone samples in control state as well as heated in vacuum to 9 temperatures from 100°C to 1000°C for 10 min were studied. An increase in nanoparticle size occurs upon heating (fig. 1) as also observed by X‐ray scattering [3]. In order to test the sensitivity of the method, we found that hydroxyapatite describes the structure well in comparison with other apatite minerals with elemental composition compatible with bone biochemistry (brushite, monetite, tuite). The structure is better described by hexagonal space group P63/m in all the temperature range, contrary to results pointing to a monoclinic to hexagonal phase transition. Furthermore, the type of carbonate substitutions which are known to occur in bone (up to 7%
w
) was investigated and could be discriminated within the limits of the method sensitivity. Additionally the probability of various ionic substitutions (F
‐
, Na
+
, Sr
2+
and Cl
‐
) in bone tissue is discussed.

Improvement of reciprocal space resolution and accessible q‐range could allow studying even more subtle crystal‐chemistry disorder in such complex materials like bone tissue. Current results are part of larger project aiming to understand the nanostructural characteristics of bone tissue and to identify key structural markers of pathological human bone [4], providing possible development of new diagnostic and pharmaceutical tools.