Electron microscopy characterisation of in vivo collagen and mineral ultra-structures, their development and pathologies
Creators: name: Klosowski, Michal Maciej
Publisher: name: Imperial College London
Year: 2014

description: The investigation of the nanostructure of mineralised tissues is challenging due to the intrinsic complexity of the hierarchical biocomposites and heterogeneity of biological samples. Despite numerous transmission electron microscopy (TEM) studies investigating a variety of mineralised tissues, details of the collagen-mineral ultrastructure and the origins of collagen and mineral formation are not fully understood. Analytical scanning transmission electron microscopy (STEM), combined with electron energy-loss spectroscopy (EELS), has the potential of identifying bone composition at a very high spatial resolution. We used these techniques to investigate the in vivo mineralisation process, and to probe the structural/compositional origins of bone-related pathologies at high spatial resolution. High-pressure freezing/freeze substitution (HPS/FS) methods were employed to preserve features of mineralisation process, specifically the structure and chemistry of the mineral and collagen phases and the distribution of diffusible mineral ions. The application of STEM-EELS in the biomineralisation studies is, however, limited, partially due to the absence of an EELS library of mineral and collagen standards. We developed an EELS spectra collection of biominerals (hydroxyapatite, carbonated hydroxyapatite, beta-tricalcium phosphate and calcite) so that biominerals can be identified by composition and coordination environment. For the first time, an extensive collection of all major elemental edges (phosphorus, carbon, calcium, oxygen) is presented and compared. We then used this library to characterise in vivo mineralisation processes. We examined turkey tendon, which calcifies with age, in order to understand the mineralisation process. We identified chemical and structural signatures representative of the non-, poorly and well mineralised tissues. In particular, a chemical signature of pyridine-based compounds was identified and a protocol was developed to assess changes in the nano-scale chemistry of the collagen-mineral matrix in disrupted tissues. We observed a change in the oxidation state of pyridine-based compounds in the collagen fibrils, which most likely occurs pror to nucleation. Mineral ions (calcium, phosphate) were delivered into the collagen matrix, either in the form of amorphous calcium phosphate vesicles or by diffusion from the body fluids. We are first to show in vivo that the mineral nucleated in the gap region of collagen fibrils in the form of ellipsoidal grains of amorphous calcium phosphate, which transformed into crystalline apatite with time. Inside the collagen fibril, pyridine-based compounds changed their oxidation stage prior, or during, the mineralisation process. We also compared healthy and abnormal (osteogenesis imperfecta or OI) mice tissues to reveal defects in the fibril architecture and mineral chemistry in the OI model. Abnormal tissues were capable of producing collagen fibrils with a characteristic banding pattern, typical for the normal collagen. However, the diameter of the abnormal fibrils was lower. Moreover, in OI-affected tissues, large regions of disorganised fibrils were seen. Defects in fibril formation have previously been predicted by bulk chromatographic and modelling studies. The morphology and crystallinity of mineral in healthy and abnormal tissues were similar. In contrast, a much stronger signal, characteristic of carbonate ion presence, was observed in the EELS spectra taken from the mineral in the OI tissue. More generally, the library of biominerals, identification of early in vivo mineralisation patterns, and identification of alterations in disease, demonstrate that STEM-EELS provides a method to identify chemical and structural features present within mineralising with unprecedented spatial resolution. Understanding the mechanisms of bone mineralisation and the nature of the collagen-mineral interaction will help in determining the source of bone’s toughness at the molecular level.