The recent application of electron tomography to the study of biomaterial interfaces with bone has brought about an awareness of nano-osseointegration and, to a further extent, demanded increasingly advanced characterization methodologies. In this study, nanoscale osseointegration has been studied via laser-modified titanium implants. The micro- and nano-structured implants were placed in the proximal tibia of New Zealand white rabbits for six months. High-resolution transmission electron microscopy (HRTEM), analytical microscopy, including energy dispersive X-ray spectroscopy (EDXS) and energy-filtered TEM (EFTEM), as well as electron tomography studies were used to investigate the degree of nano-osseointegration in two- and three-dimensions. HRTEM indicated the laser-modified surface encouraged the formation of crystalline hydroxyapatite in the immediate vicinity of the implant. Analytical studies suggested the presence of a functionally graded interface at the implant surface, characterized by the gradual intermixing of bone with oxide layer. Yet, the most compelling of techniques, which enabled straightforward visualization of nano-osseointegration, proved to be segmentation of electron tomographic reconstructions, where thresholding techniques identified bone penetrating into the nanoscale roughened surface features of laser-modified titanium. Combining high-resolution, analytical and three-dimensional electron microscopy techniques has proven to encourage identification and understanding of nano-osseointegration.
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