Introduction: Osseointegration, often understood as a direct structural and functional bone interface to an implant, is one of the key factors for the success of reconstructive and regenerative orthopaedic and dental surgery[1]. With breakthroughs in ultrastructural characterization of the human bone-implant interface, the definition of osseointegration has developed from merely a histological perspective to one that describes nanoscale integration[2]. In order to validate the observations made through experimental animal studies, it is essential to characterize the structure and composition of the bone-implant interface in human. This work uses atom probe tomography (APT), which combines sub-nanometer resolution with chemical sensitivity across the entire periodic table, for four-dimensional visualization (spatial and chemical) of osseointegration across the bone-implant interface on the atomic-scale.
Materials and Methods: The specimen used in this study was a laser-modified, dental implant (BioHelix™, Brånemark Integration AB, Mölndal, Sweden) retrieved from a 66-year-old female patient after four years in clinical function[3]. Using focused ion beam (FIB), a sample from the bone-titanium interface was sharpened into approximately 100 nm diameter tips. The sample was subsequently coated with a 15 nm thick layer of Ag and inserted into a LEAP 4000XHR (CAMECA Scientific Instruments, Madison, WI) for APT investigation. A laser pulse (λ = 355 nm, 120 pJ, 100 kHz) was used to incite field evaporation from the sample with a base temperature at 43.4 K and the chamber pressure at 4.0x10-9 Pa. The evaporation rate was maintained around 0.005 ions/pulse by controlling the direct-current potential on the sample. The reconstruction was performed using the Integrated Visualization and Analysis Software package (IVAS) v3.6.6 (CAMECA Scientific Instruments, Madison, WI) assuming the shape of a hemispherical tip on a truncated cone. The reconstruction was spatially defined by assuming the tip radius to evolve as a function of a constant specimen shank angle. The input parameters for this algorithm of initial tip radius and specimen shank angle were obtained from scanning transmission electron microscopy (STEM) images of the sample, taken both before and after the APT experiment. This is critical for ensuring the accuracy of the reconstruction.
Results and Discussion: The first APT data of a human bone-implant interface was collected (Fig. 1). It provides experimental authenticity to the atomic-scale continuum of bone and an osseointegrated implant surface. Calcium ions make direct contact with the surface oxide layer. From the proxigram (Fig. 2), it is also shown that the Ca-rich region has a high affinity for the oxide coating. Nitrogen enrichment is present between the Ti metal and the oxide layer, which may be attributable to the laser surface modification process carried out in ambient air, thereby resulting in the formation of titanium nitride. Due to high chemical sensitivity (down to 1 ppm) of APT, trace elements such as Mg and Na are also detected in bone, while Ag comes from the coating which improves the thermal conductivity of the specimen in order to decrease the thermal tail artefacts in the mass spectrum.
Conclusions: Development and expansion of characterization techniques for surfaces and interfaces of biomaterials is essential to advancing our understanding of material interactions in vivo. The current atom probe tomography demonstrates a new approach to characterizing atomic-scale osseointegration, extending and further resolving previous nanoscale descriptions of the bone-implant interface. The use of APT, preferably in combination with correlative on-axis electron and electron energy loss spectroscopy tomography, has the potential to unveil new bone bonding phenomena, whether originating from an intermediate mineral-rich or mineral-deficient zone or directly bonded to the collagen bundles.
The authors acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant program and BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Sweden.; Microscopy was performed at the Canadian Centre for Electron Microscopy at McMaster University, a facility supported by NSERC and other government agencies.
References:
[1] P-I Brånemark et al, Scand J Plast Reconstr Surg 3 (1969), p. 81.
[2] K. Grandfield, S. Gustafsson and A. Palmquist. Nanoscale, 5 (2013), p. 4032.
[3] F.A. Shah et al. Nanomedicine-Nanotechnology Biology and Medicine, 10 (2014), p. 1729.