Characterizing the Composition of Bone Formed During Fracture Healing Using Scanning Electron Microscopy Techniques.

About 5-10 % of all bone fractures suffer from delayed healing, which may lead to non-union. Bone morphogenetic proteins (BMPs) can be used to induce differentiation of osteoblasts and enhance the formation of the bony callus, and bisphosphonates help to retain the newly formed callus. The aim of this study was to investigate if scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) can identify differences in the mineral composition of the newly formed bone compared to cortical bone from a non-fractured control. Moreover, we investigate whether the use of BMPs and bisphosphonates-alone or combined-may have an effect on bone mineralization and composition. Twelve male Sprague-Dawley rats at 9 weeks of age were randomly divided into four groups and treated with (A) saline, (B) BMP-7, (C) bisphosphonates (Zoledronate), and (D) BMP-7 + Zoledronate. The rats were sacrificed after 6 weeks. All samples were imaged using SEM and chemically analyzed with EDS to quantify the amount of C, N, Ca, P, O, Na, and Mg. The Ca/P ratio was the primary outcome. In the fractured samples, two areas of interest were chosen for chemical analysis with EDS: the callus and the cortical bone. In the non-fractured samples, only the cortex was analyzed. Our results showed that the element composition varied to a small extent between the callus and the cortical bone in the fractured bones. However, the Ca/P ratio did not differ significantly, suggesting that the mineralization at all sites is similar 6 weeks post-fracture in this rat model

Characterization of the bone-metal implant interface by Digital Volume Correlation of in-situ loading using neutron tomography

Metallic implants are commonly used as surgical treatments for many orthopedic conditions. The long-term stability of implants relies on an adequate integration with the surrounding bone. Unsuccessful integration could lead to implant loosening. By combining mechanical loading with high-resolution 3D imaging methods, followed by image analysis such as Digital Volume Correlation (DVC), we aim at evaluating ex vivo the mechanical resistance of newly formed bone at the interface. X-rays tomography is commonly used to image bone but induces artefacts close to metallic components. Utilizing a different interaction with matter, neutron tomography is a promising alternative but has not yet been used in studies of bone mechanics. This work demonstrates that neutron tomography during in situ loading is a feasible tool to characterize the mechanical response of bone-implant interfaces, especially when combined with DVC. Experiments were performed where metal screws were implanted in rat tibiae during 4 weeks. The screws were pulled-out while the samples were sequentially imaged in situ with neutron tomography. The images were analyzed to quantify bone ingrowth around the implants. DVC was used to track the internal displacements and calculate the strain fields in the bone during loading. The neutron images were free of metal-related artefacts, which enabled accurate quantification of bone ingrowth on the screw (ranging from 60% to 71%). DVC allowed successful identification of the deformation and cracks that occurred during mechanical loading and led to final failure of the bone-implant interface

Neutron tomographic imaging of bone-implant interface : Comparison with X-ray tomography

Metal implants, in e.g. joint replacements, are generally considered to be a success. As mechanical stability is important for the longevity of a prosthesis, the biological reaction of the bone to the mechanical loading conditions after implantation and during remodelling determines its fate. The bone reaction at the implant interface can be studied using high-resolution imaging. However, commonly used X-ray imaging suffers from image artefacts in the close proximity of metal implants, which limit the possibility to closely examine the bone at the bone-implant interface. An alternative ex vivo 3D imaging method is offered by neutron tomography. Neutrons interact with matter differently than X-rays; therefore, this study explores if neutron tomography may be used to enrich studies on bone-implant interfaces. A stainless steel screw was implanted in a rat tibia and left to integrate for 6 weeks. After extracting the tibia, the bone-screw construct was imaged using X-ray and neutron tomography at different resolutions. Artefacts were visible in all X-ray images in the close proximity of the implant, which limited the ability to accurately quantify the bone around the implant. In contrast, neutron images were free of metal artefacts, enabling full analysis of the bone-implant interface. Trabecular structural bone parameters were quantified in the metaphyseal bone away from the implant using all imaging modalities. The structural bone parameters were similar for all images except for the lowest resolution neutron images. This study presents the first proof-of-concept that neutron tomographic imaging can be used for ex-vivo evaluation of bone microstructure and that it constitutes a viable, new tool to study the bone-implant interface tissue remodelling

An atomic force microscopy study of the growth of a calcite surface as a function of calcium/total carbonate concentration ratio in solution at constant supersaturation

Calcite growth experiments using atomic force microscopy (AFM) were conducted at two constant values of supersaturation (Qi = 5.248 and £22 = 6.457) while varying the Ca2+to CO32-concentration ratio. The calcite growth rate and the morphology of growth depend on the solution stoichiometry. At a constant degree of supersaturation, the growth rate was highest when the cation/total carbonate anion ratio, r*, was equal to 1 but decreased nonsymmetrically for higher or lower values of r*. The observed dependence of growth, rates on solution stoichiometry can be explained by nonequivalent attachment frequencies of cation and anion at ratios that differ from 1. At the same time, the morphology of the closing etch pits and of the forming nuclei was different when the rate changed, suggesting a change in the crystal growth mechanism. © 2009 American Chemical Society