Name: a sectioning, coring, and image processing guide for high-throughput cortical bone sample procurement and analysis for synchrotron micro-ct
Uploaded: 2020-06-12T14:00:00
Description: Watch this Scientific Journal Video about A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT at JoVE.com

Research described in this paper was performed at the BMIT facility at the Canadian Light Source, which is supported by the Canada Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada, and the Canadian Institutes of Health Research. The authors would like to thank the beamline scientists at the Canadian Light Source, particularly Adam Webb, Denise Miller, Sergey Gasilov, and Ning Zu for the assistance in set-up and troubleshooting of the SkyScan SR&#181;CT and white beam microscope systems. We also wish to thank Beth Dalzell from The University of Toledo College of Medicine and Life Sciences and Dr. Jeffrey Wenstrup of the Northeast Ohio Medical University for access to cadaveric samples for this study. JM Andronowski is supported through start-up research funds provided by The University of Akron and a National&#160;Institute of Justice Research and&#160;Development in Forensic Science&#160;for Criminal Justice Purposes grant (2018-DU-BX-0188).&#160;RA Davis is supported by a graduate assistantship provided by The University of Akron. Equipment and supplies used for coring and sawing were purchased by start-up funds provided by The University of Akron and NSF grant EAR-1624242 to CW Holyoke.

All specimens were sourced from embalmed cadaveric donors at the University of Toledo, College of Medicine and Life Sciences and Northeast Ohio Medical University (NEOMED), with the informed consent of the donor themselves or the donor&#39;s next-of-kin. The University of Akron Institutional Review Board for the Protection of Human Subjects (IRB) deemed these specimens exempt from full IRB review as they were not procured from living individuals. Demographic information including age, sex, and cause of death were availab...

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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beamlines+of+the+biomedical+imaging+and+therapy+facility+at+the+Canadian+light+source+-+part+3.">Beamlines of the biomedical imaging and therapy facility at the Canadian light source - part 3.</a> Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 775, 1-4 (2015).</li><li>Carter, Y., Suchorab, J. L., Thomas, C. D. L., Clement, J. G., Cooper, D. M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+variation+in+cortical+osteocyte+lacunar+parameters+in+healthy+young+males.">Normal variation in cortical osteocyte lacunar parameters in healthy young males.</a> Journal of Anatomy. 225 (3), 328-336 (2014).</li><li>Carter, Y., Thomas, C. D. L., Clement, J. G., Cooper, D. 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There has been no comprehensive, standardized protocol for procuring uniform and cylindrical cortical bone core samples for high-resolution SR&#181;CT imaging with limited FOV setups. The protocol detailed here fills&#160;that void by providing a comprehensive tutorial regarding how to procure consistently sized cortical bone core samples for SR&#181;CT imaging and the subsequent accurate visualization and extraction of microarchitectural data. We have shown that our protocol provides a more standardized and reliable met...

With recent advancements in imaging technology, it is now feasible to acquire X-ray imaging data with very high resolution. Desktop micro-CT (&#181;CT) systems are the current standard for imaging cancellous bone due to their non-destructive nature1. When imaging microstructural features of cortical bone, however, &#181;CT use has been more limited. Due to resolution constraints, desktop systems cannot attain the resolution required to image microstructural features smaller than cortical pores, such as osteocyte lacunae. For this application, SR&#181;CT is ideal owing to the greater resolution of these systems1. For example, experiments at the Canadian Light Source (CLS) on the BioMedical Imaging and Therapy (BMIT) beamlines2 have produced images with voxels as small as 0.9 &#181;m. Previous studies1,3,4,5 have used this resolution to acquire projections and subsequent three-dimensional (3D) renders from cortical bone specimens from human long bones (Figure 1) to quantify osteocyte lacunar density4,6,7,8,9 and variation in the lacunar shape and size3 across the human lifespan and between the sexes. Further studies have demonstrated the presence of osteon banding in humans10, a phenomenon previously recognized to be associated with only nonhuman mammals in the forensic anthropological literature.
In order to achieve exceptional resolution, the X-ray beam must be finely focused within the field of view (FOV), which often limits the maximum specimen size to a few millimeters in diameter. Currently, there have been no comprehensive, standardized procedures described in the literature outlining bone sample procurement that meet these restrictions. Centering specimens within the FOV is critical to ensure that 1) the sample remains centered as it rotates 180&#176; during imaging, and 2) scan artifacts are limited since there is no image truncation. In other words, no portions of the sample outside of the FOV interfere with the beam entering its focal point inside the FOV. If this occurs, the reconstruction algorithm is deprived of some of the attenuation data needed for a fully correct reconstruction. It is further worth noting that 360&#176; (full rotation) scans minimize the effects of beam hardening but increase artifacts caused by misalignment and sample movement during imaging. Thus, while a 360&#176; scan will typically generate cleaner data, imaging time is doubled and so a compromise between experimental cost and data quality must be addressed.
An important and often overlooked aspect of bone imaging experiments is the accurate and replicable specimen preparation technique performed prior to scanning. Studies that incorporate SR&#181;CT methods into their experiments briefly mention their sampling protocol, but the authors provide little to no detail regarding the particular methodology used to gather their specimens. Many such studies mention cutting rectilinear bone blocks of arbitrary dimensions, but generally provide no further information about the tools or embedding materials used3,4,10,11,12,13,14. Some researchers commonly use handheld rotary tools (e.g., Dremel) to remove rectilinear blocks of bone from a region of interest (ROI)3,4,10,11,12,13,14. This method results in nonuniformly sized samples that may be larger than the FOV, increasing the likelihood of scan artifacts and image truncation. Such specimens often require further refining using a precision diamond-wafer saw (e.g., Buehler Isomet). Procuring samples with consistent dimensions (to the two-hundredths/mm) is critical to ensure that the acquired datasets are of the highest quality and the subsequent results are replicable.
The limited reporting of sample procurement methodology adds an extra layer of difficulty when attempting to employ and/or validate methods performed in a previous study. Currently, researchers must contact authors directly for further details on their sampling procedures. The protocol detailed here provides biomedical researchers with a thoroughly documented, replicable, and cost-efficient sampling technique. The primary objective of this article is to provide a comprehensive tutorial regarding how to procure consistently sized cortical bone core samples using a mill-drill press and diamond coring bit for the accurate visualization and extraction of microarchitectural data. This method is modified from procedures used to routinely collect uniform, small-diameter (1-5 mm) cylinders from blocks of hard materials in high pressure rock mechanics15,16,17,18,19.

<table border="0" cellpadding="0" cellspacing="0" style="width:1367px;" width="1367">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">1-1/8&#34; plunge cutting carbide for composites</td>
			<td style="width:120px;">Warrior</td>
			<td style="width:133px;">61812</td>
			<td style="width:899px;">28.6mm plunge</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">70% Ethanol</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:133px;">BP8201500</td>
			<td>3.8 Liters</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Blunt-tipped forceps</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:133px;">10-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Centrifuge tubes</td>
			<td style="width:120px;">ThermoFisher</td>
			<td style="width:133px;">55398</td>
			<td></td>
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			<td height="21" style="height:21px;width:215px;">Crystalbond 509-3 Epoxy</td>
			<td style="width:120px;">Ted Pella</td>
			<td style="width:133px;">821-3</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">CTAnalyser</td>
			<td style="width:120px;">Bruker microCT</td>
			<td style="width:133px;">v.1.15.4.0</td>
			<td>Download and install at https://www.bruker.com/products/microtomography/micro-ct-software/3dsuite.html</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Dental Tool Kit</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">787269885110</td>
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			<td height="42" style="height:42px;width:215px;">Diamond wafering saw blade for composite material</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">#11-4247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Drill Press</td>
			<td style="width:120px;">Jet Mill/Drill</td>
			<td style="width:133px;">350017</td>
			<td>Model: JMD-15, benchtop drill presses are suitable substites, but typically lack a translatable machine table for positioning samples beneath the drill stem</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fine-tipped forceps</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:133px;">22-327379</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Fixturing clamps for XY machine table for mill/drill</td>
			<td style="width:120px;">MSC Industrial Supply</td>
			<td style="width:133px;">#04804571</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass microscope slides</td>
			<td style="width:120px;">Ted Pella</td>
			<td style="width:133px;">26005</td>
			<td>75x50mm slides, 1mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass slide chuck</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">#112488</td>
			<td>Large enough to hold 75x50mm glass slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Hot plate capable of reaching 140 &#176;C</td>
			<td style="width:120px;">ThermoScientific</td>
			<td style="width:133px;">HP88850105</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Incubator</td>
			<td style="width:120px;">NAPCO</td>
			<td>Model 4200</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isocut Fluid</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">111193032</td>
			<td>Lubricant; 30mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Jeweler&#39;s diamond coring drill bit</td>
			<td style="width:120px;">Otto Frei</td>
			<td style="width:133px;">#119.050</td>
			<td>2mm inner diameter hollow stem coring bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NRecon</td>
			<td style="width:120px;">Bruker microCT</td>
			<td style="width:133px;">v.1.6.10.2</td>
			<td>Download and install at https://www.bruker.com/products/microtomography.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oscillating saw</td>
			<td style="width:120px;">Harbor Freight</td>
			<td style="width:133px;">62866</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oven-safe glass dishes</td>
			<td style="width:120px;">Pyrex</td>
			<td style="width:133px;">1117715</td>
			<td>Glass food storage container</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Precision slow-speed saw (Isomet 1000)</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">111280160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Razor blades</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">25181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Shallow aluminum tins</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">B01MRWLD0R</td>
			<td>~8cm diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Specimen cups</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">616784425436 885334344729</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tergazyme detergent</td>
			<td style="width:120px;">Alconox</td>
			<td style="width:133px;">1304-1</td>
			<td>1.8kg box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ultrasonic cleaner</td>
			<td style="width:120px;">MTI Corporation</td>
			<td style="width:133px;">KJ201508006</td>
			<td></td>
		</tr>
	</tbody>
</table>

a sectioning, coring, and image processing guide for high-throughput cortical bone sample procurement and analysis for synchrotron micro-ct

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

The described method of core sampling proved to be highly effective and efficient. Coring specimens using this protocol allowed for the procurement of &#62;300 consistently sized samples for experiments on the CLS BMIT-BM beamline2, with an FOV of ~2 mm at 1.49 &#181;m voxel size. To validate the consistency of core diameter, three measurements were taken along the length (top, middle, bottom) of a subset of human anterior femoral cores (n=69). The average diameter of the cores was 1.96 &...

Watch this Scientific Journal Video about A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT at JoVE.com

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

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