We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SRµCT 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 studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (µCT) and synchrotron radiation micro-computed tomography (SRµCT) 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 SRµCT 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 SRµCT 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.
With recent advancements in imaging technology, it is now feasible to acquire X-ray imaging data with very high resolution. Desktop micro-CT (µCT) systems are the current standard for imaging cancellous bone due to their non-destructive nature1. When imaging microstructural features of cortical bone, however, µCT 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, SRµCT 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 µm. 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° 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° (full rotation) scans minimize the effects of beam hardening but increase artifacts caused by misalignment and sample movement during imaging. Thus, while a 360° 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 SRµCT 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.
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'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 available for all donors. The selected individuals did not have documented bone-affecting conditions nor exposure to treatment regimens that may have affected bone remodeling at the time of death. Cortical bone samples were obtained from femora of cadaveric modern males and females with ages ranging from 19 to 101 years of age (mean = 73.9 years). The femoral midshaft has been studied extensively including examinations of variation in cortical porosity20,21,22,23,24 and material density of bone tissue25,26,27, and has thus become a commonly used site for microstructural analyses.
1. Tissue Procurement and Maceration
2. Tissue sectioning
3. Sample coring
4. Image processing routines for evaluating bone microarchitectural parameters from cortical bone cores
The described method of core sampling proved to be highly effective and efficient. Coring specimens using this protocol allowed for the procurement of >300 consistently sized samples for experiments on the CLS BMIT-BM beamline2, with an FOV of ~2 mm at 1.49 µm 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 ± 0.11 mm, and the average thinning along the length of the core was 0.06 ± 0.06 mm/mm In order to emphasize the applicability to other hard composite materials, we attempted this method on samples of dolomite (n=32) resulting in an average diameter of 1.06 ± 0.02 mm. Thinning along the length of the core sample was recorded as 0.01 ± 0.005 mm/mm. Representative figures comparing the image processing workflow of a cored sample and one procured using a rotary tool (e.g., Dremel), as described in step 4.2.3, can be viewed in Figure 7. The sample cut using the common rotary tool exhibited an increased number of canals (Ca.N) and lacunae (Lc.N), and a decreased average canal diameter (Ca.Dm), canal volume (Ca.V), and cortical porosity (Ca.V/TV) when compared to the cored sample. While some of these differences may be due to bone microstructural variation between individuals, the higher number of canals and lacunae extracted from the rotary tool dataset were likely artificially increased due to scan artifacts and noise (Figure 7). The porosity data collected from step 4.2.3.9 for each sample is located in Table 1. It is worth noting that though the coring protocol decreases artifacts observed in SRµCT scans, the lower-quality, artifact-laden figures from the rectilinear bone block experiments (Figure 7A) represent a multi-faceted issue. Certain artifacts (e.g., phase contrast signals) may have been caused by synchrotron facility or beamline-specific issues. Scan parameters for both representative sets of experiments and the associated figures (Figures 7A, 7B) can be found in the Supplementary Materials (Tables S1, S2).
Synchrotron micro-CT images gathered from cored samples successfully suppressed scan artifacts, as demonstrated above, including streak artifacts. Subsequent image processing confirmed the potential of the technique to improve visualization of cortical bone microarchitecture. For example, mineralization differences, improved delineation of osteonal boundaries, and consistent visualization of soft tissues within vascular canals were observed (Figures 8C, 8D). The latter is critical for image processing as partial visualization of soft tissues within canals may result in inaccurate calculations of percent porosity and pore thickness, since the pores are not fully filled. The boundaries of osteocyte lacunae were also improved due to decreased birefringence, allowing for the quantification of shape parameters. The potential advantages of the described coring technique include ease of centering the specimen in the FOV, reduced analytical requirements, and consistent visualization of soft tissues within vascular canals.
Similar procedures have been used successfully to core single crystals of orthopyroxene18, polycrystalline magnesite19 and other geologic materials15,16,17 for high pressure rock deformation experiments. These experiments require cores in specific orientations relative to crystallographic axes in single crystals18 or aligned crystals in polycrystalline rocks19 in order to determine orientation-specific strengths. The approaches described above have been used to first create oriented slabs and, subsequently, collect multiple uniform, cylindrical cores for series of deformation experiments. These methods can be used to collect cores of any hard material, such as bone, ceramics or glasses. For example, the above methodology could be applied by biological anthropologists to evaluate cores from specific regions within cortical bone and their associated biomechanical (e.g., tension/compression) axes.
Figure 1. Cylindrical VOI from a left anterior human mid-shaft femur. A single SRμCT reconstructed slice of an entire core from a left anterior human mid-shaft femur (21-year-old female) (A), and 3D renders of a cylindrical VOI from superior (B) and anterior views (C) are visualized. Projections were taken at 0.9 μm, with vascular canals highlighted in red and osteocyte lacunae in grey. Scale bars denote 0.25 mm (A) and 0.02 mm (B,C). Please click here to view a larger version of this figure.
Figure 2. A mid-shaft femoral sample (5 mm thickness) mounted to a glass microscope slide with thermal epoxy (see Table of Materials) and secured to a glass slide chuck. Please click here to view a larger version of this figure.
Figure 3. Glass slide chuck with mounted specimen secured to the swivel arm of a low-speed sectioning saw (see Table of Materials) prior to sectioning. The lateral position of the swivel arm relative to the saw blade and the sectioning speed (RPM) are displayed on the top and bottom rows of the LCD display, respectively. Please click here to view a larger version of this figure.
Figure 4. A 5 mm femoral section mounted to an aluminum tin and secured to an XY mill-drill press machine table using fixturing clamps in preparation for coring. Please click here to view a larger version of this figure.
Figure 5. The mill-drill press employed in the devised protocol (A). The arrow identifies the depth limiter, which prevents the drill bit from penetrating deeply into the sample or through the bottom of the tin (B). Please click here to view a larger version of this figure.
Figure 6. A 5 mm femoral bone cross-section following core procurement from the anterior aspect. Please click here to view a larger version of this figure.
Figure 7. Single SRµCT reconstructed slices of the anterior aspect of the left femur from two individuals. Specimen (A) was sectioned using a common rotary tool and (B) was procured using the coring method described here. Each slice is compared with their segmented counterpart (C and D). Note the ease of isolating cortical porosity in the cored sample (D) as opposed to the specimen gathered with the rotary tool (C). This is evidenced further in the 3D renders of the vascular canals of each sample (E and F). Noise around the periphery of B is evident and the specimen leaves the FOV, which both result in increased challenges during image processing. The scale bar in panel (D) denotes 250 µm for panels (A-D). The scale bars in panels (E and F) denote 700 and 600 µm, respectively. Please click here to view a larger version of this figure.
Figure 8. A representative ROI from a sample procured with a rotary tool (A-C) and one cored using the method presented here (D-F). Panels (A) and (D) represent the designated ROI from the SRµCT scans. Panels (B) and (E) represent the processing stage used to isolate and extract vascular canal parameters. In the upper right of panel (B) there are extraneous objects (arrows) that have been classified as vascular canals by image processing software. Panels (C) and (F) represent the processing stage used to isolate and extract lacunae. Scale bars denote 0.1 mm for all panels. Please click here to view a larger version of this figure.
Tissue Volume (TV) | Canal Volume (Ca.V) | Canal Surface (Ca.S) | Cortical Porosity (Ca.V/TV) | Canal Surface to Tissue Volume (Ca.S/TV) | Average Canal Diameter (Ca.Dm) | Average Canal Separation (Ca.Sp) | No. of Canals (Ca.N) | No. of Lacunae (Lc.N) | Pore Density (Pores/TV) | |
Units | mm³ | mm³ | mm² | % | 1/mm | µm | µm | # | # | pores/µm³ |
Rotary Cut | 0.15861 | 0.01780 | 0.00287 | 11.23 | 0.01808 | 51.05 | 122.81 | 459 | 64662 | 0.00041 |
Cored (This method) | 0.15747 | 0.02451 | 0.00216 | 15.56 | 0.01373 | 120.73 | 145.38 | 76 | 30531 | 0.00019 |
Table 1. Representative results for step 4.2.3.9 of rotary tool and cored specimens visualized in Figure 8. Note the decreased Ca.V, Ca.V/TV, Ca.Dm, number of pores and pore density for the rotary cut sample as well as the increased number of vascular canals and lacunae. Scan artifacts partially induced by the unevenly cut sample likely contributed to an artificial increase in lacunae and cortical pores.
Supplementary Materials. Please click here to download these materials.
There has been no comprehensive, standardized protocol for procuring uniform and cylindrical cortical bone core samples for high-resolution SRµCT imaging with limited FOV setups. The protocol detailed here fills that void by providing a comprehensive tutorial regarding how to procure consistently sized cortical bone core samples for SRµCT imaging and the subsequent accurate visualization and extraction of microarchitectural data. We have shown that our protocol provides a more standardized and reliable method for procuring cortical bone cores than previous descriptions of sectioning rectilinear bone blocks of arbitrary dimensions. Thus, researchers who have relied on handheld rotary tools (e.g., Dremel) to remove irregularly sized blocks of bone likely experienced much longer sample set-up times during imaging and greater errors in thresholding and cortical pore extraction during analysis. This discrepancy highlights the need for and significance of this standardized protocol with respect to bone sample preparation, subsequent visualization and analysis, and interpreting results.
The procedure outlined here may further be adapted by researchers in related fields who commonly evaluate bone tissue such as biological anthropologists and archaeologists. No diagenetic nor archaeological/historic bone specimens, however, were cored for the described research protocol. Diagenesis, in geology, refers to alterations of a material (e.g., bone) after deposition and can encompass changes caused by physical, chemical, or biological means29,30. Ground water, fungi, and other microbial infiltration can all act as diagenetic agents and alter bone tissue micromorphology31. Such specimens may require additional procedural steps prior to coring, such as embedding in methyl methacrylate (MMA) or a two-part epoxy resin. Embedding the femoral blocks was not necessary for the described experiments due to the dense nature of femoral cortical bone, and the fact that cadaveric specimens were embalmed shortly after death. If evaluating fragile skeletal elements and their trabeculae (e.g., ribs), however, we recommend embedding the entire bone block prior to coring.
All bone tissues evaluated in this study were embalmed while fresh. The authors did not have access to the specific combination of chemicals used during the embalming process, though preservation chemicals commonly include formaldehyde, ethanol, phenol, ethylene glycol, and glutaraldehyde. Forensic anthropological data documenting changes in the microstructure of formaldehyde saturated bones is limited, though Freidlander32 demonstrated that formaldehyde fixation does not alter the morphology of certain features including Haversian canals and secondary osteons. Formaldehyde saturation, however, has documented effects on certain mechanical properties and fracture characteristics of nonhuman bone such as impact strength and fracture toughness33,34.
We have reported a method for coring cortical bone samples prior to imaging with high-resolution X-ray systems (SRµCT). This method is cost-effective, owing to the fact that materials and equipment may be sourced from local hardware stores, efficient, and ensures a uniform sample size across specimens. It is our hope that our suggestions will reduce inquiries relating to how samples should be procured, cored, and analyzed for SRµCT, as the existing literature remains sparse and lacks critical details regarding preparation and subsequent analysis. Our primary goal is to motivate researchers to apply this coring protocol as standardized procedure for high-resolution bone imaging research. We further hope that the aforementioned difficulties we experienced in developing this technique will alleviate common questions and provide guidance for troubleshooting.
The authors have nothing to disclose.
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 SRµCT 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 Institute of Justice Research and Development in Forensic Science for Criminal Justice Purposes grant (2018-DU-BX-0188). 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.
1-1/8" plunge cutting carbide for composites | Warrior | 61812 | 28.6mm plunge |
70% Ethanol | Fisher Scientific | BP8201500 | 3.8 Liters |
Blunt-tipped forceps | Fisher Scientific | 10-300 | |
Centrifuge tubes | ThermoFisher | 55398 | |
Crystalbond 509-3 Epoxy | Ted Pella | 821-3 | |
CTAnalyser | Bruker microCT | v.1.15.4.0 | Download and install at https://www.bruker.com/products/microtomography/micro-ct-software/3dsuite.html |
Dental Tool Kit | Amazon | 787269885110 | |
Diamond wafering saw blade for composite material | Buehler | #11-4247 | |
Drill Press | Jet Mill/Drill | 350017 | Model: JMD-15, benchtop drill presses are suitable substites, but typically lack a translatable machine table for positioning samples beneath the drill stem |
Fine-tipped forceps | Fisher Scientific | 22-327379 | |
Fixturing clamps for XY machine table for mill/drill | MSC Industrial Supply | #04804571 | |
Glass microscope slides | Ted Pella | 26005 | 75x50mm slides, 1mm thick |
Glass slide chuck | Buehler | #112488 | Large enough to hold 75x50mm glass slides |
Hot plate capable of reaching 140 °C | ThermoScientific | HP88850105 | |
Incubator | NAPCO | Model 4200 | |
Isocut Fluid | Buehler | 111193032 | Lubricant; 30mL |
Jeweler's diamond coring drill bit | Otto Frei | #119.050 | 2mm inner diameter hollow stem coring bit |
NRecon | Bruker microCT | v.1.6.10.2 | Download and install at https://www.bruker.com/products/microtomography.html |
Oscillating saw | Harbor Freight | 62866 | |
Oven-safe glass dishes | Pyrex | 1117715 | Glass food storage container |
Precision slow-speed saw (Isomet 1000) | Buehler | 111280160 | |
Razor blades | Amazon | 25181 | |
Shallow aluminum tins | Amazon | B01MRWLD0R | ~8cm diameter |
Specimen cups | Amazon | 616784425436 885334344729 | |
Tergazyme detergent | Alconox | 1304-1 | 1.8kg box |
Ultrasonic cleaner | MTI Corporation | KJ201508006 |