The authors would like to acknowledge the editorial reviewers as well as the two anonymous reviewers. Their suggestions and critiques were valid, much appreciated and vastly improved the original manuscript.

The protocol herein adheres to the North Carolina State University&#39;s ethics guidelines for human research.
1. Machine Preparing
NOTE: The following protocol can be broadly applied to any whole body, clinical DXA and BMD scanner.
<ol>
	<li>Perform calibration once daily prior to scanning any individuals to ensure quality control. After calibration prompts appear upon start-up of the systems&#39; software, scan a lumbar spine phantom of known density to ensure correct reading of the BMD scanner.</li>
	<li>If the scanner being utilized does not have a quality control feature in the software, compare the lumbar spine results with those recorded on the spine phantom to ensure the correct measurements. 
	NOTE: The spine phantom, should be placed in the center of the scan table and lumbar spine should be selected for quality control.</li>
	<li>Perform additional tests (e.g., radiographic uniformity) as needed. Perform radiographic uniformity every ten scans maximum to ensure that the entire scanning surface is detected by the scanner.</li>
	<li>If the scanner being utilized does not have a radiographic uniformity test in the quality control menu, select whole body scan to ensure the scanner can read the entire scanning surface. 
	NOTE: Always center the exam table following quality control and before performing exams.</li>
</ol>
2. Performing Exam
<ol>
	<li>Create patient profiles
	<ol>
		<li>Create new patient profiles for each new individual scanned to maintain chain of custody and to ensure scans are correctly associated with individual remains. If the individual being scanned is identified, proceed to step 2.1.2. If the individual is unidentified, establish the biological profile prior to scanning to employ the most accurate database references.</li>
		<li>Enter demographic information into the patient profile including estimated stature if unknown. Ensure that you select the most appropriate equation for the remains being investigated.</li>
		<li>Select scan type. For steps 2.2, select Anterior-Posterior (AP) Lumbar Spine. For step 2.3, select Left or Right Hip scans.</li>
	</ol>
	</li>
	<li>AP Lumbar Spine scan 
	NOTE: Require lumbar vertebrae (L) one to four.
	<ol>
		<li>Select Perform Exam | choose patient | Select scan type |AP Lumbar Spine | Next. Select an open container at least as large as the articulated segment of L1-L4. 
		NOTE: The one used in this study is 48.26L X 26.85W X 8.89D in cm (19 in. L X 10.57 in. W X 3.5 in. D).</li>
		<li>Fill the bottom of the container with rice as a soft tissue proxy. 
		NOTE: Any kind of rice can work as a soft tissue proxy.</li>
		<li>Place L1-L4 in anatomical position (spinous processes should be oriented downwards) in the rice with approximately 0.7 cm (0.28 in.) between each vertebral body as shown in Figure 1A. Ensure that the superior and inferior articular facts are articulated, but the vertebral bodies are not in contact with one another.</li>
		<li>Center the scanning table and place the container with L1 is oriented towards top (head) of scanning table and L4 is placed 1 cm superior to the intersecting crosshairs. The vertical laser line should be bisecting the vertebrate bodies of all four vertebrae (Figure 1B).</li>
		<li>Cover the exposed bone with rice.</li>
		<li>Select Start Scan.</li>
		<li>Proceed to analysis (step 3.1), If scanned properly (Figure 2). Repeat the scan if not all vertebrae are captured.</li>
	</ol>
	</li>
	<li>Left or Right Hip scans 
	NOTE: Figure 3 is from a left hip exam, if performing a right hip exam, positioning is mirrored.
	<ol>
		<li>Select Perform Exam | choose patient | Select scan type | Left Hip (or Right Hip) | Next. Select an open container at least as large as the articulated os coxa and femur being scanned. 
		NOTE: The one used in this study is 88.5L X 41.5W X 13.9D in cm (34.85 in. L X 16.35 in. W X 5.47 in. D).</li>
		<li>Fill the bottom of the container with rice (any kind of rice will work as a soft tissue proxy).</li>
		<li>Place the os coxa with the acetabulum and obturator foramen facing laterally with the pubic bone oriented medially. Position the ischial tuberosity beneath the femoral head as it articulates with the acetabulum (Figure 3A). 
		NOTE: Positioning of the ischial tuberosity is most important because if it extends laterally below the femoral neck it will inflate BMD estimates.</li>
		<li>Place the femur with the femoral head in the acetabulum and with the greater trochanter and femoral head in line parallel to the scanning table (i.e., in the same plane). Ensure that the femoral shaft is medially rotated with the distal condyle rotated medially and slightly higher than the medial condyle (Figure 3B).</li>
		<li>Center the scanning table, then move the position of the scanning arm and table until the laser crosshairs are oriented so that the center is directly above the subtrochanteric area of the femur with the vertical line bisecting the top-half of the femoral shaft (Figure 3A). Do not move the remains once they have been positioned. Move the table ensures that bones remain in proper anatomical position.</li>
		<li>Cover the remaining visible portion of the femoral-acetabular joint with rice.</li>
		<li>Select Start Scan.</li>
		<li>Proceed to analysis in step 3.2 if scanned properly (Figure 4). 
		NOTE: Scans should capture the alignment of the joint such that the midline of the proximal femur is in one plane. The midline should lie from the center of the femoral head to just under the greater trochanter.</li>
	</ol>
	</li>
</ol>
3. Analyzing Exams
<ol>
	<li>Analyze AP Lumbar Spine scan
	<ol>
		<li>Following the scan, an Exit Exam prompt box will appear. Select Analyze Scan. 
		NOTE: Software will separate each vertebra into their own regions to assess individual elements and total BMD when scanned properly as shown in Figure 5.</li>
		<li>Select Results in the Scan Analysis window. Select vertebral lines If the vertebrae are not properly separated for minor adjustments or directly reposition vertebrae for rescanning.</li>
		<li>Obtain both age-matched and population specific BMD reference measures to calculate a z-score when performing juvenile BMD scans.</li>
		<li>Collect results graph for visualization of the individual relative to the reference population. 
		NOTE: Figure 6 shows scan results for AP Lumbar Spine of a 31-year-old female.</li>
	</ol>
	</li>
	<li>Analyze hip scan.
	<ol>
		<li>Following the scan, an Exit Exam prompt box will appear. Select Analyze Scan. 
		NOTE: Software will automatically capture the femoral neck, Ward&#39;s triangle, and trochanteric area as shown in Figure 7, if scanned properly.</li>
		<li>Select the bone map tool to add or delete areas that are not part of the femoral neck and trochanteric region when not read exactly by the software due to slight malposition. Make midline adjustments directly on scan by selecting the neck tool and repositioning the midline.</li>
		<li>Reposition and rescan if these small adjustments do not allow the proper alignment shown in Figure 7.</li>
		<li>Select Results in the Scan Analysis window. Compare to reference data for femoral neck, trochanteric region, and the intertrochanteric region in the software for adults.</li>
		<li>Compare results with the appropriate age- and population-matched references when assessing juveniles.</li>
		<li>Use the t-score for adults as it is the most appropriate to differentially assess pathological conditions. 
		NOTE: Figure 8 shows the ideal scan results for the left hip analysis of a 31-year-old female.</li>
	</ol>
	</li>
</ol>

The authors declare no competing financial interests.

The results presented in this paper are illustrative of the applicability of BMD metrics in forensic contexts. As Figure 6 and Figure 8 show, the scanning position of living individuals for clinical BMD scans is reproducible with skeletal remains, but care must be taken to ensure proper positioning. This is especially critical for the hip examination where identifying the midline of the femoral neck require the proper angle of the femur and overestimation of BMD can occur if the iliac tuberosity is not properly positioned medially to the acetabulo-femoral joint. For the adult male discussed in Case Study 1, BMD metrics can provide the caseworker with additional information about possible pathological conditions. Without a measure of BMD, the longitudinal fractures could have been consistent with bone insufficiency. This also illustrates that BMD assessment can be advantageous over X-rays for discerning possible fracture etiologies.
Case Study 2 and 3 provide instances where BMD metrics were integral to establishing severe malnutrition that supported more commonly used methods. Juvenile cases of fatal starvation are difficult to identify and prosecute especially when remains are recovered in advanced stages of decomposition31,37,38. The addition of DXA scanning protocols when fatal starvation is suspected can provide further support for findings. In both juvenile case studies, DXA scans were applied in conjunction with standard methods to compare these individuals with living children. Indeed, in both cases the DXA results were consistent with the standard method findings illustrating its usefulness in forensic cases of fatal starvation or neglect. Overall, the three cases discussed here were bolstered by DXA analysis to either include or exclude certain inferences about each case. However, there are limitations to when this method should be applied in forensic contexts. For example, research has shown that the relationship between bone volume and bone area in juveniles varies between growth stages12,92. Ensuring that the proper methodology and normative data is being used (i.e., age-matched normative data) is imperative. When assessing infants, comparison to other methodologies, such as measurements of limb segments, should be included in the practitioner&#39;s assessment25,33.
One of the main limitations of this method is the consideration of taphonomy (i.e., diagenetic changes to skeletal composition after death). This relates to the estimation of survivorship of skeletal elements. In general, skeletal elements with higher BMD values during life will preserve more readily7,27, but this does not exclude the likelihood that the bone mineral has been altered over time. Thus, while BMD can be employed bioarchaeologically to assess general levels of survivorship it should not be interpreted as living BMD-at-death. This is because if remains have been diagenetically altered, the BMC will not be an accurate reflection of BMD during life if mineral exchange or catabolism has occurred55. For example, Ross and Juarez85 present a case where infanticide was suspected that may have been due to fatal starvation. However, traditional methods were chosen because the friability of the remains suggested extensive taphonomic alteration as the remains had been buried for approximately four years beneath a shed prior to discovery85. Thus, as mentioned previously, the taphonomic alteration would have not been an accurate reflection of the infant&#8217;s BMD at death. In closing, this method can provide support for other indicators of malnutrition or metabolic bone pathologies, however, condition of remains should be assessed before interpreting DXA results in skeletal remains.

The objective of forensic anthropological analyses relies on the practitioner&#39;s understanding of bone as a complex tissue with multiple units and variation. Bone is a hierarchical, composite tissue with both organic and inorganic components organized into a matrix of collagen and carbonated apatite1,2,3,4. The inorganic component, or bone mineral is organized in a nanocrystalline structure to provide stiffness and framework for the organic portion1,2,5. The mineral aspect comprises approximately 65% of bone by weight and its&#39; mass is influenced by both genetic and environmental factors1,2,4,6. Because bone mineral occupies a three-dimensional space, it can be measured as bone mineral density (BMD), or a function of the mass and the volume occupied7. The bulk density of bone mineral varies with age from birth into adulthood8,9,10,11,12 and has been used extensively in clinical settings as an indicator of osteoporosis and fracture risk4,13,14,15,16,17,18. Dual-energy X-ray absorptiometry (DXA) has been a widespread tool for the assessment of bone health since its introduction in 1987, particularly scans performed in the lumbar spine and hip regions11,13,19. Validation of DXA scans has been shown as the gold standard when investigating changes in BMD13,19,20,21,22,23. Subsequently, the World Health Organization (WHO) has created normative standards including t- and z-score definitions for juvenile and adult lumbar spine (L1-L4) and hips as these are the regions easily captured volumetrically11,13,19,24.
The increasing reliance on forensic anthropology in medicolegal casework has encouraged the investigation of novel techniques to better assess skeletal remains in a variety of circumstances. Among these potential techniques is the application of DXA scans to assess BMD as an indicator of bone quality in cases involving fatal starvation and neglect in juveniles25,26, identification of metabolic bone diseases, and estimating survivability of skeletal elements in taphonomic research7,27.
In the 2015 U.S. Department of Health and Human Services Child Maltreatment Report, 75.3% of the reported child abuse cases were some form of neglect with ~1,670 fatalities resulting from fatal starvation and neglect in 49 states28. Most juvenile victims of neglect fail to show signs of external physical abuse, but failure-to-thrive is seen in all cases29,30. Failure-to-thrive is defined as the inadequate nutrition intake to support growth and development. These can have different factors, one of which is neglect resulting from nutritional deprivation25,31 (see Ross and Abel32 for a more comprehensive review). Deliberate starvation that results in the death of a child or infant is much rarer and considered as the most extreme form of maltreatment25,33,34. These nutritional deficiencies have a significant effect on bone growth, particularly longitudinal growth in children as an immediate consequence of malnutrition35. Skeletal growth and mineralization primarily depend on vitamin D and calcium, and their supplementation has been linked to increased BMD25,35,36.
It is exceedingly difficult to identify or prosecute these cases even following a complete autopsy31,37,38 and special consideration to methods employed must be used. Thus, in cases where fatal starvation or malnutrition is suspected, a multidisciplinary approach is needed particularly in cases involving remains in advanced states of decomposition26. When skeletal remains are involved, bone densitometry is a useful tool in conjunction with other skeletal indicators such as dental development, measurement of the pars basilaris of the skull, and long bone lengths26. Without using the skeletal indicators mentioned above for infants and juveniles, it would not be possible to discern if low BMD is the result of an inherent metabolic disorder, malnutrition, or taphonomic process. Another concern is the estimation of body size (weight and stature) in infant or juvenile skeletal remains. Most normative data sets require information about height or weight for comparison purposes as bone growth in children is size and age dependent12. When the remains being assessed are unidentified, estimation methods should be employed. For infants under one, normative DXA data is age matched only. In juveniles over the age of 1, Ruff39 or Cowgill40 are recommended for estimating body size in skeletal remains as they are based on the Denver Growth Study sample including ages 1 - 1739,40. When age and body size are estimated, confidence intervals vary and comparison of the mean to the Center for Disease Control (CDC) produced growth curves41 should be included in the report as well as the confidence interval for the estimated body size. It is important to note that in most cases, information regarding ancestry and sex cannot be determined from juvenile skeletal remains prior to puberty, which is particularly important for adolescents as ancestry and sex are known to significantly impact BMD in adults. In these circumstances, the DXA method may not be applicable. In identified cases, biological information regarding ancestry, sex, and body size, should be obtained prior to analysis.
Bone densitometry in pediatrics has increased with the development of normative data42,43 with DXA being the most widely available technique44. Malnourished children show significantly lower levels in BMD than healthy children with mineralization correlated with severity of malnutrition45. DXA scans of the lumbar spine and hips are the most appropriate areas to assess for juveniles according to The American College of Radiology46. Reproducibility has been shown for spine, whole hip, and whole body in children throughout the growth period47. However, the lumbar spine is preferred as it is primarily composed of trabecular bone, which is more sensitive to metabolic changes during growth and has been found to be more precise than whole hip assessments25,47,48. Using DXA scans is common in pediatric assessment. However, since DXA is two-dimensional, it does not capture true volume and produces a BMD based on bone area13. In children, this is an important distinction as body and bone size vary within and between age groups in children12. Most normative data available is for comparison with DXA measurements, but care should be exercised to choose an appropriate reference population (see Binkovitz and Henwood13 for a list of commonly used DXA normative databases).
Following the scan, a z-score is calculated using an age-matched and population specific reference sample. Z-scores are more appropriate for juveniles since t-scores compare the measured BMD to a young adult sample12. A z-score between -2 to 2 indicates normal BMD for chronological age while any score below -2 indicates low BMD for chronological age49. The -2 to 2 range for both the t- and z-score represent up to two standard deviations from the mean. Plainly, if a measured BMD score is within two standard deviations above or below their reference population mean, they are considered clinically normal.
The reliance on morphological variation for the forensic anthropologist comes from many sources. One of which is the skeletal variation that arises from disease processes, including metabolic bone disorders50. The ability to identify specific disorders in skeletal remains has a two-fold advantage: 1) adding information to the biological profile making it more robust and 2) identifying if fractures are pathological or the result of inflicted trauma. There are a variety of metabolic bone disorders51,52,53, but the most relevant for BMD measures of contemporary remains is osteoporosis. Osteoporosis develops when the rate of trabecular bone loss is greater than the rate of cortical bone loss with a net loss in bone density53,54,55. Trabecular bone loss is correlated to an increased risk of fracture, especially in bones that have greater trabecular bone content (e.g., the os coxa)4,55.
Numerous studies on osteoporosis and bone mineral density in skeletal remains have been conducted on archaeological assemblages using both DXA56,57,58,59 and other methods60,61,62. However, when assessing osteoporosis in the adult skeleton from archaeological contexts, practitioners disregard that diagnosing osteoporosis clinically requires the mean of a younger reference sample contemporaneous with the individuals being assessed55,63,64. This is not an issue in forensic anthropology contexts since individuals are age- and sex- matched to modern populations with developed reference samples for both the hip and the lumbar spine, although changes in BMD through diagenesis should be considered for forensic remains. However, taphonomy is the likely factor affecting the ability to obtain legitimate BMD measures from archaeological samples. This is a consideration in forensic contexts as well, where remains recovered from burial conditions with potential postmortem intervals beyond a few months. While still of forensic interest, sufficient doubt could be raised for any BMD scores obtained from remains found in these circumstances.
Osteoporosis is clinically assessed using t-scores of BMD measures that are derived from the individuals&#8217; BMD measures in the hip or lumbar spine relative to a young adult reference sample using DXA65,66,67,68. This reference sample can be employed for identifying the occurrence of osteoporosis in the skeleton. In forensic contexts, this is useful for two reasons: 1) differentiating between fractures related to abuse-inflicted trauma in the elderly and those from increased bone fragility in osteoporotic individuals69, and 2) as a possible personal identification feature50.
Bone density has long been considered an indicator that reflects the activity and nutrition of an animal70,71. More recently it has been noted that bone density, as an intrinsic property of bone, affects its survivability during taphonomic processes7.&#160; A consequence of decomposition is the differential survivability of skeletal elements (i.e., discrete, anatomically complete units of the skeleton) and bone density can be used as a predictor of survivability, or bone strength7,70,71,72,73,74,75. This is important in forensic contexts as well as archaeological and paleontological environments in that it affects the practitioners&#8217; ability to adequately employ methods to estimate a biological profile (or age, sex, stature, and ancestry) if only certain skeletal elements are represented.
Bulk density (bone density with pore space included in the measurement) is the appropriate measurement in this situation, considering it is precisely the porous structure of bone that influences its susceptibility to taphonomic processes7. Many methods of assessing bone density have been employed including single-beam photon densitometry27,75, computed tomography76,77,78, photodensitometry72,79, and DXA80,81,82. DXA scans may be preferable to other methods as it is relatively inexpensive, whole body scans can be performed, and individual skeletal elements can be assessed separately or together during analysis. Using BMD scans before and after taphonomic research studies provides useful information on bone survivability resulting from different taphonomic factors and environments82.
This paper outlines a protocol for obtaining DXA scans of skeletal remains. The method employs the common, clinical positioning of individuals when performing lumbar spine and hip scans. This allows practitioners to compare the skeletal remains with the appropriate normative standards. The protocol outlined is applicable to both juvenile and adult remains with limitations discussed later.

<table><tbody><tr><td>QDR Discovery 4500W system</td><td>Hologic</td><td>Discovery W</td><td>All inclusive DXA whole body scanner that includes APEX software for visualization and analysis of scans. Incorporates FRAX reference data developed by WHO to provide both t- and z- scores.</td></tr><tr><td>APEX 3.2</td><td>Hologic</td><td>APEX</td><td>Software used by the DXA PC connected to the bone desitometer (QDR Discovery 4500W system) to acquire the BMD data and analyze results.</td></tr></tbody></table>

scanning skeletal remains for bone mineral density in forensic contexts

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. BMD is an important component of bone&#39;s nutritional status and in skeletal remains of both juveniles and adults, and it can provide information about bone quality. For adults remains, it can provide information on pathological conditions or when bone insufficiency may have occurred. In juveniles, it provides a useful metric to elucidate cases of fatal starvation or neglect, which are generally difficult to identify. This paper provides a protocol for the anatomical orientation and analysis of skeletal remains for scanning via dual-energy X-ray absorptiometry (DXA). Three case studies are presented to illustrate when DXA scans can be informative to the forensic practitioner. The first case study presents an individual with observed longitudinal fractures in the weight bearing bones and DXA is used to assess bone insufficiency. BMD is found to be normal suggesting another etiology for the fracture pattern present. The second case study employed DXA to investigate suspected chronic malnutrition. The BMD results are consistent with results from long bone lengths and suggest the juvenile had suffered from chronic malnutrition. The final case study provides an example where fatal starvation in a fourteen-month infant is suspected, which supports autopsy findings of fatal starvation. DXA scans showed low bone mineral density for chronological age and is substantiated by traditional assessments of infant health. However, when dealing with skeletal remains taphonomic alterations should be considered before applying this method.

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Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts | Medicine | Video

Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts

Bone mineral density (BMD) is an important factor in understanding nutritional intake. For human skeletal remains, it is a useful metric to assess quality of life in both juveniles and adults, particularly in fatal starvation and neglect cases. This paper provides guidelines for scanning human skeletal remains for forensic purposes.

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. ...

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SCIENTISTS IN ACTION

17.5K Views.  North Carolina State University. Botmineraaldichtheid (BMD) is een belangrijke factor in het begrip nutritional inname. Voor menselijk skelet blijft is het een nuttig metriek te beoordelen van de kwaliteit van leven in zowel jeugdigen en volwassenen, met name in fatale gevallen van uithongering en verwaarlozing. Dit witboek biedt richtlijnen voor het scannen van menselijke skelet resten voor forensische doeleinden.

Video: Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains

The methods presented here seek to maximize the chances for the recovery of human DNA from ancient archaeological remains while limiting input sample material. This was done by targeting anatomical sampling locations previously determined to yield the highest amounts of ancient DNA (aDNA) in a comparative analysis of DNA recovery across the skeleton. Prior research has suggested that these protocols maximize the chances for the successful recovery of ancient human and pathogen DNA from archaeological remains. DNA yields were previously assessed by Parker et al. 2020 in a broad survey of aDNA preservation across multiple skeletal elements from 11 individuals recovered from the medieval (radiocarbon dated to a period of circa (ca.) 1040-1400 CE, calibrated 2-sigma range) graveyard at Krakauer Berg, an abandoned medieval settlement near Pei&#223;en Germany. These eight sampling spots, which span five skeletal elements (pars petrosa, permanent molars, thoracic vertebra, distal phalanx, and talus) successfully yielded high-quality ancient human DNA, where yields were significantly greater than the overall average across all elements and individuals. Yields were adequate for use in most common downstream population genetic analyses. Our results support the preferential use of these anatomical sampling locations for most studies involving the analyses of ancient human DNA from archaeological remains. Implementation of these methods will help to minimize the destruction of precious archaeological specimens.

The protocol presents a series of best practice protocols for the collection of bone powder from eight recommended anatomical sampling locations (specific locations on a given skeletal element) across five different skeletal elements from medieval individuals (radiocarbon dated to a period of ca. 1040-1400 CE, calibrated 2-sigma range).

The methods presented here seek to maximize the chances for the recovery of human DNA from ancient archaeological remains while limiting input sample ...

Genetics

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

We present a robust protocol on how to carefully preserve and prepare cadaveric femora for fracture testing and quantitative computed tomography imaging. The method provides precise control over input conditions for the purpose of determining relationships between bone mineral density, fracture strength, and defining finite element model geometry and properties.

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength.  Because ex vivo biological tissues are prone to ...

Principles and Protocols of Mechanical Testing for Bone Fracture Susceptibility

Practical Considerations for the Design, Execution, and Interpretation of Studies Involving Whole-Bone Bending Tests of Rodent Bones

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care costs. The ability to understand the mechanisms underlying bone disease and the response to treatment is not only desired, but critical. Mechanical testing of bone serves as a valuable technique for understanding and quantifying a bone&#39;s susceptibility to fracture. While this method appears simple to perform, inappropriate and inaccurate conclusions may be reached if governing assumptions and key steps are disregarded by the user. This has been observed across disciplines as studies continue to be published with misuse of methods and incorrect interpretation of results. This protocol will serve as a primer for the principles associated with mechanical testing along with the application of these techniques-from considerations of sample size through tissue harvesting and storage, to data analysis and interpretation. With this in hand, valuable information regarding a bone&#39;s susceptibility to fracture may be obtained, furthering understanding for both academic research and clinical solutions.

Author Spotlight: Enhancing Accuracy and Reproducibility in Whole Bone Bending Tests 

Mechanical testing of rodent bones is a valuable method for extracting information regarding a bone's susceptibility to fracture. Lacking proper practical understanding, the results may be overinterpreted or lack validity. This protocol will serve as a guide to ensure mechanical tests are performed accurately to provide valid and functional data.

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care ...

Bioengineering

Multimodal Analysis of Bone Formation Using Explant Models

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous ...

Cortical Bone Assessment Using the Ultrasonic BDAT Device

Cortical Bone Assessment Using Ultrasonic Guided Waves: A Reproducibility Study in a Healthy Population

Fragility fractures are still a worldwide health burden in the context of population aging. In particular, the global number of hip fractures is expected to double between 2020 and 2050. Therefore, it is essential to detect patients at risk of fragility fracture at the population scale. The current golden standard is dual X-ray absorptiometry (DXA), providing the areal bone mineral density (aBMD). Ultrasonic devices, usually more portable and cheaper than X-ray devices, represent interesting DXA alternatives as screening tools. However, operator dependency is usually recognized as their main drawback. In this study, the measurement protocol of the bi-directional axial transmission (BDAT) ultrasonic device is presented in detail. The dedicated ultrasonic probe is placed at the one-third distal radius of the non-dominant forearm using conventional coupling gel. The guided interface provides in quasi-real time (about 2 Hz) four parameters of interest: velocities of the first arriving signal (vFAS) and the A0 mode (vA0),&#160;cortical thickness (Ct.Th) and porosity (Ct.Po), as well as four quality parameters. The operator moves the probe slowly at the measurement site, carefully observing the feedback provided by the interface until finding a stable position and starting a series of 10 acquisitions. When at least four consistent series are obtained, the measurement ends, and an automatic report is generated. The measurement usually takes about 5 min to complete. To determine the robustness of this protocol, a reproducibility study was conducted among 3 operators (one expert and two novices) and 14 healthy participants (6 women, 8 men, 21-53 years old). The intraclass correlation coefficients (ICC) were found good for vA0 (0.76), Ct.Po (0.80) or excellent for Ct.Th (0.87) and vFAS (0.91). The standard deviations were found to be less than 10% of the total ranges in clinical practice.

Here, we present the measurement protocol of the bi-directional axial transmission (BDAT) ultrasonic device in detail and test it in a reproducibility study, considering 14 healthy participants and 3 operators. Reliability, measured with the intraclass correlation coefficients (ICC), was good to excellent for four parameters of interest.

Fragility fractures are still a worldwide health burden in the context of population aging. In particular, the global number of hip fractures is expected ...

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

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.

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 ...

Non-invasive Skeletal Muscle Quantification in Small Animals Using Micro-computed Tomography

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the understanding of disease progression and treatment outcomes. Quantifying a live animal's lean, adipose, and skeletal mass is important in metabolic, physiology, pharmacologic, and geroscience studies. However, obtaining accurate body composition measurements, especially of lean mass, remains challenging due to the inherent limitations of conventional assessment techniques. Micro-computed tomography (micro-CT) is a non-invasive radiological technique that enables high-resolution visualization of internal structures in small animal models. A standardized micro-CT method can significantly enhance translational research with more reliable and impactful results, particularly during aging studies or at different time points within the same animal. Despite its potential, the lack of standardization in image acquisition and analysis methods significantly hinders the comparability of results across different studies. Herein, we present a comprehensive and detailed low-cost protocol for lean mass analysis using micro-CT to address these challenges and promote consistency in research involving small animal models.

This protocol uses micro-computed tomography to enable the cost-effective quantification of lean mass, including skeletal muscle and visceral tissue, adipose tissue, and skeletal tissue in small animals. It distinguishes between lean and adipose tissue, which offers significant advantages for biomedical research, particularly in translational research in small animals.

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the ...

Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts

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