Name: scanning skeletal remains for bone mineral density in forensic contexts
Uploaded: 2018-01-29T13:00:00
Description: Watch this Scientific Journal Video about Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts at JoVE.com

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 c...

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...

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 border="0" cellpadding="0" cellspacing="0" width="869">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QDR Discovery 4500W system</td>
			<td style="width:71px;">Hologic</td>
			<td style="width:119px;">Discovery W</td>
			<td style="width:463px;">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 height="21">
			<td height="21" style="height:21px;width:216px;">APEX 3.2</td>
			<td style="width:71px;">Hologic</td>
			<td style="width:119px;">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.

The methodology proposed here is commonly used in living patients and consideration of its novelty to deceased individuals should be noted. Figure 6 and Figure 8 present the results of an AP lumbar spine and left hip scan, respectively. The individual assessed in these scans is a deceased white, female, 31 years of age that is housed at the Forensic Analysis Laboratory of North Carolina State University. This individual had a tot...

Watch this Scientific Journal Video about Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts at JoVE.com

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. ...

The overall goal with this procedure is to provide information about bone quality in forensically relevant remains. This method can help answer pertinent questions for forensic anthropologists such as non-destructive assessments of juvenile fatal starvations and neglect cases. This method can also differentiate between elder abuse and pathologies, as well as distinguishing taphonomic artifacts from pathology or trauma.The main advantage of this technique is that it is reproducible and provides a quantitative assessment that can be substantiated with clinical standards. To begin this procedure, prepare the machine as outlined in the text protocol. Next, create a new patient profile for each new individual to be scanned to maintain chain of custody and to ensure scans are correctly associated.If the individual being scanned is unidentified, establish the biological profile prior to scanning. Enter the demographic information into the patient profile, including the estimated stature if the actual stature is unknown. For anterior-posterior lumbar spine scans, select Perform Scan, choose Patient, select Scan Type, AP Lumbar Spine, and then Next.Then select a container that is at least as large as the articulated segment of L1 through L4.Fill the bottom of the container with rice, which will act as a soft tissue proxy. Place the segment in anatomical position into the rice with approximately 0.7 centimeters between each vertebral body. Make sure that the superior and inferior articular facets are articulated, but that the vertebral bodies are not in contact with one another.Next, center the scanning table. Place the container on the table so that L1 is oriented toward the head of the scanning table and L4 is placed one centimeter superior to the intersecting cross hairs. The vertical laser line should be bisecting the vertebrate bodies of all four vertebrae.After this, cover the exposed bone with rice. Following the scan, an Exit Exam prompt box will appear. Select Analyze Scan.In the Scan Analysis window, select Results. If the vertebrae were not properly separated, select Vertebral Lines to make minor adjustments. Then collect the results graph for visualization of the individual relative to the reference population.To perform a left or right hip scan, first select Perform Exam, choose Patient, select Scan Type, Left Hip or Right Hip, and then Next. Select an open container that is at least as large as the articulated os coxa and femur being scanned. Then fill the bottom of the container with rice.Place the ox coxa into the rice with the acetabulum and obturator foramen facing laterally and the pubic bone oriented medially. Position the ischial tuberosity beneath the femoral head as it articulates with the acetabulum. Next, place the femurs such that the femoral head is in the acetabulum with the greater trochanter and femoral head in line parallel to the scanning table.Make sure that the femoral shaft is medially rotated with the lateral condyle rotated medially and slightly higher than the medial condyle. Then center the scanning table. Move the scanning arm and table until the laser cross hairs are oriented with the center directly above the subtrochanteric area of the femur.The laser's vertical line should bisect the top half of the femoral shaft. After this, cover the remaining visible portion of the femoral acetabular joint with rice. Select Start Scan.An Exit Exam prompt will appear after the scan is completed. Select Analyze Scan. Next, select the Bone Map tool to add or delete areas that are not part of the femoral neck and trochanteric region.Select the Neck tool and reposition the midline to realign adjustments directly on the scan. In the Scan Analysis window, select Results. For adults, compare the scan to re reference data for the femoral neck, trochanteric region, and intertrochanteric region.In this study, skeletal remains are scanned for use in forensic context. Representative scans show that the scanning positions typically used for living individuals for clinical BMD scans can be reproduced with skeletal remains. The first case studies of a 40-year old male who exhibited a rare fracture series.A DXA scan of the left hip is obtained to determine if these fractures were the results of fracture fragility or simply a post-mortem artifact from natural drying processes. The determined total BMD is 1.299 grams per square centimeter with the corresponding T-score of 1.8, indicating that bone insufficiency was not the cause. The second case study is a 13-year old girl with a suspected history of long-term abuse, recovered from a clandestine grave.The total BMD is 0.660 grams per square centimeter with the Z-score of 2.2. The Z-score is consistent with the low BMD for chronological age, supporting other methods suggesting chronic malnutrition. The third and final case study is a 14-month old infant with starvation as the suspected cause of death.The total BMD of the lumbar spine is measured at 0.190 grams per square centimeter, while the age-matched reference group's average is approximately 0.399 grams per square centimeter. The Z-score is calculated to be 5.225 with the relevant age-matched mean. These low values suggest that the infant is below the third percentile of spine BMD for 12-month olds.Once mastered, this technique can be done in one hour if it is performed properly. While attempting this procedure, it's important to remember that anatomical orientation is the primary determinate for a successful scan. After watching this video, you should have a good understanding of how to adapt clinical BMD to forensic cases.

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Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is sequentially characterized by proliferation followed by protein synthesis and ultimately bone matrix secretion. Proliferation and protein synthesis require a constant supply of amino acids. Despite this, very little is known about amino acid consumption in osteoblasts. Here we describe a very sensitive protocol that is designed to measure amino acid consumption using radiolabeled amino acids. This method is optimized to quantify changes in amino acid uptake that are associated with osteoblast proliferation or differentiation, drug or growth factor treatments, or various genetic manipulations. Importantly, this method can be used interchangeably to quantify amino acid consumption in cultured cell lines or primary cells in vitro or in isolated bone shafts ex vivo. Finally, our method can be easily adapted to measure the transport of any of the amino acids as well as glucose and other radiolabeled nutrients.

This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones.

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is ...

Technique for Salivary Gland Ultrasound Examination of Sjogren's Syndrome

Ultrasonographic Evaluation of Salivary Glands for Sjogren's Syndrome: Diagnostic and Monitoring Insights

Sjogren's syndrome (SS) is a chronic autoimmune condition commonly affecting the exocrine glands, causing oral or ocular dryness and extraglandular manifestations including arthralgia, cytopenia, and lymphoma. The presence of autoantibodies against SSA/Ro, labial salivary gland biopsy, ocular staining, Schirmer's test, or salivary flow assessment are included in the current classification criteria of SS. However, the availability and invasiveness of these procedures limit their widespread use in clinical settings. Salivary gland ultrasonography (SGUS) is a non-invasive imaging modality for the evaluation of the salivary gland parenchyma and is increasingly utilized to aid diagnosis and monitoring in SS. 
This article presents the protocol of SGUS for image acquisition at the parotid and submandibular glands. The objective is to present a standardized, reproducible, and practical approach to diagnostic SGUS for SS in daily clinical settings. Major salivary glands are scanned in a stepwise approach, beginning at the angle of the mandible for the superficial lobe of the parotid gland, followed by the deep lobe below the ramus of the mandible. Submandibular areas are then scanned for the submandibular glands. The steps in obtaining salivary gland images at each anatomical site are explained in the accompanying video. The echogenicity and echotexture at the thyroid gland are taken as a reference. The homogeneity, the presence and distribution of hypoechoic areas within the glands, and the border of the salivary glands are examined. The sizes and features of intra-/peri-glandular lymph nodes are recorded. The most distinctive sonographic feature in SS is glandular heterogeneity with the presence of hypoechoic/hyperechoic areas within the glands. 
In summary, while SGUS cannot diagnose SS on its own, it can supplement the current classification criteria of SS and guide the clinical decision for salivary gland biopsy to support the diagnosis of SS in patients with sicca syndrome or suspicious systemic features, combined with autoantibody testing.

Author Spotlight: A Focus on Standardized Salivary Gland Ultrasound Protocol in Connective Tissue Disease Research

Salivary gland ultrasonography is a promising tool for the diagnosis and evaluation of disease activity and complications in Sjogren's syndrome (SS). The scanning technique for salivary gland ultrasound examination relevant to SS is described in the article.

Sjogren's syndrome (SS) is a chronic autoimmune condition commonly affecting the exocrine glands, causing oral or ocular dryness and extraglandular ...

Economical Diatom DNA Extraction Using a Modified Kit

Extraction of Diatom DNA from Water Samples and Tissues

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

Author Spotlight: Diatom Testing for Forensic Drowning Examination 

This article describes a protocol for diatom DNA extraction using a modified common DNA extraction kit.

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. ...

Establishment and Evaluation of a Rat Femoral Box: Cavity Defect Model

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which involves the application of cells, scaffolds, and cytokines, is considered a promising solution for bone regeneration. Consequently, various animal models that simulate bone defects play a crucial role in exploring the therapeutic potential of tissue engineering for bone healing. In this study, we established a box-shaped cortical bone defect model in the mid-femur of rats, which could serve as an ideal model for assessing the function of biomaterials in promoting bone healing. This box-shaped cortical bone defect was drilled using an oral low-speed handpiece and shaped by a lathe needle. Post-operative micro-CT analysis was immediately conducted to confirm the successful establishment of the box-cavity cortical bone defect. The femurs on the operated side of the rats were then harvested at multiple time points post-surgery (0 days, 2 weeks, 4 weeks, and 6 weeks). The healing process of each sample&#39;s defect area was evaluated using micro-CT, hematoxylin and eosin (H&#38;E) staining, and Masson trichrome staining. These results demonstrated a healing pattern consistent with intramembranous ossification, with healing essentially complete by 6 weeks. The categorization of this animal model&#39;s healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

Author Spotlight: The Box-Cavity Cortical Approach for Enhanced Evaluation of Biomaterials and Bone Regeneration

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous osteogenesis.

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...