Chemical Differentiation of Osseous and Nonosseous Materials Using Scanning Electron Microscopy–Energy-Dispersive X-Ray Spectrometry and Multivariate Statistical Analysis

Identification of osseous materials is generally established on gross anatomical features. However, highly fragmented or taphonomically altered materials may be problematic and may require chemical analysis. This research was designed to assess the use of scanning electron microscopy–energy-dispersive X-ray spectrometry (SEM/EDX), elemental analysis, and multivariate statistical analysis (principal component analysis) for discrimination of osseous and nonosseous materials of similar chemical composition. Sixty samples consisting of osseous (human and nonhuman bone and dental) and non-osseous samples were assessed. After outliers were removed a high overall correct classification of 97.97% was achieved, with 99.86% correct classification for osseous materials. In addition, a blind study was conducted using 20 samples to assess the applicability for using this method to classify unknown materials. All of the blind study samples were correctly classified resulting in 100% correct classification, further demonstrating the efficiency of SEM/EDX and statistical analysis for differentiation of osseous and nonosseous materials.     

Rodents as taphonomic agents: bone gnawing by brown rats and gray squirrels

Passive infrared technology was used to film diurnal and nocturnal scavenging behavior of brown rats and gray squirrels at the University of Tennessee's Anthropological Research Facility. This direct documentation demonstrated that brown rats modified fat-laden cancellous bone while gray squirrels generally gnawed the thicker bone cortices only after fats had leached away. A case study placed in a shaded portion of the Facility indicated the postmortem interval for initial gnawing by gray squirrels was slightly over 30 months. An examination of 53 human skeletons in the William M. Bass Forensic Skeletal Collection revealed that 10 cases had gnaw marks consistent with those made by gray squirrels. One of the 10 cases had been gnawed within 16 months of time-since-death, while the remaining nine had postmortem intervals >30 months. Additional observed modifications made to nonhuman bone by gray squirrels indicate that squirrel gnaw marks on bone can serve as a minimal estimate of time-since-death in a temperate environment similar to that of East Tennessee.     

Getting Ahead: Extraction of DNA from Skeletonized Cranial Material and Teeth

Between 1990 and 2018, the Defense POW/MIA Accounting Agency submitted 2177 cranial elements and 1565 teeth to the Armed Forces Medical Examiner System—Armed Forces DNA Identification Laboratory for DNA testing. In an effort to identify missing United States service members, materials were recovered from wartime losses inclusive of World War II, the Korean War, and Southeast Asia. Using four different DNA extraction protocols, DNA testing was performed using mitochondrial DNA Sanger sequencing, modified AmpFlSTR^® Yfiler?, AmpFlSTR^® MiniFiler?, PowerPlex^® Fusion, or Next Generation Sequencing. This paper aims to provide optimal strategies for the DNA testing of skeletonized cranial materials. Cranial elements produced the most consistent results in Sanger sequencing using an organic purification; however, teeth were most successful for the same platform with an inorganic purification. The inverse is true for STR testing of cranial bones. Of the cranial elements, the temporal provided the most consistent results.     

Differentiating human bone from animal bone: a review of histological methods

This review brings together a complex and extensive literature to address the question of whether it is possible to distinguish human from nonhuman bone using the histological appearance of cortical bone. The mammalian species included are rat, hare, badger, racoon dog, cat, dog, pig, cow, goat, sheep, deer, horse, water buffalo, bear, nonhuman primates, and human and are therefore not exhaustive, but cover those mammals that may contribute to a North American or Eurasian forensic assemblage. The review has demonstrated that differentiation of human from certain nonhuman species is possible, including small mammals exhibiting Haversian bone tissue and large mammals exhibiting plexiform bone tissue. Pig, cow, goat, sheep, horse, and water buffalo exhibit both plexiform and Haversian bone tissue and where only Haversian bone tissue exists in bone fragments, differentiation of these species from humans is not possible. Other primate Haversian bone tissue is also not distinguishable from humans. Where differentiation using Haversian bone tissue is undertaken, both the general microstructural appearance and measurements of histological structures should be applied. Haversian system diameter and Haversian canal diameter are the most optimal and diagnostic measurements to use. Haversian system density may be usefully applied to provide an upper and lower limit for humans.     

丛状骨和骨单位带的种属特征及法医学应用价值

目的 探讨丛状骨和骨单位带的种属特征及其法医学应用价值。方法 人及猪、牛、羊、狗长骨中段横切磨片35张，在光学显微镜下观察骨组织中丛状骨和骨单位带的形态学特征。结果 丛状骨和骨单位带在猪、牛、羊、狗的密质骨中广泛存在，人骨组织中几乎不见；同种动物不同个体之间骨组织学结构也存在差异。结论 观察丛状骨和骨单位带是根据人骨鉴别种属的重要排除指标。     

White‐tailed Deer as a Taphonomic Agent: Photographic Evidence of White‐tailed Deer Gnawing on Human Bone

Ungulate gnawing on bone has been reported in the taphonomic and zooarchaeological literature, but there are no known reports of ungulates altering human remains. Herein, we report on the first known photographic evidence of deer gnawing human remains. As described in nonhuman scavenging literature, forking of the bone characterizes the taphonomic effect of deer gnawing in this case, which is distinct from the effect caused by other scavengers. This type of osteophagia during the winter season is consistent with previously documented behavior of deer gnawing on nonhuman bone, possibly to obtain minerals absent in their diet. In this study, we briefly discuss the distinguishing features of ungulate gnawing, the reasons for this behavior, and possible confusion with other common types of scavenging and modification. This report contributes to taphonomic literature covering the range of animal interactions with human skeletal remains.     

The Use of an Alternate Light Source for Detecting Bones Underwater

When searching underwater crime scenes or disaster scenes for fragmentary human remains, it may be advantageous for forensic divers to be able to detect the presence of bones and teeth among other marine materials (such as shells and rocks). In terrestrial environments, this can typically be accomplished by visual and instrumental methods, but underwater conditions make it difficult to employ detection and sorting techniques in these environments. This study investigates fluorescence of bones and teeth and other marine materials using a submersible alternate light source (ALS) and concludes that an ALS can be a useful tool for detecting bones and teeth in underwater searches as well in terrestrial searches and laboratory environments. The results could impact the methods and equipment used by forensic divers and forensic anthropologists when searching for skeletal remains, potentially increasing the quantity and efficiency of forensic evidence recovered.     

Applicability of Cranial Models in Urethane Resin and Foam as a Substitute for Bone: Are Synthetic Materials Reliable?

As literature is poor in functional synthetic cranial models, in this study, synthetic handmade models of cranial vaults were produced in two different materials (a urethane resin and a self‐hardening foam), from multiple bone specimens (eight original cranial vaults: four human and four swine), in order to test their resemblance to bone structure in behavior, during fracture formation. All the vaults were mechanically tested with a 2‐kg impact weight and filmed with a high‐speed camera. Fracture patterns were homogeneous in all swine vaults and heterogeneous in human vaults, with resin fractures more similar to bone fractures. Mean fracture latency time extrapolated by videos were of 0.75 msec (bone), 1.5 msec (resin), 5.12 msec (foam) for human vaults and of 0.625 msec (bone), 1.87 msec (resin), 3.75 msec (foam) for swine vaults. These data showed that resin models are more similar to bone than foam reproductions, but that synthetic material may behave quite differently from bone as concerns fracture latency times.     

Validation of X-ray fluorescence spectrometry for determining osseous or dental origin of unknown material

Forensic anthropological examinations typically involve the analysis of human skeletal remains, but in cases where samples are very small and/or physically compromised, it may first be necessary to determine whether the material is even osseous or dental in origin. X-ray fluorescence spectrometry (XRF) is a technique that reveals the elemental composition of materials and is hypothesized to have utility in such cases. XRF analysis was conducted on a variety of tissues and materials in unaltered and altered (damaged) states. With few exceptions, osseous and dental tissues in unaltered and altered conditions contained characteristic levels of calcium and phosphorus, while other materials did not. Materials could be accurately identified as osseous or dental in origin based on the calcium and phosphorus levels identified by XRF, and we therefore conclude that XRF analysis is a valid and effective means of determining osseous or dental origin of unknown material.     

The Effects of Household Corrosive Chemicals on Human Dentition*

Abstract: There is a gap in the literature concerning the chemical effects that household products may produce on human remains. The present study examines the effects of household chemical products on teeth. A total of eight chemicals were utilized for this experiment. The corrosive chemical categories include: hydrochloric acid, sulfuric acid, phosphoric acid, and sodium hydroxide. Two products with each chemical were used, each representing varying concentrations of the corrosive product. Two human teeth were allocated for emergence in the chemical throughout a 24‐h period of exposure. Results demonstrate hydrochloric acid as the most detrimental chemical to the dental samples. Sulfuric acid enacted minimal alterations to the teeth, although some etching and discoloration were noticeable. Phosphoric acid resulted in variable changes of the organic and inorganic contents of teeth. Lastly, exposure of sodium hydroxide resulted in little to no change. As hypothesized, distinct effects are observable of each chemical.     

A simple technique for imaging the human skeleton using a flatbed scanner

A simple technique for imaging the human skeleton with a flatbed scanner is presented using the auricular surface of the ilium as an example. A flatbed scanner with resolution capabilities of 600 dpi or greater allows for images of human bones. The auricular surface of the ilium was selected to demonstrate this technique as it is a fairly three-dimensional area that can be difficult to record photographically. Fifty left ilia of various ages at death from the Athens Collection were selected from which three observers (SCF, CE, and IM) scored the morphology of the auricular surface using a well-established aging method. Observations were taken of the dry bone, of digital photographs of the bone, and of scanned images of the bone, and in that sequence. Results indicate that scores of scanned images are equivalent or better than digital images of the same ilia. This technique allows for sharing data electronically with ease.     

The Potential of X‐Ray Diffraction in the Analysis of Burned Remains from Forensic Contexts*

Abstract: In view of the difficulties in extracting quantitative information from burned bone, we suggest a new and accurate method of determining the temperature and duration of burning of human remains in forensic contexts. Application of the powder X‐ray diffraction approach to a sample of human bone and teeth allowed their microstructural behavior, as a function of temperature (200–1000°C) and duration of burning (0, 18, 36, and 60 min), to be predicted. The experimental results from the 57 human bone sections and 12 molar teeth determined that the growth of hydroxylapatite crystallites is a direct and predictable function of the applied temperature, which follows a nonlinear logistic relationship. This will allow the forensic investigator to acquire useful information about the equilibrium temperature brought about by the burning process and to suggest a reasonable duration of fire exposure.     

A method for defleshing human remains using household bleach

Medical examiners and forensic anthropologists are often faced with the difficult task of removing soft tissue from the human skeleton without damaging the bones, teeth and, in some cases, cartilage. While there are a number of acceptable methods that can be used to remove soft tissue including macerating in water, simmering or boiling, soaking in ammonia, removing with scissors, knife, scalpel or stiff brush, and dermestid beetles, each has its drawback in time, safety, or potential to damage bone. This technical report using the chest plate of a stabbing victim presents a safe and effective alternative method for removing soft tissue from human remains, in particular the chest plate, following autopsy, without damaging or separating the ribs, sternum, and costal cartilage. This method can be used to reveal subtle blunt force trauma to bone, slicing and stabbing injuries, and other forms of trauma obscured by overlying soft tissue. Despite the published cautionary notes, when done properly household bleach (3-6% sodium hypochlorite) is a quick, safe, and effective method for examining cartilage and exposing skeletal trauma by removing soft tissue from human skeletal remains.     

Age estimation of Korean adults by occlusal tooth wear

The aim of the present study was to evaluate the validity of the modified Kim's scoring system as a practical method of recording occlusal tooth wear and estimating age. For this purpose, 1092 pairs of maxillary and mandibular full-arch casts were randomly selected, scored, and analyzed. The results showed that the modified Kim's scoring system had excellent reliability, and that occlusal tooth wear had a positive correlation with age. Tooth wear scores of all teeth except the two lower central incisors were higher in males than in females. Calculating tables for age estimation were designed by multiple linear regression analysis. Estimated ages were within +/-5 years of actual ages in 63.5% of male subjects, and 64.0% of female subjects. The accuracy of age estimation was increased when the subjects were divided into two age groups and data were re-analyzed. Collectively, it was shown that the modified Kim's scoring system is a reliable and accurate method for age estimation, and that the data from these 1092 individuals can be used as a standard for age estimation of Korean adults.     

Use of laser-scanning confocal microscopy in the detection of diagenesis in bone

This research demonstrates the value of laser scanning confocal microscopy (LSCM) as a research tool in osteological studies, and diagenetic studies in particular. LSCM combines properties of light and scanning electron microscopy using laser light to excite fluorophores throughout the z-axis, developing a 3-D image. Using differential staining and selecting for specific wavelengths of light, one can image targeted materials. This research is divided into two parts: visualizing bone structures such as proteins and their decompositional products and visualizing diagenesis. Part one of this study utilized pig bones as a means of testing the overall ability of LSCM to fluoresce bone. Twenty-three samples were imaged, including 13 samples from a decompositional study conducted 5 years previous, and 10 “fresh” samples collected from a commercial butcher. This part of the study determined that protein and organic components of the bone could be fluoresced and diagenetic alteration could be imaged. The second part of the study used human samples as a means of imaging and mapping diagenetic alterations. The second part of the study used 13 samples, including 4 clinical, 7 ancient, and 2 modern controls. The pig study used Basic Fuchsin and SlowFade Gold stains, while the human study used toluidine blue. Images were also taken with unstained elements. The results of the non-human study found that a fresh bone fluoresced differently than that of a 5-year subset, while the results of the human study confirmed these findings and determined that the bone diagenesis can be mapped using LSCM.     

Differentiating fragmented human and nonhuman long bone using osteon circularity

Distinguishing between human and nonhuman bone is important in forensic anthropology and archeology when remains are fragmentary and DNA cannot be obtained. Histological examination of bone is affordable and practical in such situations. This study suggests using osteon circularity to distinguish human bone fragments and hypothesizes that osteons will more closely resemble a perfect circle in nonhumans than in humans. Standard histological methods were used, and circularity was determined using an image analysis program, where circularity was controlled for by Haversian canal measurements. Homogeneity was first tested for multiple variables within human and nonhuman samples. No significant differences were found between human sexes (p = 0.657) or among nonhuman species (p = 0.553). Significant differences were found among intraskeletal elements of both humans (p = 0.016) and nonhumans (p = 0.013) and between pooled samples of humans and nonhumans (p < 0.001). Results of this study indicate that osteon circularity can be used to distinguish between fragmented human and nonhuman long bone.     

The Effects of Different Maceration Techniques on Nuclear DNA Amplification Using Human Bone

Abstract: Forensic anthropologists routinely macerate human bone for the purposes of identity and trauma analysis, but the heat and chemical treatments used can destroy genetic evidence. As a follow‐up to a previous study on nuclear DNA recovery that used pig ribs, this study utilizes human skeletal remains treated with various bone maceration techniques for nuclear DNA amplification using the standard Combined DNA Index System (CODIS) markers. DNA was extracted from 18 samples of human lower leg bones subjected to nine chemical and heat maceration techniques. Genotyping was carried out using the AmpF?STR^® COfiler^® and AmpF?STR^® Profiler Plus^® ID kits. Results showed that heat treatments via microwave or Biz/Na₂CO₃ in sub‐boiling water efficiently macerate bone and produce amplifiable nuclear DNA for genetic analysis. Long‐term use of chemicals such as hydrogen peroxide is discouraged as it results in poor bone quality and has deleterious effects on DNA amplification.     

Evaluation of Purkait's triangle method for determining sexual dimorphism

The identification of sex from the skeleton is an important demographic assessment in medicolegal investigations. Rama Purkait developed a method for estimating sex using measurements from a triangle defined by three points on the proximal end of the femur using skeletal material from Bhopal, India. This method was tested with measurements on 200 Indo-European and African American adult femora from the Terry collection using discriminant function analysis to determine if Purkait's method was valuable for determining sex in Americans. A side-by-side analysis was conducted of Purkait's "triangle method" and the maximum diameter of the femoral head to determine their relative value in assessing sexual dimorphism. In the study sample a single variable from Purkait's method provided 85.5% prediction accuracy, similar to 87% for the head diameter. Combining threshold values for a single variable from Purkait's method and the femoral head diameter raised the predictability to greater than 90% for both sexes.     

上下颌牙齿磨耗推断年龄准确性的比较

目的利用牙齿磨耗不同的分类方法分别推断上、下颌的牙齿年龄,分析各种方法推断上、下颌牙齿年龄的误差,评价上、下颌牙齿用于年龄推断的准确性。方法采用分级法与数量化法分别对164名18~46岁之间的成年人进行牙龄推断,计算出推断牙龄与实际年龄的误差,输入SPSS软件对误差的数据进行统计学分析。结果分别使用不同方法对上、下颌牙齿磨耗程度进行年龄推断的结果差别不大。结论上、下颌牙齿用于牙齿磨耗推断年龄的准确性无明显差异。     

Sexual Dimorphism of the Humerus in Contemporary Cretans—A Population‐Specific Study and a Review of the Literature*

Abstract: Sex determination is the first essential step for positive identification when a decomposed body is recovered. Taking into consideration the population aspect of sexual dimorphism of the skeleton, the present study aimed to create a sex identification technique using osteometric standards, derived from a contemporary Cretan population. A total of 168 left humeri were measured according to standard osteometric techniques. The differences between the means in males and females were significant (p < 0.0005). About 92.3% of cases were correctly classified when all measurements were applied jointly. Stepwise procedure produced an accuracy rate of 92.9%. The most effective single dimension was vertical head diameter (89.9%). The current study provides standards for a population that has not been represented so far in the existing databases. It demonstrates that the humerus is an effective bone for the estimation of sex because even in a fragmentary state it can give high classification accuracy.