Discovery and Techniques for Recovering Skeletonised Human Remains: Comprehensive Study Guide

Introduction

• Forensic anthropologists undergo rigorous training in the study of the human skeleton. Their primary objective is to assist in personal identification and reconstruct events prior to death by analyzing both osteological and non-osteological evidence.

• This specialized field requires expertise in multiple sub-fields of physical anthropology, including:

  • Osteology

  • Anthropometry

  • Growth and development

  • Physiology

  • Anthropological genetics

• The knowledge and training of these experts allow them to infer various parameters to solve medical-jurisprudence problems, such as:

  • Sex and Age

  • Ethnicity/Ancestry

  • Living stature

  • Time since death and cause of death

  • Disease conditions

Fundamentals of Human Skeletal System

• Understanding the skeletal system is essential for differentiating human remains from animal remains and for discerning demographic details from partial or debilitated remains.

• Development of Bones: Initially, the human skeleton consists of cartilages and fibrous membranes. These are subsequently replaced by bones.

• Retained Cartilages: Some cartilages are retained in adults and can assist in estimating age and sex. For example, mineralization and ossification patterns in costal cartilages (thoracic region) are predictive of sex and age (Zasshi, 1997; Rejtarová et al., 2004). However, as soft tissue, they are not always available for analysis.

• Bone Properties: Bones are the hardest tissues in the body after tooth enamel.

• Bone Count: At birth, approximately $300$ bones exist, which eventually fuse to form $206$ bones in adults.

• Classification of Bones by Location:

  • Axial Skeleton: Forms the long axis of the body; includes the skull, vertebral column, and rib cage.

  • Appendicular Skeleton: Includes bones of the upper and lower limbs, and the pelvic and pectoral girdles that attach limbs to the axial skeleton.

• Classification of Bones by Shape and Texture:

  • Long Bones: Longer than they are wide. Examples include limb bones (excluding wrist, ankle, and patella) and even the bones of the hands (metacarpals).

  • Short Bones: Cube-shaped. Examples include the wrist and ankle bones. The patella (knee cap) is a specialized short bone known as a sesamoid bone, embedded in tendons.

  • Flat Bones: Thin and slightly curved. Examples include the sternum, scapulae, ribs, and most skull bones.

  • Irregular Bones: Bones that do not fit other categories. Examples include the vertebrae and pelvic bones.

  • Texture Categories: Compact bone and Spongy bone.

Structure of Bones

Gross Anatomy

• Compact Bone: The outer, smooth layer of the bone.

• Spongy Bone: The inner layer, characterized by a honeycomb appearance of needle-like structures called trabeculae. The spaces between trabeculae are filled with yellow or red bone marrow.

• Features of a Typical Long Bone:

  • Diaphysis (Shaft): Tubular structure forming the long axis; composed of compact bone enclosing a marrow cavity.

  • Epiphysis: The expanded ends of the bone, containing spongy bone.

  • Metaphysis: The joining point between the diaphysis and epiphysis. It contains the epiphyseal line, a remnant of the hyaline disc (epiphyseal plate) that grows during childhood.

• Membranes:

  • Periosteum: A double-layered white membrane covering the outer surface of the entire bone except joints.

  • Endosteum: Covers internal bone surfaces (trabeculae of spongy bone and canals within compact bone).

  • Cells: Both membranes contain osteoblasts (bone-forming cells) and osteoclasts (bone-destroying cells).

Microscopic Structure

• Osteon (Haversian System): The basic structural unit of compact bone. These are cylindrical and arranged parallel to the long axis.

• Lamellae: Concentric layers within the osteon.

• Central (Haversian) Canal: Located at the center of the osteon; contains blood vessels and nerve fibers.

• Perforating (Volkmann's) Canal: Lies at right angles to the central canal to connect the blood/nerve supply of the periosteum to the central canals.

• Lacunae: Spaces between adjacent lamellae containing osteocytes.

• Canaliculi: Hair-like branches connecting lacunae to each other and the central canal for nutrient and waste movement.

• Spongy Bone Structure: Less organized than compact bone. It lacks osteons and is composed of irregularly arranged lamellae and osteocytes within tiny spikes of bone tissue called trabeculae.

Chemical Composition of Human Bone

• Organic Components: Includes bone cells (osteoblasts, osteocytes, osteoclasts) and the osteoid matrix (glycoproteins, proteoglycans, and collagen fibers). These provide flexibility and tensile strength.

• Inorganic Components: Constitutes $65\%$ of bone mass. Primarily composed of hydroxyapatite and mineral salts (mainly calcium phosphate). This crystalline structure provides hardness and aids in post-mortem preservation.

• Species Specificity: The combination of organic and inorganic components is species-specific, allowing for taxonomic identification.

Methods of Personal Identification: Animal versus Human Bones

• Identification Prerequisite: Forensic anthropologists must first establish if a material is bone (distinguishing it from wood, pot sheds, plastic, or stone) and then if it is human.

• Gross Morphology:

  • Epiphyseal Line: Used to rule out small animal bones that might be confused with children's bones (children have unfused epiphyseal plates).

  • Bipedalism Indicators: Human bones are generally more gracile with less robust muscle attachments and thinner cortical bone compared to large mammals due to bipedal locomotion (Christensen et al., $2014$).

  • Specific Elements: Birds have light (pneumatic) bones, a furculum (wishbone), and a synsacrum. Prey animals (deer, sheep, horses) have fused radius and ulna for weight-bearing; humans have unfused radius and ulna with narrow diaphyses for a wider range of movement.

• Dentition:

  • Herbivores: Grinding features, large incisors, small/absent canines, flat molars.

  • Carnivores: Shearing/cutting apparatus, conical canines, sharp molars, and a diastema.

  • Humans: Generalized omnivorous design. Canines are small and non-pointed; molars have rounded cusps separated by grooves.

• Histological Analysis:

  • Humans: Uneven distribution of osteons, elliptical osteon shape, thinner cortical layer, and higher porosity.

  • Animals: More compact and dense. Proximal limb cortical thickness is nearly half the total diameter. Osteons are often circular, orderly arranged, and frequently show a "plexiform" (folded-over) arrangement.

  • Metrics: Haversian canal diameters less than $50\,\mu m$ and specific counts of osteons per $1\,mm^2$ help discriminate origin (Lackey, $2000$).

• Molecular Analysis: DNA analysis (nucleotide sequencing) is highly accurate but is rarely used in typical forensic contexts due to cost, time, and its destructive nature.

Number of Individuals

• Commingled Remains: Refers to the mixing of skeletal elements of two or more individuals (Ubelaker, $2002$). Common in mass fatality incidents.

• Minimum Number of Individuals (MNI): Determined by identifying duplicated bones (e.g., two right femurs) or incompatibilities in joint articulation related to age, sex, or size configuration.

Sex Determination

Non-Metric Visual Analysis of the Pelvis

• Considered the "Gold Standard" for sexual discrimination due to female adaptations for childbirth.

• Male Pelvis: Massive, rough texture, vertical/deep Ilium, V-shaped sub-pubic angle (< 90^{\circ}), long/narrow sacrum, oval obturator foramen, narrow and deep sciatic notch.

• Female Pelvis: Light, smooth texture, laterally expanded Ilium, U-shaped sub-pubic angle ($\approx 90^{\circ}$), short/broad sacrum, triangular obturator foramen, broad and shallow sciatic notch.

Non-Metric Visual Analysis of the Skull

• Provides $98\%$ accuracy when used with the pelvis. Traits often become more masculine with age.

• Male Skull: Large and rough, sloping forehead, square orbits with rounded margins, heavy/flaring zygomatic arch, massive mastoid process, U-shaped palate.

• Female Skull: Small and gracile, vertical/rounded forehead, round orbits with sharp margins, lighter zygomatic arch, prominent parietal eminence, small/medium mastoid process, parabolic palate.

Long Bones and Other Elements

• Femur: Displays the highest sexual dimorphism. Critical discriminators include head diameter, total length, and mid-shaft circumference.

• Humerus: More robust in males. Females more frequently show perforation of the olecranon-coronoid septum. The head of the humerus is considered highly reliably dimorphic.

• Sternum: Rule of thumb (not rigorously tested) - a male manubrium is usually less than half the body length; a female manubrium is more than half.

Molecular Analysis of Sex

• PCR (Polymerase Chain Reaction): Amplifies specific DNA sequences.

• SRY Gene: Located only on the Y chromosome; presence indicates a male.

• Amelogenin (AMEL) Gene: Present on both X and Y chromosomes but varies in base pair size.

  • Females ($XX$): Show two identical bands in gel electrophoresis ($AMELX$).

  • Males ($XY$): Show two different bands ($AMELX$ and $AMELY$).

Age Estimation

Morphological Indicators

• Teeth: Process starts before birth. Deciduous teeth ($20$) are followed by permanent teeth ($32$), completing eruption by age $18$ with the 3rd molar.

  • Demirjian Method: Assessing calcification of 7 left permanent mandibular teeth (up to age $16$, modified to $18$).

  • Gustafson's 6 Changes (for adults): Attrition, root transparency, secondary dentin, cementum apposition, root resorption, periodontal ligament retraction.

• Skull Sutures: Suture fusion correlates with age. Major sutures include Sagittal, Coronal, and Lambdoid.

• Post-Cranial Bones: Estimation for those under $25$ relies on epiphyseal closure (growth plates).

• Pelvis (Pubic Symphysis): Todd ($1920$) established phase-wise morphological changes; the surface begins zigzagged and straightens/smooths with age.

• Ribs: The costo-chondral junction (specifically the 4th rib) changes from smooth/rounded to pitted/sharp with age.

Age Intervals and Fusion Timelines

• 0-1 Year: Fusion of posterior, posterior-lateral, and antero-lateral fontanelles; eruption of medial lower and upper incisors.

• 2-4 Years: Fusion of metopic suture.

• 7-8 Years: Rami of ischium and pubis join; sacral vertebrae separated by cartilage.

• 15-16 Years: Triradiate cartilage of acetabulum fuses; olecranon fuses with ulna.

• 18-20 Years: Epiphysis of wrist, knee, and lateral end of clavicle join.

• 21-25 Years: Ischial tuberosity fuses; inner epiphysis of clavicle fuses; sagittal suture begins fusing.

• 35-40 Years: Complete fusion of coronal suture.

• 60-82 Years: Fusion of occipito-mastoid, parieto-mastoid, and spheno-parietal sutures.

Microscopic and Chemical Age Methods

• Histology: Quantitative assessment of osteon density. Known as the Kerley ($1965$) method.

• Telomere Length: Length decreases with cell division (proxy for age), but is individual-specific.

• Amino Acid Racemisation: Conversion of L-amino acids to D-amino acids in proteins (e.g., eye lens, vertebral discs).

• Maillard Reaction (Browning Reaction): Yellowing/browning of tissues due to reactions between amino acids and reducing sugars.

• Radiocarbon Dating ($^{14}C$): Measuring the depletion of atmospheric isotopes in tissues like brain neurons or eye lens crystallines.

Ascertaining of Ancestry

• Challenges: Human variation is often greater within a population than between groups. Mixed ancestry does not fit established categories.

• Morphological Traits Table:

  • Caucasians: Receding frontal profile, circular/sloping orbits, elevated/angled nasal bridge, projecting nasal spine, parabolic dental arcade.

  • Africans: Long/low/narrow vault, rounded frontal profile, square orbits, broad/low nasal bridge, hyperbolic dental arcade with larger teeth, less anterior curvature of femur.

  • Asians/Amerindians: Rounded vaults, vertical frontal profile, rounded orbits, tented nasals, flared zygomatics, horseshoe-shaped dental arcade, shovel-shaped incisors, greatest anterior bowing of femur.

• Metric Analysis: Uses software like FORDISC, CRANID, or 3D ID to compare measurements against known databases.

• Molecular Analysis: Ancestry Informative Markers (AIMs) like SNPs, STRs, VNTRs, and In-Dels. Mitochondrial DNA (mtDNA) tracks maternal lineage, and Y-chromosome markers track paternal lineage.

Stature Estimation

• Mathematical vs. Anatomical: Anatomical methods involve summing the heights of all skeletal elements contributing to height. Mathematical methods use regression equations for single bones.

• Fully’s Anatomical Method: Sum of basion-to-bregma height, total vertebral column height, first sacral segment, femoral length, tibial length, and tarsal height.

  • Formula: $\text{Stature} = 0.98 \times (\text{total skeletal height}) + 14.63 \pm 2.05\,\text{cm}$

• Regression for Humerus: Stature is roughly $5$ times the length of the humerus. If the bone is fragmented, researchers measure specific segments:

  • Segment 1: Proximal head to distal circumference of head.

  • Segment 2: Distal head circumference to proximal margin of olecranon fossa.

  • Segment 3: Olecranon fossa measurement.

  • Segment 4: Distal margin of olecranon fossa to distal trochlea.

Questions & Discussion

1) Illustrate the difference between human and non-human bones.

• Differences exist in morphology, histology, and molecular structure. Humans are more gracile and less dense. For example, birds have a furculum (wishbone), which humans lack. Histologically, humans have elliptical osteons and thinner cortical bone, while animals often show a circular, plexiform pattern. DNA analysis can also provide taxonomic profiling.

2) Differentiate between male and female using non-metric visual analysis of the human skeleton.

• The pelvis and skull provide $98\%$ accuracy. Pelvic features (sub-pubic angle, sciatic notch) vary significantly. Skull features like the shape of the forehead, orbits, and mastoid processes are key markers. Male bones are generally more robust and show more muscular impressions.

3) Explain the molecular method of determining sex of an individual from skeletal remains.

• This involves DNA extraction and PCR amplification. The AMELX/AMELY genes and the SRY gene are targets. Females (XX) show identical bands on gel electrophoresis, while males (XY) show two different bands because of the Y chromosome's specific version of the amelogenin gene (AMELY) and the presence of the SRY gene.

4) List three most common chemical methods of age determination from skeletal remains and describe the principle behind them.

• Amino Acid Racemisation: Based on the shift of L-type amino acids to D-type over time post-protein synthesis.

• Maillard Reaction: A browning reaction between amino acids and reducing sugars indicating time lapse.

• Radiocarbon Method: Based on $^{14}C$ concentrations in tissues matching atmospheric levels until death, then depleting at a known rate.

5) Why is ancestry attribution considered a challenging task for forensic anthropologists?

• Intra-population variation exceeds inter-population variation, making exclusive categories difficult. Biological markers are rare, and mixed ancestry individuals do not fit distinct profiles. Age and sex factors must also be considered first as they influence bone development.

6) Describe the procedure of estimating stature using regression formulae for the upper arm humerus bone.

• First, the complete length of the bone is determined. If fragmented, segments (Head to olecranon fossa, etc.) are measured and converted to a total estimated bone length using regression. This length is then plugged into population-specific regression formulas that account for the individual's sex and ancestry to provide a height range.