THE FORENSIC APPLICATION OF COMPARATIVE

MAMMALIAN BONE HISTOLOGY

by

HARALD HORNI, B.A.

A THESIS

IN

ANTHROPOLOGY

Submitted to the Graduate Faculty of Texas Tech University in

Partial Fulfillment of the Requirements for

the Degree of

MASTER OF ARTS

Approved

© 2002 Harald Homi

ACKNOWLEDGEMENTS

This project would not have been completed without the help, support, and

guidance of several people. First, I would like to thank Drs. Robert R. Paine and Richard

A. Nisbett (my thesis committee). Their guidance and editorial comments have been

instrumental to this thesis. I am especially thankful to Dr. Paine for his comments and

suggestions throughout this creative process. Thanks to Drs. Grant D. Hall and Claude B

Lobstein for supplying this research with much needed skeletal material To Dr. Robert P.

Mensforth, aka Dr. Bob, thanks for showing me the anthropological light. During my

undergraduate years I could not have asked for a greater mentor than you (you have

taught me well).

Words cannot describe how thankful I am for the love, patience, and support my

wife, Jo, has shown me during the past three years. Academically, I would not have been

where I am today without her. To my parents, Ame and Ragnhild Homi, I am eternally

grateful for your unconditional love and support. Thanks for letting me chase my dreams.

11

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

LIST OF TABLES

LIST OF FIGURES vi

CHAPTER

v

I INTRODUCTION 1

II BACKGROUND 5

Bone Biology 5 Bone Cells 9 Bone Formation and Repair 12 Bone Microstructure 17

III MATERIALS AND METHODS 21

Materials 21 Preparation of Bone Samples 21 Skeletal Samples 23

Methods 25 Labeling of Bone Samples 25 Preparation of Bone Samples Prior to Cutting 26 Histological Data Collection 27

IV RESULTS 30

Secondary Osteonal Osteocyte Count to Secondary Osteon Size Ratio 30 Average Secondary Osteonal Osteocyte Density 33 Average Secondary Osteon Area 39 Average Osteocyte Density 44 Average Haversian Canal Area 45

111

V DISCUSSION 51

Secondary Osteonal Osteocytes 54 Secondary Osteon Size 55 Osteocyte Density 57 Haversian Canal Size 58 Identification 59

VI CONCLUSIONS 61

AppUcations 63

Future Research 64

BIBLIOGRAPHY 66

APPENDIX: SPECIES SPECIFIC DATA BY QUADRANT, LOCATION, AND BONE 69

IV

LIST OF TABLES

3.1 Species Names 23

3.2 Skeletal Samples 24

4.1 Average Secondary Osteonal Osteocyte Count to

Secondary Osteon Size Ratio 31

4.2 Secondary Osteonal Osteocyte Count by Species at lOOX Magnification 34

4.3 Secondary Osteon Area of the Femur in mm by Species at lOOX Magnification 39

4.4 Secondary Osteon Area of the Humerus in mm by Species at lOOX Magnification 41

4.5 Haversian Canal Area of the Femur in mm by Species at lOOX Magnification 45

4.6 Haversian Canal Area of the Humems in mm^ by Species at lOOX Magnification 46

5.1 Secondary Osteon Size in mm Calculations Based Upon Jowsey's 1966 Study 56

A. 1 Species Specific Osteocyte Density Data By Quadrant, Bone, Location, and Area (2.16mm^) at 1OOX Magnification 70

LIST OF FIGURES

2.1 Microstructure of Cortical Bone 20

3.1 Color-and Quadrant-Coded Cross Section of a Femur 26

4.1 Secondary Osteonal Osteocyte Count to Secondary Osteon Size Ratio 32

4.2 Average Secondary Osteonal Osteocytes by Species at lOOX 37

4.3 Average Secondary Osteonal Osteocytes in the Humerus and

Femur by Species at lOOX 38

4.4 Combined Secondary Osteon Area by Species at lOOX 40

4.5 Average Secondary Osteon Area in mm of the Femur by Species at 1 OOX 42

4.6 Average Secondary Osteon Area in mm of the Humems by Species at 1 OOX 43

4.7 Average Haversian Canal Area in mm of the Femur by Species at 1 OOX 47

4.8 Average Haversian Canal Area in mm of the Humems

by Species at 1 OOX 48

4.9 Average Haversian Canal Area in mm by Species at lOOX 49

A. 1 Average Osteocyte Count by Species at lOOX 74

VI

CHAPTER I

INTRODUCTION

Bone examined histologically can offer us useful information, which can aid us in

the identification of human skeletal remains discovered in a forensic context. Despite this

analytical potential most forensic anthropological research is conducted at the gross

anatomical level. The purpose of this thesis is to microscopically examine and compare

histological features found m non-human mammals to the features found in human bone.

The goal is to develop a method that would be able to distinguish between human and

non-human bone at the microscopic level, with special emphasis on the identification of

fragmentary pieces of cortical bone specific to the forensic context and to archaeological

remains.

When fragmentary pieces of bone are found at a scene, law enforcement agencies

could benefit from a method that would enable them to easily distinguish between human

and non-human bone. This distinction is cmcial; a positive identification for human

skeletal remains may mean a homicide investigation is required. Owsley et al. (1985) did

develop a method to histologically distinguish between fragmentary human and deer

bone. However, a more accurate method to distinguish human from non-human bone is

needed.

This research is an attempt to broaden the spectmm of applicable methods

forensic anthropologists can use in their identification process of human remains In

circumstances where few and fragmentary pieces of bone, with similar gross morphology

to those of human bones, are found, the identification process should be aided by the

results generated by this research.

Depending on the location of the bone, histological features differ within an

individual (Pfeififer, 1998). The Haversian systems are, on average, larger in the femur

compared to those m the rib for example. What happens when skeletal remains of

unknown origin are presented for histological analysis? To date, histological techniques

are not available or widely used. Clearly a method for solving whether or not the sample

is human bone or not must be developed, a method that does not require a specific bone

or a specific location in order to make an assessment of its origin. My thesis research

will deal with both parts of this problem, is it possible to determine the origin (human vs.

non-human) if only fragmentary pieces of bone are available for analysis?

Previous histological research performed on mammalian bone has primarily

focused on the Haversian system itself (Burr et al, 1990; Enlow, 1962: Enlow and Brown

1956, 1957, 1958; Frost, 1987; Georgia et al, 1982; Harsanyi, 1993; Jowsey, 1966;

Mulhera and Ubelaker, 2001; Pfeiffer, 1998). Features within Haversian systems need to

be studied as well including the secondary osteon size. Haversian canal size, and the

lacunae/osteocyte numbers. These features could provide important information in the

taxa identification process.

The lacunae are important vascular portions in bone, as they house the osteocytes

These are osteoblasts, the bone forming cells, which have become trapped in bone matrix

(Cormack, 2001: 179). I am hypothesizing that Haversian systems with an increased

number of lacunae will indicate the prior capability of producing bone more rapidly than

bone with Haversian systems with fewer lacunae. In addition, bone turnover, or

remodeling, would be faster where more osteogenic cells are available to secrete bone.

Furthermore, I also hypothesize that human and non-human species differ in their lacunae

makeup because of differential stressors (i.e., dietary, environmental, and biomechanical)

exerted on the bones. Lacunae makeup may also be due to phylogenetic relationships

between taxa, and among them. Is it possible to distinguish these histological features at

the taxa level? If they are distinguishable, as I hypothesize, they may provide a new

method of proving the presence of human skeletal remains at crime scene locations. The

field of forensic science (including forensic anthropology) needs a method that is able to

distinguish faunal remains from human remains when only fragmentary remains are

present. If some simple test can determine human from animal bone by microscopy,

forensic scientists will have another valuable tool on their side in their quest for positive

identification. Forensic anthropologists can then subject bone fragments of any location,

and taxa that is given to them to this test to determine, with higher level of confidence

than currently available methods can provide (Harsanyi, 1993; Jowsey, 1966; Mulhern

and Ubelaker, 2001; Owsley et al., 1985), if the bone fragment is human or not.

Potentially, this can mean the difference between a medico-legal investigation or a

natural deposition of animal remains.

Human variation is vast and this is also the case with regards to histological

features of bone (Pfeififer, 1998). Haversian systems vary in size, shape, and density, and

it is here where the potential problems lie: intra versus inter species/taxa variation To

overcome these problems, data on bone histology must be collected, analyzed, and

compared to previous findings. Most anthropological histology research on bone has

dealt with the Haversian system, or the secondary osteon, and some it its characteristics,

size and shape mainly (Burr et al, 1990; Pfeififer, 1998). However, there are other

histological features that must be investigated in the future. These features include

fragmentary osteon numbers, the density of lacunae within the osteons, and the size of

osteonal lamellae to name a few. In the past, these features have more or less been

overlooked. My research will mainly deal with the lacunae (which contain the osteocytes)

that are embedded in the bone matrix both vdthin and outside of the secondary osteon.

The reasons for conducting forensic focused histological research on human and

non-human bone are plentiful. Osseous tissue has yet to be fully explored. The

knowledge we do possess is considerable, and we do have reasonable understanding of

the form, structure, and function of bone. However, our goal should always be to

augment our knowledge with new research, and thus broaden our understanding of not

only skeletal tissue but ourselves as well. To the physical/forensic anthropologist, the

microscopic study of bone ought to be of interest as both anthropological and biological

answers still He in the microscopic anatomy of skeletal tissue. The following research is

an attempt to broaden anthropological horizons on the issue of human skeletal

identification.

CHAPTER II

BACKGROUND

The purpose of this part of the thesis is to review basic information about the

histological features of mammalian bone, as well as to review the taxa comparative

histological research, and how this work pertains to the forensic anthropologist.

Histological research of prehistoric human populations has been performed (Burr et al,

1990: Stout, 1978; Stout and Teifiebaum, 1976); however, the focus of this thesis is on

bone of modem human populations and mammalian species.

Bone Biology

Osseous tissue is a cormective tissue. There are organic and inorganic components

to bone; collagen fibers are the predominant part of the organic component while the

inorganic part is primarily composed of hydroxyapatite (Cormack, 2001: 182). The

hydroxyapatite is responsible for the rigidity and hardness in bone and constitutes

roughly two-thirds of the bone's weight. The strength and elasticity is due to the collagen

fibers (Martin et al, 1998:39).

Cancellous (spongy) and cortical (compact) bone are two types of bone that can

be distinguished macroscopically. Cancellous bone consists of trabeculae, which unite

with other trabeculae, and form a meshwork, which gives it its spongy appearance

(Martin et al, 1998: 32). Bone marrow fills the spaces between the trabeculae. The

compact bone seems to be a solid matter, except it actually exhibits microscopically small

spaces (i.e., canahcuH, Haversian canals, Volkmann's canals, and Howship lacunae). The

cancellous and cortical osseous tissues differ merely in the number and size of the spaces

and the quantity of sohd matter. The make-up is the same in both types, however, blood

vessels usually do not traverse cancellous bone (Krause, 1996; 68). In every bone, with

few exceptions, both cancellous and cortical bone is present. However, the distribution

and quantity of each may vary considerably. The diaphysis, or the shaft, of a typical long

bone is primarily comprised of cortical bone. The shaft surrounds the medullary cavity.

The epiphysis, the proximal and distal ends, is for the most part cancellous bone, only

covered by a shell of cortical bone. In flat bones, such as bones of the cranial vault, the

cancellous bone, or diploe, is enclosed by two plats of cortical bone (Ham and Cormack,

1979: 387). The irregular bones (e.g., vertebrae) are primarily cancellous bones covered

in a sheath of cortical bone.

There are two different types of bone that can be distinguished microscopically,

lamellar and woven bone. Lamellar bone is highly organized consisting of parallel

lamellae, or layers, and is formed slowly. Woven bone appears to have random and

poorly organized tissue (Martin et al., 1998: 35-36). The production of woven bone is

quicker than what is seen in the lamellar bone and because of this woven bone is weaker

than lamellar bone. However, "woven bone may become more highly mineralized than

lamellar bone, which, mechanically speaking, may help compensate for its lack of

organization" (Martin et al, 1998: 36).

Lamellar, compact bone, can fiirthermore be divided into primary and secondary

bone. Primary bone is produced by bone being laid down onto a pre-existing bone surface

during growth, such as the periosteal surface. Secondary bone, on the other hand is the

immediate replacement of new bone where previous bone has been resorbed (Martin et

al, 1998: 37-38). Primary bone can also be divided into two types: circumferential

lamellar (primary lamellar) and plexiform bone. Primary lamellar bone is laid down

parallel to the bone surface. Blood vessels are integrated into the lamellae, and each

vessel in surrounded by circular lamellae. These circular lamellae form the primary

osteon, and in its center is the primary Haversian canal (Martin et al., 1998: 37).

Plexiform bone is a combination of bone woven and lamellar bone. This type of bone is

produced by continually creating trabecular networks (woven bone) on bone surfaces

where lamellar bone fills the gaps between the trabeculae. Plexiform bone has the

appearance of stacked bricks; this is caused by the rectilinear vascular spaces between

each layer of bone (Martin et al, 1998: 38). In addition, plexiform bone is more

mineralized then woven bone and is structurally stronger than then both primary and

secondary bone. Its growth is faster than what is seen in primary and secondary bone, and

is primarily seen in fast growing herbivores such as deer and horses.

Where bone is being resorbed, secondary bone is formed. In both cortical and

cancellous bone, bone is resorbed continually in order for the bones to remodel

Cylindrical features known as Haversian systems or secondary osteons make up

secondary bone (Martin et al, 1998: 38). The secondary osteons are bound at the

periphery by a cement line, as it acts as a border to the surrounding bone. At the center of

the secondary osteon is the Haversian canal Even though cancellous bone is also

considered secondary bone, it does not frequently produce secondary osteons because

they rarely fit inside individual trabeculae (Martin et al, 1998: 38).

Periosteum is a fibrous membrane, which covers the outer surface of bone, except

in the articulating surfaces. The periosteum is a membrane of specialized connective

tissue, and this membrane nourishes bone, as it is highly vascularized. It also anchors

muscles to bone. There are two layers of periosteum; a dense outer layer, which contains

blood vessels, and an inner layer, the osteogenic layer, which consists of a more loose

connective tissue (Ham and Cormack, 1979: 389). It is the collagenous fibers of this layer

that enter the bone as Sharpey's fibers. The osteogenic cells of this inner layer become

active upon stimulation (i.e., by fracture). Nearly all bone formation occurring during

development and growth is due to the osteoprogenitor cells in the osteogenic layer

(Cormack, 2001; 185). The periosteum is said to be resting when neither resorption nor

appositional growth is occurring. Similarly, the marrow cavities. Haversian canals,

Volkmann's canals, and the canaliculi are lined by the endosteum, a similar membrane as

the periosteum. Both membranes are said to be osteogenic, or able to produce bone

(Martin et al, 1998:64-65).

The endosteal membrane lines cancellous bone, the Haversian canal, and the

marrow cavities (Ham and Cormack, 1979: 389). It has both osteogenic and hemopoietic

properties. The osteogenic cells in the Haversian canals aid in the formation of new

Haversian systems where old ones have been resorbed. Osteoclasts, which resorb bone

matrix, are found in the endosteum. The body's need for calcium may also trigger an

osteoclastic resorption from the inner bone surface ((jartner and Hiatt, 2001: 152). This

leads to bone loss, or an increase in the diameter of the medullary cavity.

Bone consists of an inter-cellular calcified material, often referred to as bone

matrix. This matrix is organized into lamellae, layers, which can have varying

arrangements. The areas between different lamellae, or mterstitial substance, have

lacunae (cavities enclosed in bone matrix) in which osteocytes are located. Canaliculi

extend form each lacuna, and these narrow channels coimect different lacunae together

and enter neighboring lamellae to unite with adjacent lacunae. This results in the

intercoimecting of lacunae with microscopic systems of channels (Cormack, 2001: 180).

Bone Cells

Four types of bone cells are recognized, osteoprogenitor cells, osteoblastic cells,

osteocytes, and osteoclastic cells. Osteoprogenitor cells are derived from mesenchyme

(Cormack, 2001: 124), and consist of stem cells with mitotic potential and the ability to

transform into mature bone cells. Commonly, they are located in the inner portion of the

periosteum, in the endosteum, and in the vascular canals of cortical bone. There are two

forms of osteoprogenitor cells; the pre-osteoblast, and the pre-osteoclast. Stem cells in the

mesenchyme form the osteoblasts (Martin et al, 1998: 101), while blood monocytes give

rise to the osteoclasts (Cormack, 2001: 185).

Osteoblasts are responsible for osteogenesis, or bone formation. They produce,

package, and transport organic constituents of bone matrix Bone marrow, endosteum,

periosteum, and the periodontal membrane are able to give rise to osteoblasts (Kessel,

1998: 147). The osteoblasts line bone surfaces, and they appear elongated when they are

active. When bone matrix formation is initiated, the matrix is secreted onto pre-existing

bone. The osteoblasts then become encased within their own matrix, and become

inactive. The inactive osteoblast is smaller and more oval in shape than the active one.

When the osteoblasts become trapped and encased by the bone matrix, they are called

osteocytes (Martin et al, 1998: 47).

Osteocytes reside in lacunae; these are the small cavities within the interstitial

substance (bone matrix). The canalicuU, which extend throughout the bone matrix, form a

canalicular circulatory system providing the osteocyte with nourishment (Cormack, 2001

180). The canalicuH extending from one osteocyte touch those of other osteocytes,

communication junctions (or gaps) are formed and ions and small molecules can pass

between osteocytes. An osteocyte cannot survive if it is located more than 0.2 millimeters

from a capillary (Kessel, 1998: 149). To bring blood vessels closer to the osteocyte a

Haversian system may be needed. On the surface of bone there are grooves and ridges,

and blood vessels run along these grooves. When osteogenic cells of the periosteum are

transformed into osteoblasts the ridges eventually fuse together, thus leaving a canal with

blood vessels inside of it; a new Haversian system is formed.

The osteoclasts are large and muhinucleated cells, and are only found in

mineralized tissues. These cells are responsible for bone resorption, and are only present

on bone where resorption is taking place. Howship's lacunae, or resorption bays, host the

osteoclasts and they are the sites where bone erosion takes place (Cormack, 2001 183)

The osteoclasts secrete a variety of products, including lysosomal and non-lysosomal

10

enzymes, and some proteins. In the ruflfled border of the osteoclast, Na^/K^-ATPase is

present. This enzyme acts as a hydrogen exchanger (for sodium and calcium) and thus

regulates the acidity in the region (Kessel, 1998: 150).

The intercellular substance, or bone matrix, has a well-organized stmcture. Its

organic component is mainly osteocollagenous fibers, resembling collagenous fibers of

loose connective tissue (Ham and Cormack, 1979: 396). A cementing substance unites

the fibers consisting of protein-polysaccharides (glycosaminoglycans). Compared to

cartilage the ground substance consists of less sulfated polysaccharides, or chondroitin

sulfates (Ham and Cormack, 1979: 397). Unlike cartilage matrix, bone matrix is thus

acidophil (cartilage matrix is basophil). The inorganic portion, which comprises around

65% of the weight of the bones, is primarily mineral crystals of calcium phosphate

(similar to hydroxyapatite). These minerals deposit in alignment with the collagen fibers,

as dense particles. A special layer of organic cement borders the canaliculi and lacunae.

The absence of fibrils makes this layer different from the rest of the bone matrix. The

lamellar arrangement of the bone matrix is what characterizes it. The thickness of each

layer, lamella, is generally three to seven millimeters (Leeson et al, 1985: 136). The

fibers in each lamella are parallel and take a heUcal course. In neighboring lamella the

pitch of this helical course changes, such that an angle of roughly 90 degrees is formed

between the fibers. It is this arrangement of ahering fiber direction that makes the

lamellae appear so distinct from one another.

The structure of cancellous bone is simple. It consists of trabeculae which form a

network, and the pattern of this network is determined by the mechanical function(s) of

11

the individual bone (Leeson et al, 1985: 136) The trabeculae consist of a number of

lamellae, with lacunae which contain osteocytes, and a canaliculi system. The lamellae

are indistinct in prenatal cancellous bone, as the network of osteocollagenous fibers is

highly irregular. This is also characteristic of woven bone, bone in which the fiber

bundles run irregularly through the matrix, interiacing and crossing without apparent

order or system (Martin et al, 1998: 36), and occurs in aduhs in patches associated with

fracture repair.

Bone Formation and Repair

The lamellae are regularly organized in cortical bone. The blood vessels

nourishing the bone determine the arrangement of the lamellae. Haversian canals pass

through bone in a longitudinal fashion. Volkmaim's canals enter the bone from the

periosteal and endosteal surfaces of the bone, and connect the Haversian systems together

(Cormack, 2001: 194). Thus, this system of canals forms a complex and continuous set of

channels containing the nerves and blood vessels (and sometimes lymph) of the bone

The Haversian system (osteon) consists of a number of concentric lamellae, the bone

matrix and the central canal (Haversian canal). This makes up the stmctural unit of

compact bone. Lacunae of the Haversian system communicate with the Haversian canal

by means of canaliculi (Martin et al., 1998: 47). Generally, canaliculi at the periphery of

the Haversian system do not communicate with adjacent systems. Interstitial lamellae or

partly destroyed Haversian systems fill the spaces between Haversian systems On the

internal and at the peripheral surfaces, lamellae are parallel with the surface, and are

12

referred to as endosteal (inner) and periosteal (outer) circumferential lamellae. Here

canaliculi open freely onto the endosteal and periosteal surfaces. Neighboring Haversian

systems are contained by the cement line, which is a thin layer of modified matrix

Sharpey's fibers are also contained within the lamellae in the periosteal layer of the bone.

The function of these fibers is to secure the periosteum to the bone. Where ligaments and

tendons insert Sharpey's fibers are especially numerous (Cormack, 2001: 203).

Endochondral, intramembranous and appositional growth are the three main types

of bone growth. Ectopic growth may also occur; however, this is abnormal If bone

develops within or directly on a membrane it is intramembranous growth; enchondral

growth occurs within cartilage. Before ossification can occur this cartilage must be

removed.

In the long bones, proper growth in length is dependent upon an orderiy sequence

of events that occur within, and between, the cells of the epiphyseal disk, or growth plate.

Bone must be laid down on some stmcmred surface, either fibrous tissue or cartilage, as

bone cells have no intrinsic capacity to produce three-dimensional form. In case of

endochondral growth, bone is laid down on a cartilage template (Martin et al, 1998: 60).

The growth plate is located between the metaphyses and the epiphyses at the ends of each

growing long bone during subaduh years. When growth is completed in the adult the

growth plate undergoes dissolution and resorption. This resuhs in synostosis (ftision) of

the epiphyses and the metaphyses of the long bones. The epiphyseal line (which may be

visible on x-ray 5 to 10 years after bone growth has ceased) marks where this bony ftision

occurred.

13

Intramembranous ossification takes place in cranial bones and the clavicle. A

highly vascularized area and the presence of mesenchymal cells are required for

intramembranous ossification to take place. Mesenchyme cells, which are typically

branched, or irregular in shape, aggregate and clusters are formed where bone is to be

formed (Kessel, 1998: 167). Pericytes, undifferentiated mesenchyme cells, which can

give rise to dififerentiated cells involved in regeneration and repair, associated with

capillaries provide fibroblasts and osteogenic cells. When the mesenchymal cell division

increases the number of available cells, which can be dififerentiated, some become

osteoprogenitor cells. The osteoprogenitor cells acquire a prominent Golgi complex and

an extensive rER (rough Endoplasmic Reticulum), and these signal the osteoprogenitor

cells to differentiate into osteoblasts, and to synthesize, package and export the bone

matrix (Kessel, 1998: 167).

A spicule is initially formed, as bone matrix is surrounded by osteoblasts, and it

grows in size by appositional growth. The osteoblasts become trapped in the lacunae, and

no longer secrete bone matrix (but do have cellular extensions in the canaliculi),

osteoclasts may appear on the spicule shortly after their formation (indication of eariy

remodeUng). As bone deposition continues, the spicule enlarges, and the trabeculae are

formed. The trabeculae anastomose with other trabeculae, resulting in the spongy

appearance of cancellous bone. In cancellous bone the osteocytes are nourished, by

diflRision, from marrow vessels. Osteoclasts residing on the trabeculae can directly

remodel this type of bone (Leeson et al, 1985: 139). The action of the osteoclasts, and

the continued secretion of bone matrix, resuh in two plates of compact bone, which is

14

separated by the diploe, as seen in the flat bones of the cranium. Cancellous bone can be

converted into compact, cortical, bone by continued secretion by the osteoblasts, however

the development of a Haversian system is required for this conversion.

The diameter of long bones increases by appositional growth. Osteoclasts in the

endosteum resorb bone from the inside, making the cross-section of the bone thinner

However, osteoblasts in the periosteum redeposit bone on the outside of the bone. Thus,

the normal thickness of the cross-section is maintained, but the diameter of the bone has

increased.

When ossification occurs somewhere in the body where it does not belong it is

referred to as ectopic osteogenesis (Ham and Cormack, 1979: 393). However, this is not a

common occurrence. Bone has been observed in kidneys and in the walls of arteries,

though ectopic ossification is not regarded as pathological.

Fractures of bone do occur, usually due to too high stress loads or because of

some pathological condition. A fracture mptures arteries and veins in the periosteum,

endosteum, and in the Haversian canals. A hematoma forms around the site of the

fracture, and the blood vessels are sealed off. At the fracture site, the periosteum is pulled

ofif of the broken ends of the bone, and its osteogenic layer forms a callus. The broken

ends are tied together by fibrous connecting tissue; the callus is mineralized, and becomes

woven bone when the osteoblasts respond. Over time, this woven bone callus becomes

lamellar bone in the process of fracture repair.

Bone can only be remodeled by resorption from and by addition to the surfaces of

bone (Martin et al, 1998: 60), and these processes take place under different

15

circumstances, structural modehng and internal remodeling. Stmctural modeling occurs:

(1) when bones grow in length and width (enchondral and appositional growth), (2) when

a bone gets an increase in work load, which may be due to increased use of that particular

bone, and (3) mainly during ontogenetic development. When aduU size and shape is

reached, molding is no longer required and it ceases.

Internal remodeling is required as Haversian systems do not last throughout an

adult's life-span due to the fact that bone must continually be renewed. Haversian

systems are thus continually bemg resorbed, or at lest parts of them, and new ones being

produced in the channels (Howship's lacunae) made during resorption. The removal of

bone from bony surfaces is due to osteoclastic activity.

As noted by Martin et al. (1998), the weakness in bone can be due to the

mechanical characteristics of the Haversian systems (secondary osteons). Their presence

is essential to the health of bone; however, they are also the Achilles heel of bones.

Haversian systems represent minute holes in the bone, and these holes act as stress

concentrators during both compressive and tensile loads. Thus, fractures will always

propagate from osteon to osteon (Martin et al, 1998: 181). The same problem occurs in

other building materials too, and to engineers it is well known that any hole in any

structural material represents the weakest point of that particular material. Preventative

actions, or precautions, may of course be taken to reduce the loads around these holes.

Should a failure take place, however, it will occur around these stress concentrators Bone

is a good example of how it tries to reduce the risk of failures around the osteons, and this

is a good example of how Wolffs Law is appUed to bone. According to Wolffs Law

16

(Wolfif, 1892), bone will be deposited where high mechanical loads are placed upon the

bone (increase in bone mass), whereas bone will be removed from areas with decreased

loads and less bone is required to manage the loads placed upon the bone in these areas

(Martin et al, 1998: 31). As age increases, there is a corresponding increase in the

number of Haversian systems, thus making aging bone a more likely target for increased

failures (i.e., fractures). In younger bone, however, there are fewer Haversian systems

compared to older bone. In addition, the amount of interstitial and circumferential

lamellae is higher in younger individuals. These non-osteonal lamellae decrease the risk

of failures, and are thus a bony mechanism of reducing stress-related failures. As age

progresses and bone remodeling slows down, the lamellar portion of the bone decreases

and Haversian systems slowly replace the lamellar bone, and the stmctural integrity of

the bone is weakened.

Bone Micro stmcture

The Haversian system (secondary osteon) has a central canal, the Haversian canal,

around which concentric lamellae of bony matrix are located, giving the osteon a

cylindrical shape (Martin et al, 1998: 33). Inside the Haversian canal there are blood

vessels (capillaries and venules), lymphatic vessels, endosteum, and some have nerves

Mature compact bone (in aduhs) is mostly comprised of secondary osteons. Secondary

osteons differ from primary osteons, as they are solely a resuh of remodeling Frost

(1985; 215) defines remodeling as a fimction that " signifies a package or quantized

turnover of hard tissue. In the 10 (Intermediary Organization) mechanisms that provide it.

17

some activating event marshalls osteoclasts, supporting cells, and a capillary in an

organized unit which resorbs a packet of bone. The 'clasts then disappear, and the 'blasts

replace them to form new bone and refill the resorption cavity " In addition, secondary'

osteons are defined at the periphery by a cement Une or the reversal hne (Frost, 1985:

216). Primary osteons do not exhibit this feamre. Primary osteons do have a central canal,

which suppUes blood and other nutrients to the bone, and osteocytes arranged around this

canal. However, since the primary osteon is laid down on pre-existing bone the primary

osteons lack the cement line. The cement line is a resuh of bone remodeling.

Osteons may range from three to five millimeters in length, are approximately 0 3

millimeters in diameter, and are comprised of several lamellae of bone. For increased

strength, collagen fibrils in lamellae adjacent to each other mn in different directions

Haversian canals are interconnected by Volkmann's canal, and these vascular canals

communicate with endosteal and periosteal surfaces (Ham and Cormack, 1979: 437)

Osteoc5^es are present in the lacunae, spaces within the bone matrix itself, and the

canaUculi extend from the osteocytes. These intercoimect the Haversian canal with the

osteoc3^es in the osteonal lamellae. The cement line encircles the Haversian system,

representing areas where bone resorption ceased and the commencement of new bone

formation.

As bone is not a static material, it has to be remodeled throughout life. This

remodeUng process is continues throughout life, and does not cease until death occurs

However, there are histological changes in the stmcture of bone throughout the life cycle

With age these changes manifest themselves in several areas. Evans (1976) reported

changes in the mechanical and physical properties, break area of cortical bone, and a

change in osteon density and overall osteon area (mm )̂ with increased age. Amprino

(1958) showed that the rate of remodeling slows down with age, and that the amount of

primary bone decreases, while Rogers et al. (1952) demonstrated a decrease in the

organic/inorganic ratio. Currey (1964) had comparable results in sunilar smdies.

Furthermore, Currey's findings supplemented the previous resuhs since he did not find

any connection between the osteon size and its lumen (Haversian canal). The Ha\ ersian

canal stays the same size throughout life; rather it is the osteon itself that decreases in size

with age. However, during the formation of Haversian systems, the Haversian canal

varies in size.

19

1. Periosteal Surface 2. Cement Line 3. Secondary Osteon 4. Primary Bone 5. Secondary Bone 6. Primary Osteon

7. Haversian Canal 8. Endosteal Surface (Marrow Cavity) 9. Osteocyte (Osteocyte Lacuna)

10. Haversian Canal 11. Volkmaim's Canal

Figure 2.1 Microstructure of Cortical Bone

20

CHAPTER III

METHODS AND MATERIALS

Materials

Preparation of Bone Samples

Histological preparation of samples utilized standard equipment (Stout and Paine,

1994). This includes an Isomet bone-cutting saw (Buehler, Lake Bluff, IL) equipped with

diamond-tipped saw blades, an Ecomet®3 variable speed polisher/grinder (Buehler, Lake

Bluff, IL), an Olympus BX51 light microscope, sandpaper discs, petrographic slides,

microscopic sUdes, sUp-covers, Permount (Fisher Scientific, Fair Lawn, NJ), xylene

solution, sonic cleaner, hand tally lap counter, pipettes, metal forceps, glass jars, and

notebook.

A Buehler Isomet low speed saw was used to cut the bone sections. The samples

to be cut were mounted onto the saw's lever arm using clamps to secure the bones

themselves. Buehler's diamond-tipped blades were used to cut the bone cross-sections

The five inch blade was used to section human femora, and other large diameter bone,

while the four inch blade was used on smaller diameter sections Matching blade size to

the cross-section of the sample provide better cutting and thinner sections

Thin sections had to be transparent before they provided micro-anatomical data

Though the Isomet bone-saw can cut thin wafers of bone, approximately 125-100

microns thick. The desired reading thickness of 75 microns was preferred for reading

histological features. To get the samples thin enough, they had to be ground down using a

Buehler Ecomet®3 variable speed grinder-polisher with 400- and 600-grit sandpaper discs

(Buehler, Lake Bluff, IL). By holding the bone samples directly down on the sandpaper

surface while the grinder was spinning, the remainder of bone tissue could be removed to

achieve a readable thickness.

Petrographic and microscopic slides were also needed, as the bone samples had to

be mounted onto these before the cross-sections can be either ground down or viewed

through the microscope. The bone samples had to be held in place when ground, so a

strong adhesive is necessary to mount the samples onto these slides. Permount (Fisher

Scientific, Fair Lawn, NJ), a histological mounting solution (a clear dying glue) was used

to adhere the thin sections onto the slides. Petrographic slides were used to mount the

samples before the bones were ground down, while microscopic slides were utilized for

the final mounting. Cover slips were used to protect the bone samples on the microscopic

sHdes once they were mounted.

As the thin sections were prepared for histological reading they were cleaned

using a xylene solution and a sonic cleaner. This final step in the preparation process

provided cleaned sections, which were easy to read.

To view the samples of bone cross-sections, a standard light microscope with the

ability to magnify these samples at least lOOX was needed. This research used a light

microscope. The lOX objectives and lOX oculars were employed during the gathering of

histological data. For area counts, one of the oculars was fitted with a standard counting

reticule. This lens made it possible to calculate area, as it shows a grid pattern in the line

of view. This one in particular, when using lOOX magnification, would square off an area

22

of bone of 2.16 mml This squared off area was used in this research as the limiting

parameters when counting osteons and osteocytes.

A lap counter was used to count osteocytes within the squared-ofif areas, as well

as for counting the osteocytes within some of the osteons. The lap counter was used as an

aid to not lose count. The data collected was recorded in a notebook.

Skeletal Samples

The skeletal remains of seven mammalian species, including armadillo, cat,

coyote, deer, goat, human, and wolf bones, were cut into cross-sections and prepared for

histological study. Non-human mammalian bone samples were obtained from Dr. Cjrant

D. Hall (Texas Tech University), Dr. Robert P. Mensforth (Cleveland State University),

and Dr. Robert R. Paine (Texas Tech University). The skeletal samples used in this

research are summarized in Table 3.2.

Table 3.1 Species Names

Common Name Latin Name Domesticated Cat Coyote Wolf White Tailed Deer Armadillo Domesticated Goat Human

Felis cattus Canis latrans Canis lupus Odocoileus virginianus Dasypus novemcinctus Capra hircus Homo sapiens

23

species Cat Coyote Wolf /Armadillo Deer Goat Human

Number

3 8

Age aduh aduh adult aduh aduh

sub-aduh adult

Table 3.2 Skeletal

Humems R+L

X

R X

R L R

Samples

Ulna R+L

X

X

X

X

X

R

Radius R+L

X

X

X

X

R+L R

Femur R+L L L R+L R

R+L R

Tibia R+L X

X

R+L X

R+L R

R = Right L = Left X = Absent

Permission to harvest human bone samples from the long bones was provided by

Dr. Claude B. Lobstein, coordinator of anatomical services at Texas Tech University

Heahh Sciences Center. The samples were taken from deceased individuals who had

donated their bodies to science. However, only bone-plugs from the mid-shaft of the long

bones were allowed to be removed. Consequently, the entire human thin-sections were

from the mid-shaft. Proximal or distal end sections were not obtained for the human

samples. Eight individuals (three adult females and five adult males) made up the human

sample collection. Each of the bone-plugs was approximately one inch in length Five

bone-plugs were harvested from each individual from each of the long bones, except the

fibulae. Furthermore, permission to remove whole bones was not granted and hence,

samples were only removed from the right long bones of these individuals. From the

human bones, forty thin-sections were prepared for histological viewing.

Histological research was conducted on an adult wolf Dr Robert P Mensforth,

associate professor at Cleveland State University's anthropology department, made the

24

right humerus and the left femur of the wolf available. Six thin-sections were prepared

from the wolf sample; proximal, midshaft, and distal ends from both the femur and the

humems.

The total number of thin-sections prepared for this research was 112. Each of

these thin-sections was microscopically examined as outlined in the methods section.

Methods

Labeling of the Bone Samples

Anatomical orientations of the samples were recorded. This was achieved by

using four different colors of permanent markers; red, green, black, and blue The medial

side of each bone was labeled with red ink, the lateral part green, the anterior portion

black, and the posterior side was colored blue. By color-coding the bones, the chance of

misreading the bones under the microscope was eliminated. It also allowed for each

cross-section of bone to be divided into four different quadrants: I ~ anterior-medial, II -

anterior-lateral. III ~ posterior-medial, and IV ~ posterior-lateral (see Figure 3 1) The

quadrants were then used as different locations for the collection of the data used in this

research. Once the bone samples were cleaned, labeled, and inked, the samples were

ready to be cut into thin cross-sections and mounted onto microscopic slides

Additionally, each non-human mammal specimen was sampled at the proximal,

mid-shaft, and distal ends. This division was used to assure a representative analysis of

the bones than what a single cross-section would provide

25

Lateral

Left

Anterior

Medial

Right

Anterior

Lateral

Posterior Posterior

Figure 3.1 Color- and Quadrant-Coded Cross Section of a Femur

Preparation of Bone Samples Prior to Cutting

Where it was needed, the soft tissue covering the bones was removed In addition,

as some of the samples were of fresh bone, the samples also needed to be boiled Boiling

the bones eliminated potential bone marrow seepage The bones were de-greased, and

decomposition of soft tissue was prevented. The bones were placed in a large vat filled

with approximately seven gallons of water. To the water, two bottle caps of bleach were

added. The bones were then lowered into the vat and were boiled on low heat for eight

hours.

26

Histological Data Collection

In order to collect the necessary data proposed for this research, some basic tools

were needed. First and foremost, a standard light microscope was used, employing the

lOX objectives and the lOX oculars. One of the oculars was also fitted with a counting

reticule, making area calculations possible when looking through the microscope.

Secondly, a Sportsman® officials hand tally lap counter was used in order to make

general counting the numbers of osteocytes across the entire cross-section less diflficuh.

In essence, it prevented restarting the counting if the count was lost as the numbers

increased.

For each of the cross-sectional quadrants, a single random area was chosen to be

read histologically. This was achieved by placing the slides that were to be read under the

microscope, and without looking, an area within the cross-section of the bone was picked

Random areas were chosen because it is difficuh to determine the origin of very small

bone fragments. Collecting data from four randomly sampled areas within each cross-

section and from three levels within each bone (where possible) would result in twelve

areas of data collection, representing a "tme" cross-sectional average of the entire bone.

This method greatly increases the chance that data collected from a fragment of bone

would fall within the numbers collected from the entire bone sample(s).

Once the location was known, an area was chosen by randomly moving the thin

section around the stage of the microscope, though making sure the view never left the

quadrant where the data was to be collected Stopping the slide randomly while moving it

around on the stage, guaranteed a random selection of the area to be histologically read

27

In each area the numbers of primary and secondary osteons were counted. By adding

these two numbers, the total number of Haversian canals was calculated. The numbers of

haversian canals were also used to calculate the density of osteons (primary and

secondary) per unit area. The unit area was calculated using an ocular fitted with a

reticular lens, and at lOOX magnification the area enclosed by the reticule was 2.16 mm

Within the squared-ofif areas, the total number of lacunae (the housing for the

osteocytes) was counted. The total osteocyte number was then divided by the unit area,

giving the total osteocyte density. Furthermore, an average sized secondary osteon was

selected among all the osteons within the square, and its osteocytes were counted. This

average sized secondary osteon was chosen by comparing all the osteons within the

reticule, and one that was of average size (not the smallest and not the largest) was

picked. The secondary osteonal osteocytes (osteocytes within the secondary osteon) were

then counted. The numbers generated were then compared with the results of the humems

and femur secondary osteon size data, to see if the ratio of secondary osteonal osteocytes

per average secondary osteon differed among the species examined.

This procedure was used in the collection of all the data for the different species

the bones came from. Using the same data collection methods for all the species ensured

continuity as well as a reduction of possible inconsistencies that could, in the long mn,

lead to misinterpretation of the data itself The data was recorded in tables for easy access

and for ease of reading the numbers generated.

When the data had been recorded and collected, secondary osteonal osteocyte

density, secondary osteon size, average Haversian canal size, osteocyte density average.

28

and secondary osteon averages per unit area were calculated. These were calculated for

each of the species and compared to each other, and all the animal species were compared

to the human averages and densities. The differences between these numbers were the

basis for the differentiation between human bone and bone from other mammaUan taxa.

The Haversian canal areas and secondary osteon areas were calculated using

Optimas (Media Cybernetics, Silver Springs, MD). This computer program allows the

image that is seen in the microscope to be frozen on the computer screen, and by using

the mouse the outline of the secondary osteon could be traced. Once the cement line has

been traced all around the secondary osteon, Optimas calculated the area within the

traced area. The Haversian canal area measurements were also collected using this

technique. The collection of Haversian canal area and secondary osteon area

measurements was done by taking ten samples from both the humeri and the femora of

all the species. The humeri and the femora were used as most of the species had both of

these bones present.

29

CHAPTER IV

RESULTS

The seven mammahan species studied (cat, coyote, wolf, armadillo, deer, goat,

and human) were all subjected to histological examination. The resuhs from these

observations are presented in this chapter in the form of tables and figures. These include

tables of the raw data for taxa specific secondary osteonal osteocyte to secondary osteon

size ratio (Table 4.1 and Figure 4.1), secondary osteonal osteocyte count (Table 4.2 and

Figures 4.2 and 4.3), secondary osteon area measurements (Tables 4.3 and 4.4 and

Figures 4.4, 4.5, and 4.6), and Haversian canal area measurements (Tables 4.5 and 4.6

and Figures 4.7, 4.8, and 4.9). The appendix includes a table and a figure (Table A. 1 and

Figure A l ) representing the average osteocyte densities for each of the respective

mammalian taxa.

Secondary Osteonal Osteocyte Count to Secondarv Osteon Size Ratio

To provide a clearer assessment of this data, a ratio of the number of osteocytes

per secondary osteon to the average secondary osteon size can be calculated This ratio is

shown in Table 4.1 and in Figure 4.1. Since average secondary osteonal area

measurements were only available for the femur and the humems, the average osteocyte

counts from the humeri and the femora were the only to be calculated for this ratio This

data was taken from Table 4.1. Figure 4.1 cleariy shows the vast differences this ratio is

between the seven species examined during this research. The human average secondary

30

osteonal osteocyte to secondary osteon size ratio is by far the smallest at 984.0. The

average secondary osteonal osteocyte to average secondary osteon size ratio is calculated

in the following maimer:

(humems + femur') average = 1015.4 + 952.6 = 984.0 . number of averages 2

Table 4.1 Average Secondary Osteonal Osteocyte Count to Secondary Osteon Size Ratio

Species Bone Secondary Osteonal Osteocytes/Secondary Osteon Size Ratio Cat

Coyote

Wolf

Humems Femur

Femur

Humems Femur

Armadillo Femur

35.4/0.02700mm^= 1311.1 37.9/0.02109mm^-1797.1

44.2/0.02361mm'= 1802.1

62.0/0.02514mm^ = 2466.2 61.5/0.02787mm^ = 2206.7

35.9/0.02353mm^= 1525.7

Deer

Goat

Human

Humems Femur

Humems Femur

Humems Femur

52.3/0.02058mm^ = 2541.3 41.2/0.02233mm^= 1845.1

67.0/0.02016mm^ = 3323.4 64.5/0.02822mm^ = 2285.6

49.5/0.04875mm^ = 1015.4 SI l/0 05364mm^= 952.6

The human average, 984.0, is 541.7 lower than in the second lowest average of

1525.7, found in the armadillo. The goat has the highest average ratio at 2804 51, and it is

2.84 times that of the human ratio. The human range is cleariy removed from ranges seen

in the other taxa.

31

3400-

3200 -

3000

2800 -

2600 --

2400 -

2200-

CO

o

cyt

o <u <o O

nal

o <u «4-» V)

O

2000-

1800-

1600 -

1400 -

1200 -

(

1

1000 -

fvn -

-H igh

- L o w

• Average

Cat

1797.06

1311.11

1554.09

Coyote

1802.08

1802.08

1802.08

Wolf

2466.19

2206.67

2336.42

Species

Armadillo

1525.71

1525.71

1525.71

Deer

2541.3

1845.05

2193.18

Goat

3323.41

2285.61

2804.51

{

Human

1019.49

952.65

984

The graph represents the range, not the standard deviation Cat N=l, Coyote N=l, Wolf N=l, Armadillo N=l, Deer N=l, Goat N=3, Human N=8

Figure 4.1 Secondary Osteonal Osteocyte Count to Secondary Osteon Size Ratio

32

Average Secondarv Osteonal Osteocvte Density

Table 4.2 represents the average of the secondary osteonal osteocyte density

averages found for each taxa. The secondary osteonal osteocyte averages presented in

Figures 4.2 and 4.3 also shows how variable the osteocyte count is within a secondary

osteon. The combined average of 61.8 osteocytes within the secondary osteon is the

highest (though there are single osteocyte counts that are higher), and was recorded in the

goat. The lowest of the combined averages, 35.9, was recorded in the armadillo. The

human average of 50.3 is the third highest.

Even though these averages are highly variable, one thing must be kept in mind:

the secondary osteon size among the species also varies. This means, an osteon that is

large and has a large amount of osteocytes within it can have the same osteonal osteocyte

density as a small osteon with few osteocytes within it.

33

Table 4.2 Secondary Osteonal Osteocyte Count by Species at I OOX Magnification

Quadrant Species Bone I II III IV Average Cat Humems 48 * 25 * (N=l) 29 35 49 43

31 27 36 13 44 36 41 16 51 34 39 48 35.4

Una 40 38 37 59 46 33 44 27 21 * 36 29 41 29 26 28 33 * 27 28

Radius

Femur

Tibia

52 29 31 41 33 *

42 23 *

40 42 25 24 46 44 29 21

57 *

40 64 29 *

43 *

63 55 33 38 27 38 52 37 45

35 *

54 41 24 31 35 25 42 43 36 *

30 40 22 37 42

59 *

37 31 30 10 *

32 37 42 34 *

57 *

26 35 94

37.7

37.6

37.9

37.6 Combined Humems and Femur Average 36.5

Coyote Femur 44 40 31 41 (N=l) 54 43 48 38

49 45 48 49 44.2 Combined Humems and Femur Average 44 2

Wolf (N=l)

Humems 68 59 74

47 68 70

50 55

37

80 80

56 62.0

34

Table 4.2 continued

I 69 64 57

(

n 35 63 49

Quadrant m 55 71 80

IV 68 70 57

Species Bone I n III IV Average Wolf Femur

61.5 Combined Humems and Femur Average 61.8

Armadillo Femur 47 23 50 * (N=l) * * 34 *

23 * 51 * 29 35 48 * 35.9

Tibia 30 36 48 34 * 42 22 38 42 72 * 47 20 52 32 30 43 * 25 52 39.1

Combined Humems and Femur Average 35.9

Deer Humems * * 58 57 (N=l) 41 53 '̂ * 52.3

Femur 35 * * 47 * 40 * * 3g * * * 412

Combined Humems and Femur Average 46 8

Goat Humems 106 * 26 * 67.0 (N=3) Radius * 56 * 45

* * 39 53 41 56 20 38 * 60 42 54 44.9

Femur * 128 36 * * * 53 * * * 41 * 64.5

Tibia 53 * * 27 * 46 * * 42.0

Combined Humems and Femur Average 65 8

Human Humems 43 53 33 58 (N=8) 46 56 60 41

45 50 38 48 46 49 37 45

" i ^

Table 4.2 continued

Species Human

Bone

Ulna

Radius

Femur

Tibia

I 54 44 56 50 69 70 57 38 56 64 61 76 60 38 69 60 66 42 68 44 52 67 49 43 43 62 43 38 56 55 57 42 33 36 46 57

Quadrant II

50 45 42 59 50 37 59 45 62 55 74 65 32 39 56 56 62 57 53 47 33 30 57 45 70 40 66 57 50 52 52 44 42 39 43 49

rnmhined Humems

III

38 73 47 48 59 61 63 41 63 54 65 77 51 92 61 42 49 41 77 71 47 80 59 53 42 50 66 62 46 60 51 48 30 50 40 64

IV 39 47 64 85 42 47 86 52 38 48 41 49 32 51 58 43 58 39 58 65 61 43 38 37 47 48 42 64 58 56 67 37 34 62 27 48

Averase

49.5

57.0

54.3

51.1

47.5 1 and Femur Average 50.3

* indicates no data

36

70 T

65 -

60 -

55 -

50 -

o

w O 75 45 + c o V) O

40

35 {

Species 30 -

-High

-Low

• Average

Cat

39.6

35.4

37.6

Coyote

54

31

44.2

Wolf

62

61.5

61.8

Armadillo

39.1

35.9

37.5

Deer

52.3

41.2

46.8

Goat

67

42

54.6

Human

57

47.9

52

The graph represents the range, not the standard deviation Cat N=l, Coyote N=l, Wolf N=l, Armadillo N=l, Deer N=l, Goat N=3, Human N=8

Figure 4.2 Average Secondary Osteonal Osteocytes by Species at lOOX

37

70 T

65 {

60 -

55 -

50 -V)

o

"OT

O lo 45 c o o V) O

40 -

35 {

{

?0 -

-High

-Low

• Average

Cat

37.9

35.4

36.7

Coyote

44.2

44.2

44.2

Wolf

62

61.5

61.8

Species

Armadillo

35.9

35.9

35.9

Deer

52.3

41.2

46.8

Goat

67

64.5

65.8

Human

51.1

49.5

50.3

The graph represents the range, not the standard deviation Cat N=l, Coyote N=l, Wolf N=l, Armadillo N=l, Deer N=l, Goat N=3, Human N=8

Figure 4.3 Average Secondary Osteonal Osteocytes in the Humems and Femur by Species at lOOX

38

Average Secondarv Osteon Area

Tables 4.3 and 4.4 show the resuhs of the average secondary osteon area found ir

the femur and humems, respectively. They are separated into the average area

measurements by species of the femur (Figure 4.4) and the humems (Figure 4.5), and the

combined averages by species (Figure 4.6).

Table 4.3 Secondary Osteon Area of the Femur in mm^ by Species at lOOX Magnification

Cat 0.02651 0.01934 0.03132 0.02321 0.01719 0.02778 0.01274 0.02140 0.02211 0.00936

Ave. 0.02110

Covote 0.02599 0.02815 0.01692 0.02644 0.02151 0.02755 0.02591 0.02148 0.01908 0.02310 0.02361

Wolf 0.03485 0.01983 0.02725 0.02510 0.04114 0.03323 0.02256 0.02211 0.02233 0.03033 0.02536

Armadillo 0.02728 0.01557 0.02573 0.01568 0.01009 0.03263 0.03173 0.02154 0.03396 0.02113 0.02353

Deer 0.01803 0.01913 0.02444 0.02485 0.02020 0.01817 0.02361 0.02728 0.02274 0.02489 0.02233

Goat 0.04032 0.02159 0.04656 0.01385 0.02135 0.02080 0.02513 0.03277 0.02234 0.03747 0.02822

Human 0.05120 0.04933 0.06018 0.04972 0.04024 0.06223 0.05161 0.06301 0.04992 0.05899 0.05364

Figure 4.6 shows the distribution of the average secondary osteon area of the

femur in each respective species. The human average, 0.05120 mm , is the highest

average and is well above all the averages of the non-human mammalian taxa, though

some overiap in ranges occurs at its lower values. The overiap is with the high values of

the goat, armadillo, and wolf The goat average, 0.02419 mm^ is 0.00728 mm^ smaller

that the lowest secondary osteon area recorded in the human sample. The lowest average

secondary osteon area, 0.02146 mm^ was recorded in the deer; however, the lowest

secondary osteon area measurement, 0.00936 mm^ was recorded in the cat

39

E E _c (0 0)

0.07 T

0.06 -

0.05 -

0.04 -

0.03

0.02

0.01

1 1 1

0 -

-High

-Low

• Average |

Cat

0.03132

0.00936

0.02405

Coyote

0.02815

0.01692

0.02361

Wolf

0.03492

0.01683

0.02651

Species

Armadillo

0.03396

0.01009

0.02353

Deer

0.03074

0.01514

0.02146

Goat

0.04656

0.01385

0.02419

Human

0.06097

0.03147

0.0512

The graph represents the range, not the standard deviation Cat N=l, Coyote N=l, Wolf N=l, Armadillo N=l, Deer N=l, Goat N=3, Human N=8

Figure 4.4 Combined Secondary Osteon Area by Species at lOOX

40

Table 4.4 Secondary Osteon Area of the Humems in mm^ by Species at lOOX Magnification

Cat 0.02931 0.02138 0.02775 0.02484 0.02626 0.02344 0.02061 0.03024 0.02836 0.01765

Avg. 0.02498

Covote *

*

*

*

*

*

*

*

*

*

*

Wolf 0.02075 0.02341 0.03237 0.02831 0.01683 0.02580 0.02438 0.03492 0.02280 0.02187 0.02514

Armadillo *

*

*

*

*

*

*

*

*

*

*

Deer 0.01931 0.02224 0.02047 0.03074 0.01514 0.01926 0.01936 0.02272 0.01947 0.01705 0.02058

Goat 0.02080 0.02116 0.02114 0.01679 0.01881 0.02716 0.02406 0.01926 0.01506 0.01731 0.02016

Human 0.05676 0.05815 0.05444 0.04233 0.03147 0.05082 0.04236 0.05150 0.06097 0.03871 0.04875

* mdicates no data

Figure 4.5 depicts the average secondary osteon area of the humems of the

species examined. Again, the human average value, 0.04875 mm , is the highest of all the

species averages. The lowest human area, 0.030147 mm , is barely overiapping with the

highest value of the wolf, 0.03492 mm .̂ The difference between the human low value

and the highest wolf value is a mere 0.00345 mm .̂ The lowest average, 0.02016 mm^

and the lowest value, 0.01506 mm^ were found in the goat. This is in stark contrast to the

values found for the goat in the femur, where they were among the high values recorded

In addition, the humems of the coyote and the armadillo were not present, and no values

for their humeri could be recorded. By comparing their respective values from the femur,

it is unlikely they would have been in the human range, and h is probable they would

have been in the lower ranges along with the other species.

41

E E

(0

0.07 -r

0.06

0.05

0.04

0.03 -

0.02

0.01 1

n -u

-High

-Low

• Average

Cat

0.03132

0.00936

0.02109

Coyote

0.02815

0.01692

0.02361

Wolf

0.03485

0.01983

0.02787

Species

Armadillo

0.03396

0.01009

0.02353

Deer

0.02728

0.01803

0.02233

Goat

0.04656

0.01385

0.02822

Human

0.06018

0.04024

0.05364

The graph represents the range, not the standard deviation Cat N=l, Coyote N=l, Wolf N=l, Armadillo N=l, Deer N=l, Goat N=3, Human N=8

Figure 4.5 Average Osteon Area in mm of the Femur by Species at lOOX

42

0.08

0.06

0.04

E E

(0

0.02

Cat Coyote Wolf Species

Armadillo Deer Goat Human

•High

•Low

Average

0.03024

0.01765

0.027

0.03492

0.01683

0.02514

0.03074 0.01514 0.02058

0.02716

0.01506

0.02016

0.06097

0.03147

0.04875

The graph represents the range, not the standard deviation Cat N=l. Coyote N-1, Wolf N=l, Amiadillo N=l, Deer N=l, Goat N=3. Human N=8

Figure 4.6 Average Secondary Osteon Area in mm^ of the Humems by Species at lOOX

43

In Figure 4.6, the femur and humems secondary osteon areas are combined mto

one average and range. Again the human area measuremems are the highest with the

average secondary osteon area of 0.05120 mm^ The closest average value recorded,

0.02651 mm^ is found in the wolf This is 51.7% of the human value, i.e., the human

average is ahnost twice as large as the second largest average secondary osteon area, and

more than twice as large in area compared to the remaining species.

Average Osteocvte Density

The average osteocyte densities are summarized in Table A. 1 and Figure A. 1. All

the bones were combined to show this figure. Quadrant, bone, and location-specific data

for all the species are available in the appendix. Figure A. 1 shows there is considerable

variation in the osteoc3^e counts among the different taxa. The highest average osteocyte

count per 2.16 mm^ is 2178.08, and is found in the wolf The wolf also had the highest

osteocyte count of 2753. The range of average osteocyte counts in the non-human species

is 1359.29 - 2178.08. The human average osteocyte count of 1153.94 osteocytes per 2.16

mm^ is the lowest. However, the cat had the lowest single osteocyte count of 803 This

suggests that humans have, on average, a lower osteocyte count than the other species

examined, even if there is overlap in their ranges.

44

Average Haversian Canal Area

The resuhs of the average Haversian canal area measurements are represented in

Figures 4.7, 4.8, and 4.9. Figure 4.7 presents the average Haversian canal area of the

femur for every species; Figure 4.8 shows the same measurements from the humems,

while the combined average of the femur and humems for aU the respective species is

given in Figure 4.9.

Table 4.5 Haversian Canal Area of the Femur in mm^ by Species at lOOX Magnification

Cat 0.00048 0.00063

0.00070 0.00068

0.00030 0.00052

0.00052 0.00049

0.00045 0.00028

Ave. 0.00051

Covote 0.00044 0.00066 0.00077 0.00074

0.00039 0.00064

0.00072 0.00046

0.00033

0.00033 0.00055

Wolf

0.00188 0.00101

0.00079 0.00097 0.00091 0.00157

0.00056 0.00032 0.00168 0.00116 0.00109

Armadillo 0.00097 0.00204 0.00112 0.00144 0.00106 0.00253 0.00257 0.00112

0.00128 0.00124 0.00151

Deer

0.00102 0.00099 0.00086 0.00121 0.00108 0.00083 0.00107 0.00086 0.00081 0.00105 0.00098

Goat 0.00229 0.00301 0.00366 0.00057 0.00105 0.00227

0.00273 0.00092 0.00082 0.00142 0.00187

Human 0.00169 0.00149

0.00219 0.00214 0.00188 0.00294

0.00102 0.00317 0.00224 0.00355 0.00223

Figure 4.7 shows that the human Haversian canal average of the femur, 0.00225

mm ,̂ is the highest, however, only by a slight margin. The goat average is only 0.00039

mm^ smaller than what is recorded for the human. Furthermore, the full range of the

human Haversian canal area is completely within the range of the goat's range In

addition, the human average is lower than the high value recorded in the armadillo at

0.00257 mm^ There is substantial overiap in the ranges in the area of the Haversian

45

canals in humans and those of the other species. The lowest average, 0.00051 mm^ was

recorded in the cat, as was the low value of 0.00028 mm .̂

tive In Figure 4.8, the average Haversian canal area of the humems in the respecti^

species is presented. In clear contrast to the findings in the femur, there are no overiaps in

ranges between the human and any of the non-human species. Though there are overiaps

in the non-human species. The human average of 0.00226 mm' is the highest average,

while the goat's average of 0.00064 mm' is the smallest. There is no comparable data for

the coyote and the armadillo.

Table 4.6 Haversian Canal Area of the Humems in mm' by Species at lOOX Magnification

Cat Covote Wolf Armadillo Deer Goat Human 0.00076

0.00082 0.00079

0.00052 0.00063

0.00058 0.00072 0.00113 0.00052

0.00061

*

*

*

*

*

*

*

*

*

*

0.00052 0.00078 0.00107 0.00113 0.00062

0.00061 0.00061 0.00073 0.00069

0.00083

*

*

*

*

*

*

*

*

*

*

0.00076 0.00051 0.00104

0.00142 0.00132 0.00132 0.00046 0.00075 0.00064 0.00077

0.00059 0.00078 0.00059

0.00058 0.00068 0.00100 0.00040 0.00066 0.00066 0.00042

0.00306 0.00180 0.00228 0.00241 0.00144 0.00214 0.00217 0.00272 0.00214 0.00245

Ave. 0.00071 0.00076 * 0.00090 0.00064 0.00257

46

0.004 T

E E

CO 9i

0.0035

0.003

0.0025 -

0.002

0.0015

0.001 -

0.0005 - 1 I 1

n u

-High

-Low

• Average

Cat

0.0007

0.00028

0.00051

Coyote

0.00077

0.00033

0.00055

Wolf

0.00188

0.00032

0.00108

Species Armadillo

0.00257

0.00097

0.00154

Deer

0.00121

0.00081

0.00098

Goat

0.00366

0.00057

0.00187

Human

0.00355

0.00102

0.00223

The graph represents the range, not the standard deviation Cat N=l, Coyote N=l, Wolf N=l, Armadillo N=l, Deer N=l, Goat N=3, Human N=8

Figure 4.7 Average Haversian Canal Area in mm' of the Femur by Species at lOOX

47

0.004 T

0.003 -

E E

0.002 -

1 0.001 -

n -

-H igh

- L o w

• Average

Cat

0.00113

0.00052

0.00071

Coyote Wolf

0.00113

0.00052

0.00076

Species Armadillo Deer

0.00142

0.0004

0.00081

Goat

0.001

0.0004

0.00064

Human

0.00306

0.00144

0.00226

The graph represents the range, not the standard deviation Cat N=l, Coyote N=l, Wolf N=I, Armadillo N=l, Deer N=l, Goat N=3, Human N=8

Figure 4.8 Average Haversian Canal Area in mm' of the Humems by Species at lOOX

48

0.004 T

0.0035

0.003

0.0025 -

E E c en

0.002-

0.0015 -

0.001 -

0.0005 - 1 Species

-H igh

- L o w

Cat

0.00113

0.00028

• Average | 0.00061

Coyote

0.00077

0.00033

0.00055

Wolf

0.00188

0.00032

0.00092

Armadillo

0.00257

0.00097

0.00154

Deer

0.00142

0.0004

0.0009

Goat

0.00366

0.0004

0.00126

Human

0.00355

0.00102

0.00255

The graph represents the range, not the standard deviation Cat N=I, Coyote N=l, Wolf N=l, Armadillo N=l, Deer N=l, Goat N=3, Human N=8

Figure 4.9 Average Haversian Canal Area by Species at lOOX

49

The combined Haversian canal area is shown in Figure 4.9. As with the resuhs

from the femur, there are substantial overlaps in the ranges of the Haversian canal area

measurement. However, the averages are not as close as they were between the goat and

the human in the femur. The goat is no longer has the closest average, the armadillo does

(with no available data for the humems). In human the average Haversian canal measures

0.00255 mm', and it is the highest average of all the species, while the smallest average

Haversian canal was 0.00055 mm , and was found in the coyote

The data shows there are differences among all these species at the histological

level, which could be an indication that a separation between human and non-human

remains could be ascertained by microscopy.

50

CHAPTER V

DISCUSSION

The histological study of skeletal remains can offer us substantial information

with regards to the identification of bone fragments. However, this area of anthropology

does not appear to be the topic of interest to many researchers. Admittedly, this may be

due to the low frequency of forensic cases with fragmentary skeletal remains. The

majority of cases include remains that can readily be identified using gross anatomical

methods when analyzing for sex, age, and height. However, by studying the

microanatomy of bone, additional methods can be devised for the use of physical/forensic

anthropologists when they are analyzing skeletal remains. Histological methods have

been developed for age assessing uidividuals (Keriey, 1965), ahhough this is not the

objective of this study.

A forensic case based solely on fragmentary remains, age, sex, and height criteria

are not the first estimations that must be confirmed. Determining whether the fragments

are human is the first priority. In such cases, when the origin of the bones cannot be

identified readily, histological analysis can be used as an effective method for

determining their origins. The goal of this thesis is exactly that, the development of a

method by which human remains can be distinguished from animal remains by

physical/forensic anthropologists. There are only a few basic methods can be utilized for

this purpose (Harsanyi, 1993; Jowsey, 1966; Owsley et al., 1985). Developing more

methods that can be used for determining species origin will decrease inaccuracies in

51

current methods. Hence, we will also be better prepared to identify fragmentary remains

as that of human or non-human origins.

This research began as an attempt to develop a method by which remains can be

distinguished at the histological level. Previous research focusing on the identification of

human remains by means of microscopy has primarily focused on the size of the

secondary osteon, the arrangement of the secondary osteons, as well as the size of the

Haversian canal within the secondary osteon (Harsanyi, 1993; Jowsey, 1966; Mulhern

and Ubelaker, 2001). For comparison purposes, ten secondary osteons measurements

from the midshaft of the femora and humeri of each taxa were taken. A bridge between

this work and that of previous literature (Harsanyi, 1993; Jowsey, 1966) is provided.

As reported by Jowsey (1966) and Harsan)^ (1993), both the secondary osteon

size and Haversian canal size among species vary. This information can be used to

determine whether or not the fragmentary bone is human. However, secondary osteon

size and Haversian canal size overlap among human and non-human mammalian species

even though the averages appear to be different. This fact makes it difficult to make

clear-cut decisions about their taxa origins. Using only two criteria (secondary osteon

size and Haversian canal size) the identification can end up being less accurate. To

supplement these two criteria for human identification, the probability of incorrect

distinction between human versus non-human bone could be reduced. The gap of

misidentification is thus bridged and the accuracy of determining the origins of bone

fragments has increased.

52

I am testing the hypothesis that non-human mammahan taxa will exhibit

differential osteocyte densities than what is seen in human bone. This hypothesis assumes

that the osteoblasts and osteocytes in different taxa should be activated depending on the

stressors (such as, mechanical, environmental and dietary) placed upon their bones.

Species with a relatively high stress levels will likely have a higher bone mmover rate

compared to a species with lower stress levels. This higher bone turnover, or remodeling,

requires bone to be produced more frequently as well as at a more rapid pace. Both the

frequency and speed in which new bone would have to be formed would require

heightened osteoblastic activity.

Bone producing osteoblasts would occur in a higher frequency in taxa that are

exposed to higher stress levels. As the bone is laid down, the osteoblasts become

entrapped in the secreted bone matrix, and their numbers can be recorded as osteocyte

denshies. As the osteocytes represent the osteoblastic activity, their densities should

indicate to us the levels of stressors the bones are subjected to. Higher stress levels

automatically put higher loads on the bones, and according to Wolffs law (Wolff, 1892)

where there are loads put upon the bone, bone will be formed. Conversely, bone will not

be formed where it is not needed. As less bone is needed where less stress is placed upon

the bone, bone formation is not needed as frequently and the osteoblastic activity is not as

active. The resuh in the density of the osteocytes will be different between, higher in a

species with high stress levels while it would be lower in a species with less stressors

forced upon them.

53

The speed in which the secondary osteons are made can also be a contributmg

factor. As reported in this research, the secondary osteons in humans are larger than those

found in non-human taxa. Sigma (a), the time it takes for a secondary osteon (BMU) to

be formed, is approximately three months in humans (Frost, 1986: 73). The fact that

larger osteons take longer time to be formed, or Sigma is higher, than smaller ones should

indicate that osteoblasts within larger osteons do not produce bone matrix quicker than

osteoblasts in a smaller one. However, as it takes less time for smaller secondary osteons

to be formed, larger quantities can be formed within the same time frame compared to

bigger secondary osteons. This results in more secondary osteons per unit area as well as

an increased number of osteocytes (as there are more secondary osteons present, and they

all have osteocytes within) and, in fact, that is exactly what appears to be the case by

analyzmg the data collected for this study.

Secondarv Osteonal Osteocvtes

The secondary osteonal osteocyte density provides very valuable information As

the density of osteocytes depends on the size of the secondary osteon, a small secondary

osteon can have fewer osteocytes than a large secondary osteon but both can have the

same osteonal osteocyte density. To differentiate between the secondary osteon size and

how many osteocytes it contains, the ratio between the number of secondary osteonal

osteocytes and the size of the secondary osteon is calculated. Surprisingly, this ratio

proved to be very different among the respective species used in this smdy The human

values are lower than any other animal by a margin of more than 50% This is indicative

54

of the difference between the human and non-human samples. It could very well be one

of the most effective criterion telling fragmentary human skeletal remains apart from the

non-human remains to come out of this thesis project. This needs to be investigated

fiirther.

Secondarv Osteon Size

The secondary osteons among all the species used during this research differ in

their overall area measurements (see Tables 4.2 and 4.3). The average area of a human

osteon measures 0.05120 mm', while 0.02389 mm' is the combined osteon average area

of non-human species. This is a difference of 0.02731 mm'. Thus, the size variation is

114.3 % of the non-human average or 53.3 % of the total human average.

On average, the human secondary osteons are more than twice the size of osteons

found in non-human species. However, when the ranges of osteon size are taken into

account, osteons overlap in size as well The lowest human secondary osteon area

recorded was 0.03147 mm , and this value is lower than the highest non-human osteon

size. The highest non-human osteon measured 0.04656 mm . The high value for non-

human osteons is, however, lower than the average human osteon size. Furthermore, the

low value for human osteons (0.03147 mm') is well above the average of the non-human

osteon size (0.02389 mm'). In other words, human osteons are on average larger in size

than the largest non-human osteon, and the non-human osteons are on average smaller

than the smallest human osteon. Though this is the trend seen in this research, the ov erlap

in ranges should not be overlooked.

•̂>

By comparing these findings with previous pubHcations fiirther distinctions can

be made. Jowsey (1966) calculated the diameters of secondary osteons. The diameter can

be used to calculate the approximate size of the secondary osteon measured in her study

The secondary osteons are circular is shape (though not completely), and by using the

formula m the approximate size can be calculated. Secondary osteon size based upon

Jowsey's findings are Usted in Table 5.1

Table 5.1 Secondary Osteon Size in mm' Calculations Based Upon Jowsey's 1966 Study

Species High. Low Average Cat Dog Human Cow Rabbit Rat

0.03904 0.02894 0.05851 0.06602 0.01130 0.00581

0.00830 0.01056 0.02349 0.03462 0.00453 0.00264

0.02086 0.01860 0.04906 0.04906 0.00754 0.00407

The only major difference between Jowsey's study and the observations reported

on in this study is the size of the secondary osteons in the cow. The herbivores used in

this research, goat and deer, do not exhibit the same secondary osteon size as seen the

cow. The cow's secondary osteons are much larger, more than double that of the goat's

and deer's average. In comparison to the human averages in this smdy, the cow's average

secondary osteon size is only 0.00214 mm' smaller than what is reported in the humans

Furthermore, the range of the secondary osteon size in the cow extends beyond the range

of the humans.

56

Further testing is needed to differentiate between the cow and the human bone.

However, though no cow bone was available during this smdy, if plexiform bone is

present, as it is in most large and fast-growing herbivores, h can be used as a

distinguishing criterion. In addition, the secondary osteonal osteocyte to secondary osteon

size ratio could also be used to distinguish between the bones of these two taxa. In fijture

research, this ratio must be calculated for the cow in order to make the distinction.

Using only secondary osteon size as a criterion to make the distinction is possible.

First, if a secondary osteon is larger than approximately 0.047 mm it is either cow or

human. A distinction between these two must then be made. Secondly, if a secondary

osteon is smaller than approximately 0.023 mm' the sample is most likely to be of non-

human origin as this is beyond the lowest area measurement seen in the human samples.

Osteocvte Densitv

In this study, the average human osteocyte density is 1153.94/2.16 mm

(534.23/mm') while the combined average osteocyte density of non-human taxa is

1706.74/2.16 mm' (790.16/mm'). The difference between these two averages is 552.8

osteocytes per 2.16 mm' (255.93/mm'), see Table A l in the appendix. This difference is

significant as the difference itself is 47.9 % (almost half) of the total osteocyte density

found in humans by the same area measurement. In addition, this difference is also

approximately 300 osteocytes less than the lowest osteocyte count observed in humans It

is important to note, however, that the ranges of both human and non-human osteocMe

denshies overiap, and the low-to-high numbers of human osteocytes are within all the

57

non-human ranges. Generally speaking, the human average osteocyte density is lower

than any of the averages seen in the non-human mammaUan taxa.

The armadillo average (1359.29) comes closest to the human average (1153.94),

but is still higher by a margin of 205.35 osteocytes per 2.16 mm' (95.07/mm'), see Table

A.I. This margin of 205.35 osteocytes is 17.8 % of the total human average; it is, in

essence, one fifth of the total human osteocyte count. This gap is certainly an indication

of the differences between the two species. On the other end of the continuum is the

highest osteocyte density. In the wolf (Table Al ) , the average osteocyte count is 2178.08

per 2.16 mm' (1008.37/mm'). The difference between wolf and human averages is a

staggering 1014.14 osteocytes per 2.16 mm' (474.14/mm'). This difference is 87.9 % of

the total osteocyte density observed m humans, and is almost twice as high as the human

average. These are substantial differences that cannot be overiooked

Haversian Canal Size

The Haversian canal size is yet another feature that can be measured to determine

taxa origin. The average human Haversian canal size measured 0.00225 mm while

0.00096 mm' is the average Haversian canal size of non-human taxa (see Tables 4.4 and

4.5). This is a difference of 0.00129 mm'. The variation in Haversian canal size is ever

larger than what is seen in the osteon size. 0.00129 mm' is 57.3 % of the total human

average, and 0.00225 mm' (human) is 234.4 % larger than 0.00096 mm' (non-human)

As it was seen in the osteon size, the human Haversian canals are more than twice the

size of those found in non-human species.

58

However, the human range (0.00102 - 0.00355 mm') is entirely within the non-

human range (0.00028 - 0.00366 mm'). The highest Haversian canal value was recorded

in the goat, and the lowest value from the cat. All the non-human species were of similar

size with regards to their low Haversian canal sizes (except the armadillo, which was

closer to the human low value). With the exception of the small differences in their low

values, all the non-human samples also had their ranges within the range of the high and

low values seen in the goat.

Though the ranges overiap, the averages do not. All the non-human species have

average Haversian canal sizes well below the human average. The combined average for

the non-human species (0.00096 mm') is less than half the size of the average seen in

human Haversian canals (0.00225 mm'), and the closest non-human average (0.00154

mm ) was recorded in the armadillo, not the goat. Hence, the law of averages is still

applicable in this sample.

Identification

When the identification process is impeded by few and fragmentary bones,

histological examination and analysis is a useful tool. Described above are methods that

can be used during this identification process. However, by themselves, neither method

can be used exclusively to determine the origins of the fragments. The reason why either

method cannot be used is simple: the overiaps in ranges. With small test samples the

fragmentary bone may not correctly represent the whole sample, and several techniques

59

should be used to increase accuracy. The accuracy will increase if aU the methods are

utilized together.

If all three methods reflect values that are in the non-human range, the hkelihood

of the samples being human is reduced, and in all likelihood the sample has a non-human

origin. Conversely, if all three indicate averages in the human range, the most probable

answer is that the sample is human. This is the goal of this research, to improve the

accuracy of identifying human remains and, by introducing this method of calculating the

secondary osteonal osteocyte to secondary osteon size ratio, as well as the secondary

osteon size differences, the accuracy of the identification does increase

Though the accuracy has been demonstrated to increase by adding the secondary

osteonal osteocyte to secondary osteon size ratio as a criterion, this research is not

conclusive, and can only suggest the trends presented. The sample sizes in this research

were small, most species were only represented by one individual, and any attempt to

statistically analyze the result would have been inaccurate. That is not to say the results

found are invalid, but they suggest further testing is necessary to statistically validate the

resuhs. However, this research has been able to provide evidence that human and non-

human bone are different at the microscopic level, and using several methods to

distinguish the origin of fragmentary bone increases the accuracy of such an assessment,

and that the trends suggest human bone can be distinguished from those of non-human

origin by means of microscopy.

60

CHAPTER VI

CONCLUSIONS

The sample size used for this project was small. As a resuh, the findings of this

thesis are suggestive and indicative of the difference among mammalian species at the

histological level. The resuhs do indicate a strong link between the secondary osteonal

osteocyte count/secondary osteon size ratio to their species origin. Currently, no one else

has developed this identification criterion and there is no evidence in the literature to

suggest otherwise. There are, however, other methods that have been developed to

identify human and non-human remains at the histological level (Harsanyi, 1993, Jowsey,

1966; Owsley et al., 1985). The results offered by this thesis research should be strong

evidence for the need of fiirther research within this area of histology.

In order to make further distinctions between the histological make-up of

mammalian species (as well as species belonging to other taxa), the need for larger

sample sizes is required. The data collected on larger samples will corroborate on the

findings generated during this research, and the increased sample size will also

statistically vaUdate the resuhs that are generated in such an experiment.

The species examined during this study all exhibited at least two different types of

bone at the microscopic level, primary lamellar and secondary bone The primary

lamellar bone contains primary osteons, while secondary bone has the easily

distinguishable secondary osteons. However, in two species, deer and goat, a third type of

bone also appeared: plexiform bone. This type of bone has a very distinct appearance.

61

and is reminiscent of layers or stacks of bricks, and no secondary osteons appear within

this type of bone. In both the deer and goat, the plexiform bone was distributed

throughout the cross-sectional area of the bones, and the cortical area had very little

primary lamellar or secondary bone. However, in both species, scattered at the periosteal

and endosteal surfaces there were small amounts of both primary lamellar and secondary

bone present. These two types of bones were in limited quantities, and in order to collect

data these areas had to be actively sought.

The challenge in determining the origm of fragmentary bone comes at the

histological level when both primary lamellar and secondary bone are present and

plexiform bone is absent. When this is the case, the fragments cannot be discarded as

being human, and fiirther testing is required. As described, this research has developed a

new method that can be apphed to make this distinction more accurate Counting

osteocytes, and calculating their densities, within a set area of a cross-section of a bone

fragment can be utilized in conjunction with osteon size and Haversian canal size to

determine the origin (human vs. non-human) of the bone fragment. Furthermore, the ratio

of osteonal osteocytes to the secondary osteon size can also be used in the origin

assessment of fragmentary skeletal remains. However, the obvious differences in values

between the humems and the femur Haversian canal size cannot be explained at this time

A check of the original data shows that the correct values have been used. Additional

research is needed in order to make statistical valid decisions regarding these

inconsistencies.

62

Though plexiform bone was only found in the goat and deer bones during this

project, it does rarely occur in humans, primarily in early childhood and infancy. With

this in mind, aided by secondary osteonal osteocyte coums, secondary osteon size,

secondary osteon osteocyte to secondary osteon size ratio. Haversian canal size, and

osteocyte count/density it can readily be determined whether or not fragmentary bone

with plexiform, primary lamellar, and secondary bone present is human

Apphcations

This research has primarily been geared towards the forensic application of the

comparative study of mammalian cortical bone, and in all likelihood this is the aspect in

which it could have its greatest contribution. That is not to say it cannot be applied to

other fields in anthropology, it can in all actuality be applied to three of the four sub-

fields of anthropology: archaeology, cuhural, and physical^iological/fo^ensic.

Physical anthropologists can benefit in more than just the forensic application of

this research. It can be applied to paleopathological assessment of skeletal populations, or

at least make these assessments more accurate (as suggested by Schultz, 2001).

Furthermore, fossil remains associated with human evolution can be subjected to

histological analyses, and help in the reconstmction of our family tree.

As archaeologists study past cuhures, skeletal remains are frequently recovered

from archaeological sites. These remains can, when properiy interpreted, aid the

archaeologists in their analyses of the site from where the remains were recovered When

artifacts, such as pottery, are recovered, the pottery temper can be analyzed

63

microscopicaUy. In temper where fragmented bones have been utihzed, histological

methods can be applied to tell what kind of bone is present in the pottery (Paine et al.,

ND). The distinction between human and animal bone within the pottery will allow

anthropologists to make predictions about how different peoples utihzed their

environment, especially faunal resources.

Future Research

The findings presented in this research need to be elaborated on in flimre research

project as these findings certainly are intriguing, especially the secondary osteonal

osteocyte to secondary osteon size ratio. This ratio appears to be the least overiapping of

all the presented methods, and it could offer the most accurate assessment of the origins

of fragmentary skeletal remains. In order to statistically validate this ratio, larger sample

sizes are necessary. Each specimen must be represented in numbers and in age ranges

that would allow the results to be statistically valid. Furthermore, future research into this

filed must also incorporate the sex differences among the samples. Differences among

more mammahan species must also be included, such as rodents, as well as other animals

belonging to taxonomic classes other than Mammalia.

In addition, when plexiform bone is encountered, its stmcture needs to be studied

more thoroughly. Currently, there are no methods available to distinguish between such

bone in animals in which it frequently appears. There might be minute differences in the

plexiform bone that is yet to be discovered, and histological studies can be used for thi

purpose.

lis

64

The more we know about the microscopic stmcture of all types of bone,

physical/forensic/archaeological anthropologists wiU be able to determme with greater

accuracy what types of bone that is present. This could in turn yield greater

understanding of past populations, and their life ways, and most critically, it could aid

law enforcement agencies solve potential homicide cases.

The applications of skeletal histology within the field of anthropology are

plentifiil, though very few anthropologists (Paine, Pfeiffer, Schultz, Stout, and Ubelaker)

are actively engaged in histological research. With the basic understanding of bone

biology, interpreting histological slides ought to be of interest to the majority of physical

anthropologists as it has such a wide application within this sub-field. The most probable

reason why so few engage in this type of research is the destmctive nature of histological

analysis of skeletal remains. With current technology bones have to be cut in order to be

viewed microscopically. With further research, like this one, maybe more

anthropologists will come to appreciate the full potential of osteological histology

research, and the beneficial applications histological research can offer the field of

anthropology.

65

BIBLIOGRAPHY

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Burr D, Ruff CB, and Thompson DD. 1990. Patterns of Skeletal Histological Change Through Time: Comparison of an Archaic Native American Population Whh Modem Populations. Anat. Rec, 226: 307-313.

Currey JD. 1964. Some Effects of Aging in Human Haversian Systems. J. Anat Lond , 1:69-75.

Cormack DH. 2001. Essential Histology. Lippincott Wilhams & Wilkins, Philadelphia, PA

Enlow DH. 1962. Function of the Haversian System. Am. J. Anat., 110: 269-282.

Enlow DH and Brown SO. 1956. A Comparative Histological Study of Fossil and Recent Bone Tissue. Part I. Tx. J. Sci., 8: 405-443.

Enlow DH and Brown SO. 1957. A Comparative Histological Study of Fossil and Recent Bone Tissue. Part II. Tx. J. Sci., 9: 186-214.

Enlow DH and Brown SO. 1958. A Comparative Histological Study of Fossil and Recent Bone Tissue. Part III. Tx. J. Sci., 10: 187-230

Evans FG. 1976. Mechanical Properties and Histology of Cortical Bone From Younger and Older Men. Anat. Rec, 185: 1-12.

Frost HM. 1985. The "New Bone": Some Anthropological Potentials. Yrbk. Am. J. Phys Anthropol., 28:211-226.

Frost HM. 1986. Intermediary Organization of the Skeleton, Vol. I. CRC Press Inc., Boca

Raton, FL.

Frost HM. 1987. Secondary Osteon Populations: An Algorithm for Determining Mean Bone Tissue Age. Yrbk. Phys. Anthropol., 30: 221-238.

Gartner LP and Hiatt JL. 2001. Color Textbook of Histology. W.B. Saunders Company,

Philadelphia, PA.

66

Georgia R, Albu I, Sicoe M, and Georoceanu M. 1982. Comparative Aspects of the Density and Diameter of Haversian Canals in the Diaphyseal Compact Bone of Man and Dog. Rev. Roum. Morphol. Embryol. Physiol., 28: 11-14.

Ham AW and Cormack DH. 1979. Histophysiology of Cartilage, Bone, atid Joints Lippincott, Philadelphia, PA.

Harsanyi L. 1993. Differential Diagnosis of Human and Animal Bone. In: Histology of Ancient Bone: Methods and Diagnosis. Gmpe G and Garland AN (eds.). Springer-Veriag Beriin.

Jowsey J. 1966. Studies of Haversian Systems in Man and Some Animals. J Anat Lond 100: 857-864.

Keriey ER. 1965. The Microscopic Determination of Age in Human Bone. Am. J. Phys. Anthropol, 23: 149-164

Kessel RG. 1998. Basic Medical Histology. Oxford University Press, Inc New York NY.

Krause WJ. 1996. Essentials of Human Histology. Little, Brown and Company Inc , Boston, MA.

Leeson CR, Leeson TS, and Paparo AA. 1985. Atlas of Histology. Saunders, Philadelphia, PA.

Martin RB, Burr DB, and Sharkey NA. 1998. Skeletal Tissue Mechanics. Springer-Verlag, New York, NY.

Mulhern DM and Ubelaker DH. 2001. Differences in Osteon Banding Between Human and Nonhuman Bone. J. Forensic Sci., 46: 220-222.

Owsley DW, Mires AM, and Keith MS. 1985. Case Involving Differentiation of Deer and Human Bone Fragments. J. Forensic Sci., 30:572-578

Paine RR, Walter T, and Homi H (No Date) Bone Tempered Pottery from Archaeological Site 41 VTl 1. Manuscript TTU.

Pfeiffer S. 1998. Variability of Osteon Size in Recent Human Populations. Am. J. Phys Anthropol., 106: 219-227.

Rogers HJ, Weidmann SM, and Parkinson A. 1952. The Collagen Content of Bones From Rabbits, Oxen, and Humans. Biochem. J , 50 537-542

67

Schultz M. 2001. Paleohistopathology of Bone: A New Approach to the Study of Ancient Diseases. Yrbk. Am. J. Phys. Anthropol., 44: 106-147

Stout SD. 1978. Histological Stmcture and its Preservation m Ancient Bone. Curr. Anthropol., 19: 601-603.

Stout SD and Paine RR. 1992. Brief Communication: Histological Age Estimation using Rib and Clavicle. Am. J. Phys. Anthropol., 87: 111-115.

Stout SD and Paine RR. 1994. Brief Communication: Bone Remodeling Rates: A Test of an Algorithm for Estimating Missing Osteons. Am. J. Phys. Anthropol., 93: 123-129.

Stout SD and Teitelbaum SL. 1976. Histomorphometric Determination of Formation Rates of Archaeological Bone. Calcif Tiss. Res., 21: 163-169.

Thompson DD. 1980. Age Changes in Bone Mineralization, Cortical Thickness, and Haversian Canal Area. Calcif Tiss. Int., 31: 5-11.

Thompson DD and Galvin CC. 1983. Estimation of Age at Death by Tibial Osteon Remodeling. Int. J. Forensic Sci., 22: 203-211.

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68

APPENDIX

SPECIES SPECIFIC DATA BY QUADRANT, LOCATION,

AND BONE

69

Table A. 1 Species Specific Osteocyte Density Data by Quadrant, Bone, Location,

and Area (2.16mm') at lOOX Magnification

Species Cat (N=l)

Coyote (N=l)

Wolf (N=l)

Bone Location L F P l L F M l LFD 1 R F P l R F M l RFD 1 L H P l L H M l LHD 1 RHP I R H M l R H D l LRP 1 L R M l L R D l RRP I R R M l R R D l LTP 1 L T M l LTD 1 RTP 1 R T M l R T D l L U P l L U M l LUD 1 R U P l R U M l R U D l

L F P l LFM 1 LFD 1

L F P l LFM 1 L F D l

Quadrant I II

1175 1076 1246 1250 1222 1377 1794 2029 1546 1775 1161 1243 1351 1236 1571 1294 1310 1021 1245 1568 1044 1008 1246 1222 1790 1317 1237 1350 1513 1647 1236 1592 1385 1232 1777 1740 1560 1169 899 1150

1395 1260 1287 1515 1244 1776 1486 1341 1616 1199 1195 970 983 1171

1669 1814 1331 1063 892 833

1931 2003 825 1973

2011 1717

2065 2289 1854 2120 1980 1731

III IV 1676 1680 1107 1266 1562 1062 1673 1468 1511 1663 1266 1247 1534 1208 1514 1638 1490 1168 1586 1477 1001 964 1280 1563 1486 1883 1593 1246 1199 1182 1600 1201 1213 1397 1966 1712 1790 1735 1323 2238 1119 1083 1342 1408 1881 1583 1680 1528 1357 1566 1311 1266 1145 1300 1394 1293 808 1215 948 1058

2087 2466 2129 1950

2148 1860

1981 1824

2338 2178 2030 1957

70

Table A. 1 contmued

Species Wolf

Bone Location R H P l R H M l R H D l

I Quadrant n m IV

1942 1526 2753 2281 2184 2587 2658 2491 2286 2450 2205 2564

Armadillo (N=l)

Deer (N=l)

Goat (N=3)

LFP 1 L F M l LFD 1 R F P l R F M l RFD 1 LTP 1 L T M l R T P l R T M l R T D l

R F P l R F M l R F D l RHP 1 R H M l RHD 1

L F M l R F M l R F M 2 RFM3 RFD 1 RFD2 L H P l L H M l LHM2 L H D l LHD 2 L R P l L R M l RRP I RRD 1 L T M l L T M 2

1304 1270 1253 1546 1597 1420 1198 1260 1128 1446 1400

1895 2020 1788 1887 1430 1456

1141 1322 1488 1051 1500 1814 1230 2073 1577 1926 1599 1368 1417 1209 1589 1100 1615

1429 1248 1388 1662 1550 1732 1190 1376 nil 1175 1164

2161 1592 1924 2062 1829 1743

1447 1224 1700 1370 1512 1878 1354 1349 1564 1553 2030 1276 1249 1776 1447 1323 1791

1371 1222 1185 1511 1580 1286 1369 1137 929 1037 1661

2097 1410 1825 1996 2037 1740

1244 1536 2043 1194 1678 1356 1340 1512 1471 1270 1987 1073 1643 1523 1703 1212 2057

1644 1343 1246 1488 1425 1483 1565 1483 1193 1030 1503

2100 1395 2176 1830 1600 2012

1054 1663 1420 1445 1282 1183 1207 1580 1447 1671 1513 1310 1498 1350 1327 1425 1366

71

Table A. 1 continued

Species Bone Location I Quadrant II ni IV

Goat

Human (N=8)

RTD 1

R F M l R F M 2 RFM3 RFM4 RFM5 RFM6 RFM7 RFM8 R H M l R H M 2 RHM3 RHM4 RHM5 R H M 6 R H M 7 R H M 8 R R M l RRM2 RRM3 R R M 4 RRM5 R R M 6 R R M 7 R R M 8 R T M l R T M 2 R T M 3 R T M 4 R T M 5 R T M 6 R T M 7 R T M 8 R U M l RUM 2 RUM 3 RUM 4 RUM 5 RUM 6

1633 1359 1767 1607

1206 1047 1381 1181 940 1130 1077 1398 888 862 1251 1036 1239 1174 1302 1376 945 877 1116 1061 1405 1190 1185 1199 1074 1680 1365 1099 953 884 958 1356 1130 1217 1027 940 1084 1243

987 1000 1275 1126 1419 1093 1069 1490 1265 940 1188 1092 1047 1082 1269 1471 968 996 1218 1010 1191 1260 945 1255 1301 1343 1430 965 1008 1236 1021 1300 911 1076 1205 1062 1050 1358

978 1239 1254 1276 1168 1081 1118 1479 1135 1014 1235 1229 1174 1057 1480 1351 981 951 1369 1109 1138 1396 1292 1402 1255 1211 1396 1184 962 1067 1064 1253 1007 1213 1104 1234 1286 1164

1040 1166 1208 1052 1196 1032 1171 1432 971 985 1066 1073 1120 1329 1193 1449 847 970 1211 1217 1182 1067 1190 1482 1146 989 1417 1367 1350 995 1368 1271 892 968 1180 1020 1165 1100

72

Table A. 1 continued

Quadrant Species Bone Location I II III IV Human RUM 7 1397 1007 1313 1258

RUMS 1363 1311 1496 1540 LF= Left Femur, RF= Right Femur, LH= Left Humerus, RH= Right Humerus, LR= Left Radius, RR= Right Radius, LT= Left Tibia, RT= Right Tibia, LU= Left Ulna, RU= Right Ulna, P= Proximal end, M= Mid-shaft, D- Distal end, 1= Specimen # 1, 8= Specimen #8

73

3000 T

2500 -

2000

c rj o O 0)

o 0 ) *-» CO O

1500 --

1000

1 500

n -

-H igh

- L o w

• Average

Cat

2238

803

1373.5

Coyote

2466

1717

2008.3

Wolf

2753

1526

2178.1

Species

Amiadillo

1732

929

1359.3

Deer

2176

1395

1833.5

Goat

2073

1054

1487.7

Human

1680

847

1153.9

The graph represents the range, not the standard deviation Cat N=l, Coyote N=l, Wolf N=l, Amiadillo N=l, Deer N=l, Goat N=3, Human N=8

Figure A. 1 Average Osteocyte Count by Species at lOOX

74

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