Review

The TEM characterization of the lamellar structure

of osteoporotic human trabecular bone

Matthew Aaron Rubin, Iwona Jasiuk*

Department of Mechanical and Industrial Engineering, Concordia University, 1455 de Maisonneuve Blvd. West, Montreal, Que., Canada H3G 1M8

Received 14 June 2005; revised 18 July 2005; accepted 25 July 2005

Abstract

The lamellar structure of osteoporotic human trabecular bone was characterized experimentally by means of transmission electron

microscopy (TEM). More specifically, the TEM was used to determine if trabecular bone exhibits similar lamellar structural motifs as

cortical bone by analyzing unmineralized, mineralized and demineralized bone, and to study the influence of the osteocyte network on the

lamellar structure of osteoporotic trabecular bone. Comparison with normal trabecular bone is included. This paper summarizes partial

results of a larger study, which addressed the characterization of the hierarchical structure of normal versus osteoporotic human trabecular

bone [Rubin, M.A., 2001. Multiscale characterization of the ultrastructure of trabecular bone in osteoporotic and normal humans and in two

inbred strains of mice. MS Thesis, Georgia Institute of Technology.] at several structural scales.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Trabecular bone; Lamellar level; Osteoporosis; Transmission electron microscopy

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653

2. Methods and preparation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 657

2.1. Calcified bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658

2.2. Decalcification of bone samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658

3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658

3.1. Unmineralized lamellar structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658

3.2. Mineralized lamellar structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660

3.3. Demineralized lamellar structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660

4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663

1. Introduction

Bone is a natural composite material with a hierarchical

structure (e.g. Lakes, 1993; Rho et al., 1998; Weiner and

Traub, 1992; Hoffler et al., 2000). Five structural levels can

0968-4328/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micron.2005.07.010

* Corresponding author. Tel.: C1 514 848 2424x3143; fax: C1 514 848

3175.

E-mail address: ijasiuk@encs.concordia.ca (I. Jasiuk).

be distinguished in bone’s structure: (1) macrostructural

level: whole bone; (2) mesostructural level: trabecular

and cortical bone; (3) microstructural (or lamellar) level

(10–500 mm): single osteons and trabeculae (trabecular

pockets); (4) sub-microstructural level (1–10 mm): single

lamellae; and (5) nanostructural level (below 1 mm):

collagen fibrils and apatite crystals.

In this paper we study the structure of osteoporotic

human trabecular bone at the microstructural level using

transmission electron microscopy (TEM). Osteoporosis is

a disease caused by abnormal bone metabolism. It is

characterized by low bone mass and microarchitectural

Micron 36 (2005) 653–664

www.elsevier.com/locate/micron

Fig. 1. TEM micrograph of the characteristic arcing pattern in partially mineralized twisted or rotated plywood motif in osteoporotic bone. Two black bands

(black arrows) are folds in the section due to specimen preparation. Unmineralized collagen (Uc), mineralized region (M).

Fig. 2. TEM micrograph of the characteristic arcing pattern of unmineralized twisted or rotated plywood motif in osteoporotic bone. A successive transition of

longitudinally (L), obliquely (O) and transversely sectioned fibrils (T) is apparent. Unmineralized collagen (Uc), mineral zone (M).

M.A. Rubin, I. Jasiuk / Micron 36 (2005) 653–664654

M.A. Rubin, I. Jasiuk / Micron 36 (2005) 653–664 655

deterioration of bone tissue with a consequent increase in

bone fragility and susceptibility to fracture (e.g. Rai and

Behari, 1986; Parafitt and Chir, 1987; Morita et al., 1994;

Werner et al., 1996; Myers and Wilson, 1997; National

Institute of Health, 2000).

Electron microscopy has been used successfully to

analyze bone’s structure (Bloom and Fawcett, 1975; Kessel

and Kardon, 1979; Eriksen et al., 1994). For example,

morphological investigations employing scanning electron

microscopy (SEM) have led to a better understanding of

the mesostructure of osteoporotic trabecular bone (e.g.

Lozupone and Favia, 1990; Mosekilde, 1990; Takita et al.,

1992; Jayasinghe et al., 1993; Morita et al., 1994; Marks

et al., 1996). SEM has shown that with aging and

osteoporosis, trabecular bone undergoes morphological

changes, including thinning of trabeculae, removal and

disconnection of trabecular elements, loss of trabecular

contiguity and reduction in density (e.g. Jayasinghe et al.,

1993; Snyder et al., 1993; Amling et al., 1996). However,

the effect of osteoporosis on the microstructural, sub-

Fig. 3. TEM micrograph of an orthogonal plywood motif in osteoporotic bo

(T) sectioned unmineralized fibrils are evident.

microstructural or nanostructural levels of trabecular bone

has received little attention.

TEM has proven to be a valuable tool for analyzing

lower structural levels in cortical bone and the mineralized

turkey leg tendon (MTLT) (e.g. Ascenzi and Benvenuti,

1986; Giraud-Guille, 1988; Traub et al., 1989; Landis and

Song, 1993; Marotti, 1993; Prostak and Lees, 1996;

Weiner et al., 1997). Recently, TEM has been employed

by us to evaluate normal and osteoporotic bone structure

at the nanostructural level (Rubin et al., 2003). The present

study is focused on the TEM characterization of

osteoporotic human trabecular structure at the microstruc-

tural level.

Electron microscopy studies that examined the micro-

structure (i.e. lamellar structure) in bone are limited. For

example, researchers have used SEM to study the

lamellar structure in cortical bone observed on fractured

surfaces (e.g. Marotti, 1993; Raspanti et al., 1996; Weiner

et al., 1997), or to study the two-dimensional aspects of

the lamellar structure in bone (e.g. Lips et al., 1978;

ne. Successive layers of alternating longitudinally (L) and transversely

M.A. Rubin, I. Jasiuk / Micron 36 (2005) 653–664656

Darby and Meunier, 1981; Cane et al., 1982; Sato et al.,

1986; Giraud-Guille, 1988; Marotti, 1993; Kragstrup

et al., 1983; Kargstrup and Melsen, 1983; Weiner et al.,

1997, 1999; Ziv et al., 1996; Eyden and Tzaphildou,

2001). The majority of these investigators suggested that

the lamellar organization in cortical bone is best

represented by the plywood-type structures (orthogonal,

twisted, and rotated) (e.g. Giraud-Guille, 1988; Weiner

et al., 1997).

Giraud-Guille (1988) has shown that within the lamellae

of compact bone there coexist two different kinds of plywood

structures—the orthogonal plywood and the twisted plywood

motifs. The orthogonal plywood structure corresponds to the

classical view (Gerbhardt, 1906) in which the layers of

collagen fibrils are oriented in the same direction in an

individual lamella, while the layers of collagen fibrils in the

successive lamella are oriented 908 to the previous direction.

In the twisted plywood model, the parallel layers of collagen

fibrils continuously rotate from plane to plane forming a

helical structure, so that in a sense, there is no individual

Fig. 4. TEM micrograph of unmineralized and mineralized zones in osteoporotic

longitudinally sectioned fibrils (black arrows) near the mineral front represents the

in the mineral zone. Dark black band in mineral region is a fold in the section due t

mineralized region (M).

lamella (Hollister, 2001). However, the bone still shows

a lamellar structure because as the orientation of the collagen

fibrils rotates through 1808 cycles, the fibril orientation

repeats itself (Martin et al., 1998).

A more recent model by Weiner et al. (1997) suggested

that lamellar bone should be viewed as a series of ‘lamellar

units’, each of which is made up of five sub-layers, differing

by 308. Fibrils in successive sub-layers are ideally oriented

at 08, 308, 608, 908, and 1208, with the fourth layer being

orthogonal to the first, and the fifth sub-layer being ideally

608 with respect to the first sub-layer of the next set of fibrils.

An alternate model proposed by Marotti (1993) suggested

that lamellar bone is made up of alternating collagen-rich

(dense, thinner lamellae) and collagen-poor (loose, thicker

lamellae) layers, all having an interwoven arrangement of

fibrils. In other words, the collagen fibrils are not oriented

parallel to each other, but have random orientations.

Regardless of the model, all of these studies have used

cortical bone to observe the lamellar structure, rather than

trabecular bone.

bone showing mainly transversely sectioned fibrils. The parallel array of

successive lamellar layer. The canaliculi (white arrows) are seen traversing

o specimen preparation (dotted white arrow). Unmineralized collagen (Uc),

M.A. Rubin, I. Jasiuk / Micron 36 (2005) 653–664 657

The objective of this paper is to characterize the lamellar

structure (microstructural level) of the osteoporotic human

trabecular bone by means of TEM. To our knowledge this is

the first such study.

2. Methods and preparation techniques

Trabecular (cancellous) bone was extracted from femurs

of three osteoporotic human males and females (79–91 years

old). Bone samples were obtained from Georgia Baptist

Hospital in Atlanta, Georgia. Bone mineral density (BMD)

measurements were used to determine whether individuals

had osteoporosis. The average BMD for osteoporotic bone

was 0.244 mg/mm3 (in contrast to 0.587 mg/mm3 for normal

bone). The tissue was cut into 1 cm cubes and stored in 90%

ethanol solution. The experimental protocol for the collec-

tion of tissue was approved by the IRB at the Atlanta Medical

Center.

A JEOL JEM-1210 Analytical TEM operated at 90 kV

was used to view the calcified and decalcified human

Fig. 5. TEM micrograph of an atypical unmineralized collagen structure showin

numerous dark circular mineral clusters (dotted arrows) in osteoporotic bone.

successively alternating transverse and longitudinally sectioned fibrils (thick arro

trabecular bone sections. To view the lamellar structure

more clearly, some bone samples were decalcified, i.e. bone

mineral was removed. This process exposed the collagen

framework and any organizational structure they possessed

while mineralized. The remaining bone samples stayed

calcified and were then used to compare lamellar structures

with the demineralized specimens. TEM images were

photographed at low (2000!) to intermediate (20,000!)

magnifications to best describe these features. The negatives

were then scanned into a computer to generate digitized

images. These images were processed and analyzed using

PC-based Adobe Photoshop software. In Adobe Photoshop

we measured microstructural features using the measure tool

(up to the three decimal place accuracy), which allowed us to

measure the x- and y-coordinates of the starting location, the

horizontal and vertical distances traveled from the x- and y-

axes, the angle measured relative to the axis, and the total

distance between two objects. We also used the Levels

histogram to correct the image’s tonal range by adjusting the

intensity levels of the image’s shadows, midtones, and

highlights.

g a collection of loosely scattered collagen fibrils (thin black arrows) and

Only the region at the left edge shows any discernable resemblance of

w).

M.A. Rubin, I. Jasiuk / Micron 36 (2005) 653–664658

2.1. Calcified bone

Bone specimens were postfixed in 1% osmium tetroxide,

dehydrated in an acetone series (30, 50, 70, 80, 90, 100%),

then infiltrated with a graded series of acetone and three

changes of Spurr resin, and finally embedded in fresh Spurr

resin in labeled Beeme capsules. Ultrathin sections (90–

100 nm) of bone were then cut with a diamond knife on a

RMC MT 7000 Ultramicrotome and picked up on 300-mesh

copper and Formvare coated, single slot, copper grids.

2.2. Decalcification of bone samples

Bone specimens were decalcified in 0.5 M of 10%

EDTA, pH 8.0 (Tris), and 0.01% Sodium Azide solution

at room temperature for 4 days. The EDTA was replaced

with fresh solution every 24 h for 4 days. After 4 days,

the specimens were rinsed and then submerged in an

isotonic saline solution for 24 h at 4 8C. The samples

were then post-fixed in osmium tetroxide and the

remaining procedures were the same as for calcified

bone.

Fig. 6. TEM micrograph of an unmineralized fragmented plywood structure in ost

and transversely sectioned (T) fibrils are seen, though the structure is not comple

3. Results and discussion

TEM was used to investigate the osteoporotic human

trabecular bone at the microstructural level. More

specifically it was used to: (1) determine if trabecular

bone exhibits similar lamellar structural motifs as cortical

bone by analyzing unmineralized, mineralized and

demineralized bone, and (2) study the influence of the

osteocyte network on the lamellar structure of osteoporotic

trabecular bone.

3.1. Unmineralized lamellar structures

The collagen structures (Figs. 1 and 2) showed the

characteristic arcing pattern of the incompletely mineralized

fibrils, which acted as a template for a developing lamellar

unit. The successive direction of longitudinal, oblique, and

transverse fibrils, indicative of the twisted or rotated

plywood structure, was clearly seen (Fig. 2). The occurrence

of this structural appearance was fully described in

Giraud-Guille (1988). Fibrils appear as dots when cut

transversely and as parallel segments, short or long, when

eoporotic bone. A successive transition of longitudinally (L), obliquely (O)

te.

M.A. Rubin, I. Jasiuk / Micron 36 (2005) 653–664 659

the fibrils were cut obliquely or longitudinally (Fig. 2).

Fig. 3 showed characteristics of the orthogonal plywood

motif, such that the successive layers of alternating

longitudinally and transversely sectioned unmineralized

fibrils were evident. However, this orthogonal disposition

was not as discernable in Fig. 4, as mainly transversely

sectioned fibrils were seen in both unmineralized and

mineralized zones. However, parallel arrays of long-

itudinally sectioned fibrils, marked by black arrows, existed

near the mineral front and they most likely represented the

successive lamellar units, which were orthogonal to

transversely sectioned fibrils. Also, these thin arrays could

possibly serve as unmineralized collagen templates for the

canaliculi seen traversing the mineral zone. The mineralized

counterparts of the canaliculi appeared as dark, thin bands of

100–300 nm in thickness. The thickness of canaliculi was

measured using the measure tool in Adobe Photoshop.

Thickness measurements were done based on the distance

between the discernable outer edges of the thin bands of

longitudinally sectioned collagen fibrils at mutliple points

along its length.

Additionally, these thin arrays of longitudinally

sectioned fibrils appeared to be associated with

Fig. 7. TEM micrograph of a repeating mineralized lamellar structure with dark a

lamella units tend to blend into one another, making it difficult to distinguish bou

the formation of collagen bundles, approximately 2–

3 mm in diameter in transverse cross-section, seen in

unmineralized region bordering the mineral front. Each

bundle was separated or traversed by these thin arrays of

longitudinally sectioned fibrils, giving rise to gaps (light

regions) on sides of each bundle. These arrays appeared to

wrap around the bundles.

However, not all of the micrographs displayed the

typical plywood motifs commonly described in bone. For

instance, Fig. 5 showed a collection of loosely scattered

collagen fibrils (denoted by thin arrows) and numerous

dark circular mineral clusters (marked by dotted arrows)

speckled haphazardly throughout the unmineralized

region. Parallel arrays of longitudinally, as well as

transversely and obliquely sectioned fibrils traversed the

entire unmineralized regions, though no complete struc-

tures were seen within the majority of these regions. Only

the regions at the lower left of Fig. 5 showed any

discernable resemblance of successively alternating trans-

versely and longitudinally sectioned fibrils (denoted by

thick arrows). A rather fragmented plywood structure

was observed in Fig. 6 with numerous gaps between fibrils

and mineral clusters. Despite the successive transition of

nd light bands in osteoporotic bone. In lower right region the edges of the

ndaries between dark and light bands.

M.A. Rubin, I. Jasiuk / Micron 36 (2005) 653–664660

longitudinally, obliquely and transversely sectioned fibrils

seen in Fig. 6, the unmineralized collagen fibrils were

sparse and thus, the lamellar structure appeared incom-

plete. With the formation of large mineral clusters along

the mineral front and large separations between arrays of

unmineralized fibrils, it almost appeared like the unminer-

alized and mineralized regions had been stretched or torn

apart from each other. This torn appearance was a result of

collagen deficiency in that region.

3.2. Mineralized lamellar structures

In the mineralized region, the unmineralized collagen

framework observed previously was seen as a repeating

lamellar structure of dark and light bands (Fig. 7). Closer

examination revealed that dark regions corresponded to

disordered arrangements of generally longitudinally sec-

tioned fibrils, while the lighter bands corresponded to a

region with more obliquely sectioned fibrils. Other

lamellar structures such as distinct, alternating, transverse

and longitudinal fibrils, as well as arcing patterns between

Fig. 8. TEM micrograph showing the characteristic arcing pattern of a deminera

transition of longitudinally (L), obliquely (O) and transversely (T) sectioned fibri

dark bands also appeared (Fig. 7). In some regions (e.g.

lower right of Fig. 7) the edges of the lamellar units

tended to blend into one another, making it almost

impossible to distinguish the boundaries between the

dark and light bands.

3.3. Demineralized lamellar structures

The ubiquitous plywood lamellar structures observed

in the mineralized regions of osteoporotic bone were also

evident in the demineralized osteoporotic bone (Figs. 8–11).

The lamellar structure tended to be disrupted and/or tangled

with the extensive networking of the radial fibril arrays

(Figs. 9 and 10). As also observed in the normal decalcified

bone micrographs (Rubin, 2001), these radial arrays were

associated with canaliculi, which could be seen traversing

more or less perpendicularly to lamellar boundaries

(Figs. 8–10). The canaliculi, denoted by black arrows

(Figs. 8 and 9) appeared as white, elliptically shaped bodies,

approximately 1–3 mm long, some either with or without

longitudinally sectioned fibrils trailing from one end.

lized twisted or rotated plywood motif in osteoporotic bone. A successive

ls is apparent. The canaliculi is shown by an arrow. Close-up of Fig. 9.

Fig. 9. TEM micrograph of a demineralized osteoporotic bone microstructure showing little lamellar disturbance from the radial arrays. These radial arrays

correspond to canaliculi (arrows), which are traversing more or less perpendicularly to lamellae boundaries. Several canaliculi are extending from arbitrary

sides of the osteocyte (Os).

M.A. Rubin, I. Jasiuk / Micron 36 (2005) 653–664 661

Several canaliculi could be seen extending from arbitrary

sides of the osteocyte denoted by the symbol Os in Fig. 9.

Lamellar thickness measurements were done based on

the distance between the central regions of the longitudinal

arrays of collagen fibrils at multiple points along their length

(i.e. distance between the midpoints of two adjacent ‘dark

bands’ at multiple spots along the array). Thus, the average

lamellar thickness between successive lamellae was about

5.8G0.3 mm, with a maximum of about 8 mm in the regions

containing osteocytes. These measurements are in agree-

ment with Kragstrup et al. (1983).

In addition to well-defined plywood structures, devi-

ations of the plywood structures were also evident in the

osteoporotic micrographs (Figs. 10 and 11). We made

similar observations about the microstructure of normal

trabecular bone (Rubin, 2001). For instance, a clear

distinction between the longitudinally sectioned fibrils and

the obliquely sectioned fibrils was lost among the

convoluted lamellar structure seen in center region of

Fig. 11. An ordered lamellar structure was more evident at a

higher magnification, however, longitudinally sectioned

fibrils were still seen traversing in arbitrary directions.

This unequal spacing was also observed in Fig. 10, where

many lamellar layers were seen to be considerably smaller

than other ones. A diverging lamellar unit seen on the

bottom portion of Fig. 10 indicated that lamellar layers

might not always be exactly parallel to each other. Even

though faint traces of two arrays of longitudinally sectioned

fibrils could be seen on either side the diverging layer, the

nature of the curving lamella is surprising. It is quite

possible that this divergence in lamellar layers could result

from the scalloped-like trabecular packet geometry.

4. Summary

The major results on the microstructure of the

osteoporotic human trabecular bone study presented in

this paper are summarized as follows:

1. Trabecular and cortical bone had equivalent collagen

organizations within the lamellae at the microstructural

level. This was determined by analyzing decalcified

lamellar structures in trabecular bone and comparing

Fig. 10. TEM micrograph of a disrupted lamellar structure resulting from the extensive networking of the radial fibril arrays. The lamellar spacing between the

bands of longitudinally sectioned fibrils varies considerably, with no two being the same. A diverging lamellar unit (arrow) is seen on the bottom-right portion

of the image.

M.A. Rubin, I. Jasiuk / Micron 36 (2005) 653–664662

them with published results on the cortical bone

structure.

2. Atypical lamellar structures were evident in osteoporotic

trabecular bone. These atypical structures, which were

mainly seen in the unmineralized regions, suggested that

the lamellar pattern in bone was much more complex

than envisioned.

3. There was evidence of lamellar structure alteration by

the osteocyte network.

The above conclusions summarize the results presented

in this paper for human osteoporotic trabecular bone.

However, they are more general as they hold for both

normal and osteoporotic trabecular bone. The results of the

normal trabecular bone study were reported elsewhere

(Rubin, 2001). In the next paragraphs we present the

summary pertaining to both normal and osteoporotic bone,

for completeness. The information on normal bone we draw

from (Rubin, 2001).

In summary, the TEM analysis of the microstructure of

human trabecular bone confirmed many of the results found

in cortical bone and MTLT, as well as surfaced a few new

questions. This investigation showed that the plywood-like

structures routinely observed in cortical bone, twisted,

rotated or orthogonal, existed in both normal and

osteoporotic human trabecular bone. However, there were

some structural differences in the lamellar motifs between

normal and osteoporotic bone. One of these differences

involved a more pristine lamellar organization in normal

trabecular bone. Observations of atypical lamellar structures

found in the mineralized and unmineralized regions of both

normal and osteoporotic trabecular bone suggested that

bone exhibited much more complicated structures than

previously reported.

The osteocyte network was shown to have some two-

dimensional structural significance on the lamellar organ-

ization in both normal and osteoporotic bone. However, it

appeared that the lamellar structure of osteoporotic bone

was more affected by this vast network, as the canaliculi

tended to be more pronounced and abundant.

Finally, the overall objectives of this research were to

investigate the ultrastructural characteristics of osteoporotic

Fig. 11. TEM micrograph from osteoporotic demineralized bone showing deviations from the plywood structures. A clear distinction between the

longitudinally sectioned fibrils and the obliquely sectioned fibrils is lost among the convoluted lamellar structure.

M.A. Rubin, I. Jasiuk / Micron 36 (2005) 653–664 663

and normal human trabecular bone by means of TEM and to

provide the characterization of the normal and osteoporotic

human trabecular bone hierarchical structure from the

mesoscale down to the nanoscale. They are summarized in

Rubin (2001). The results presented in this paper are a

subset of this research. The comparison of normal and

osteoporotic bone at sub-microstructure and nanostructure

levels was presented by us in Rubin et al. (2003).

Acknowledgements

We would like to thank Dr Robert Apkarian and

Jeannette Taylor from Integrated Microscopy and Micro-

analytical Facility at Emory University for their assistance

with TEM specimen preparation and viewing of images and

to Dr Timothy Ganey from Atlanta Medical Center for

supplying us with the bone tissue.

References

Amling, M., Posl, M., Ritzel, H., Hahn, M., Vogel, M., Wening, V.J.,

Delling, G., 1996. “Architecture and distribution of cancellous bone

yield vertebral fracture clues: A histomorphometric analysis of the

complete spinal column from 40 autopsy specimens. Arch. Orthop

Trauma Surg 115, 262–269.

Ascenzi, A., 1986. Benvenutti A Orientation of collagen fibers at the

boundary between two successive osteonic lamellae and its mechanical

interpretation. J. Biomech 19, 455–463.

Bloom, W., Fawcett, D., 1975. Bone, A Textbook of Histology, ten th 1975

(Chpt 11) pp 244–261.

Cane, V., Marotti, G., Volpi, G., Zaffe, D., Palazzini, S., 1982. “Size and

density of steocyte lacunae in different regions of long bones. Calcif.

Tissue Int. 34, 558–563.

Darby, A.J., Meunier, P.J., 1981. “Mean wall thickness and formation

periods of trabecular bone packets in idiopathic osteoporosis. Calcif.

Tissue Int. 33, 199–204.

Eriksen, E.F., Axelrod, D.W., Melsen, F., 1994. Bone Histomorphometry.

Raven Press, New York.

Eyden, B., Tzaphildou, M., 2001. Structural variations of collagen in

normal and pathological tissues: role of electron microscopy. Micron

32, 287–300.

Gebhardt, W., 1906. Uber funktionell wichtige Anordnungsweisen der

feineren und groberen Bauelemente des Wirbeltierknochens.II

Spezieller Teil. I. Arch. Entw. Mech. Org. 20, 187–322.

Giraud-Guille, M.M., 1988. Twisted plywood architecture of collagen

fibrils in human compact bone osteons. Calcif. Tissue Int. 42,

167–180.

Hoffler, C.E., McCreadie, B., Smith, E., Goldstein, S., 2000. A hierarchical

approach to exploring bone mechanical properties. In: An, Draughn

(Eds.), Mechanical Testing of Bone and the Bone-Implant Interface,

pp. 133–149 (Chpt. 8).

M.A. Rubin, I. Jasiuk / Micron 36 (2005) 653–664664

Hollister S (2001), From: www.engin.umich.edu/class/bme456/

bonestructure.

Jayasinghe, J., Jones, S.J., Boyde, A., 1993. Scanning electron microscopy

of human lumbar vertebral trabecular bone surfaces. Virchows Archiv

A Pathol. Anat. 422, 25–34.

Kessel, R., Kardon, R., 1979. Tissue and Organs: A Text Atlas of Scanning

Electron Microscopy. W.H. Freeman and Company.

Kragstrup, J., Melsen, F., 1983. Three dimensional morphology of

trabecular bone osteons reconstructed from serial sections. Metab.

Bone Dis. Rel. Res. 5, 127–130.

Kragstrup, J., Melsen, F., Mosekilde, L., 1983. Thickness of lamellae

in normal human iliac trabecular bone. Metab. Bone Dis. Rel. Res. 4,

291–295.

Lakes, R., 1993. Materials with structural hierarchy. Nature 361, 511–

515.

Landis, Song, 1993. Mineral and organic matrix interaction in normally

calcifying tendon visualized in three dimensions by high voltage

electron microscopic tomography and graphic image reconstruction.

J. Struct. Biology 110, 39–54.

Lips, P., Courpron, P., Meunier, P.J., 1978. Mean wall thickness of

trabecular bone packets in the human iliac crest: changes with age.

Calcif. Tissue Int. 26, 13–17.

Lozupone, E., Favia, A., 1990. The structure of the trabeculae of cancellous

bone. 2. Long Bones and Mastiod. Calcif. Tissue Int. 46, 367–372.

Marks, S., Cielinski, M., Sundquist, K., 1996. Bone surface

morphology reflects local skeletal metabolism. Micro. Res. And

Tech. 33, 121–127.

Marotti, G., 1993. A new theory of bone lamellation. Calcif. Tissue Int. 53,

S47–S56.

Martin, B., Burr, D., Sharkey, N., 1998. Skeletal Tissue Mechanics.

Springer-Verlag, New York.

Morita, M., Ebihara, A., Itoman, M., Sasada, T., 1994. Progression of

osteoporosis in cancellous bone depending on trabecular structure. Ann.

of Biomed. Eng. 22, 532–539.

Mosekilde, L., 1990. Consequences of the remodeling process for vertebral

trabecular bone-structure - A scanning electron-microscopy study

(uncoupling of unloaded structures). Bone Miner. 10, 13–35.

Myers, E.R., Wilson, S.E., 1997. Biomechanics of osteoporosis and

vertebral fracture. Spine 22 (24S), 25S–31S.

National Institutes of Health, The Osteoporosis and Related Bone Diseases

wNational Resource Center (2000), From: http//www.osteo.org/docs/

183.199143826.htm.

Parafitt, A., Chir, B., 1987. Bone remodeling and bone loss: Understanding

the pathophysiology of osteoporosis. Clin. Obstet. Gynecol. 30 (4),

789–811.

Prostak, K.S., Lees, S., 1996. Visualization of crystal matrix structure. In

Situ demineralization of mineralized turkey tendon and bone. Calcif.

Tissue Int. 59, 474–479.

Rai, D.V., Behari, J., 1986. Biophysical characterization of osteoporotic

bone. Env. Res. 40, 68–83.

Raspanti, M., Guizzardi, S., Strocchi, R., Ruggeri, A., 1996. Collagen fibril

patterns in compact bone: Preliminary ultrastructural observations. Acta

Anatomica 155, 249–256.

Rho, J.Y., Kuhn-Spearing, L., Zioupos, P., 1998. Mechanical properties and

the hierarchical structure of bone. Med Eng & Phys 20, 92–102.

Rubin, M. A (2001), Multiscale characterization of the ultrastructure of

trabecular bone in osteoporotic and normal humans and in two inbred

strains of mice, M.S. thesis, Georgia Institute of Technology.

Rubin, M.A., Jasiuk, I., Taylor, J., Rubin, J., Ganey, T., Apkarian, R.P.,

2003. TEM analysis of the nanostructure of normal and osteoporotic

human trabecular bone. Bone 33, 270–282.

Sato, K., Wakamatsu, E., et al., 1986. Histomorphometric study of

trabecular channels in normal iliac bone. Calcif. Tissue Int. 39, 2–7.

Snyder, B., Piazza, S., Edwards, W., Hayes, W., 1993. Role of trabecular

morphology in the etiology of age-related vertebral fractures. Calcif.

Tissue Int. 53 (1), S14–S22.

Takita, T., Naguro, T., Yamamoto, K., 1992. Fine structure of remodeling

sites on iliac cancellous bone in senile osteoporosis: A study by

scanning electron microscopy using an improved organic specimen

preparation method. Cells and Materials 2 (2), 135–142.

Traub, W., Arad, T., Weiner, S., 1989. Three-dimensional ordered

distribution of crystals in turkey in tendon collagen fibers. Proc. Natl.

Acad Sci. USA 86, 9822–9826.

Weiner, S., Traub, W., 1992. Bone structure: from angstroms to microns.

FASEB 6, 879–885.

Weiner, S., Arad, T., Sabanay, I., Traub, W., 1997. Rotated plywood

structure of primary lamellar bone in the rat: orientations of the collagen

fibril arrays. Bone 20 (6), 509–514.

Weiner, S., Traub, W., Wagner, H., 1999. Lamellar bone: Structure-

function relations. J. Struc. Bio. 126, 241–255.

Werner, H.J., Martin, H., Behrend, D., Schmitz, K.P., Schober, H.C., 1996.

The loss of stiffness as osteoporosis progresses. Med. Eng. Phys. 18,

601–606.

Ziv, V., Wagner, H., Weiner, S., 1996. Transitional structures in lamellar

bone. Microsc. Res. Tech. 33, 203–213.