BIOE 4710 / 5710

Soft and Hard Tissue Biomechanics

(Bone)
Bone Histology

Dr. Edward Nyman, Jr., Ph.D.

Research Assistant Professor

Engineering Center for Orthopaedic Research Excellence (ECORE)
Departments of Bioengineering & Orthopaedic Surgery
The University of Toledo, NI 5040
edward.nyman@utoledo.edu

Overview
I. Basic Principles of Histology
II. Histology Methods Related to Bone Imaging
III. Staining and Dying
IV. Bone Samples / Applications
V. Quantification of Bone Remodeling

Methods of Bone and Tissue Imaging

Microscopy
X-Ray
Ultrasound
SPECT & Gamma Camera
CT
NMR & MRI
PET

Microscopy
Processing bone tissue requires highly
specialized techniques, unique equipment, and
technical expertise.
Histological sections may be obtained on fresh
frozen bones, decalcified and paraffinembedded bones, or on non-decalcified,
plastic-embedded bones.

Processing and Embedding

Fixation
Washing
Dehydration
Infiltration
Embedment

Sample Preparation: Fixation

Stabilize structures (micro-anatomical
arrangement of tissue elements)
Retain constituents (stabilize cellular inclusions)
Negate autolysis and decay (putrefication)
Improve contrast (enhance the refractive index
of tissues)
Harden tissue (render cell constituents insoluble
and resistant to subsequent processes)

Types of Fixation
Mechanical (Physical) fixation

Heating or freezing

Chemical fixation

Immersion
Perfusion
Vapor
Phase-partition

Processing
Biological issues have a high concentration of
water
Embedding media are usually nonpolar
Water must be removed via dehydration with
alcohol or acetone
Replaced with either wax or plastic (resin)

Microtomy
Microtomy (small cuts) takes practice and
experience to produce quality microscope
preparations.
4 critical requirements for sample success

Practice and experience

A sharp microtome knife
A proper microtome
Well prepared tissue

https://www.youtube.com/watch?v=O0D2fW1a39Y

Types of Microtomes

Handheld
Cambridge rocking microtome
Rotary microtomes
Base-sledge and sliding microtomes
Freezing microtomes
Cryostats
Vibrotomes

Rotary Microtomes
Most widely used type of microtome

Stationary knife
Sample placed on a ball-joint
Cutting stroke simply down/up motion

Produces flat sections

Staining and Dying

Principle of creating differential contrast
Can be used to detect specific tissue
constituents
Can be used to determine cell viability
Can be used to determine relative pH

Common Laboratory Stains

Stain

Common
use

Red
Nucle Cytopla Blood
us
sm
Cells
(RBC)

General staining
Hematoxylin when paired
Blue
with Eosin
General staining
when paired
Eosin
N/A
with
Haematoxylin
Toluidine
General staining Blue
blue

Collag
en
Specifically stains
Fibers
N/A

Nucleic acids - Blue

blue eER (ergastoplasm) - Blue

N/A

N/A

Pink

Orange/Red Pink

Elastic fibers - pink,

reticular fibers - pink

Blue

Blue

Blue

Mast cells granules purple

Muscle Fibers Red

Gomori's
trichrome
stain

Connective and Gray/Blu

Red
muscle tissue
e

Red

Green

Masson's
trichrome
stain

Connective
tissue

Red

Blue/Gre Cartilage - Blue/green, Muscle fibers

en
Red

Mallory's
trichrome
stain

Connective
tissue

Orange

Deep
Blue

Black

Red

Red/Pink

Pale Red

Keratin - Orange,
Cartilage - Blue, Bone matrix - Deep
Blue, Muscle fibers - Red

Table sourced from Michael H. Ross, Wojciech Pawlina, (2006). Histology: A Text
and Atlas. Hagerstwon, MD: Lippincott Williams & Wilkins. ISBN 0-7817-5056-3

Unstained Section

Goldners Trichrome Stain

Iron Stain

Von Kossa Stain

Tetracycline

Microscopes
Brightfield Light Microscopy (optical)

Phase contrast
Polarized
Differential contrast
Reflection

Fluorescence Microscopy
Confocal Microscopy
Multiphoton
Transmission Electron Microscopy
Scanning Electron Microscopy
Scanning Transmission Electron Microscopy
Atomic Force & Scanning Tunneling

Optical Microscopy
Optical or light microscopy involves passing visible light
transmitted through, or reflected from, the sample
through a single or multiple lenses to allow a magnified
view of the sample.
The resulting image can be detected directly by the eye,
imaged on a photographic plate or captured digitally.
The single lens with its attachments, or the system of
lenses and imaging equipment, along with the
appropriate lighting equipment, sample stage and
support, makes up the basic light microscope.

Resolution versus Magnification

Fluorescence Microscopy
Fluorescent molecule = fluorochrome
absorbs light of specific wavelength
When excited by absorption, fluorochrome
emits light of longer wavelength.
Every fluorochrome has an absorption and
emission spectra.

Electron Microscopy
Developed in 1930s
Uses electron beams instead of visible light.
Because of the much shorter wavelength of the electron beam than
of light, resolution is far greater.
TYPES:
Transmission electron microscopy (TEM) is principally quite similar to
the compound light microscope, by sending an electron beam
through a very thin slice of the specimen.

The resolution limit is ~0.03 nanometer (~2.5 nm practically for biological samples)

Scanning electron microscopy (SEM) visualizes details on the

surfaces of cells and particles and gives a very nice 3D view.

The magnification is in the lower range than that of the transmission electron
microscope (~ 30 nm for bio samples).

Transmission Electron
Microscopy (TEM)

Very thin sections

Beam of electrons
(=0.05)

Electromagnetic lenses

Stain with metals

Stain: electron dense = dark
Unstained: light

Scanning Electron Microscopy (SEM)

Surface structure
Sectioning not required

Metal coating of specimen

Electron scattering
Primary electrons
Secondary electrons
Detector

http://www.chm.bris.ac.uk/pt/diamond/jamespthesis/chapter2_files/image002.gif

Scanning Electron Microscopy (SEM)

Black Ant

Neuron growing on astroglia

House Fly

Human stem cells

Human red blood cells

Neurons CNS

Scanning Probe Microscopes

Employ a sharp tip that is scanned over a
sample surface to measure some property
Two Types:

Scanning Tunneling Microscope (STM)

Atomic Force Microscope (AFM)

Scanning Probe Microscopes

STM (1980s):

can image individual atoms

very sharp metallic wire brought to within a few angstroms
of a surface and scanned over that surface
tunneling current is distance dependent - can actually be
used to manipulate atoms one at a time

AFM (1985):

uses tiny diamond-tip glued onto a thin piece of gold that

acts like a spring
actually in contact with the sample - can manipulate a
sample

Bone Histology Samples

optical

SEM

Lamellar Bone

Picture courtesy Gwen Childs, PhD.

Haversian System
Osteon with
central Haversian
canal containing

osteocyte

osteon

Cells
Vessels
Nerves

Volkmanns canal

Connects osteons
Picture courtesy Gwen Childs, PhD.

Haversian
canal

Volkmanns
canal

Osteoblasts

Picture courtesy Gwen Childs, Ph.D.

Osteocytes

Picture courtesy Gwen Childs, Ph.D.

Osteoclasts

Picture courtesy Gwen Childs, Ph.D.

Cartilage

Hyaline

Fibrocartilage

Elastic

Bone Remodeling: Quantification

Histomorphometry: measurement and analysis of
bone structure and bone remodeling. Usually performed
on cancellous bone from transiliac biopsies.
Isotropic (randomly oriented) nature of trabeculae in iliac
bone is assumed.
2D measurements (of area) converted to 3D (volume)
measurements. This is a fundamental stereologic
principle used in histomorphometry.

Bone Remodeling: Quantification

Using computer graphics,
multiple fields of known
medullary area/volume are
analyzed. Bone tissue volume
(TV) is the sum of field volumes
Trabeculae within each field are
graphically outlined and
trabecular bone volume (Tb.V),
and total trabecular bone surface
(Tb.S) are calculated.

Bone Remodeling: Quantification

Trabecular bone volume, (Tb.V) = relative volume of
total cancellous bone measured (TV) (expressed as
%) that is occupied by trabeculae.

Tb.V is about 20% in women and 22% in men.

Tb.V is related to cancellous bone mass and declines with
age and with bone loss

Note: Tb.V is also commonly referred to as Bone Volume /

Total Volume (BV/TV)

Bone Remodeling: Quantification

Bone Formation Rates (BFR/BV and BFR/BS):
calculated rates at which cancellous bone surface and
bone volume are being replaced annually.
Derived from estimates of:

Mineral Appositional Rate (MAR), (interlabel distance (4/)

(labeling interval) in m/Day x 365.
Relative Mineralizing Surface (MS), Bone Surfaces (BS) or
Bone Volume (BV)

BFR = MAR(MS/BS)
BFR= MAR(MS/BV)

Bone Remodeling: Quantification

Bone formation rates are expressed as:
BFR/BV in (mm/mm/yr)
BFR/BS in (mm/mm/yr)

Bone Remodeling: Quantification

NORMAL MEAN VALUES
Parameter

Female mean Male mean

Wall Thickness (W.Th)

49.8 m

Mineral Apposition Rate (MAR)

0.88 m/d

0.89 m/d

Bone formation Rate

Surface (BFR/BS) (mm/mm/yr)
Volume (BFR/BV) (mm/mm/yr)

0.019
0.250

0.009
0.131

Mineralization Lag Time (M.Lt)

21.1 d

27.6 d

Activation Frequency (Ac.f)

0.42 y

Review
I. Basic Principles of Histology
II. Histology Methods Related to Bone Imaging
III. Staining and Dying
IV. Bone Samples / Applications
V. Quantification of Bone Remodeling