what-when-how
In Depth Tutorials and Information
amino acid chains. This does not argue against a role
for the noncollagenous proteins, as they too influence
hydroxyapatite crystal aggregation and shape (e.g.,
15-17
)
as established by scanning and transmission electron
microscopy, but it suggests collagen itself can have a sig-
nificant effect in the mineralization process. The purpose
of this chapter is to review the methods that can be used
to study the effects of the matrix on bone properties in
OI, and to use data so derived to describe our view of
mineralization mechanisms in OI.
can be made that describe the bone histology and can be
used for comparisons between experimental groups. For
example, for “area” one can measure bone volume, oste-
oid volume, percentage of surface; for “distance” one can
measure trabecular size, intertrabecular distance, cortical
thickness; and for “number” osteoblasts, osteoclasts, blood
vessels, etc., can be counted. All of these measurements
and what they mean have been described by the ASBMR
Nomenclature Committee.
26
The measurements based on
the fluorescent labels are indicated as dynamic measure-
ments and measurements based purely on histological
appearance are called static measurements. A good illus-
tration of these methods is seen in a study of dynamic and
static histomorphometry in children with OI.
27
The processing of bone samples for histology is
determined by the final use for the sections. For para-
fin embedding and sectioning and subsequent stain-
ing to characterize the organic matrix, the bone samples
are decalcified with either ethylenediaminetetraacetic
acid (EDTA) or a dilute acid (
Figure 4.1
). For immuno-
staining purposes, EDTA is preferred as the decalcify-
ing agent since it permits a positive response to most of
the antibodies available. For calcified bone samples, the
tissues are frequently fixed in 70-80% ethyl alcohol if
fluorescent labeling is used to collect dynamic measure-
ments. Formalin may be used instead of alcohol; how-
ever, formalin tends to induce background non-specific
autofluorescence in the bone matrix which makes the
fluorescence analysis more difficult. The processing and
embedding procedure of Erben
28
for methyl-methacry-
late embedding of calcified tissues is recommended. This
procedure includes polymerization of the final blocks at
−20°C, which keeps the heat of polymerization to a mini-
mum which helps preserve tissue immunogenicity and
histochemistry. The blocks of embedded bone are sec-
tioned at 2-8 microns thick using a heavy duty sliding
microtome with a tungsten carbide blade. Sections are
collected on coated slides, plastic dissolved and stained
with Goldner's trichrome stain (for matrix) or von Kossa
stain (for mineral).
Some of the earliest histology and descriptive morphol-
ogy of OI came out of Johns Hopkins School of Medicine.
In 1952, Dr. Follis
29
described the pathology of osteogen-
esis imperfecta congenita for all of the tissues obtained
from a single still-born infant. He indicated that cartilage
and primary ossification centers were formed normally,
and fractures which were present appeared to be heal-
ing in a normal fashion. There were deposits of reticu-
lum within the osteoid which were distinguished by their
argyrophilia, to distinguish from collagen which does not
stain with silver. There were also significant deposits of
metachromatic material which could not be further iden-
tified. Dr. McKusick extended the study of OI
30
and also
clarified the differences between morphology of the con-
genital and the tarda forms of the disease. The conclusions
METHODOLOGY
Tissue Sources
The tissues available for analysis of the mineral and
matrix in OI come from studies of naturally occur-
ring models in mouse, such as the oim
/
oim,
18
cattle,
19,20
dogs
21-23
and other large animals,
24
cell and organ cultures
derived from patients with OI, mouse models engineered
to have an OI phenotype (see Chapter 21: Animal Models
of OI (Charlotte Phillips, Stephanie Carleton and Bettina
Gentry)), genetically modified zebra fish,25
25
and to a lim-
ited extent, tissue obtained from human subjects, gener-
ally during surgical procedures.
Analyses and How they Inform About Normal
Vs. OI Bone as a Function of Age
Matrix and Whole Tissue Analysis
HISTOLOGY
The histology and morphometry of bone is unique in
that this tissue consists of a calcified collagenous matrix
as well as bone-forming and bone-resorbing cell popula-
tions. Each of these components of bone can be measured
by morphometric means to provide a quantitative histol-
ogy for normal and pathological bone. For example, live
animals can be injected with fluorescent compounds such
as calcein (green fluorescence), doxycycline (yellow luo-
rescence) or xylenol orange (red orange fluorescence),
which localize specifically to new bone-forming areas. By
measuring the distance in microns between two differ-
ent fluorescent labels and dividing by the number of days
between injections, the mineral apposition rate (MAR)
can be calculated. Using the same samples, the amount of
bone surface labeled with fluorescent label divided by the
total bone surface within the microscopic field will indi-
cate the percentage of bone labeled. Knowing the percent
labeled surface and the MAR, one can then calculate the
bone formation rate (BFR). These values strongly indi-
cate how much bone is being produced, where it is being
deposited and the rate of new bone formation for each
animal within a study. There are many measurements that
