fixed in the cartilage matrix by their ability to bind to
hyaluronan, a long-chain, unbranched protein.
study population consisted of six human subjects (ages, 2, 4, 11,
5, 39, and 57 y). Data on sex of the patients and duration from
4
1
time of injury to excision of stenosis were not controlled.
The authors recognize that a large selection bias is inherent
in the present study because only specimens severe enough to
warrant surgical intervention were used as the basis for the data.
No determination can be made regarding each age group’s indi-
vidual tolerance to injury or biochemical response to injury as a
function of duration of injury. Furthermore, appropriate age-
matched control sections do not exist because normal, unaffected
trachea is not usually resected and archived.
Collagen II is the principal collagen secreted by chon-
drocytes and is specific to cartilage. It is a marker for
proliferating, differentiated chondrocytes and serves to
distinguish the chondrocytes from their mesenchymal pre-
cursors, which secrete collagen type I. Collagen II struc-
ture comprises three identical polypeptide chains (alpha
chains) twisted into a right-handed helix. Each helix is
further bonded both internally and to other collagen II
helices. The end result is a tightly bound lattice of collagen
II fibrils. Collagen II provides the tensile strength of the
cartilage structure; hence, a balance exists between the
intrinsic tendency of the cartilage to swell, because of its
hydrophilic aggrecan molecules, and the rigidity attribut-
Samples
Gross specimens were examined to ensure that the area to
be studied included the tracheal injury. Specimens were sec-
tioned in the axial plane at a thickness of 6 m, baked, and
deparaffinized. One section from each age group was stained with
hematoxylin and eosin (H&E); the remainder of the sections
underwent immunohistochemical staining as described in “Im-
munohistochemistry.” Areas of experimental interest consisted of
the sites of cartilage ring fracture relative to the remaining,
grossly normal ring. It is likely that the entire ring was exposed
to the inflammatory process; however, on H&E staining, the
areas of fracture represented a relatively greater loss of cartilage
matrix and were adjacent to other, better-preserved sections of
the ring.
5,6
able to the collagen II lattice.
Collagen I is the fibrillar collagen of bone, tendon,
dentin, and dermis. The distribution pattern of collagen I
suggests that it plays a major supporting role in tissues
that normally exhibit little distensibility under mechani-
cal stress, similar to collagen II in articular cartilage.
Collagen I is thought to be produced by the mesenchymal
precursors of chondrocytes; however, it is known to re-
main in the extracellular matrix after conversion of the
2
precursor cells to mature chondrocytes. It has a structure
similar to that of collagen II in that it is composed of three
polypeptide chains; however, not all three are identical
5
(
two alpha-1[I] and one alpha-2[I]). Like collagen II, col-
Immunohistochemistry
lagen I fibrils possess the ability to cross-link, thereby
forming a rigid, lattice-like structure.
Antibodies. The antibody II-II6B3 (Developmental Studies
Hybridoma Bank, Iowa City, IA) is a monoclonal antibody that
recognizes the triple helix of collagen II. It is specific for collagen
II without cross-reactivity to any of the other known collagens.
Antibody was used at a dilution of 25 g/mL in 2% goat serum in
phosphate-buffered saline (PBS).
5,6
Because collagen I is the predominant component of
scar tissue, an area of investigation has centered around
whether articular cartilage is able to heal with predomi-
nantly type I or type II collagen. The collagen II lattice has
a specific tensile strength for load bearing, and this carti-
lage characteristic may be altered by replacement with a
collagen I lattice. To date, the results have been conflict-
ing; however, in general, articular hyaline cartilage was
able to regenerate with collagen II if the defect was in
proximity to the marrow or if the cartilage was relatively
The antibody 5-D-4 (lot 99401, Seikagaku Corp., Falmouth,
MA) is a monoclonal antibody that recognizes the keratan sulfate
region of the aggrecan molecule. This antibody was selected be-
cause it recognizes an epitope in and adjacent to the main site of
proteolytic cleavage by many of the matrix metalloproteinases.
Antibody was used at a dilution of 5 g/mL in 2% goat serum in
PBS.
Rabbit anti-human collagen I (lot 4129, Caltag Laborato-
ries, Burlingame, CA) is a polyclonal rabbit antibody that is
specific for collagen I antigenic determinants with no cross-
reactivity to collagen types II, III, IV, and V. Antibody was used
at a dilution of 20 g/mL in 2% goat serum in PBS.
Secondary antibodies consisted of goat anti-mouse and goat
anti-rabbit antibodies linked to fluorescein (Jackson ImmunoRe-
search, Westgrove, PA). Antibodies were used at a dilution of 15
g/mL in 2% goat serum in PBS.
4
7
immature. The present study investigates levels of colla-
gen types I and II in the areas of tracheal cartilage frac-
ture or attenuation to provide insight into which protein
provides structural support to the cartilage ring, which
proteins are most susceptible to the inflammatory process,
and which may be regenerated. These findings will also
provide information regarding the state of differentiation
of the chondrocyte because cell function can be correlated
with the production of distinct collagen types. In addition,
we seek to identify a potential developmental difference in
patterns of protein loss.
Staining. For specimens stained for aggrecan and collagen
II, bovine testicular hyaluronidase at a concentration of 1,600
units/mL acetate buffer was incubated with the tissue sections for
3
0 minutes at room temperature. For specimens stained for col-
lagen I, a pepsin digestion was performed using 0.4% pepsin in
.01 N HCl for 30 minutes. Slides were washed three times in
MATERIALS AND METHODS
0
Study Population
Tris-buffered saline and blocked in 2% goat serum for 1 hour at
room temperature. Incubations for the primary antibodies were
performed overnight at room temperature. Secondary antibodies
were incubated for 45 minutes at room temperature. Negative
controls for all antibodies consisted of tissue sections treated as
described earlier in the present study, without the use of the
primary antibody.
Archival, paraffin-embedded tissue blocks were obtained in
accordance with the Internal Review Board from the Pathology
Department of Massachusetts General Hospital (Boston, MA).
Specimens consisted of segments of human trachea excised for
repair of subglottic and tracheal stenosis because of intubation
injury. Gross specimens were required to have gross cartilage
integrity or fracture, or both, to be included in the study. The
Laryngoscope 112: June 2002
Mankarious et al.: Human Tracheal Stenosis
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026