Download Morphology: Allograft Heart Valves

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Morphology: Allograft Heart Valves
Stephen L. Hilbert, Frederick J. Schöen, and Victor J. Ferrans
Aortic and Pulmonary Valves
General Morphologic Features
Aortic and pulmonary valves are referred to
collectively as semilunar valves. The normal
aortic valve is non-obstructive when open, competent when closed, non-thrombogenic, noninjurious to blood cells, durable, resistant to
infection and, capable of continuously remodeling its extracellular matrix and repairing itself
when injured. The dilated pockets of aortic root
behind the valve cusps bulge with each systolic
ejection of blood and are called sinuses of Valsalva.1 Normally, the three aortic valve cusps
fold back into their respective sinuses of
Valsalva when the left ventricular (LV) pressure exceeds that in the aortic root (in ventricular systole).When aortic root pressure exceeds
that in the LV cavity, the cusps fall back across
the outflow tract. Prolapse into the LV is prevented by their concave semilunar shape and
coaptation of cuspal free edges (in ventricular
diastole).
Aortic valve function normally relies on its 3
cusps, the annular fibrous tissue, and aortic
root/sinuses of Valsalva. The aortic valve cusps
attach to the aortic wall in a crescentic fashion,
ascending to sites where adjacent cusps are
separated by only a small distance (commissures) and descending to the trough of each
cusp.1 The three commissures are spaced circumferentially approximately 120° apart and
occupy the three points of a triradiate crown.
The aortic valve cusps are named for their rela-
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tionship to the coronary artery ostia; thus, a
normal valve has right, left and non-coronary
cusps. The structure of the pulmonary valve and
surrounding tissues is similar to that of the
aortic valve, except for a thinner structure, lack
of well-developed sinuses behind the cusps and
absence of coronary arterial orifices.
The three aortic cusps have a similar shape,
resembling that of a half-moon (frequently
referred to as semilunar cusps), but are usually
unequal in size. The thin, crescentic region of
each cusp between its free edge and closing
edge, termed the lunula, defines the coaptive
region of the cusps. During valve closure, the
individual halves of the lunulas of one cusp
contact the corresponding regions of both adjacent cusps, thereby effecting a competent seal.
A fibrous mound, known as the nodule of Arantius (nodulus Arantii), is located on the ventricular surface, in the middle of the free edge of
each cusp.1 Visible semilunar ridges 2 to 3 mm
from the cuspal free edge define the lower edge
of the lunula and rise to meet the nodule of
Arantius. Coaptation of the three nodules
ensures complete central closure of the valve
orifice during ventricular diastole. Since the
cross-sectional area of the aortic root is smaller
than the total surface area of the cusps, normal
aortic valve cusps overlap as much as 40% of
their area in the closed position.
Although it is common to have fenestrations
(holes) near the free edges as a developmental
or degenerative abnormality, this generally has
no functional significance, since the lunular
tissue does not contribute to separating aortic
22. Morphology: Allograft Heart Valves
201
Figure 22.1. Light micrograph
illustrating the characteristic histologic appearance of a porcine
aortic valve. Three distinct regions
are present: the ventricularis (v),
the spongiosa (s), and the fibrosa
(f). H&E stain. ¥ 100.
from ventricular blood during diastole. In contrast, fenestrations below the lunula cause
incompetence and they suggest a previous or
active infection.
The aortic and pulmonary valves have a well
defined histologic structure1 and lack intrinsic
blood vessels, since they are sufficiently thin to
receive nutrients by diffusion from surrounding
blood. The presence of nerve fibers and nerve
terminals have recently been demonstrated in
aortic valve cusps, although the functional significance of this finding is unknown.2
The cusps of the semilunar valves consist of
three distinct histologic layers (from the inflow
surface): the ventricularis, the spongiosa and
the fibrosa (Figure 22.1). These layers are
similar in distribution in all semilunar valves;
however, the pulmonary valve is thinner and
more delicate in appearance.1 Ultrastructural
studies of the pulmonary valve have been
limited.3 Thus, the description which follows is
based primarily on studies of the aortic valve in
humans and in various animal species.4–13
Among the latter, porcine aortic valves have
been extensively studied, due to their use as
bioprosthetic heart valves. The only significant
morphologic difference between the human
and the porcine aortic valve involves the right
coronary cusp, which in swine is larger than the
other two cusps and contains a “muscle shelf”
located in the basal one-third to one-half of the
cusp. The “muscle shelf” consists of cardiac
myocytes and their vascular supply and represents an extension of the ventricular septal
muscle into the basal region of the cusp. This
muscle layer also may be present, but to a lesser
extent, in the non-coronary cusp.6 Thus, the
hemodynamic implications of the presence of a
“muscle shelf” (such as an asynchronous,
delayed opening of the cusp), a reduced effective orifice area, and the propensity of this
region to postimplantation degenerative calcification, are of no concern with the use of
human allograft valves.
Cellular Components
Four major types of cells are present in cardiac
valves: 1) endothelial cells; 2) interstitial connective cells; 3) mononuclear cells derived from
the blood, and 4) interstitial dendritic cells. The
endothelial cells form a continuous monolayer
that completely lines the surfaces of the valves
and is contiguous with the endothelial cell layer
of adjacent regions of the endocardium and/or
great vessels. These cells are flattened, have
single, centrally located nuclei, contain actinlike and intermediate (10 nm) filaments and are
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connected by junctional complexes9 that
provide a permeability barrier to the diffusion
of substances from the blood into the valvular
tissue. The interstitial connective tissue cells are
present throughout all layers of the valve. Cell
counts have demonstrated a relatively uniform
distribution of cells within specific histologic
regions of porcine aortic valve cusps, (e.g., a
mean +/- S.D. of 1760 +/- 312 nuclei/mm2 of
tissue section in the spongiosa and 1960≤68
in the fibrosa).14 These interstitial cells usually
are referred to as “valvular fibroblasts”, even
though they actually show a spectrum of morphologic differentiation.10–12 This spectrum
includes: 1) clearly fibroblastic cells (with relatively extensively developed rough-surfaced
endoplasmic reticulum); 2) intermediate forms,
such as myofibroblasts (with less abundant
endoplasmic reticulum, more developed actinlike filaments and peripherally located dense
bodies that serve as attachment sites for the
filaments) and 3) typical smooth muscle cells,
with few cisterns of endoplasmic reticulum but
very abundant actin-like filaments, peripherally
located dense bodies and well developed basement membranes. These cells have been shown
to form a network in which they are interconnected by gap junctions.12 Small numbers of
macrophages and lymphocytes (mostly T-
S.L. Hilbert et al.
lymphocytes) are also present throughout the
various layers of the valves. In addition, interstitial dendritic cells also have been demonstrated to be present in heart valves. These cells
are thin, uninucleated and have very slender,
elongated cytoplasmic processes, but lack basement membranes and actin filaments. They are
similar to cells of this type that are present
throughout a variety of other tissues, including
the heart.13 By the use of immunohistochemical
techniques, they can be identified specifically
and distinguished from smooth muscle cells and
other types of connective tissue cells.15,15a These
cells are presumed to function, as they do in
other tissues, in the presentation and processing of antigens.
The ventricularis is an extension of the ventricular endocardium. It is subjacent to the
spongiosa and is in direct contact with the
endothelial cell layer lining the inflow surface
of the cusp. The ventricularis contains connective tissue cells (as described above), multidirectionally oriented collagen fibers and an
extensive network of elastic fibers. The most
prominent elastic fibers are oriented perpendicular to the free edge of the cusp (Figure 22.2).
The ventricularis is thickened in the region of
cuspal coaptation at the nodulus Arantii in the
aortic valve (nodulus Morgagni in the pul-
Figure 22.2. Histologic section
of a porcine aortic valve demonstrating abundant, radially oriented elastic fibers (arrowhead)
within the ventricularis. The
inflow and outflow surfaces of the
leaflet are lined by endothelial
cells. Movat pentachrome stain.
¥ 200.
22. Morphology: Allograft Heart Valves
203
Figure 22.3. Transmission electron micrograph of the outflow
surface of a native porcine
aortic valve. Note the presence
of an intact endothelial cell
layer, a cell-cell junctional
complex (arrowhead) and reduplication of the basement membrane (arrow). Fibroblasts, collagen fibrils and elastic fibers
(stained black) are seen in the
fibrosa. Kajikawa stain. ¥ 9,000.
monary valve).1 As a practical note, the ultrastructural study of cardiac valves involves a
number of unusually difficult problems, particularly the proper identification of the inflow
and outflow regions of the tissue specimen
being examined. For this purpose, an elastic
fiber stain (Kajikawa) (Figure 22.3) is preferable to routine electron microscopic stains,
since it facilitates the identification of the
ventricularis.6
The central layer of the cusp is referred to as
the spongiosa.This layer is histologically similar
in semilunar and in atrioventricular valves
and is composed of loosely arranged collagen
fibers, fibroblasts and other types of connective
tissue cells embedded in an extracellular matrix
rich in proteoglycans. The spongiosa is particularly prominent in the basal aspect of
the cusp and does not extend to the free
edge, which consists of only the fibrosa and the
ventricularis.
The fibrosa is composed of fibroblasts, other
types of connective tissue cells and dense collagen bundles, which serve as the major structural component of the outflow region of the
cusp. The collagen bundles approaching the
commissures are arranged into densely packed
cords, which are integrated into the valvular
ring. A few connective tissue cells, including
elongated fibroblasts and myofibroblasts,10–12
and small numbers of elastic fibers are present
in the fibrosa. Elastic fibers are prominently
seen in the basal region of the cusp near the
outflow surface and appear as a distinct histologic layer, referred to as the arterialis. A single
layer of endothelial cells lines the outflow
surface of the cusp. Reduplication of the
endothelial basement membrane is a unique
ultrastructural marker that serves to identify
the outflow surface of the porcine aortic valve
cusp (Figure 22.3).6
Morphologic studies of the aortic valve
surface have provided insights into the effects
of the physical forces (e.g., pressure, tension)
applied to the valvular tissue at the time of histologic fixation.4 The inflow surface is much
smoother than the outflow surface, demonstrating radially oriented fine striations that
correspond to the elastic fibers present in the
ventricularis. The outflow surface has a corrugated appearance, due to the presence of
coarse, circumferentially arranged collagen
bundles in the fibrosa (Figure 22.4). These
bundles consist of densely packed collagen
fibrils, which appear wavy or crimped when in
the relaxed state. As increasing pressure is
204
S.L. Hilbert et al.
Figure 22.4. Scanning electron micrograph of the surface
of a cryopreserved pulmonary
valve allograft, demonstrating
the corrugated appearance
produced by the underlying
collagen bundles and the
marked retention of collagen
crimp. ¥ 750.
applied to the valvular tissue, the collagen
fibrils become elongated or straightened, with
consequent loss of crimp (Figure 22.5). Polarized light microscopy is particularly well suited
for the assessment of the extent of collagen
crimp (Figure 22.6). It is noteworthy that pressure gradients as small as 2 mm Hg are sufficient
to significantly alter the magnitude of collagen
crimp (Figure 22.7).16,17
Extracellular Matrix
Cardiac valves are primarily composed of
extracellular matrix components of connective
Figure
22.5. Transmission
electron micrograph demonstrating the loss of collagen
crimp, as shown by collagen
fibril elongation or straightening. High pressure-fixed porcine aortic valve. ¥ 2,900.
22. Morphology: Allograft Heart Valves
205
Figure 22.6. Polarized light
micrographs depicting the morphologic appearance of the
collagen bundles and collagen
cords of a porcine aortic valve.
(A) Characteristic birefringent
banding pattern of collagen
bundles in which crimp is
retained. (B) Loss of collagen
crimp (straightening) resulting
from tissue elongation by the
application of a 100 gram load
to the leaflet. ¥ 100.
tissue. Maintained by the interstitial cells, these
components consist mainly of collagen fibrils,
elastic fibers and proteoglycans, which represent the bulk of the physical mass of the cusp.
As described above, the histologic organization
of these components into three distinct layers
Figure 22.7. Histologic section
illustrating the effects of pressure fixation on the morphologic appearance of a porcine
aortic valve. Note the presence
of three distinct histologic
regions (ventricularis, spongiosa, fibrosa); however, due to
the loss of collagen crimp, the
collagen bundles have become
elongated, resulting in the
overall linear appearance of
the valvular tissue. Compare
with Figure 6.4. Toluidine blue
stain. ¥ 200.
determines the unique biomechanical properties and resultant functional characteristics of
cardiac valves.9,18–22 Type I and Type III collagen
are the most abundant extracellular proteins in
cardiac valves; however, Type V collagen is also
present as a minor component.23 Recent studies
206
S.L. Hilbert et al.
characterizing the types of collagen in cartilage have demonstrated that collagens may be
present as either homogeneous fibrils or as
mixed fibrils.24 Similar studies of cardiac valve
collagens have not been made.
The functional effects of proteoglycancollagen interactions may involve the regulation of extracellular polymerization of collagen
fibrils.25,26 Domains of high positive charge
density are located within the collagen fibril
at sites of overlap of adjacent collagen molecules (i.e., carboxyl- and amino-terminal
end of the polypeptide chain; staggered overlap region). These sites are involved in the
formation of intermolecular cross-links in the
collagen fibril. The extent of the cross-linking
of the collagen fibrils may be regulated by
the presence of proteoglycan glucosaminoglycan side chains and at these cationic sites.25,26
These concepts are of theoretical and practical
significance concerning the direction of future
research on the viability of cells in cryopre-
served allografts and on the mechanisms of
renewal of the extracellular matrix of allograft
valves.
In addition to the various morphologic characteristics of allograft valves, a variety of
gender and age-related anatomic and histologic
changes occur in human aortic valves. The following changes are most frequently observed
in valves harvested from male donors: 1)
degeneration of collagen fibers; 2) a fibroelastic “spur” along the coaptation surface; 3) a
decrease in the number of cuspal fibroblasts
and in the amount of proteoglycans; 4) accumulation of extracellular lipid (Figure 22.8) and
5) calcific deposits (Figure 22.9).27–29 Because of
the prevalence of these changes, current donor
criteria for allograft heart valves include an age
restriction and a negative medical history of
previous cardiac surgery, uncontrolled hypertension, functional cardiac murmurs, rheumatic
fever and malignant, autoimmune or vascular
diseases.29
Figure 22.8. Explanted aortic valve demonstrating
lipid accumulation, collagen fiber degeneration and
the loss of typical valvular histologic features. The
cleft-shaped inclusions indicate that cholesterol
esters were present in the valvular tissue. H&E stain.
¥ 200.
22. Morphology: Allograft Heart Valves
207
Figure 22.9. Histologic section of a calcific nodule
within the spongiosa of a porcine aortic valvular bioprothesis. This nodule has resulted in deformation
and disorganization of the collagen bundles in the
fibrosa. Note the loss of the histologic layering of the
valve and the insudation of plasma proteins (arrow)
and lipids (arrowhead). Toluidine blue stain. Glycol
methacrylate. ¥ 100.
Mitral Valve
semilunar valves, this layer dips into the insertion sites of the chordae tendineae.
The fibrosa contains fibroblasts and other
types of connective tissue cells, as well as a
continuous layer of crimped collagen bundles
that are oriented parallel (circumferential) to
the ventricular surface and continue into the
chordae tendineae. The latter structures consist
of an outer layer lined by endothelial cells and
an inner core. The outer layer has a subendothelial region containing a few collagen and
elastic fibers. As mentioned above, the inner
core is a continuation of the valvular fibrosa
and consists of longitudinally oriented, crimped
collagen bundles. Fibroblasts and myofibroblasts are interspersed between the collagen
bundles in these areas.
The ventricularis represents a continuation
of the ventricular endocardium. It is covered by
an endothelial cell layer with a subendothelial
region rich in proteoglycans and elastic fibers.
The elastic fibers in the ventricularis of the posterior cusp are shorter than those in the auricularis. The ventricularis of the anterior cusp, in
General Morphologic Features
The anterior mitral valve leaflet is significantly
larger than the posterior leaflet; in addition,
there are a few histologic differences.1 The histologic appearance of the mitral valve leaflet is
similar to that previously described for the
semilunar valve cusps.
The auricularis of the mitral valve consists of
an elastic lamina with interspersed collagen
fibers and smooth muscle cells lying beneath a
continuous layer of endothelial cells. The auricularis may comprise as much as 20% of the
leaflet structure and represents a continuation
of the atrial endocardium, extending over approximately 60% of the leaflet surface. At the
free edge of the leaflet, all the histologic regions
merge, and the elastic lamellae are less superficial and are covered by a layer of connective
tissue containing collagen fibers and few cells.
The spongiosa is histologically similar to that
of aortic and pulmonary valves. In contrast to
208
S.L. Hilbert et al.
contrast to that of the posterior cusp, is a continuation of the subaortic endocardium and can
be readily identified by the presence of dense
elastic fibers, which may be more prominent
than those in the auricularis.
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