Transcript Advantages Of Homograft

Homograft, Xenograft
& Bioprosthesis
Seoul National University Hospital
Department of Thoracic & Cardiovascular Surgery
Valved Homografts
Introduction
• Valved homografts, introduced in the 1960s, have
used for reconstruction of the right ventricular outflow
tract .
• Advantages of homografts, such as ease of handling and
improved hemostasis, have been major contributing
factors
• Viable endothelial cells and perhaps also viable
fibroblasts, thus contributing to an antigenically active
cells, which could lead to a more intense host response.
• This increased immunogenicity could perhaps be an
important contributing factor for accelerated
fibrocalcifications, particularly in very young patients.
Homograft
Processing
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Cryopreservation
Antibiotics
Fetal calf serum
RPMI media
DMSO
Thawing
Harvest of aortic,
mitral, pulmonary
valve & others
Sterilization for Homograft
Antibiotics for uses
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Modified Hanks solution or TCM
Cefoxitin sodium ( 240mg/L )
Vancomycin hydrochloride ( 50mg/L)
Lincomycin hydrochloride ( 120mg/L)
Polymixin B sulfate ( 100mg/L)
Amphotericin B ( 25mg/L )
Homograft Processing
Essence
1. Possible procurement on beating heart, but
non-beating heart within 24 hrs after death
2. Reduce antibiotic incubation time to 6 hrs
3. Fibroblast is important in the maintenance
of the valve matrix.
* Viable cells persist, produce collagen and
repair damaged matrix.
4. Low dose antibiotics and no amphotericin B
Homograft
Laboratory evaluation
• Hematoxylin-eosin & elastica van Gieson
• Elastic fiber of vessel wall
• Muscle actin positive cell
• Immunohistochemical staining
• Flow cytometry
• Antibodies for inflammatory cell
• CD3 for all T lymphocytes
• CD4 for TH lymphocytes and macrophages
• CD8 for TC lymphocytes and NK cells
• Scanning EM for endothelial integrity
Homograft
Advantages
1. Technical ease of implantation because they are soft
& mold easily with patient’s tissue, resulting in
better hemostasis in complex operations
2. Better hemodynamics than porcine-valved dacron
conduit, which improves the RV function after
operation.
3. The branch of the homograft may be used to patch
distal pulmonary artery stenoses
Homograft
Durability of aortic valve
• Homograft can last 20 years, essentially as a dead piece
of tissue(become acellular within a few months), free
from any mechanical reinforcement by crosslinking
agents.
• Unknown exactly what features of the native valve
tissue give it such remarkable durability than other
bioprosthesis, the importance of its internal complexity
is being appreciated more and more.
• Presence of interconnected sheets of collagen, layers
and tubes of elastin, highly nonlinear mechanics,
anisotropy, and viscoelasticity endow the valve tissue
with is unique longevity.
Cryopreservation
Methods
• Treatment with antibiotic agents at 4 ℃ for 24 hours or
culture medium with antibiotics
• Frozen in tissue culture medium(TCM) 199 solution
containing 10% calf serum, 10% DMSO, and 5%
HEPES buffer, with a cooling velocity at a rate of -1 ℃ /
min to -80 ℃ with programmable controlled-rate
freezer.
• Frozen xenograft is put into sealed package and
preserved in vapor phase of liquid nitrogen at -196 ℃
• The cryopreserved xenograft is thawed in a water bath
at 37 ℃ quickly
Cryopreservation
Protocol
• Transport
Maximum 24 h at 4°C
RPMI nr. 1640 300 mL
• Sterilization
14–18 h at 4°C
RPMI nr. 1640 300 mL
24 mg Mefoxin. 12 mg Lincomycine
5 mg Vancomycine. 100000 UI Colymicine
• Storage
–150°C until implant
90 g Albumine 4%
10 mL DMSO
DMSO = dymethylsulphoxide;
RPMI = Roswell Park Memorial Institute.
Cryopreservation
Influence on immunity
• Cryopreservation technique might attenuate
allograft immunogenicity by reducing the
viability and cellularity of the endothelium or
by diminishing the expression of leukocyte
adhesion molecule
• Cryopreservation technique reduces the
xenogeneic immunogenicity of endothelial cells
as in allograft
• Cryopreservation technique could not reduce
the immunogenicity of other cellular &
extracellular matrices of xenograft
Cryopreservation
Effects on cells
• Cytosolic and mitochondrial functions of endothelial
cells were damaged seriously during cryopreservation,
especially in thawing process, and those process may
cause a latent cytosolic and mitochondrial injury, even
in the fibroblast
• Viability of donor fibroblast which can remodel the
matrix assembly is damaged by cryopreservation and
thawing, that may cause collagenolytic activation and
degradation of collagen synthesis in a cryopreserved
valve, which will lead to destruction of matrix
Cryopreservation
Effects on valve tissue
• The valvular extracellular matrix(ECM ) contains a
variety of structures, underlies and surrounds the
interstitial cells, and performs many essential functions,
including mechanical support and physical strength
• ECM exerts profound influences on cell adherence,
migration, and differentiation as well as the pattern of
gene expression of the cells in contact with it
• The quality of the structural matrix at implantation
may predetermine durability or failure of a
cryopreserved heart valve (collagenous bundles and
elastin-containing fibers) based on different techniques
before implantation
Cryopreservation
Effects on valve matrix
• Conventional approaches to cryopreservation of heart
valves have a detrimental and destructive effect on
crucial leaflet matrix elements.
• Serious alteration and significant deterioration of
collagenous and elastic fiber structures, accompanied
by a general damage of the leaflet histoarchitecture
caused by extracellular ice formation.
• Alternative preservation techniques proposed for
clinical use, such as vitrification, D-hydro-, or glyceroltreatment, are crucial.
Cryopreservation
Mechanisms of structural alteration
• Related to an activation of metalloproteinases (MMPs)
upon thawing, and disruption of normal extracellular
matrix metabolism by the valve interstitial cells, a
function shown to be impaired in cryopreserved tissue
• Side specific cell phenotypes and differences in
extracellular matrix organization may account for the
heterogeneity in extracellular matrix alteration.
• The lack of crimp in the thawed cryopreserved valves
suggests that the biomechanical properties of the
ventricularis layer have been altered
Cryopreserved Homografts
Examination parameters
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Tissue structure
Tissue viability
Cell proliferative capacity
Metabolic function
Identification of cell-specific antigens
( Immunohistochemistry)
• Flow cytometry( MHC I and II antibody)
Cryopreserved Homograft
 Viability test
1. Radiolabeled thymidine
2. Collagen synthesis
3. Protein synthesis
4. Dye uptake & dye exclusion
5. Autoradiography
Homograft Viability Test
Methods for viability
• Cytometry using propidium iodure and
fluorescent diacetate (FDA).
• Double staining with fluorescein
diacetate-propidium iodure (FDA-PI) is
reported to be a rapid method for
assessing cell viability compared with the
trypan blue dye exclusion method
Cryopreserved Homografts
Structural fate
• Viable fibroblasts synthesize main constituents of
extracellular matrix: collagen, elastin, reticulin, and
mucopolysaccharides, therefore longevity of the
implantation is likely related to the viability of
fibroblasts in the implanted valves
• However, fibroblast in the donor allograft were unable
to survive long, because of apoptosis.
• Endothelial cells exhibit strong antigenicity, but can not
survive under ischemic conditions and moreover,
during the cryopreservation process endothelial cells
lose their ability to proliferate.
• The homograft will lose its endothelial cells and
fibroblasts , and eventually become a non-viable tissue.
Cryopreserved Homograft
Standard model
• This demonstrates preservation of endothelial & valve
architecture & viability
• This viability may not be a positive attribute, as initial
thought, it contributes to immunogenicity and elicits a
more vigorous immunologic reaction from the recipient.
• Heightened immune response contributes to accelerated
degeneration of allograft valve
• An immunologically neutral graft has therefore been
considered desirable, leading to new development that
is decellularized to avoid introduction of immunogenic
cells into the recipient
Cryopreservation
Effects on immune reaction
• There were donor-specific immune responses evoked by
allograft insertion, and on the other hand , clinically
explanted allografts showed no evidence of rejection
• Cryopreservation causes the greatest delay & diminuation
of the expression of leukocyte adhesion molecules
• Cryopreservation attenuates the allograft immunogenicity
by reducing the viability and cellularity of endothelium
• But it seems to be impossible to reduce xenogeneic
immunogenicity with cryopreservation because of the
marked destruction of other cellular and extracellular
components
Aortic Valve Replacement
Advantages of homograft
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Good hemodynamics,
Fewer thromboembolic complications
Avoidance of anticoagulation
Suitability in the presence of infection
Disadvantages of homograft
• Risk of early failure due to technical error
• Limited durability and availability
Homograft Implantation
Surgical indication of endocarditis
• Hemodynamic instability
• Microbiology resistance, usually involved in active aortic
endocarditis including vegetations, cusp destruction, and
periannular extensions.
• Aortic vegetations, especially when their diameter is
superior to 10 mm, may be responsible for systemic
embolization
• Anatomic lesions progression are traditional
complications leading to early surgery
• Aortic periannular extension can degenerate in abscesses,
discontinuity, fistulas, aberrant communications and
false aneurysm, increasing congestive heart failure, and
mortality rate
Homografts Implantation
Aortic position
• pulmonary autograft is not recommended for young
rheumatic aortic valve patients, particularly with
mitral valve involvement
• Ross procedure could be performed in young aortic
valve patients if their mitral and pulmonary valves are
intact
• HAVR should be tailored to individual patients so as to
choose the scalloped subcoronary or root replacement
• In view of the long-term prognosis, the surgeon should
use, if possible, the subcoronary replacement technique,
which makes the reoperation much easier
Homografts Implantation
Pulmonary position
• Superior conduit durability in Ross patients has
emphasized the placement of the homograft in the
orthotopic pulmonary position and less sternal
compression of anteriorly placed conduits
• The predominant mechanism of homograft stenosis was
a poorly understood inflammatory reaction based on
their noting of early onset of stenosis, rapid clinical
progression
• Stretching and lengthening of the homograft causing
release of tissue factors was one possibility suggested
and the extent of that phenomenon might relate to the
degree of peripheral vascular distortion present
Homograft Failure
Etiologic factors
• Factors unrelated to immunologic injury would include
mechanical factors, such as sternal compression, or
oversizing or undersizing of conduits with anatomic
distortion, in addition to ischemic injury.
• Factors related to immunologic injury include ABO
incompatibility and human leukocyte antigens (HLA)
incompatibility
• Tissue processing steps such as warm ischemic time,
antibiotic disinfection, cryopreservation, and thawing
all affect viability of the grafts
• More recently, investigators have demonstrated
significant cellular infiltration with T-cells and B-cells
in failed explanted valved allografts in children
Homograft Failure
Specials in young
• The most important factors are size of conduit & age
at implantation.
• Primary reason, growth of young child over time
• Secondary reason, decrease in the functional lumen
of the conduit due to calcification or immune related
• Anatomic type of the conduit, aortic or pulmonary
• Heightened immune reaction to the implanted tissue
• There is evidence that children produce a virulent Tcell response, where adults mount a much weaker
response
Homograft Failure
Risk Factors
1. Younger recipient age
2. Aortic homograft related to higher elastin
& intrinsic calcium content
3. Small homograft valve size
4. Long aortic cross-clamp time at operation
5. Pulmonary hypertension
6. Distal pulmonary artery disease
7. Younger donor age ( increased immunogenecity )
8. Technical factors
1) Hood extension of proximal suture line
2) Anatomic versus nonanatomic placement
3) Compression of conduit
Homograft Failure
Alternatives to homograft
• After a period of 10 years, 30% of the children initially
corrected with allografts and 70% of the patients with
xenografts had undergone replacement of their initial
conduits.
• We have avoided using an allograft or heterograft
conduit in most patients with tetralogy of Fallot or
pulmonary atresia with ventricular septal defect.
• We have preferred to construct a transannular patch
with an underlying PTFE monocusp
• Until recently, cryopreserved allografts have been the
best valved conduits and the limited durability of the
allograft conduit became evident within a few years
Implanted Allografts
Failure process
• Transplanted cryopreserved homografts rapidly
lose their cellular components during the first year of
implantation while the normal tissue architecture is
damaged.
• The allografts are capable of eliciting a cellular and
humoral immune response, it is not yet clear what exact
role the immune response plays in ECM damage
• After transplantation, indicators of immune-mediated
injury as persistent up-regulation of leukocyte adhesion
molecules, the presence of neutrophyl granulocytes,
depositions of antibodies and complement are missing in
early phase after implantation.
Implanted Allografts
Loss of cellularity
• Immunologic & chemical injury
Allografts are eliciting a cellular &
humoral immune response, not yet clear
what exact role the immune response
• Hypoxia during valve processing
• Reperfusion injury at implantation
Homograft
Immune responses
• Children & adults respond differently to homograft.
• Younger donor age related to increased
immunogenicity
• There is evidence that children produce a virulent
T-cell response, where adults mount a much weaker
response.
• In addition, laboratory studies have demonstrated
that allograft valve destruction is T-cell mediated.
• Other HLA loci and nonimmunologic factors,
including growth, degeneration, and technique, play
a role in homograft failure
Implanted Allografts
Immune responses
• All allograft recipients are likely to form IgG- and Tcell-mediated reaction to donor HLA antigens
• Donor-specific T cells are likely to be the main agents of
allograft injury, effected through secretion of high
levels of cytokines
• The location of induction and amplication of the
immune response to the allograft remains unknown.
• Dendritic cells, which is identified in human great
vessels, together with endothelial cells are capable of
presenting foreign HLA class I and II antigens to
recipient T cells
Implanted Allografts
• Immune responses
• Valve leaflet cellularity was demonstrated to be
significantly reduced with time
• Donor-specific immune activation, some early
infiltration of immune effector cells, such as
macrophages and T lymphocytes is demonstrated
• Most of the evidence for immune-mediated damage in
this animal model occurred in the first 2 weeks after
implantation, and by 4 weeks the valves were largely
acellular
• This observation may in fact represent evidence of
immune-related valve injury, despite the absence of
up-regulation of cell adhesion molecules
Homograft Failure
Fate of implanted homograft
• Virtually all cryopreserved homografts demonstrate
acellularity within a year of implantation
• The lack of cells and cellular function limits tissue
growth, performance, and reparative capacity; such
"tissues" typically scar and then mineralize, which
often leads to dysfunction.
• Chronic rejection of cells in classically cryopreserved
allograft tissues promotes the migration of
inflammatory cells, exacerbating tissue degeneration,
fibrosis, and functional failure
Homograft Implantation
Reduce immune response
• One would be to match all allografts by blood
type and HLA type.
• Another would be to somehow remove the
immunogenicity of valved allograft tissue,
perhaps by removing the antigen presenting
cells on the allograft
• The third option would be to immunosuppress
the recipient in order to abrogate the immune
response of the recipient to the donor allograft.
Blood Cells
Production
• In children, blood cells are produced in the
marrow of all bones.
• After the age of 20 years, only the marrow of the
vertebrae, sternum, and ribs remain significantly
active (red marrow) in the production of
erythrocytes, many leukocytes, and platelets.
• Active marrow contains pluripotential stem cells
that are capable of replacing bone marrow
completely (self-renewal) or can be diverted from
self-renewal toward separate pools of committed
progenitor cells
Stem Cells & Progenitor
Cells
Characteristics
• Progenitor cells : (1) they have lost the
capacity for self renewal and (2) they are not
pluripotential but are committed to produce
(under the proper growth conditions) daughter
cells of a particular type.
During maturation, each cell line, promoted by a
variety of stimulating factors, acquires distinctive
properties
• Pluripotent stem cell can differentiate in a few
divisions into one of six classes of progenitor
cells
Stem Cells & Progenitor
Cells
A pluripotent stem cell can differentiate in a few divisions into one of six
classes
of progenitor cells that go on to produce blast cells. Blast cells
are the earliest morphologically distinct precursors of specific cell types.
EPO = erythropoietin; GCSF= granulocyte colony-stimulating factor; GMCSF = granulocyte-macrophage colony-stimulating factor; IL = interleukin;
M-CSF = monocyte colony-stimulating factor; PMNs = polymorphonuclear
Red Blood Cell
Immunologic properties
• The red cells of different individuals differ to a very small
extent in the structure of some carbohydrates that are part
of membrane glycolipids
• These differences bestow antigenic properties on red blood
cells and cause red cell agglutination
 ABO antigens
• The A and B antigens are the most important of the more
than 100 different blood group antigens that have been
identified
• In neonatal life, antibodies quickly develop against the
antigens that are not present on our red cells, and these
antibodies, called agglutinins, are carried in plasma.
• The antigens are called agglutinogens and are carried on
the red cells in the blood as well as on cells in many other
Red Blood Cell
Rh antigens
• Within the Rh system, the C, D, and E antigens
are most important. They are found only in red
cells.
• D is the most antigenic component, and the
presence or absence of D is designated as “Rhpositive” or “Rh-negative,” respectively.
• 85 % of Caucasians & more than 99% of Asians
are Rh+
• Anti-D develops only when the blood of a D–
individual is exposed to D+ red cells.
• This can occur as a result of transfusion or when
Rh+ fetal blood mixes with the circulation of an
Rh– mother
Antigens of ABO group
A, H antigen that is present in individuals with type O blood. B, A
antigen (type A blood) has a terminal N-acetylgalactosamine (NAG). C,
B antigen (type B blood) has a terminal galactose (Gal). Cer = ceramide;
Fuc = fucose; Gal = galatose; Glu = glucose.
Human Blood
Human Blood
Components of plasma
• The major plasma proteins are albumin (4.5 g/dL),
several globulins (2.5 g/dL), and fibrinogen(0.3
g/dL).
• Most are synthesized by the liver, and they have
five major functions: (1) carriers for hormones,
trace metals, or drugs; (2) proteolytic agents in
the cleavage of various hormonal or enzymatic
precursors; (3) protease inhibitors; (4) source of
plasma colloid osmotic pressure;(5) source of the
humoral immunity portion of the immune system.
Arachidonic Acid Derivatives
• Derivatives of arachidonic acid play a crucial role in platelet adhesion
to the endothelium, and the clotting process can be disrupted by
interruption of arachidonic acid metabolism.
Vascular Endothelium
Roles in
hemostasis
Complement System
Natures
• A system of 11 plasma enzymes identified as C1 to
C9; C1 consists of the three subunits C1q, C1r, and
C1s.
The enzymes circulate in the inactive form
but can be activated to lyse foreign cells. The
activation proceeds in a step-like fashion, each
activated enzyme hydrolyzing a peptide bond in the
next inactive enzyme
• Classical pathway is triggered when
immunoglobulin G or M binds to cell surface
antigens
• Alternate pathway of complement activation does
not require binding of immunoglobulins to cell
surface antigens and triggered
Complement System
 Activation
• Classical pathway ; The consequent activation
cascade eventually leads to (a) activated C3, a
cell surface-associated factor that promotes
opsonization, and (b) activated (C5, C6, C7, C8,
C9), which is associated with production of
chemotactic substances, release of histamine,
and insertion of perforins into the plasma
membrane.
• Alternate pathway ; It is triggered when the
circulating protein factor I attaches to specific
surface polysaccharides in a bacterium or virus.
This pathway also leads to activation of C3 and
(C5, C6, C7, C8, C9) and their associated
opsonization or cell lysis.
Complement Activation
Monocytes
Function
• Monocytes are formed in the bone marrow, enter
the blood, and circulate for about 3 days before
they enter the tissues by diapedesis and become
tissue macrophages that differentiate to perform
specific functions in different tissues.
• They are phagocytic cells and perform many of
the same actions that are performed by
neutrophils
• By secreting a large number of lysosomal,
chemotactic, complement-activating, and
pyrogenic factors, they are key effectors in the
elimination of microorganisms and play an
important role in immunity and blood clot
formation.
Mast Cells
Function and role
• Mast cells are found in tissues, and although they
resemble basophils in some respects, they are different
and derive from a different marrow stem cell.
• They are markedly granulated and are frequently
found under epithelial surfaces.
• They are especially rich in heparin and histamine, and
the granules containing these substances are released
when immunoglobulin E (IgE)–coated antigens bind to
receptors on the mast cell surface.
• They trigger hypersensitivity reactions and participate
in inflammatory responses.
Granulocytes
Formation of granulocytes
• They arise from three populations of committed
stem cells in the bone marrow and arrive in the
tissues fully differentiated as eosinophils,
basophils, or neutrophils
• Eosinophils are especially abundant in mucosal
tissues of the respiratory, lower urinary, and GI
tracts. Their major role is to attack parasites and
also involved in allergic reactions
• Basophils are rich in histamine and heparin and
release inflammatory mediators when activated.
• Neutrophils are the first line of defense against
infections and play a crucial role in inflammation.
Granulocytes
Functions of granulocytes
• Granulocytes, especially neutrophil, contain
mechanisms by which they can progress rapidly
from a harmless circulating intravascular cell to a
specific phagocytic cell and killer of foreign
particles & bacteria
• In acute inflammation, neutrophils are captured
and mobilized within minutes to hours and
accumulate locally to form the initial defense in a
locally restricted area. They are followed by
monocytes within 1 day and by lymphocytes
within several days.
• Neutrophil involvement in acute inflammation can
be broken down into eight distinct phases:
Functions of Granulocytes
 Inflammation
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Recognition
Expression of adhesion molecules and inflammatory mediators
Hydrodynamic margination, capture, and rolling of neutrophils
Activation, adhesion, and spreading
Diapedesis
Migration
Phagocytosis
Apoptosis and elimination of neutrophils
 Phagocytosis
• Process of immobilization, ingestion, and digestion of foreign
agents by granulocytes and monocytes.
• A vital first step in phagocytosis is the activation of the
complement system
Acute Inflammation Process
 Recognition
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When tissue macrophages recognize foreign
particles that have invaded tissue, they release
a variety of inflammatory mediators, including
tubular necrosis factor alpha (TNF- ), colonystimulating factors for granulocytes (G-CSF) or
granulocyte-macrophages (GMCSF),
leukotriene B4 (LTB4), complement fragment
C5a, interleukin-8 (IL-8), and others.
These soluble mediators can act as priming or
activating factors for neutrophils and vascular
endothelial cells.
Acute Inflammation Process
Expression of adhesion
molecules & inflammatory
mediators
• Neutrophils are captured & drawn toward the
margins of the flowing stream and performed by
a variety of adhesion molecules, expressed
cooperatively on the surface of endothelial cells
and activated neutrophils
• Adhesion molecules involved in leukocyte–
endothelium interactions include,
immunoglobulins, integrins, selectins, & other
molecules like CD44 and VAP-1.
• They mediate cell-to-cell and cell-to-substrate
Acute Inflammation Process
Hydrodynamic margination,
capture, & rolling of neutrophils
• Radial displacement (hydrodynamic margination)
& retardation of neutrophils by the endothelium
are required as initial steps in the inflammatory
response.
• Contact is made primarily through the E- and Pselectins, adhesion molecules that are induced
(E-selectin) or translocated (P-selectin) to the
surface of endothelial cells in postcapillary
venules by a number of chemical signals.
• Their ligands are complex carbohydrates that are
constitutively expressed on the neutrophil
surface. Intermittent breaking of these contacts
Acute Inflammation Process
Activation, adhesion, and spreading
• Neutrophil activation is enhanced by exposure to the
endothelium, and further expression of adhesion
molecules leads to firm neutrophil adhesion to and
spreading across the endothelial cell.
Diapedesis
• Adhesion and spreading are prerequisites and lead to
migration of the activated neutrophil through the
intercellular junctions of neighboring endothelial cells .
Neutrophils and monocytes migrate preferentially from
postcapillary venules.
Acute Inflammation Process
Migration
• After passing through the endothelial junction and the
basement membrane, the activated neutrophils, guided
by chemotactic stimuli, migrate toward the foreign
particles that initiate the inflammatory response.
• This migration is guided by interactions between
adhesion molecules on the neutrophil surface and
elements of the interstitial matrix.
Acute Inflammation Process
Phagocytosis
• Once the neutrophils reach the foreign particles, they attach to
the opsonized surface of the agent (if it is large), or they engulf
the agent within a phagocytic vacuole.
• Destruction of foreign material is chiefly by reactive oxygen
metabolites (superoxide radicals) and granule contents including
elastases, cathepsin G, proteases, and others.
Apoptosis and elimination of neutrophils
• Among the β2 integrins that are activated in the inflammatory
response are those that trigger apoptosis of activated neutrophils
so that they can be eliminated.
Apoptotic neutrophils are specifically recognized and eliminated
by macrophages.
Neutrophils in Inflammation
Lymphocytes
Origin & role
• Lymphoid precursor cells migrate in fetal or early
postnatal life to either the thymus or lymph nodes
and spleen.
• Cells originating from thymus-routed precursors
become T cells whereas the others become B cells.
 Production of lymphocytes
• A single lymphocyte carries only one unique
specificity
• If it is triggered to increase the number of its
unique receptors ( T cells) or antibodies ( B cells),
the increase can be accomplished only by clonal
multiplication of the original cell.
B cells (B-Lymphocyte)
Function
• B cells carry immunoglobulins as surface receptors.
• Antigens can stimulate these cells to clone into plasma
cells that synthesize and secrete large quantities of a
specific immunoglobulin antibody, different from the
antibodies synthesized by all other B-cell clones
• At the cellular level, an immune response is
initiated when a sufficient number of B cells or T
cells have bound an antigen
• Although there are some immune responses that
occur by way of B cells alone, without the
involvement of T cells, the vast majority of B-cell
activations require help from T cells.
Immunoglobulins
Structure
• Immunoglobulins consist of two identical
“heavy” amino acid chains and two
identical “light” chains, assembled into a
Y-shaped molecule
• There are five different heavy chains,
determining whether the immunoglobulin
isotype is IgA, D, E, G, or M and differing
from one another by the variable domain of
the pair of heavy chains.
• There are only two variants of the light
chain.
Immunoglobulins
Function
• While Ig bound to the B-cell surface act as
receptors, freely circulating Ig can be
antibodies, and as such, they recognize
and bind antigens in order to (1)
precipitate antigen from solution or (2)
attach antigen to phagocytic/cytotoxic
cells for subsequent destruction.
T Cells ( T-Lymphocyte)
Characteristics
• T cells are grouped into two classes, (1) cytotoxic T
cells, which destroy hostile cells; and (2) helper T cells,
which assist B cells in their immunologic tasks.
• Helper T cells are further divided into two subclasses: (i)
TH1 cells secrete IL-2 and γ-interferon and help
cytotoxic T cells and macrophages, and (ii) TH2 cells
secrete IL-4, IL-5, and IL-6, which promote B-cell
activation, and IL-10, which is an inhibitory cytokine in
many settings
T Lymphocytes
Classification
T-cell Receptor Proteins
The T-cell receptor in normally functioning T cells is closely
associated with CD3, a complex consisting of five subunits
that are formed by five different peptides, labeled , , ,  , and
 . CD3 functions to transmit to the cell interior the signal that
was received by the T-cell receptor.
B & T Lymphocytes
Responses
T Cells Receptor Proteins
Types and characteristics
• T cells carry two types of receptor proteins on their
surfaces, named T-cell receptor & CD molecules.
• These receptors consist of two chains (α and β) that are
anchored in the plasma membrane, and extracellular
region of each is folded into two domains
• The most distal tip of the chains forms the recognition
and binding sites (called the paratope).
• The T-receptor paratope recognizes as an epitope only a
specific portion of the molecules of the major
histocompatibility complex (MHC) on the surface of
other cells
T Cells Receptor Proteins
CD(Cluster differntiation)
• CD molecules also recognize a specific portion of the
MHC on other cells, but it is a different portion from
that recognized by the T-cell receptor.
• CD molecules are polypeptides with a membranespanning domain, they and a variety of co-receptors
cooperate with the T receptor
• CD3 is present in all classes of T cells and Helper T cells
carry only CD4, Cytotoxic T cells carry only CD8.
• The specific association of CD4 with TH and CD8 with
TC helps ensure discrimination in the association of T
cells with other cells.
Immune Responses
General principles
• Immune responses consist of defense
mechanisms, characterized by recognition of
nonself, specificity, and memory.
• Natural immunity reside in circulating
components, such as interferon or properdin,
capable of acting directly & immediately on
foreign matter.
• Acquired immunity are normally dormant but can
be activated in response to specific stimuli
• Active acquired immunity, derived from
circulating lymphocytes, mature within the
organs of the immune system (bone marrow,
lymph nodes, spleen) with a delay of 5 to 10 days,
Immune Responses
T-cell-independent
• There are some antigens that can stimulate B
cells to proliferate and differentiate into antibodysecreting plasma cells without the involvement of
T cells.
• These antigens characteristically bind the B-cell
receptors at several points and are capable of
generating a sufficiently strong signal to activate
some B cells.
• However, these reactions do not produce memory
B cells and generally lead to the production of
only low-affinity IgM antibodies rather than the
full Ig complement
Immune Responses
T-cell-dependent
• When T cells are involved, the cells to which they
attach are either being assisted or destroyed,
depending on whether attaching T cell is a helper
or cytotoxic cell.
• Cells that are generally useful for body defense
mechanisms carry MHC-II, and MHC-II binds only
helper T cells.
• Other cells carry MHC-I, and MHC-I binds
cytotoxic T cells.
• Helper T cells, suppressor T cells, cytotoxic T
cells are involved
Helper T cells
Function
• Activated TH stimulate macrophages to make
them more effective destroyers of pathogens and
help other lymphocytes to respond to antigen.
• Activation of helper T cells: The usual pathway is
that a microbe is ingested by an antigenpresenting cell, such as a macrophage, digested,
and degraded by cytosolic lysosomes.
• The resulting protein fragments of 10 to 15 amino
acids are then bound to MHC-II that was
synthesized in the endoplasmic reticulum of the
antigen-presenting cell.
• The MHC-II/antigen unit is transported to the
surface of the antigen-presenting cell, where it
can be recognized by the T receptor of a helper T
Helper T cells
Activation of B cells by helper T cells
1. The foreign molecule (antigen) is recognized and bound by
the specific immunoglobulin receptor on the outside of the
B cell.
2. The receptor/antigen combination is internalized and
degraded
into peptide fragments that can be
bound to MHC-II proteins.
3. The MHC-II/peptide fragment unit is transported to the
surface
of the B cell so that it can become an
antigen-presenting cell recognizable by the T receptor of a
helper T cell.
4. Once a TH has been activated, it directs at least some of its
membranebound and secreted products toward the surface
of
the antigen-presenting B cell. Among these is a
ligand for the CD40 transmembrane molecule on the B-cell
Cytotoxic T Cells
Function
• Cytotoxic T cells (TC) act directly to kill
infected cells or eliminate microorganisms, such
as viruses, that proliferate inside cells where they
cannot be detected by antibodies. Once TC are
activated, they destroy the target cell by
mechanisms that induce apoptosis.
• Activation of cytotoxic T cells by infected cells:
All proteins in a cell, including viral proteins, are
continuously degraded.
• Activation of cytotoxic T cells by helper T cells:
Interleukin secretion by activated helper T cells
is an important signal for T-cell proliferation
Major Histocompatibility
Complex
Nature & function
 MHC molecules are proteins that are anchored
to the extracellular surface of cells.
 Their function is to bind antigen for
presentation to T cells and they have two classes
of MHC molecules
• MHC-I are present on practically all nucleated
human cells and they are epitopes only for
cytotoxic T cells.
• MHC-II molecules are normally confined to
specialized cells, such as B cells, macrophages,
and other antigen-presenting cells that take up
antigens from the extracellular space and MHC-II
Major Histocompatibility
Antigen
Characteristics in human
 In humans the major histocompatibility complex is
defined by the human leukocyte antigen system (HLA)
• Human MHC, clustered on the short arm of
chromosome 6, is operationally divided into genes
which code for class I and class II antigen.
• Class I antigens are detected by selorogic technique,
these disparities cause agglutination and /or lysis of
lymphocytes.
• Class II antigen originally defined by by mixed
lymphocyte culture and now selorogic technique, in
which disparities caused lymphocyte proliferation
Major Histocompatibility
Antigen
Structures
• The chemical structures of both class I and II antigens
display variable and constant regions
• Class I antigens of HLA system are encoded by genes
which include A, B and C loci
• Class II antigen of HLA system are encoded by genes
which include the Dq, Dr and Ds loci
• Each locus consists of at least 20 alternative expressions
as alleles or variants
• Each individual expresses two alleles codominantly at
each locus; one allele is the representative from each
paraenteral chromosome.
Human Panel-reactive Antibody
 Determined by a flow cytometry crossmatch
• HLA class I antibody
HLA-A
HLA-B
HLA-C
•
HLA class II antibody
HLA-DR
HLA-DQ
HLA-DS
Human Panel-reactive Antibody
Implications
• Cellular and humoral immune responses against donorspecific HLA class I and II antigens on implanted
cryopreserved allografts
• Panel-reactive antibody is expressed as the percentage
of lymphocyte panel members against which each
patient's serum reacts and therefore reflects the
breadth of allosensitization against the potential donor
population.
• A PRA of less than 10% is nonreactive, PRA of 11% to
50% low reactive, and PRA over 50% high reactive.
• The antibody of HLA-DR antigens is intriguing and
may suggest some residual cells, notably highly
immunogenic, HLA class II –expressing dendritic cells
resistant to the decellularization process.
Human Panel-reactive Antibody
Determination
• HLA-A, HLA-B, and HLA-C loci serotyping by means
of the standard complement-dependent cytotoxicity test
• HLA-DR/DQ antibodies were evauated by means of a
flow cytometry technique.
• RPA was determined by means of the sensitive antihuman kappa light-chain immunoglobulin
cytotoxicity(AHG-CDC) technique against a frozen Tlymphocyte panel composed of 40 individuals of divere
HLA type and racial background.
• RPA was expressed as the percentage of lymphocyte
panel members against which the patient’s serum
reacts and thus against which the patient has HLA class
I antibody.
Tissue Engineering
Tissue Engineering
Introduction
• Concept of tissue engineering was developed to alleviate
the shortage of donor organs.
• Objective of tissue engineering is to develop laboratorygrown tissue or organs to replace or support the
function of defective or injured body parts.
• Tissue engineering is an interdisciplinary approach that
relies on the synergy of cell biology, materials
engineering, & reconstructive surgery to achieve its goal
• Fundamental hypothesis underlying tissue engineering
is that dissociated healthy cells will reorganize into
functional tissue when given the proper structural
support and signals
Tissue Engineering
Recent myocardial graft
• 3-D contractile cardiac grafts using gelatin sponges and
synthetic biodegradable polymers.
• Formation of bioengineered cardiac grafts with 3-D
alginate scaffolds.
• Use of extracellular matrix (ECM) scaffolds.
• 3-D heart tissue by gelling a mixture of cardiomyocytes
and collagen.
• Culturing cell sheets without scaffolds using a
temperature-responsive polymer.
• Creating sheets of cardiomyocytes on a mesh consisting
of ultrafine fibers.
Tissue Engineering
Definition
• The application of scientific principles to the
design, construction, modification, growth and
maintenance of living tissue
• The application of principles and methods of
engineering and life sciences to obtain a
fundamental understanding of structurefunction relationships in novel and pathological
mammalian tissues and the development of
biological substitutes to restore, maintain or
improve tissue function.
Tissue Engineering
Current issues
• Goal of heart valve tissue engineering is the
development of a valve prosthesis that combines
unlimited durability with physiologic blood flow
pattern and biologically inert surface properties
• Major problems are the first, mechanical tissue
properties deteriorate when cells are removed & the
tertiary structure of fibrous valve tissue constituents
is altered during the decellularization process, and
the second, open collagen surfaces are highly
thrombogenic, because collagen directly induces
platelet activation as well as coagulation factor XII.
Tissue-engineered Valve
Two main approaches
• Regeneration involves the implantation of a resorbable
matrix that is expected to remodel in vivo and yield a
functional valve composed of the cells and connective
tissue proteins of the patient.
• Repopulation involves implanting a whole porcine
aortic valve that has been previously cleaned of all pig
cells, leaving an intact, mechanically sound connective
tissue matrix.
• The cells of the patients are expected to repopulate and
revitalize the acellular matrix, creating living tissue
that already has the complex microstructure necessary
for proper function and durability
Tissue-engineered Valve
Development
 Three approaches
• Acellular matrix xenograft
• Bioresorbable scaffold
• Collagen-based constructs containing
entrapped cells
• Other substrates in early development
 Hybrid approaches
 Stem cells and other future prospects
Tissue-engineered Valve
Development
• Seeding a biodegradable valve matrix with autologous
endothelial or fibroblast cells
• Seeding a decellularized allograft valve with vascular
endothelial cells or dermal fibroblast
• Use of a decellularized allograft with maintained
structural integrity as a valve implant that will be
repopulated by adaptive remodeling
• A possible alternative to the acellular valve and the
bioresorbable matrix approaches is the fabrication of
complex structures by manipulating biological
molecules. With sufficient fidelity, one could potentially
fabricate structures as complex as aortic valve cusps
Tissue-engineered Valve
Problems
• Decellularization process render all allograft valves
immunologically inert ?
• What will happen to xenogeneic decellularized graft
immunologically ?
• Seeded vascular endothelial cell penetrate matrix and
differentiate into fibroblast and myo-fibroblast that are
biologically active ?
• Regenerate the collagen & elastin matrix of the
allograft such that valve will maintain structural
integrity ?
• Utilization on other cardiac valves such as aortic valve ,
which has significant structural difference ?
Heart Valve Tissue Engineering
Developing steps
• The initial approach was based on the fabrication of
the entire valve scaffold from biodegradable polymers,
followed by in vitro seeding with autologous cells
• The complex three-dimensional structure of the native
valve can hardly be achieved with current techniques,
and the structural and mechanical properties of the
various polymers are not ideal.
• In vitro seeding and conditioning with cells of the
future recipient is a time-consuming process, and it
remains unclear whether the cells actually adhere to the
scaffold after implantation
• More recently, natural xenogenic or allogenic heart
valve tissue has been propagated as a scaffold.
Tissue-engineered Heart Valve
Cryopreserved human umbilical cord cells
Tissue-engineered Heart Valve
Stereolithographic model
Three-dimensional reconstructed stereolithographic model from the inside of an
aortic homograft. (B) Trileaflet heart valve scaffold from porous poly-4hydroxybutyrate including sinus of Valsalva (seen from the aortic side) fabricated
from the stereolithographic model.
Allograft Tissue Engineering
Immunogenicity
• Allogrft tissue stimulates a profound cell-mediated
immune response with diffuse T cell infiltrates and
progressive failure of the allograft valve has been
attributed to this alloreactive immune response
• The role of humoral response in allograft failure is less
clear, recently, evidence has been accumulating that
allograft tissue used in congenital cardiac surgery also
stimulates a profound humoral response
• As previously mentioned, it is believed that the cellular
elements are the antigenic stimulus for the alloreactive
immune response, and thus decellularization has been
proposed to reduce the antigenicity of these tissues.
Tissue Procurement
Processing
• Hearts were transported on wet ice in Roswell Park
Memorial Institute (RPMI) 1640 medium
supplemented with polymyxin B. Warm ischemic time
was less than 3 hours, and cold ischemic time didn't
exceed 24 hours.
• Tissue conduits were dissected from the heart and
truncated immediately distal to the leaflets. They were
then placed in RPMI 1640 supplemented with
polymyxin B, cefoxitin, lincomycin, and vancomycin at
4°C for 24 ± 2 hours.
• Representative 1 cm2 tissue sections were placed in
phosphate buffered water and vigorously vortexed, and
8 mL was injected into anaerobic and aerobic bottles
and analyzed for 14 days for bacterial or fungal growth.
Decellularization
Introduction
• In an attempt to reduce the antigenic response,
decellularization processes have been introduced for
cryopreserved tissue.
• Experimental and clinical experience with this
decellularization process has been gained with porcine
vena cava porcine tissue, porcine aortic and
pulmonary valve conduits, ovine pulmonary valve
conduits, and, subsequently, human femoral vein and
human pulmonary valve conduits.
• There has also been experimental evidence that the
decellularized matrix becomes populated with
functional recipient cells.
Decellularization
Basic concepts
• Detergent/enzyme decellularization methods remove
cells and cellular debris while leaving intact structural
protein “ scaffolds ”
• Identified as biologically and geometrically potential
extracellular matrix scaffold which to base recellulazed
tissue-engineered vascular and valvular substitutes
• Decreased antigenicity and capacity to recellularize
suggests that such constructs may have favorable
durability
Acellular Matrix Tissue
Approach to generate
• First break apart the cell membranes through lysis in
hyper- and hypotonic solutions, followed by extraction
with various detergents
• The detergents include the anionic Sodium dodecyl
sulfate, the zwitterionic CHAPS and CHAPSO, and the
nonionic BigCHAP, Triton X-100, and Tween family of
agents.
• The enzymes that have accompanied these detergent
treatments have focused mainly on cleaving and
removing the DNA that is part of the cellular debris.
Decellularization
Rationale
• A persistent immunoreactivity against donor
antigens has been implicated.
• Early calcification and stenosis from an intense
inflammatory reaction may be manifestations
of this immune response.
• Early structural failure has been shown to be
more prevalent in younger patients, perhaps
because of a more aggressive immune response
Decellularization Process
Methods
• Decellularization method utilizes an anionic detergent,
recombinant endonuclease, and ion exchange resins to
minimize processing reagent residuals in the tissues.
• Acellular vascular scaffolds macroscopically appear
similar to native tissue but are devoid of intact cells
and contain virtually no residual cellular debris.
• Decellularized tissues should avoid pronounced
immune responses and nonspecific inflammation with
consequential scarring and ultimately, mineralization,
the avoidance of which allows recellularization of the
scaffold
Decellularization Process
Recent status
• Multistep detergent–enzymatic extraction, Triton
detergent, or trypsin/ethylenediaminetetraacetic acid.
• A more recent protocol using sodium dodecyl sulfate
(SDS) in the presence of protease inhibitors was
successful for aortic valve conduit decellularization
• Histological analysis showed that the major structural
components seemed to be maintained.
• The effect of cell removal on different types of ECM
molecules and the remodeling of the ECM in the
transplanted aortic valve.
Decellularization Procedures
Methods
Treatment
•
•
•
•
Concentration
Triton X-100
Trypsin
Trypsin/Triton X-100
SDS
Duration ation (h)
1%–5%
0.5%
0.5%/1%–5%
0.1%–1%
• SDS, Sodium dodecyl sulfate
24
0.5–1.5
0.5–1.5/24
24
Acellularization Procedures
Enzymatic process
• Valve or conduits were harvested under sterile
condition and stored at 4°C.
• Within 30 minutes the conduits were acellularized in a
bioreactor.
• The bioreactor was filled with 0.05% trypsin and
0.02% ethylenediamine tetraacetic acid (EDTA) for 48
hours, followed by phosphate-buffered saline (PBS)
flushing for 48 hours to remove cell debris.
• All steps were conducted in an atmosphere of 5% CO2
and 95% air at 37°C with the bioreactor rotating at a
speed of 7 rpm.
Decellularization Procedures
 Enzymatic process
• The entire construct was washed for 30 minutes at
room temperature in povidone-iodine solution and
sterile PBS, followed by another overnight incubation
at 4°C in an antibiotic solution
• After this decontamination procedure, the valves were
placed in a solution of 0.05% trypsin and 0.02% EDTA
(Biochrom AG) at 37°C and 5% CO2 for 12 hours
during continuous 3-dimensional shaking.
• After removal of the trypsin-EDTA, the constructs were
washed with PBS for another 24 hours to remove
residual cell detritus.
Depopulated Allografts
Processing
• Transported in iced physiologic buffer for
depopulation processing and cryopreservation.
• The steps included cell lysis in hypotonic
solution, enzymatic digestion of nucleic acid,
and washout in an isotonic neutral buffer.
• Once depopulated, the allografts were
cryopreserved and stored in liquid nitrogen
until implantation
Homograft Decellularization
Nature
• Processing allograft tissues with detergents and
enzymes may provide scaffolds that have the necessary
biological and geometric recellularization potential
• Adequate decellularization should decrease antigenicity,
avoid allosensitization, and remove cellular remnants
that may serve as nidi for calcification and its
associated consequences.
• Physical, metabolic, and synthetic characteristics of
migrating autologous cells (recellularization of acellular
tissues) theoretically should provide the necessary
structural and functional characteristics to sustain
engineered tissue longevity and durability.
Homograft Decellularization
Cell free or nonimmunogenic
• Less viable cellular element
No immune cell infiltration
No donor-specific immune activation
• Well preserved ultrastructure
• Positive effect on survival and
functionality of the valve
Decellularization
Characteristics
• The resulting acellular vascular scaffolds
macroscopically appear similar to native tissue but
are devoid of intact cells and contain virtually no
residual cellular debris.
• Adequately decellularized tissues should avoid
pronounced immune responses and nonspecific
inflammation with consequential scarring and
ultimately, mineralization
• Perhaps the absence of allosensitization by vascular
human leukocyte antigens may help avoid both
humoral and cell-mediated chronic rejection
Decellularization Process
Immunologic response
• HLA class I & II antibodies are known to be elevated in children
receiving homografts, and it seems that HLA class II is
particularly important
• The antibody elicited in these grafts toward HLA-DR
antigens is intriguing and may suggest some residual
cells, notably highly immunogenic, HLA class II –
expressing dendritic cells that may be more resistant to
the decellularization process.
• Decellularized tissue scaffolds (whether preceded by
classic cryopreservation or not) demonstrated the
smallest detectable amounts of MHC I and II antigen
and also provoked little or no PRA response.
Decellularized Bioprosthesis
Main process
• Decellularization process involves cell lysis in a
hypotonic sterile water and equilibrated in water
and treated by enzymatic digestion of nucleic acids
with a combined solution of ribonuclease and
deoxyribonucease
• The resulting allograft have a 99% reduction in
staining of endothelial & interstitial cellular elements
• This process is claimed to leave valve biologic matrix
and structure intact
• Marked reduction in staining for class I & II
histocompatibility antigens
Incomplete Decellularization
Implications
• Incomplete decellularization with an excess of cellular
debris, however, can provoke significant immunemediated inflammation, resulting in functional failure
• If residual cytokines remain in the extracellular matrix
after decellularization, they can potentially promote
nonspecific inflammatory responses during reperfusion,
exacerbating the scar & foreign-body healing responses,
which in turn might promote immune responses and
ultimate failure of the tissue-engineered construct
• Demonstrations of acellularity with routine staining
methods, absence of retained donor DNA are
insufficient evidence of adequate reduction of
antigenicity by putative decellularization methods.
Reendothelization Process
Implications
• A functioning endothelium requires an appropriate
matrix cell population for communication, leading
to cell and tissue functionality as well as providing
appropriate triggers for cell population maintenance,
migration, and proliferation.
• The endothelium is likely responsible for being
responsive to sheer stress and then "signals" the
myofibroblast cell population to synthesize more
structural protein such as collagen and elastin in
response to the sheer stress or higher pressures.
• Reendothelization of tissue-engineered vascular
constructs will, in part, depend upon the restoration
of an appropriate interstitial matrix cell population.
Seeding of Endothelial Cells
 Endothelialization of porcine glutaraldehydefixed valves
• Poor cell adhesion on glutaraldehyde-fixed porcine
surfaces was also a result of a change in the physicochemical properties caused by the cross-linking.
• Reduced hydrophily prevented the cells to attach
properly.
• This could be changed by introducing a strong
hydrophilic substance through the way of a chemical
salt formation on the surface
• Citric acid or ascorbic acid, which are both strong
organic acids used and no signs for any structural
weakening due to the citric acid pretreatment
Endothelial Cell Seeding
 On porcine glutaraldehyde-fixed valves
• After incubation with serum-supplemented M-199 for
24 hours at 4°C, the valves were incubated with citric
acid (10% by weight) for 5 minutes at a pH of 3 to 3.5.
• This pretreatment increases hydrophilsm of the surface,
thus improving cell adhesion and attachment
• The pretreated, but unseeded valves exhibited a cellfree surface of free collagen fibers prior to cell seeding
• Thereafter, the prostheses were rinsed 3 times and
buffered to a physiologic pH using PBSB buffer.
• After the final washing procedure, the valves were preseeded with myofibroblasts, followed by endothelial cell
Recellularization
Lavoratory evidence
• Stains for T-cell surface antigen, CD4, and CD8
yielded negative results.
• Neoendothelial cells stained for factor VIII.
• Smooth muscle cells in arteriole walls stained
for smooth muscle actin, and cells scattered in
the adventitia stained for procollagen type I.
• Leaflet explants had no detectable
inflammatory cells and were repopulated with
fibrocytes and smooth muscle cells
Decellularized Porcine Valve
Synergraft failure
• In early phase, blood contact to the collagen matrix
activates a multitude of the events which lead to
thrombocyte activation, liberation of chemotaxic and
proliferative stimulating factors and within hours to
polymorphnuclear neutrophil granulocyte and
macrophage influx
• This early inflammatory response may be responsible
for significant weakening of the matrix structure of the
wall and be the cause of the graft rupture
• In human implant, there was no repopulation of the
matrix with fibroblast and myofibroblasts, lined with
fibrous sheath & disorganized pseudointima
Decellularized Heart Valve
Synergraft(decellularization)
• Since not repopulated with cells before implantation,
it does not represent a true tissue engineered product
• The decellularized porcine heart valve is hypothesized
that this will significantly reduce antigenicity and will
ideally allow for repopulation of the graft with recipient
autologous cells and creat a living tissue
• By concept the matrix would be degraded and the
recipient cells would generate a new matrix.
• In human implant, fibroblasts seem unable to invade
the matrix which is virtually instead encapsulated
Recellularization
Reendothelization process
• A functioning endothelium requires an appropriate
matrix cell population for communication, leading
to cell and tissue functionality as well as providing
appropriate triggers for cell population maintenance,
migration, and proliferation.
• The endothelium is likely responsible for being
responsive to sheer stress and then "signals" the
myofibroblast cell population to synthesize more
structural protein such as collagen and elastin in
response to the sheer stress or higher pressures.
• Reendothelization of tissue-engineered vascular
constructs will, in part, depend upon the restoration
of an appropriate interstitial matrix cell population.
Recellularization
Processing
• Slower recellularization in the luminal side, suggesting
that cells migrate into the matrix primarily from the
adventitial aspect rather than the lumen
• Migrating fibroblast-like cells were found to stain
positively for -smooth muscle actin, which is consistent
with the dual phenotype of vascular and valve leaflet
myofibroblasts
• This seems to indicate that a decellularized matrix can
be conducive to autologous recellularization
• Well-functioning endothelium requires an appropriate
matrix cell population for communication, leading to
cell and tissue functionality as well as providing
appropriate triggers for cell population maintenance,
migration, and proliferation
Decellularization
Preparation
• Graft obtain
• Storage in a nutrient solution with
antibiotics for at least 7 days
• Decellularization of graft immersed in
solution for 24hours in room temperature
• Keep in physiologic saline solution until
implantation
Decellularization Process
Commonly used agents
• 1 % tetra-octylphenyl-polyoxyeyhylene
( Triton X ) with 0.02% EDTA in phosphate
buffered saline
• 1 % deoxicholic acid and 70% ethanol for
24hours under constant agitation
• Trypsin/ethylenediaminetetraacetic acid
• Sodium dodecyl sulfate ( 0.1% SDS ) in the
presence of protease inhibitors, Rnase and
Dnase
• Detergent ( N-lauroylsarcosinate ), benzonase
endonuclease solution, polymyxin B
Decellularization
Process methods
•
•
•
•
•
Samples were placed in hypotonic Tris buffer (10 mmol/L, pH 8.0)
containing phenylmethylsulfonyl fluoride (0.1 mmol/L) and
ethylenediamine tetraacetic acid (5 mmol/L) for 48 hours at 4°C.
Next, samples were placed in 0.5% octylphenoxy polyethoxyethonal
(Triton X-100, Sigma) in a hypertonic Tris-buffered solution (50
mmol/L, pH 8.0; phenylmethylsulfonyl fluoride, 0.1 mmol/L;
ethylenediamine tetraacetic acid, 5 mmol/L; KCl, 1.5 mol/L) for 48
hours at 4°C.
Samples were then rinsed with Sorensen’s phosphate buffer (pH 7.3)
and placed in Sorensen’s buffer containing DNase (25 µg/mL), RNase
(10 µg/mL), and MgCl2 (10 mmol/L) for 5 hours at 37°C.
Samples were then transferred to Tris buffer (50 mmol/L, pH 9.0;
Triton X-100 0.5%) for 48 hours at 4°C.
Finally, all samples were washed with phosphate-buffered saline at 4°C
for 72 hours, changing the solution every 24 hours.
Immunohistochemistry
Methods
• Tissue was harvested for histology at 1, 2, and 4 weeks. Samples
were formalin fixed (10%), paraffin embedded, and serially
sectioned (5 µm) for histologic and immunohistochemical
examination, ensuring valve leaflets were visualized in all sections.
• Immunohistochemistry involved standard staining techniques with
biotinylated secondary antibodies, a peroxidase avidin-biotin
complex, and 3.3' diaminobenzidene as the chromogen.
• Primary monoclonal antibodies for T cells (anti-CD3; sc1127) and
cytotoxic T cells (anti-CD8; sc7970) were used.
• Allogeneic nondecellularized grafts were associated with
significant CD3+ and CD8+ T cell infiltrates in aortic valve leaflets
by 1 week after transplantation, rapidly decreasing in the following
weeks.
Histology & Immunohistology
Examination
• Explanted tissue specimens were studied as
hematoxylin/eosin, elastica van Gieson, and von Kossa
stained paraffin or immunostained frozen sections.
• The antibodies for immunohistochemistry included
monoclonal antibodies against CD31, -smooth muscle
actin , and vimentin , and a polyclonal antibody against
von Willebrand factor
• Expression of von Willebrand factor (vWF), vascular
endothelial growth factor (VEGF), vascular smooth
muscle -actin 2 (ACTA2), smooth muscle 22 (SM22 ),
and vimentin were determined with quantitative realtime RT-PCR
Homograft Decellularization
Cell free or nonimmunogenic
• Less viable cellular element
No immune cell infiltration
No donor-specific immune activation
• Well preserved ultrastructure
Heterograft Decellularization
Current status
• The use of a decellularized matrix of a xenograft is
preferred because synthetic scaffolds are not only
expensive and potentially immunogenic, they also
suffer from toxic degradation and inflammatory
reaction.
• Recently, nonseeded allogenic and xenogenic matrices
have been implanted in animals.
• These matrices are expected to be covered with host
cells, as observed in experimental animals.
• But, a so-called pseudointima can be seen, which is far
from being a functional endothelial cell layer, this and
the naked collagen structures are the potential
thrombogenicity
Xenografts Decellularization
Porcine
• Nonenzymatic process on the basis of osmotic shock,
detergent cell extraction (0.1% sodium dodecylsulfate),
and antiproteases recently, which induced complete
decellularization without major impairment of the
structural proteins
• Under that process, valve conduit tissues had equal
strength compared with fresh tissues and only modest
changes in extensibility in vitro.
• In addition, this process was recently demonstrated to
remove xenoantigens.
Xenograft Matrix
Goal of seeding
• Sheathing(intimal proliferation) eventually will lead to
retraction or complete immobilization of the cusp and
induce thrombogenicity in the valves (sheathing
originates from fibrin deposition and thrombus
organization)
• The first reason for not implanting an acellular matrix
in animals as the outgrowth of endothelial cells is
higher in animal models than in human
• The second reason for coating the acellular matrix with
endothelial cells was to reduce immunologic reactions,
Decellularization of Biomatrix
Advantages
• Enzymatically decellularized extracellular matrix
without tanning-induced crosslinks possesses epitopes
for cellular adhesion receptors, facilitating
repopulation with tissue-specific cell types but also
inflammatory cells
• Nonautologous matrix constituents such as collagen,
elastin, and proteoglycans have little antigenicity, given
that cellular components are entirely removed.
• Mismatch of HLA-DR & ABO antigens on endothelial
cells in unmodified valve allografts is associated with
accelerated valve failure
Decellularization of Biomatrix
Disadvantages
• The mechanical tissue properties deteriorate when the
cells are removed and the tertiary structure of fibrous
valve tissue constituents is altered during the
decellularization
• The mechanical properties do not allow for implantation
in the high pressure system by aggressive enzymatic
digestion
• Open collagen surfaces are highly thrombogenic, because
collagen directly induces platelet activation as well as
coagulation factor XII
Autologous Recellularization
Rationale
• Autologous recellularization with in-migration of
phenotypically appropriate matrix cells and formation
of neointima, suggesting actual biological incorporation
into the host matrix structure.
• These attributes may portend prolonged durability,
resistance to prosthetic endocarditis, cell-mediated
tissue repair, and protein renewal functions that
approach the goals of tissue-engineered constructs but
without preseeding or the need for extensive in vitro
bioreactor manipulations.
• Decellularization of allograft tissue results in removal
of cells and proteins, potentially creating defects, spaces,
or voids within the collagen matrix that may lead to
structural compromise
Bioprosthetic Valve & Conduit
Ideal Valved Conduit
Indications
• The ideal conduit for replacement of the
pulmonary artery and valve remains elusive,
particularly for the child or young adult
• A prosthesis that achieves the characteristics of
durability, ease of implantation, availability
readiness in small sizes, freedom from the need
for anticoagulation, limited antigenicity, low
risk of thromboembolic complications, and
growth potential remains to be realized.
Heart Valve Substitutes
Types of valve
• Heart valve substitutes are of two principal types:
mechanical prosthetic valves with components
manufactured of nonbiologic material (eg, polymer,
metal, carbon) or tissue valves which are constructed,
at least in part, of either human or animal tissue
• Tissue valves have been used since the early 1960s
when aortic valves obtained fresh from human
cadavers were transplanted to other individuals
(homografts). A decade later, chemically preserved
stent-mounted tissue bioprosthetic valves (generally
termed bioprostheses) were commercially produced
and implanted
Bioprosthetic Heart Valves
General introduction
• Bioprosthetic valves have excellent hemodynamic profiles,
and also they do not require permanent anticoagulation
• The relatively short durability of bioprosthetic valves
limits their application to patients with either a
contraindication to anticoagulation or to an elderly age
group with a low likelihood for reoperation.
• The mechanisms by which biologic valves degrade are
considered multifactorial, with involvement of
immunologic rejection, mechanical wear out, calcification,
and enzymatic digestion, with subsequent loss of the
original histologic integrity.
Prosthetic Heart Valve
Indications
• The criteria of ideal valve
: durability, nonthrombogenicity,
easy implantability and availability
• Types of valve
: mechanical valves, stented/stentless
xenograft, cryopreserved aortic or
pulmonic homograft, autograft
Heart Valves
Characteristics
• Mechanical prosthetic valves have a substantial risk of
systemic thromboemboli and thrombotic occlusion, and
the chronic anticoagulation therapy required in all
mechanical valve recipients potentiates hemorrhagic
complications. Nevertheless, contemporary mechanical
prostheses are durable.
• Tissue valves have a low rate of thromboembolism
without anticoagulation, owing to a central pattern of
flow similar to that of the natural heart valves and
cusps composed of valvular or nonvalvular animal or
human tissue. However, a high rate of valve failure with
structural dysfunction owing to progressive tissue
deterioration undermines their attractiveness .
Heart Valve Replacement
Perspectives
• Approximately 85,000 substitute valves are implanted
in the US and 275,000 worldwide each year, of which
we presently estimate that approximately half are
mechanical and half are tissue, suggesting a shift
toward increasingly greater usage of tissue valves over
the last decade.
• Within 10 years postoperatively, prosthesis-associated
problems overall necessitate reoperation or cause death
in at least 50% - 60% of patients with substitute valves.
• The rate is similar for mechanical prostheses and
bioprostheses; however, the frequency and nature of
specific valve-related complications vary with the
prosthesis type, model, site of implantation, and certain
characteristics of the patient.
Bioprosthetic Heart Valves
Fate of bioprosthesis
• Structural dysfunction is the major cause of failure of
bioprosthetic heart valves.
• The principal underlying pathologic process is cuspal
calcification; secondary tears frequently precipitate
regurgitation. Calcification can also cause pure stenosis
owing to cuspal stiffening.
• Calcific deposits are usually localized to cuspal tissue
(intrinsic calcification), but calcific deposits extrinsic to
the cusps may develop in thrombi or endocarditic
vegetations (extrinsic calcification).
• Progressive collagen deterioration, independent of
calcification, is also a likely important contributor to
the limited durability of bioprosthetic valves.
Bioprosthetic Heart Valves
Immune response
• Preformed antibodies against [alpha]-Gal cause
opsonization within the valve tissue
• Blood contact to collagen matrix activates & leads to
thrombocyte activation, liberation of chemotaxic and
proliferative stimulating factors and within hours to
PM neutrophil, granulocyte and macrophage influx
• Degeneration begins with the penetration of
immunoglobulines (IgG/IgM) into the valve-matrix.
• Subsequently, macrophages are deposited on the valve
surface and erythrocytes penetrate into the valve.
Finally, collagen breakdown and calcification
Bioprosthetic Heart Valves
Mitroflow pericardial valve
• This is a second generation bioprosthesis made
of a single sheet of glutaraldehyde-preserved
bovine pericardium mounted on the outside of
a flexible Dacron-covered Delrin stent.
• Due to its design characteristics, an unimpeded
leaflet opening and blood flow occurs, which
results in an excellent hemodynamic
performance and a proven superiority when
compared with other pericardial bioprostheses
Valved Conduit Manufacturing
Process flowchart(Biocor)
•
•
•
•
•
•
•
•
•
•
Tissue procurement
Dissection, collection and transportation
Inspection and rinsing
Tissue fixation and bioburden reduction & storage
Conduit manufacturing & inspection
Bioburden reduction and storage
Quality control and final review
Chemical sterilization
Aseptic transfer
Package and final inspection
Shelhigh Valved Conduit
Valve tissue fixation
• Glutaraldehyde fixation of the tissue occurs with 24
hours of receipt of tissue.
• Lot integrity is maintained during the fixation and the
valve are bathed in 0.35% glutaraldehyde and a
hydrostatic closing pressure of <4mmHg is applied to
the valves.
• This pressure is maintained by the glutaraldehyde
recirculation rate.
• The valves are detatched from the fixation plug and
placed together in a labelled container 0.35%
glutaraldehyde
Shelhigh Valved Conduit
• Pericardial tissue fixation
• The pericardial sac are placed flat in a shallow
container of freshly prepaired glutaraldehyde solution
• The pericardia are fixed at room temperature,
unstressed for at least 2 weeks.
• The glutaraldehyde solution is changed between 2 and
4 hours and again at 24 hours.
• Fixed tissue quarantine bioburden reduction process is
performed to reduce bioburden of of the valves in the
event organisms resistant to 0.35& glutarakdehyde are
present
Shelhigh Valved Conduit
Process flowchart
•
•
•
•
•
•
•
•
•
Procurement
Dissection
Tissue fixation
Inspection
Fixed dissection
Prolapse test
Mounting
Sterilization & detoxification
Packaging
Shelhigh Valved Conduit
Histopathology
• Intimal fibrosis, neointimal proliferation, and neointimal peel
formation; chronic inflammation with histiocytic demarcation of
the xenograft tissues, as well as a granulomatous reaction with the
presence of giant cells of foreign-body type and acute granulocytic
inflammation, and rarely calcifications
Shelhigh Valved Conduit
Histopathology
• Neointimal proliferation included all through the
conduit & valvular cusps encased by reactive
neointimal layer, with immobility, fibrous contraction
• Chronic inflammation with a marked histiocytic
component with a maximum inflammation between the
xenograft & reactive neointimal layer
• Immunohistochemistry revealed predominantly
histiocytic and T-lymphocytic infiltrates, with a sparse
B-lymphocytic component.
• Histiocytic & granulocytic reactions most pronounced
surrounding the porcine myocardium,and with an
acute inflammatory component with PML
Shelhigh Valved Conduit
Immunohistochemical study
• Paraffin sections were routinely stained with
hematoxylin and eosin and Verhoeff's van
Gieson elastic tissue stain
• Immunohistochemical staining for CD3, CD4,
CD8 (for T-cell subsets), CD20 (B-cell subset),
and CD68 (for macrophages) was performed to
characterize the inflammatory infliltrate
• Naphtol AS-D-chloracetate esterase staining
was used to visualize granulocytic infiltrates.
Chemical Tissue Fixation
Principles
• Aldehydes are the most commonly used
tissue treatment agents
• Tissue fixation with aldehydes is a well
established and widely accepted process
Formaldehyde Fixation
Charasteristics
• When applied to tissue, aldehydes like
formaldehyde form cross-links with tissue
proteins and produce water as a by-product
• Aldehydes like formaldahyde, however, may
require heating and may react slowly with
tissue proteins
Glutaraldehyde Fixation
Principles
• Glutaraldehyde has become a popular fixing agent
because it offers two aldehyde groups and therefore
greater cross-linking potential than does formaldehyde.
• Glutaraldehyde offers so many CHO groups that many
aldehyde groups are unbound in the treated tissue.
• These toxic radical groups may cause inflammation in
the surrounding tissue after implantation, leading to
calcification of the implant.
Glutaraldehyde Fixation
 Adverse effect
1. Making biologic material stiff & hydrophobic
2. Release of residual cytotoxicity induce the
foreign body reaction
3. No endothelial cell lining onto the cytotoxic
treated area
Glutaraldehyde Fixation
Use as valve prostheses
• As a biologic extracellular matrix scaffold, porcine
heart valves for their well-known good hemodynamic
behavior and unlimited availability.
• Porcine scaffolds are usually treated with
glutaraldehyde to improve mechanical properties and
to limit the xenogeneic rejection process.
• Glutaraldehyde treatment profoundly modifies the
extracellular matrix structure and makes it improper
to support cell migration, recolonization, and the
matrix-renewing process
Glutaraldehyde Fixation
No-react neutralization
• The proprietary No-react tissue treatment
process begin with proven glutaraldehyde
fixation, but then adds a heparin wash process
that renders the unbound aldehyde sites
inactive
Genipin Fixation
Characteristics
• Naturally occurring cross-linking agent
• Genipin & related iridoid glucosides extracted
from the fruit of Gardenia Jasminoides as an
antiphlogistics & cholagogues in herbal medicine
• React with free amino groups of lysine,
hydroxylysine or arginine residues within
biologic tissue
• Blue pigment products from genipin &
methylamine, the simplest primary amine
Autologous Pericardium
Fates of fresh pericardium
• Fibrotic & retracted
• Progressive thinning with dilatation &
aneurysmal formation
• Incorporated into the surrounding host
tissue with growth potential
• Common feature is tissue thinning with
reduction in connective cells or degenerative
nucleic change
Bioprosthetic Heart Valves
Use of animal valve prostheses
• The use of animal valve prostheses was first proposed and tested
during the 1940s and 1950s
• Fixatives were introduced not as a method of decreasing
antigenicity but as a sterilizing and stiffening agent.
• Duran and Gunningt sterilized porcine graft by immersion in
liquid ethylene oxide.
• Binet and colleagues placed non-sterile pig in a mercurial
solution" for 3 days.
• The first formaldehyde preservation of porcine valves was
performed by O’Brien, who used the solution in 1967 for "both
preservation and sterilization.
Conditioning of Heterografts
• Biologic factors affecting durability
• Diagramatic representation of different stages of method
for conditioning heterografts
Glutaraldehyde Treatment
Action on pericardium
• The treatment with glutaraldehyde solutions allows the
simultaneous fixation/shaping and decontamination of
the bovine pericardium
• The glutaraldehyde is a cross-linking agent, employed
in the tanning of biological tissues; covalent bonds
produced in the cross-linking process are both
chemically and physically strong
• Although the specific action of glutaraldehyde is still
unclear, it is believed that it stabilizes the collagen
fibers against proteolytic degradation
Glutaraldehyde Treatment
Action on tissues
• Glutaraldehyde mechanism of action
Glutaraldehyde Preservation
Mechanism
• Devitalizes the native cell population
• Denaturizes antigenic protein domains
• Changes the scaffold protein architecture
rendering in vivo repopulation with
recipient cells impossible
• No potential for growth, limiting their use
in infants and children.
Glutaraldehyde Fixation
Action & adverse effects
• Glutaraldehyde (GA) is currently the standard reagent
for preservation and biochemical fixation
• It imparts intrinsic tissue stability (biodegradation
resistance) and reduces the antigenicity of the material.
• Recent reports have suggested a detrimental role of
aldehyde-induced intra- and intermolecular collagen
cross-linkages in initiating tissue mineralization
• GA has been implicated in devitalization of the intrinsic
connective tissue cells of the bioprosthesis, thus
resulting in breakdown of transmembrane calcium
regulation and hence contributing to cell-associated
calcific deposits
Glutaraldehyde Preservation
Fate of bioprosthesis
• Reduced immunologic recognition & resistance
to degradative enzymes
• limited durability and structural deterioration;
nonviable tissues and inability of cell to migrate
through extracellular matrix
• Stiffened valve;
abnormal stress pattern causing accelerated
calcification
Calcification of Bioprosthesis
Etiology
• Tissue valve calcification is initiated primarily within residual cells
that have been devitalized, usually by glutaraldehyde pretreatment.
• The mechanism involves reaction of calcium-containing
extracellular fluid with membrane-associated phosphorus to yield
calcium phosphate mineral deposits.
• Calcification is accelerated by young recipient age, valve factors
such as glutaraldehyde fixation, and increased mechanical stress.
• The most promising preventive strategies have included binding of
calcification inhibitors to glutaraldehyde fixed tissue, removal or
modification of calcifiable components, modification of
glutaraldehyde fixation, and use of tissue cross linking agents
other than glutaraldehyde.
Tissue Valve Preparation
Principles
• Ensure reproducibility, desired tissue
biomechanics, desired surface chemistry,
matrix stability, and resistance to calcification
• A variety of treatments have been used
clinically as well as experimentally
• They may be broken down into two broad
categories: modifications to glutaraldehyde
processed tissue and nonglutaraldehyde
processes.
Calcification of Bioprosthesis
Preventive methods(lipid)
• Calcium phosphate crystals containing Na, Mg, and
carbonate nucleate due to devitalization of the cells and
thus inactivation of the calcium pump
• Membrane-bound phospholipids have also been
associated with calcification nucleation due to alkaline
phosphatase hydrolysis
• Ethanol has been used to remove phospholipids and
mitigate calcification, yet phospholipids have also been
removed with chloroform-methanol yielding
• Lipid extraction can also be performed through tissue
processing with detergent compounds such as sodium
dodecyl sulfate.
Calcification of Bioprosthesis
Preventive methods(aldehyde)
• Free aldehyde within the tissue matrix has been
thought to be an initiator for calcification as well.
• This is supported by studies that demonstrate that
aldehyde-binding agents such as alpha-amino oleic acid
(AOA; Biomedical Design, Marietta, Ga), L-glutamic
acid, & aminodiphosphonate prevent cusp calcification.
• Yet, post treatment with the amino acid lysine does not
prevent cuspal calcification. and emphasizes the
multiplicity of pathways by which calcification can
initiate.
Tissue Valve Preparation
Processes
• The modifications to glutaraldehyde processed tissue
with detergents such as sodium dodecyl sulfate and
Tween-80 to remove phospholipids, ethanol
preincubation to remove phospholipids, covalently
bound AOA, L-glutamic acid and aminodiphosphonate
to bind free aldehydes, and detoxification processes
using urazole and homocysteic acid.
• Nonglutaraldehyde processes include but are not
limited to epoxy compounds, dye-mediated photooxidative reactions including PhotoFix and
carbodiimide compounds including Ultifix
Pericardial Bioprostheses
Characteristics
• The pericardial bioprostheses are fabricated using
bovine pericardium, which is sewn into a valvular
configuration on a stented frame.
• The first commercially available pericardial valve, the
Ionescu-Shiley valve was abandoned in 1988 due to a
high incidence of valvular deterioration characterized
by leaflet tears and valve incompetence.
• Of the second-generation pericardial valves, introduced
in 1982, the Carpentier-Edwards pericardial valve has
shown better results than the other valve in its class, the
Pericarbon pericardial bioprosthesis
Pericardial Bioprostheses
SOPRANOTM heart valve
• The functional component consists of three leaflets of
bovine pericardium fixed by treatment in buffered
glutaraldehyde solutions
• The tissue leaflets are sutured to the inside of a flexible
support, the stent; this is formed by a core in acetalic
resin(polyoxymethylene) covered with a knitted
polyester fabric(polyethylene tereftalate, PET); this
includes a radio opaque silicone rubber insert which is
positioned at the same level as the sinusoidal border at
the entrance to the valve; the fabric is coated with
Carbofilm, a thin film of turbostratic carbon aimed to
enhance the biocompatibility
TM
SOPRANO
Heart Valve
General features
TM
SOPRANO
Heart Valve
Manufacture flow chart
TM
SOPRANO
Heart Valve
Technical features
• Assembly of the two pericardial sheets functional component with
the supporting stent
SOPRANOTM Heart Valve
Manufcturing materials
• The pericardium undergo glutaraldehyde-based
process in which the stabilizing agent react under
dynamic conditions.
• The valve prosthesis is treated for the elimination of
glutaraldehyde residues and stored in a buffered
solution without aldehydes
• All the surfaces not formed by biologic tissue, including
the suture threads, are coated with Carbofilm, a thin
firm of carbon with turbostratic structure,
• The valve support incorporates a tantalium wire
rimming the edge of the inflow side of the valve so that
the implanted valve can be seen on radiography
TM
SOPRANO
Heart Valve
Major materials
•
•
•
•
•
Bovine pericardium for valve functional component
Polyester(PET) suture thread for sewing
Acetalic resin for valve support
Polyester(PET) knitted fabric for sewing ring
Tantalium wire for radiopaque marker embedded in
the valve support
• Carbofilm for biocompatible coating
• Silicone loaded with BaSO4 for additional radiopaque
sewing ring filler
TM
SOPRANO
Heart Valve
Procedures of pericardium
•
•
•
•
•
Animal selection
Pericardium retrieval operation
Dedicated personnel selection & training
Record keeping
Traceability
SOPRANOTM Heart Valve
Pericardium properties
• Bioburden of shipment medium
less than 105 CFU( colony forming units)/ml
• Thickness of pericardium
Valve size 19-23
0.30-0.42mm
Valve size 25-27
0.34-0.49mm
Valve size 29-33
0.40-0.53mm
• Morphology
Absence of flaws such as, vascularised areas, traumas,
superficial non-uniformity areas, inflammations
TM
SOPRANO
Heart Valve
Glutaraldehyde treatment
• Operating conditions, concentration, time &
temperature of cross-linking treatment, settled up
• After a first treatment with low glutaraldehyde(0.2%)
solution, the pericardial sheets are joined together.
• The biological component is then assembled with the
support structure and another fixation step with 0.5%
glutaraldehyde solution is accomplished
• A subsequent treatment with high glutaraldehyde
(0.5%) solution is aimed at the achieving the sterility
SOPRANOTM Heart Valve
Detoxification treatment
• As a results of glutaraldehyed fixation, tissue valves
may contain residual amount of unbounded aldehyde
group, which is responsible for blood cell damage
• Moreover the collagen affinity to binding with calcium
ions is enhanced in presence of unsaturated
glutaraldehyde groups, and this could facilitate
calcification process.
• A neutralizing post-fixation treatment based on the
action of homocysteic acid is performed, and this
molecule reacts with unsaturated aldehyde group, thus
neutralizing them
TM
SOPRANO
Heart Valve
 Sterilization of biological component
• Acceptance bioburden limit of incoming pericardium less than
100.000CFU/ml
• Aseptic processing and cleanroom technology rules are adopted
• The sterility of the pericardial component is reached by means
of a chemical treatment with low concentration glutaraldehyde
solution for prolonged times
 Sterilization of non-biologic components
• Valve stent is sterilized by chemical treatment
• Container, lid, holder, ID, sutures, equipments are sterilized
by Ethylene oxidem
• Chemical solutions are sterilized by means of filtration on
0.22um membrane
Aortic Porcine Bioprosthesis
CryoLife-O'Brien bioprosthesis
• O'Brien Stentless Aortic Porcine Bioprosthesis (CryoLife
Inc, Kennesaw, Ga) composite design was to optimize
hemodynamic performance and durability of valves.
• Three noncoronary leaflets are prepared by low-pressure
glutaraldehyde fixation process of less than 2 mm Hg.
• The scalloped design and the absence of xenograft tissue
below the leaflet hinge allow implantation with a single
suture line.
• Because there are no synthetic materials other than the
suture, the risk of endocarditis is expected to be reduced
Stentless Bioprosthesis
Specification
• Elimination of a rigid sewing ring, the dynamic nature
of the aortic root may be maintained after AVR with
this device.
• Maintenance of normal aortic root function may at
least in part be responsible for the excellent
hemodynamic performances of both stentless valves, as
well as aortic homografts.
• Medtronic Freestyle stentless bioprostheses and St Jude
Medical Toronto stentless porcine valve bioprostheses,
O'Brien Stentless Aortic Porcine Bioprosthesis
Determinants of Mineralization
 The determinants of bioprosthetic valve and
other biomaterial mineralization include
factors related to
(1) host metabolism,
(2) implant structure and chemistry,
(3) mechanical factors.
• Natural cofactors and inhibitors may also play
a role Accelerated calcification is associated
with young recipient age, glutaraldehyde
fixation, and high mechanical stress.
Calcification Process
Hypothesis
Bioprosthetic Heart Valves
Mechanism of calcification
• Mineralization process in the cusps of bioprosthetic
heart valves is initiated predominantly within
nonviable connective tissue cells that have been
devitalized but not removed by glutaraldehyde
pretreatment procedures
• This dystrophic calcification mechanism involves
reaction of calcium-containing extracellular fluid with
membrane-associated phosphorus, causing calcification
of the cells.
• This likely occurs because the normal extrusion of
calcium ions is disrupted in cells that have been
rendered nonviable by glutaraldehyde fixation.
Prevention of Calcification
 Three generic strategies have been investigated
for preventing calcification of biomaterial
implants:
• Systemic therapy with anticalcification agents;
• Local therapy with implantable drug delivery
devices;
• Biomaterial modifications, such as removal of
a calcifiable component, addition of an
exogenous agent, or chemical alteration.
Antimineralization
Strategies
 Systemic drug administration
 Localized drug delivery
 Substrate modification
•
•
•
•
Inhibitors of calcium phosphate mineral formation
Biphosphonates, trivalent metal ions, Amino-oleic acid
Removal/modification of calcifiable material
Surfactants, Ethanol, Decellularization
Improvement/modification of glutaraldehyde fixation
Fixation in high concentrations of glutaraldehyde
Reduction reactivity of residual chemical groups
Modification of tissue charge
Incorporation of polymers
Use of tissue fixatives other than glutaraldehyde
Epoxy compounds , Carbodiimides, Acyl azide,
Photooxidative preservation
Glutaraldehyde Preservation
Actions & limitation
• Reduced immunologic recognition and
resistance to degradative enzymes
• limited durability & structural deterioration;
nonviable tissues & inability of cell to migrate
through extracellular matrix
• Stiffened valve leaflets : abnormal stress
pattern causing accelerated calcification
Bioprosthetic Heart Valve
Prevention of calcification
• Several antimineralization pretreatments, such as
amino-oleic acid, surfactants, or bisphosphonates have
been investigated.
• Ethanol prevents mineralization of the cusps by
removal of cholesterol and phospholipids and major
alterations of collagen intrahelical structural
relationships.
• Aluminum chloride pretreatment prevents aortic wall
calcification by inhibition of elastin mineralization due
to the following mechanisms: binding of Al to elastin
resulting in a permanent protein-structural change
conferring calcification resistance, inhibition of alkaline
phosphatase activity, diminished upregulation of the
extracellular matrix protein, tenascin C, and inhibition
of matrix metalloproteinase-mediated elastolysis.
Bioprosthesis Calcification
Prevention
• Inhibitors of hydroxyapatite formation
Bisphosphonates
Trivalent metal ions
• Calcium diffusion inhibitor ( amino-oleic acid )
• Removal or modification of calcifiable material
Surfactants
Ethanol
Decellularization
• Modification of glutaraldehyde fixation
• Use of other tissue fixatives
• Problems created by an exposed aortic wall
Bioprosthetic Valved Conduit
New bioprosthesis
• By Medtronic, Inc. (Minneapolis, MN), the Contegra
conduit is a 0.25% glutaraldehyde-fixed segment of
bovine jugular vein, containing a venous valve; the
whole xenograft is fixed under minimal pressure, less
than 3 mm Hg
• Other stentless xenografts have been developed during
the last years, including the following: the LabCor from
Sulzer Carbomedics (Austin, TX); the Biocor from St
Jude Medical (Belo Horizonte, Brasil), which is a
bovine pericardial conduit with an aortic porcine valve;
and the Shelhigh pulmonic conduit (Union, NJ), which
consists of a pericardial bovine tube with a stentless
porcine pulmonary valve.
Bioprosthetic Valved Conduit
Characteristics
• By Medtronic, Inc. (Minneapolis, MN), the Contegra
conduit is a 0.25% glutaraldehyde-fixed segment of
bovine jugular vein, containing a venous valve; the
whole xenograft is fixed under minimal pressure, less
than 3 mm Hg
• Other stentless xenografts have been developed during
the last years, including the following: the LabCor from
Sulzer Carbomedics (Austin, TX); the Biocor from St
Jude Medical (Belo Horizonte, Brasil), which is a
bovine pericardial conduit with an aortic porcine valve;
and the Shelhigh pulmonic conduit (Union, NJ), which
consists of a pericardial bovine tube with a stentless
porcine pulmonary valve.
Bioprosthetic Valved Conduit
Recent development
• The Contegra conduit is a heterologous bovine jugular
vein graft with a trileaflet venous valve. The conduit is
fixed with 0.25% glutaraldehyde under zero pressure
condition. No additional anticalcification treatment is
used.
• Shelhigh No-react graft is porcine pulmonary valve
conduit, strikingly unsupported conduit.
• The typical finding of the explanted conduits was
prominent intimal peel formation at the distal
anastomosis without calcification, but little histologic
evidence of immunologic activity
Mechanical Valved Conduit
Controversies
• Mechanical PVR for patients who have had multiple
prior operations, or who are on warfarin
• In general, we have not advised mechanical PVR in
children, but mechanical PVR, if anticoagulation is
required for a mechanical prosthesis
• An adequate sized pulmonary prosthesis can be
inserted with no prosthetic material or by using only an
anterior patch of pericardium
• Recently, it has been reported that tilting-disc valves in
the pulmonary position may perform better than
bileaflet valves
Valved Conduit Implantation
General aspects
• Conduit implantation for the RVOT reconstruction
constitutes approximately 15% to 20% of all congenital
cardiac operations
• Homografts have shown the best results so far, with
overall survivals of appoximately 84% and 31% at 5 and
15 years, respectively
• Young age at implantation, and the need for a small
homograft have consistently been recognized as risk
factors for accelerated failure
• Problem to use homografts is the scarcity of suitably
sized homografts and the consistency of its quality.
• Xenograft aortic or pulmonary valves & conduits have
failed to match the results of the homograft conduits
Valved Conduit
Replacement
Indications for replacement
• In asymptomatic patients is near systemic or
systemic pressure of the RV and a peak
instantaneous Doppler systolic gradient greater
than 65–70 mmHg, compared to 40–50 mmHg in
the majority
• Transcatheter interventions such as balloon
dilatation or stent implantation when indicated,
may prolong the RV–PA conduit lifespan and
delay the first reoperation for conduit obstruction.
• Use of an autologous alternative like ‘Reparation
a` l’e´tage Ventriculaire’ (REV procedure) or other
technical options
Valved Conduit Implantation
Sizing & shaping
• The main pulmonary artery was always completely
transected to allow a harmonious end-to-end
anastomosis of the graft to the pulmonary bifurcation
• The diameter of the valved conduit was mostly
determined according to the diameter of the PA
bifurcation, although the distance between the conal
septum and pulmonary bifurcation was also taken into
consideration at times
• Locate the valve as cranial as possible immediately
below the bifurcation of the pulmonary artery to avoid
geometrical distortion of the valve at the site of
proximal implantation in the right ventricle.
Valved Conduit Implantation
 Technical aspect
•
•
•
•
A better size matching of the graft to the pulmonary
artery diameter may reduce local turbulent flow and
subsequent development of the fibrotic membrane
Relative profile of the stenotic membrane becomes
critical in small conduits(<14mm )
Plane of valve annulus should remain perpendicular
to the graft direction.
Short bevel results in traction on the convex part of
graft with caudal rotation of the annular plane &
compression at the leaflet level
Mechanical Valved Conduit
Four main criteria
• First, older age to avoid outgrowth of the
prosthesis
• Second, multiple previous operations with an
increased reoperation-related morbidity
• Third, current use of anticoagulants
• Fourth, patient compliance with anticoagulant
therapy.
Valved Conduit Implantation
Technique for small conduit
• The distal anastomosis is made in diagonal
shape to enlarge the anastomosis
• 7-0 prolene sutures in multiple continuous
interrupted sutures
• Start the anastomosis always from the outside
the lumen
• Anticoagulation for at least 2-3 months and
aspirin for life
Valved Conduit Implantation
Sizing
• Attempt to keep the pulmonary bifurcation intact
• The main pulmonary artery was always completely
transected to allow a harmonious end-to-end
anastomosis of the graft to the pulmonary bifurcation
• The diameter of the valved conduit was mostly
determined according to the diameter of the PA
bifurcation, although the distance between the conal
septum and pulmonary bifurcation was also taken into
consideration at times
• Locate the valve as cranial as possible immediately
below the bifurcation of the pulmonary artery
Bioprosthetic Conduit
Causes of early stenosis
• Chronic movement at the level of the anastomosis could
be an ongoing local trigger for peel formation.
• The intensive narrowing process may be in part the
result of a strong cellular immunologic reaction against
the xenograft
• Additional factors such as endothelial lesions made
during conduit suture, residual glutaraldehyde release
from the implant, and shear stresses related to
hemodynamic conditions at the level of the anastomosis
might amplify this proliferative reaction.
Bioprosthetic Conduit Stenosis
Medical prevention
• Heparin infusion (10 IU/kg/h ) in early postoperatively,
followed by low molecular weight heparin (until
hospital discharge).
• Aspirin is given postoperatively for 3 months, starting
from the first postoperative day
• Aspirin plays a protective role for the endothelium and
influences neointima formation
• Use aspirin for long periods to avoid thrombosis, as
well as anti-inflammatory therapy to prevent chronic
rejections
Shelhigh No-react Graft
Histopathology of explant
• Intimal fibrosis, neointimal proliferation, and
neointimal peel formation; chronic inflammation with
histiocytic demarcation of the xenograft tissues, as well
as a granulomatous reaction with the presence of giant
cells of foreign-body type.
• Immunohistochemistry revealed predominantly
histiocytic and T-lymphocytic infiltrates, with a sparse
B-lymphocytic component.
• The granulomatous and histiocytic demarcation
resulted in an incomplete or complete neointimal
"peel" formation in most conduits.
Xenograft Valve or Conduit
Xenoreactive antigen
• Immunogenic [alpha]-Gal-epitope was founf on
fibrocytes interspersed in the connective tissue of
porcine bioprosthetic valves
• Patients with a bioprostheses had developed a
significant increase of naturally occurring cytotoxic
IgM antibodies directed towards [alpha]-Gal
• Patients after the implantation of bioprostheses
demonstrated an increased cytotoxicity against [alpha]Gal-bearing PK-15 cells
• The specificity of the cytotoxic effects was proven as
soluble Gal[alpha]1–3Gal[beta]1–4GlcNAc markedly
inhibited cell death of [alpha]-Gal-bearing PK15 cells
Difference of Gal Expression
Mechanisms
• Decreased α -Gal expression on valve endothelium could
be due to either decreased glycosylation or decreased
surface glycoproteins on leaflet endothelium.
• Protein expression is closely regulated in endothelial
cells, and flow rate can clearly modulate endothelial
gene expression.
• Expression of proteins, such as major histocompatibility
complex antigen and vascular cell adhesion molecule 1,
are all influenced by the amount of shear force exerted
on the endothelium.
Xenograft Valve or Conduit
Xenoreactive immulogic acivity
• Younger patients evidenced increased valve
degeneration, indicating increased immunologic
activity directed against the xenograft
• Degeneration of bioprostheses begins with the
penetration of immunoglobulines (IgG/IgM) into the
valve-matrix.
• Subsequently, macrophages are deposited on the valve
surface and erythrocytes penetrate into the valve.
• Finally, collagen breakdown calcification takes place
• Thus, the deposition of immunoglobulins represents the
initial immunological trigger for calcification in
bioprostheses
Xenograft Valve or Conduit
Investigation of xenoantigen
• Presence of [alpha]-Gal-epitope on native and
fixed porcine valves
• Implantation of bioprostheses elicits increased
formation of cytotoxic anti [alpha]-Gal IgM
antibodies
• Potentially increased lysis of [alpha]-Galbearing PK15 porcine cells from serum
obtained prior and after bioprostheses
implantation.
Xenoreactive Valve or Conduit
Immunohistochemistry
• Labelling with IB4, and antibody against von Willebrand Factor
• Then the diluted primary antibody against vWF [1: 300] was
applied & rinsed in PBS and incubated with a secondary antibody
• After rinsing, finally, sections were rinsed again in PBS and
mounted in 60% glycerine in Tris buffer: double-fluorescence
labelling was applied
• Sections were incubated in the primary antibody against vimentin
[1: 100] and then stained with the secondary antibody
• Finally, sections were labelled with IB4 and in addition, sections were
coincubated with Gal[alpha]1–3Gal[beta]1–4GlcNAc (5 mg mL-1 and
1 mg mL-1, to demonstrate galactose [alpha]1,3-galactose specificity
with IB4-isolectin staining.
Xenoreactive Valve or Conduit
Enzyme-linked immunoabsorbent assays
• ELISA assay performed to compare the
incidence of naturally occurring cytotoxic anti
α-Gal IgM antibodies
Cytolysis of PK-15 cells
• Cytotoxic activity of the anti α -Gal IgM
antibodies, PK-15 (ATCC CCL-33) (20·000/96
round-bottom plate well) were incubated with
a serum pool diluted.
• Cell death determined by trypan-blue staining
Xenoreactive Valve or Conduit
Removal of α-gal epitopes
• Green coffee bean α -galactosidase can cleave the
terminal α -galactose(α-Gal) on oligosaccharides .
• α-galactosidase, 1) functions at a range of temperature
and pH relevent to clinical organ transplantation, 2) is
effective in removing the terminal α-Gal sugar from
pig vessel and, 3) results in prolonging survival of
porcine vessels
• The tissues were subsequently immersed into a
phosphate-citrate-sodium chloride buffer containing
100 U/ml α-galactosidase and incubated for 4 hr at 426°C with gentle shaking
Xenotransplantation
Xenotransplantation
Clinical application
•
•
•
•
•
•
•
Selection of a donor species
Hyperacute rejection
Acute vascular rejection
Accommodation
Cellular rejection
Physiologic limitations
Zoonosis
Xenotransplantation
Biologic responses
• Transplantation into an unmanupulated recipient would
give rise to hyperacute rejection
• If hyperacute rejection is avoided, as for example by
inhibition of complement, the graft becomes subject to
acute vascular rejection
• If antidonor antibodies are depleted from the recipient,
the graft may undergo accommodation
• If acute vascular rejection is avoided, the graft may
undergo cellular rejection or chronic rejection
Xenotransplantation
Endothelial activation of rejection
• Resting or quiscent endothelial cells form a tight
monolayer that poses an effective barrier to blood cells
and plasma proteins
• Exposure of endothelial cells to agents such as
interleukin-1, tumor necrosis factor, or endotoxin,
causes the cell to undergo a series of metabolic and
structural changes, known as activation.
• Activated endothelial cells promote platelet aggregation,
fibrin generation, and neutrophil adhesion, and a
monolayer of activated endothelial cells is permeable to
plasma protein and blood cells
Xenotransplantation
Biologic responses
Xenotransplanrtation
Inhibit complement
Acute vascular
rejection
Accomodation
Deplete Ab
Hyperacute rejection
Cellular
rejection
Chronic
rejection
Xenotransplantation
Hyperacute rejection
• Hyperacute rejection is characterized clinically by the
development and rapid progression of discoloration
and ecchymosis and with severe organ failure over a
period of minutes to hours
• The histology of hyperacute rejection is characterized
by hemorrhage and widespread platelet thrombi
• Hyperacute rejection would be initiated by binding to
the endothelial lining of blood vessels in that organ of
xenoreactive natural antibodies ( antigen, Galα1-3Gal)
• Antibody binding activates the complement system,
and the action of complement on blood vessels causes
the devastating picture of hyperacute rejection
Xenotransplantation
Hyperacute rejection
• It is characterized by widespread hemorrhage,
edema, thrombosis, and a relative lack of a
cellular infiltrates.
• HAR is triggered by the xenoreactive
antibodies in the host circulation which
recognize and bind donor endothelium
• It is the subsequent activation of host
complement that mediates HAR, leading to
immediate graft destruction
Xenotransplantation
Hyperacute Rejection
• Synthesis and inhibition of synthesis of Galα1-3Gal,
major target antigen in cardiac xenograft rejection
Xenotransplantation
Prevention of hyperacute rejection
• Prevention of hyperacute rejection can be achieved by
either interfering with antibody binding to the graft or
inhibiting activation of the complement system
• Extracorporeal perfusion of blood or separated plasma
through a column bearing Galα1-3Gal, and by lowering
the antigen expression by genetic means
• Inhibition of activation of complement using cobra
venom factor, soluble complement receptor type 1,
gamma globulin, expression of human complement
regulatory protein ( decay accelerating factor )
Porcine Xenotransplantation
Prevention of hyperacute rejection
• The prominent epitope is a terminal carbohydrate
moiety, Galα1-3Galβ1-4GlcNAc-R(α Gal ) which is
found on various glycoproteins and glycolipids on
porcine cells.
• HAR can be prevented by continual systemic inhibition
of complement through the use of genetically modified
porcine organs that express human membrane-bound
complement regulatory proteins, by immunoapheresis
to remove all circulating antibody, or by selective
immunoapheresis that removes only antigal antibodies,
and synthesis of αGal tricsaccharide that can neutralize
circulating anti-Gal antibodies
Xenotransplantation
Acute vascular rejection
• Acute vascular rejection( delayed xenograft rejection)
may begin within 24 hours of reperfusion and generally
destroys the graft over a period of days to weeks
• The histologic features include endothelial swelling,
ischemia, and diffuse intervascular thrombosis, with
thrombi consisting mainly of fibrin and is much like the
picture of acute vascular rejection of allograft
• The cause may be the antidonor antibodies that bind to
a xenograft that precipitate endothelial injury by
activating the complement system or by perturbing
endothelial cell surface molecules, and also facilitate
injury by interacting with inflammatory cells
Xenotransplantation
Acute Vascular Rejection
• Role of antidonor antibodies in the pathogenesis of acute vascular
rejection. Antidonor antibodies may directly perturb endothelial
cells or ( C ) trigger activation of complement
Xenotransplantation
Accommodation
• If hyperacute rejection and acute vascular
rejection are temporarily averted, a state of
accommodation may occur in which the graft
appears inured to the presence of antidonor
antibodies in circulation
• The mechanisms are uncertain, although they
may involve a change in the antibody
repertoire, change in antigen, or the acquired
resistance of endothelium to humoral injury
Xenotransplantation
Cellular rejection(1)
• Difference between the cellular response to a
xenograft and to an allograft may be how to T cells
recognize foreign tissue.
• In xenotransplantation, there is evidence that T cells
may not always recognize xenogeneic cells directly, in
part because the foreign accessory signals are
incompatible ( indirect recognition )
• In allotransplantation, T cells recognize foreign major
histocompatibility antigens expressed on the foreign
cells ( direct recognition ).
• Binding antidonor antibodies heighten cellular
immune response by causing activation of antigen
presenting cells
Xenotransplantation
Cellular rejection(2)
• The extent to which the direct versus indirect pathways
of T cell recognition might utilized in cellular responses
to xenotransplantation has therapeutic implication
• If direct pathway predominates, as it does in
allotransplantation, then genetic engineering of donor
might help to limit that response, but, on the other
hand, if the indirect pathway predominates, anti-CD4
antibodies might be useful
• Natural killer cells are lymphopcytes that lack of T cell
receptor but that recognize foreign or virus-infected
cells and in so doing release inflammatory cytokines
and mediate cytotoxicity
Xenotransplantation
Cellular Rejection
• Interactions of T lymphocytes of a graft recipient
with antigen-presenting cells
Vascularized Xenograft
Rejection
• Vascularized xenografts are referred as concordant or
discordant according to the timing and the immune
effectors depositing on graft capillary endothelial cells.
• Concodant xenografts are rejected within days of
engraftments when the immunoglobulins specific to the
graft have been induced, and discordant xenografts are
rejected within minutes
• Hyperacute rejection in discordance is mediated by the
reaction of natural antibodies (classic pathway) or the
complement system activated by alternative pathway
( antibody-independent pathway of complement
activation)
Vascularized Xenograft
Rejection
• The capillary rejection of a vascularized xenograft
coincides with the onset of graft dysfunction & these
changes are limited to the endothelium
• Then the medial smooth muscle cells are subjected to
immune effectors after the endothelial cells have been
rejected
• There is also inflammation of the medial extracellular
matrix which is probably elicited by immunogenicity
of the xenogeneic extracellular matrix
Xenotransplantation
Complement-regulatory proteins
• The beta-actin-CD59 transgene contains a chick betaactin promoter that controls expression of a cDNA for
human CD59. In this transgene, the hCD59 cDNA is
embedded in a human alpha-globin gene expression
cassette that provides splicing and polyadenylation
signal sequences.
• The H2K-DAF transgene contains a cDNA for human
DAF regulated by the mouse H2K promoter and H2K
polyadenylation signal sequences
• Probes for DNA and RNA hybridizations were derived
from the cDNA of each of the genes
Xenotransplantation
Complement-regulatory proteins
• Normally, the vasculature is protected from autologous
complement-mediated damage by several membranebound complement-regulatory proteins, such as
CD59(protectin), decay accelerating factor(CD55,
DAF), or membrane cofactor protein(CD46)
• Each CRPs of species have been shown to be most
effective at controlling the activation of homologous
complement, and the complement of closely related
species, but they are much less effective regulators of
complement from more divergent species
• In the heart, hCD59(protectin) expression was detected
on the endothelial cells and on the myocytes
Xenotransplantation
Complement-regulatory proteins
• There exist a family of complement-regulatory proteins
which , under physiologic conditions, down-reguate the
complement cascade to protect cells from damage by
autologous complement
• CRPs are found in both soluble and membrane-bound
forms.
• CRPs exhibit homologous restriction so that CRPs
from one species are relatively inefficient at regulating
complement from a distant species
• Expression of human membrane-bound complementregulatory proteins could provide local protection from
complement-mediated damage
Xenotransplantation
Complement-regulatory proteins
• A lack of effective complement regulation sensitizes the
porcine organ to complement-mediated damage, and
thereby, contributes to hyperacute rejection.
• Then the introduction of of human CRP function, even
at lower levels, may significantly effect the humoral
rejection of porcine xenograft
• Hypothesis, by enhancing the resistance of xenogenic
vascular endothelium to complement-mediated damage
through expression of human CRP gene products in
transgenic animal
Xenotransplantation
Generation of transgenic pigs
• A 60 kh genomic construct encompassing the
human CD46 gene, or hDAF, or
CD59(protectin) was isolated from a P1 phase
library and microinjected into the male
pronuclei of fertilized porcine oocytes to
generate transgenic pig
• Transgenic animals were identified by
Southern blot analysis of DNA from tail
biopsies
Xenotransplantation
Transgenic pigs
• A line of transgenic pigs that express the human
complement-regulatory proteins human CD59 and
human decay-accelerating factor
• This specificity is evident in transgenic organs in which
low levels of CD59 and human decay-accelerating
factor(CD55) expression significantly effect the
humoral immune response that causes xenograft
rejection
• Transgenic organs with high levels of human
complement-regulatory protein expression will be
sufficient to alleviate the humoral immunologic
barriers
Xenotransplantation
 Reconsideration in concordant or discordant
• It is focused on the presence or absence of hyperacute
rejection to classify species combination, at least three
factors might be considered
• The presence or absence of natural antibodies directed
against endothelial cells of a xenogeneic donor
• The function, or lack thereof, of complement regulatory
proteins of recipient in the “environment” of the donor
organ
• The relative effectiveness of cell membrane-associated
complement inhibitory proteins of the donor against
the complement system of the recipient
Xenotransplantation
Pig model
• The animal of choice currently considered to be the pig
because of its size, physiologic compatibility, and
breeding characteristics and the potential for genetic
modification.
• The current limitation to clinical xenotransplantation is
immunologic rejection.
• Achieving this goal is to define immunosuppressive
regimens that control the xenograft immune response
by using non–life-supporting heterotopic transplants.
Xenotransplantation
Anti-xenoreactive antibody
• Xenoreactive IgG/IgM antibodies were detected in the
sera of primates, including man, against the
oligosaccharid residue [alpha]-Gal, which is present on
pig tissue
• In primates the [alpha]-Gal-specific IgM-antibodies
lead to complement activation and hyperacute rejection
of pig xenografts within minutes to hours
• About 1% of the circulating immunoglobulines are
specific for [alpha]-Gal in humans and their natal
formation is presumably owing to exposure to gut flora
expressing the [alpha]-Gal-epitope
Xenotransplantation
Xenoreactive antigen
• [alpha]-Gal is an unique carbohydrate structure, which
has been evolutionarily conserved in most mammalian
species except humans, apes and Old World monkeys
• [alpha]-Gal is widely expressed on the cell surface of
mammals and highly expressed in capillaries as
compared to large vessels, due to differential gal α 1, 3
galactosyltransferase expression endothelium
• Since humans lack the α- galactosyltransferase, they
have natural anti-Gal antibodies.
• About 1% of B cell clones in humans produce natural
anti-Gal
Anti-Gal Antibodies
Measurement
• Antibody levels were measured by enzyme-linked immunosorbent
assay (ELISA) using HSA-[alpha]1,3 Gal as a specific antigen and
human serum albumin (HSA)
• RIA/EIA 96-well plates were coated with 30 µg/ml of either HSA[alpha]1,3 Gal or HSA alone, blocked with 0.1% Tween 20/1%
HSA-phosphate-buffered saline (PBS), and then incubated with
serial dilutions of serum at 4°C.
• After washing plates, wells were incubated with streptavidinalkaline phosphatase-conjugated goat anti-human µ-chain, or
streptavidin-alkaline phosphatase-conjugated goat anti-human
[gamma]-chain, and developed with Sigma Fast pNPP tablets
• The absorbance at 405 nm was determined using a Molecular
Dynamics Plate Reader and Softmax Pro software
Anti-α-Gal Antibodies
Measurement
• Isotype-specific determination of anti-α-Gal antibodies
was performed by enzyme-linked immunosorbent assay
• Binding of xenoreactive IgM to cultured porcine aortic
endothelial cells (anti-PAEC IgM) was determined by
ELISA
• Antipig antural antibody levels were measured by
ELISA using porcine platelet extracts (PPE) as antigen
Xenotransplantation
Recent porcine trial
• Porcine human model
• Produce genetically engineered pigs that
---- Lack Gal epitope( humans have circulating
antiGal Ab)
---- Express human dacay accelerating factor
(hDAF) ( presence of hDAF protects against
complement mediated hyperacute rejection
• Nuclear transfer to shorten span required to
produce identical herds of genetically altered
animals
Immunofluorescent Studies
Reagents
• Biopsy tissues were stained for IgG, IgM, C3, C4, C5b
neoantigen, MAC, properdin, fibrinogen,
polymorphonuclear neutrophils, and platelets
• Affinity-isolated, fluorescein isothiocyanate (FITC)
goat anti-human antibodies against IgG, IgM, C4, C3,
and C5b
• Properdin and fibrinogen using FITC-conjugated
rabbit anti-human antibodies
• MAC using murine monoclonal antibodies against a
neoantigen of MAC
• Monocytes and/or granulocytes using murine antihuman CD11b/CD18 (OKM1) and platelets using
murine anti-human CD9
Xenotransplantation
Recent porcine trial
• The enzyme alpha1,3-galactosyltransferase(alpha1,3GT
or GGTA1) synthesizes alpha 1,3-galatose epitopes,
which are the major xenoantigens causing hyperacute
rejection , and acute vascular rejection
• Reported earlier the targeted disruption of one allele of
the alpha 1,3 GT gene in cloned pig.
• Based on a bacterial toxin was used to select for cells in
which the second allele of gene was knocked out.
• Sequencing analysis demonstrated that knocked out of
the second allele of the alpha 1,3 GT gene was caused
by a T-to-G single point mutation at the second base of
exon 9
Xenotransplantation
Prevention of Gal expression
• Breeding of pigs that do not express Gal, achieved
by knocking out the gene for the enzyme, α1,3galactosyltransferase( nuclear transfer/embryo
transfer techniques)
• Reducing Gal expression on pig cells, by cleaving Gal
from the underlying substrate, or replacing Gal with
an alternative, innocuous oligosaccharide by a process
that has been termed ‘competitive glycosylation’ to
reduce cell-surface expression of several
oligosaccharides
Xenotransplantation
• Pig organs, when transplanted to primates, are
rapidly rejected because of the presence of recipient
antibody that recognizes a carbohydrate epitope,
galactose 1–3 galactose ( -Gal), which is present on
glycoproteins and glycolipids on the pig endothelium.
• This rejection process, hyperacute rejection, can be
routinely overcome by the removal of anti-Gal antibody
or the inhibition of the complement cascade by the use
of transgenic pigs that express human complement
regulatory proteins.
• When hyperacute rejection is blocked, grafts are
usually lost within a few days to weeks through a
process called delayed xenograft rejection (DXR).
Xenotransplantation
Pig to primate model
• Hyperacute rejection occurs in the discordant pig-toprimate model using vascularized porcine organs, and
in ABO-mismatched renal and cardiac allografts, due
to the binding of preformed natural antibodies to cell
surface carbohydrate structures, and subsequent
complement activation.
• To date, control of the anti-Gal immune response in
pig-to-primate xenotransplantation model has been
attempted using phamachological immunosuppression,
tolerance induction, or antibody removal using
plasmapheresis, organ perfusion, or immunoadsorption.
Anti-gal Immune Response
Control methods
• To date, in pig-to-primate xenotransplant model has
been attempted using pharmacological
immunosuppression, tolerance induction, or antibody
removal using plasmapheresis, organ perfusion, or
immunoadsorption and transgenic animal.
• None of these methodologies has been consistently
successful in the primate model, and all are associated
with a high protocol-related morbidity, especially
strategies that involve the use of immunoapheresis,
which is particularly challenging due to the small size
of the primate recipients
Xenotransplantation
Immunosuppression & medical therapy
•
•
•
•
•
•
•
•
•
Splenectomy
TPC(α-galactosyl-polyethylene glycol conjugate)
Tacrolimus
Sirolimus
Corticosteroid
Rituximab(anti-CD20)
Lovenox(low molecular weight heparin)
RATG
Ganciclovir, valganciclovir, bactrim
Xenotransplantation
Complications of immunosuppression
• PCR for detection of cytomegalovirus & gamma
herpesvirus
• Antirejection therapy
• Immunosuppressants, ganciclovir, ATG
• Infectious complicationof virus
• Posttransplant lymphoproliferative disease
(Reactivation of of host lymphotropic virus)
Vascularized Xenograft
Rejection
• The capillary rejection of a vascularized xenograft
coincides with the onset of graft dysfunction & these
changes are limited to the endothelium
• Then the medial smooth muscle cells are subjected to
immune effectors after the endothelial cells have been
rejected
• There is also inflammation of the medial extracellular
matrix which is probably elicited by immunogenicity
of the xenogeneic extracellular matrix
Xenotransplantation
 Reconsideration in concordant or discordant
• It is focused on the presence or absence of hyperacute
rejection to classify species combination, at least three
factors might be considered
• The presence or absence of natural antibodies directed
against endothelial cells of a xenogeneic donor
• The function, or lack thereof, of complement regulatory
proteins of recipient in the “environment” of the donor
organ
• The relative effectiveness of cell membrane-associated
complement inhibitory proteins of the donor against
the complement system of the recipient
Xenotransplantation
Transgenes of CRP
• The beta-actin-CD59 transgene contains a chick betaactin promoter that controls expression of a cDNA for
human CD59. In this transgene, the hCD59 cDNA is
embedded in a human alpha-globin gene expression
cassette that provides splicing and polyadenylation
signal sequences.
• The H2K-DAF transgene contains a cDNA for human
DAF regulated by the mouse H2K promoter and H2K
polyadenylation signal sequences
• Probes for DNA and RNA hybridizations were derived
from the cDNA of each of the genes
Xenotransplantation
Complement-regulatory proteins(CRP)
• Normally, the vasculature is protected from autologous
complement-mediated damage by several membranebound complement-regulatory proteins, such as
CD59(protectin), decayaccelerating factor(CD55, DAF),
or membrane cofactor protein(CD46)
• Each CRPs of species have been shown to be most
effective at controlling the activation of homologous
complement, and the complement of closely related
species, but they are much less effective regulators of
complement from more divergent species
• In the heart, hCD59(protectin) expression was detected
on the endothelial cells and on the myocytes
Xenotransplantation
Complement-regulatory proteins
• There exist a family of complement-regulatory proteins
which , under physiologic conditions, down-reguate the
complement cascade to protect cells from damage by
autologous complement
• CRPs are found in both soluble and membrane-bound
forms.
• CRPs exhibit homologous restriction so that CRPs
from one species are relatively inefficient at regulating
complement from a distant species
• Expression of human membrane-bound complementregulatory proteins could provide local protection from
complement-mediated damage
Xenotransplantation
Transgenic animal
• A lack of effective complement regulation sensitizes the
porcine organ to complement-mediated damage, and
thereby, contributes to hyperacute rejection.
• Then the introduction of of human CRP function, even
at lower levels, may significantly effect the humoral
rejection of porcine xenograft
• Hypothesis, by enhancing the resistance of xenogenic
vascular endothelium to complement-mediated damage
through expression of human CRP gene products in
transgenic animal
Xenotransplantation
Generation of transgenic pigs
• A 60 kh genomic construct encompassing the
human CD46 gene, or hDAF, or
CD59(protectin) was isolated from a P1 phase
library and microinjected into the male
pronuclei of fertilized porcine oocytes to
generate transgenic pig
• Transgenic animals were identified by
Southern blot analysis of DNA from tail
biopsies
Xenotransplantation
Transgenic pigs
• A line of transgenic pigs that express the human
complement-regulatory proteins human CD59 and
human decay-accelerating factor
• This specificity is evident in transgenic organs in which
low levels of CD59 and human decay-accelerating
factor(CD55) expression significantly effect the
humoral immune response that causes xenograft
rejection
• Transgenic organs with high levels of human
complement-regulatory protein expression will be
sufficient to alleviate the humoral immunologic
barriers
Xenotransplantation
Pig model
• The animal of choice currently considered to be the pig
because of its size, physiologic compatibility, and
breeding characteristics and the potential for genetic
modification.
• The current limitation to clinical xenotransplantation is
immunologic rejection.
• Achieving this goal is to define immunosuppressive
regimens that control the xenograft immune response
by using non–life-supporting heterotopic transplants.
Anti-α-Gal Antibodies
Measurement
• Isotype-specific determination of anti-α-Gal antibodies
was performed by enzyme-linked immunosorbent assay
• Binding of xenoreactive IgM to cultured porcine aortic
endothelial cells (anti-PAEC IgM) was determined by
ELISA
• Antipig antural antibody levels were measured by
ELISA using porcine platelet extracts (PPE) as antigen
Immunofluorescent Studies
Reagents
• Biopsy tissues were stained for IgG, IgM, C3, C4, C5b
neoantigen, MAC, properdin, fibrinogen,
polymorphonuclear neutrophils, and platelets
• Affinity-isolated, fluorescein isothiocyanate (FITC)
goat anti-human antibodies against IgG, IgM, C4, C3,
and C5b
• Properdin and fibrinogen using FITC-conjugated
rabbit anti-human antibodies
• MAC using murine monoclonal antibodies against a
neoantigen of MAC
• Monocytes and/or granulocytes using murine antihuman CD11b/CD18 (OKM1) and platelets using
murine anti-human CD9
Xenoreactive Antigen
Anti-xenoreactive antibody
• Xenoreactive IgG/IgM antibodies were detected in the
sera of primates, including man, against the
oligosaccharid residue [alpha]-Gal, which is present on
pig tissue
• In primates the [alpha]-Gal-specific IgM-antibodies
lead to complement activation and hyperacute rejection
of pig xenografts within minutes to hours
• About 1% of the circulating immunoglobulines are
specific for [alpha]-Gal in humans and their natal
formation is presumably owing to exposure to gut flora
expressing the [alpha]-Gal-epitope
Xenoreactive Antigen
Characteristics
• [Alpha]-Gal is an unique carbohydrate structure, which
has been evolutionarily conserved in most mammals
except humans, apes & Old World monkeys
• [Alpha]-Gal is widely expressed on the cell surface of
mammals and highly expressed in capillaries as
compared to large vessels, due to differential gal α 1, 3
galactosyltransferase expression among different
endothelium
• Since humans lack the α- galactosyltransferase, they
have natural anti-Gal antibodies and about 1% of B cell
clones in humans produce natural anti-Gal
Anti-Gal Antibodies
Measurement
• Antibody levels were measured by enzyme-linked immunosorbent
assay (ELISA) using HSA-[alpha]1,3 Gal as a specific antigen and
human serum albumin (HSA)
• RIA/EIA 96-well plates were coated with 30 µg/ml of either HSA[alpha]1,3 Gal or HSA alone, blocked with 0.1% Tween 20/1%
HSA-phosphate-buffered saline (PBS), and then incubated with
serial dilutions of serum at 4°C.
• After washing plates, wells were incubated with streptavidinalkaline phosphatase-conjugated goat anti-human µ-chain, or
streptavidin-alkaline phosphatase-conjugated goat anti-human
[gamma]-chain, and developed with Sigma Fast pNPP tablets
• The absorbance at 405 nm was determined using a Molecular
Dynamics Plate Reader and Softmax Pro software
Xenotransplantation
Pig to primate model
• Hyperacute rejection occurs in the discordant pig-toprimate model using vascularized porcine organs, and
in ABO-mismatched renal and cardiac allografts, due
to the binding of preformed natural antibodies to cell
surface carbohydrate structures, and subsequent
complement activation.
• To date, control of the anti-Gal immune response in
pig-to-primate xenotransplantation model has been
attempted using phamachological immunosuppression,
tolerance induction, or antibody removal using
plasmapheresis, organ perfusion, or immunoadsorption.
Xenotransplantation
Recent porcine trial
• Porcine human model
• Produce genetically engineered pigs that
---- Lack Gal epitope( humans have circulating
antiGal Ab)
---- Express human dacay accelerating factor
(hDAF) ( presence of hDAF protects against
complement mediated hyperacute rejection
• Nuclear transfer to shorten span required to
produce identical herds of genetically altered
animals
Xenotransplantation
Recent porcine trial
• The enzyme alpha1,3-galactosyltransferase(alpha1,3GT
or GGTA1) synthesizes alpha 1,3-galatose epitopes,
which are the major xenoantigens causing hyperacute
rejection , and acute vascular rejection
• Reported earlier the targeted disruption of one allele of
the alpha 1,3 GT gene in cloned pig.
• Based on a bacterial toxin was used to select for cells in
which the second allele of gene was knocked out.
• Sequencing analysis demonstrated that knocked out of
the second allele of the alpha 1,3 GT gene was caused
by a T-to-G single point mutation at the second base of
exon 9
Xenotransplantation
• Pig organs, when transplanted to primates, are
rapidly rejected because of the presence of recipient
antibody that recognizes a carbohydrate epitope,
galactose 1–3 galactose ( -Gal), which is present on
glycoproteins and glycolipids on the pig endothelium.
• This rejection process, hyperacute rejection, can be
routinely overcome by the removal of anti-Gal antibody
or the inhibition of the complement cascade by the use
of transgenic pigs that express human complement
regulatory proteins.
• When hyperacute rejection is blocked, grafts are
usually lost within a few days to weeks through a
process called delayed xenograft rejection (DXR).
Antixenoreactive Response
Control methods
• To date, in pig-to-primate xenotransplant model has
been attempted using pharmacological
immunosuppression, tolerance induction, or antibody
removal using plasmapheresis, organ perfusion, or
immunoadsorption
• None of these methodologies has been consistently
successful in the primate model, and all are associated
with a high protocol-related morbidity, especially
strategies that involve the use of immunoapheresis,
which is particularly challenging due to the small size
of the primate recipients
Xenotransplantation
Immunosuppression therapy
•
•
•
•
•
•
•
•
•
Splenectomy
TPC(α-galactosyl-polyethylene glycol conjugate)
Tacrolimus
Sirolimus
Corticosteroid
Rituximab(anti-CD20)
Lovenox(low molecular weight heparin)
RATG
Ganciclovir, valganciclovir, bactrim
Xenotransplantation
Immunosuppression
• PCR for detection of cytomegalovirus &
gamma herpesvirus
• Antirejection therapy
• Immunosuppressants, ganciclovir, ATG
• Infectious complicationof virus
• Posttransplant lymphoproliferative disease
(Reactivation of of host lymphotropic virus)