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Organ Preservation & Tissueengineering
Seoul National University Hospital
Department of Thoracic & Cardiovascular Surgery
Organ Preservation
Glutaraldehyde Fixation
Principles
• Ultrastructural integrity is important for
prevention of tissue calcification.
• Immediate fixation with higher concentrations
of GA at low temperature significantly
preserves tissue integrity.
• It may be postulated that higher concentrations
of GA lead to a lower degree of calcification.
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
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.
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
Crosslinking
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
Aspects of calcific degeneration
* Excess aldol condensates in the tissue
* Autolytic tissue damage
* Changes of proteoglycan content of the tissue
* Continual enzyme activity
* Insufficiently suppressed immunogenicity
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 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
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
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.
Calcification of Bioprosthesis
Heat treatment
• Heat may facilitate extraction and denaturation of the
phospholipids and proteins involved in the process of
calcification
• The tissues obtained at the slaughterhouse were
immediately placed in the 0.625% glutaraldehyde
solution.
• After 15 days of fixation in this solution, submitted to
heat treatment
• Glass bottles containing tissues in glutaraldehyde
solution were placed in an oven at 50°C for 2 months
with permanent agitation by a rotator machine (3
rotations/minute), then the glutaraldehyde solution was
replaced by a fresh solution.
Bioprosthesis Mineralization
 Determinants
• 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.
Bioprosthesis Calcification
Prevention
• 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
Prevention of Mineralization
Residual glutaraldehyde reduction
• Reaction between epsilon amino groups of collagen
lysine and aldehyde residues on the glutaraldehyde
molecules results in the formation of a Schiff base
(Amino acid neutralization)
• Glutaraldehyde polymerizes, creating new covalent
bonds with the bioprosthetic tissue, and subsequent
degradation of polymeric glutaraldehyde cross-links
leads to a cytotoxic reaction.
• Improvement of spontaneous endothelialization as well
as mitigation of mineralization has been achieved by
post-fixation detoxification with the various amino acid
solutions
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
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
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 ?
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
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.
Bioengineered Vascular Graft
Requisite for small caliber graft
• A synthetic small caliber graft should be
resistant to thrombosis and biocompatible,
resembling a native artery
• The graft should have excellent biomechanical
stability, and be able to withstand the long-term
hemodynamic stress of the arterial circulation
• Suturability and handling are also important
factors in minimizing operative time and risk.
Bioengineered Vascular Graft
Recent progress
• Synthetic materials such as Dacron or expanded
polytetrafluoroethylene have been used successfully in
peripheral revascularization but failed in coronary
revascularization
• Dacron grafts lead to thrombosis and neointimal
thickening in low blood flow & the ePTFE grafts also
fail owing to surface thrombogenicity for small vessels
• Endothelial cell seeded grafts might be more effective
for anticoagulation compared with nonseeded grafts.
However, the manufacturing process is complex, time
consuming, and costly.
Allograft Immunogenicity
Alloreactive response
• 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.
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
Decellularization
Methods of process
• 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
Recellularization
Process
• Recellularization of decellularized tissues seemed to
occur in a time-dependent fashion.
• Slower recellularization in the luminal side, suggesting
that cells migrate into the matrix primarily from the
adventitial aspect rather than the lumen and indicating
that local cells, rather than circulating pluripotent
progenitor cells, are the likely source of infiltrating
myofibroblasts.
• 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.
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.
Decellularization of Allograft
Methods
• Decellularized cryopreserved allograft will eliminate
the immune response and, it is hoped, allow host cell
ingrowth and better durability
• Decellularization process that first involves cell lysis in
hypotonic sterile water solution, after that, equilibrated
in buffer and treated by enzymatic digestion of nucleic
acids with a combined solution of ribonuclease and
deoxyribonuclease and then then undergoes a multiday
washout in isotonic neutral buffer, then cryopreserved
according to a controlled rate freezing protocol.
• The resulting decellularized cryopreserved allografts
have been shown to have approximately a 99%
reduction in staining of endothelial and interstitial
cellular elements, especially the fibroblast
Decellularization Agents
Agents for decellularization
• 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
Method of process
•
•
•
•
•
•
Valves were rinsed with saline solution, and stored in Tris buffer (pH 8.0, 50
mmol/L, on ice) for transport & stored in CMRL solution (90 mL, Gibco), fetal
bovine serum (FBS; 10 mL, Sigma), and penicillin-streptomycin solution
(penstrep; 0.5 mL, Sigma) for 24 hours at 4°C.
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 X100, 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 of evaluation
• 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, Santa Cruz
Biotechnology) and cytotoxic T cells (anti-CD8; sc7970, Santa Cruz
Biotechnology) 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
Decellularized Bioprosthesis
Process & results
• Decellularization process involves cell lysis in a
hypotonic sterile water and equilibrated in water &
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
Heterograft Decellularization
Characteristics
• 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
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 celltypes 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
Vascular Graft Tissue Engineering
Endothelial progenitor cells
• A potentially promising cell source is endothelial
progenitor cells (EPCs), a subpopulation of stem cells
in human peripheral blood.
• EPCs are a unique circulating subtype of bone marrow
cells differentiated from hemangioblasts, a common
progenitor for both hematopoetic and endothelial cells.
• These cells manifest the potential to differentiate into
mature endothelial cells.
• EPCs have been investigated for the repair of injured
vessels, neovascularization or regeneration of ischemic
tissue, coating of vascular grafts, endothelialization of
decellularized grafts
Tissue Engineering
Endothelial progenitor cells
• The umbilical cord blood is a known source for
endothelial progenitor cells differentiated from
haemangioblasts, a common progenitor for both
haematopoetic and endothelial cells
• These cells have the potential to differentiate into
mature endothelial cells and have been successfully
utilized in non-tissue engineering applications such as
for the repair of injured vessels, neo-vascularization or
regeneration of ischemic tissue as well as coating of
synthetic vascular grafts.
• Recently, animal derived EPCs have been used for the
endothelialization of decellularized grafts in animal
models and for seeding of hybrid grafts.
Biodegradable Vascular Scaffolds
Scaffold characteristics
• The tissue scaffold was composed of a polyglycolic acid
mesh sheet sandwiched between 2 sheets of a copolymer
of polylactic acid and -caprolactone at a 50:50 ratio.
• The polymer matrix had more than 80% porosity with
a pore diameter of 20 to 50 µm before seeding.
• It loses its strength in approximately 16 weeks and is
degraded by hydrolysis in vivo after approximately 24
weeks.
• These polymers were fabricated into a hybrid tubular
scaffold 8 mm in diameter, 15 mm long, and 0.6 mm
thick.
Tissue Engineering
Biodegradable scaffold
(A)
(B)
Formation of a biodegradable scaffold reinforced with woven polylactic
acid mesh (arrow) cross-linked with collagen-microsponge (A). Scanning
electron microscopy image of the tissue-engineered patch shows the
uniformly distributed and interconnected pore structure (pore size 50–150
µm) of the collagen-microsponge (B) (magnification 40x).
Tissue Engineering
Biodegradable vascular scaffolds
• Biodegradable polyurethane foam, porosity > 95%,
0.5 cm diameter, 2.5 cm length
Tissue Engineering
Technique
• Venous wall cells were isolated and explanted in vitro
and seeded on a biodegradable polymer scaffold,
Heart Valve Tissue Engineering
Biomaterial/polymer composite materials
• Extraction of a porcine heart valve and removal of all
xenogenic cells by enzymatic digestion without altering
the biological properties of valve matrix components
• Penetration of decellularized matrix with biodegradable
polymer to enhance the mechanical characteristics of
the porous valve scaffold and to cover thrombogenic
matrix components.
• Coating with poly (hydroxybutyrate) does indeed
improve biocompatibility and mechanical properties
in vitro, and that such hybrid tissue heals in well and
developed the morphologic characteristics of a native
aortic valve.
Tissue-engineered Prosthesis
Limitations
• Can’t be prepared for emergency operation
• Sufficient cell proliferation can’t be
accomplished in all patients
• Can’t be used in systemic circulation now
• Small diameter vascular graft may occlude
Tissue-engineered Prosthesis
Graft compliance test
• Static, internal, and volumetric compliances were
determined by increasing fluid volume incrementally
and recording pressure.
• Percent radial compliance was calculated using the
formula: % Compliance = (R – R0)/ P x 100, where R =
graft radius, R0 = initial graft radius, and p = pressure
changes.
• Internal radius was calculated from the volume with
the assumption that the length remained constant.
Tissue-engineered Prosthesis
Graft tensile strength test
• After grafts were removed, two 5-mm segments were
cut from the midportion of the graft for tensile strength
testing
• Two dowel pins were inserted within each 5-mm sample
and secured with custom fixtures to a Chatillon test
stand (Model TCD200; Chatillon, Largo, FL) and 2pound load cell (Model DFGS 2; Chatillon).
• The pins were then pulled apart at a rate of 50 mm/min.
Maximum force was recorded and ultimate tensile
strength (UTS) was calculated as: UTS = Max Load/(2
x thickness x length).
Tissue-engineered Prosthesis
Histologic & immunohistochemistry
• Graft patency, neointima formation, endothelialization
of the graft, and tissue ingrowth and angiogenesis in the
graft wall were examined histologically and
immunohistochemically.
• The explanted graft was fixed in 10% buffered
formalin
• Immunohistochemical studies were performed for
Ram-11, von Willebrand factor (vWF), and -actin
• Luminal surface fibrin/platelet aggregation,
endothelialization, and cellular infiltration of the grafts
were graded from grade 0 to 4.