organ preservation & tissue- engineering seoul national university hospital department of...

Organ Preservation & Tissue-engineering

Seoul National University Hospital

Department of Thoracic & Cardiovascular Surgery

Organ Preservation

Glutaraldehyde Fixation

Principles• Ultrastructural integrity is important for preve

ntion 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 ti

ssue treatment agents

• Tissue fixation with aldehydes is a well established and widely accepted process

Glutaraldehyde FixationPrinciples• Glutaraldehyde has become a popular fixing agent beca

use 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 formalde

hyde 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


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.


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


Action & adverse effects• Glutaraldehyde (GA) is currently the standard reagent for

preservation and biochemical fixation • It imparts intrinsic tissue stability (biodegradation resistanc

e) and reduces the antigenicity of the material.• Recent reports have suggested a detrimental role of aldehyd

e-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


Adverse effect1. Making biologic material stiff & hydrophobic

2. Release of residual cytotoxicity induce the for

eign body reaction

3. No endothelial cell lining onto the cytotoxic t

reated area


Use as valve prostheses• As a biologic extracellular matrix scaffold, porcine hear

t 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


No-react neutralization • The proprietary No-react tissue treatment proc

ess 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 fr

om 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 & aneury

smal formation• Incorporated into the surrounding host

tissue with growth potential• Common feature is tissue thinning with reduct

ion in connective cells or degenerative nucleic change

Conditioning of HeterograftsBiologic 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


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 biomecha

nics, 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 BioprosthesisPreventive methods(lipid)• Calcium phosphate crystals containing Na, Mg, and carbonat

e 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 though

t to be an initiator for calcification as well. • This is supported by studies that demonstrate that alde

hyde-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 BioprosthesisHeat treatment• Heat may facilitate extraction and denaturation of the phos

pholipids 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 bio

material 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 A

ccelerated calcification is associated with young recipient age, glutaraldehyde fixation, and high mechanical stress.

Calcification ProcessHypothesis

Bioprosthetic Heart Valves

Mechanism of calcification• Mineralization process in the cusps of bioprosthetic hea

rt 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 f

or preventing calcification of biomaterial implants:

• Systemic therapy with anticalcification agents; • Local therapy with implantable drug delivery de

vices; • Biomaterial modifications, such as removal of a

calcifiable component, addition of an exogenous agent, or chemical alteration.

AntimineralizationStrategies 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


Actions & limitation• Reduced immunologic recognition and resista

nce 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 amin

o-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 CalcificationPrevention• Inhibitors of hydroxyapatite formation Bisphosphonates

Trivalent metal ions• Calcium diffusion inhibitor ( amino-oleic acid )• Removal or modification of calcifiable material

SurfactantsEthanol

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 s

hortage of donor organs. • Objective of tissue engineering is to develop laboratory-grow

n 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 s

ynthetic biodegradable polymers. • Formation of bioengineered cardiac grafts with 3-D algin

ate scaffolds. • Use of extracellular matrix (ECM) scaffolds. • 3-D heart tissue by gelling a mixture of cardiomyocytes a

nd collagen. • Culturing cell sheets without scaffolds using a temperatu

re-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 mat

rix 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


Development Three approaches • Acellular matrix xenograft• Bioresorbable scaffold• Collagen-based constructs containing entrap

ped cells

• Other substrates in early development Hybrid approaches Stem cells and other future prospects


Development • Seeding a biodegradable valve matrix with autologous endot

helial or fibroblast cells• Seeding a decellularized allograft valve with vascular endoth

elial cells or dermal fibroblast• Use of a decellularized allograft with maintained structural i

ntegrity 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 im

munologically inert ?• What will happen to xenogeneic decellularized graft i

mmunologically ?• Seeded vascular endothelial cell penetrate matrix and d

ifferentiate 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 ?


Development • Seeding a biodegradable valve matrix with auto

logous endothelial or fibroblast cells• Seeding a decellularized allograft valve with va

scular endothelial cells or dermal fibroblast• Use of a decellularized allograft with maintaine

d structural integrity as a valve implant that will be repopulated by adaptive remodeling

Tissue-engineered Valve Problems• Decellularization process render all allograft valves im

munologically inert ?• What will happen to xenogeneic decellularized graft i

mmunologically ?• Seeded vascular endothelial cell penetrate matrix and d

ifferentiate 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 enti

re 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 ValveStereolithographic model

Three-dimensional reconstructed stereolithographic model from the inside of an aortic homograft. (B) Trileaflet heart valve scaffold from porous poly-4-hydroxybutyrate 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 immu

ne 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 ProcurementProcessing• Hearts were transported on wet ice in Roswell Park Mem

orial 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, decellulariz

ation 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 ce

lls 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 h

yper- 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 an

tigens 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 deter

gent, or trypsin/ethylenediaminetetraacetic acid.• A more recent protocol using sodium dodecyl sulfate (S

DS) 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

Treatment Concentration Duration ation (h)

• Triton X-100 1%–5% 24

• Trypsin 0.5% 0.5–1.5

• Trypsin/Triton X-100 0.5%/1%–5% 0.5–1.5/24

• SDS 0.1%–1% 24

• SDS, Sodium dodecyl sulfate

Methods

Acellularization ProceduresEnzymatic process• Valve or conduits were harvested under sterile condition an

d stored at 4°C.• Within 30 minutes the conduits were acellularized in a biore

actor. • The bioreactor was filled with 0.05% trypsin and 0.02% eth

ylenediamine 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 te

mperature 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 depo

pulation processing and cryopreservation. • The steps included cell lysis in hypotonic solutio

n, 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 enzyme

s 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.


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 macroscopi

cally 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 ProcessImmunologic response• HLA class I & II antibodies are known to be elevated in children receiv

ing 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 hypot

onic 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 d

ebris, however, can provoke significant immune-mediated 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 ProcessImplications• A functioning endothelium requires an appropriate ma

trix 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 glutaraldehyde-fixed

valves• Poor cell adhesion on glutaraldehyde-fixed porcine surfaces

was also a result of a change in the physico-chemical properties caused by the cross-linking.

• Reduced hydrophily prevented the cells to attach properly. • This could be changed by introducing a strong hydrophilic s

ubstance 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 h

ours 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 cell-free 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 pre-seeded with myofibroblasts, followed by endothelial cell

Recellularization

Lavoratory evidence• Stains for T-cell surface antigen, CD4, and CD8 y

ielded 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 activ

ates 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 ma




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 antibi

otics for at least 7 days• Decellularization of graft immersed in sol

ution for 24hours in room temperature• Keep in physiologic saline solution until i

mplantation

Decellularization ProcessCommonly 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 pre

sence of protease inhibitors, Rnase and Dnase• Detergent ( N-lauroylsarcosinate ), benzonase e

ndonuclease solution, polymyxin B

Decellularization Process methods• Samples were placed in hypotonic Tris buffer (10 mmol/L, pH 8.0) cont

aining 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 resista

nt 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 polytetra

fluoroethylene 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 immu

ne 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 ce

lls 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

RecellularizationProcess• Recellularization of decellularized tissues seemed to occ

ur 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 ma




Decellularization of AllograftMethods• Decellularized cryopreserved allograft will eliminate the immu

ne 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 ) wit

h 0.02% EDTA in phosphate buffered saline• 1 % deoxicholic acid and 70% ethanol for 24hours und

er constant agitation• Trypsin/ethylenediaminetetraacetic acid• Sodium dodecyl sulfate ( 0.1% SDS ) in the presence

of protease inhibitors, Rnase and Dnase• Detergent ( N-lauroylsarcosinate ), benzonase endonucl

ease solution, polymyxin B

DecellularizationMethod of process • Valves were rinsed with saline solution, and stored in Tris buffer (pH 8.0, 50 mm

ol/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 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 of evaluation• Tissue was harvested for histology at 1, 2, and 4 weeks. Samples wer

e 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/eos

in, 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 real-time RT-PCR


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 hypoto

nic 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 DecellularizationCharacteristics• The use of a decellularized matrix of a xenograft is pref

erred 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 BiomatrixAdvantages• Enzymatically decellularized extracellular matrix with

out 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 ce

lls 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 proge

nitor 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

Endothelial progenitor cells• The umbilical cord blood is a known source for endothe

lial 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 mo

dels and for seeding of hybrid grafts.

Tissue Engineering

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.

Biodegradable scaffold

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).

(A) (B)

Tissue Engineering

Biodegradable vascular scaffolds

• Biodegradable polyurethane foam, porosity > 95%,

0.5 cm diameter, 2.5 cm length

Tissue Engineering

Tissue Engineering

• Venous wall cells were isolated and explanted in vitro

and seeded on a biodegradable polymer scaffold,

Technique

Heart Valve Tissue EngineeringBiomaterial/polymer composite materials• Extraction of a porcine heart valve and removal of all x

enogenic 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 accomplish

ed in all patients• Can’t be used in systemic circulation now• Small diameter vascular graft may occlude


Graft compliance test• Static, internal, and volumetric compliances were deter

mined 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.


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 2-pound 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).


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, endotheliali

zation, and cellular infiltration of the grafts were graded from grade 0 to 4.

organ preservation & tissue- engineering seoul national university hospital department of...

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