Organ preservation tissue engineering
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Organ Preservation & Tissue-engineering. Seoul National University Hospital Department of Thoracic & Cardiovascular Surgery. Organ Preservation. Glutaraldehyde Fixation. Principles Ultrastructural integrity is important for prevention of tissue calcification.

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Organ preservation tissue engineering

Organ Preservation & Tissue-engineering

Seoul National University Hospital

Department of Thoracic & Cardiovascular Surgery


Organ preservation tissue engineering

Organ Preservation


Glutaraldehyde fixation

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

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 fixation1

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

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 fixation2

Glutaraldehyde Fixation

  • Crosslinking


Glutaraldehyde preservation

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 fixation3

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 fixation4

Glutaraldehyde Fixation

  • Action & adverse effects

  • Glutaraldehyde(GA) is currently the standard reagent for preservation andbiochemical fixation

  • It imparts intrinsic tissue stability (biodegradation resistance)and reduces the antigenicity of the material.

  • Recent reports have suggested a detrimental role of aldehyde-inducedintra- and intermolecular collagen cross-linkages in initiatingtissue mineralization

  • GA has beenimplicated in devitalization of the intrinsic connective tissuecells of the bioprosthesis, thus resulting in breakdown of transmembranecalcium regulation and hence contributing to cell-associatedcalcific deposits


Glutaraldehyde fixation5

Glutaraldehyde Fixation

  • Adverse effect

  • Making biologic material stiff & hydrophobic

  • Release of residual cytotoxicity induce the foreign body reaction

  • No endothelial cell lining onto the cytotoxic treated area


Glutaraldehyde fixation6

Glutaraldehyde Fixation

  • Use as valve prostheses

  • As a biologic extracellular matrix scaffold, porcineheart valves for their well-known good hemodynamic behaviorand unlimited availability.

  • Porcine scaffolds are usually treated with glutaraldehyde toimprove mechanical properties and to limit the xenogeneic rejectionprocess.

  • Glutaraldehyde treatment profoundly modifiesthe extracellular matrix structure and makes it improper tosupport cell migration, recolonization, and the matrix-renewingprocess


Glutaraldehyde fixation7

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

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

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

Conditioning of Heterografts

  • Biologic factors affecting durability

  • Diagramatic representation of different stages of method

  • for conditioning heterografts


Glutaraldehyde treatment

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 treatment1

Glutaraldehyde Treatment

  • Action on tissues

  • Glutaraldehyde mechanism of action


Glutaraldehyde preservation1

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

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

Tissue Valve Preparation

  • Principles

  • Ensure reproducibility, desired tissuebiomechanics, desired surface chemistry, matrix stability, andresistance to calcification

  • A variety of treatments have been used clinicallyas well as experimentally

  • They may be broken down into twobroad categories: modifications to glutaraldehyde processedtissue and nonglutaraldehyde processes.


Calcification of bioprosthesis1

Calcification of Bioprosthesis

  • Preventive methods(lipid)

  • Calciumphosphate crystals containing Na, Mg, and carbonatenucleate due to devitalization of the cells and thus inactivationof the calcium pump

  • Membrane-bound phospholipids have also been associated withcalcification nucleation due to alkaline phosphatase hydrolysis

  • Ethanol has been used to remove phospholipids and mitigatecalcification, yet phospholipids have also been removed withchloroform-methanol yielding

  • Lipid extraction can also be performedthrough tissue processing with detergent compounds such as sodiumdodecylsulfate.


Calcification of bioprosthesis2

Calcification of Bioprosthesis

  • Preventive methods(aldehyde)

  • Free aldehyde within thetissue matrix has been thought to be an initiator for calcificationas well.

  • This is supported by studies that demonstrate thataldehyde-binding agents such as alpha-amino oleic acid (AOA;Biomedical Design, Marietta, Ga), L-glutamic acid, & aminodiphosphonateprevent cusp calcification.

  • Yet, post treatment withthe amino acid lysine does not prevent cuspal calcification. and emphasizes the multiplicity of pathways by which calcificationcan initiate.


Calcification of bioprosthesis3

Calcification of Bioprosthesis

  • Heat treatment

  • Heat may facilitate extractionand denaturation of the phospholipids and proteins involvedin the process of calcification

  • The tissues obtained at the slaughterhouse were immediatelyplaced in the 0.625% glutaraldehyde solution.

  • After 15 daysof fixation in this solution, submitted to heat treatment

  • Glass bottles containingtissues in glutaraldehyde solution were placed in an oven at50°C for 2 months with permanent agitation by a rotatormachine (3 rotations/minute), then the glutaraldehyde solution was replaced bya fresh solution.


Bioprosthesis mineralization

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 mechanicalstress.


Calcification process

Calcification Process

  • Hypothesis


Bioprosthetic heart valves

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

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

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

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 preservation2

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

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 calcification1

Bioprosthesis Calcification

  • Prevention

  • Inhibitors of hydroxyapatite formation BisphosphonatesTrivalent 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


Tissue engineering1

Tissue Engineering

  • Introduction

  • Concept of tissue engineering was developed to alleviatethe shortage of donor organs.

  • Objective of tissue engineeringis to develop laboratory-grown tissue or organs to replace orsupport the function of defective or injured body parts.

  • Tissue engineering is an interdisciplinary approach that relieson the synergy of cell biology, materials engineering, & reconstructivesurgery to achieve its goal

  • Fundamental hypothesis underlying tissue engineering isthat dissociated healthy cells will reorganize into functionaltissue when given the proper structural support and signals


Tissue engineering2

Tissue Engineering

  • Recent myocardial graft

  • 3-D contractile cardiac grafts using gelatin spongesand synthetic biodegradable polymers.

  • Formation of bioengineered cardiac grafts with3-D alginate scaffolds.

  • Use ofextracellular matrix (ECM) scaffolds.

  • 3-D heart tissue by gelling a mixture of cardiomyocytesand collagen.

  • Culturingcell sheets without scaffolds using a temperature-responsivepolymer.

  • Creating sheets of cardiomyocytes on a mesh consisting ofultrafine fibers.


Tissue engineering3

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

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 valve1

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 valve2

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 valve3

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 valve4

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 valve5

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

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

Tissue-engineered Heart Valve

  • Cryopreservedhuman umbilical cord cells


Tissue engineered heart valve1

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-4-hydroxybutyrate including sinus of Valsalva (seen from the aortic side) fabricated from the stereolithographic model.


Allograft tissue engineering

Allograft Tissue Engineering

  • Immunogenicity

  • Allogrft tissue stimulates a profound cell-mediated immune responsewith diffuse T cell infiltrates and progressive failure of the allograftvalve 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 allografttissue used in congenital cardiac surgery also stimulates aprofound humoral response

  • As previouslymentioned, it is believed that the cellular elements are theantigenic stimulus for the alloreactive immune response, andthus decellularization has been proposed to reduce the antigenicityof these tissues.


Tissue procurement

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

Decellularization

  • Introduction

  • In an attempt to reduce the antigenic response, decellularizationprocesses have been introduced for cryopreserved tissue.

  • Experimental and clinical experience with this decellularizationprocess has been gained with porcine vena cava porcine tissue, porcine aortic and pulmonary valve conduits, ovinepulmonary valve conduits, and, subsequently, humanfemoral vein and human pulmonary valve conduits.

  • There has alsobeen experimental evidence that the decellularized matrix becomespopulated with functional recipient cells.


Decellularization1

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

Acellular Matrix Tissue

  • Approach to generate

  • First break apart the cell membranesthrough 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.


Decellularization2

Decellularization

  • Rationale

  • Apersistent immunoreactivity against donor antigens has beenimplicated.

  • Early calcification and stenosis froman intense inflammatory reaction may be manifestations of thisimmune response.

  • Early structural failure has been shown tobe more prevalent in younger patients, perhaps because of amore aggressive immune response


Decellularization process

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 process1

Decellularization Process

  • Recent status

  • Multistep detergent–enzymaticextraction, Triton detergent, or trypsin/ethylenediaminetetraaceticacid.

  • A more recent protocol using sodium dodecyl sulfate(SDS) in the presence of protease inhibitors was successfulfor aortic valve conduit decellularization

  • Histologicalanalysis showed that the major structural components seemedto be maintained.

  • The effect of cell removal on different types of ECM moleculesand the remodeling of the ECM in the transplanted aortic valve.


Decellularization procedures

Decellularization Procedures

  • Methods

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


Acellularization procedures

Acellularization Procedures

  • Enzymatic process

  • Valve or conduits were harvested under sterile condition and stored at 4°C.

  • Within 30 minutes the conduitswere acellularized in a bioreactor.

  • The bioreactor was filledwith 0.05% trypsin and 0.02%ethylenediamine tetraacetic acid (EDTA) for 48 hours,followed by phosphate-buffered saline (PBS) flushing for 48hours to remove cell debris.

  • All steps were conducted in anatmosphere of 5% CO2 and 95% air at 37°C with the bioreactorrotating at a speed of 7 rpm.


Decellularization procedures1

Decellularization Procedures

  • Enzymatic process

  • The entire construct waswashed for 30 minutes at room temperature in povidone-iodinesolution and sterile PBS, followed by another overnight incubationat 4°C in an antibiotic solution

  • After thisdecontamination procedure, the valves were placed in a solutionof 0.05% trypsin and 0.02% EDTA (Biochrom AG) at 37°C and5% CO2 for 12 hours during continuous 3-dimensional shaking.

  • After removal of the trypsin-EDTA, the constructs were washedwith PBS for another 24 hours to remove residual cell detritus.


Depopulated allografts

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

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 decellularization1

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


Decellularization3

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 process2

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

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

Incomplete Decellularization

  • Implications

  • Incomplete decellularization with an excess of cellular debris, 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 process

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

Seeding of Endothelial Cells

  • Endothelialization of porcine glutaraldehyde-fixed valves

  • Poor cell adhesion on glutaraldehyde-fixed porcine surfaceswas also a result of a change in the physico-chemical propertiescaused by the cross-linking.

  • Reduced hydrophilyprevented the cells to attach properly.

  • This could be changedby introducing a strong hydrophilic substance through the wayof a chemical salt formation on the surface

  • Citric acid or ascorbic acid, which are both strong organicacids used and no signsfor any structural weakening due to the citric acid pretreatment


Endothelial cell seeding

Endothelial Cell Seeding

  • On porcine glutaraldehyde-fixed valves

  • After incubationwith serum-supplemented M-199 for 24 hours at 4°C, the valveswere incubated with citric acid (10% by weight) for 5 minutesat a pH of 3 to 3.5.

  • This pretreatment increases hydrophilsmof the surface, thus improving cell adhesion and attachment

  • The pretreated, but unseeded valves exhibited a cell-freesurface of free collagen fibers prior to cell seeding

  • Thereafter, the prostheses were rinsed 3 times and bufferedto a physiologic pH using PBSB buffer.

  • After the final washingprocedure, the valves were pre-seeded with myofibroblasts, followedby endothelial cell


Recellularization

Recellularization

  • Lavoratory evidence

  • Stains for T-cell surfaceantigen, CD4, and CD8 yielded negative results.

  • Neoendothelialcells stained for factor VIII.

  • Smooth muscle cells in arteriolewalls stained for smooth muscle actin, and cells scattered inthe adventitia stained for procollagen type I.

  • Leaflet explants had no detectable inflammatory cellsand were repopulated with fibrocytes and smoothmuscle cells


Decellularized porcine valve

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

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


Recellularization1

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.


Recellularization2

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


Decellularization4

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 process3

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


Decellularization5

Decellularization

  • Process methods

  • Samples were placed in hypotonic Tris buffer (10 mmol/L, pH8.0) containing phenylmethylsulfonyl fluoride (0.1 mmol/L) andethylenediamine tetraacetic acid (5 mmol/L) for 48 hours at4°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’sphosphate buffer (pH 7.3) and placed in Sorensen’s buffercontaining DNase (25 µg/mL), RNase (10 µg/mL), andMgCl2 (10 mmol/L) for 5 hours at 37°C.

  • Samples were thentransferred to Tris buffer (50 mmol/L, pH 9.0; Triton X-1000.5%) for 48 hours at 4°C.

  • Finally, all samples were washedwith phosphate-buffered saline at 4°C for 72 hours, changingthe solution every 24 hours.


Bioengineered vascular graft

Bioengineered Vascular Graft

  • Requisite for small caliber graft

  • A synthetic small caliber graft should be resistant to thrombosisand biocompatible, resembling a native artery

  • The graft shouldhave excellent biomechanical stability, and be able to withstandthe long-term hemodynamic stress of the arterial circulation

  • Suturability and handling are also important factors in minimizingoperative time and risk.


Bioengineered vascular graft1

Bioengineered Vascular Graft

  • Recent progress

  • Synthetic materialssuch as Dacron or expandedpolytetrafluoroethylene have been used successfullyin peripheral revascularization but failed in coronary revascularization

  • Dacron grafts lead to thrombosis and neointimal thickeningin low blood flow & the ePTFE grafts also fail owing to surfacethrombogenicity for small vessels

  • Endothelial cell seededgrafts might be more effective for anticoagulation comparedwith nonseeded grafts. However, the manufacturing processis complex, time consuming, and costly.


Allograft immunogenicity

Allograft Immunogenicity

  • Alloreactive response

  • Allogrft tissue stimulates a profound cell-mediated immune responsewith diffuse T cell infiltrates and progressive failure of the allograftvalve 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 allografttissue used in congenital cardiac surgery also stimulates aprofound humoral response

  • As previouslymentioned, it is believed that the cellular elements are theantigenic stimulus for the alloreactive immune response, andthus decellularization has been proposed to reduce the antigenicityof these tissues.


Decellularization6

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


Decellularization7

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


Recellularization3

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

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

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

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


Decellularization8

Decellularization

  • Method of process

  • Valves were rinsed with salinesolution, 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-streptomycinsolution (penstrep; 0.5 mL, Sigma) for 24 hours at 4°C.

  • Samples were placed in hypotonic Tris buffer (10 mmol/L, pH8.0) containing phenylmethylsulfonyl fluoride (0.1 mmol/L) andethylenediamine tetraacetic acid (5 mmol/L) for 48 hours at4°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’sphosphate buffer (pH 7.3) and placed in Sorensen’s buffercontaining DNase (25 µg/mL), RNase (10 µg/mL), andMgCl2 (10 mmol/L) for 5 hours at 37°C.

  • Samples were thentransferred to Tris buffer (50 mmol/L, pH 9.0; Triton X-1000.5%) for 48 hours at 4°C.

  • Finally, all samples were washedwith phosphate-buffered saline at 4°C for 72 hours, changingthe solution every 24 hours.


Immunohistochemistry

Immunohistochemistry

  • Methods of evaluation

  • Tissue was harvested for histology at 1, 2, and 4 weeks. Sampleswere formalin fixed (10%), paraffin embedded, and serially sectioned(5 µm) for histologic and immunohistochemical examination,ensuring valve leaflets were visualized in all sections.

  • Immunohistochemistryinvolved standard staining techniques with biotinylated secondaryantibodies, a peroxidase avidin-biotin complex, and 3.3' diaminobenzideneas the chromogen. Primary monoclonal antibodies for T cells(anti-CD3; sc1127, Santa Cruz Biotechnology) and cytotoxic Tcells (anti-CD8; sc7970, Santa Cruz Biotechnology) were used.

  • Allogeneic nondecellularizedgrafts were associated with significant CD3+ and CD8+ T cellinfiltrates in aortic valve leaflets by 1 week after transplantation,rapidly decreasing in the following weeks.


Histology immunohistology

Histology & Immunohistology

  • Examination

  • Explanted tissue specimens were studied as hematoxylin/eosin,elastica van Gieson, and von Kossa stained paraffin or immunostainedfrozen sections.

  • The antibodies for immunohistochemistry includedmonoclonal antibodies against CD31, -smooth muscle actin , and vimentin, and a polyclonal antibody against von Willebrandfactor

  • Expression of von Willebrandfactor (vWF), vascular endothelial growth factor (VEGF), vascularsmooth muscle -actin 2 (ACTA2), smooth muscle 22 (SM22 ), andvimentin were determined with quantitative real-time RT-PCR


Homograft decellularization2

Homograft Decellularization

  • Cell free or nonimmunogenic

  • Less viable cellular element

    No immune cell infiltration

    No donor-specific immune activation

  • Well preserved ultrastructure


Decellularized bioprosthesis1

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

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

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

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 biomatrix1

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

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


Endothelial progenitor cells

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

Biodegradable VascularScaffolds

  • 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

Tissue Engineering

Biodegradable scaffold

(B)

(A)

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).


Biodegradable vascular s caffolds

Tissue Engineering

Biodegradable vascular scaffolds

  • Biodegradable polyurethane foam, porosity > 95%,

  • 0.5 cm diameter, 2.5 cm length


Tissue engineering4

Tissue Engineering

  • Technique

  • Venous wall cells were isolated and explanted in vitro

  • and seeded on a biodegradable polymer scaffold,


Heart valve tissue engineering1

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

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 prosthesis1

Tissue-engineered Prosthesis

  • Graft compliance test

  • Static, internal, and volumetric complianceswere determined by increasing fluid volume incrementally andrecording pressure.

  • Percent radial compliance was calculatedusing the formula: % Compliance = (R – R0)/ P x 100, whereR = graft radius, R0 = initial graft radius, and p = pressurechanges.

  • Internal radius was calculated from the volume withthe assumption that the length remained constant.


Tissue engineered prosthesis2

Tissue-engineered Prosthesis

  • Graft tensile strength test

  • After grafts were removed, two 5-mm segments were cut from themidportion of the graft for tensile strength testing

  • Two dowel pins were inserted within each 5-mm sampleand secured with custom fixtures to a Chatillon test stand (ModelTCD200; Chatillon, Largo, FL) and 2-pound load cell (Model DFGS2; Chatillon).

  • The pins were then pulled apart at a rate of50 mm/min. Maximum force was recorded and ultimate tensile strength(UTS) was calculated as: UTS = Max Load/(2 x thickness x length).


Tissue engineered prosthesis3

Tissue-engineered Prosthesis

  • Histologic & immunohistochemistry

  • Graft patency, neointima formation, endothelialization of thegraft, and tissue ingrowth and angiogenesis in the graft wallwere examined histologically and immunohistochemically.

  • Theexplanted graft was fixed in 10% buffered formalin

  • Immunohistochemical studies were performed for Ram-11, von Willebrandfactor (vWF), and -actin

  • Luminal surface fibrin/platelet aggregation, endothelialization,and cellular infiltration of the grafts were graded from grade0 to 4.


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