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Homograft, Xenograft & Bioprosthesis Seoul National University Hospital Department of Thoracic & Cardiovascular Surgery.

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Presentation on theme: "Homograft, Xenograft & Bioprosthesis Seoul National University Hospital Department of Thoracic & Cardiovascular Surgery."— Presentation transcript:

1 Homograft, Xenograft & Bioprosthesis Seoul National University Hospital Department of Thoracic & Cardiovascular Surgery

2 Valved Homografts  Introduction Valved homografts, introduced in the 1960s, have used for reconstruction of the right ventricular outflow tract. Advantages of homografts, such as ease of handling and improved hemostasis, have been major contributing factors Viable endothelial cells and perhaps also viable fibroblasts, thus contributing to an antigenically active cells, which could lead to a more intense host response. This increased immunogenicity could perhaps be an important contributing factor for accelerated fibrocalcifications, particularly in very young patients.

3 Homograft  Processing Cryopreservation Antibiotics Fetal calf serum RPMI media DMSO Thawing Harvest of aortic, mitral, pulmonary valve & others

4 Sterilization for Homograft  Antibiotics for uses Modified Hanks solution or TCM Cefoxitin sodium ( 240mg/L ) Vancomycin hydrochloride ( 50mg/L) Lincomycin hydrochloride ( 120mg/L) Polymixin B sulfate ( 100mg/L) Amphotericin B ( 25mg/L )

5 Homograft Processing  Essence 1. Possible procurement on beating heart, but non-beating heart within 24 hrs after death 2. Reduce antibiotic incubation time to 6 hrs 3. Fibroblast is important in the maintenance of the valve matrix. * Viable cells persist, produce collagen and repair damaged matrix. 4. Low dose antibiotics and no amphotericin B

6 Homograft  Laboratory evaluation Hematoxylin-eosin & elastica van Gieson Elastic fiber of vessel wall Muscle actin positive cell Immunohistochemical staining Flow cytometry Antibodies for inflammatory cell CD3 for all T lymphocytes CD4 for TH lymphocytes and macrophages CD8 for TC lymphocytes and NK cells Scanning EM for endothelial integrity

7 Homograft  Advantages 1. Technical ease of implantation because they are soft & mold easily with patient’s tissue, resulting in better hemostasis in complex operations 2. Better hemodynamics than porcine-valved dacron conduit, which improves the RV function after operation. 3. The branch of the homograft may be used to patch distal pulmonary artery stenoses

8 Homograft  Durability of aortic valve Homograft can last 20 years, essentially as a dead piece of tissue(become acellular within a few months), free from any mechanical reinforcement by crosslinking agents. Unknown exactly what features of the native valve tissue give it such remarkable durability than other bioprosthesis, the importance of its internal complexity is being appreciated more and more. Presence of interconnected sheets of collagen, layers and tubes of elastin, highly nonlinear mechanics, anisotropy, and viscoelasticity endow the valve tissue with is unique longevity.

9 Cryopreservation  Methods Treatment with antibiotic agents at 4 ℃ for 24 hours or culture medium with antibiotics Frozen in tissue culture medium(TCM) 199 solution containing 10% calf serum, 10% DMSO, and 5% HEPES buffer, with a cooling velocity at a rate of -1 ℃ / min to -80 ℃ with programmable controlled-rate freezer. Frozen xenograft is put into sealed package and preserved in vapor phase of liquid nitrogen at -196 ℃ The cryopreserved xenograft is thawed in a water bath at 37 ℃ quickly

10 Cryopreservation  Protocol Transport Maximum 24 h at 4°C RPMI nr mL Sterilization 14–18 h at 4°C RPMI nr mL 24 mg Mefoxin. 12 mg Lincomycine 5 mg Vancomycine UI Colymicine Storage –150°C until implant 90 g Albumine 4% 10 mL DMSO DMSO = dymethylsulphoxide; RPMI = Roswell Park Memorial Institute.

11 Cryopreservation  Influence on immunity Cryopreservation technique might attenuate allograft immunogenicity by reducing the viability and cellularity of the endothelium or by diminishing the expression of leukocyte adhesion molecule Cryopreservation technique reduces the xenogeneic immunogenicity of endothelial cells as in allograft Cryopreservation technique could not reduce the immunogenicity of other cellular & extracellular matrices of xenograft

12 Cryopreservation  Effects on cells Cytosolic and mitochondrial functions of endothelial cells were damaged seriously during cryopreservation, especially in thawing process, and those process may cause a latent cytosolic and mitochondrial injury, even in the fibroblast Viability of donor fibroblast which can remodel the matrix assembly is damaged by cryopreservation and thawing, that may cause collagenolytic activation and degradation of collagen synthesis in a cryopreserved valve, which will lead to destruction of matrix

13 Cryopreservation  Effects on valve tissue The valvular extracellular matrix ( ECM ) contains a variety of structures, underlies and surrounds the interstitial cells, and performs many essential functions, including mechanical support and physical strength ECM exerts profound influences on cell adherence, migration, and differentiation as well as the pattern of gene expression of the cells in contact with it The quality of the structural matrix at implantation may predetermine durability or failure of a cryopreserved heart valve (collagenous bundles and elastin-containing fibers) based on different techniques before implantation

14 Cryopreservation  Effects on valve matrix Conventional approaches to cryopreservation of heart valves have a detrimental and destructive effect on crucial leaflet matrix elements. Serious alteration and significant deterioration of collagenous and elastic fiber structures, accompanied by a general damage of the leaflet histoarchitecture caused by extracellular ice formation. Alternative preservation techniques proposed for clinical use, such as vitrification, D-hydro-, or glycerol- treatment, are crucial.

15 Cryopreservation  Mechanisms of structural alteration Related to an activation of metalloproteinases (MMPs) upon thawing, and disruption of normal extracellular matrix metabolism by the valve interstitial cells, a function shown to be impaired in cryopreserved tissue Side specific cell phenotypes and differences in extracellular matrix organization may account for the heterogeneity in extracellular matrix alteration. The lack of crimp in the thawed cryopreserved valves suggests that the biomechanical properties of the ventricularis layer have been altered

16 Cryopreserved Homografts  Examination parameters Tissue structure Tissue viability Cell proliferative capacity Metabolic function Identification of cell-specific antigens ( Immunohistochemistry) Flow cytometry( MHC I and II antibody)

17 Cryop reserved Homograft  Viability test 1. Radiolabeled thymidine 2. Collagen synthesis 3. Protein synthesis 4. Dye uptake & dye exclusion 5. Autoradiography

18 Homograft Viability Test  Methods for viability Cytometry using propidium iodure and fluorescent diacetate (FDA). Double staining with fluorescein diacetate-propidium iodure (FDA-PI) is reported to be a rapid method for assessing cell viability compared with the trypan blue dye exclusion method

19 Cryopreserved Homografts  Structural fate Viable fibroblasts synthesize main constituents of extracellular matrix: collagen, elastin, reticulin, and mucopolysaccharides, therefore longevity of the implantation is likely related to the viability of fibroblasts in the implanted valves However, fibroblast in the donor allograft were unable to survive long, because of apoptosis. Endothelial cells exhibit strong antigenicity, but can not survive under ischemic conditions and moreover, during the cryopreservation process endothelial cells lose their ability to proliferate. The homograft will lose its endothelial cells and fibroblasts, and eventually become a non-viable tissue.

20 Cryopreserved Homograft  Standard model This demonstrates preservation of endothelial & valve architecture & viability This viability may not be a positive attribute, as initial thought, it contributes to immunogenicity and elicits a more vigorous immunologic reaction from the recipient. Heightened immune response contributes to accelerated degeneration of allograft valve An immunologically neutral graft has therefore been considered desirable, leading to new development that is decellularized to avoid introduction of immunogenic cells into the recipient

21 Cryopreservation  Effects on immune reaction There were donor-specific immune responses evoked by allograft insertion, and on the other hand, clinically explanted allografts showed no evidence of rejection Cryopreservation causes the greatest delay & diminuation of the expression of leukocyte adhesion molecules Cryopreservation attenuates the allograft immunogenicity by reducing the viability and cellularity of endothelium But it seems to be impossible to reduce xenogeneic immunogenicity with cryopreservation because of the marked destruction of other cellular and extracellular components

22 Aortic Valve Replacement  Advantages of homograft Good hemodynamics, Fewer thromboembolic complications Avoidance of anticoagulation Suitability in the presence of infection  Disadvantages of homograft Risk of early failure due to technical error Limited durability and availability

23 Homograft Implantation  Surgical indication of endocarditis Hemodynamic instability Microbiology resistance, usually involved in active aortic endocarditis including vegetations, cusp destruction, and periannular extensions. Aortic vegetations, especially when their diameter is superior to 10 mm, may be responsible for systemic embolization Anatomic lesions progression are traditional complications leading to early surgery Aortic periannular extension can degenerate in abscesses, discontinuity, fistulas, aberrant communications and false aneurysm, increasing congestive heart failure, and mortality rate

24 Homografts Implantation  Aortic position pulmonary autograft is not recommended for young rheumatic aortic valve patients, particularly with mitral valve involvement Ross procedure could be performed in young aortic valve patients if their mitral and pulmonary valves are intact HAVR should be tailored to individual patients so as to choose the scalloped subcoronary or root replacement In view of the long-term prognosis, the surgeon should use, if possible, the subcoronary replacement technique, which makes the reoperation much easier

25 Homografts Implantation  Pulmonary position Superior conduit durability in Ross patients has emphasized the placement of the homograft in the orthotopic pulmonary position and less sternal compression of anteriorly placed conduits The predominant mechanism of homograft stenosis was a poorly understood inflammatory reaction based on their noting of early onset of stenosis, rapid clinical progression Stretching and lengthening of the homograft causing release of tissue factors was one possibility suggested and the extent of that phenomenon might relate to the degree of peripheral vascular distortion present

26 Homograft Failure  Etiologic factors Factors unrelated to immunologic injury would include mechanical factors, such as sternal compression, or oversizing or undersizing of conduits with anatomic distortion, in addition to ischemic injury. Factors related to immunologic injury include ABO incompatibility and human leukocyte antigens (HLA) incompatibility Tissue processing steps such as warm ischemic time, antibiotic disinfection, cryopreservation, and thawing all affect viability of the grafts More recently, investigators have demonstrated significant cellular infiltration with T-cells and B-cells in failed explanted valved allografts in children

27 Homograft Failure  Specials in young The most important factors are size of conduit & age at implantation. Primary reason, growth of young child over time Secondary reason, decrease in the functional lumen of the conduit due to calcification or immune related Anatomic type of the conduit, aortic or pulmonary Heightened immune reaction to the implanted tissue There is evidence that children produce a virulent T- cell response, where adults mount a much weaker response

28 Homograft Failure  Risk Factors 1. Younger recipient age 2. Aortic homograft related to higher elastin & intrinsic calcium content 3. Small homograft valve size 4. Long aortic cross-clamp time at operation 5. Pulmonary hypertension 6. Distal pulmonary artery disease 7. Younger donor age ( increased immunogenecity ) 8. Technical factors 1) Hood extension of proximal suture line 2) Anatomic versus nonanatomic placement 3) Compression of conduit

29 Homograft Failure  Alternatives to homograft After a period of 10 years, 30% of the children initially corrected with allografts and 70% of the patients with xenografts had undergone replacement of their initial conduits. We have avoided using an allograft or heterograft conduit in most patients with tetralogy of Fallot or pulmonary atresia with ventricular septal defect. We have preferred to construct a transannular patch with an underlying PTFE monocusp Until recently, cryopreserved allografts have been the best valved conduits and the limited durability of the allograft conduit became evident within a few years

30 Implanted Allografts  Failure process Transplanted cryopreserved homografts rapidly lose their cellular components during the first year of implantation while the normal tissue architecture is damaged. The allografts are capable of eliciting a cellular and humoral immune response, it is not yet clear what exact role the immune response plays in ECM damage After transplantation, indicators of immune-mediated injury as persistent up-regulation of leukocyte adhesion molecules, the presence of neutrophyl granulocytes, depositions of antibodies and complement are missing in early phase after implantation.

31 Implanted Allografts  Loss of cellularity Immunologic & chemical injury Allografts are eliciting a cellular & humoral immune response, not yet clear what exact role the immune response Hypoxia during valve processing Reperfusion injury at implantation

32 Homograft  Immune responses Children & adults respond differently to homograft. Younger donor age related to increased immunogenicity There is evidence that children produce a virulent T-cell response, where adults mount a much weaker response. In addition, laboratory studies have demonstrated that allograft valve destruction is T-cell mediated. Other HLA loci and nonimmunologic factors, including growth, degeneration, and technique, play a role in homograft failure

33 Implanted Allografts  Immune responses All allograft recipients are likely to form IgG- and T- cell-mediated reaction to donor HLA antigens Donor-specific T cells are likely to be the main agents of allograft injury, effected through secretion of high levels of cytokines The location of induction and amplication of the immune response to the allograft remains unknown. Dendritic cells, which is identified in human great vessels, together with endothelial cells are capable of presenting foreign HLA class I and II antigens to recipient T cells

34 Implanted Allografts Immune responses Valve leaflet cellularity was demonstrated to be significantly reduced with time Donor-specific immune activation, some early infiltration of immune effector cells, such as macrophages and T lymphocytes is demonstrated Most of the evidence for immune-mediated damage in this animal model occurred in the first 2 weeks after implantation, and by 4 weeks the valves were largely acellular This observation may in fact represent evidence of immune-related valve injury, despite the absence of up-regulation of cell adhesion molecules

35 Homograft Failure  Fate of implanted homograft Virtually all cryopreserved homografts demonstrate acellularity within a year of implantation The lack of cells and cellular function limits tissue growth, performance, and reparative capacity; such "tissues" typically scar and then mineralize, which often leads to dysfunction. Chronic rejection of cells in classically cryopreserved allograft tissues promotes the migration of inflammatory cells, exacerbating tissue degeneration, fibrosis, and functional failure

36 Homograft Implantation  Reduce immune response One would be to match all allografts by blood type and HLA type. Another would be to somehow remove the immunogenicity of valved allograft tissue, perhaps by removing the antigen presenting cells on the allograft The third option would be to immunosuppress the recipient in order to abrogate the immune response of the recipient to the donor allograft.

37 Blood Cells  Production In children, blood cells are produced in the marrow of all bones. After the age of 20 years, only the marrow of the vertebrae, sternum, and ribs remain significantly active (red marrow) in the production of erythrocytes, many leukocytes, and platelets. Active marrow contains pluripotential stem cells that are capable of replacing bone marrow completely (self-renewal) or can be diverted from self-renewal toward separate pools of committed progenitor cells

38 Stem Cells & Progenitor Cells  Characteristics Progenitor cells : (1) they have lost the capacity for self renewal and (2) they are not pluripotential but are committed to produce (under the proper growth conditions) daughter cells of a particular type. During maturation, each cell line, promoted by a variety of stimulating factors, acquires distinctive properties Pluripotent stem cell can differentiate in a few divisions into one of six classes of progenitor cells

39 Stem Cells & Progenitor Cells A pluripotent stem cell can differentiate in a few divisions into one of six classes of progenitor cells that go on to produce blast cells. Blast cells are the earliest morphologically distinct precursors of specific cell types. EPO = erythropoietin; GCSF= granulocyte colony-stimulating factor; GM- CSF = granulocyte-macrophage colony-stimulating factor; IL = interleukin; M-CSF = monocyte colony-stimulating factor; PMNs = polymorphonuclear monocytes.

40 Red Blood Cell  Immunologic properties The red cells of different individuals differ to a very small extent in the structure of some carbohydrates that are part of membrane glycolipids These differences bestow antigenic properties on red blood cells and cause red cell agglutination  ABO antigens The A and B antigens are the most important of the more than 100 different blood group antigens that have been identified In neonatal life, antibodies quickly develop against the antigens that are not present on our red cells, and these antibodies, called agglutinins, are carried in plasma. The antigens are called agglutinogens and are carried on the red cells in the blood as well as on cells in many other tissues

41 Red Blood Cell  Rh antigens Within the Rh system, the C, D, and E antigens are most important. They are found only in red cells. D is the most antigenic component, and the presence or absence of D is designated as “Rh- positive” or “Rh-negative,” respectively. 85 % of Caucasians & more than 99% of Asians are Rh+ Anti-D develops only when the blood of a D– individual is exposed to D+ red cells. This can occur as a result of transfusion or when Rh+ fetal blood mixes with the circulation of an Rh– mother

42 Antigens of ABO group A, H antigen that is present in individuals with type O blood. B, A antigen (type A blood) has a terminal N-acetylgalactosamine (NAG). C, B antigen (type B blood) has a terminal galactose (Gal). Cer = ceramide; Fuc = fucose; Gal = galatose; Glu = glucose.

43 Human Blood

44  Components of plasma The major plasma proteins are albumin (4.5 g/dL), several globulins (2.5 g/dL), and fibrinogen(0.3 g/dL). Most are synthesized by the liver, and they have five major functions: (1) carriers for hormones, trace metals, or drugs; (2) proteolytic agents in the cleavage of various hormonal or enzymatic precursors; (3) protease inhibitors; (4) source of plasma colloid osmotic pressure;(5) source of the humoral immunity portion of the immune system.

45 Arachidonic Acid Derivatives Derivatives of arachidonic acid play a crucial role in platelet adhesion to the endothelium, and the clotting process can be disrupted by interruption of arachidonic acid metabolism.

46 Vascular Endothelium  Roles in hemostasis

47 Complement System  Natures A system of 11 plasma enzymes identified as C1 to C9; C1 consists of the three subunits C1q, C1r, and C1s. The enzymes circulate in the inactive form but can be activated to lyse foreign cells. The activation proceeds in a step-like fashion, each activated enzyme hydrolyzing a peptide bond in the next inactive enzyme Classical pathway is triggered when immunoglobulin G or M binds to cell surface antigens Alternate pathway of complement activation does not require binding of immunoglobulins to cell surface antigens and triggered

48 Complement System  Activation Classical pathway ; The consequent activation cascade eventually leads to (a) activated C3, a cell surface-associated factor that promotes opsonization, and (b) activated (C5, C6, C7, C8, C9), which is associated with production of chemotactic substances, release of histamine, and insertion of perforins into the plasma membrane. Alternate pathway ; It is triggered when the circulating protein factor I attaches to specific surface polysaccharides in a bacterium or virus. This pathway also leads to activation of C3 and (C5, C6, C7, C8, C9) and their associated opsonization or cell lysis.

49 Complement Activation

50 Monocytes  Function Monocytes are formed in the bone marrow, enter the blood, and circulate for about 3 days before they enter the tissues by diapedesis and become tissue macrophages that differentiate to perform specific functions in different tissues. They are phagocytic cells and perform many of the same actions that are performed by neutrophils By secreting a large number of lysosomal, chemotactic, complement-activating, and pyrogenic factors, they are key effectors in the elimination of microorganisms and play an important role in immunity and blood clot formation.

51 Mast Cells  Function and role Mast cells are found in tissues, and although they resemble basophils in some respects, they are different and derive from a different marrow stem cell. They are markedly granulated and are frequently found under epithelial surfaces. They are especially rich in heparin and histamine, and the granules containing these substances are released when immunoglobulin E (IgE)–coated antigens bind to receptors on the mast cell surface. They trigger hypersensitivity reactions and participate in inflammatory responses.

52 Granulocytes  Formation of granulocytes They arise from three populations of committed stem cells in the bone marrow and arrive in the tissues fully differentiated as eosinophils, basophils, or neutrophils Eosinophils are especially abundant in mucosal tissues of the respiratory, lower urinary, and GI tracts. Their major role is to attack parasites and also involved in allergic reactions Basophils are rich in histamine and heparin and release inflammatory mediators when activated. Neutrophils are the first line of defense against infections and play a crucial role in inflammation.

53 Granulocytes  Functions of granulocytes Granulocytes, especially neutrophil, contain mechanisms by which they can progress rapidly from a harmless circulating intravascular cell to a specific phagocytic cell and killer of foreign particles & bacteria In acute inflammation, neutrophils are captured and mobilized within minutes to hours and accumulate locally to form the initial defense in a locally restricted area. They are followed by monocytes within 1 day and by lymphocytes within several days. Neutrophil involvement in acute inflammation can be broken down into eight distinct phases:

54 Functions of Granulocytes  Inflammation RecognitionRecognition Expression of adhesion molecules and inflammatory mediatorsExpression of adhesion molecules and inflammatory mediators Hydrodynamic margination, capture, and rolling of neutrophilsHydrodynamic margination, capture, and rolling of neutrophils Activation, adhesion, and spreadingActivation, adhesion, and spreading DiapedesisDiapedesis MigrationMigration PhagocytosisPhagocytosis Apoptosis and elimination of neutrophilsApoptosis and elimination of neutrophils  Phagocytosis Process of immobilization, ingestion, and digestion of foreign agents by granulocytes and monocytes. A vital first step in phagocytosis is the activation of the complement system

55 Acute Inflammation Process  Recognition When tissue macrophages recognize foreign particles that have invaded tissue, they release a variety of inflammatory mediators, including tubular necrosis factor alpha (TNF-  ), colony- stimulating factors for granulocytes (G-CSF) or granulocyte-macrophages (GMCSF), leukotriene B4 (LTB4), complement fragment C5a, interleukin-8 (IL-8), and others. These soluble mediators can act as priming or activating factors for neutrophils and vascular endothelial cells.

56 Acute Inflammation Process  Expression of adhesion molecules & inflammatory mediators Neutrophils are captured & drawn toward the margins of the flowing stream and performed by a variety of adhesion molecules, expressed cooperatively on the surface of endothelial cells and activated neutrophils Adhesion molecules involved in leukocyte– endothelium interactions include, immunoglobulins, integrins, selectins, & other molecules like CD44 and VAP-1. They mediate cell-to-cell and cell-to-substrate interactions by recognizing and binding specific ligands, such as other adhesion molecules.

57 Acute Inflammation Process  Hydrodynamic margination, capture, & rolling of neutrophils Radial displacement (hydrodynamic margination) & retardation of neutrophils by the endothelium are required as initial steps in the inflammatory response. Contact is made primarily through the E- and P- selectins, adhesion molecules that are induced (E-selectin) or translocated (P-selectin) to the surface of endothelial cells in postcapillary venules by a number of chemical signals. Their ligands are complex carbohydrates that are constitutively expressed on the neutrophil surface. Intermittent breaking of these contacts causes rolling

58 Acute Inflammation Process  Activation, adhesion, and spreading Neutrophil activation is enhanced by exposure to the endothelium, and further expression of adhesion molecules leads to firm neutrophil adhesion to and spreading across the endothelial cell.  Diapedesis Adhesion and spreading are prerequisites and lead to migration of the activated neutrophil through the intercellular junctions of neighboring endothelial cells. Neutrophils and monocytes migrate preferentially from postcapillary venules.

59 Acute Inflammation Process  Migration After passing through the endothelial junction and the basement membrane, the activated neutrophils, guided by chemotactic stimuli, migrate toward the foreign particles that initiate the inflammatory response. This migration is guided by interactions between adhesion molecules on the neutrophil surface and elements of the interstitial matrix.

60 Acute Inflammation Process  Phagocytosis Once the neutrophils reach the foreign particles, they attach to the opsonized surface of the agent (if it is large), or they engulf the agent within a phagocytic vacuole. Destruction of foreign material is chiefly by reactive oxygen metabolites (superoxide radicals) and granule contents including elastases, cathepsin G, proteases, and others.  Apoptosis and elimination of neutrophils Among the β2 integrins that are activated in the inflammatory response are those that trigger apoptosis of activated neutrophils so that they can be eliminated. Apoptotic neutrophils are specifically recognized and eliminated by macrophages.

61 Neutrophils in Inflammation

62 Lymphocytes  Origin & role Lymphoid precursor cells migrate in fetal or early postnatal life to either the thymus or lymph nodes and spleen. Cells originating from thymus-routed precursors become T cells whereas the others become B cells.  Production of lymphocytes A single lymphocyte carries only one unique specificity If it is triggered to increase the number of its unique receptors ( T cells) or antibodies ( B cells), the increase can be accomplished only by clonal multiplication of the original cell.

63 B cells (B-Lymphocyte)  Function B cells carry immunoglobulins as surface receptors. Antigens can stimulate these cells to clone into plasma cells that synthesize and secrete large quantities of a specific immunoglobulin antibody, different from the antibodies synthesized by all other B-cell clones At the cellular level, an immune response is initiated when a sufficient number of B cells or T cells have bound an antigen Although there are some immune responses that occur by way of B cells alone, without the involvement of T cells, the vast majority of B-cell activations require help from T cells.

64 Immunoglobulins  Structure Immunoglobulins consist of two identical “heavy” amino acid chains and two identical “light” chains, assembled into a Y-shaped molecule There are five different heavy chains, determining whether the immunoglobulin isotype is IgA, D, E, G, or M and differing from one another by the variable domain of the pair of heavy chains. There are only two variants of the light chain.

65 Immunoglobulins  Function While Ig bound to the B-cell surface act as receptors, freely circulating Ig can be antibodies, and as such, they recognize and bind antigens in order to (1) precipitate antigen from solution or (2) attach antigen to phagocytic/cytotoxic cells for subsequent destruction.

66 T Cells ( T-Lymphocyte)  Characteristics T cells are grouped into two classes, (1) cytotoxic T cells, which destroy hostile cells; and (2) helper T cells, which assist B cells in their immunologic tasks. Helper T cells are further divided into two subclasses: (i) TH1 cells secrete IL-2 and γ-interferon and help cytotoxic T cells and macrophages, and (ii) TH2 cells secrete IL-4, IL-5, and IL-6, which promote B-cell activation, and IL-10, which is an inhibitory cytokine in many settings

67 T Lymphocytes  Classification

68 T-cell Receptor Proteins The T-cell receptor in normally functioning T cells is closely associated with CD3, a complex consisting of five subunits that are formed by five different peptides, labeled , , , , and . CD3 functions to transmit to the cell interior the signal that was received by the T-cell receptor.

69 B & T Lymphocytes  Responses

70 T Cells Receptor Proteins  Types and characteristics T cells carry two types of receptor proteins on their surfaces, named T-cell receptor & CD molecules. These receptors consist of two chains (α and β) that are anchored in the plasma membrane, and extracellular region of each is folded into two domains The most distal tip of the chains forms the recognition and binding sites (called the paratope). The T-receptor paratope recognizes as an epitope only a specific portion of the molecules of the major histocompatibility complex (MHC) on the surface of other cells

71 T Cells Receptor Proteins  CD(Cluster differntiation) CD molecules also recognize a specific portion of the MHC on other cells, but it is a different portion from that recognized by the T-cell receptor. CD molecules are polypeptides with a membrane- spanning domain, they and a variety of co-receptors cooperate with the T receptor CD3 is present in all classes of T cells and Helper T cells carry only CD4, Cytotoxic T cells carry only CD8. The specific association of CD4 with TH and CD8 with TC helps ensure discrimination in the association of T cells with other cells.

72 Immune Responses  General principles Immune responses consist of defense mechanisms, characterized by recognition of nonself, specificity, and memory. Natural immunity reside in circulating components, such as interferon or properdin, capable of acting directly & immediately on foreign matter. Acquired immunity are normally dormant but can be activated in response to specific stimuli Active acquired immunity, derived from circulating lymphocytes, mature within the organs of the immune system (bone marrow, lymph nodes, spleen) with a delay of 5 to 10 days, and they exhibit memory

73 Immune Responses  T-cell-independent There are some antigens that can stimulate B cells to proliferate and differentiate into antibody- secreting plasma cells without the involvement of T cells. These antigens characteristically bind the B-cell receptors at several points and are capable of generating a sufficiently strong signal to activate some B cells. However, these reactions do not produce memory B cells and generally lead to the production of only low-affinity IgM antibodies rather than the full Ig complement

74 Immune Responses  T-cell-dependent When T cells are involved, the cells to which they attach are either being assisted or destroyed, depending on whether attaching T cell is a helper or cytotoxic cell. Cells that are generally useful for body defense mechanisms carry MHC-II, and MHC-II binds only helper T cells. Other cells carry MHC-I, and MHC-I binds cytotoxic T cells. Helper T cells, suppressor T cells, cytotoxic T cells are involved

75 Helper T cells  Function Activated TH stimulate macrophages to make them more effective destroyers of pathogens and help other lymphocytes to respond to antigen. Activation of helper T cells: The usual pathway is that a microbe is ingested by an antigen- presenting cell, such as a macrophage, digested, and degraded by cytosolic lysosomes. The resulting protein fragments of 10 to 15 amino acids are then bound to MHC-II that was synthesized in the endoplasmic reticulum of the antigen-presenting cell. The MHC-II/antigen unit is transported to the surface of the antigen-presenting cell, where it can be recognized by the T receptor of a helper T cell.

76 Helper T cells  Activation of B cells by helper T cells 1. The foreign molecule (antigen) is recognized and bound by the specific immunoglobulin receptor on the outside of the B cell. 2. The receptor/antigen combination is internalized and degraded into peptide fragments that can be bound to MHC-II proteins. 3. The MHC-II/peptide fragment unit is transported to the surface of the B cell so that it can become an antigen-presenting cell recognizable by the T receptor of a helper T cell. 4. Once a TH has been activated, it directs at least some of its membranebound and secreted products toward the surface of the antigen-presenting B cell. Among these is a ligand for the CD40 transmembrane molecule on the B-cell surface. CD40 & its ligand are crucial for normal TH–B cell interaction.

77 Cytotoxic T Cells  Function Cytotoxic T cells (TC) act directly to kill infected cells or eliminate microorganisms, such as viruses, that proliferate inside cells where they cannot be detected by antibodies. Once TC are activated, they destroy the target cell by mechanisms that induce apoptosis. Activation of cytotoxic T cells by infected cells: All proteins in a cell, including viral proteins, are continuously degraded. Activation of cytotoxic T cells by helper T cells: Interleukin secretion by activated helper T cells is an important signal for T-cell proliferation

78 Major Histocompatibility Complex  Nature & function  MHC molecules are proteins that are anchored to the extracellular surface of cells.  Their function is to bind antigen for presentation to T cells and they have two classes of MHC molecules MHC-I are present on practically all nucleated human cells and they are epitopes only for cytotoxic T cells. MHC-II molecules are normally confined to specialized cells, such as B cells, macrophages, and other antigen-presenting cells that take up antigens from the extracellular space and MHC-II are epitopes only for helper T cells.

79 Major Histocompatibility Antigen  Characteristics in human  In humans the major histocompatibility complex is defined by the human leukocyte antigen system (HLA) Human MHC, clustered on the short arm of chromosome 6, is operationally divided into genes which code for class I and class II antigen. Class I antigens are detected by selorogic technique, these disparities cause agglutination and /or lysis of lymphocytes. Class II antigen originally defined by by mixed lymphocyte culture and now selorogic technique, in which disparities caused lymphocyte proliferation

80 Major Histocompatibility Antigen  Structures The chemical structures of both class I and II antigens display variable and constant regions Class I antigens of HLA system are encoded by genes which include A, B and C loci Class II antigen of HLA system are encoded by genes which include the Dq, Dr and Ds loci Each locus consists of at least 20 alternative expressions as alleles or variants Each individual expresses two alleles codominantly at each locus; one allele is the representative from each paraenteral chromosome.

81 Human Panel-reactive Antibody  Determined by a flow cytometry crossmatch HLA class I antibody HLA-A HLA-B HLA-C HLA class II antibody HLA-DR HLA-DQ HLA-DS

82 Human Panel-reactive Antibody  Implications Cellular and humoral immune responses against donor-specific HLA class I and II antigens on implanted cryopreserved allografts Panel-reactive antibody is expressed as the percentage of lymphocyte panel members against which each patient's serum reacts and therefore reflects the breadth of allosensitization against the potential donor population. A PRA of less than 10% is nonreactive, PRA of 11% to 50% low reactive, and PRA over 50% high reactive. The antibody of HLA-DR antigens is intriguing and may suggest some residual cells, notably highly immunogenic, HLA class II –expressing dendritic cells resistant to the decellularization process.

83 Human Panel-reactive Antibody  Determination HLA-A, HLA-B, and HLA-C loci serotyping by means of the standard complement-dependent cytotoxicity test HLA-DR/DQ antibodies were evauated by means of a flow cytometry technique. RPA was determined by means of the sensitive anti- human kappa light-chain immunoglobulin cytotoxicity(AHG-CDC) technique against a frozen T- lymphocyte panel composed of 40 individuals of divere HLA type and racial background. RPA was expressed as the percentage of lymphocyte panel members against which the patient’s serum reacts and thus against which the patient has HLA class I antibody.

84 Tissue Engineering

85  Introduction Concept of tissue engineering was developed to alleviate the shortage of donor organs. Objective of tissue engineering is to develop laboratory- grown 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

86 Tissue Engineering  Recent myocardial graft 3-D contractile cardiac grafts using gelatin sponges and synthetic biodegradable polymers. Formation of bioengineered cardiac grafts with 3-D alginate scaffolds. Use of extracellular matrix (ECM) scaffolds. 3-D heart tissue by gelling a mixture of cardiomyocytes and collagen. Culturing cell sheets without scaffolds using a temperature-responsive polymer. Creating sheets of cardiomyocytes on a mesh consisting of ultrafine fibers.

87 Tissue Engineering  Definition The application of scientific principles to the design, construction, modification, growth and maintenance of living tissue The application of principles and methods of engineering and life sciences to obtain a fundamental understanding of structure- function relationships in novel and pathological mammalian tissues and the development of biological substitutes to restore, maintain or improve tissue function.

88 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.

89 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

90 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

91 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

92 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 ?

93 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.

94 Tissue-engineered Heart Valve  Cryopreserved human umbilical cord cells

95 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.

96 Allograft Tissue Engineering  Immunogenicity Allogrft tissue stimulates a profound cell-mediated immune response with diffuse T cell infiltrates and progressive failure of the allograft valve has been attributed to this alloreactive immune response The role of humoral response in allograft failure is less clear, recently, evidence has been accumulating that allograft tissue used in congenital cardiac surgery also stimulates a profound humoral response As previously mentioned, it is believed that the cellular elements are the antigenic stimulus for the alloreactive immune response, and thus decellularization has been proposed to reduce the antigenicity of these tissues.

97 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 cm 2 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.

98 Decellularization  Introduction In an attempt to reduce the antigenic response, decellularization processes have been introduced for cryopreserved tissue. Experimental and clinical experience with this decellularization process has been gained with porcine vena cava porcine tissue, porcine aortic and pulmonary valve conduits, ovine pulmonary valve conduits, and, subsequently, human femoral vein and human pulmonary valve conduits. There has also been experimental evidence that the decellularized matrix becomes populated with functional recipient cells.

99 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

100 Acellular Matrix Tissue  Approach to generate First break apart the cell membranes through lysis in hyper- and hypotonic solutions, followed by extraction with various detergents The detergents include the anionic Sodium dodecyl sulfate, the zwitterionic CHAPS and CHAPSO, and the nonionic BigCHAP, Triton X-100, and Tween family of agents. The enzymes that have accompanied these detergent treatments have focused mainly on cleaving and removing the DNA that is part of the cellular debris.

101 Decellularization  Rationale A persistent immunoreactivity against donor antigens has been implicated. Early calcification and stenosis from an intense inflammatory reaction may be manifestations of this immune response. Early structural failure has been shown to be more prevalent in younger patients, perhaps because of a more aggressive immune response

102 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

103 Decellularization Process  Recent status Multistep detergent–enzymatic extraction, Triton detergent, or trypsin/ethylenediaminetetraacetic acid. A more recent protocol using sodium dodecyl sulfate (SDS) in the presence of protease inhibitors was successful for aortic valve conduit decellularization Histological analysis showed that the major structural components seemed to be maintained. The effect of cell removal on different types of ECM molecules and the remodeling of the ECM in the transplanted aortic valve.

104 Decellularization Procedures Treatment Concentration Duration ation (h ) Triton X-100 1%–5% 24 Trypsin 0.5% 0.5–1.5 Trypsin/Triton X %/1%–5% 0.5–1.5/24 SDS 0.1%–1% 24 SDS, Sodium dodecyl sulfate  Methods

105 Acellularization Procedures  Enzymatic process Valve or conduits were harvested under sterile condition and stored at 4°C. Within 30 minutes the conduits were acellularized in a bioreactor. The bioreactor was filled with 0.05% trypsin and 0.02% ethylenediamine tetraacetic acid (EDTA) for 48 hours, followed by phosphate-buffered saline (PBS) flushing for 48 hours to remove cell debris. All steps were conducted in an atmosphere of 5% CO 2 and 95% air at 37°C with the bioreactor rotating at a speed of 7 rpm.

106 Decellularization Procedures  Enzymatic process The entire construct was washed for 30 minutes at room temperature in povidone-iodine solution and sterile PBS, followed by another overnight incubation at 4°C in an antibiotic solution After this decontamination procedure, the valves were placed in a solution of 0.05% trypsin and 0.02% EDTA (Biochrom AG) at 37°C and 5% CO 2 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.

107 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

108 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.

109 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

110 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

111 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.

112 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

113 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.

114 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.

115 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 substance through the way of a chemical salt formation on the surface Citric acid or ascorbic acid, which are both strong organic acids used and no signs for any structural weakening due to the citric acid pretreatment

116 Endothelial Cell Seeding  On porcine glutaraldehyde-fixed valves After incubation with serum-supplemented M-199 for 24 hours at 4°C, the valves were incubated with citric acid (10% by weight) for 5 minutes at a pH of 3 to 3.5. This pretreatment increases hydrophilsm of the surface, thus improving cell adhesion and attachment The pretreated, but unseeded valves exhibited a 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

117 Recellularization  Lavoratory evidence Stains for T-cell surface antigen, CD4, and CD8 yielded negative results. Neoendothelial cells stained for factor VIII. Smooth muscle cells in arteriole walls stained for smooth muscle actin, and cells scattered in the adventitia stained for procollagen type I. Leaflet explants had no detectable inflammatory cells and were repopulated with fibrocytes and smooth muscle cells

118 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

119 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

120 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.

121 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

122 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

123 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

124 Decellularization  Process methods Samples were placed in hypotonic Tris buffer (10 mmol/L, pH 8.0) containing phenylmethylsulfonyl fluoride (0.1 mmol/L) and ethylenediamine tetraacetic acid (5 mmol/L) for 48 hours at 4°C. Next, samples were placed in 0.5% octylphenoxy polyethoxyethonal (Triton X-100, Sigma) in a hypertonic Tris-buffered solution (50 mmol/L, pH 8.0; phenylmethylsulfonyl fluoride, 0.1 mmol/L; ethylenediamine tetraacetic acid, 5 mmol/L; KCl, 1.5 mol/L) for 48 hours at 4°C. Samples were then rinsed with Sorensen ’ s phosphate buffer (pH 7.3) and placed in Sorensen ’ s buffer containing DNase (25 µ g/mL), RNase (10 µ g/mL), and MgCl 2 (10 mmol/L) for 5 hours at 37°C. Samples were then transferred to Tris buffer (50 mmol/L, pH 9.0; Triton X %) 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.

125 Immunohistochemistry  Methods Tissue was harvested for histology at 1, 2, and 4 weeks. Samples were formalin fixed (10%), paraffin embedded, and serially sectioned (5 µ m) for histologic and immunohistochemical examination, ensuring valve leaflets were visualized in all sections. Immunohistochemistry involved standard staining techniques with biotinylated secondary antibodies, a peroxidase avidin-biotin complex, and 3.3' diaminobenzidene as the chromogen. Primary monoclonal antibodies for T cells (anti-CD3; sc1127) and cytotoxic T cells (anti-CD8; sc7970) were used. Allogeneic nondecellularized grafts were associated with significant CD3 + and CD8 + T cell infiltrates in aortic valve leaflets by 1 week after transplantation, rapidly decreasing in the following weeks.

126 Histology & Immunohistology  Examination Explanted tissue specimens were studied as hematoxylin/eosin, elastica van Gieson, and von Kossa stained paraffin or immunostained frozen sections. The antibodies for immunohistochemistry included monoclonal antibodies against CD31, -smooth muscle actin, and vimentin, and a polyclonal antibody against von Willebrand factor Expression of von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), vascular smooth muscle -actin 2 (ACTA2), smooth muscle 22 (SM22 ), and vimentin were determined with quantitative real- time RT-PCR

127 Homograft Decellularization  Cell free or nonimmunogenic Less viable cellular element No immune cell infiltration No donor-specific immune activation Well preserved ultrastructure

128 Heterograft Decellularization  Current status The use of a decellularized matrix of a xenograft is preferred because synthetic scaffolds are not only expensive and potentially immunogenic, they also suffer from toxic degradation and inflammatory reaction. Recently, nonseeded allogenic and xenogenic matrices have been implanted in animals. These matrices are expected to be covered with host cells, as observed in experimental animals. But, a so-called pseudointima can be seen, which is far from being a functional endothelial cell layer, this and the naked collagen structures are the potential thrombogenicity

129 Xenografts Decellularization  Porcine Nonenzymatic process on the basis of osmotic shock, detergent cell extraction (0.1% sodium dodecylsulfate), and antiproteases recently, which induced complete decellularization without major impairment of the structural proteins Under that process, valve conduit tissues had equal strength compared with fresh tissues and only modest changes in extensibility in vitro. In addition, this process was recently demonstrated to remove xenoantigens.

130 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,

131 Decellularization of Biomatrix  Advantages Enzymatically decellularized extracellular matrix without tanning-induced crosslinks possesses epitopes for cellular adhesion receptors, facilitating repopulation with tissue-specific cell types but also inflammatory cells Nonautologous matrix constituents such as collagen, elastin, and proteoglycans have little antigenicity, given that cellular components are entirely removed. Mismatch of HLA-DR & ABO antigens on endothelial cells in unmodified valve allografts is associated with accelerated valve failure

132 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

133 Autologous Recellularization  Rationale Autologous recellularization with in-migration of phenotypically appropriate matrix cells and formation of neointima, suggesting actual biological incorporation into the host matrix structure. These attributes may portend prolonged durability, resistance to prosthetic endocarditis, cell-mediated tissue repair, and protein renewal functions that approach the goals of tissue-engineered constructs but without preseeding or the need for extensive in vitro bioreactor manipulations. Decellularization of allograft tissue results in removal of cells and proteins, potentially creating defects, spaces, or voids within the collagen matrix that may lead to structural compromise

134 Bioprosthetic Valve & Conduit

135 Ideal Valved Conduit  Indications The ideal conduit for replacement of the pulmonary artery and valve remains elusive, particularly for the child or young adult A prosthesis that achieves the characteristics of durability, ease of implantation, availability readiness in small sizes, freedom from the need for anticoagulation, limited antigenicity, low risk of thromboembolic complications, and growth potential remains to be realized.

136 Heart Valve Substitutes  Types of valve Heart valve substitutes are of two principal types: mechanical prosthetic valves with components manufactured of nonbiologic material (eg, polymer, metal, carbon) or tissue valves which are constructed, at least in part, of either human or animal tissue Tissue valves have been used since the early 1960s when aortic valves obtained fresh from human cadavers were transplanted to other individuals (homografts). A decade later, chemically preserved stent-mounted tissue bioprosthetic valves (generally termed bioprostheses) were commercially produced and implanted

137 Bioprosthetic Heart Valves  General introduction Bioprosthetic valves have excellent hemodynamic profiles, and also they do not require permanent anticoagulation The relatively short durability of bioprosthetic valves limits their application to patients with either a contraindication to anticoagulation or to an elderly age group with a low likelihood for reoperation. The mechanisms by which biologic valves degrade are considered multifactorial, with involvement of immunologic rejection, mechanical wear out, calcification, and enzymatic digestion, with subsequent loss of the original histologic integrity.

138 Prosthetic Heart Valve  Indications The criteria of ideal valve : durability, nonthrombogenicity, easy implantability and availability Types of valve : mechanical valves, stented/stentless xenograft, cryopreserved aortic or pulmonic homograft, autograft

139 Heart Valves  Characteristics Mechanical prosthetic valves have a substantial risk of systemic thromboemboli and thrombotic occlusion, and the chronic anticoagulation therapy required in all mechanical valve recipients potentiates hemorrhagic complications. Nevertheless, contemporary mechanical prostheses are durable. Tissue valves have a low rate of thromboembolism without anticoagulation, owing to a central pattern of flow similar to that of the natural heart valves and cusps composed of valvular or nonvalvular animal or human tissue. However, a high rate of valve failure with structural dysfunction owing to progressive tissue deterioration undermines their attractiveness.

140 Heart Valve Replacement  Perspectives Approximately 85,000 substitute valves are implanted in the US and 275,000 worldwide each year, of which we presently estimate that approximately half are mechanical and half are tissue, suggesting a shift toward increasingly greater usage of tissue valves over the last decade. Within 10 years postoperatively, prosthesis-associated problems overall necessitate reoperation or cause death in at least 50% - 60% of patients with substitute valves. The rate is similar for mechanical prostheses and bioprostheses; however, the frequency and nature of specific valve-related complications vary with the prosthesis type, model, site of implantation, and certain characteristics of the patient.

141 Bioprosthetic Heart Valves  Fate of bioprosthesis Structural dysfunction is the major cause of failure of bioprosthetic heart valves. The principal underlying pathologic process is cuspal calcification; secondary tears frequently precipitate regurgitation. Calcification can also cause pure stenosis owing to cuspal stiffening. Calcific deposits are usually localized to cuspal tissue (intrinsic calcification), but calcific deposits extrinsic to the cusps may develop in thrombi or endocarditic vegetations (extrinsic calcification). Progressive collagen deterioration, independent of calcification, is also a likely important contributor to the limited durability of bioprosthetic valves.

142 Bioprosthetic Heart Valves  Immune response Preformed antibodies against [alpha]-Gal cause opsonization within the valve tissue Blood contact to collagen matrix activates & leads to thrombocyte activation, liberation of chemotaxic and proliferative stimulating factors and within hours to PM neutrophil, granulocyte and macrophage influx Degeneration begins with the penetration of immunoglobulines (IgG/IgM) into the valve-matrix. Subsequently, macrophages are deposited on the valve surface and erythrocytes penetrate into the valve. Finally, collagen breakdown and calcification

143 Bioprosthetic Heart Valves  Mitroflow pericardial valve This is a second generation bioprosthesis made of a single sheet of glutaraldehyde-preserved bovine pericardium mounted on the outside of a flexible Dacron-covered Delrin stent. Due to its design characteristics, an unimpeded leaflet opening and blood flow occurs, which results in an excellent hemodynamic performance and a proven superiority when compared with other pericardial bioprostheses

144 Valved Conduit Manufacturing  Process flowchart(Biocor) Tissue procurement Dissection, collection and transportation Inspection and rinsing Tissue fixation and bioburden reduction & storage Conduit manufacturing & inspection Bioburden reduction and storage Quality control and final review Chemical sterilization Aseptic transfer Package and final inspection

145 Shelhigh Valved Conduit  Valve tissue fixation Glutaraldehyde fixation of the tissue occurs with 24 hours of receipt of tissue. Lot integrity is maintained during the fixation and the valve are bathed in 0.35% glutaraldehyde and a hydrostatic closing pressure of <4mmHg is applied to the valves. This pressure is maintained by the glutaraldehyde recirculation rate. The valves are detatched from the fixation plug and placed together in a labelled container 0.35% glutaraldehyde

146 Shelhigh Valved Conduit Pericardial tissue fixation The pericardial sac are placed flat in a shallow container of freshly prepaired glutaraldehyde solution The pericardia are fixed at room temperature, unstressed for at least 2 weeks. The glutaraldehyde solution is changed between 2 and 4 hours and again at 24 hours. Fixed tissue quarantine bioburden reduction process is performed to reduce bioburden of of the valves in the event organisms resistant to 0.35& glutarakdehyde are present

147 Shelhigh Valved Conduit  Process flowchart Procurement Dissection Tissue fixation Inspection Fixed dissection Prolapse test Mounting Sterilization & detoxification Packaging

148 Shelhigh Valved Conduit Intimal fibrosis, neointimal proliferation, and neointimal peel formation; chronic inflammation with histiocytic demarcation of the xenograft tissues, as well as a granulomatous reaction with the presence of giant cells of foreign-body type and acute granulocytic inflammation, and rarely calcifications  Histopathology

149 Shelhigh Valved Conduit  Histopathology Neointimal proliferation included all through the conduit & valvular cusps encased by reactive neointimal layer, with immobility, fibrous contraction Chronic inflammation with a marked histiocytic component with a maximum inflammation between the xenograft & reactive neointimal layer Immunohistochemistry revealed predominantly histiocytic and T-lymphocytic infiltrates, with a sparse B-lymphocytic component. Histiocytic & granulocytic reactions most pronounced surrounding the porcine myocardium,and with an acute inflammatory component with PML

150 Shelhigh Valved Conduit  Immunohistochemical study Paraffin sections were routinely stained with hematoxylin and eosin and Verhoeff's van Gieson elastic tissue stain Immunohistochemical staining for CD3, CD4, CD8 (for T-cell subsets), CD20 (B-cell subset), and CD68 (for macrophages) was performed to characterize the inflammatory infliltrate Naphtol AS-D-chloracetate esterase staining was used to visualize granulocytic infiltrates.

151 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

152 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

153 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.

154 Glutaraldehyde Fixation  Adverse effect 1.Making biologic material stiff & hydrophobic 2.Release of residual cytotoxicity induce the foreign body reaction 3.No endothelial cell lining onto the cytotoxic treated area

155 Glutaraldehyde Fixation  Use as valve prostheses As a biologic extracellular matrix scaffold, porcine heart valves for their well-known good hemodynamic behavior and unlimited availability. Porcine scaffolds are usually treated with glutaraldehyde to improve mechanical properties and to limit the xenogeneic rejection process. Glutaraldehyde treatment profoundly modifies the extracellular matrix structure and makes it improper to support cell migration, recolonization, and the matrix-renewing process

156 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

157 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

158 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

159 Bioprosthetic Heart Valves  Use of animal valve prostheses The use of animal valve prostheses was first proposed and tested during the 1940s and 1950s Fixatives were introduced not as a method of decreasing antigenicity but as a sterilizing and stiffening agent. Duran and Gunningt sterilized porcine graft by immersion in liquid ethylene oxide. Binet and colleagues placed non-sterile pig in a mercurial solution" for 3 days. The first formaldehyde preservation of porcine valves was performed by O’Brien, who used the solution in 1967 for "both preservation and sterilization.

160 Conditioning of Heterografts Biologic factors affecting durability Diagramatic representation of different stages of method for conditioning heterografts

161 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

162 Glutaraldehyde Treatment  Action on tissues Glutaraldehyde mechanism of action

163 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.

164 Glutaraldehyde Fixation  Action & adverse effects Glutaraldehyde (GA) is currently the standard reagent for preservation and biochemical fixation It imparts intrinsic tissue stability (biodegradation resistance) and reduces the antigenicity of the material. Recent reports have suggested a detrimental role of aldehyde-induced intra- and intermolecular collagen cross-linkages in initiating tissue mineralization GA has been implicated in devitalization of the intrinsic connective tissue cells of the bioprosthesis, thus resulting in breakdown of transmembrane calcium regulation and hence contributing to cell-associated calcific deposits

165 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

166 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.

167 Tissue Valve Preparation  Principles Ensure reproducibility, desired tissue biomechanics, desired surface chemistry, matrix stability, and resistance to calcification A variety of treatments have been used clinically as well as experimentally They may be broken down into two broad categories: modifications to glutaraldehyde processed tissue and nonglutaraldehyde processes.

168 Calcification of Bioprosthesis  Preventive methods(lipid) Calcium phosphate crystals containing Na, Mg, and carbonate nucleate due to devitalization of the cells and thus inactivation of the calcium pump Membrane-bound phospholipids have also been associated with calcification nucleation due to alkaline phosphatase hydrolysis Ethanol has been used to remove phospholipids and mitigate calcification, yet phospholipids have also been removed with chloroform-methanol yielding Lipid extraction can also be performed through tissue processing with detergent compounds such as sodium dodecyl sulfate.

169 Calcification of Bioprosthesis  Preventive methods(aldehyde) Free aldehyde within the tissue matrix has been thought to be an initiator for calcification as well. This is supported by studies that demonstrate that aldehyde-binding agents such as alpha-amino oleic acid (AOA; Biomedical Design, Marietta, Ga), L-glutamic acid, & aminodiphosphonate prevent cusp calcification. Yet, post treatment with the amino acid lysine does not prevent cuspal calcification. and emphasizes the multiplicity of pathways by which calcification can initiate.

170 Tissue Valve Preparation  Processes The modifications to glutaraldehyde processed tissue with detergents such as sodium dodecyl sulfate and Tween-80 to remove phospholipids, ethanol preincubation to remove phospholipids, covalently bound AOA, L-glutamic acid and aminodiphosphonate to bind free aldehydes, and detoxification processes using urazole and homocysteic acid. Nonglutaraldehyde processes include but are not limited to epoxy compounds, dye-mediated photo- oxidative reactions including PhotoFix and carbodiimide compounds including Ultifix

171 Pericardial Bioprostheses  Characteristics The pericardial bioprostheses are fabricated using bovine pericardium, which is sewn into a valvular configuration on a stented frame. The first commercially available pericardial valve, the Ionescu-Shiley valve was abandoned in 1988 due to a high incidence of valvular deterioration characterized by leaflet tears and valve incompetence. Of the second-generation pericardial valves, introduced in 1982, the Carpentier-Edwards pericardial valve has shown better results than the other valve in its class, the Pericarbon pericardial bioprosthesis

172 Pericardial Bioprostheses  SOPRANO TM heart valve The functional component consists of three leaflets of bovine pericardium fixed by treatment in buffered glutaraldehyde solutions The tissue leaflets are sutured to the inside of a flexible support, the stent; this is formed by a core in acetalic resin(polyoxymethylene) covered with a knitted polyester fabric(polyethylene tereftalate, PET); this includes a radio opaque silicone rubber insert which is positioned at the same level as the sinusoidal border at the entrance to the valve; the fabric is coated with Carbofilm, a thin film of turbostratic carbon aimed to enhance the biocompatibility

173 SOPRANO TM Heart Valve  General features

174 SOPRANO TM Heart Valve  Manufacture flow chart

175 SOPRANO TM Heart Valve Assembly of the two pericardial sheets functional component with the supporting stent  Technical features

176 SOPRANO TM Heart Valve  Manufcturing materials The pericardium undergo glutaraldehyde-based process in which the stabilizing agent react under dynamic conditions. The valve prosthesis is treated for the elimination of glutaraldehyde residues and stored in a buffered solution without aldehydes All the surfaces not formed by biologic tissue, including the suture threads, are coated with Carbofilm, a thin firm of carbon with turbostratic structure, The valve support incorporates a tantalium wire rimming the edge of the inflow side of the valve so that the implanted valve can be seen on radiography

177 SOPRANO TM Heart Valve  Major materials Bovine pericardium for valve functional component Polyester(PET) suture thread for sewing Acetalic resin for valve support Polyester(PET) knitted fabric for sewing ring Tantalium wire for radiopaque marker embedded in the valve support Carbofilm for biocompatible coating Silicone loaded with BaSO4 for additional radiopaque sewing ring filler

178 SOPRANO TM Heart Valve  Procedures of pericardium Animal selection Pericardium retrieval operation Dedicated personnel selection & training Record keeping Traceability

179 SOPRANO TM Heart Valve  Pericardium properties Bioburden of shipment medium less than 10 5 CFU( colony forming units)/ml Thickness of pericardium Valve size mm Valve size mm Valve size mm Morphology Absence of flaws such as, vascularised areas, traumas, superficial non-uniformity areas, inflammations

180 SOPRANO TM Heart Valve  Glutaraldehyde treatment Operating conditions, concentration, time & temperature of cross-linking treatment, settled up After a first treatment with low glutaraldehyde(0.2%) solution, the pericardial sheets are joined together. The biological component is then assembled with the support structure and another fixation step with 0.5% glutaraldehyde solution is accomplished A subsequent treatment with high glutaraldehyde (0.5%) solution is aimed at the achieving the sterility

181 SOPRANO TM Heart Valve  Detoxification treatment As a results of glutaraldehyed fixation, tissue valves may contain residual amount of unbounded aldehyde group, which is responsible for blood cell damage Moreover the collagen affinity to binding with calcium ions is enhanced in presence of unsaturated glutaraldehyde groups, and this could facilitate calcification process. A neutralizing post-fixation treatment based on the action of homocysteic acid is performed, and this molecule reacts with unsaturated aldehyde group, thus neutralizing them

182 SOPRANO TM Heart Valve  Sterilization of biological component Acceptance bioburden limit of incoming pericardium less than CFU/ml Aseptic processing and cleanroom technology rules are adopted The sterility of the pericardial component is reached by means of a chemical treatment with low concentration glutaraldehyde solution for prolonged times  Sterilization of non-biologic components Valve stent is sterilized by chemical treatment Container, lid, holder, ID, sutures, equipments are sterilized by Ethylene oxidem Chemical solutions are sterilized by means of filtration on 0.22um membrane

183 Aortic Porcine Bioprosthesis  CryoLife-O'Brien bioprosthesis O'Brien Stentless Aortic Porcine Bioprosthesis (CryoLife Inc, Kennesaw, Ga) composite design was to optimize hemodynamic performance and durability of valves. Three noncoronary leaflets are prepared by low-pressure glutaraldehyde fixation process of less than 2 mm Hg. The scalloped design and the absence of xenograft tissue below the leaflet hinge allow implantation with a single suture line. Because there are no synthetic materials other than the suture, the risk of endocarditis is expected to be reduced

184 Stentless Bioprosthesis  Specification Elimination of a rigid sewing ring, the dynamic nature of the aortic root may be maintained after AVR with this device. Maintenance of normal aortic root function may at least in part be responsible for the excellent hemodynamic performances of both stentless valves, as well as aortic homografts. Medtronic Freestyle stentless bioprostheses and St Jude Medical Toronto stentless porcine valve bioprostheses, O'Brien Stentless Aortic Porcine Bioprosthesis

185 Determinants of Mineralization  The determinants of bioprosthetic valve and other biomaterial mineralization include factors related to (1) host metabolism, (2) implant structure and chemistry, (3) mechanical factors. Natural cofactors and inhibitors may also play a role Accelerated calcification is associated with young recipient age, glutaraldehyde fixation, and high mechanical stress.

186 Calcification Process  Hypothesis

187 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.

188 Prevention of Calcification  Three generic strategies have been investigated for preventing calcification of biomaterial implants: Systemic therapy with anticalcification agents; Local therapy with implantable drug delivery devices; Biomaterial modifications, such as removal of a calcifiable component, addition of an exogenous agent, or chemical alteration.

189 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

190 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

191 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.

192 Bioprosthesis Calcification  Prevention Inhibitors of hydroxyapatite formation Bisphosphonates Trivalent metal ions Calcium diffusion inhibitor ( amino-oleic acid ) Removal or modification of calcifiable material Surfactants Ethanol Decellularization Modification of glutaraldehyde fixation Use of other tissue fixatives Problems created by an exposed aortic wall

193 Bioprosthetic Valved Conduit  New bioprosthesis By Medtronic, Inc. (Minneapolis, MN), the Contegra conduit is a 0.25% glutaraldehyde-fixed segment of bovine jugular vein, containing a venous valve; the whole xenograft is fixed under minimal pressure, less than 3 mm Hg Other stentless xenografts have been developed during the last years, including the following: the LabCor from Sulzer Carbomedics (Austin, TX); the Biocor from St Jude Medical (Belo Horizonte, Brasil), which is a bovine pericardial conduit with an aortic porcine valve; and the Shelhigh pulmonic conduit (Union, NJ), which consists of a pericardial bovine tube with a stentless porcine pulmonary valve.

194 Bioprosthetic Valved Conduit  Characteristics By Medtronic, Inc. (Minneapolis, MN), the Contegra conduit is a 0.25% glutaraldehyde-fixed segment of bovine jugular vein, containing a venous valve; the whole xenograft is fixed under minimal pressure, less than 3 mm Hg Other stentless xenografts have been developed during the last years, including the following: the LabCor from Sulzer Carbomedics (Austin, TX); the Biocor from St Jude Medical (Belo Horizonte, Brasil), which is a bovine pericardial conduit with an aortic porcine valve; and the Shelhigh pulmonic conduit (Union, NJ), which consists of a pericardial bovine tube with a stentless porcine pulmonary valve.

195 Bioprosthetic Valved Conduit  Recent development The Contegra conduit is a heterologous bovine jugular vein graft with a trileaflet venous valve. The conduit is fixed with 0.25% glutaraldehyde under zero pressure condition. No additional anticalcification treatment is used. Shelhigh No-react graft is porcine pulmonary valve conduit, strikingly unsupported conduit. The typical finding of the explanted conduits was prominent intimal peel formation at the distal anastomosis without calcification, but little histologic evidence of immunologic activity

196 Mechanical Valved Conduit  Controversies Mechanical PVR for patients who have had multiple prior operations, or who are on warfarin In general, we have not advised mechanical PVR in children, but mechanical PVR, if anticoagulation is required for a mechanical prosthesis An adequate sized pulmonary prosthesis can be inserted with no prosthetic material or by using only an anterior patch of pericardium Recently, it has been reported that tilting-disc valves in the pulmonary position may perform better than bileaflet valves

197 Valved Conduit Implantation  General aspects Conduit implantation for the RVOT reconstruction constitutes approximately 15% to 20% of all congenital cardiac operations Homografts have shown the best results so far, with overall survivals of appoximately 84% and 31% at 5 and 15 years, respectively Young age at implantation, and the need for a small homograft have consistently been recognized as risk factors for accelerated failure Problem to use homografts is the scarcity of suitably sized homografts and the consistency of its quality. Xenograft aortic or pulmonary valves & conduits have failed to match the results of the homograft conduits

198 Valved Conduit Replacement  Indications for replacement In asymptomatic patients is near systemic or systemic pressure of the RV and a peak instantaneous Doppler systolic gradient greater than 65 – 70 mmHg, compared to 40 – 50 mmHg in the majority Transcatheter interventions such as balloon dilatation or stent implantation when indicated, may prolong the RV–PA conduit lifespan and delay the first reoperation for conduit obstruction. Use of an autologous alternative like ‘Reparation a` l’e´tage Ventriculaire’ (REV procedure) or other technical options

199 Valved Conduit Implantation  Sizing & shaping The main pulmonary artery was always completely transected to allow a harmonious end-to-end anastomosis of the graft to the pulmonary bifurcation The diameter of the valved conduit was mostly determined according to the diameter of the PA bifurcation, although the distance between the conal septum and pulmonary bifurcation was also taken into consideration at times Locate the valve as cranial as possible immediately below the bifurcation of the pulmonary artery to avoid geometrical distortion of the valve at the site of proximal implantation in the right ventricle.

200 Valved Conduit Implantation  Technical aspect A better size matching of the graft to the pulmonary artery diameter may reduce local turbulent flow and subsequent development of the fibrotic membrane Relative profile of the stenotic membrane becomes critical in small conduits(<14mm ) Plane of valve annulus should remain perpendicular to the graft direction. Short bevel results in traction on the convex part of graft with caudal rotation of the annular plane & compression at the leaflet level

201 Mechanical Valved Conduit  Four main criteria First, older age to avoid outgrowth of the prosthesis Second, multiple previous operations with an increased reoperation-related morbidity Third, current use of anticoagulants Fourth, patient compliance with anticoagulant therapy.

202 Valved Conduit Implantation  Technique for small conduit The distal anastomosis is made in diagonal shape to enlarge the anastomosis 7-0 prolene sutures in multiple continuous interrupted sutures Start the anastomosis always from the outside the lumen Anticoagulation for at least 2-3 months and aspirin for life

203 Valved Conduit Implantation  Sizing Attempt to keep the pulmonary bifurcation intact The main pulmonary artery was always completely transected to allow a harmonious end-to-end anastomosis of the graft to the pulmonary bifurcation The diameter of the valved conduit was mostly determined according to the diameter of the PA bifurcation, although the distance between the conal septum and pulmonary bifurcation was also taken into consideration at times Locate the valve as cranial as possible immediately below the bifurcation of the pulmonary artery

204 Bioprosthetic Conduit  Causes of early stenosis Chronic movement at the level of the anastomosis could be an ongoing local trigger for peel formation. The intensive narrowing process may be in part the result of a strong cellular immunologic reaction against the xenograft Additional factors such as endothelial lesions made during conduit suture, residual glutaraldehyde release from the implant, and shear stresses related to hemodynamic conditions at the level of the anastomosis might amplify this proliferative reaction.

205 Bioprosthetic Conduit Stenosis  Medical prevention Heparin infusion (10 IU/kg/h ) in early postoperatively, followed by low molecular weight heparin (until hospital discharge). Aspirin is given postoperatively for 3 months, starting from the first postoperative day Aspirin plays a protective role for the endothelium and influences neointima formation Use aspirin for long periods to avoid thrombosis, as well as anti-inflammatory therapy to prevent chronic rejections

206 Shelhigh No-react Graft  Histopathology of explant Intimal fibrosis, neointimal proliferation, and neointimal peel formation; chronic inflammation with histiocytic demarcation of the xenograft tissues, as well as a granulomatous reaction with the presence of giant cells of foreign-body type. Immunohistochemistry revealed predominantly histiocytic and T-lymphocytic infiltrates, with a sparse B-lymphocytic component. The granulomatous and histiocytic demarcation resulted in an incomplete or complete neointimal "peel" formation in most conduits.

207 Xenograft Valve or Conduit  Xenoreactive antigen Immunogenic [alpha]-Gal-epitope was founf on fibrocytes interspersed in the connective tissue of porcine bioprosthetic valves Patients with a bioprostheses had developed a significant increase of naturally occurring cytotoxic IgM antibodies directed towards [alpha]-Gal Patients after the implantation of bioprostheses demonstrated an increased cytotoxicity against [alpha]- Gal-bearing PK-15 cells The specificity of the cytotoxic effects was proven as soluble Gal[alpha]1–3Gal[beta]1–4GlcNAc markedly inhibited cell death of [alpha]-Gal-bearing PK15 cells

208 Difference of Gal Expression  Mechanisms Decreased α -Gal expression on valve endothelium could be due to either decreased glycosylation or decreased surface glycoproteins on leaflet endothelium. Protein expression is closely regulated in endothelial cells, and flow rate can clearly modulate endothelial gene expression. Expression of proteins, such as major histocompatibility complex antigen and vascular cell adhesion molecule 1, are all influenced by the amount of shear force exerted on the endothelium.

209 Xenograft Valve or Conduit  Xenoreactive immulogic acivity Younger patients evidenced increased valve degeneration, indicating increased immunologic activity directed against the xenograft Degeneration of bioprostheses begins with the penetration of immunoglobulines (IgG/IgM) into the valve-matrix. Subsequently, macrophages are deposited on the valve surface and erythrocytes penetrate into the valve. Finally, collagen breakdown calcification takes place Thus, the deposition of immunoglobulins represents the initial immunological trigger for calcification in bioprostheses

210 Xenograft Valve or Conduit  Investigation of xenoantigen Presence of [alpha]-Gal-epitope on native and fixed porcine valves Implantation of bioprostheses elicits increased formation of cytotoxic anti [alpha]-Gal IgM antibodies Potentially increased lysis of [alpha]-Gal- bearing PK15 porcine cells from serum obtained prior and after bioprostheses implantation.

211 Xenoreactive Valve or Conduit  Immunohistochemistry Labelling with IB4, and antibody against von Willebrand Factor Then the diluted primary antibody against vWF [1: 300] was applied & rinsed in PBS and incubated with a secondary antibody After rinsing, finally, sections were rinsed again in PBS and mounted in 60% glycerine in Tris buffer: double-fluorescence labelling was applied Sections were incubated in the primary antibody against vimentin [1: 100] and then stained with the secondary antibody Finally, sections were labelled with IB4 and in addition, sections were coincubated with Gal[alpha]1–3Gal[beta]1–4GlcNAc (5 mg mL -1 and 1 mg mL -1, to demonstrate galactose [alpha]1,3-galactose specificity with IB4-isolectin staining.

212 Xenoreactive Valve or Conduit  Enzyme-linked immunoabsorbent assays ELISA assay performed to compare the incidence of naturally occurring cytotoxic anti α- Gal IgM antibodies  Cytolysis of PK-15 cells Cytotoxic activity of the anti α -Gal IgM antibodies, PK-15 (ATCC CCL-33) (20·000/96 round-bottom plate well) were incubated with a serum pool diluted. Cell death determined by trypan-blue staining

213 Xenoreactive Valve or Conduit  Removal of α-gal epitopes Green coffee bean α -galactosidase can cleave the terminal α -galactose(α-Gal) on oligosaccharides. α-galactosidase, 1) functions at a range of temperature and pH relevent to clinical organ transplantation, 2) is effective in removing the terminal α-Gal sugar from pig vessel and, 3) results in prolonging survival of porcine vessels The tissues were subsequently immersed into a phosphate-citrate-sodium chloride buffer containing 100 U/ml α-galactosidase and incubated for 4 hr at 4- 26°C with gentle shaking

214 Xenotransplantation

215  Clinical application Selection of a donor species Hyperacute rejection Acute vascular rejection Accommodation Cellular rejection Physiologic limitations Zoonosis

216 Xenotransplantation  Biologic responses Transplantation into an unmanupulated recipient would give rise to hyperacute rejection If hyperacute rejection is avoided, as for example by inhibition of complement, the graft becomes subject to acute vascular rejection If antidonor antibodies are depleted from the recipient, the graft may undergo accommodation If acute vascular rejection is avoided, the graft may undergo cellular rejection or chronic rejection

217 Xenotransplantation  Endothelial activation of rejection Resting or quiscent endothelial cells form a tight monolayer that poses an effective barrier to blood cells and plasma proteins Exposure of endothelial cells to agents such as interleukin-1, tumor necrosis factor, or endotoxin, causes the cell to undergo a series of metabolic and structural changes, known as activation. Activated endothelial cells promote platelet aggregation, fibrin generation, and neutrophil adhesion, and a monolayer of activated endothelial cells is permeable to plasma protein and blood cells

218 Xenotransplantation  Biologic responses Xenotransplanrtation Inhibit complement Acute vascular Cellular Chronic rejection rejection rejection Accomodation Deplete Ab Hyperacute rejection

219 Xenotransplantation  Hyperacute rejection Hyperacute rejection is characterized clinically by the development and rapid progression of discoloration and ecchymosis and with severe organ failure over a period of minutes to hours The histology of hyperacute rejection is characterized by hemorrhage and widespread platelet thrombi Hyperacute rejection would be initiated by binding to the endothelial lining of blood vessels in that organ of xenoreactive natural antibodies ( antigen, Galα1-3Gal) Antibody binding activates the complement system, and the action of complement on blood vessels causes the devastating picture of hyperacute rejection

220 Xenotransplantation  Hyperacute rejection It is characterized by widespread hemorrhage, edema, thrombosis, and a relative lack of a cellular infiltrates. HAR is triggered by the xenoreactive antibodies in the host circulation which recognize and bind donor endothelium It is the subsequent activation of host complement that mediates HAR, leading to immediate graft destruction

221 Xenotransplantation Hyperacute Rejection Synthesis and inhibition of synthesis of Galα1-3Gal, major target antigen in cardiac xenograft rejection

222 Xenotransplantation  Prevention of hyperacute rejection Prevention of hyperacute rejection can be achieved by either interfering with antibody binding to the graft or inhibiting activation of the complement system Extracorporeal perfusion of blood or separated plasma through a column bearing Galα1-3Gal, and by lowering the antigen expression by genetic means Inhibition of activation of complement using cobra venom factor, soluble complement receptor type 1, gamma globulin, expression of human complement regulatory protein ( decay accelerating factor )

223 Porcine Xenotransplantation  Prevention of hyperacute rejection The prominent epitope is a terminal carbohydrate moiety, Galα1-3Galβ1-4GlcNAc-R(α Gal ) which is found on various glycoproteins and glycolipids on porcine cells. HAR can be prevented by continual systemic inhibition of complement through the use of genetically modified porcine organs that express human membrane-bound complement regulatory proteins, by immunoapheresis to remove all circulating antibody, or by selective immunoapheresis that removes only antigal antibodies, and synthesis of αGal tricsaccharide that can neutralize circulating anti-Gal antibodies

224 Xenotransplantation  Acute vascular rejection Acute vascular rejection( delayed xenograft rejection) may begin within 24 hours of reperfusion and generally destroys the graft over a period of days to weeks The histologic features include endothelial swelling, ischemia, and diffuse intervascular thrombosis, with thrombi consisting mainly of fibrin and is much like the picture of acute vascular rejection of allograft The cause may be the antidonor antibodies that bind to a xenograft that precipitate endothelial injury by activating the complement system or by perturbing endothelial cell surface molecules, and also facilitate injury by interacting with inflammatory cells

225 Xenotransplantation Acute Vascular Rejection Role of antidonor antibodies in the pathogenesis of acute vascular rejection. Antidonor antibodies may directly perturb endothelial cells or ( C ) trigger activation of complement

226 Xenotransplantation  Accommodation If hyperacute rejection and acute vascular rejection are temporarily averted, a state of accommodation may occur in which the graft appears inured to the presence of antidonor antibodies in circulation The mechanisms are uncertain, although they may involve a change in the antibody repertoire, change in antigen, or the acquired resistance of endothelium to humoral injury

227 Xenotransplantation  Cellular rejection(1) Difference between the cellular response to a xenograft and to an allograft may be how to T cells recognize foreign tissue. In xenotransplantation, there is evidence that T cells may not always recognize xenogeneic cells directly, in part because the foreign accessory signals are incompatible ( indirect recognition ) In allotransplantation, T cells recognize foreign major histocompatibility antigens expressed on the foreign cells ( direct recognition ). Binding antidonor antibodies heighten cellular immune response by causing activation of antigen presenting cells

228 Xenotransplantation  Cellular rejection(2 ) The extent to which the direct versus indirect pathways of T cell recognition might utilized in cellular responses to xenotransplantation has therapeutic implication If direct pathway predominates, as it does in allotransplantation, then genetic engineering of donor might help to limit that response, but, on the other hand, if the indirect pathway predominates, anti-CD4 antibodies might be useful Natural killer cells are lymphopcytes that lack of T cell receptor but that recognize foreign or virus-infected cells and in so doing release inflammatory cytokines and mediate cytotoxicity

229 Xenotransplantation Cellular Rejection Interactions of T lymphocytes of a graft recipient with antigen-presenting cells

230 Vascularized Xenograft  Rejection Vascularized xenografts are referred as concordant or discordant according to the timing and the immune effectors depositing on graft capillary endothelial cells. Concodant xenografts are rejected within days of engraftments when the immunoglobulins specific to the graft have been induced, and discordant xenografts are rejected within minutes Hyperacute rejection in discordance is mediated by the reaction of natural antibodies (classic pathway) or the complement system activated by alternative pathway ( antibody-independent pathway of complement activation)

231 Vascularized Xenograft  Rejection The capillary rejection of a vascularized xenograft coincides with the onset of graft dysfunction & these changes are limited to the endothelium Then the medial smooth muscle cells are subjected to immune effectors after the endothelial cells have been rejected There is also inflammation of the medial extracellular matrix which is probably elicited by immunogenicity of the xenogeneic extracellular matrix

232 Xenotransplantation  Complement-regulatory proteins The beta-actin-CD59 transgene contains a chick beta- actin promoter that controls expression of a cDNA for human CD59. In this transgene, the hCD59 cDNA is embedded in a human alpha-globin gene expression cassette that provides splicing and polyadenylation signal sequences. The H2K-DAF transgene contains a cDNA for human DAF regulated by the mouse H2K promoter and H2K polyadenylation signal sequences Probes for DNA and RNA hybridizations were derived from the cDNA of each of the genes

233 Xenotransplantation  Complement-regulatory proteins Normally, the vasculature is protected from autologous complement-mediated damage by several membrane- bound complement-regulatory proteins, such as CD59(protectin), decay accelerating factor(CD55, DAF), or membrane cofactor protein(CD46) Each CRPs of species have been shown to be most effective at controlling the activation of homologous complement, and the complement of closely related species, but they are much less effective regulators of complement from more divergent species In the heart, hCD59(protectin) expression was detected on the endothelial cells and on the myocytes

234 Xenotransplantation  Complement-regulatory proteins There exist a family of complement-regulatory proteins which, under physiologic conditions, down-reguate the complement cascade to protect cells from damage by autologous complement CRPs are found in both soluble and membrane-bound forms. CRPs exhibit homologous restriction so that CRPs from one species are relatively inefficient at regulating complement from a distant species Expression of human membrane-bound complement- regulatory proteins could provide local protection from complement-mediated damage

235 Xenotransplantation  Complement-regulatory proteins A lack of effective complement regulation sensitizes the porcine organ to complement-mediated damage, and thereby, contributes to hyperacute rejection. Then the introduction of of human CRP function, even at lower levels, may significantly effect the humoral rejection of porcine xenograft Hypothesis, by enhancing the resistance of xenogenic vascular endothelium to complement-mediated damage through expression of human CRP gene products in transgenic animal

236 Xenotransplantation  Generation of transgenic pigs A 60 kh genomic construct encompassing the human CD46 gene, or hDAF, or CD59(protectin) was isolated from a P1 phase library and microinjected into the male pronuclei of fertilized porcine oocytes to generate transgenic pig Transgenic animals were identified by Southern blot analysis of DNA from tail biopsies

237 Xenotransplantation  Transgenic pigs A line of transgenic pigs that express the human complement-regulatory proteins human CD59 and human decay-accelerating factor This specificity is evident in transgenic organs in which low levels of CD59 and human decay-accelerating factor(CD55) expression significantly effect the humoral immune response that causes xenograft rejection Transgenic organs with high levels of human complement-regulatory protein expression will be sufficient to alleviate the humoral immunologic barriers

238 Xenotransplantation  Reconsideration in concordant or discordant It is focused on the presence or absence of hyperacute rejection to classify species combination, at least three factors might be considered The presence or absence of natural antibodies directed against endothelial cells of a xenogeneic donor The function, or lack thereof, of complement regulatory proteins of recipient in the “environment” of the donor organ The relative effectiveness of cell membrane-associated complement inhibitory proteins of the donor against the complement system of the recipient

239 Xenotransplantation  Pig model The animal of choice currently considered to be the pig because of its size, physiologic compatibility, and breeding characteristics and the potential for genetic modification. The current limitation to clinical xenotransplantation is immunologic rejection. Achieving this goal is to define immunosuppressive regimens that control the xenograft immune response by using non–life-supporting heterotopic transplants.

240 Xenotransplantation  Anti-xenoreactive antibody Xenoreactive IgG/IgM antibodies were detected in the sera of primates, including man, against the oligosaccharid residue [alpha]-Gal, which is present on pig tissue In primates the [alpha]-Gal-specific IgM-antibodies lead to complement activation and hyperacute rejection of pig xenografts within minutes to hours About 1% of the circulating immunoglobulines are specific for [alpha]-Gal in humans and their natal formation is presumably owing to exposure to gut flora expressing the [alpha]-Gal-epitope

241 Xenotransplantation  Xenoreactive antigen [alpha]-Gal is an unique carbohydrate structure, which has been evolutionarily conserved in most mammalian species except humans, apes and Old World monkeys [alpha]-Gal is widely expressed on the cell surface of mammals and highly expressed in capillaries as compared to large vessels, due to differential gal α 1, 3 galactosyltransferase expression endothelium Since humans lack the α- galactosyltransferase, they have natural anti-Gal antibodies. About 1% of B cell clones in humans produce natural anti-Gal

242 Anti-Gal Antibodies  Measurement Antibody levels were measured by enzyme-linked immunosorbent assay (ELISA) using HSA-[alpha]1,3 Gal as a specific antigen and human serum albumin (HSA) RIA/EIA 96-well plates were coated with 30 µg/ml of either HSA- [alpha]1,3 Gal or HSA alone, blocked with 0.1% Tween 20/1% HSA-phosphate-buffered saline (PBS), and then incubated with serial dilutions of serum at 4°C. After washing plates, wells were incubated with streptavidin- alkaline phosphatase-conjugated goat anti-human µ-chain, or streptavidin-alkaline phosphatase-conjugated goat anti-human [gamma]-chain, and developed with Sigma Fast pNPP tablets The absorbance at 405 nm was determined using a Molecular Dynamics Plate Reader and Softmax Pro software

243 Anti-α-Gal Antibodies  Measurement Isotype-specific determination of anti-α-Gal antibodies was performed by enzyme-linked immunosorbent assay Binding of xenoreactive IgM to cultured porcine aortic endothelial cells (anti-PAEC IgM) was determined by ELISA Antipig antural antibody levels were measured by ELISA using porcine platelet extracts (PPE) as antigen

244 Xenotransplantation  Recent porcine trial Porcine human model Produce genetically engineered pigs that ---- Lack Gal epitope( humans have circulating antiGal Ab) ---- Express human dacay accelerating factor (hDAF) ( presence of hDAF protects against complement mediated hyperacute rejection Nuclear transfer to shorten span required to produce identical herds of genetically altered animals

245 Immunofluorescent Studies  Reagents Biopsy tissues were stained for IgG, IgM, C3, C4, C5b neoantigen, MAC, properdin, fibrinogen, polymorphonuclear neutrophils, and platelets Affinity-isolated, fluorescein isothiocyanate (FITC) goat anti-human antibodies against IgG, IgM, C4, C3, and C5b Properdin and fibrinogen using FITC-conjugated rabbit anti-human antibodies MAC using murine monoclonal antibodies against a neoantigen of MAC Monocytes and/or granulocytes using murine anti- human CD11b/CD18 (OKM 1 ) and platelets using murine anti-human CD9

246 Xenotransplantation  Recent porcine trial The enzyme alpha1,3-galactosyltransferase(alpha1,3GT or GGTA1) synthesizes alpha 1,3-galatose epitopes, which are the major xenoantigens causing hyperacute rejection, and acute vascular rejection Reported earlier the targeted disruption of one allele of the alpha 1,3 GT gene in cloned pig. Based on a bacterial toxin was used to select for cells in which the second allele of gene was knocked out. Sequencing analysis demonstrated that knocked out of the second allele of the alpha 1,3 GT gene was caused by a T-to-G single point mutation at the second base of exon 9

247 Xenotransplantation  Prevention of Gal expression Breeding of pigs that do not express Gal, achieved by knocking out the gene for the enzyme, α1,3- galactosyltransferase( nuclear transfer/embryo transfer techniques) Reducing Gal expression on pig cells, by cleaving Gal from the underlying substrate, or replacing Gal with an alternative, innocuous oligosaccharide by a process that has been termed ‘competitive glycosylation’ to reduce cell-surface expression of several oligosaccharides

248 Xenotransplantation Pig organs, when transplanted to primates, are rapidly rejected because of the presence of recipient antibody that recognizes a carbohydrate epitope, galactose 1–3 galactose ( -Gal), which is present on glycoproteins and glycolipids on the pig endothelium. This rejection process, hyperacute rejection, can be routinely overcome by the removal of anti-Gal antibody or the inhibition of the complement cascade by the use of transgenic pigs that express human complement regulatory proteins. When hyperacute rejection is blocked, grafts are usually lost within a few days to weeks through a process called delayed xenograft rejection (DXR).

249 Xenotransplantation  Pig to primate model Hyperacute rejection occurs in the discordant pig-to- primate model using vascularized porcine organs, and in ABO-mismatched renal and cardiac allografts, due to the binding of preformed natural antibodies to cell surface carbohydrate structures, and subsequent complement activation. To date, control of the anti-Gal immune response in pig-to-primate xenotransplantation model has been attempted using phamachological immunosuppression, tolerance induction, or antibody removal using plasmapheresis, organ perfusion, or immunoadsorption.

250 Anti-gal Immune Response  Control methods To date, in pig-to-primate xenotransplant model has been attempted using pharmacological immunosuppression, tolerance induction, or antibody removal using plasmapheresis, organ perfusion, or immunoadsorption and transgenic animal. None of these methodologies has been consistently successful in the primate model, and all are associated with a high protocol-related morbidity, especially strategies that involve the use of immunoapheresis, which is particularly challenging due to the small size of the primate recipients

251 Xenotransplantation  Immunosuppression & medical therapy Splenectomy TPC(α-galactosyl-polyethylene glycol conjugate) Tacrolimus Sirolimus Corticosteroid Rituximab(anti-CD20) Lovenox(low molecular weight heparin) RATG Ganciclovir, valganciclovir, bactrim

252 Xenotransplantation  Complications of immunosuppression PCR for detection of cytomegalovirus & gamma herpesvirus Antirejection therapy Immunosuppressants, ganciclovir, ATG Infectious complicationof virus Posttransplant lymphoproliferative disease (Reactivation of of host lymphotropic virus)

253 Vascularized Xenograft  Rejection The capillary rejection of a vascularized xenograft coincides with the onset of graft dysfunction & these changes are limited to the endothelium Then the medial smooth muscle cells are subjected to immune effectors after the endothelial cells have been rejected There is also inflammation of the medial extracellular matrix which is probably elicited by immunogenicity of the xenogeneic extracellular matrix

254 Xenotransplantation  Reconsideration in concordant or discordant It is focused on the presence or absence of hyperacute rejection to classify species combination, at least three factors might be considered The presence or absence of natural antibodies directed against endothelial cells of a xenogeneic donor The function, or lack thereof, of complement regulatory proteins of recipient in the “environment” of the donor organ The relative effectiveness of cell membrane-associated complement inhibitory proteins of the donor against the complement system of the recipient

255 Xenotransplantation  Transgenes of CRP The beta-actin-CD59 transgene contains a chick beta- actin promoter that controls expression of a cDNA for human CD59. In this transgene, the hCD59 cDNA is embedded in a human alpha-globin gene expression cassette that provides splicing and polyadenylation signal sequences. The H2K-DAF transgene contains a cDNA for human DAF regulated by the mouse H2K promoter and H2K polyadenylation signal sequences Probes for DNA and RNA hybridizations were derived from the cDNA of each of the genes

256 Xenotransplantation  Complement-regulatory proteins(CRP) Normally, the vasculature is protected from autologous complement-mediated damage by several membrane- bound complement-regulatory proteins, such as CD59(protectin), decayaccelerating factor(CD55, DAF), or membrane cofactor protein(CD46) Each CRPs of species have been shown to be most effective at controlling the activation of homologous complement, and the complement of closely related species, but they are much less effective regulators of complement from more divergent species In the heart, hCD59(protectin) expression was detected on the endothelial cells and on the myocytes

257 Xenotransplantation  Complement-regulatory proteins There exist a family of complement-regulatory proteins which, under physiologic conditions, down-reguate the complement cascade to protect cells from damage by autologous complement CRPs are found in both soluble and membrane-bound forms. CRPs exhibit homologous restriction so that CRPs from one species are relatively inefficient at regulating complement from a distant species Expression of human membrane-bound complement- regulatory proteins could provide local protection from complement-mediated damage

258 Xenotransplantation  Transgenic animal A lack of effective complement regulation sensitizes the porcine organ to complement-mediated damage, and thereby, contributes to hyperacute rejection. Then the introduction of of human CRP function, even at lower levels, may significantly effect the humoral rejection of porcine xenograft Hypothesis, by enhancing the resistance of xenogenic vascular endothelium to complement-mediated damage through expression of human CRP gene products in transgenic animal

259 Xenotransplantation  Generation of transgenic pigs A 60 kh genomic construct encompassing the human CD46 gene, or hDAF, or CD59(protectin) was isolated from a P1 phase library and microinjected into the male pronuclei of fertilized porcine oocytes to generate transgenic pig Transgenic animals were identified by Southern blot analysis of DNA from tail biopsies

260 Xenotransplantation  Transgenic pigs A line of transgenic pigs that express the human complement-regulatory proteins human CD59 and human decay-accelerating factor This specificity is evident in transgenic organs in which low levels of CD59 and human decay-accelerating factor(CD55) expression significantly effect the humoral immune response that causes xenograft rejection Transgenic organs with high levels of human complement-regulatory protein expression will be sufficient to alleviate the humoral immunologic barriers

261 Xenotransplantation  Pig model The animal of choice currently considered to be the pig because of its size, physiologic compatibility, and breeding characteristics and the potential for genetic modification. The current limitation to clinical xenotransplantation is immunologic rejection. Achieving this goal is to define immunosuppressive regimens that control the xenograft immune response by using non–life-supporting heterotopic transplants.

262 Anti-α-Gal Antibodies  Measurement Isotype-specific determination of anti-α-Gal antibodies was performed by enzyme-linked immunosorbent assay Binding of xenoreactive IgM to cultured porcine aortic endothelial cells (anti-PAEC IgM) was determined by ELISA Antipig antural antibody levels were measured by ELISA using porcine platelet extracts (PPE) as antigen

263 Immunofluorescent Studies  Reagents Biopsy tissues were stained for IgG, IgM, C3, C4, C5b neoantigen, MAC, properdin, fibrinogen, polymorphonuclear neutrophils, and platelets Affinity-isolated, fluorescein isothiocyanate (FITC) goat anti-human antibodies against IgG, IgM, C4, C3, and C5b Properdin and fibrinogen using FITC-conjugated rabbit anti-human antibodies MAC using murine monoclonal antibodies against a neoantigen of MAC Monocytes and/or granulocytes using murine anti- human CD11b/CD18 (OKM 1 ) and platelets using murine anti-human CD9

264 Xenoreactive Antigen  Anti-xenoreactive antibody Xenoreactive IgG/IgM antibodies were detected in the sera of primates, including man, against the oligosaccharid residue [alpha]-Gal, which is present on pig tissue In primates the [alpha]-Gal-specific IgM-antibodies lead to complement activation and hyperacute rejection of pig xenografts within minutes to hours About 1% of the circulating immunoglobulines are specific for [alpha]-Gal in humans and their natal formation is presumably owing to exposure to gut flora expressing the [alpha]-Gal-epitope

265 Xenoreactive Antigen  Characteristics [Alpha]-Gal is an unique carbohydrate structure, which has been evolutionarily conserved in most mammals except humans, apes & Old World monkeys [Alpha]-Gal is widely expressed on the cell surface of mammals and highly expressed in capillaries as compared to large vessels, due to differential gal α 1, 3 galactosyltransferase expression among different endothelium Since humans lack the α- galactosyltransferase, they have natural anti-Gal antibodies and about 1% of B cell clones in humans produce natural anti-Gal

266 Anti-Gal Antibodies  Measurement Antibody levels were measured by enzyme-linked immunosorbent assay (ELISA) using HSA-[alpha]1,3 Gal as a specific antigen and human serum albumin (HSA) RIA/EIA 96-well plates were coated with 30 µg/ml of either HSA- [alpha]1,3 Gal or HSA alone, blocked with 0.1% Tween 20/1% HSA-phosphate-buffered saline (PBS), and then incubated with serial dilutions of serum at 4°C. After washing plates, wells were incubated with streptavidin- alkaline phosphatase-conjugated goat anti-human µ-chain, or streptavidin-alkaline phosphatase-conjugated goat anti-human [gamma]-chain, and developed with Sigma Fast pNPP tablets The absorbance at 405 nm was determined using a Molecular Dynamics Plate Reader and Softmax Pro software

267 Xenotransplantation  Pig to primate model Hyperacute rejection occurs in the discordant pig-to- primate model using vascularized porcine organs, and in ABO-mismatched renal and cardiac allografts, due to the binding of preformed natural antibodies to cell surface carbohydrate structures, and subsequent complement activation. To date, control of the anti-Gal immune response in pig-to-primate xenotransplantation model has been attempted using phamachological immunosuppression, tolerance induction, or antibody removal using plasmapheresis, organ perfusion, or immunoadsorption.

268 Xenotransplantation  Recent porcine trial Porcine human model Produce genetically engineered pigs that ---- Lack Gal epitope( humans have circulating antiGal Ab) ---- Express human dacay accelerating factor (hDAF) ( presence of hDAF protects against complement mediated hyperacute rejection Nuclear transfer to shorten span required to produce identical herds of genetically altered animals

269 Xenotransplantation  Recent porcine trial The enzyme alpha1,3-galactosyltransferase(alpha1,3GT or GGTA1) synthesizes alpha 1,3-galatose epitopes, which are the major xenoantigens causing hyperacute rejection, and acute vascular rejection Reported earlier the targeted disruption of one allele of the alpha 1,3 GT gene in cloned pig. Based on a bacterial toxin was used to select for cells in which the second allele of gene was knocked out. Sequencing analysis demonstrated that knocked out of the second allele of the alpha 1,3 GT gene was caused by a T-to-G single point mutation at the second base of exon 9

270 Xenotransplantation Pig organs, when transplanted to primates, are rapidly rejected because of the presence of recipient antibody that recognizes a carbohydrate epitope, galactose 1–3 galactose ( -Gal), which is present on glycoproteins and glycolipids on the pig endothelium. This rejection process, hyperacute rejection, can be routinely overcome by the removal of anti-Gal antibody or the inhibition of the complement cascade by the use of transgenic pigs that express human complement regulatory proteins. When hyperacute rejection is blocked, grafts are usually lost within a few days to weeks through a process called delayed xenograft rejection (DXR).

271 Antixenoreactive Response  Control methods To date, in pig-to-primate xenotransplant model has been attempted using pharmacological immunosuppression, tolerance induction, or antibody removal using plasmapheresis, organ perfusion, or immunoadsorption None of these methodologies has been consistently successful in the primate model, and all are associated with a high protocol-related morbidity, especially strategies that involve the use of immunoapheresis, which is particularly challenging due to the small size of the primate recipients

272 Xenotransplantation  Immunosuppression therapy Splenectomy TPC(α-galactosyl-polyethylene glycol conjugate) Tacrolimus Sirolimus Corticosteroid Rituximab(anti-CD20) Lovenox(low molecular weight heparin) RATG Ganciclovir, valganciclovir, bactrim

273 Xenotransplantation  Immunosuppression PCR for detection of cytomegalovirus & gamma herpesvirus Antirejection therapy Immunosuppressants, ganciclovir, ATG Infectious complicationof virus Posttransplant lymphoproliferative disease (Reactivation of of host lymphotropic virus)


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