Organ Preservation & Tissue-engineering

1. Organ Preservation & Tissue-engineering Seoul National University Hospital Department of Thoracic & Cardiovascular Surgery

2. Organ Preservation

3. Glutaraldehyde Fixation • Principles • Ultrastructural integrity is important for prevention of tissue calcification. • Immediate fixation with higher concentrations of GA at low temperature significantly preserves tissue integrity. • It may be postulated that higher concentrations of GA lead to a lower degree of calcification.

4. 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

5. 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.

6. 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

7. Glutaraldehyde Fixation • Crosslinking

8. 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.

9. Glutaraldehyde Fixation • Aspects of calcific degeneration * Excess aldol condensates in the tissue * Autolytic tissue damage * Changes of proteoglycan content of the tissue * Continual enzyme activity * Insufficiently suppressed immunogenicity

10. Glutaraldehyde Fixation • Action & adverse effects • Glutaraldehyde(GA) is currently the standard reagent for preservation andbiochemical fixation • It imparts intrinsic tissue stability (biodegradation resistance)and reduces the antigenicity of the material. • Recent reports have suggested a detrimental role of aldehyde-inducedintra- and intermolecular collagen cross-linkages in initiatingtissue mineralization • GA has beenimplicated in devitalization of the intrinsic connective tissuecells of the bioprosthesis, thus resulting in breakdown of transmembranecalcium regulation and hence contributing to cell-associatedcalcific deposits

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

12. Glutaraldehyde Fixation • Use as valve prostheses • As a biologic extracellular matrix scaffold, porcineheart valves for their well-known good hemodynamic behaviorand unlimited availability. • Porcine scaffolds are usually treated with glutaraldehyde toimprove mechanical properties and to limit the xenogeneic rejectionprocess. • Glutaraldehyde treatment profoundly modifiesthe extracellular matrix structure and makes it improper tosupport cell migration, recolonization, and the matrix-renewingprocess

13. 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

14. 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

15. 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

16. Conditioning of Heterografts • Biologic factors affecting durability • Diagramatic representation of different stages of method • for conditioning heterografts

17. 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

18. Glutaraldehyde Treatment • Action on tissues • Glutaraldehyde mechanism of action

19. 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

20. 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.

21. Tissue Valve Preparation • Principles • Ensure reproducibility, desired tissuebiomechanics, desired surface chemistry, matrix stability, andresistance to calcification • A variety of treatments have been used clinicallyas well as experimentally • They may be broken down into twobroad categories: modifications to glutaraldehyde processedtissue and nonglutaraldehyde processes.

22. Calcification of Bioprosthesis • Preventive methods(lipid) • Calciumphosphate crystals containing Na, Mg, and carbonatenucleate due to devitalization of the cells and thus inactivationof the calcium pump • Membrane-bound phospholipids have also been associated withcalcification nucleation due to alkaline phosphatase hydrolysis • Ethanol has been used to remove phospholipids and mitigatecalcification, yet phospholipids have also been removed withchloroform-methanol yielding • Lipid extraction can also be performedthrough tissue processing with detergent compounds such as sodiumdodecylsulfate.

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

24. Calcification of Bioprosthesis • Heat treatment • Heat may facilitate extractionand denaturation of the phospholipids and proteins involvedin the process of calcification • The tissues obtained at the slaughterhouse were immediatelyplaced in the 0.625% glutaraldehyde solution. • After 15 daysof fixation in this solution, submitted to heat treatment • Glass bottles containingtissues in glutaraldehyde solution were placed in an oven at50°C for 2 months with permanent agitation by a rotatormachine (3 rotations/minute), then the glutaraldehyde solution was replaced bya fresh solution.

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

26. Calcification Process • Hypothesis

27. 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.

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

29. 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

30. Prevention of Mineralization • Residual glutaraldehyde reduction • Reaction between epsilon amino groups of collagen lysine and aldehyde residues on the glutaraldehyde molecules results in the formation of a Schiff base (Amino acid neutralization) • Glutaraldehyde polymerizes, creating new covalent bonds with the bioprosthetic tissue, and subsequent degradation of polymeric glutaraldehyde cross-links leads to a cytotoxic reaction. • Improvement of spontaneous endothelialization as well as mitigation of mineralization has been achieved by post-fixation detoxification with the various amino acid solutions

31. 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

32. 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.

33. Bioprosthesis Calcification • Prevention • Inhibitors of hydroxyapatite formation BisphosphonatesTrivalent metal ions • Calcium diffusion inhibitor ( amino-oleic acid ) • Removal or modification of calcifiable material  SurfactantsEthanol Decellularization • Modification of glutaraldehyde fixation • Use of other tissue fixatives • Problems created by an exposed aortic wall

34. Tissue Engineering

35. Tissue Engineering • Introduction • Concept of tissue engineering was developed to alleviatethe shortage of donor organs. • Objective of tissue engineeringis to develop laboratory-grown tissue or organs to replace orsupport the function of defective or injured body parts. • Tissue engineering is an interdisciplinary approach that relieson the synergy of cell biology, materials engineering, & reconstructivesurgery to achieve its goal • Fundamental hypothesis underlying tissue engineering isthat dissociated healthy cells will reorganize into functionaltissue when given the proper structural support and signals

36. Tissue Engineering • Recent myocardial graft • 3-D contractile cardiac grafts using gelatin spongesand synthetic biodegradable polymers. • Formation of bioengineered cardiac grafts with3-D alginate scaffolds. • Use ofextracellular matrix (ECM) scaffolds. • 3-D heart tissue by gelling a mixture of cardiomyocytesand collagen. • Culturingcell sheets without scaffolds using a temperature-responsivepolymer. • Creating sheets of cardiomyocytes on a mesh consisting ofultrafine fibers.

37. 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.

38. 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

39. 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

40. 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

41. 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 ?

42. 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

43. 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 ?

44. 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.

45. Tissue-engineered Heart Valve • Cryopreservedhuman umbilical cord cells

46. 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.

47. Allograft Tissue Engineering • Immunogenicity • Allogrft tissue stimulates a profound cell-mediated immune responsewith diffuse T cell infiltrates and progressive failure of the allograftvalve has been attributed to this alloreactive immune response • The role of humoral response in allograft failure is less clear, recently, evidence has been accumulating that allografttissue used in congenital cardiac surgery also stimulates aprofound humoral response • As previouslymentioned, it is believed that the cellular elements are theantigenic stimulus for the alloreactive immune response, andthus decellularization has been proposed to reduce the antigenicityof these tissues.

48. Tissue Procurement • Processing • Hearts were transported on wet ice in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with polymyxin B. Warm ischemic time was less than 3 hours, and cold ischemic time didn't exceed 24 hours. • Tissue conduits were dissected from the heart and truncated immediately distal to the leaflets. They were then placed in RPMI 1640 supplemented with polymyxin B, cefoxitin, lincomycin, and vancomycin at 4°C for 24 ± 2 hours. • Representative 1 cm2 tissue sections were placed in phosphate buffered water and vigorously vortexed, and 8 mL was injected into anaerobic and aerobic bottles and analyzed for 14 days for bacterial or fungal growth.

49. Decellularization • Introduction • In an attempt to reduce the antigenic response, decellularizationprocesses have been introduced for cryopreserved tissue. • Experimental and clinical experience with this decellularizationprocess has been gained with porcine vena cava porcine tissue, porcine aortic and pulmonary valve conduits, ovinepulmonary valve conduits, and, subsequently, humanfemoral vein and human pulmonary valve conduits. • There has alsobeen experimental evidence that the decellularized matrix becomespopulated with functional recipient cells.

50. 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