2.20 Decellularized Scaffolds

2.20 Decellularized Scaffolds EM Srokowski, University of Toronto, Toronto, ON, Canada KA Woodhouse, University of Toronto, Toronto, ON, Canada and Queen's University, Kingston, ON, Canada r 2017 Elsevier Ltd. All rights reserved.

2.20.1 2.20.1.1 2.20.1.2 2.20.2 2.20.2.1 2.20.2.2 2.20.2.2.1 2.20.2.2.2 2.20.2.2.3 2.20.2.2.3.1 2.20.2.2.3.2 2.20.2.2.3.3 2.20.2.2.3.4 2.20.2.2.4 2.20.2.2.5 2.20.2.3 2.20.2.4 2.20.2.5 2.20.2.5.1 2.20.2.5.2 2.20.3 2.20.3.1 2.20.3.2 2.20.3.3 2.20.3.4 2.20.3.5 2.20.4 References

Introduction What is Decellularization Benefits and Challenges of Decellularization Decellularization Methodology Physical Methods Chemical Methods Solvent extraction Alkaline and acid treatments Detergent extraction Nonionic detergents Ionic detergents Cationic detergents Zwitterionic detergents Hypotonic and hypertonic treatment Enzymatic treatment Additional Reagents Additional Processing Important Considerations Characterization of decellularization Host response to decellularized scaffolds Origin of Decellularized Scaffolds and Their Applications Small Intestine Dermis Urinary Bladder Vascular and Cardiac Tissue Embryoid Body Matrix Concluding Remarks

Abbreviations a-Gal epitope Gala1-3Galb1-4GlcNAc-R Ab Antibody ACL Anterior cruciate ligament ADM Acellular dermal matrix anti-a-Gal Galactose-a-1,3-galactose antibody BAM Bladder acellular matrix BAMG Bladder acellular matrix graft bFGF Basic fibroblast growth factor C-ECM Cardiac extracellular matrix CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1propanesulfonate CS Chondroitin sulfate CS-A Chondroitin sulfate A CS-B Chondroitin sulfate B CTAB Cetyltriethylammonium bromide DNA Deoxyribonucleic acid DNase Deoxyribonuclease DSC Differential scanning calorimetry EBs Embryoid bodies ECM Extracellular matrix

452

453 453 453 454 456 456 456 457 457 457 458 458 458 458 459 459 459 461 461 462 463 463 464 465 466 467 467 467

EDTA Ethylenediaminetetraacetic acid ESCs Embryonic stem cells ETO Ethylene oxide GAGs Glycosaminoglycans HA Hyaluronan H&E Hematoxylin and Eosin HS Heparan sulfate Ig Immunoglobulins LS Lauroyl sarcosinate MGP Methyl green-pyronine MHC Major histocompatibility complex PAA Peracetic acid PDM Placental decellular matrix PES Ethanol peracetic acid solution PMSF Phenylmethylsulfonylfluoride RGD Amino acid residues arginine-glycine-asparagine (Arg-Gly-Asp) RNA Ribonucleic acid RNase Ribonuclease SB-10 Sulfobetaine-10 SB-16 Sulfobetaine-16

Comprehensive Biomaterials II, Volume 2

doi:10.1016/B978-0-08-100691-7.00055-0

Decellularized Scaffolds

SDS Sodium dodecyl sulfate SD Sodium deoxycholate (deoxycholic acid) SEM Scanning electron microscopy SIS Small intestinal submucosa TEM Transmission electron microscopy TGF-b1 Transforming growth factor beta 1

2.20.1

453

TnBP Tri(n-butyl)phosphate R X-100 Octyl phenol ethoxylate Triton UBM Urinary bladder matrix UBS Urinary bladder submucosa VEGF Vascular endothelial growth factor

Introduction

Organ demand is a major healthcare issue that is growing in magnitude, globally. Although organ donations are increasing, the number of patients waiting for a transplant is increasing more rapidly. In 2006, the world market for organ replacement therapy was estimated to be in excess of $350 billion and expected to surpass $500 billion within 20 years.1 An area of study that holds great potential to combat this ever-increasing crisis is tissue engineering. Tissue engineering applies the principles of cell transplantation, material science, and engineering to develop biological substitutes that can restore and maintain normal function.2 The pioneering work of Robert Langer, Joseph Vacanti, and colleagues introduced the concept of using a degradable three-dimensional (3D) scaffold as a temporary substitute for the extracellular matrix (ECM).3–6 The ECM is, by definition, nature's ideal biologic scaffold material.7 The composition and ultrastructure of the ECM consists of a complex combination of structural and functional proteins, glycosaminoglycans (GAGs), glycoproteins, and small molecules all arranged in a unique, tissue-specific 3D architecture.8 In addition to providing structural support, the ECM modulates cellular behavior by regulating the activity of growth factors, cytokines, matrixdegrading enzymes, and their inhibitors. More importantly, the ECM is in a state of dynamic equilibrium with its surrounding microenvironment.8 Consequently, the ECM facilitates the bidirectional flow of information between adjacent cells and the external environment, which is crucial in a number of biological processes such as embryonic development, wound repair, and hemostasis. Briefly, native ECM is composed primarily of collagen (collagen type I being the predominant type), providing the necessary mechanical strength to the scaffold. Elastin is another dominant structural protein found in the ECM that provides elasticity and resilience to connective tissues. Fibronectin, a glycoprotein, is also found in abundance within the ECM, in particular in the basement membrane, submucosal structures, and in interstitial tissues. Fibronectin is important in mediating cell–matrix communication, due to its rich content of Arg-Gly-Asp (RGD) subunits that are important for cell adhesion.8 Laminin, another adhesion protein found abundantly in the basement membrane of the ECM, plays an important role in early embryonic development as well as a critical role in the process of cell and tissue differentiation.8 In addition, the ECM contains various mixtures of GAGs such as chondroitin sulfate A (CS-A) and B (CS-B), heparin, heparan sulfate (HS), and hyaluronan (HA). The GAGs bind various growth factors and cytokines in the ECM, as well as promote water retention and contribute to the gel properties of the ECM.8,9 Moreover, although native ECM contains very small quantities of growth factors and cytokines, they are vital in modulating a variety of cellular activities (i.e., cell migration, proliferation, differentiation, and maturation).8,9 An important characteristic of native ECM that distinguishes it from the variety of other scaffolds used in tissue engineering applications is its diversity of structural and functional proteins. In general, the scaffold approach in tissue engineering falls under two categories: (1) the use of synthetic matrices; and (2) the use of natural matrices, which include decellularized matrices. The objective of this chapter is to provide an overview of the current knowledge surrounding decellularized matrices including the various methods used to decellularize tissues, the manufacturing effects of decellularization, and lastly the origin and applicability of decellularized matrices in a therapeutic tissue engineering context.

2.20.1.1 What is Decellularization Decellularization is the process of removing the allogeneic or xenogeneic cellular antigens from a tissue that would initiate an immune response while leaving behind an intact ECM comprising a mixture of structural and functional molecules. Decellularization results in a decellularized matrix also referred to as acellular or devitalized matrix in the literature. The particular terminology used to denote a decellularized matrix in this chapter has been kept consistent as that referred to the referenced publication. Decellularization strategies vary depending on the tissue being processed. In general, the manufacturing process involves a series or combination of physical, chemical, and enzymatic treatments that ideally removes all cellular and nuclear materials without adversely affecting the composition, organization, biological activity, and mechanical integrity of the native ECM tissue. The efficiency of decellularization is a factor of both the processing methodology and the nature of the tissue.10,11

2.20.1.2 Benefits and Challenges of Decellularization Decellularization of tissue has been widely shown to be an attractive method of obtaining scaffolds for tissue engineering. An alternative to decellularization is the use of synthetic scaffold materials. These materials are typically characterized by uniform and reproducible mechanical and material properties. However, synthetic materials can lack the bioinductive and constructive remodeling properties inherently characteristic of ECM-derived scaffolds.10,12–14

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Table 1 Comparison of the benefits and challenges of decellularization Benefits Minimizes immunological response upon implantation Allows for the use of xenogeneic materials Ability to conserve the mechanical integrity of native tissue Ability to retain the biochemical composition of native tissue Ability to retain the three-dimensional microarchitecture of native tissue Retains the bioinductive properties (cryptic peptides) to facilitate tissue remodeling Widely applicable and economical Long-term storage capability Potential for an off-the-shelf product Challenges Requires optimization of decellularization process for each tissue source Immunological response from incomplete cellular removal Decellularization efficiency dependent on tissue source, thickness, and architecture Bioactivity and structural integrity of tissue may be impaired during manufacturing process Difficulty in characterizing efficiency of decellularization Scaffold properties constrained by the tissue source characteristics Reliance on host cell availability and infiltration Rapid in vivo degradation before recellularization

The use of decellularized scaffolds for a tissue engineering approach provides a number of benefits, as listed in Table 1. First, removal of allogeneic or xenogeneic cellular antigens not only minimizes the source of immunogenicity on implantation, but also preserves the ECM components, which are highly conserved among species allowing the use of xenogeneic materials.15 Moreover, decellularized scaffolds can retain a more physiologically relevant composition and microenvironment to the native ECM tissue. For instance, the ultrastructure and 3D architecture of the tissue can be preserved throughout the decellularizing process, retaining defining structures such as laminin and collagen type IV of the basement membrane in urinary bladder matrix (UBM)16 and placental decellular matrix (PDM),17 which are crucial in modulating cellular behavior. Moreover, decellularization may preserve the alignment and organization of collagen fibers,10,18 as well as elastic fibers,14,19 that aid in retaining not only elasticity to the scaffold but also biological activity.20,21 Consequently, the functionality and mechanical stability of decellularized scaffolds can provide the closest match to that of native tissues.22 Decellularized scaffold may also function as resorbable, provisional matrices whose degradation products release cryptic peptides such as endostatin, angiostatin, and canstatin, which actively participate in long-term tissue remodeling.7,23 Lastly, decellularized scaffolds present a widely applicable yet economical option with the potential for an off-the-shelf product with long-term storage capabilities for clinical applications.8,24–29 Despite the numerous benefits decellularized scaffolds provide, some challenges still exist. First, the decellularization process needs to be optimized for each tissue source, as the ECM harvested from the tissue contains a unique arrangement and composition of structural and functional components specific to that particular tissue or organ.11 As a result, incomplete cellular removal may elicit immune reactions on implantation. In addition, tissue variability may exist in terms of tissue thickness or the age of the tissue, which further complicates the efficacy of decellularization.7,11,12,15,30 Likewise, depending on the methods and/or reagents used in the manufacturing process, the bioactivity and structural integrity of the scaffold may be impaired. Therefore, a systematic and perhaps lengthy approach may be needed to fine tune the decellularization strategy. Challenges in characterizing the decellularized scaffold also exist in terms of accurately identifying and quantifying both the bioactive components and residual decellularizing agents in the matrix.11,15 Moreover, the material properties of the decellularized scaffold are inherently constrained by the characteristics of the tissue source including shape, and mechanical behavior.10 Thus, additional processing such as crosslinking may need to be introduced to improve the versatility of the scaffold. Decellularized scaffolds, by definition, also rely on cellular infiltration by host cells whose availability in vivo may be limited or whose infiltration may be inhomogeneous.29,31 Lastly, decellularized scaffold can degrade quickly in vivo, however, before complete recellularization.23,25,32

2.20.2

Decellularization Methodology

In 1975, Meezan et al. described the first decellularization strategy for extracting cells from the ECM through a multistep chemical and enzymatic process in preparation of basement membranes from several tissues.33 In recent years, there have been a number of methods developed to manufacture biologic scaffolds derived from decellularized tissues and organs, as summarized in Table 2. The resulting decellularized ECM-derived scaffolds have been broadly applied to a multitude of applications including adipose,17

Decellularized Scaffolds

Table 2

455

Common decellularization methods and reagents

Method Physical Snap freezing & lyophilization

Mode of action

Effects on ECM

Examples of tissue/organ

References

Intracellular ice crystal formation resulting in permeabilization of cell membrane

ECM is disrupted or fractured during rapid freezing

Ligament; cardiac valves; pericardium; bone matrix; amniotic membrane; embryoid bodies; liver Heart valves leaflets; meniscus; ligament; nerve; embryonic chick knee; dermis; embryoid bodies SIS; UBM; UBS; aorta; tendon; meniscus

34–41

Heart valves; bladder; heart; placenta; umbilical veins

17,19,53–59

Repeated freeze–thaw

Mechanical force (delamination and pressure) Mechanical flow (agitation and perfusion) Chemical Solvent extraction (ethanol, xylene, butanol, acetone) Tri(n-butyl)phosphate

Alkaline and acidic treatments

Detergent extraction Nonionic detergents (Tritons X-100, Tweens 20, Tweens 80) Ionic detergents (SDS, sodium deoxycholate, Tritons X-200, lauroyl sarcosinate)

Cationic detergents (CTAB) Zwitterionic detergents (CHAPS, Empigen BB, SB-10, SB-16) Hypotonic and hypertonic solutions Enzymatic Trypsin

Endonucleases

Exonucleases

40,42–48

Pressure can burst cells and tissue layer removal eliminates cells Facilitates chemical exposure and removal of cellular debris; may lyse cells

Mechanical force can damage ECM components

Facilitates membrane lipid removal, specifically triglycerides and cholesterol Organic solvent that disrupts protein–protein interactions

Prolonged exposure may induce Blood vessels; heart collagen structural changes in valves; umbilical veins the ECM Variable outcome with cell Articular cartilage; tendon; removal and disruption to ligament ECM SIS; UBM; UBS; blood Potential oxidative damage to vessels; gallbladder; GAGs such as HA, as well as microscopic structural changes embryoid bodies to collagen

12,56,60,61

Variable outcome dependent on tissue type

Ligaments; tendons; cartilage; aortic valves; embryoid bodies

62,63,65,68–70

Variable outcome dependent Cartilage; ligament; on tissue type, detergent tendon; pericardium; concentration, and treatment meniscus; heart valves; duration; potential loss of blood vessels; bladder; GAG content, collagen skin; placenta structural change, and change to mechanical integrity Structural damage to ECM Nerve proteins Sufficient cell removal and Umbilical arteries; nerve minimal ECM disruption

17,33,53,62,63, 71–82

Cell lysis by osmotic shock

Effective at cell lysis but requires additional processing for removal of cellular debris

22,53,58,74,77, 79,81

Serine protease, cleaves proteins at the C-side of Arg and Lys residues; chelating agents (EDTA) typically used with trypsin Cleaves the interior phosphodiester bond of ribonucleotide and deoxyribonucleotide chains Cleaves the terminal phosphodiester bond of ribonucleotide and deoxyribonucleotide chains

Longer incubations may disrupt Heart valves; blood ECM structure and composition vessels; pericardium

12–14,19,70, 76,82

Difficult to remove from the tissue and could invoke an immune response

17,53,58,68,69,73, 74,77,80,82

Solubilizes cytoplasmic components of cells; disrupts nucleic acids

Disrupts lipid–lipid and lipid–protein interactions but leaves protein–protein interactions intact Solubilizes cytoplasmic and nuclear cellular membranes; tends to denature proteins

Disrupts cellular membrane and solubilizes cellular content Exhibits properties of both nonionic and ionic detergents

Aggressive conditions may harm ECM components

Bladder; blood vessels; aortic valves; meniscus; ligament

Heart valves; pericardium; bladder; placenta; embryoid bodies

Adapted and reprinted with permission from Gilbert, W.; Sellaro, L.; Badylak, F. Biomaterials 2006, 27, 3675–3683. © Elsevier.

16,49–52

62–65

50,60,66–68

83 27,83,84

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Decellularized Scaffolds

bladder,53,77,85–88 nerve,83,89–92 and cardiovascular27,57,69,73,93–100 tissues. The most robust and effective decellularization protocols include a combination of physical, chemical, and enzymatic treatments.11 In general, a typical decellularization protocol contains the following series of steps: (1) lysis of cell membranes by physical treatments or ionic solutions, (2) separation of cellular component from the ECM using enzymatic treatments, (3) solubilization of cytoplasmic and nuclear cellular components by detergent treatment, and finally (4) rinsing and removal of cellular remnants from the ECM. Each of these steps may be coupled with mechanical agitation, and repeated a number of times to increase the effectiveness. The effectiveness of decellularization and preservation of the native ECM can be evaluated at each of the steps by a variety of methods, as will be discussed. Nonetheless, it is important to acknowledge that tissues depending on their origin, composition, structure, and thickness may respond variably to a decellularization process. Decellularization strategies must therefore be optimized for each tissue by controlling the concentration and duration of each treatment, and subsequently analyzing their effects on the composition, architecture, as well as function of the remaining decellularized ECM.68,101

2.20.2.1 Physical Methods To facilitate the decellularization of tissue, physical methods such as lyophilization, repeated freeze-thawing, sonication, direct pressure, and agitation may be used. The premise for each method employed is to disrupt the cell's plasma membrane resulting in cell lysis and removal from the tissue. These methods provide a direct and rapid means of decellularization. However, these methods typically need to be used in conjugation with chemical extraction treatments to increase their effectiveness.40 Snap freezing followed by lyophilization has been utilized to decellularize a variety of tissues prior to therapeutic application, including ligaments,36,37,41 cardiac valves,34 pericardium,38,102 bone matrix,35 amniotic membrane,103 and more recently embryoid bodies.40 Rapid freezing of the tissue or matrices results in intracellular ice crystal formation, disrupting the cellular membrane and thereby cell lysis. Subsequent removal of water molecules by freeze-drying may also facilitate removal of cellular debris. Freeze–thaw cycling is another physical technique consisting of submerging the tissue in liquid nitrogen (snap freezing) followed by room temperature thawing in a buffered aqueous solution. Multiple tissues have been decellularized by freeze–thaw cycling including peripheral nerve grafts,43,92 meniscal tissue,46,47,79 embryonic chick knees,48 and human dermis.42 In both techniques, the rate of temperature change needs to be optimized for each tissue or matrix in order to prevent damage to ECM components.11 Application of direct pressure is another physical technique,11,16,49,51,52,104 but this technique is only effective for tissues that are not characterized by densely organized ECM (e.g., liver, lung); otherwise, the treatment may leave cellular debris behind thereby inducing potential immunogenic reactions.11,78 Recently, supercritical carbon dioxide containing a small amount of ethanol has been used as an extraction medium to decellularize porcine aortas.52 Physical separation or delamination of tissue layers is a common technique reported by the Badylak group16,50,105,106 in creation of the small intestinal submucosa (SIS)-ECM as well as the urinary bladder submucosa (UBS) and urinary bladder matrix (UBM)-ECM. Delamination has proven to be an effective physical technique in decellularization of the tissue without disruption to the native ECM structure and architecture.11,16,50,107,108 Mechanical agitation using a magnetic stir plate or an orbital shaker and the introduction of sonication are among other common physical techniques used in decellularization procedures.11 In many decellularization protocols, physical techniques are employed in conjugation with chemical treatments to assist in decellularizing the tissue. The latter strategies are largely based on passive diffusion, which require extensive washing steps to not only remove the cellular debris from the ECM, but also remove any residual chemical compounds. In addition, the optimal speed, volume of reagent, and length of agitation all affect the efficiency of decellularization, which in turn is dependent on the composition, architecture, and thickness of the tissue.11,56 More recently in the literature,17,54,56,57,59 the application of convective flow or perfusion has been commonly reported as an effective decellularization strategy, especially in thick tissues. For instance, Flynn et al. used a perfusion system to aid decellularization of human placenta,17 while Ott et al. decellularized whole rat hearts using a modified Langendorff apparatus.57

2.20.2.2 Chemical Methods A variety of chemical treatments can be used independently or in a multistep extraction process to decellularize tissues. As summarized in Table 2, the reagents used to decellularize different tissues can be generally categorized into solvents for extraction, alkaline or acidic detergents, hypotonic and hypertonic solutions, and enzymes.11 The choice of reagent(s) is largely dependent on the suitability to effectively treat the tissue source, in terms of conserving the native ECM's biochemical and structural properties and also simultaneously removing the undesirable cellular elements.68 It is important to recognize, however, that the extraction process may result in cell damage and death, which can consequently produce divergent secondary signals on implantation. For instance, capase found on the cell surface of apoptotic cells can degrade elastin, where the resulting peptides can signal through the elastin–laminin receptor in the presence of transforming growth factor b 1 (TGF-b1) to ultimately produce bone.109 Therefore, as with each decellularization treatment, care must be taken to optimize the extraction process best suited for the tissue of interest.

2.20.2.2.1

Solvent extraction

Solvent extraction through the use of reagents such as ethanol,12,56,60,61 xylene, and butanol12 as well as acetone56 has been reported to facilitate decellularization of blood vessels,12,60 heart valves,110 and umbilical veins.56 Predominantly used in

Decellularized Scaffolds

457

combination with other chemical treatments, this pretreatment has been reported to extract membrane-bound lipids such as phospholipids, triglycerides, and cholesterol while minimizing tissue calcification.12,61 In comparison to proteins, the chemistry of the phospholipid molecules, for example, allows for greater solubilization by organic solvents. However, disruptions to the ECM structural components and biomechanical behavior have been observed post solvent extraction treatment.12,61 Tri(n-butyl)phosphate (TnBP) is another commonly used organic solvent, reported to aid in the extraction of cells from blood derivatives as well as lipid-enveloped viruses,111,112 articular cartilage,63 tendon,62 and anterior cruciate ligament (ACL).64,65 Unlike other solvents, TnBP disrupts protein–protein interactions.11 To date, mixed outcomes on the effectiveness of cellular removal and disruption to the ECM with TnBP treatment have been reported. Decellularization of rat tail tendon with 1% TnBP for 48 h yielded complete removal of nuclear remnants and preserved the natural structure and mechanical properties of the tissue.62 On the other hand, treatment of articular cartilage with 2% TnBP had a minimal effect on tissue decellularization but exhibited a decrease in GAG content and change in mechanical integrity in comparison to native tissue.63 Similarly, decellularization of an ACL graft with a Triton–TnBP treatment showed equivalent levels of cellular extraction to that with Triton–sodium dodecyl sulfate (SDS) in the midsubstance of the graft; however, incomplete decellularization was observed at the femoral insertion.65 Moreover, ACL treatment with TnBP preserved both the ECM structure and mechanical behavior of the tissue better than other extraction techniques, although disruption to the GAG content and collagen structure was reported.64,65

2.20.2.2.2

Alkaline and acid treatments

Alkaline and acidic treatments have been used to decellularize tissues such as SIS, UBM, UBS,50,66 blood vessels,60 the cholecyst wall (gallbladder),67 and, more recently, mineralized human primary osteoblast-derived matrices.113 Alkaline extraction procedures use alkaline salts such as those of sodium or potassium as well as alkaline earth metals such as calcium.60 Acidic treatments may involve the use of acetic acid, peracetic acid (PAA), hydrochloric acid, and sulfuric acid.11 These treatments not only disrupt the cellular membrane but can also solubilize major cytoplasmic components of the cells (i.e., organelles), which aids in the removal of nucleic acids from the matrices.11 Goissis and colleagues60 devitalized small and large blood vessels from beagle dogs using a multistep process centered on an alkaline extraction process, and showed that the efficiency of cell removal is dependent on the segment and the type of blood vessel. Moreover, despite the harsh alkaline conditions used, no denaturation of the collagen matrix was found and the collagen and elastic fibers were preserved. On the other hand, Badylak et al.50,66 have decellularized a variety of porcine tissues including SIS, UBM, and UBS using PAA at concentrations of B0.10–0.15% (w/v).11 The PAA treatment has been reported to effectively decellularize thin intact ECM matrices11 while simultaneously disinfecting the matrices by penetrating into microorganisms and oxidizing microbial enzymes.114,115 Despite the oxidative nature of PAA, the treatment has been found to retain both structural and functional molecules of the native ECM such as collagen,16 sulfated GAGs (HA, heparin, HS, CS-A, and dermatan sulfate),9,116 and various growth factors (vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), TGF-b1).9,116–118 However, oxidative damage to HA in SIS after PAA treatment117 and microscopic changes to the structural integrity of collagen fibers are issues that have been raised in the literature.50,116,117

2.20.2.2.3

Detergent extraction

Detergents are water-soluble molecules that have the ability to incorporate themselves into the lipid bilayer of the cellular membrane. Consequently, they solubilize the lipid membrane by disrupting the lipid–lipid and lipid–protein interactions and encapsulate proteins within detergent micelles at certain concentrations.11,119 A variety of detergent extraction techniques have been utilized to decellularize tissues, which can range from the use of nonionic, ionic, cationic, or zwitterionic detergents.11,119 Differences in the size, charge, denaturing ability, and critical micelle concentration may determine the tissue penetration, membrane disruption, and pattern of protein–detergent interaction, all of which may ultimately influence the effect on the ECM.119 Regardless of the type of detergent used in the decellularization protocol, steps must be taken to efficiently remove residual detergent from the matrices to avoid pronounced immune responses.75,120 2.20.2.2.3.1 Nonionic detergents Nonionic detergents, such as octyl phenol ethoxylate (Tritons X-100), Tweens 20, and Tweens 80, are mild or nondenaturants capable of breaking lipid–lipid and protein–lipid interactions but leaving protein–protein interactions intact.11,119 Tritons X-100 is the most widely studied nonionic detergent used to decellularize a variety of tissues such as ligaments,65 tendons,62 cartilage,63 bladder,24,53 nerve,83 liver,39 aortic valves,69,70 blood vessels121 and embryoid bodies.122 However, the effectiveness of Tritons X-100 in terms of complete cellular and nuclear removal as well as preservation of ECM components is variable, depending on the nature of the tissue. For instance, similar cellularity and nuclear content to controls were observed after 1% Tritons X-100 treatment of carotid arteries121 and tendons62 while complete removal of cellular remnants from aortic valve leaflets has been observed with the same treatment.70,74 Mixed results on collagen density and structure, GAG content, and mechanical integrity of tissues after Tritons X-100 treatment have also been reported. For example, Tritons X-100 treatment of aortic valves showed a reduction in the collagen density,69,70 as well as loss of GAG content (especially CS) and loss of adhesion molecules, laminin, and fibronectin.70 Conversely, treatment of ACL with Tritons X-100 showed no effect on the collagen and GAG content, as well as on mechanical properties. As such, the effectiveness of using a mild, nonionic detergent to decellularize largely depends on both the type of tissue as well as the subsequent decellularization treatments used.

458

Decellularized Scaffolds

2.20.2.2.3.2 Ionic detergents Ionic detergents such as sodium dodecyl sulfate (SDS), sodium deoxycholate (SD), Tritons X-200, and more recently lauroyl sarcosinate (LS) are commonly used in decellularization protocols as they are effective in solubilizing both cytoplasmic and nuclear cellular membranes.11 In general, ionic detergents are denaturants. They disrupt the noncovalent bonds within proteins, causing them to lose their native conformation,119 and in terms of the ECM, potentially disrupt the integrity of collagen.56 In a number of extraction protocols, sufficient levels of cell removal from tissues such as cartilage,63 tendon,62 ligament,22 skin,72 and heart valves74,76 have been achieved through the use of the surfactant SDS. As an amphipathic molecule, SDS effectively binds to proteins via its hydrophobic domain, resulting in an increased negative charge that can lead to both swelling of the tissue and destabilization of the collagen triple helical domain.73,76 In the literature, there have been concerns about the use of SDS in extraction protocols because of its effects on collagen stability, potential toxicity, and retention within the matrix.62,64,76,123 However, recent reports suggest SDS to be an effective decellularization agent with minimal ECM disruption but favorable cellular repopulation that can, in part, be linked to detergent concentration, treatment duration, and the nature of the tissue.22,63,78,79,120,124 Gratzer et al.22 found that residual SDS in decellularized porcine ACL tissue did not influence the viability and repopulation of autologous ACL fibroblasts. However, treatment with 1% SDS for 48 h significantly reduced GAGs levels and caused structural changes to collagen while no significant change in mechanical behavior was noted in the decellularized ACL tissue. On the other hand, Lichtenberg et al.124 re-endothelialized decellularized ovine pulmonary valves post 0.5% SDS treatment, and not only maintained a confluent layer of endothelial cells under physiological pulmonary flow conditions, but also found preservation of the ECM structure and mechanical integrity of the decellularized scaffold to be comparable to that of the native tissue. Lastly, Elder et al.63 found that the duration of SDS treatment in decellularizing articular cartilage determined the level of biochemical and ultrastructural change in the tissue. For instance, treatment with 2% SDS for 1 or 2 h resulted in a 33% reduction in DNA content but maintained the biochemical (i.e., GAG and collagen content) and biomechanical properties of the tissues. In contrast, 2% SDS treatment for 6 or 8 h resulted in a greater reduction in DNA content, and significantly decreased both the GAG content and compressive properties of the tissue. SD is another ionic detergent that is very effective in removing cellular remnant, but tends to have even more significant impact on the ECM structure than SDS.11 The specific effect of SD on decellularized ECM is unclear to date11 as it is typically used in conjunction with other decellularizing agents.76,82 2.20.2.2.3.3 Cationic detergents In general, for decellularization, the class of cationic detergents is typically deemed to be that of the most cytotoxic as well as too strong detergents that cause structural proteins in the native ECM to denature.83 Cetyltriethylammonium bromide (CTAB) is a cationic detergent used as part of a detergent-based decellularization method for nerve tissue. Although CTAB was found to be effective at removing cellular components from the nerve tissue, it was unable to preserve the ECM structure.83 2.20.2.2.3.4 Zwitterionic detergents A variety of zwitterionic or amphoteric detergents have also been incorporated in decellularization methodologies, such as 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), as well as sulfobetaine-10 (SB-10) and -16 (SB-16). Zwitterionic detergents exhibit properties of both nonionic and ionic detergents, but have a greater tendency to denature proteins than nonionic detergents.11 In the literature, zwitterionic detergents are typically used in combination with an ionic detergent that allows for tighter packing of the detergent micelles.83 More specifically, the use of both a zwitterionic and an ionic detergent allows the neutrally charged zwitterionic molecules to intersperse between the ionic molecules, lowering the repulsion between head groups and thus, increasing the packing density of the micelles. A tighter packing density is presumed to result in smaller, more robust micelles that are able to better penetrate into the tissue and mediate the removal of more hydrophobic materials (i.e., cell membranes).83 Zwitterionic detergents have been used in decellularization strategies for nerves83 and umbilical arteries.27,84 In general, zwitterionic detergents are considered to be mild detergents as they yield sufficient cell removal and preserve the ECM to some extent.11,83

2.20.2.2.4

Hypotonic and hypertonic treatment

To enhance the action of detergents or as a measure on its own, osmotic shock may be incorporated into the decellularization strategy as a means to effectively lyse cells. Osmotic shock has been used in decellularization strategies for a number of tissues including bladder,53,58,77 blood vessels,81 placenta,17 aortic valves,74 meniscus,79 and ligament.22 Its introduction generally involves treatment with a hypotonic solution followed by treatment with a hypertonic solution. Osmotic lysis with a hypotonic solution causes the cells to swell and burst. Swelling of the cell, caused by the inward movement of water, may also enhance the uptake of detergents into the cellular membrane. The opposite effect occurs in a hypertonic solution where water moves outward from the cell causing cellular dehydration and shrinkage, which aids in cellular death and detachment from the matrix.119 Although this strategy is highly effective at lysing cells, additional processing is required to remove the resultant cellular debris from the tissue, especially residual nucleic acids. Therefore, an additional nuclease enzyme digestion step typically follows the osmotic shock treatment.11,119

Decellularized Scaffolds 2.20.2.2.5

459

Enzymatic treatment

Enzymatic treatments, involving proteases and nucleases, are incorporated into the decellularization strategy to aid in the removal of cellular proteins and nucleic remnants. Trypsin is one of the most commonly used proteolytic enzymes in decellularization methodologies, and is typically used in combination with the calcium chelating agent, ethylenediaminetetraacetic acid (EDTA).11,119 EDTA is used to inactivate intracellular proteases that are released by the trypsinized cells and are capable of degrading the ECM constituents. Trypsin is a serine protease that cleaves proteins at the carboxyl side of lysine and arginine residues except when followed by proline, as is the case with collagen. It is predominantly used in decellularization protocols to aid in the detachment of cells from the surrounding ECM proteins by disrupting protein interactions, resulting in a more open matrix that can facilitate the removal of cellular debris.11,119 Trypsin has been part of enzymatic treatments for decellularization of a number of tissues including heart valves,13,19,70,76 vascular grafts,12,14 and pericardium.82 Nonetheless, variable cell loss has been reported in the literature following trypsin treatment.13,19,76 Schenke-Layland and colleagues reported insufficient cell removal from pulmonary valves but preservation of ECM components (GAG, collagen, and elastin) following shorter trypsin-EDTA incubation times (5 and 8 h). At a longer incubation period (24 h), complete cellular removal was reported. However, the ECM biochemical composition (especially in terms of o-sulfated GAGs as well as acid and salt soluble collagens) and ECM integrity were negatively impacted in a time-dependent manner.19 On the other hand, complete cellular removal and well-conserved and arranged collagen and elastin fibers were found by Teebken et al. who used a multistep enzymatic extraction method with trypsinEDTA, deoxyribonuclease (DNase), and ribonuclease (RNase) to decellularize thoracic aortas.14 Therefore, the nature of the tissue as well as the duration of enzymatic digestion must be taken into consideration when incorporating trypsin-EDTA into a decellularization strategy.11,12,76 Nucleases such as endonucleases and exonucleases may also be incorporated in enzymatic treatments.11 DNases and RNases have been commonly used in decellularization methodologies in combination with detergent extraction treatments to aid in the removal of nuclear debris. Many of the nucleases used in decellularization protocols are extracted from bovine tissues running the risk for prion disease transmission.11 An alternative may be to use recombinant nucleases. As with protease treatment, the duration of nuclease treatment must also be optimized in order to effectively remove cellular antigens without harming the ECM composition and structure.68,122 As such, decellularization protocols using either proteases or nucleases must incorporate effective steps for the removal of residual enzymes in the tissue prior to in vivo implantation.

2.20.2.3 Additional Reagents Lysing of cellular membranes during decellularization may also release a number of cellular proteases into the environment, causing additional, yet unintentional damage to the ECM. Moreover, maintaining the tissue above 4 1C for any length of time after removal can result in tissue breakdown.109 In order to account for these disruptions, a number of lengthy decellularization processes are run in cooler conditions and include protease inhibitors such as phenylmethylsulfonylfluoride (PMSF), aprotonin, and leupeptin.11 In addition, decellularization may be conducted in buffered solutions of pH 7–8, as proteases function optimally in acidic conditions. Lastly, many decellularization protocols include antibiotic solutions such as those of penicillin, streptomycin, or amphotericin to prevent bacterial contamination.11 However, special attention must be taken to ensure that residual antibiotic does not remain in the decellular matrix prior to in vivo implantation; otherwise regulatory approval for clinical use will be difficult.11

2.20.2.4 Additional Processing Following physical, chemical, and/or enzymatic treatments, decellularized ECM scaffolds are typically disinfected and/or terminally sterilized before being used or stored. Decellularized scaffolds are commonly disinfected using oxidizing agents such as hydrogen peroxide, sodium peroxide, PAA, or an ethanol–peracetic acid solution (PES).25,50,114,116,125 However, long exposure periods of these disinfectants may cause oxidative damage to collagen fibers and GAGs, ultimately reducing the structural integrity of the matrix.26,50,116 Prior to in vivo implantation, decellularized scaffolds are also subjected to terminal sterilization usually by ethylene oxide (ETO) treatment, gamma irradiation, or electron beam irradiation.25,50,117,126 Several studies have shown that these sterilants can have detrimental effects on the mechanical behavior of the scaffolds. However, certain sterilants can cause more significant damage to the matrix than others. For instance, Freytes and colleagues found that in comparison to the ionizing radiation treatments, ETO treatment had the least detrimental effect on the mechanical properties of a lyophilized form of the UBM.25 Moreover, they also suggested that the overall effect of sterilization depends on the relative amounts, density, and distribution of the ECM constituents, as well as dosage of the sterilant.25 Decellularized ECM scaffolds may also take on a variety of different forms upon additional processing steps to not only improve their shelf life but also expand their clinical applicability, as recently reviewed elsewhere.10 For instance, decellularized scaffolds can be hydrated or dehydrated,127 comminuted into a powdered ECM form,128,129 or solubilized into a liquid or gel form.130,131 Table 3 summarizes some of the commercially available decellularized scaffolds that have been packaged and stored under various conditions, including hydrated (Pelvicol™, Bard), laminated and vacuum dried (Restore™, Depuy Orthopedics), and freeze-dried (Bards Dermal Allografts, Bard).7,10,127 Storage of processed ECM scaffolds in a hydrated state helps to maintain the native tissue architecture. However, continuous leaching and/or hydrolysis of structural (i.e., collagen) and nonstructural (i.e., growth factors) components may disrupt the functionality and shelf life of the scaffold.10,127 Dehydration of processed ECM

460

Decellularized Scaffolds

Table 3

Commercially available devices composed of naturally derived extracellular matrix

Product

Company

Material

Chemical modification

Form

Use

Acell Vets

Acell, Inc.

Natural

Powder

Treatment of canine arthritic hips

AlloDerms/Cymetras

LifeCell Corp.

Porcine urinary bladder Human dermis

Natural

Dry sheet/gel

AlloPatch HD™ Axis™ dermis Bards Dermal Allograft

MTF Sports Medicine Mentor Bard

Natural Natural Natural

Dry sheet Dry sheet Dry sheet

Hernia, abdominal wall repair and breast reconstruction, soft tissue defects Tendon augmentation Pelvis organ prolapse Pelvic floor reconstruction

CardioGRAFTs CuffPatch™ CryoValves SG

LifeNet Health Arthrotek CryoLife, Inc.

DurADAPT™

Synovis Life Technologies, Inc. Synovis Life Technologies, Inc. Cook SIS TEI Biosciences Bard Ethicon 360, Inc.

Dura-Guards Durasiss Durepairs FasLatas FlexHDs

Human fascia lata Human dermis Cadaveric human dermis Human cardiac tissue Porcine SIS Human pulmonary valve Equine pericardium

Natural Cross-linked Natural

Hydrated Hydrated

Cardiovascular repair Soft tissue reinforcement Cardiac valve repair

Cross-linked

Dry sheet

Repair dura matter

Bovine pericardium

Cross-linked

Hydrated

Spinal and cranial repair

Porcine SIS Fetal bovine skin Cadaveric fascia lata Human dermis

Natural Natural Natural

Dry sheet Dry sheet Dry sheet Hydrated

Human dermis Porcine pulmonary valve Urinary bladder matrix Porcine SIS

Natural Natural

Dry sheet

Repair dura matter Repair of cranial or spinal dura Urological repair Connective and soft tissue reinforcement Foot ulcers Pulmonary valve reconstruction

Natural Natural

Dry sheet or powder Dry sheet

Equine pericardium

Cross-linked

Dry sheet

Porcine dermis Bovine pericardium

Cross-linked Cross-linked

Hydrated Dry sheet

Partial and full thickness wounds & superficial and second degree burns Rotator cuff and Achilles tendon repair Pelvic floor reconstruction Pericardial and soft tissue repair

Graft Jackets MatrixP/MatrixP plus™ MatriStems

Wright Medical Tech AutoTissueGmbH

Oasiss

Healthpoint

OrthADAPT™

Synovis Life Technologies, Inc. Bard Synovis Life Technologies, Inc. Tissue Science Laboratories TEI Biosciences Edwards Lifesciences CryoLife, Inc. DePuy Ortho Tech Cook SIS LifeCell Corp.

Porcine dermis

Cross-linked

Hydrated

Soft connective tissue repair

Fetal bovine skin Porcine heart valve Bovine pericardium Porcine SIS Porcine SIS Porcine dermis

Cross-linked Cross-linked Natural Natural Natural Natural

Hydrated Dry sheet Hydrated Dry sheet Dry sheet Dry sheet

Wound management Valve reconstruction Soft tissue reconstruction Soft tissue treatment Urinary incontinence treatment Soft tissue reinforcement

TEI Biosciences Cook SIS

Fetal bovine skin Porcine SIS

Natural Natural

Dry sheet Dry sheet

Human fascia lata Fetal bovine skin Bovine pericardium

Natural Natural Cross-linked

Dry sheet Dry sheet Dry sheet

Bovine pericardium

Cross-linked

Hydrated

Soft tissue repair

Xelma™

Mentor TEI Biosciences Synovis Life Technologies, Inc. Synovis Life Technologies, Inc. Molnlycke

Soft tissue membrane repair Soft tissue repair and reinforcement Urethral sling Rotator cuff reconstruction Vascular reconstruction

Gel

Venous leg ulcer repair

Xenform™ Zimmer Collagen Patchs

TEI Biosciences Tissue Science Laboratories

Dry sheet Hydrated

Soft tissue reenforcement Rotator cuff repair

Pelvicols Peri-Guards Permacol™ PriMatrix™ Prima™ ProPatchs Restores Orthobiologic Stratasiss Strattice™ (formerly XenoDerm) SurgiMend™ Surgisiss Suspend™ TissueMends Vascu-Guards Veritass

ACell, Inc.

Amelogenins in propylene glycol alginate and water Fetal bovine skin Porcine dermis

Natural Cross-linked

Adapted and reprinted with permission from Badylak, S. F.; Freytes, D. O.; Gilbert, T. W. Acta Biomater. 2009, 5, 1–13. © Elsevier.

Soft tissue reconstruction

Decellularized Scaffolds

461

scaffolds, by lyophilization or by vacuum pressing, may minimize hydrolytic degradation during storage. However, dehydration may also result in scaffold ultrastructural changes that can consequently affect cellular attachment, infiltration, and remodeling.10,127 Powdered or gelated forms of decellularized scaffolds can potentially conform to any 3D shape as well as have the capability of being delivered to a site of interest by minimally invasive techniques.129,130 Regardless of the manufacturing process, the effect of storage duration and temperature on the scaffold's structural and functional properties must also be taken into consideration in order to have a therapeutically relevant material.26

2.20.2.5 Important Considerations 2.20.2.5.1

Characterization of decellularization

Decellularization strategies aim to remove all cellular debris while preserving the composition, mechanical integrity, and biological activity of the remaining ECM. Therefore, decellularization efficiency and reproducibility are characterized not only by the cellular constituents that are removed, but also by the desired components of the ECM that are retained. In this chapter, there is very little information comparing the effectiveness of different decellularization methods in treating a tissue of interest.82 Nonetheless, it is important to recognize that the decellularization process needs to be optimized for each harvested tissue to account for tissue variability. A number of different techniques can be used to characterize a decellularized scaffold that in general fall under the following categories: (a) histological, immunohistochemical, and biochemical assessment; (b) scaffold architecture assessment; (c) biomechanical assessment; (d) biofunctionality assessment; and (e) microbiological assessment, as summarized in Table 4. A simple yet effective initial test that is commonly performed to detect any residual nucleated cells or cell fragments throughout the decellularization process is the histological stain Hematoxylin and Eosin (H&E). A variety of other histological stains such as Masson's Trichrome, Movat's Pentachrome, and methyl green-pyronine (MGP) can be used to evaluate the intracellular components of the decellularized tissue, as depicted in Fig. 1 for a PDM.17 Immunohistochemical assessment for collagen, laminin, actin, and fibronectin can be used to gain a qualitative understanding of the biological composition, while a combination of biochemical assays such as for DNA content, GAG content, and/or collagen content can be used to quantify the biological Table 4

Summary of decellularization characterization techniques

A. Histological, Immunohistochemical, Biochemical Assessment Histological evaluation: Hematoxylin and Eosin (H&E) – detects residual nucleated cells or cell fragments Masson's Trichrome – characterizes collagen structure Van Gieson – differentiates between collagen and elastin Miller's Elastin stain – detects elastin Picrosirius Red stain – detects collagen Hoechst – detects DNA Methyl Green-Pyronine (MGP) – detects DNA and RNA Movat's Pentachrome – detects collagen, elastin, nuclei, GAGs Alizarin stain – detects calcification Gomori Trichrome stain – detects connective tissue Herovici stain – discriminates between collagen type I and III Immunohistochemical evaluation: Various markers – collagen type I, III, and IV; laminin; chondroitin sulfate; peripherin; van Willebrand factor; fibronectin; a-Gal; major histocompatibility complex (MHC) molecules Biochemical evaluation: DNA quantification – Hoechst; DAPI; propidium iodide; PicoGreen Total protein content – Bradford method; Bicinchoninic acid Collagen content – Hydroxyproline assessment; Sircol Assay Denatured collagen content – Hydroxyproline assessement post digestion GAG content – Dimethylene blue; Blyscan Assay Elastin content – Fastin Elastin Assay, Hydroxyproline assessment post digestion Triglyceride content: Triglyceride SL Assay B. Scaffold Ultrastructure Assessment SEM, TEM, Near-infrared multiphoton laser scanning microscopy DSC – collagen denaturation temperature, tissue shrinkage temperature C. Biomechanical Assessment Uniaxial tensile properties Biaxial tensile properties Viscoelastic behavior D. Biofunctionality Assessment In vivo/in vitro biocompatibility – cytotoxicity, cellular infiltration E. Microbiological Assessment Effectiveness of sterilization – detection of microorganism (bacterial/fungi) viability and growth

17,58,59,75,80,82

17,58,65,70,79

11,12,19,22,27,40,59,63,68,73–75,77,80,124

60,73,82,132

25,50,59,73,77,80,130

17,53,80,120,133–135 77,120

462

Decellularized Scaffolds

Untreated placenta

PDM

Hematoxylin and eosin Light pink: Collagen Dark pink/purple: Cells or cell fragments

Methyl green-pyronine Green: DNA Pink: RNA

Masson’s trichrome Green: Collagen Red/black: Cells or cell fragments

Movat’s pentachrome Yellow: Collagen Purple/black: Cells, cell fragments or elastin

Fig. 1 Histological assessment of placental decellular matrix (PDM) following the completion of decellularization, and untreated placenta. Hematoxylin and Eosin, Masson's trichrome, and Movat's pentachrome stainings indicate that there are no cells or cellular debris in the PDM following processing. The MGP staining shows that the PDM is free of residual RNA and DNA. Original magnification  50. Adapted and reprinted with permission from Flynn, L.; Semple, J. L.; Woodhouse, K. A. J. Biomed. Mater. Res. A 2006, 79, 359–369. © Wiley InterScience.

composition of the decellularized tissue in comparison to its native form. Assessment of both scaffold ultrastructure and biomechanical behavior provides insight into the structural integrity of the decellularized scaffold. Assessment of collagen denaturation temperature and tissue shrinkage temperature has also been routinely performed to determine the structural stability of collagen.60,73,74,82 The biofunctionality of the decellularized scaffold can be evaluated through in vivo and/or in vitro biocompatibility markers such as cytotoxicity and cellular infiltration.17,53,80,120,133 Lastly, prior to their use, decellularized scaffolds are commonly disinfected and/or terminally sterilized; therefore, the effectiveness of the sterilization procedure should be evaluated against microbial viability and growth.77,120

2.20.2.5.2

Host response to decellularized scaffolds

Decellularization aims to remove cellular antigens that are recognized as foreign by the host in response to the ECM-derived scaffold, which if unsuccessfully decellularized can ultimately result in an adverse inflammatory response or immunemediated rejection on implantation.7,10,136 With decellularized scaffolds, the host is presented with a different scenario from what is normally encountered with autogenous, allogenous, or xenogeneic whole organ grafts. In a typical tissue graft, immune recognition of cellular antigens occurs followed by the production of proinflammatory mediators and cytotoxicity, ultimately resulting in organ rejection.8,137 However, in the case of the decellular scaffold, only the constituent proteins of the ECM and their degradation products are present, eliciting a different inflammatory and immunologic response. Unlike whole organ transplantation, the mechanisms of the host immune response to allogeneic or xenogeneic decellular scaffolds are neither well studied nor well understood.137 In the literature, decellularized scaffolds have been noted to be immunologically tolerated as allografts for dural or dermis,138 heart valve,13,139 and nerve28,91 replacement. However, concerns over the use of decellular xenogeneic grafts have been raised.139–143 A comprehensive analysis of the immune response to biologic materials has been recently reviewed by Badylak and Gilbert; therefore, the reader is directed to this literature for a more thorough discussion.136

Decellularized Scaffolds

463

In brief, one of the major obstacles facing transplantation of living animal tissue to humans (i.e., xenotransplantation) is the interaction between the human natural galactose-a-1,3-galactose antibody (anti-a-Gal) and the a-Gal epitope (Gala1-3Galb14GlcNAc-R), the latter being found in high density as a cell-surface molecule in nonprimate mammals and New World monkeys.144,145 In humans, apes, and Old World monkeys, the a-Gal epitope is not expressed because of two frameshift mutations in the a1-3-galactosyl-transferase gene. Instead, they produce large amounts of anti-Gal antibodies (Ab) constituting approximately 1% of circulating immunoglobulins (Ig) such as IgG, IgM, and IgA, which consequently mediate hyperacute or delayed rejection of xenotransplants.140,145,146 A number of studies have attempted to identify and/or quantify the presence of the a-Gal epitope post decellularization including in porcine medial meniscus,79 porcine heart valves,143 porcine pericardium,141 and porcine SIS.147 In the decellularized scaffold of porcine pericardium, the decellularization process was able to completely remove the a-Gal epitope, some of which incorporated the use of a-galactosidase to aid in xenogeneic antigen removal.141 In contrast, porcine SIS has been found to contain small amounts of the a-Gal epitope,147 but its presence was unable to either activate the complement pathway in human plasma or influence its ability to serve as a bioscaffold for tissue remodeling following implantation.148 More specifically, implantation of SIS was found to elicit an immune response but the response was restricted to the Th-2 pathway and not to the Th1 pathway, which allowed acceptance and remodeling of the xenogeneic graft.149 Another area of concern with decellular xenogeneic grafts, in particular porcine grafts, is the presence of porcine DNA remnants. It has been suggested that these remnants may be the cause of ‘inflammatory reactions’ following implantation in orthopedic applications.150 However, a number of commercially available decellularized scaffolds such as Restores (porcine SIS), GraftJacket™ (human dermis), and TissueMend™ (bovine dermis) contain trace amounts of DNA remnants.150–152 In a recent study by Gilbert and colleagues, it was found that in all of the decellularized products investigated, small amounts of remnant DNA were present. However, the remaining DNA was present as small fragments (o300 bps) in most of the scaffolds with the exception of GraftJacket™ that contained full DNA strands.152 Even with the most rigorous decellularization protocol, complete cellular removal is very unlikely.152 Nonetheless, despite the presence of DNA remnants, the investigated commercially available decellularized scaffolds have been clinically effective for their intended applications.136,152

2.20.3

Origin of Decellularized Scaffolds and Their Applications

Decellularization of tissues has yielded a variety of naturally derived decellular matrices, many of which are commercially available to treat an assortment of conditions such as rotator cuff repair, bladder repair, and spinal and cranial repair, as listed in Table 3. Decellularized scaffolds have a rich history and this section will highlight the origin of some tissues used to produce decellularized matrices and their applicability to tissue engineering.

2.20.3.1 Small Intestine SIS is a resorbable xenogeneic bioscaffold derived from porcine small intestine, as shown in Fig. 2.105 SIS is prepared by mechanically removing the tunica mucosa, serosa, and tunica muscularis from the inner and outer surfaces of the intestine. The material is thoroughly rinsed in water, treated with peracetic acid, and then rinsed with buffered saline or water to yield a neutral pH. The resultant thin, translucent matrix (0.1 mm wall thickness) is composed primarily of 90% collagen (mostly collagen type I), fibronectin, laminin, proteoglycans (decorin), glycoproteins (biglycan and entactin), GAGs (HA, heparin, HS, CS-A, CS-C, dermatan sulfate), and growth factors (bFGF, VEGF, TGF-b).10,105 This milieu of structural and functional molecules establishes a unique 3D scaffold that may assist in cell migration, cell-to-cell interactions, as well as cell growth and differentiation during the regenerative process. The exact cellular mechanisms involved in remodeling following SIS implantation are not completely understood. However, the remodeling process has been strongly associated with angiogenesis, cell migration, and differentiation,

cm

1

2

3

4

5

Fig. 2 A cross section of intact small intestine with an attached piece of mesenteric adipose tissue (about 7 O'clock position) is shown on the right side of this figure. The tissue on the left is a longitudinal view of the SIS graft material after the superficial mucosa, the serosa and mesenteric connective tissue, and the muscularis externa have been removed. This material is very flexible and strong and is partially transparent when viewed against the light. Reprinted with permission from Badylak, S. F.; Lantz, G. C.; Coffey, A.; Geddes, L. A. J. Surg. Res. 1989, 47, 74–80. © Elsevier Ltd.

464

Decellularized Scaffolds

Table 5

Applications of SIS in tissue engineering

Application

Model

References

Vascular Cardiac Dura mater Abdominal wall Bladder Bowel Esophagus Tendon Ligament Cartilage (Meniscus) Dermal Bone

Canine, porcine Canine, porcine Rodent, canine Canine, rodent Rabbit, canine, rodent Canine Canine Canine, rabbit Canine Rabbit, canine Rodent Rodent

105,155–161 162–164 165,166 167,168 108,169–173 174 175 107,176 177 178–180 168 181,182

and deposition of extensive ECM.9 SIS grafts also have the ability to rapidly and completely degrade within a short term following implantation.32,153,154 The capacity of SIS to induce site-specific in vivo cellular repopulation and regeneration without in vitro cell seeding in a variety of applications has been well documented throughout the literature, as summarized in Table 5. Despite its xenogeneic nature, implanted SIS grafts are capable of surviving without signs of rejection in a number of animal systems including rat, mice, dog, and rabbit as well as in human patients.7,136,140,147 Early studies found SIS to function well as arterial and venous grafts with rapid replacement by native tissue.105,156,157 Despite SIS's significant potential as vascular grafts, challenges of endothelialization and long-term vascular patency rates (418 months) have been raised.105,156,183 SIS has also been extensively used in the genitourinary system. Kropp et al.108,171 found successful histological and functional regeneration of both rodent and canine bladders using SIS in a partial (40%) cystectomy model. For instance, at 15 months postoperative, histological analysis of the SIS augmented bladders showed significant regeneration of all three major layers of the bladder wall (urothelium, smooth muscle, and serosa) with no evidence of scar formation. In vitro contractility studies on SIS regenerated bladder strips demonstrated that the contractile activity of the augmented bladder tissue matched that of the native tissue. Similarly, in vitro tensile testing demonstrated no significant differences in the stress–strain properties of the graft tissue versus native bladder. The regenerated bladders also showed functional nerve regeneration and innervation comparable to native bladder.171,172 Nonetheless, recent SIS studies have been inconsistent with regard to bladder regeneration.184,185 For instance, significant morphological and histological differences between distal and proximal segments of SIS in bladder regeneration and calcification within the regeneration bladder have been observed.184,185 Larger SIS grafts in a subtotal cystectomy (90%) model were unable to induce the same quality and quantity of bladder regeneration as previously observed with a partial (40%) cystectomy model.186 Lastly, long-term bladder reconstitution with SIS augmented bladders has shown variable outcomes with regard to fibrosis, capacity, and compliance.187–189 Despite mixed results with certain applications of SIS grafts between various studies, a multitude of positive clinical outcomes have been reported. In addition, as of 2007, more than one million human patients have received an SIS graft to reconstruct a variety of tissues.7 To date, the reasons for the disparate results are unknown but may be related to patient selection, surgical technique, and/or lack of our understanding regarding optimal use of an inductive scaffold for reconstruction of certain tissues.7

2.20.3.2 Dermis Decellularized dermal tissue or acellular dermal matrix (ADM) can be derived from full or split-thickness skin from which the cellular components (keratinocytes, sweat glands, sebaceous glands, fibroblasts, vascular endothelium, and smooth muscle) are removed or extracted.135 Studies with an ADM, AlloDerms (LifeCell Corp.), processed from banked human cadaver skin have shown the material to be safe and efficacious in a variety of surgical procedures.190 These procedures include treatment of fullthickness burns,135,191 use as a substrate for oral resurfacing and periodontics,192 a substrate for dural replacement,190 a smallcaliber vessel substitute,193 as well as a soft tissue filler in plastic surgery.194,195 A porcine equivalent of AlloDerms, XenoDerm (LifeCell Corp.), has also shown efficacy as a viable dura substitute in a mini-pig model.138 In both cases, porcine and human dermis are decellularized by first removing the epidermis by incubating the skin in 1 M sodium chloride (NaCl) at 37 1C for 8 h. Dermal fibroblast and epithelial cells are then removed by incubating the material in 2% SD containing 10 mM EDTA at room temperature overnight. The dermis is then cryoprotected with a combination of 35% maltodextrin and 10 mM disodium-EDTA and packaged as a freeze-dried sheet until it is rehydrated at the time of surgery.138,190 This decellularization process has shown to reduce major histocompatability complex (MHC) Class I and Class II molecules, as well as preserve the normal architecture of the dermis.138,190,196 Decellularized dermis has been noted to be rich in collagen type I, type IV, and type VII as well as in elastin content, providing the graft with favorable elastic properties.183 Grafted acellular dermal matrix has also been shown to support fibroblast infiltration, neovasculation, and epithelialization in the absence of an inflammatory response.135

Decellularized Scaffolds

465

A number of other methods have been attempted to decellularize dermal tissue using a combination of extraction (i.e., Tritons X-100, SDS) and enzymatic (i.e., trypsin, dispase, DNase/RNase) treatments resulting in varying degrees of decellularization efficiency.72,197–200 For instance, Walter et al. prepared an ADM by two different methods using either dispase followed by Tritons X-100 treatment (dispase–Triton) or hypertonic NaCl solution followed by SDS (NaCl–SDS) treatment. Both methods completely removed the epidermis and most cellular components from the dermis while preserving the basic dermal architecture. However, the NaCl–SDS method retained a greater amount of cell-surface antigens as well as ECM components in comparison to the dispase–Triton method.200 Moreover, despite the clinical effectiveness of allogeneic decellularized dermis, challenges such as limited availability of cadaver skin, high cost of commercially available human ADM, and the risk of disease transmission (i.e., Cruetzfeldt–Jakob disease) exist.197,198 Lastly, variable outcomes have been reported in the literature regarding the histological and biocompatible differences between ADM xenografts and allografts,197,198,201 warranting further investigation into the underlying mechanisms.

2.20.3.3 Urinary Bladder The bladder is a soft tissue organ and despite its relative structural simplicity, it is one of the most distensible and strongest muscles in the body.202 Early work by Badylak et al. showed promising results for esophageal repair using UBS produced by mechanical delamination, resulting in only the tunica submucosa.175 More recently, decellularized bladder tissue, in the form of UBM, has been used for esophageal repair203,204 as well as for treatment as a cardiac patch,162,164,205,206 and thoracic wall repair,207 displaying great potential for tissue restoration. UBM is prepared by first trimming the residual external connective tissue and soaking the urothelial layer in saline before the remaining bladder tissue is mechanically delaminated to remove the epithelium, serosa, muscularis externa, and the tunica submucosa. The remaining basement membrane of the tunica mucosa and the subjacent tunica propria, collectively termed UBM, is disinfected in a PES solution followed by a saline solution rinse. Pioneering work by Tanagho208–211 and Atala,24,212,213 has driven the use of decellularized bladder tissue to a number of urological applications such as urethral, ureteral, bladder wall, and vaginal replacement, as shown in Fig. 3.214 Tanagho and colleagues208,215–219 decellularized bladder tissue, termed bladder acellular matrix graft (BAMG), according to an adapted methodology described by Meezan et al.33 The decellularization process consisted of a combination of hypotonic cell lysis, DNase digestion, and lipid membrane solubilization by an anionic detergent. This process has been applied to a variety of species’ bladder tissue such as hamster, rabbit, dog, rat, pig, and human bladder tissue, and subsequently evaluated for bladder regeneration in homologous or heterogenous bladder augmentation. Early studies showed promising results of morphologic and functional rat bladder regeneration following homologous BAMG augmentation cystoplasty.208,209,218 Findings by Sutherland et al. confirmed these results with a similar rat BAMG homologous in vivo model.220 However, concerns of bladder stone formation and clinical relevancy were raised over the rat model. Subsequently, homologous BAMG augmentation in a canine model was performed and showed that the augmented bladders were functionally compliant and closely resembled native bladder with only minimal scar formation.219 Work in our own and collaborator's laboratory53,55,58 decellularized porcine bladder tissue, termed bladder acellular matrix (BAM), using a multistep detergent-enzymatic extraction process originally established by Wilson et al.99 to decellularize smalldiameter blood vessels. Initial work demonstrated that BAM, used in an in vivo short-term (12 week) homologous model of bladder augmentation, promoted the ingrowth of urothelium, smooth muscle, and blood vessels. By 12 weeks postoperatively, the augmented bladders also exhibited capacity and compliance comparable to normal bladders.58 However, in a follow-up study evaluating BAM at a longer time frame (22 weeks), histological results were less encouraging indicating a fibroproliferative response

(a)

(b)

(c)

(d)

Fig. 3 Tissue engineering of the urethra using an acellular collagen matrix obtained and processed from porcine bladder submucosa. (a) Representative case of a patient with a bulbar stricture. (b) During the urethral repair surgery, strictured tissue is excised, preserving the urethral plate on the left side, and matrix is anastamosed to the urethral plate in an onlay fashion on the right. (c) Urethrogram 6 months after repair. (d) Cystoscopic view of urethra before surgery on the left side, and 4 months after repair on the right side. Reprinted with permission from Atala, A. Curr. Opin. Biotechnol. 2009, 20, 575–592. © Elsevier Ltd.

466

Decellularized Scaffolds

resulting in gross graft contracture and decrease in elasticity. As with many decellularized scaffolds, the mechanisms promoting regeneration with restoration of bladder tissue function have not been fully elucidated. A desire to understand the dynamic and reciprocal dialogue between the cells and their extracellular microenvironment has prompted studies to incorporate cell-based models and bioactive molecules as a means to potentially improve in vivo organ restoration following implantation.133,221–223

2.20.3.4 Vascular and Cardiac Tissue Decellularization of vascular tissue is an encouraging approach for treatment of cardiovascular disease, including coronary artery and peripheral vascular disease. A number of vascular tissues have been decellularized including umbilical arteries,27,56 carotid arteries,12,14,224 pulmonary arteries,75 and iliac arteries60,71 for the intended application as either small or large caliber vascular grafts. Although decellularized vascular grafts provide the ease of procurement and availability, concerns of incomplete cellular repopulation75 as well as susceptibility of the elastin-based scaffolds to vascular calcification225–227 have been raised. A number of studies report decellularization of cardiac valves including pulmonary valves13,76,81,124,132,228 and aortic valves.69,70,74,81,96,229–232 Cardiac valves from a wide variety of species have also been used, such as canine, porcine, ovine, and rodent, all for their intended organ-specific application. Challenges with these particular decellularized scaffolds include maintenance of hemodynamic function,233 control of early calcification,228 cellular repopulation, and matrix generation.132,234 Moreover, the importance of ensuring complete decellularization has been exemplified with the Synergraft™ (CryoLife, Inc.) decellularized porcine aortic valves. Although in vitro and in vivo experimental results with Synergraft™ were encouraging, early clinical performance in young children was not successful. Macroscopically and histologically, the explanted grafts showed fibrous overgrowth and a strong inflammatory response, initially attributed to a foreign-body-type reaction.235 In this follow-up study, not only was incomplete decellularization confirmed using the Synergraft™ method, but also detection of the a-Gal epitope suggested that it might have triggered hyperacute rejection.235 However, to what extent and quantity the presence of a-Gal epitope in heart valve bioprostheses triggers a host inflammatory response needs to be further investigated. Recently, as a means to meet the growing demand for a bioartificial heart for end-stage heart failure, Ott and colleagues decellularized whole rat hearts by retrograde coronary perfusion (Langendorff) using a SDS-Tritons X-100 extraction method to generate a biocompatible whole-heart scaffold with a perfusable vascular tree, patent valves, and a four-chamber geometry.57 Subsequently, the whole-heart scaffolds were recellularized with cardiac and endothelial cells and allowed to mature under physiologically relevant conditions in a bioreactor. By 28 days in perfused organ culture, whole heart constructs showed electric and contractile response as well as pump function equivalent to 2% of adult or 25% of 16-week fetal heart function. On a larger scale, Badylak and colleagues have recently decellularized in a reproducible and time-effective manner (o10 h) porcine whole hearts by pulsatile retrograde aortic perfusion to generate cardiac-ECM (C-ECM).59 The perfused decellularization process consists of an enzymatic (trypsin), nonionic detergent (Tritons X-100), ionic detergent (deoxycholic acid), and acid solution (PES) treatment with systematic hypotonic and hypertonic rinses to remove cellular debris, as depicted in Fig. 4. The resultant C-ECM preserved the composition and complex 3D architecture of the native heart. Moreover, C-ECM was found to be a suitable substrate for chicken embryonic cardiomyocyte attachment and maintenance of phenotype. Collectively, both investigations identify the significant potential this novel whole organ decellularization approach may bring to the field for tissue engineering. Not only is this technique potentially scalable to hearts of human size and complexity, but also adaptable to a wide range of other organs such as lung, liver, and kidney.

(a)

(c)

(b)

(d)

(e)

Fig. 4 Representative images of the gross appearance of intact porcine hearts subjected to decellularization by retrograde perfusion. (a) Before decellularization, (b) after 0.02% trypsin, (c) after 3% Tritons X-100, (d) after 4% sodium deoxycholate, and (e) after 0.1% peracetic acid/4% ethanol. Reprinted with permission from Wainwright, J. M.; Czajka, C. A.; Patel, U. B.; et al. Tissue Eng. C Methods 2009, 16 (3), 525–532. © Mary Ann Liebert, Inc.

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467

2.20.3.5 Embryoid Body Matrix New to the area of decellularization, ECM has recently been isolated from differentiating mouse embryonic stem cells (ESCs), termed embryoid bodies (EBs), as a means to recapitulate the dynamic stem cell niche.40,68,122 Recent evidence suggests that the spatiotemporal composition of biomolecular factors synthesized and released by ESCs in vivo plays an indispensible role of regulating stem cell behavior and thus, regenerative events.236–240 Studies by the McDevitt group have decellularized EBs in a scalable manner at different time-points along their differentiation pathway by two different strategies: (1) chemical treatment consisting of Tritons X-100 in combination with DNase68,122, and (2) physical disruption by either lyophilization or freeze–thaw cycling in combination with DNase treatment.40 For both strategies, decellularization efficiency was assessed on the basis of cell viability, DNA removal, ultrastructure assessment, and protein retention. Not only were the EBs successfully decellularized, but retention and distribution of adhesive ECM proteins (i.e., collagen type IV, laminin, fibronectin) and the sulfated GAG hyaluronan were found to vary as a function of differentiation time in the acellular EB matrices in a similar fashion as native EBs. Moreover, the acellular EB matrices permitted exogenous repopulation and attachment of fibroblast in vitro.40 Many of the existing decellularized scaffolds originate from adult tissue sources that have limited regenerative capacity. The concept of using decellular matrices from stem cells actively undergoing morphogenic differentiation provides a unique and inherently flexible route to deliver morphogenic factors, in particular morphogens and mitogens, to an injured or diseased adult tissue that can potentially mimic early stages of embryogenesis and promote functional regeneration.

2.20.4

Concluding Remarks

The area of decellularization has contributed significantly to the progression of where tissue engineering is today and perhaps more importantly, toward its future direction. In comparison to other tissue engineering approaches that use synthetic matrices or single purified ECM matrices (e.g., collagen, elastin, or fibrin-based matrices), decellularized scaffolds offer the advantage of a more physiologically relevant composition of ECM components that is organ and tissue specific. With an optimized decellularization process, decellularized scaffolds can preserve the intricate complexities of the spatial and temporal ECM microenvironment that plays an integral role in modulating cellular behavior such as migration, proliferation, and differentiation. Moreover, decellularization offers the possibility of using xenogeneic tissues under the condition that all cellular antigens have been effectively removed to minimize an adverse immunologic response. Despite the significant advancements made in the area of decellularization to date, questions remain unanswered.241 These include the following: Is there an optimal decellularization strategy for each tissue in the body? Is there an optimal method of storage/preservation so that an ‘off-the-shelf’ product is achievable? Is it necessary for recellularization of the bioscaffold prior to implantation? Although these questions exist, it is encouraging to realize from the results observed both experimentally and clinically from the vast number of different decellularized tissues and more recently embryonic stems cells and whole organs that our ultimate goal of functional tissue restoration is achievable.

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