Chapter II.6.16
Tissue Engineering with Decellularized Tissues
Introduction
Materials for the repair or reconstruction of injured, missing or weakened tissues can be composed of either synthetic or naturally occurring components. The optimal material for each application should be selected based upon criteria such as the structural and mechanical requirements of the intended application, the type of response expected from the adjacent host tissue, and the ability of the material to support normal tissue growth and function over the long-term. Unlike conventional surgical repair procedures, in which a biomaterial mesh simply needs to have sufficient strength to hold adjacent tissues together and avoid an adverse host response, tissue engineering and regenerative medicine approaches utilize “scaffold” materials to facilitate the restoration of the normal structure and function of the tissue or organ of interest. The present chapter will focus exclusively upon the use of decellularized tissues (i.e., extracellular matrix) as scaffold materials in tissue engineering and regenerative medicine applications.
Every tissue and organ is composed of cells and extracellular matrix (ECM). The ECM consists of the secreted products, both structural and functional, of the resident cells of each tissue and organ. The composition and ultrastructure of the ECM is determined by factors that influence the phenotype of its resident cells, including mechanical forces, biochemical milieu, oxygen requirements, pH, and inherent gene expression patterns. In turn, the ECM influences the attachment, migration, proliferation, and three-dimensional organization of cells, as part of a process of dynamic reciprocity (Bissell et al., 1982; Boudreau et al., 1995; Ingber, 1991). The ECM is essentially an “information highway” between its embedded cells and serves a critical function in tissue and organ homeostasis, as well as response to injury.
For these same reasons, multiple forms of allogeneic and xenogeneic ECM have been isolated, processed into application-appropriate configurations, and investigated as scaffolds for tissue engineering and regenerative medicine purposes in multiple body systems. Tissue engineering and regenerative medicine approaches utilizing ECM scaffolds have been successfully used in both pre-clinical and clinical studies. Examples of ECM scaffold sources, configurations, and tissues that have been reconstructed using ECM scaffolds are listed in Table II.6.16.1, and examples of commercially available ECM scaffold materials, their source tissue, and configuration are provided in Table II.6.16.2.
TABLE II.6.16.1 Partial List of ECM Scaffold Donor Sources, Configurations, and Applications
TABLE II.6.16.2 Partial List of Commercially Available Scaffold Materials Composed of Extracellular Matrix
The success of ECM scaffolds in tissue engineering and regenerative medicine applications may be attributed in large part to their ability to modulate the default mechanisms of tissue repair. In general, the response following implantation of an acellular non-chemically cross-linked ECM scaffold has been described as “constructive remodeling.” That is, ECM scaffolds are capable of inducing the formation of new tissue structures that are arranged in a spatially appropriate pattern for the tissue of interest. For example, ECM scaffolds have been shown to be capable of inducing the formation of functionally innervated muscular tissue in a model of abdominal wall reconstruction (Agrawal et al., 2009; Valentin et al., 2010). This is in direct contrast to the default mechanism of mammalian response to injury that involves inflammation and scarring.
The objective of this chapter is to provide a rationale for the use of ECM as a scaffold for tissue engineering and regenerative medicine applications, an overview of the methods used in the preparation of ECM materials, a description of the composition and structure of such scaffold materials, and the mechanisms by which these biologic materials function as inductive scaffolds for tissue reconstruction.
Rationale for the Decellularization of Tissues and Organs and the Use of Decellularized Tissues as Scaffolds in Tissue Engineering and Regenerative Medicine
Individual ECM components such as collagen I, laminin, and fibronectin have been isolated, purified, and processed for use in cell culture, as biomaterial coatings, and as three-dimensional scaffolds for tissue engineering and regenerative medicine applications (Dejana et al., 1987; Macarak and Howard, 1983; Glowacki and Mizuno, 2008). These approaches have typically been successful for such applications. However, an advantage of utilizing ECM materials that have been derived through the decellularization of intact tissues or organs as substrates or scaffolds for cell growth and differentiation is the presence of tissue-specific structural and functional molecules (and their inhibitors) in the same relative amounts that exist in nature, and in their native three-dimensional ultrastructure. The ECM is capable of presenting these factors efficiently to resident or migrating cell surface receptors, protecting growth factors from degradation, and modulating the synthesis of new ECM molecules (Entwistle et al., 1995; Bonewald, 1999; Kagami et al., 1998; Roberts et al., 1988; Freise et al., 2009). If one considers the ECM as a substrate for in vivo cell growth, migration, and differentiation, it is reasonable to think of the ECM as a temporary (i.e., degradable) controlled release vehicle for naturally derived functional molecules. Many groups have attempted to create ECM analogs using individual ECM components and/or synthetic materials (Causa et al., 2007; Alini et al., 2003; Koh et al., 2008); however, the diversity and complex structure of the molecules that make up the ECM predict the difficulty of creating such a scaffold in vitro. It is for these reasons that the isolation of ECM through the decellularization of tissues and organs is an effective method for the production of materials to be used in tissue engineering and regenerative medicine approaches to tissue reconstruction.
Methods of Decellularization
The tissues from which ECM scaffold materials are harvested, the species of origin, the decellularization method, and the methods by which the material is sterilized can vary widely. Antigenic epitopes associated with cell membranes and intracellular components of tissues and organs are the primary cause of the adverse immunologic response elicited by allogeneic and xenogeneic tissue transplants (Erdag and Morgan, 2004; Gock et al., 2004; Ross et al., 1993), while the molecules that constitute the extracellular matrix are generally conserved across species lines, and are well-tolerated even by xenogeneic recipients (Bernard MPC et al., 1983; Bernard MPM et al., 1983; Constantinou CDJ, 1991; Exposito JYDA et al., 1992). Therefore, a suitable ECM scaffold material must be effectively treated to remove cellular material or chemically cross-linked to mask the antigens prior to implantation. The most commonly used chemical cross-linking agents are gluteraldehyde, carbodiimide, genipin, and hexamethylene-diisocyanate (Bhrany et al., 2006; Billiar et al., 2001; Harper, 2001). Although a description of the specific effects of chemical cross-linking on ECM scaffold materials is not within the scope of this chapter, the choice to decellularize or chemically cross-link the material has important effects on the host tissue response, which will be discussed later in the chapter.
Although it seems logical that the decellularization process will by definition affect the structure and composition of the extracellular matrix, the ultimate goal of any decellularization protocol is to remove all cellular material without adversely affecting the biochemical composition, mechanical behavior, topographical ligand landscape, and eventual biologic activity of the remaining scaffold material (Crapo et al., 2011). Decellularization processes generally begin with exposure of the tissue to ionic solutions to disrupt the cell membrane, followed by separation of cellular components from the ECM using enzymatic treatments (e.g., trypsin or nucleases), solubilization of cytoplasmic and nuclear components using detergents (e.g., most commonly sodium dodecyl sulfate, sodium deoxycholate or Triton™ X-100), and ultimately removal of cellular debris from the ECM scaffold material. Alkaline and acidic solutions are also commonly used to remove nucleic acids from ECM scaffolds, and to disinfect the material prior to sterilization. All of these steps can be coupled with physical treatments (e.g., freezing, direct pressure, sonication, and agitation) to increase their effectiveness. Following decellularization, all residual chemicals must be removed by thorough rinsing to avoid an adverse host–tissue response to the decellularization agents.
Most commercially available biologic scaffold materials are manufactured by first processing the organ of interest into a sheet prior to decellularization. However, due to the density, mass, and three-dimensional architecture of many whole organs such as the heart, liver, and kidney, these approaches are not effective for removing cellular material from these tissues (Crapo et al., 2011). Recent reports have described methodology for decellularizing intact hearts by perfusion of the same reagents described above through the existing vascular network (Ott et al., 2008; Wainwright et al., 2010). Although additional work is still needed, these preliminary reports suggest that the native three-dimensional architecture and biochemical composition of the tissue can be largely preserved, thus providing a promising approach for complex organ engineering (Badylak et al., 2012).
Despite the goal of preserving the intact ECM after the decellularization process, each treatment has an effect on the tissue. Detergents have been shown to disrupt the collagen that is present within certain tissues, thereby decreasing the mechanical strength of the resulting ECM scaffold. The same detergent may, however, have no effect on the collagen present within another tissue (Cartmell and Dunn, 2004; Woods and Gratzer, 2005). This is likely due to the diversity of the types of collagen known to exist within tissues and organs, many of which serve tissue-specific functions (van der Rest and Garrone, 1991). Studies have also shown that removal of glycosaminoglycans (GAGs) from the scaffold can adversely affect scaffold remodeling and the viscoelastic behavior of the scaffold (Cartwright et al., 2006; Lovekamp et al., 2006). Therefore, decellularization methods require optimization for each tissue and organ to remove cellular material without compromising the structural and functional properties of the remaining ECM.
Despite efforts to fully decellularize tissues, most commonly used methods are insufficient to achieve complete decellularization, as most if not all ECM scaffold materials retain some amount of DNA (Derwin et al., 2006; Farhat et al., 2008; Gilbert et al., 2009). In addition, antigens such as the galactosyl alpha 1,3 galactose (i.e., gal-epitope) have been shown to be present in porcine ECM. Fortunately, the gal-epitope in ECM scaffolds does not activate complement or bind IgM antibody, presumably because of the small amount and widely scattered distribution of antigen (McPherson et al., 2000; Raeder et al., 2002). So, while the full removal of cellular content remains the goal of the decellularization process, it appears that there is a threshold below which the amount or integrity of the cellular material is insufficient to invoke immune-mediated rejection. The presence and effects of these molecules will be discussed more fully later in the chapter.
ECM Configuration
Following the decellularization of a tissue or organ, the resulting ECM may take on a variety of shapes and sizes which are dependant on the particular architecture of the decellularized organ of interest or the methods used in the decellularization process (Gilbert et al., 2006). As mentioned previously, many tissues and organs such as dermis, small intestine, and urinary bladder are typically processed into a sheet-like configuration prior to decellularization. The sheet form may be insufficient in its mechanical properties and/or three-dimensional morphology (i.e., shape and size) depending on the application of interest. Therefore, a number of methods have been utilized for the processing of ECM scaffolds into a variety of application-specific shapes and sizes. Intact ECM scaffolds have been molded and vacuum pressed into shapes that include tubes (Badylak et al., 2005), cones (Nieponice et al., 2006), and multi-laminate sheets, among others (Freytes et al., 2004). These scaffolds have been utilized in applications ranging from esophageal repair (tubular) (Badylak et al., 2005) to gastro-esophageal junction repair (cone-shaped) (Nieponice et al., 2006), and orthopedic applications (multi-laminate sheets) (Dejardin et al., 2001). ECM materials have also been comminuted to create a powder form of the scaffold, which is of interest for injectable and space-filling applications (Gilbert et al., 2005). A hydrogel form of ECM has also been produced via enzymatic degradation of whole ECM scaffolds (Freytes et al., 2008). The ability of ECM scaffolds to be formed into varied shapes and sizes further add to their utility as scaffolds for tissue engineering and regenerative medicine applications.
Composition of Extracellular Matrix
Most of the component molecules of the ECM are well-recognized and form a complex, tissue-specific meshwork of proteins, glycosaminoglycans, glycoproteins, and small molecules (Laurie et al., 1989; Baldwin, 1996; Martins-Green and Bissel, 1995). The logical division of the ECM into structural and functional molecules is not possible, because many of the component molecules have both structural and functional roles in health and disease. For example, both collagen and fibronectin, molecules that were once considered to exist purely for their “structural” properties, are now known to have a variety of “functional” moieties, with properties ranging from cell adhesion and motility to angiogenesis or inhibition of angiogenesis. These “bimodal” or multifunctional molecules provide a hint of the diverse cryptic peptide sequences that exist within certain parent molecules and which, in themselves, have biologic effects that significantly affect the ECM scaffold remodeling process (Agrawal et al., 2010, 2011a,b). This section will highlight a number of the molecules that are known to exist within many of the ECM scaffolds described in this chapter. However, it should be noted that the ECM of each tissue and organ is unique, and the exact composition depends largely on the resident cell population and the function of the organ of interest. Chapter I.2.7 also discusses many natural biomolecules and their use as biomaterials.
Collagen
There are more than 20 distinct types of collagen, each with a unique biologic function. These proteins account for nearly 90% of the dry weight of the ECM of most tissues and organs (van der Rest and Garrone, 1991). Type I collagen is the major structural protein present in tissues, and is found ubiquitously throughout the plant and animal kingdoms. Type I collagen is particularly abundant in ligamentous, tendinous, and connective tissue structures, and provides the strength necessary to accommodate the uniaxial and multiaxial mechanical loading to which these tissues are commonly subject. These same tissues provide a convenient source of collagen for many medical device applications. For example, bovine type I collagen is harvested from Achilles tendon, and is perhaps the most commonly used xenogeneic ECM component intended for therapeutic applications.
Other collagen types also exist in the ECM of most tissues, but typically in much lower quantities. These alternative collagen types provide distinct, tissue-specific, physical and mechanical properties to the ECM, while simultaneously acting as ligands that interact with the resident cell populations. For example, type III collagen is found within the submucosal tissue of selected organs such as the urinary bladder; a location in which tissue flexibility and compliance are required for appropriate function, as opposed to the more rigid properties required of a tendon or ligament supplied by type I collagen (Piez, 1984). Type IV collagen is present within the basement membrane of most vascular structures, and within tissues that contain an epithelial cell component (Piez, 1984; Barnard and Gathercole, 1991; Yurchenco et al., 1994). The ligand affinity of type IV collagen for endothelial cells is the reason for its use as a biocompatible coating for medical devices intended to have a blood interface. Type VI collagen is a relatively small molecule that serves as a connecting unit between glycosaminoglycans and larger structural proteins such as type I collagen, thus providing a gel-like consistency to the ECM (Yurchenco et al., 1994), and has recently been shown to sequester latent forms of matrix metalloproteinases (Freise et al., 2009). Type VII collagen is found within the basement membrane of the epidermis, and functions as an anchoring fibril to protect the overlying keratinocytes from sheer stresses (Yurchenco et al., 1994). In nature, collagen is intimately associated with glycosylated proteins, growth factors, and other structural proteins such as elastin and laminin to provide unique tissue properties (Yurchenco et al., 1994). Each of these types of collagen exists within many of the ECM scaffolds used in tissue engineering and regenerative medicine applications.
Fibronectin
Fibronectin, second only to collagen in quantity within the ECM of most tissues, was the first primarily “structural” molecule identified to also possess a functional motif. Fibronectin is a dimeric molecule of 250,000 MW subunits, and exists both in tissue and soluble isoforms and possesses ligands for adhesion of many cell types (McPherson and Badylak, 1998; Miyamoto et al., 1998; Schwarzbauer, 1991, 1999). The ECM of submucosal structures, basement membranes, and interstitial tissues all contain abundant fibronectin (McPherson and Badylak, 1998; Schwarzbauer, 1999). The cell-friendly characteristics of this protein have made it an attractive substrate for in vitro cell culture. Fibronectin has also been used as a coating for synthetic scaffold materials to promote host biocompatibility. Fibronectin is rich in the Arg-Gly-Asp (RGD) subunit, a tripeptide that is important in cell adhesion via the α5β1 integrin (Yurchenco et al., 1994), and is found at an early stage within the ECM of developing embryos. Fibronectin is critical for normal biologic development, especially the development of vascular structures. The importance of this molecule and its interactions with other matrix components cannot be overstated with regard to cell–matrix communication.
Laminin
Laminin is a complex adhesion protein found in the ECM, especially within the basement membrane (Schwarzbauer, 1999). This protein plays an important role in early embryonic development, and is perhaps the best studied of the ECM proteins found within embryonic bodies (Li et al., 2002). Laminin is a trimeric cross-linked polypeptide that exists in numerous forms dependent upon the particular mixture of peptide chains (e.g., α1, β1, γ1) (Timpl, 1996; Timpl and Brown, 1996). The prominent role of laminin in the formation and maintenance of vascular structures is particularly noteworthy when considering the ECM as a scaffold for tissue reconstruction (Ponce et al., 1999; Werb et al., 1999). The crucial role of the beta-1 integrin chain in mediating hematopoietic stem cell interactions with fibronectin and laminin is well-established (Ponce et al., 1999; Werb et al., 1999). Loss of the beta-1 integrin receptors in mice results in intrapartum mortality. This protein appears to be among the first and most critical ECM factors in the process of cell and tissue differentiation. The specific role of laminin in tissue reconstruction when ECM is used as a scaffold for tissue engineering and regenerative medicine applications is unclear; however, its importance in normal development suggests that this molecule is essential for self-assembly of cell populations, and for organized functional tissue development, as opposed to scar tissue formation.
Glycosaminoglycans
A mixture of glycosaminoglycans (GAGs) is present within native ECM, with the amount and relative distribution dependent upon the tissue location of the ECM in the host, the age of the host, and the microenvironment. These GAGs promote water retention, bind growth factors and cytokines, and contribute to the gel properties of the ECM. The heparin-binding properties of numerous cell surface receptors and of many growth factors (e.g., fibroblast growth factor family, vascular endothelial cell growth factor) make the heparin-rich GAGs important components of naturally occurring substrates for cell growth. The glycosaminoglycans present in ECM include chondroitin sulfates A and B, heparin, heparan sulfate, and hyaluronic acid (Entwistle et al., 1995; Hodde et al., 1996). Hyaluronic acid has been extensively investigated as a scaffold material for tissue reconstruction and as a carrier for selected cell populations in therapeutic tissue engineering applications (Chapter I.2.7). The concentration of hyaluronic acid within ECM is highest in fetal and newborn tissues (greater than 20% of total GAGs), and has been associated with desirable healing properties. The specific role, if any, of hyaluronic acid upon progenitor cell proliferation and differentiation during adult wound healing is unknown.
Growth Factors
A characteristic of ECM scaffolds that clearly distinguishes them from other scaffolds for tissue reconstruction is the diversity of the structural and functional proteins that contribute to its composition. The bioactive molecules that reside within the ECM and their unique spatial distribution patterns provide a reservoir of biologic signals. Although the quantity of cytokines and growth factors present within ECM is very small, these molecules act as potent modulators of cell behavior. The list of growth factors found within ECM is extensive, and includes the fibroblast growth factor (FGF) family, vascular endothelial cell growth factor (VEGF), stromal-derived growth factor (SDF-1), transforming growth factor beta (TGF-beta), keratinocyte growth factor (KGF), epithelial cell growth factor (EGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), and bone morphogenetic protein (BMP), among others (Bonewald, 1999; Kagami et al., 1998; Roberts et al., 1988). These factors exist in multiple isoforms, each with a unique biologic activity. Purified forms of growth factors have been investigated in recent years as therapeutic methods of encouraging blood vessel formation (e.g., VEGF), stimulating deposition of granulation tissue (PDGF), and bone (BMP), and encouraging epithelialization of wounds (KGF). However, this therapeutic approach has been disappointing, because of the difficulty in determining optimal dose and methods of delivery, the ability to sustain and localize the growth factor release at the desired site, and the inability to turn the factor “on” and “off” as needed during the course of tissue repair. An important function of the ECM is its role as a reservoir for latent forms of many growth factors and its ability to release these factors during the process of in vivo degradation (Freise et al., 2009).
Mechanisms by Which ECM Scaffolds Function as Inductive Templates for Tissue Reconstruction
The mechanisms by which decellularized tissue (i.e., the ECM remaining following decellularization) scaffolds support a constructive remodeling process and the formation of new functional tissue when appropriately utilized in tissue engineering and regenerative medicine applications are not fully-understood. However, several biologically important events have been clearly associated with positive outcomes. Among these events are: (1) a robust innate immune response consisting of neutrophils at early time points, changing to a predominantly mononuclear cell/macrophage cell population during the 2–3 week period following implantation (Chapter II.2.2); (2) rapid and complete scaffold degradation and the release of bioactive matricryptic peptides from parent molecules within the ECM; (3) abundant angiogenesis which accompanies the deposition of new ECM by the cells that have infiltrated the scaffold; and (4) spatial and temporal remodeling which is clearly respondent to mechanical forces. Each of these events will be discussed separately, but it should be understood that they are occurring simultaneously and are interdependent upon each other for a constructive remodeling outcome following the implantation of an ECM scaffold.
Host Cell and Immune Responses to Implanted ECM Scaffolds
The mechanisms of the host cellular and humoral response to whole organ transplantation are reasonably well-understood. Xenogeneic and allogeneic cellular antigens are recognized by the host, elicit immune activation, and cause the production of pro-inflammatory mediators with downstream cytotoxicity and transplant tissue rejection. The mechanisms of the host immune response to acellular scaffolds derived from ECM, either allogeneic or xenogeneic, are neither as well-studied nor as well-understood as whole organ and tissue transplantation. The preparation of ECM scaffolds for tissue engineering and regenerative medicine applications involves the decellularization of the tissue or organ from which the ECM is to be harvested (Gilbert et al., 2006). The removal of the cellular component produces a different type of “tissue graft” than is typically presented with autogeneic, allogeneic or xenogeneic whole organ grafts. An ECM scaffold consists primarily of the ECM constituent molecules, many of which have been found to be conserved across species (van der Rest and Garrone, 1991), thus mitigating many adverse components of the host immune response (Allman et al., 2001).
Potential Immune Activating Molecules within ECM Scaffolds
Following the removal of resident cells from the tissue of interest, the majority of the components of the remaining ECM scaffold are conserved across species and are, therefore, largely non-immunogenic. However, many ECM scaffolds have been shown to contain a number of components that are thought to induce adverse host immune- and/or rejection-type responses when present in large quantities. These components include the α-Gal epitope and DNA. The α-Gal epitope is known to cause hyperacute rejection of organ transplants (Collins et al., 1994; Cooper et al., 1993; Galili et al., 1985; Oriol et al., 1993). However, studies of α-Gal-positive ECM scaffold implantation have not shown adverse responses that can be attributed to the α-Gal epitope (Raeder et al., 2002; Daly et al., 2009). A recent study investigated the effects of the presence of the α-Gal epitope upon the remodeling of ECM scaffolds in a non-human primate model (Daly et al., 2009). The study compared the host response to ECM derived from allogeneic, xenogeneic porcine, and xenogeneic α-Gal−/− porcine sources. The results of the study showed that although those animals implanted with an ECM scaffold containing the α-Gal epitope exhibited an increase in serum anti-Gal antibodies, there were no adverse effects of the α-Gal epitope upon the remodeling response. Several studies have shown the presence of DNA fragments remaining within ECM scaffolds following the decellularization and sterilization processes (Roberts et al., 1988; Derwin et al., 2006; Zheng et al., 2005). A recent study examined the presence of DNA within a number of commercially available ECM scaffolds (Gilbert et al., 2008), some of which are listed in Table II.6.16.2. The results of the study showed that, although all of the products tested contained small amounts of DNA, the remnants generally consisted of fragments of less than 300 bp. Despite the presence of small amounts of both the α-Gal epitope and DNA within ECM scaffolds, adverse clinical effects have not been observed. This is likely due to the minute amounts of these components present, and the rapid degradation of the ECM scaffold. It has been shown that the presence of large amounts of cellular material within an implanted ECM scaffold lead to scar tissue formation, as opposed to the modulation of the host response towards a constructive remodeling outcome (Brown et al., 2008). Therefore, it is probable that there is a threshold amount of these components required to induce adverse effects upon the remodeling response.
Innate Immune Response to ECM Scaffolds
In general, innate immune cells (neutrophils and macrophages) are the first cells to encounter and respond to implanted biomaterials (also see Chapter II.2.2). The immediate cellular response observed following the implantation of an ECM scaffold consists almost exclusively of neutrophils, as one might expect, but there is also a significant mononuclear cell component. In the absence of large amounts of cellular debris within the scaffold, chemical cross-linking or an excess of contaminants such as endotoxin, the neutrophil infiltrate diminishes almost entirely within 72 hours and is replaced by a mononuclear cell population. This type of response, characterized by a large infiltration of innate immune cells, has been conventionally interpreted as either acute or chronic inflammation with associated negative implications. However, the presence of these cells, especially mononuclear macrophages, has been shown to be essential to the formation of the type of constructive remodeling response that has been observed following the implantation of ECM scaffolds (Brown et al., 2008, 2012; Valentin et al., 2006, 2009; Badylak et al., 2008).
A histologically similar population of neutrophils and macrophages is observed following the implantation of ECM scaffolds which either have or have not been processed using chemical cross-linking agents such as glutaraldehyde or carbodiimide; however, the tissue remodeling outcome observed following the implantation of chemically cross-linked ECM scaffolds is distinctly different than that observed with the use of non-cross-linked scaffolds (Valentin et al., 2006). The host tissue response typically observed following implantation of an acellular ECM scaffold that has not been chemically cross-linked is characterized by a dense infiltration of neutrophils at early time-points, changing to primarily mononuclear cells thereafter. This infiltrate of innate immune cells is accompanied by rapid degradation of the ECM scaffold, and replacement with organized, site-specific, functional host tissue (Valentin et al., 2006; Badylak et al., 2002; Gilbert et al., 2007c). If the scaffold has been processed using chemical cross-linking agents such as glutaraldehyde or carbodiimide, the host response is characterized by a similar presence of a large number of neutrophils and macrophages, but results in a more typical pro-inflammatory response consisting of dense fibrous tissue encapsulation and the prolonged presence of a multinucleate cell population (Valentin et al., 2006). Although histologically similar populations of neutrophils and macrophages are present in the host response to either scaffold type, studies have linked the differences observed in remodeling outcomes, in part, to differences in the phenotype of the host innate immune cells which participate in the host response to implanted ECM scaffolds (Brown et al., 2008; Badylak et al., 2008).
Mononuclear macrophages are plastic innate immune cells that are capable of changing their phenotype in response to local stimuli during the process of wound healing. The macrophages participating in the host response following implantation of a biomaterial are exposed to multiple stimuli, including cytokines and effector molecules secreted by cells (including other macrophages) participating in the host response, microbial agents, epitopes associated with the implanted biomaterial, and the degradation products (if any) of the biomaterial, among others. Recently, macrophages have been characterized by differential receptor expression, cytokine, and effector molecule production, and function as having either an M1 or M2 phenotype (Mantovani et al., 2004; Mills et al., 2000). M1 or classically activated macrophages, produce pro-inflammatory cytokines and effector molecules, possess the ability to efficiently present antigen, and are inducer and effector cells in Th1-type inflammatory responses (Gordon and Taylor, 2005; Mosser, 2003; Verreck et al., 2006; Mantovani et al., 2005). M2 or alternatively activated macrophages, are generally characterized by minimal production of pro-inflammatory cytokines, their involvement in Th2-type responses, and their ability to facilitate tissue repair and regeneration. The overall M1/M2 profile of the host macrophage response to an implanted material will fall somewhere along a continuum between the M1 and M2 extremes (Mills et al., 2000). It should be noted that M1 and M2 macrophages are indistinguishable by the routine histologic methods that are generally used to evaluate the host response to a biomaterial. A full description of the activating signals, characteristic surface markers, gene expression, effector molecule production, and biologic activity associated with M1 and M2 macrophages is beyond the scope of this chapter; however, this subject has been reviewed extensively elsewhere (Martinez et al., 2008).
Recent studies of the M1/M2 profile of the macrophages responding to implanted ECM scaffolds have shown that acellular, non-cross-linked ECM scaffolds elicit an enhanced M2-type macrophage response, and result in constructive tissue remodeling (Brown et al., 2008, 2012; Badylak et al., 2008). Chemically cross-linked ECM scaffolds, however, elicit a predominantly M1-type macrophage response and result in a more typical foreign-body-type of response that includes the deposition of dense collagenous connective tissue, and a lack of constructive remodeling. Autograft controls also exhibited a predominance of the M1 phenotype, and resulted in scarring. The exact mechanisms by which acellular non-cross-linked ECM scaffolds are capable of modulating the default host macrophage response are, as of yet, unknown. However, it is increasingly clear that the M1/M2 polarization profile of the macrophages that participate in the host response to ECM scaffolds is related to the downstream outcome associated with their implantation (Brown et al., 2012). Further, characterization and control of the M1/M2 phenotype may provide a tool by which a constructive and functional tissue remodeling outcome can be predicted and/or promoted.
T-Cell-Mediated Immune Response to ECM Scaffolds
In addition to eliciting a robust, but friendly, host innate immune response, acellular non-cross-linked ECM scaffolds have consistently been shown to evoke a Th2-type T-cell response (Allman et al., 2001, 2002). The Th2 response is generally associated with transplant acceptance. One study utilized a mouse model of subcutaneous implantation to examine the T-cell response to xenogeneic muscle tissue, syngeneic muscle tissue, and an acellular ECM scaffold (Allman et al., 2001). Results showed that the xenogeneic tissue implant was associated with a response consistent with rejection. That is, the xenogeneic muscle implant showed signs of necrosis, granuloma formation, and encapsulation. The syngeneic tissue and the ECM scaffold elicited an acute inflammatory response that resolved with time, and resulted in an organized tissue morphology at the remodeling site. Tissue cytokine analysis revealed that the ECM group elicited expression of IL-4, and suppressed the expression of IFN-γ compared to the xenogeneic tissue implants. The ECM group elicited the production of an ECM-specific antibody response; however, it was restricted to the IgG1 isotype. Reimplantation of the mice with another ECM scaffold led to a secondary anti-ECM antibody response that was also restricted to the IgG1 isotype, and there was no evidence of the formation of a Th1-type response. Further investigation confirmed that the observed responses were in fact T-cell-dependent. Finally, it has been shown that, while both T- and B-cells respond to ECM scaffolds, they are not required for acceptance or constructive remodeling of an ECM implant (Allman et al., 2001). This further indicates the importance of the host innate immune response in driving/determining the downstream remodeling outcome following implantation of an ECM scaffold.
Degradation of ECM Scaffolds
ECM scaffolds are rapidly degraded in vivo. A recent study showed that 10 layer 14C-labeled ECM scaffolds were 60% degraded at 30 days post-implantation and 100% at 90 days post-surgery in a model of canine Achilles tendon repair (Gilbert et al., 2007c). During this period, the scaffold was populated and degraded by host cells, and resulted in the formation of site-specific functional host tissue. The major mechanism of excretion of the degraded scaffold was found to be via hematogenous circulation and elimination by the kidneys, urine, and exhaled CO2 (Record et al., 2001). The mechanisms of in vivo degradation of ECM scaffolds are complex, and include both cellular and enzymatic pathways. The process is mediated by inflammatory cells, such as macrophages, which produce oxidants as well as proteolytic enzymes that aid in the degradation of the matrix. Another study utilizing 14C-labeled ECM scaffolds showed that peripheral blood monocytes are required for the early and rapid degradation of both ECM scaffolds, and that cross-linked ECM scaffolds are resistant to macrophage-mediated degradation (Valentin et al., 2009).
ECM scaffolds have also been degraded in vitro by chemical and physical methods. Recent findings suggest that the degradation products of ECM scaffolds are bioactive (Brennan et al., 2006, 2008; Haviv et al., 2005; Li et al., 2004; Reing et al., 2009; Sarikaya et al., 2002). Studies have shown antimicrobial activity associated with the degradation products of ECM scaffolds; however, in the absence of degradation, antimicrobial activity was not seen, suggesting that some of the bioactive properties of the ECM are derived from its degradation products, rather than from whole molecules present in the ECM (Holtom et al., 2004). Degradation products of ECM scaffolds have also been shown to be chemoattractants for progenitor and non-progenitor cell populations (Brennan et al., 2008; Haviv et al., 2005; Li et al., 2004; Reing et al., 2009). An ECM scaffold that cannot degrade (i.e., is chemically cross-linked) may not release bioactive degradation products, including those bioactive molecules that may be responsible for modulating the host response towards constructive remodeling. Furthermore, chemical cross-linking may alter ligand–receptor interactions important in determining cell–scaffold interactions.
One of the biologic effects of ECM degradation is the recruitment of host stem and progenitor cells to the site of degradation. A study of ECM scaffold remodeling in a mouse Achilles tendon model examined the ability of ECM scaffolds and autograft tissue to recruit bone marrow-derived cells (Zantop et al., 2006). Bone marrow-derived cells were observed in the sites of remodeling associated with both ECM scaffolds and autograft control tissue, among what appeared to be predominantly mononuclear cells at early time points (1 and 2 weeks) post-surgery. Both scaffold types remodeled into tissue resembling the native Achilles tendon; however, by 16 weeks the presence of bone marrow-derived cells was observed only in the ECM treated group. Another study, also utilizing a model of mouse Achilles tendon, examined the ability of ECM scaffold explants to cause the chemotaxis of progenitor cells after 3, 7, and 14 days of in vivo remodeling (Beattie et al., 2009). The results of the study showed greater migration of progenitor cells towards tendons repaired with ECM scaffolds, compared to tendons repaired with autologous tissue and uninjured normal contralateral tendon. The mechanisms by which ECM scaffolds recruit progenitor cell populations during in vivo remodeling and the specific phenotype of the cells recruited remains unknown, and the effects of the recruited cells upon the scaffold remodeling process is also not well-understood.
Angiogenesis and New ECM Deposition
Angiogenesis is implicit to the success of many tissue engineering and regenerative medicine strategies involving biomaterials. The in-growth of vessels into a tissue-engineered construct provides a means for nourishing the tissue growing on or within the implanted material. In the absence of angogenesis, many implanted biomaterials may fail to integrate with the surrounding tissue, and/or fail to support cell populations that have been seeded onto or migrate into the material. The process of angiogenesis within the native extracellular matrix of tissues and organs has been studied in-depth. The mechanisms by which ECM scaffolds facilitate angiogenesis are not clear. Angiogenesis has, however, been shown to be a prominent feature of ECM scaffold remodeling, and is commonly observed within the first 1–3 days following scaffold implantation. These vessels remain within the implantation site, and continue to provide a blood supply to the remodeling tissue throughout the remodeling process. It has been shown that certain ECM scaffolds contain bioactive VEGF (a potent angiogenic factor) and basic fibroblast growth factor (bFGF) (Chun et al., 2007). Factors such as these may account, in part, for the ability of ECM scaffolds to promote angiogenesis at early time points following implantation.
Response to Mechanical Stimuli
Several studies have shown that early site-appropriate mechanical loading facilitates the remodeling of ECM scaffold materials into site-specific tissue (Boruch et al., 2009; Hodde et al., 1997). In a recent pre-clinical study, partial cystectomies repaired with an ECM scaffold material were exposed to long-term catheterization and prevention of bladder filling, with an associated lack of cyclic distention and decrease in maximal bladder distention, compared to bladders that experienced an early return to normal micturition following ECM scaffold implantation (Boruch et al., 2009). The presence of physiologic amounts of mechanical loading in the early timeframe promoted remodeling of the ECM scaffold material into tissue that had a highly differentiated transitional urothelium, vasculature, innervation, and islands of smooth muscle cells. Delayed return of normal mechanical loading was insufficient to overcome the lack of early mechanical signals, and resulted in degradation of the ECM scaffold material without constructive remodeling.
Similar results were observed in a study of Achilles tendon repair in rabbits, with and without post-surgical immobilization (Hodde et al., 1997). With early mechanical loading, the SIS was rapidly infiltrated by mononuclear cells and new, highly aligned host collagenous connective tissue eventually replaced the ECM scaffold. In contrast, after a period of immobilization, cell infiltration into the scaffold was limited to the periphery of the graft, and little or no ECM remodeling was observed. The lack of cellular infiltration in the immobilized Achilles tendon differed from the abundant mononuclear cell population observed in the early catherization group in the study described above, possibly due to the inadvertent pressure on the bladder from surrounding tissues. It is possible that non-specific mechanical loading may have contributed to the cell infiltration, but site-appropriate mechanical loading is necessary for site-specific remodeling.
When ECM was used for vascular reconstruction, the host remodeling response was different in the arterial system than in the venous system (Lantz et al., 1993; Sandusky Jr. et al., 1992). In the arterial system, a smooth muscle layer developed in the adventitia of the remodeling ECM, while the remodeling response after repair of the superior vena cava led to the formation of primarily collagenous tissue (Lantz et al., 1993; Sandusky Jr. et al., 1992). These differences in the remodeling response are thought to be due to the different mechanical environments present in the high pressure arterial circulation and low pressure venous circulation.
The mechanisms by which ECM scaffolds promote site-specific remodeling in the presence of site-appropriate mechanical loading have been partially elucidated. Static and cyclic stretching of cells seeded on an ECM scaffold in vitro modulated the collagen expression by the cells, enhanced the cell and collagen alignment, and improved in the mechanical behavior of the scaffold (Almarza et al., 2008; Androjna et al., 2007; Gilbert et al., 2007b; Nguyen et al., 2009; Wallis et al., 2008). In addition, during in vivo remodeling, it is thought that the progenitor cells that are recruited to the site of ECM remodeling by matricryptic peptides formed during scaffold degradation will differentiate into site-appropriate cells in the presence of local mechanical cues (Reing et al., 2009; Zantop et al., 2006; Badylak et al., 2001; Beattie et al., 2009; Crisan et al., 2008). Several in vitro studies have shown that mechanical loading can induce progenitor cells to differentiate into fibroblasts, smooth muscle cells, and osteoblasts (Altman et al., 2002; Matziolis et al., 2006; Nieponice et al., 2007).
Conclusion
ECM scaffolds have been isolated from a number of tissues and organs for the purpose of tissue engineering and regenerative medicine. The methods by which these scaffolds are derived are highly dependent on the structure of the organ of interest, and may include both chemical and physical methods. Decellularization processes should be designed such that the majority of the antigenic cellular components are removed while attempting to maintain the highly complex, tissue-specific combination of structural and functional molecules present within the ECM. Following decellularization, ECM scaffolds can be processed into application-specific configurations. These scaffolds have been used successfully in a wide variety of clinical and preclinical settings. The success of ECM scaffolds in these applications is due, in large part, to their ability to modulate the default mammalian host response from scar tissue formation to constructive remodeling. The ability of an ECM scaffold to promote constructive remodeling has been related to a number of biologic events that occur during the remodeling process. Among these are the ability of the scaffold to elicit a friendly host innate immune response, degrade rapidly and fully while releasing bioactive peptides from ECM parent molecules, promote angiogenesis at early time-points following implantation, and respond to the mechanical microenvironment in which it is placed. These processes occur concurrently through the course of scaffold remodeling, and are likely interrelated. Failure of an ECM scaffold to undergo any one of these processes may result in degradation of the scaffold without constructive remodeling, fibrous encapsulation of the implanted material and a lack of scaffold degradation or the formation of scar tissue within the implant site.
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