The Interplay of ECM-Based Graft Materials and Mechanisms of Tissue Remodeling

Wound healing is a complex natural process that involves the recruitment of cells, the renewal of tissue composition, and the reinforcement of structural tissue architecture. Following ischemic injury or chronic disease, wound healing is delayed, and can often result in chronic inflammation or permanent morbidity. Tissue engineering strategies to harness the wound healing process include the use of naturally derived extracellular matrix (ECM) scaffolds with inherent bioactivity to both passively facilitate and actively direct healing toward a successful resolu-tion. As the body heals, the properly designed ECM scaffold is gradually remod-eled and integrated into the body, leaving behind organized tissue that provides long-term strength. Herein we explain the interplay of the ECM (i.e., its complex composition and bioactivity) with the cells of the body throughout the process of tissue remodeling, thus explaining how even a tissue-engineered xenograft material can direct the body to restore itself.


Introduction: extracellular matrix as an implantable graft material
Biologic materials used to repair soft tissue defects must be strong and easy to handle during implantation, but they must also be able to support tissue integration and maturation once implanted. ECM-based biologic grafts have been widely used in surgery over the last two decades. They are a good choice for surgeons because they can be safely implanted in contaminated settings where synthetic materials are contraindicated. Even though synthetic mesh materials continue to be favored in general surgical practice because of their versatility and low cost, they remain susceptible to chemical degradation over time, can create physical tissue erosion due to mismatches in their mechanical properties with the surrounding tissues, and may undergo encapsulation following placement because the body views them as foreign materials [1]. Of critical importance in many applications, synthetics can provide a nidus for microorganism growth; therefore, if they become infected when in the body, they typically need to be removed [2].
ECM biomaterials derived from natural tissue sources, however, have generally provided adequate strength, resistance to infection, and stability over time such that they make adequate materials for soft tissue reconstruction [3].
These materials can be obtained as autografts or allografts, but autografts result in donor site morbidity, while cadaveric allograft tissues may transmit disease, are inherently inconsistent, and are typically quite expensive.
Recent years have seen the advent of multiple off-the shelf tissue-based ECM biomaterials that claim to provide an optimal healing environment for soft tissues. They can be obtained from a wide variety of mammalian tissues, processed using a wide range of chemicals and cross-linking agents, or can be provided in such a way that retains the information-rich scaffold into which adjacent cells migrate to create a replacement tissue ( Table 1). Many studies have shown constructive, functional tissue remodeling with partial restoration of siteappropriate tissue using these graft materials [4][5][6][7], yet this is not always the case. Less favorable outcomes include the accumulation of serous fluid at the implant site, rapid degradation of the graft material with associated mechanical failure, or a lack of biomaterial integration with the patient's tissues, resulting in a foreign body response [8,9]. These less-than-favorable outcomes typically have been associated with variations in manufacturing methods that result in the failure of the material to maintain nature's natural composition and three-dimensional architecture that makes the extracellular matrix (ECM) the ideal template for tissue repair and regeneration.
Materials that are minimally processed most closely recapitulate the structure and function of the original tissue while providing a safe, biocompatible material for soft tissue reconstruction. The natural ECM, when retained in its complex arrangement of matrix proteins and associated factors, can provide the key extracellular signals and inherent bioactivity needed to restore damaged tissues to their natural state [7]. This complexity allows the naturally occurring biologic graft to completely integrate with the recipient's tissues and cells to ultimately form a vascularized, highly organized tissue structure that resembles the native tissue structure and architecture [4,7,10,11].

Extracellular matrix as bioactive structure
The ECM is a three-dimensional network of extracellular macromolecules, such as collagens, glycoproteins, proteoglycans, and glycosaminoglycans, that provides structural and biochemical support to surrounding cells. Because of different structural and mechanical requirements, the composition of ECM varies from tissue to tissue; however, providing a structure for cell adhesion, directing cell-tocell communication, and regulating cell processes such as growth, migration and differentiation are common functions of the ECM [12].
Regardless of the source, ECM is a complex three-dimensional scaffold consisting of structural and functional proteins and components arranged in a tissuespecific orientation [12]. The ECM components directly interact with fibroblasts, endothelial cells, and macrophages to maintain a natural and functional homeostatic environment through a process known as dynamic reciprocity (Figure 1) [13]. When injury occurs and the natural equilibrium is disrupted, the dynamic environment that exists between the ECM and cells orchestrates acute inflammation, wound healing and tissue remodeling to regain function and restore homeostasis. After injury occurs and the ECM is damaged, a biologic graft can be implanted to provide a surrogate matrix structure that allows dynamic reciprocity to begin immediately, ultimately achieving tissue restoration via the process of constructive tissue remodeling.
Endogenous ECM functions as the intended bioactive structure when normal tissue turnover is taking place or when no significant tissue loss is encountered. The body has a remarkable ability to self-renew, in large part due to the instructional nature of the ECM, but in the presence of significant tissue loss, large areas of trauma, or surgical reconstructions, there is a need for an exogenous material to augment and to bring order to somewhat chaotic processes. An exogenous ECM can serve as this bioactive, instructive, and even mechanical blueprint for a constructive tissue remodeling process [7,14,15].

Extracellular matrix and constructive tissue remodeling
Constructive tissue remodeling is more than just another word for wound healing or for tissue repair. The stages of wound healing include initial hemostasis, characterized by clot formation; inflammation, characterized by the deposition of inflammatory and progenitor cells, leading to the formation of granulation tissue; proliferation, where resident cells secrete growth factors and cytokines and collagen deposition occurs; and remodeling, where the newly formed tissue matures and collagen strength increases to meet the demands of the body [16] (Figure 2). Tissue repair results in the formation of scar tissue, which is known to be less strong than native tissue and can therefore be more susceptible to reinjury [5].
Unlike the tissue repair process that occurs in the absence of a biologic graft material, the constructive tissue remodeling process that can be directed by an ECM graft leads to a more natural healing process in the recipient that is characterized by the deposition of organized connective tissue, rather than just chaotic scar [17]. The ideal ECM graft is characterized by an open matrix structure to allow for rapid cellular ingrowth. It is also characterized by the presence of structural collagens and non-collagen ECM components (such as messenger nucleic acids, growth factors, glycoproteins, proteoglycans, and glycosaminoglycans), which act to facilitate the renewal of natural dynamic reciprocity [18]. When tissue homeostasis is disrupted, the biologic graft plays the role of the recipient's natural ECM and works to bridge the recipient's cells across the wound to ultimately restore a homeostatic environment. The restoration of homeostasis following injury in the presence of a biologic graft occurs through the constructive process of tissue remodeling.
Tissue remodeling is a process of tissue restoration that improves upon the scar tissue outcome typically achieved by tissue repair. It can be divided into three separate phases: 1) Cell recruitment; 2) Tissue renewal; and 3) Tissue reinforcement.
During cell recruitment, the remodeling process starts when the body's inflammatory and progenitor cells populate the biologic graft and release cytokines and growth factors that bind to the graft and recruit collagen-secreting fibroblasts [18,19]. In this phase, the graft primarily acts as a scaffold material to support the population of the open ECM structure by the patient's own cells.
As remodeling progresses, the patient's macrophages and fibroblasts in the newly populated matrix work together with matrix-bound signaling factors to renew the tissue through the complementary processes of phagocytosis, collagen deposition, and angiogenesis. In this phase, the biologic graft is gradually replaced by the patient's own tissue and cells [18,19].
Over the medium to long term, the resident fibroblasts secrete cytokines and growth factors to signal reinforcement of the deposited tissue through the processes of additional collagen deposition and maturation, resulting in a strong, repaired tissue [10,[20][21][22]. In this phase, the biologic graft is no longer needed as the patient's own collagen has gradually matured into a stable structure that has long-term strength but is entirely the patient's own [20][21][22]. The resulting tissue structure is mature, organized and strong, and can withstand (and is even driven by) the natural physiological forces that it encounters [17,23]. A biologic graft with the correct composition and three-dimensional architecture directs the patient's body to replace itself -to completely remodel -rather than to heal through a tissue repair process that results in chaotic, weak, and ineffective scar tissue formation [20][21][22]. By providing the correct cues to help the body restore itself, the graft provides both an essential temporary structure and the local tissue instructions to lead the patient to achieve a natural repair (Figure 3).

Mechanisms of action for ECM-directed tissue remodeling
An ECM-based biologic graft that has been optimally processed to harness the tissue remodeling properties of nature acts more than just a mechanical tissue reinforcement device. While mechanical reinforcement is still the primary mechanism of action for these materials, additional mechanisms of action include: providing a porous tissue scaffold matrix structure to allow for fibroblast infiltration and population; altering the surrounding wound environment by modulating local cytokine activity; and, optimally, acting as a reservoir for growth factors and signaling molecules that can be used by the patient as tissue renewal and reinforcement progress ( Table 2).

Mechanical reinforcement during surgical repair
Poor wound healing after trauma, surgery, or due to chronic disease is the consequence of a poorly regulated tissue repair response that directly effects the processes of inflammation, angiogenesis, matrix deposition, and cell recruitment [24]. As a result, tissue healing typically takes a significant time to achieve in patients with advanced age or with comorbidities. Prolonged mechanical reinforcement is often needed to get proper approximation of the wound edges and to bolster the anatomy until tissue ingrowth is sufficient to achieve the required strength to maintain tissue integrity. This mechanical reinforcement mechanism is the primary (and often only) means by which most implantable devices achieve their effect. For example, synthetic mesh materials, such as polypropylene or polytetrafluoroethylene, derive their reinforcement benefit from the strength of their fibers at implant but never completely integrate with the patient's tissues over time [25]. Synthetic materials are often recognized as foreign by the body -as a material that needs to be removed or expunged [26]. When this occurs, an inflammatory response is initiated by the patient's immune system, setting up a chronic inflammatory state that never resolves and can result in chronic pain and fibrosis [26].  For a well-designed biologic ECM graft, the mechanical means of tissue support remains its primary mechanism of action. The ECM graft must allow the passage of suture and reinforce the area of weakness under significant pull-out force. It must also provide tensile strength and mechanical compliance commensurate with the surrounding tissues. Unlike synthetic or even many biosynthetic materials, such as poly-4-hydroxybutyrate (P4HB), ECM-based biologic devices are not meant to be static implants but are designed to fully integrate with the patient over time. Their mechanical properties change after implant as they undergo interaction with the patient's cells, tissues, and the local wound environment [27] and must therefore be designed to retain their mechanical integrity even while actively participating in the process of tissue renewal. The dynamic process of tissue remodeling is a balance of ECM graft degradation with the formation of new patient-derived collagen, meaning that an ECM graft must be designed with known strength requirements and degradation rates to keep the repair intact during all phases of tissue remodeling: 1) Cell recruitment; 2) Tissue renewal; and 3) Tissue reinforcement (Figure 4) [20,28].

Providing a tissue scaffold matrix structure
When foreign materials are implanted into the body, they are quickly recognized by the immune system as something either to rapidly destroy or to compartmentalize [29]. The body accomplishes these activities by secreting inflammatory enzymes and pH modifiers or by recruiting an army of macrophages to form a scarified wall around the implant. While permanent synthetic materials and crosslinked biologic grafts are typically walled off by the recipient because they are resistant to degradation [30], biosynthetic matrices are often hydrolyzed or otherwise degraded over time without allowing complete tissue integration and permanent reinforcement to occur [31].
Purified biologic ECM grafts typically contain few of the naturally occurring macromolecules of the complex ECM because they have been deconstructed with chemicals and then "purified" into single-component constructs or reconstituted into single-component implants. While this type of graft material can still act as a matrix structure to support cell ingrowth, the lack of complex signaling macromolecules from the natural ECM and its susceptibility to matrix-degrading enzymes, such as collagenases, limits its ability to actively promote fibroblast and endothelial cell proliferation and secretion of new ECM [32,33]. Non-crosslinked biologic ECM grafts that have been processed to retain the composition and architecture of healthy ECM are neither encapsulated nor degraded upon implant [7]. Instead, they contain the complex information of the natural ECM that makes them an ideal scaffold environment upon which cells can move and proliferate, allowing for colonization of fibroblasts and endothelial cells, the eventual secretion of growth factors, and the deposition of a collagen matrix [10]. The porous nature of the ECM scaffold provides not only the structure and interstices for ingrowth but also the recognition and binding sites that facilitate cellular attachment and migration [10]. During the process of tissue renewal, the porous matrix structure of the non-crosslinked ECM graft allows for angiogenesis and ultimately the removal of byproducts of cellular metabolism, facilitating the process of tissue remodeling that is essential to obtaining a long-lasting, strong, and permanent repair [10,34].

Modulating endogenous cytokine activity
The local wound environment is characterized by a dynamic milieu of signaling factors designed to shepherd an injury through the four phases of wound healing and to ultimately restore tissue strength and homeostasis [16]. In most instances this occurs in a well-defined series of events leading to complete tissue restoration that is modulated directly by the local ECM. Because the ECM is laden with macromolecules that explicitly bind cytokines and alter their half-lives, bioactivities, and concentrations, the presence of a healthy ECM in the local wound environment is essential for tissue remodeling to occur. When the ECM is corrupt, it cannot support tissue restoration and chronic inflammation results [35].
Chronic, non-healing wounds are characterized by increased levels of proinflammatory cytokines, increased levels of MMPs, and low levels of growth factors known to stimulate wound closure [36,37]. They are highly inflamed and proteolytic, have become stalled in the inflammation stage of wound healing, and cannot support fibroblast function [38]. In cases such as this, replacing the damaged ECM with a healthy ECM-based biologic graft can alter the local wound environment by modulating the endogenous cytokine profile of the injured area and stimulating normal fibroblast and endothelial cell function [39].
This tertiary mechanism of action for ECM-directed tissue remodeling, endogenous cytokine modulation, harnesses the natural structure and composition of the ECM to direct tissue remodeling down a productive pathway [37]. Unlike synthetic and biosynthetic materials that contain no ECM-binding sites and cannot directly influence the composition of the natural wound environment; unlike crosslinked ECM biologic graft materials which have had their binding sites obscured by the crosslinking process; and unlike purified biologic ECM grafts that are limited in the types of cytokines that can interact with them; well-designed, non-crosslinked, biologic ECM graft materials have been shown to positively alter the local environment and lead to constructive tissue remodeling and wound healing [10,37,39,40].

Acting as a cytokine reservoir
Matrix biologists have long regarded the ECM as a repository for latent bioactivity in the form of growth factors, cytokines, and more recently, messenger nucleic acid depots. Even in their dehydrated state, these factors retain their potency and structure because they are tightly bound to proteins that protect them from degradation [41,42]. Also, recently, science has uncovered the remarkable ability for these embedded matrix molecules to modulate cellular activity across species and after long periods of dormancy. Porcine growth factors can activate human cells, and vice-versa, with predictable potency and expected effects, even after dehydration and sterilization [41,42]. It is this growth factor and cytokine repository that separates a complex biologic ECM graft from other types of non-instructional implant materials.
After implantation, a complex biologic ECM graft plays the role of the innate ECM, interacting with the patient's cells through dynamic reciprocity to direct tissue repair down a positive, active state of wound healing and toward an organized repair that resembles native tissue structure and architecture rather than scar tissue. When its role has been fully realized, an ECM graft becomes completely replaced by patient tissue and removed from the body through the normal process of matrix turnover, leaving no graft components behind [43]. In many ways it is the repository of latent bioactivity that allows the well-designed ECM graft to stimulate transformation of itself, by the patient's cells, into a new, complex and complete, functional tissue.

Summary: extracellular matrix past, present, and future
ECM graft materials have been used surgically for decades, but historically they have been enzymatically stripped of their biological information, chemically cross-linked to enhance their durability (while quite effectively silencing their biological activity), or otherwise adulterated in such a way as to act much more like synthetic mesh than a truly instructive matrix [34,44]. A more modern approach to ECM graft design can capitalize on the inherent complexity and instructiveness of natural ECM to build an implant with multi-factorial mechanisms of action that harmonize with healing, serve as a surrogate ECM in the wound, and stimulate the processes of dynamic reciprocity toward renewed homeostasis. Such an implant can guide the patient's cells through a series of cellular recruitment, renewal of lost matrix structures, and reinforcement of tissue strength while undergoing complete turnover and disappearance of the original implant.
The current state of the art for ECM grafts has been described. These materials have shown remarkable success in a wide variety of clinical applications [3,7]. However, there is still room for improvement. Naturally occurring biologic ECM graft materials can be enhanced or fortified to accelerate some of these biological functions, stimulate cellular phenotype selection, or even create inherent antimicrobial activities that will better withstand infection. Ultimately, the goal of such "next generation" implants must be one of synergizing with natural biology and improving upon the complex interaction of the graft with the patient to allow tissue repair, remodeling, and regenerative processes to proceed unhindered.