Consequences of ineffective decellularization of biologic scaffolds on the host response
Introduction
The use of biologic scaffolds derived from decellularized mammalian tissues is commonplace. Such scaffolds are composed of extracellular matrix (ECM) and have been used to repair or replace a variety of damaged or diseased tissues including cardiac [1], [2], [3], esophageal [4], [5], dermal [6], and musculotendinous tissues [7], [8], [9], [10], [11], among others. These materials are typically regulated as devices and marketed as surgical mesh products; however, these ECM-based scaffolds can also serve as an inductive template for tissue repair and regeneration [12], [13], [14]. Numerous commercial products composed of allogeneic or xenogeneic ECM are now available for clinical use (Table 1).
Results of preclinical and clinical studies with biologic scaffolds have varied from very successful [15], [16], [17], [18], [19] to complete failure [20], [21], [22], [23]. The host response to these materials can be attributed to factors such as the source species (e.g., human, porcine, equine, or bovine), the tissue from which the ECM is isolated (e.g., dermis, small intestine, or pericardium), mechanical loading [24], [25], and the niche factors to which the scaffold is exposed following implantation. The decellularization, disinfection, and sterilization methods used during the manufacturing process can markedly influence the tissue remodeling response and functional outcome [26], [27]. Despite these known variations in functional outcome, no quantitative criteria by which decellularization can be assessed have been suggested until recently [26] and as a result, the amount of retained cellular material varies widely among commercial products composed of decellularized tissues [28]. The consequences of ineffective or incomplete decellularization upon the host response have not been systematically investigated.
The innate and acquired immune response to non-autologous cells is well established and understood by the tissue and organ transplantation community. However, the response to acellular xenogeneic or allogeneic biologic scaffold materials is less well understood. The macrophage represents a key component of the host response. Macrophages are activated in response to tissue damage, infection, or the presence of foreign antigens and subsequently release a variety of cytokines and chemokines [29]. Macrophages are now recognized to assume a variety of phenotypes characterized by distinct functional properties, surface markers, and their secreted cytokine profile [30]. Polarized macrophages are referred to as either M1 or M2 cells, mimicking the Th1/Th2 nomenclature. Classically activated, M1 proinflammatory macrophages express IL-12high, IL-23high, IL-10low; metabolize arginine; produce high levels of inducible nitric oxide synthetase (iNOS); secrete toxic reactive oxygen and nitric oxygen intermediates and inflammatory cytokines such as IL-1β, IL-6, and TNF. M1 macrophages are inducer and effector cells in Th1 type inflammatory responses. In contrast, M2, alternatively activated macrophages are induced by exposure to a variety of signals including the cytokines IL-4, IL-13, and IL-10, immune complexes, and glucocorticoid or secosteroid (vitamin D3) hormones. M2 activated macrophages express IL-12low, IL-23low, and IL-10high; have high levels of scavenger, mannose, and galactose receptors; produce arginase in the place of arginine which results in the secretion of ornithine and polyamines; are involved in polarized Th2 reactions; and possess the ability to facilitate tissue repair and constructive remodeling [29], [31], [32].
Previous studies have demonstrated the importance of macrophages in tissue repair and regeneration, particularly in applications involving biologic scaffolds [33], [34], [35], [36], [37]. Macrophages are responsible, in part, for the degradation of ECM scaffolds. Depletion of circulating macrophages severely attenuates scaffold degradation and the associated constructive remodeling response [37]. Non-crosslinked biologic scaffolds promote the expression of an M2 macrophage phenotype at the site of remodeling although a population of macrophages expressing markers of an M1 phenotype persists [34], [36]. Tidball has demonstrated that it is the switch from an M1 to M2 phenotype that stimulates progenitor cell differentiation and constructive tissue remodeling while an early M1 response stimulates progenitor cell recruitment and proliferation [33]. The ability of biologic scaffolds to promote expression of an M2 macrophage phenotype may therefore be critical to their ability to promote constructive tissue remodeling. However, the mechanism by which these biologic scaffolds promote M2 macrophage expression is currently unknown.
It is known that cell death incites a series of events that typically results in the classic cascade of inflammatory processes including polymorphonuclear leukocyte (PMN) and mononuclear cell accumulation, edema, fibroblast infiltration, and eventual scar tissue formation [38], [39], [40]. The presence of intact cells within a biologic scaffold material has been shown to elicit a greater proinflammatory response than use of the acellular biologic scaffold alone [36]. It is logical, therefore, that cell remnants within a partially decellularized tissue could elicit a proinflammatory response that would adversely affect a constructive tissue remodeling outcome. In fact, it has been shown that the presence of intact cells within implanted scaffolds can be associated with adverse remodeling [36]. In contrast, thoroughly decellularized biologic ECM scaffolds are known to promote a host response that is polarized towards the M2 macrophage phenotype and is associated with constructive tissue remodeling [34], [36], [37]. These two responses sit at the polar extremes of the host response to an implanted biologic scaffold. Little is known about which cell components stimulate an M1 macrophage response or if a threshold level for cellular material exists below which the M2 macrophage phenotype predominates. It has been suggested that the presence of mitochondria or mitochondrial DNA may be a stimulator of M1 macrophages given their primitive bacterial origin [41]. In light of the heterogeneity with regard to the amount and efficacy of decellularization in commercially available ECM scaffolds [28], a more thorough understanding of the effects of cell remnants upon the host response is needed.
The objective of the present study was to evaluate the effect of cell remnants (i.e., ineffective tissue decellularization) within biologic scaffolds upon in vitro and in vivo outcome measures. Three different methods were used to decellularize porcine small intestinal submucosal ECM (SIS-ECM). The amount of cellular material remaining was quantified by the amount and fragmentation of DNA. The polarization profile of activated macrophages in response to these ECM scaffolds was then assessed in vitro and in vivo using a rodent model of body wall repair.
Section snippets
Harvest and preparation of ECM from porcine small intestine
Preparation of small intestinal submucosa (SIS) ECM has been previously described [42], [43]. Briefly, jejunum was harvested from market weight (240–260 lbs.) pigs and split longitudinally. The superficial layers of the tunica mucosa were mechanically removed. Likewise, the tunica serosa and tunica muscularis externa were mechanically removed, leaving the tunica submucosa and basilar portions of the tunica mucosa. To produce ECM with differing amounts of remnant cellular material, three
DNA concentration and fragmentation in scaffolds
The amount of tissue decellularization following the three preparation methods was assessed using previously established guidelines for decellularization [26]. Intact nuclei were visible by H&E and DAPI staining on samples prepared with PBS washing only (Fig. 1A and B). No intact nuclei were seen by H&E staining on samples treated with PAA for either 1 hr (Fig. 1C) or 2 h (Fig. 1E) although potential fragments of DNA were seen attached to the ECM fibers in DAPI stained samples following 1 hr
Discussion
The present study attempts to determine the association between decellularization efficacy and host response by qualitative and quantitative methods. Quantitative criteria of decellularization have not been described until recently [26]. The presence of xenogeneic DNA within biologic scaffold materials has been suggested as a possible cause of an “inflammatory response” [20] in patients. Indeed, many commercial biologic scaffolds contain varying amounts of remnant DNA [28], [46]. This remnant
Conclusion
The results of this study show that decellularization efficacy of biologic scaffold materials is at least one determinant of the macrophage phenotype response. Although a cause-effect relationship between macrophage phenotype and remodeling outcome has not been definitely shown, a clear association exists. Effective decellularization remains an important component in the production of ECM-based scaffolds for therapeutic applications.
Acknowledgments
Funding for this study was provided through a grant from the National Institutes of Health (R01 AR054940).
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These authors contributed equally.