ReviewAn overview of tissue and whole organ decellularization processes
Introduction
Biologic scaffolds composed of extracellular matrix (ECM) are commonly used for a variety of reconstructive surgical applications and are increasingly used in regenerative medicine strategies for tissue and organ replacement. The ECM represents the secreted products of resident cells of each tissue and organ, is in a state of dynamic reciprocity with these cells in response to changes in the microenvironment, and has been shown to provide cues that affect cell migration, proliferation, and differentiation [1], [2], [3], [4], [5], [6], [7]. Preservation of the native ultrastructure and composition of ECM during the process of tissue decellularization is highly desirable [8], [9], [10], [11], [12], [13], [14]. A review of tissue decellularization techniques and their effect upon ECM properties was published in 2006 [15]. However, the development of new decellularization techniques and the advent of three-dimensional whole organ decellularization have since emerged. In addition, the deleterious in vivo effects of residual cellular material are becoming more recognized [16], [17], [18], [19]. The rapid diversification of both decellularization methods and source tissues, and the expanding list of clinical applications suggest that cell residues in ECM should be evaluated objectively against a quantitative definition. The objectives of the present manuscript are (1) to provide an updated overview of tissue and organ decellularization techniques and their expected effects on the mechanical and biological properties of the remaining ECM as determined by systematic investigations [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45] and (2) to define a decellularization standard that has been shown to avoid adverse host responses following implantation and is associated with constructive host responses.
Section snippets
Clinical relevance and rationale for the use of ECM as a biologic scaffold
The use of ECM derived from decellularized tissue is increasingly frequent in regenerative medicine and tissue engineering strategies, with recent applications including the use of three-dimensional ECM scaffolds prepared by whole organ decellularization [8], [9], [10], [46], [47]. Clinical products such as surgical mesh materials composed of ECM are harvested from a variety of allogeneic or xenogeneic tissue sources, including dermis, urinary bladder, small intestine, mesothelium, pericardium,
Decellularization agents
The most effective agents for decellularization of each tissue and organ will depend upon many factors, including the tissue’s cellularity (e.g. liver vs. tendon), density (e.g. dermis vs. adipose tissue), lipid content (e.g. brain vs. urinary bladder), and thickness (e.g. dermis vs. pericardium). It should be understood that every cell removal agent and method will alter ECM composition and cause some degree of ultrastructure disruption. Minimization of these undesirable effects rather than
Techniques to apply decellularization agents
The optimal application of decellularization agents is dependent upon tissue characteristics such as thickness and density, the agents being used, and the intended clinical application of the decellularized tissue. Prior to applying decellularization agents, undesirable excess tissue may be removed to simplify the cell removal process [29], [97]. Tissue removal may focus on retention of key ECM components such as basement membrane. Direct force may also be applied to tissue to aid in
Sterilization of decellularized ECM
It is necessary to sterilize biologic scaffolds composed of ECM prior to implantation or in vitro use, including depyrogenation to eliminate endotoxins and intact viral and bacterial DNA that may be present. Biological scaffolds may be sterilized by simple treatments such as incubation in acids [64] or solvents [60], but such methods may not provide sufficient penetration or may damage key ECM constituents [65]. However, sterilization methods such as ethylene oxide exposure, gamma irradiation,
Evaluation of decellularized ECM
Residual cellular material within ECM may contribute to cytocompatibility problems in vitro and adverse host responses in vivo upon reintroduction of cells [16], [17], [18], [19]. Although decellularization techniques cannot remove 100% of cell material, it is possible to quantitatively assay cell components such as double-stranded DNA (dsDNA), mitochondria, or membrane-associated molecules such as phospholipids. The threshold concentration of residual cellular material within ECM sufficient to
Conclusion
The preparation of biologic scaffold materials composed of mammalian ECM requires decellularization of source tissues. Such decellularization typically involves exposure to selected non-physiologic chemical and biologic agents such as detergents and enzymes and physical forces that unavoidably cause disruption of the associated ECM. Since the source tissues for biologic scaffolds are typically allogeneic or xenogeneic in origin, maximal decellularization is desirable. The choice of
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