Elsevier

Methods

Volume 84, August 2015, Pages 25-34
Methods

Review Article
Methods of tissue decellularization used for preparation of biologic scaffolds and in vivo relevance

https://doi.org/10.1016/j.ymeth.2015.03.005Get rights and content

Highlights

  • Current decellularization techniques and agents are described for the preparation of ECM bioscaffolds.

  • Maintaining ECM structure while removing cellular components is key.

  • Considerations for selecting decellularization agents are discussed.

  • The effect of decellularization on the host response to ECM is discussed.

Abstract

Biologic scaffolds composed of extracellular matrix (ECM) are widely used in both preclinical animal studies and in many clinical applications to repair and reconstruct tissues. Recently, 3-dimensional ECM constructs have been investigated for use in whole organ engineering applications. ECM scaffolds are prepared by decellularization of mammalian tissues and the ECM provides natural biologic cues that facilitate the restoration of site appropriate and functional tissue. Preservation of the native ECM constituents (i.e., three-dimensional ultrastructure and biochemical composition) during the decellularization process would theoretically result in the ideal scaffold for tissue remodeling. However, all methods of decellularization invariably disrupt the ECM to some degree. Decellularization of tissues and organs for the production of ECM bioscaffolds requires a balance between maintaining native ECM structure and the removal of cellular materials such as DNA, mitochondria, membrane lipids, and cytosolic proteins. These remnant cellular components can elicit an adverse inflammatory response and inhibit constructive remodeling if not adequately removed.

Many variables including cell density, matrix density, thickness, and morphology can affect the extent of tissue and organ decellularization and thus the integrity and physical properties of the resulting ECM scaffold. This review describes currently used decellularization techniques, and the effects of these techniques upon the host response to the material.

Introduction

Biologic scaffold materials composed of extracellular matrix (ECM), and derived via decellularization of source mammalian tissues, are widely used for clinical applications that involve repair and reconstruction of musculoskeletal tissues, cardiovascular structures, lower urinary tract, gastrointestinal tract, and central nervous system, among others [1], [2], [3], [4], [5], [6]. Such scaffold materials have the potential to promote and facilitate “constructive remodeling” processes that form vascularized, innervated, functional tissue. Mechanisms by which these constructive remodeling events occur include the recruitment and differentiation of stem/progenitor cells [7], [8], [9], [10] and modulation of the innate immune response [11], [12], [13]. The effector molecules responsible for these processes represent a combination of sequestered cytokines and chemokines within the matrix, and matricryptic peptides generated or exposed during the process of ECM degradation [8], [14], [15], [16]. Thorough decellularization of source tissues to generate the ECM scaffolds is critical for realization of the full potential of these ECM mediated events. Failure to effectively decellularize the tissue results in retained cell remnants, which can act in a manner similar to damage associated molecular pattern molecules (DAMPs) following tissue injury. Ineffective decellularization has been shown to be associated with an intense inflammatory response which can mitigate or completely inhibit a constructive remodeling outcome [17].

All methods of decellularization disrupt the structure and composition of the ECM. The goal of tissue decellularization is thorough removal of cells and cell remnants while retaining the three dimensional ultrastructure and composition of the native ECM to the extent possible. Optimal methods of decellularization vary among tissues and organs due to tissue-specific factors such as cell density, matrix density, and geometric considerations including tissue thickness and shape. Complete removal of all cell remnants is not possible, and decellularization processes inevitably and invariably cause some disruption of matrix architecture, orientation, and surface ligand landscape. The specific cellular elements that elicit adverse and proinflammatory responses are only partially known. One objective of the present manuscript is to provide guidance as to the effects of various decellularization methods upon both the resulting biologic scaffold and the associated host remodeling response and outcome. Decellularization methods described include mechanical, chemical, detergent, and enzymatic techniques, or combinations thereof. Table 1 provides an overview of decellularization processes used for various tissues and organs and the effects of those processes on ECM constituents and the host response.

Section snippets

Decellularization agents and techniques

Decellularization protocols have been described for nearly every tissue in the body. The decellularization methods must be tuned to the tissue of interest and ultimately the intended use of the decellularized tissue. It is not uncommon for vastly different protocols, with varied detergents and delivery methods, to be described for decellularization of the same tissue.

Establishing metrics for effective decellularization

While it is unlikely that any decellularization protocol will completely remove all cell remnants, cell components that do remain (e.g., DNA, phospholipids) can be quantitatively assayed. Until recently, no quantitative metric has been suggested to evaluate the efficacy of a decellularization protocol [38]. It is important to note that for most studies reviewed in the present manuscript, no objective criteria were used to assess degree of decellularization. Only one study to date has related

Regulatory requirements for sterilization

There are many examples of biologic scaffold products composed of decellularized tissues. These include dermis, small intestine, urinary bladder, mesothelium, pericardium, and heart valve. These products are used for repair applications in soft tissue, tendon, chronic wounds, breast reconstruction, opthalmology, dentistry, valve replacement and others. Biologic scaffolds from decellularized xenogenic source tissue are typically regulated by the Food and Drug Administration (FDA) as medical

Conclusion

The potential benefits provided by biologic scaffold materials for replacement and reconstruction of damaged or missing tissues and organs are noteworthy. However, the effects of such materials upon stem/progenitor cell recruitment and the innate immune response are critically dependent upon the methods used to manufacture these materials; decellularization and terminal sterilization being among the most important. With respect to decellularization, one size does not fit all when considering

Acknowledgments

This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1247842 (to T.J.K.). Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the National Science Foundation.

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