Xenogeneic and allogeneic cellular antigens are recognized as foreign by the host and therefore induce an inflammatory response or an immune-mediated rejection of the tissue. However, the components of ECM are generally conserved among species and are tolerated well, even by xenogeneic recipients [5, 6]. ECM from a variety of tissues, including heart valves [7, 8], blood vessels [9, 10], skin [11], nerves [12], skeletal muscle [13], tendons [14], ligaments [15], small intestinal submucosa [16], urinary bladder [17], and liver [18], has been reported for use in tissue engineering and regenerative medicine applications. The purpose of decellularization protocols, though minimizing adverse effects on the mechanical integrity of the remaining ECM, is to efficiently remove all cells and nuclear material (Fig. 6.2).

With the purpose of removing the cells, any of the processing steps changes the native three-dimensional structure of ECM. The most common method used for the decellularization of an organ involves a combination of physical and chemical processing. Physical treatment can include agitation or sonication, mechanical massage and pressure, or a freeze–thaw process. Using these methods, the cell contents are removed, the cell membrane is easily destroyed by subsequent rinsing and removal of the cell content from the ECM. However, although these physical processes achieve complete decellularization, they are insufficient, and must be combined with chemical treatment. Enzymatic treatments, such as trypsin, ionic detergents and chemical treatments, can disrupt charge coupling among the cell membrane, cell, and extracellular component. Tissue is composed of both cell materials, and ECM has a variable degree of miniaturization according to the tissue source. ECM is sufficient to allow proper exposure of all the cells to the chaotropic agents. To provide a path for removal from the organ, the cellular material must be destroyed during the decellularization process. The purpose of the main part of the decellularization process is to retain the original mechanical and biological properties and minimize disruption.

The most robust and effective physical decellularization protocol uses a combination of chemicals, and includes an enzymatic approach. Decellularization protocols, typically enzyme treatment, surface-active agents, cytoplasmic, solubilization of cellular components of the nucleus, and the final removal of cellular debris from tissue, cellular components from the ECM, involve physical treatment or ionic solution. Following separation, the dissolution of the cell membrane begins. To enhance effectiveness, these steps can be combined with mechanical stirring. Following decellularization, all the residual chemicals must be removed to avoid the response of chemically adverse host tissue. The efficiency of the storage of decellularization and ECM can be assessed using one of several methods.

Facilitating the replacement of the ECM graft with functional host tissue is considered a constructive remodeling response. ECM scaffold changes during such a remodeling response are dependent on factors such as the local tissue microenvironment, the rate of scaffold degradation, forces present within the mechanical environment, and the rate and extent to which the infiltrating cells deposit new ECM [48, 49]. Decellularized scaffolds retain a variety of collagens, growth factors, glycosaminoglycans, and other matrix proteins, and the arrangement of the proteins is minimally disturbed through the process [50–53]. Upon implantation, mononuclear cells migrate into the decellularized scaffold and degrade it, while concomitantly depositing new site-appropriate tissue [54, 55]. The structural changes that occur during in vivo remodeling of ECM scaffolds are associated with marked changes in scaffold strength. In the early phase of remodeling, degradation occurs quite rapidly, before the newly deposited ECM can become fully organized. However, once the infiltrating cells have established residence and begin producing new site-specific ECM, the remodeling process progresses over time toward a tissue construct that has similar mechanical properties very similar to those of native tissue [54, 56].

Clinical application of a decellularized scaffold requires donor scaffold sterilization. The sterilization process primarily eliminates endotoxins and intact viral and bacterial DNA that may induce unwanted inflammation upon scaffold implantation [57]. Decellularized scaffolds may be sterilized using simple treatments such as incubation in acids [58] or solvents [59], but such methods may not provide sufficient penetration or may damage key ECM constituents [60]. Sterilization methods such as ethylene oxide exposure, gamma irradiation, and electron beam irradiation achieve effective sterilization, but are known to alter ECM microstructure and mechanical properties [61, 62].

The major concerns of all decellularization protocols remain ECM disruption, immunogenicity, and thrombogenicity. The effectivity of decellularization and the alterations to the ECM vary according to the source, composition, and density of the tissue, and to other factors. Decellularization agents that are very efficient at removing cellular components can also cause damage to the collagen structure and remove growth factors and other important ECM components. However, because of insufficient decellularization, residual cellular components within ECM may contribute to in vitro cytotoxicity and adverse host responses in vivo upon reintroduction of cells [71–73]. There are conflicting results on the effect of decellularization techniques. Grauss et al. [74] found that chemically induced decellularization using Triton X-100 or trypsin resulted in changes in the ECM constitution, which could lead to problems in valve functionality and cell growth and migration, whereas Schenke-Layland et al. [75] used a decellularization method, in which pulmonary valves were dissected from porcine hearts, placed in a solution of trypsin-EDTA, incubated at 37°C for 24 h, followed by a 24-h wash in phosphate-buffered solution (PBS), and showed acceptable removal of cell components from a porcine pulmonary valve. Cartmell and Dunn [14] compared the methods of decellularization for rat tail tendon using three extraction chemicals (t-octylphenoxypolyethoxyethanol [Triton X-100], tri(n-butyl)phosphate [TnBP], and sodium dodecyl sulfate [SDS]). They showed that treatment of tendons with 1% Triton X-100 for 24 h disrupted the collagen fiber structure, but did not remove cells. Treatment with 1% SDS for 24 h or 1% TnBP for 48 h resulted in an acellular tendon matrix with retention of near-normal structure and mechanical properties. Woods and Gratzer [15] also compared the effect of decellularization using one of the three protocols incorporating the surfactant lauryl sulfate (SDS), Triton X-100, and/or an organic solvent, tri(n-butyl)phosphate (TnBP). They showed that all three treatments were effective at removing cells and preserving the mechanical properties of the graft, with subtle, but notable, differences. Most detergents, when used as agents for decellularization, cause some removal of GAG from the scaffold, which has varying degrees of a negative effect on the tensile viscoelasticity of the scaffold [76]. Another factor that can influence the mechanical properties of the scaffold is the duration of exposure to decellularization agents [77]. There is practically no consensus regarding the effects of any of the decellularization methods. Therefore, it is very important to optimize the decellularization method for obtaining acceptable cell removal according to the source, composition and density of the tissue, and other factors.

Evaluation of the residual materials and cellular components within the decellularized ECM is a pivotal step in translating them into a clinically practicable, transplantable substance. There are a number of methods available to determine the effectivity of the elimination of cellular material from tissues. Although the decellularization process cannot remove 100% of cell material, it is crucial in quantifying cellular components such as double-stranded DNA (dsDNA), mitochondria, or membrane-associated molecules such as phospholipids in a decellularized scaffold. Crapo et al. [57] suggested the following minimal criteria sufficient to satisfy the intent of decellularization: (1) no visible nuclei upon histological evaluation, (2) the remaining dsDNA content should not exceed 200 base pairs in length, and (3) the amount of dsDNA should not exceed 50 ng/mg of dry weight of the material. The first criterion is easily evaluated using standard histological staining with hematoxylin and eosin or immunofluorescent methods such as DAPI or Hoechst. The second and third criteria are easily evaluated using commercially available dsDNA intercalators, such as PicoGreen^®, propidium iodide, or bisbenzimide, and by gel electrophoresis respectively. Jackson and Simon [78] demonstrated the fate of DNA from allografts after transplantation using a DNA probe technique that clearly distinguished donor cells from host cells and could also be utilized to evaluate whether any DNA is present in the decellularized scaffold in the Spanish goat model. Electron microscopic methods or polymerase chain reaction (PCR) are usable, but not typically used to evaluate the presence of residual nuclear material or cytoplasmic debris because of the technical complexity and expense of these procedures for routine work [79]. Schmitt et al. [80] studied the cell viability, cell migration parameters, and biomechanical properties of human adipose-derived stem cells following reseeding on human tendon scaffolds in vivo using bioluminescent imaging and immunohistochemistry. They showed that reseeded cells remained viable on the implanted constructs at up to 4 weeks using bioluminescent imaging, and histological evaluation showed host cell invasion and proliferation of the repair sites. Biomechanical testing revealed no significant difference in ultimate load to failure and a 2-mm gap force. This movement of host cells and human adipose-derived stem cells into the scaffold suggests the biointegration of the allograft. Lin et al. [81] evaluated the biocompatibility of the decellularized scaffolds using enzyme–detergent methods for the cell removal of mouse skeletal muscle tissue by histological staining (sections stained with H&E, Van Gieson’s, and DAPI) and DNA quantification. They showed that mouse skeletal muscles combined with an enzyme–detergent mixture (trypsin and Triton X-100) can yield an intact matrix devoid of cells, depleting more than 93% of the nuclear component and exhibiting comparable biomechanical properties to those of native tissue, and that infiltration of inflammatory cells increased into the scaffold initially and then decreased gradually until day 30. Although the above methods provide important information regarding the effectiveness of decellularization methods, the biologic consequences of small amounts of nuclear material or cytoplasmic debris within the decellularized scaffold are unclear. Crapo et al. [57] suggested that a standard for tissue decellularization might provide numerous benefits, including (1) allowing investigators and ECM product manufacturers to evaluate the effectiveness of a protocol when reporting new decellularization techniques or describing products composed of ECM derived from decellularized tissue; (2) enabling congruous comparison of different ECM products; (3) eliminating variations in cell and host responses to ECM products caused by variations in residual DNA, thereby facilitating interpretation and comparison of in vitro and in vivo results; and (4) promoting the rapid and effective development of additional clinical applications for ECM products within the field of regenerative medicine and tissue engineering.

The decellularization methods involve a wide variety of chemicals used because of their capability to damage cells. The strong chemicals can thoroughly remove the cell components, but the internal fiber scaffold structure may also be seriously damaged, whereas mild chemicals can preserve the internal fiber scaffold structure. However, the removal effect of cell components is far from satisfying, which is the messy problem of chemical extraction. The presence of some residual decellularization agents within ECM biomaterials is toxic to host cells and may be responsible for discouraging cellular ingrowth [43, 82]. There is a need for the development of assays to quantify the presence of residual chemicals in the decellularized scaffold material and the removal methods of residual chemicals.