Enzymatic biologic agents include proteases (e.g., trypsin, dispases), esterases (phospholipase A2), and nucleases (e.g., DNase, RNase), and are advantageous because of their specificity for biologic substrate.
Trypsin selectively cleaves cell adherent proteins, on the carboxyl side of the amino acids arginine or lysine to detach cells from the tissue surface. Trypsin has been shown to be effective for completely decellularizing aortic valves and porcine skin, but long incubation times damage the collagen matrix (Brown et al., 2011; Prasertsung et al., 2008; Meyer et al., 2006).
Dispase II is a bacterial protease that selectively cleaves fibronectin and collagen IV in the basement membrane zone of the skin, to separate epithelial sheets from the substratum (Stenn et al., 1989; Gonzalez-Andrades et al., 2011). Dispase II can effectively decellularize porcine skin, with minimal degradation of collagen and laminin, but usually requires associated treatment with other agents such as detergent or trypsin to achieve adequate decellularization (Walter et al., 1998; Prasertsung et al., 2008).
DNase and RNase are endonucleases that hydrolyze deoxyribonucleotide and ribonucleotide chains, respectively. Typically, these enzymatic agents are added to detergent treatments if decellularization criteria are not initially met with the detergent alone, to help remove residual DNA (Grauss et al., 2005; Fitzpatrick et al., 2010; Liao et al., 2007; Rieder et al., 2004). When a 24-hour SDS treatment of rat aortic valves still had remaining nuclei, Grauss et al. (2005) added an additional 1-hour step of DNase (20 μg/mL) and RNase (0.2 mg/mL) to produce a completely acellular material based on hematoxylin and eosin staining. Major structural components (elastin, collagen, GAGs) were retained, but with loss of chondroitin sulfate and fibronectin.