Helliwell JA, Thomas DS, Papathanasiou V, Homer-Vanniasinkam S, Desai A, et al.
Journal of tissue engineering •
The aim of this study was to adapt a proprietary decellularisation process for human dermis for use with porcine skin. Porcine skin was subject to: sodium chloride (1 M) to detach the epidermis, trypsin paste to remove hair follicles, peracetic acid (0.1% v/v) disinfection, washed in hypotonic buffer and 0.1% (w/v) sodium dodecyl sulphate in the presence of proteinase inhibitors followed by nuclease treatment. Cellular porcine skin, decellularised porcine and human dermis were compared using histology, immunohistochemistry, GSL-1 lectin (alpha-gal epitope) staining, biochemical assays, uniaxial tensile and in vitro cytotoxicity tests. There was no microscopic evidence of cells in decellularised porcine dermis. DNA content was reduced by 98.2% compared to cellular porcine skin. There were no significant differences in the biomechanical parameters studied or evidence of cytotoxicity. The decellularised porcine dermis retained residual alpha-gal epitope. Basement membrane collagen IV immunostaining was lost following decellularisation; however, laminin staining was retained.
Derham C, Yow H, Ingram J, Fisher J, Ingham E, et al.
Tissue engineering. Part A •
This study aimed to investigate a biocompatible, biomechanically functional, small-diameter (<6 mm) scaffold for tissue engineering a vascular graft using acellular porcine ureters. Porcine ureters were decellularized and sterilized using sequential treatment with hypotonic Tris buffer, sodium dodecyl sulphate 0.1% w/v (plus proteinase inhibitors), nuclease solution (RNase and DNase), and peracetic acid. The scaffold was compared with fresh ureter according to histology, immunocytochemistry, quantitative determination of alpha-galactosyl (alpha-Gal), and biochemistry. The biomechanical properties of the scaffold were compared with those of fresh ureters and human saphenous vein. The biocompatibility of decellularized ureters was assessed using in vitro contact and extract cytotoxicity tests. The in vivo biocompatibility was investigated using a mouse model. The histioarchitecture of the acellular ureteric scaffolds was preserved with some loss of basement membrane proteins while showing no evidence of cellularity. There was no evidence of residual alpha-Gal epitope present in acellular ureter. The ultimate tensile strength, compliance, and burst pressures of the acellular ureters were not compromised, compared with fresh tissues (p > 0.05), and the results compared favorably with fresh human saphenous vein samples (p > 0.05). The decellularized scaffolds were shown to be biocompatible with porcine smooth muscle and endothelial cells in vitro. One month after subcutaneous implantation in mice, explants were analyzed immunohistochemically using anti-CD3, Factor VIII, F4/80 (macrophage), and alpha-smooth muscle actin antibodies. The fresh tissue controls had a significantly thicker capsule (of inflammatory cells and fibrous tissue) than decellularized implants (p < 0.05). Decellularized explants were infiltrated with a combination of fibroblast-like cells and macrophages, indicating a healthy repair process. This study has demonstrated the potential of acellular porcine ureteric scaffolds in tissue engineering small-diameter living vascular grafts.
