Development of a porcine bladder acellular matrix with well-preserved extracellular bioactive factors for tissue engineering.
In this study, we compared four decellularization protocols and finally developed an optimized one through which a porcine bladder acellular matrix (BAM) with well-preserved extracellular bioactive factors had been prepared. In this protocol, the intact bladder was treated with trypsin/ethylenediaminetetraacetic acid to remove the urothelium, then with hypotonic buffer and Triton X-100 in hypertonic buffer to remove the membranous and cytoplasmic materials, and finally with nuclease to degrade the cellular nuclear components. Bladder distention and mechanical agitation were simultaneously used to facilitate cell removal. Meanwhile, several preservative techniques, including limitation of wash time, supplement with inhibitors of proteinase, control of the pH value and temperature of the wash buffer, ethylene oxide sterilization, and lyophilization of the scaffold for storage, were used to protect the extracellular bioactive factors. This decellularization protocol had completely removed the cellular materials and well preserved the extracellular collagen, sulfated glycosaminoglycan (GAG), and bioactive factors. The preserved bioactive factors had a great potential of promoting the proliferation and migration of both human bladder smooth muscle cell and human umbilical vein endothelial cell. It was also found that the amount of two representative bioactive factors, platelet-derived growth factor BB and vascular endothelial growth factor, was positively correlated with the sulfated GAG content in the porcine BAM, implying that the amount of sulfated GAG might be a determinant for preservation of bioactive factors in the decellularized tissues. In conclusion, the porcine BAM with well-preserved extracellular bioactive factors might be a favorable scaffold for tissue engineering applications.
Yang
Bin
B
Department of Urology, Affiliated Drum Tower Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu, PR China.
Zhang
Yifen
Y
Zhou
Liuhua
L
Sun
Zeyu
Z
Zheng
Junhua
J
Chen
Yun
Y
Dai
Yutian
Y
D000818:Animals; D002465:Cell Movement; D049109:Cell Proliferation; D017077:Culture Media, Conditioned; D004247:DNA / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D007150:Immunohistochemistry; D013552:Swine; D023822:Tissue Engineering; D001743:Urinary Bladder / Q000378:metabolism
D017077:Culture Media, Conditioned; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D004247:DNA