Formation of corporal tissue architecture in vivo using human cavernosal muscle and endothelial cells seeded on collagen matrices.

摘要

We explored the feasibility of developing corporal tissue, consisting of human cavernosal smooth muscle and endothelial cells in vivo, using three-dimensional acellular collagen matrices, which are similar in architecture to native corpora. Acellular collagen matrices were derived from processed donor rabbit corpora, using cell lysis techniques. Human corpus cavernosal muscle and endothelial cells were seeded on the acellular matrices. A total of 80 matrices, 20 without cells and 60 with cells, were implanted subcutaneously in athymic mice. An additional 36 matrices seeded with cells were maintained in culture for up to 4 weeks. Hydroxyproline quantification, Western blot analysis, RT-PCR, and scanning electron microscopy of the matrices, with and without cells, were performed at various time points. Animals were killed 3 days and 1, 2, 3, 4, 6, and 8 weeks after implantation. Immunocytochemical and histological analyses were performed to confirm the muscle and endothelial phenotype. Organ bath studieswere performed in order to determine the degree of tissue contraction. Western blot analysis detected alpha-actin, myosin, and tropomyosin proteins from human corporal smooth muscle cells. Expression of muscarinic acetylcholine receptor (mAChR) subtype m4 mRNA was demonstrated by RT-PCR from corporal muscle cells before and 8 weeks after seeding. The implanted matrices showed neovascularity into the sinusoidal spaces by 1 week after implantation. Increasing organization of smooth muscle and endothelial cells lining the sinusoidal walls was observed at 2 weeks and continued with time. The matrices were covered with the appropriate cell architecture 4 weeks after implantation. The matrices showed a stable collagen concentration over 8 weeks, as determined by hydroxyproline quantification. Immunocytochemical studies using alpha-actin and factor VIII antibodies confirmed the presence of corporal smooth muscle and endothelial cells, both in vitro and in vivo, at all time points. There was no evidence of cellular organization in the control matrices. Organ bath studies showed that the cell-seeded corporal tissue matrices responded to electrical field stimulation, whereas the unseeded implants failed to respond. This study demonstrates that human cavernosal smooth muscle and endothelial cells seeded on three-dimensional acellular collagen matrices derived from donor corpora are able to form well-vascularized corporal tissues in vivo.