Decellularized tissue-engineered blood vessel as an arterial conduit

Abstract

Arterial tissue-engineering techniques that have been reported previously typically involve long waiting times of several months while cells from the recipient are cultured to create the engineered vessel. In this study, we developed a different approach to arterial tissue engineering that can substantially reduce the waiting time for a graft. Tissue-engineered vessels (TEVs) were grown from banked porcine smooth muscle cells that were allogeneic to the intended recipient, using a biomimetic perfusion system. The engineered vessels were then decellularized, leaving behind the mechanically robust extracellular matrix of the graft wall. The acellular grafts were then seeded with cells that were derived from the intended recipient--either endothelial progenitor cells (EPC) or endothelial cell (EC)--on the graft lumen. TEV were then implanted as end-to-side grafts in the porcine carotid artery, which is a rigorous testbed due to its tendency for graft occlusion. The EPC- and EC-seeded TEV all remained patent for 30 d in this study, whereas the contralateral control vein grafts were patent in only 3/8 implants. Going along with the improved patency, the cell-seeded TEV demonstrated less neointimal hyperplasia and fewer proliferating cells than did the vein grafts. Proteins in the mammalian target of rapamycin signaling pathway tended to be decreased in TEV compared with vein grafts, implicating this pathway in the TEV's resistance to occlusion from intimal hyperplasia. These results indicate that a readily available, decellularized tissue-engineered vessel can be seeded with autologous endothelial progenitor cells to provide a biological vascular graft that resists both clotting and intimal hyperplasia. In addition, these results show that engineered connective tissues can be grown from banked cells, rendered acellular, and then used for tissue regeneration in vivo.

Fig. 1.

Tissue-engineered vessel from porcine SMC. (A) Porcine tissue-engineered vessel after 10 wk in culture. (B) Histology of the tissue-engineered vessel by H&E staining. H&E of the tissue-engineered vessel before (C) and after (D) decellularization with a loss of nuclei. Masson's Trichrome stain before (E) and after (F) decellularization demonstrating preservation of collagen (blue) throughout the matrix. (Scale bars: B, 500 μm; C–F, 100 μm.)

Fig. 2.

Characterization of porcine EPCs from peripheral blood. (A) EPCs exhibit typical cobblestone endothelial cell morphology. Immunofluorescent detection of CD31 (B), CD144 (C), vWF (D) demonstrate endothelial cell phenotype. Functional properties of the EPCs included incorporation of aLDL (E) and formation of capillary-like tubes on Matrigel (F). (Scale bars: 50 μm.) Flow cytometry is positive for CD31 (G) and negative for CD45 (H) (black peak is antibody stained). (I) EPC-derived EC and differentiated aortic EC response to shear stress on a decellularized porcine artery by immunoblotting for eNOS protein: low shear = 1 dyne/cm²; high shear = 15 dynes/cm².

Fig. 3.

Implantation of vessels. (A) TEV anastomosed as an end-to-side bypass in the porcine carotid artery. (B) Angiogram of a vein graft (left, arrows at anastomoses) and an EPC-seeded TEV (right, arrowheads at anastomoses) 30 d after implantation. (C) Low magnification cross-section of an explanted EPC-seeded engineered vessel with cellular repopulation of the matrix. Patent vein graft (D) and an occluded vein graft (E), both demonstrating intimal hyperplasia. Immunohistochemical staining of explanted cell-seeded grafts for vWF on the luminal surface (arrow) (F), SMC α-actin expressing cells (arrow) on the periphery of the residual engineered matrix (G), and vimentin-positive cells (arrow) populating the residual graft matrix (H). (I) CD45 (arrow) stain for leukocytes. (J) The preimplant engineered matrix seeded with GFP⁺-labeled EPCs. After 30 d of implantation, GFP⁺-labeled EPCs were detected on the lumen of the engineered vessel (K). (Scale bars: C–E, 500 μm; F–K, 100 μm.)

Fig. 4.

Characterization of explanted grafts. The luminal area (A) and intimal area (B) were measured for the cell-seeded tissue-engineered vessels (TEV) (n = 5) and the vein grafts (VG) [n = 6, patent vein grafts (n = 3) and occluded by intimal hyperplasia (n = 3)]. (C) Number of PNCA+ nuclei per high power field of the engineered graft neointima and vessel wall compared with vein graft intima and media. Immunoblots of engineered cell-seeded vessels (n = 5) or vein grafts (n = 6) for phosphorylated p70S6K and quantified and normalized by β-actin expression (D and E). TEV, 3,920 ± 1,096 OD; VG, 14,124 ± 4,795 OD. Data represents mean ± SEM.