Tissue-engineered graft constructed by self-derived cells and heterogeneous acellular matrix

Abstract

Background: Endothelial and smooth muscle cells were used as seeding cells and heterogeneous acellularized matrix was used as scaffold to construct the tissue-engineered graft.

Methods: A 2 weeks piglet was selected as a donor of seeding cells. Two-centimetre length of common carotid artery was dissected. Endothelial cells and smooth muscle cells were harvested by trypsin and collagenase digestion respectively. The isolated cells were cultured and expanded using routine cell culture technique. An adult sheep was used as a donor of acellularized matrix. The thoracic aorta was harvested and processed by a multi-step decellularizing technique to remove the original cells and preserve the elastic and collagen fibers. The cultured smooth muscle cells and endothelial cells were then seeded to the acellularized matrix and incubated in vitro for another 2 weeks. The cell seeded graft was then transplanted to the cell-donated piglet to substitute part of the native pulmonary artery.

Results: The cultured cells from piglet were characterized as endothelial cells by the presence of specific antigens vWF and CD31, and smooth muscle cells by the presence of specific antigen alpha-actin on the cell surface respectively with immunohistochemical technique. After decellularizing processing for the thoracic aorta from sheep, all the cellular components were extracted and elastic and collagen fibers kept their original morphology and structure. The maximal load of acellular matrix was decreased and 20% lower than that of untreated thoracic aorta, but the maximal tensions between them were not different statistically and they had similar load-tension curves. Three months after transplantation, the animal was sacrificed and the graft was removed for observation. The results showed that the inner surfaces of the graft were smooth, without thrombosis and calcification. Under microscopy, a great number of growing cells could be seen and elastic and collagen fibers were abundant.

Conclusion: Cultured self-derived endothelial and smooth muscle cells could be used as seeding cells and heterogeneous acellularized matrix could be used as scaffold in constructing tissue-engineered graft.

Fig. 1

Seven days primary cultured ECs. The cells were polygonal or round-liked and formed a confluent layer, exhibiting a slabstone or cobblestone-liked morphology (LM×40)

Fig. 2

Immunohistochemical analysis showed the positive reaction of the cultured cells to CD31 monoclonal antibody which is specific to ECs (LM×100)

Fig. 3

Immunohistochemical analysis showed the positive reaction of the cultured cells to vWf monoclonal antibody which is specific to ECs (LM×200)

Fig. 4

Seven days primary cultured SMCs. The cells were fusiform-liked and showed the “hills and valleys” phenotype (LM×100)

Fig. 5

Immunohistochemical analysis showed the positive reaction of the cultured cells to α-actin monoclonal antibody which is specific to SMCs (LM×100)

Fig. 6

The decellularized matrix, no cells remained, but the collagen and elastic fibers kept their original porous morphology and structure, HE staining (LM×40)

Fig. 7

Seven days after cells seeding, many SMC-liked cells could be seen in the decellularized matrix, but the arrangement of cells were not very regular, HE staining (LM×40)

Fig. 8

Seven days after cells seeding, ECs-liked cells could be seen at the surface of the decellularized matrix. Silver nitrate staining (LM×40)

Fig. 9

Three months after transplantation, the inner surface of the graft was smooth and was laied with endothelial-like tissue. Neither thrombosis nor calcification could be seen

Fig. 10

Three months after transplantation, many growing cells could be seen in the graft. Right upper graph was the neighboring pulmonary artery tissue. HE staining (LM×40)

Fig. 11

Three months after transplantation, elastic fibers in the graft were abundant. Right upper graph was the neighboring pulmonary artery tissue and right lower graph was the acellularized matrix. Elastic staining (LM×40)