Engineering of Vascular Grafts With Genetically Modified Bone Marrow Mesenchymal Stem Cells on Poly (Propylene Carbonate) Graft

Abstract

Bone marrow mesenchymal stem cells (MSCs) have demonstrated their pluripotency to differentiate into different cell lineages and may be an alternative cell source for vascular tissue engineering. The objective of this study is to create small diameter vessels by seeding and culture of genetically modified MSCs onto a synthetic polymer scaffold produced by an electrospinning technique. A tubular scaffold (2 mm in diameter) with a microstructure of nonwoven fibers was produced by electrospinning of poly (propylene carbonate) (PPC). Rat MSCs obtained from bone marrow were expanded in culture and modified with vasculoprotective gene endothelial nitric oxide synthase (eNOS) or marker gene green fluorescent protein (GFP). These MSCs were seeded onto the electrospun fibrous grafts (internal diameter = 2 mm), and cultured in 5% CO(2) at 37 degrees C. The growth of MSCs in the scaffold was analyzed with scanning electron microscopy (SEM) and hematoxylin and eosin (H&E) staining. The gene transfer and transgenic gene expression were examined with fluorescence-activated cell sorting (FACS), immunochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR), and western blot. The production of nitric oxide (NO) by the engineered vessels was measured with an NO detection kit. Our data showed that the seeded cells integrated with the microfibers of the scaffold to form a three-dimensional cellular network, indicating a favorable interaction between this synthetic PPC scaffold with MSCs. High transduction efficiency was obtained with the use of concentrated retrovirus in the gene transfection of MSCs. The eNOS gene transcripts and protein were detected in the grafts seeded with eNOS-modified MSCs by RT-PCR and immunochemical staining. The amount of NO produced by grafts seeded with eNOS-modified MSCs was comparable to that produced by native blood vessels, and it was significantly higher than that in the grafts seeded with nonmodified MSCs. In summary, the vascular graft produced by culture of eNOS gene-modified MSCs onto the electrospun tubular scaffolds shows promising results in terms of function. The use of MSCs and therapeutic genes in tissue engineering of blood vessels could be helpful in improving vessel regeneration and patency.

Porous Polymeric Bioresorbable Scaffolds for Tissue Engineering

“…Histochemical staining of these cross-sections gives the possibility to study cell infiltration into the porous structure which is a key issue for the integration of the implanted material into the recipient tissue [139,146,147].…”

“…-Western Blotting for semiquantative detection of specific protein expression [139,141,142]. After cell detachment from ES scaffold, proteins are extracted from cells and they are separated by using gel elecrophoresis.…”

“…-Real Time Polymerase Chain Reduction (PCR) for determining the abundance of different RNA molecules within the cells in order to assess regulation of gene expression [137,[139][140][141].…”

“…The process was invented at the beginning of 1900 by Cooley and Morton [136,137] and it was mainly developed thanks to the contribution of Formhals' patents [138][139][140][141][142] for textile applications. However, only at the beginning of this century ES began to be used for scaffold fabrication [143][144][145][146][147] and nowadays it is considered a powerful technique that is employed to this aim by many researchers all around the world.…”

Tissue‐Engineered Blood Vessels With Endothelial Nitric Oxide Synthase Activity

“…16 Synthetic polymer [poly(propylene carbonate)] small-diameter (internal diameter: 2 mm) vascular grafts seeded with MSCs transfected with eNOS gene showed the potential for patency improvement in vitro. 43 Thus, TEVGs with hybrid polymer (PGA/PLCL) scaffolds and BMCs could be used to replace diseased blood vessels with small diameter.…”