Mitomycin-Treated Endothelial and Smooth Muscle Cells Suitable for Safe Tissue Engineering Approaches

Abstract

In our previous study, we showed that discarded cardiac tissue from the right atrial appendage and right ventricular myocardium is an available source of functional endothelial and smooth muscle cells for regenerative medicine and tissue engineering. In the study, we aimed to find out what benefits are given by vascular cells from cardiac explants used for seeding on vascular patches engrafted to repair vascular defects in vivo. Additionally, to make the application of these cells safer in regenerative medicine we tested an in vitro approach that arrested mitotic division to avoid the potential tumorigenic effect of dividing cells. A tissue-engineered construction in the form of a patch based on a polycaprolactone-gelatin scaffold and seeded with endothelial and smooth muscle cells was implanted into the abdominal aorta of immunodeficient SCID mice. Aortic patency was assessed using ultrasound, MRI, immunohistochemical and histological staining. Endothelial and smooth muscle cells were treated with mitomycin C at a therapeutic concentration of 10 μg/ml for 2 h with subsequent analysis of cell proliferation and function. The absence of the tumorigenic effect of mitomycin C-treated cells, as well as their angiogenic potential, was examined by injecting them into immunodeficient mice. Cell-containing patches engrafted in the abdominal aorta of immunodeficient mice form the vessel wall loaded with the appropriate cells and extracellular matrix, and do not interfere with normal patency. Endothelial and smooth muscle cells treated with mitomycin C show no tumorigenic effect in the SCID immunodeficient mouse model. During in vitro experiments, we have shown that treatment with mitomycin C does not lead to a decrease in cell viability. Despite the absence of proliferation, mitomycin C-treated vascular cells retain specific cell markers, produce specific extracellular matrix, and demonstrate the ability to stimulate angiogenesis in vivo. We pioneered an approach to arresting cell division with mitomycin C in endothelial and smooth muscle cells from cardiac explant, which prevents the risk of malignancy from dividing cells in vascular surgery. We believe that this approach to the fabrication of tissue-engineered constructs based on mitotically inactivated cells from waste postoperative material may be valuable to bring closer the development of safe cell products for regenerative medicine.

Keywords: endothelial cells; mitomycin C; polycaprolactone; smooth muscle cells; tissue-engineered vascular graft; vascular patch.

FIGURE 1

Scanning electron microscopy image of a scaffold slice with a low-permeability layer (A) magnification 1,000X, the inner surface of the scaffold (B) magnification 2.500X.

FIGURE 2

Blood flow and wall thickness in the operated mouse aorta. A,B Ultrasound examination of the linear blood flow velocity in the area of the implanted vascular patch and around it at the 4th (A) and 24th (B) weeks after implantation. At the 4th week after implantation n = 6 for cell-seeded patch, n = 6 for unseeded patch; at the 24th week after implantation n = 6 for cell-seeded patch, n = 3 for unseeded patch. (C) Wall thickness of vascular patches before (n = 6 for cell-seeded patch, n = 6 for unseeded patch) and 24 weeks after implantation (n = 6 for cell-seeded patch, n = 3 for unseeded patch).

FIGURE 3

MRI assessment of aortic patency. “Experiment” and “control” are mouse groups implanted with cell-seeded and unseeded patches, respectively. N = 6 for each group at each time point. Red arrows indicate the patent abdominal aorta, green arrows indicate the patent iliac arteries.

FIGURE 4

Immunofluorescent staining of cell-seeded patches with antibodies to endotheliocyte (hCD31, mCD31) and smooth muscle (SMMHC) cell markers at control points of the experiment. The borders of the patch are shown with a dashed line. Scale bar 50 μm.

FIGURE 5

Immunofluorescent staining of unseeded patches with antibodies to endotheliocyte (mCD31) and smooth muscle (SMMHC) cell markers at control points of the experiment. The borders of the patch are shown with a dashed line. Scale bar 50 μm.

FIGURE 6

Extracellular matrix components in cell-seeded and unseeded patches. The borders of the patch are shown with a dashed line. Scale bar 50 μm.

FIGURE 7

Hematoxylin and eosin staining of cryosectioned patches. Magnification ×100 and 400. 1—inner side of the patch in contact with the bloodstream; 2—the outer side of the patches in contact with the surrounding tissues; 3—low-permeability inner layer; 4—suture material (Premilene 10/0).

FIGURE 8

Proliferation dynamics and viability of MMC-treated (MMC+) and untreated (MMC-) endothelial and smooth muscle cells. (A) XTT values indirectly reflect the number of viable cells. XTT values dynamics during 8 days of cultivation demonstrate the proliferation of MMC + cells and its absence in MMC- cells. N = 9 for each group at each time point. (B) Percentage of viable cells stained with Propidium Iodide and Annexin V according to flow cytometry. For each group, 10⁴ events were estimated.

FIGURE 9

Properties of MMC-treated (MMC+) and untreated (MMC-) endothelial cells. (A) MMC + endothelial cells reveal CD31⁻and vWF-positive staining and produce extracellular matrix (collagen IV- and fibronectin-positive staining). (B) MMC+ and MMC- endothelial cells show no difference in the ability to form capillary-like structures in Matrigel. Scale bar 100 μm.

FIGURE 10

MMC-treated (MMC+) and untreated (MMC-) smooth muscle reveal αSMA- and SMMHC-positive staining and produce extracellular matrix elastin. Scale bar 100 μm.

FIGURE 11

Evaluation of functional properties of MMC + cells in vivo. (A) Visualization of the injected mix (Matrigel and cells stained with the vital dye MitoTracker Deep Red FM) after 14 days with a Kodak In-Vivo Multispectral Imaging System device. N = 2 (on the left). Vasculature in cryosections of the Matrigel plug at day 14 after injection of Matrigel & MMC + endothelial and smooth muscle cells, Matrigel & MMC- endothelial and smooth muscle cells, and Matrigel only (control w/o cells) is detected by isolectin B4 Alexa 594 conjugate staining (red). N = 5 for each group. Scale bar 100 μm. (B) Diagrams representing the quantitative parameters of vessels positive for isolectin B4 Alexa 594 conjugate staining, imaged on a fluorescent microscope in 10 random fields of view.

FIGURE 12

Mitotically inactivated (MMC+) endothelial (EC) and smooth muscle (SMC) cells show no tumorigenic effect in vivo. Histological sections show the absence of a tumorigenic effect 12 weeks after injection of Matrigel with MMC+ (n = 5 for MMC + EC, n = 4 for MMC + SMC) or MMC- cells (n = 5 for MMC- EC, n = 4 for MMC- SMC) or Matrigel only (w/o cells; n = 5). The tumor (shown in red dotted line) is detected only after injection of the highly-tumorigenic HEK293FT cell line (n = 5). Magnification—50X.