Tissue-engineered blood vessel substitute by reconstruction of endothelium using mesenchymal stem cells induced by platelet growth factors Matheus Bertanha,

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Presentation transcript:

Tissue-engineered blood vessel substitute by reconstruction of endothelium using mesenchymal stem cells induced by platelet growth factors Matheus Bertanha, MSc, Andrei Moroz, PhD, Rodrigo Almeida, BSc, Flavia Cilene Alves, PhD, Michele Janegitz Acorci Valério, PhD, Regina Moura, PhD, Maria Aparecida Custódio Domingues, PhD, Marcone Lima Sobreira, PhD, Elenice Deffune, PhD Journal of Vascular Surgery Volume 59, Issue 6, Pages 1677-1685 (June 2014) DOI: 10.1016/j.jvs.2013.05.032 Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 1 Mesenchymal stem cells (MSCs) morphology observed at phase-contrast inverted microscopy and characterization by flow cytometry. A, Initial cell adhesion to the culture flasks (arrows), low confluence. MSCs features fibroblastic-like morphology, well-defined nucleus, and two nucleoli (scale bar = 50 μm). B, Cells proliferated and colonies are obtained, featuring the same morphologic characteristics (scale bar = 100 μm). C, After the third passage, cells are cultivated until 100% confluence for CD90 cell characterization (scale bar = 200 μm). D, CD90 positive cells, recognized as MSCs, constitute all cells in the gated area (M1) of the flow cytometry analysis. Journal of Vascular Surgery 2014 59, 1677-1685DOI: (10.1016/j.jvs.2013.05.032) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 2 Native and sodium dodecyl sulfate (SDS)-decellularized vein fragments after histologic processing and Masson's trichrome staining. A, Naturally occurring vein morphology with lumen (*), well preserved endothelial cells (arrows), smooth muscle cells, and multiple nuclei at the inner collagenous tissue (arrowheads) (scale bar = 120 μm). B, Decellularized vein morphology displaying its lumen (*), cell-free endothelium (arrows), and cell-free inner collagenous tissue (crosses). Note that the thick red layer on A, comprised by endothelial cells and smooth muscle cells, is totally absent on the decellularized vein fragment. All cells (rejection inductor) were successfully removed (scale bar = 120 μm). Journal of Vascular Surgery 2014 59, 1677-1685DOI: (10.1016/j.jvs.2013.05.032) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 3 Immunofluorescence analysis of the bioscaffold seeded with mesenchymal stem cells (MSCs) after 60 minutes of initial seeding. MSCs were stained with Qtracker 605 Cell Labeling Kits (Molecular Probes; Life Technologies) and diluted at the PuraMatrix peptide hydrogel (BD Biosciences). The stained cells/PuraMatrix solution was applied at the lumens of the decellularized vein scaffolds, with repetitive pipetting. A, Note the accumulation of cells lined in the vessel lumen wall (arrow) (scale bar = 300 μm). B, Again, cells lined on the lumen (arrow) can be seen alongside with cells in the inner portions of the scaffold (arrowheads) (scale bar = 300 μm). C, A transversal section of the scaffold showing cells inside the PuraMatrix peptide solution inside the vessel lumen can be seen (*; scale bar = 100 μm). D, Another section showing cells lined in the lumen (arrow) and inside the inner collagenous portion of the scaffold (arrowhead) (scale bar = 100 μm). Journal of Vascular Surgery 2014 59, 1677-1685DOI: (10.1016/j.jvs.2013.05.032) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 4 Mesenchymal stem cells (MSCs) seeded in decellularized vein scaffolds, cultivated with endothelial inductor growth factor (EIGF) supplementation, observed at phase-contrast inverted microscopy. A and B, Note the initial cells adhering in the lumen of the decellularized scaffold (arrows) (scale bar = 20 μm). C and D, After 1 week of cell cultivation, the initial adhering cells seen at the previous figures presents a morphology shift, from rounded-shaped cells to scamous/flattened epithelial-shaped cells (arrowheads) (scale bar = 40 μm). Journal of Vascular Surgery 2014 59, 1677-1685DOI: (10.1016/j.jvs.2013.05.032) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 5 Histologic sections of mesenchymal stem cell (MSC)-seeded vein fragments after the third week of endothelial differentiation using endothelial inductor growth factor (EIGF) supplementation. A, Cells with evident endothelial cell morphology lined on the lumen of the decellularized scaffolds (arrows) (low magnification; hematoxylin-eosin staining; scale bar = 20 μm). B, Transversal section showing a capillary-like structure (*) composed of three endothelial cells (arrowheads) (hematoxylin-eosin staining; scale bar = 20 μm). C, Anti-fascin monoclonal antibody staining of cells lined in the scaffold's lumen (arrows) (scale bar = 10 μm). D, Quantification of the von Willebrand factor (vWF) on the conditioned medium (CM) of cells cultivated in the decellularized scaffolds, with regular cell culture medium and EIGF supplemented medium. While the CM of cells cultivated under regular medium presents low levels of vWF, the CM of cells cultivated with EIGF supplementation presents high detectable levels of this endothelial function marker. Data are expressed as the mean ± standard deviation of seven individual enzyme-linked immunosorbent assay readings. * Statistically significant values with P < .05. Journal of Vascular Surgery 2014 59, 1677-1685DOI: (10.1016/j.jvs.2013.05.032) Copyright © 2014 Society for Vascular Surgery Terms and Conditions