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. 2018 Mar;12(3):e1325-e1336.
doi: 10.1002/term.2512. Epub 2017 Nov 27.

Prevascularization of natural nanofibrous extracellular matrix for engineering completely biological three-dimensional prevascularized tissues for diverse applications

Lijun Zhang  1   2 , Zichen Qian  2 , Mitchell Tahtinen  2 , Shaohai Qi  1 , Feng Zhao  2
Affiliations

Prevascularization of natural nanofibrous extracellular matrix for engineering completely biological three-dimensional prevascularized tissues for diverse applications

Lijun Zhang et al. J Tissue Eng Regen Med. 2018 Mar.

Abstract

Self-sustainability after implantation is one of the critical obstacles facing large engineered tissues. A preformed functional vascular network provides an effective solution for solving the mass transportation problem. With the support of mural cells, endothelial cells (ECs) can form microvessels within engineered tissues. As an important mural cell, human mesenchymal stem cells (hMSCs) not only stabilize the engineered microvessel network, but also preserve their multi-potency when grown under optimal culture conditions. A prevascularized hMSC/extracellular matrix (ECM) sheet fabricated by the combination of hMSCs, ECs and a naturally derived nanofibrous ECM scaffold offers great opportunity for engineering mechanically strong and completely biological three-dimensional prevascularized tissues. The objective of this study was to create a prevascularized hMSC/ECM sheet by co-culturing ECs and hMSCs on a nanofibrous ECM scaffold. Physiologically low oxygen (2% O2 ) was introduced during the 7 day hMSC culture to preserve the stemness of hMSCs and thereby their capability to secrete angiogenic factors. The ECs were then included to form microvessels under normal oxygen (20% O2 ) for up to 7 days. The results showed that a branched and mature vascular network was formed in the co-culture condition. Angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiopoietin-1 (Ang-1) were significantly increased by low-oxygen culture of hMSCs, which further stabilized and supported the maturation of microvessels. A differentiation assay of the prevascularized ECM scaffold demonstrated a retained hMSC multi-potency in the hypoxia cultured samples. The prevascularized hMSC/ECM sheet holds great promise for engineering three-dimensional prevascularized tissues for diverse applications.

Keywords: angiogenesis; extracellular matrix scaffold; mesenchymal stem cell; microvessel; nanofibrous scaffold; prevascularization.

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Figures

Figure 1
Figure 1
Vasculature formation on ECM scaffolds. a) Low and high magnification images of microvessels at different time points. The hypoxia groups (H-groups) had higher density and more branches than the normoxia groups (N-groups) at day 5 and day 7. The microvessels were stained with CD31 primary antibody (green). The cell nuclei were stained with DAPI (blue). Quantification of microvessel total (b) and average (c) length, branches (d), surface area fraction (e), lumens (f), and average diameter (g) for both H-groups and N-groups. Hypoxia enhanced the vessel structures in all categories. (*p < 0.05, **p < 0.01)
Figure 2
Figure 2
Cell proliferation in both H-groups and N-groups at day 7. a) Ki 67 (red) staining showed that more active cells were present after hypoxia culture. b) BrdU (green) positive staining showed more proliferative cells surrounding the microvessels in the H-group. Vessels were labeled with CD31 (red). c) Quantification of Ki 67 showed that the active cell ratio was significantly higher in the H-group than the N-group. d) Quantification of BrdU showed that the proliferating cell ratio was significantly higher in the H-group. (**p < 0.01)
Figure 3
Figure 3
(a) Dextran permeability assay of hypoxia and normoxia samples at day 7. Accumulated dextran dye was observed surrounding the boundary of the microvessels in hypoxia samples, as pointed by the yellow arrows. In comparison, dextran particles were found inside the microvessels in normoxia samples. (b) Confocal images of α-SMA-expressing hMSCs around the vasculatures. The side view of the vasculature showed α-SMA-expressing hMSCs supported the microvessel lumen in the H-groups, but not in the N-groups. Immunofluorescent staining of CD146 pericyte marker for H-group and N-group (c) demonstrated that both groups have positively stained vessel structure. CD146 expression (d) was performed by western blot and quantified (e) to show that more CD146 was expressed in the H-group. (*p < 0.05)
Figure 4
Figure 4
Secretion of angiogenic growth factors (a) VEGF, (b) bFGF, (c) TGF-β1, (d) Ang-1, and (e) Ang-2 at day 7. The concentrations of VEGF, bFGF, and Ang-1 were increased in H-groups (only when ECM was present), while the TGF-β1 and Ang-1 (ECM absent) decreased in the H-groups. (*p < 0.05, **p < 0.01)
Figure 5
Figure 5
Effects of hypoxia on angiogenic growth factor secretion (a–c) and gene expression (d–h) on pre-vascularized samples at day 7. The gene expression of VEGFA, bFGF, and HIF-1α were significantly higher in the H-groups. The protein secretion of VEGFR2 was significantly higher in the H-groups. But gene expression of VEGFR2 was lowered at day 7. (**p < 0.01)
Figure 6
Figure 6
Osteogenic differentiation (14 days) and adipogenic differentiation (21 days) of hMSCs after vascularization. a) Von Kossa staining of the differentiated groups and undifferentiated control. b) Quantification of ALP activity. c) Quantification of Ca2+ concentration. d) Expression of osteogenic and stemness genes. The hypoxia samples showed enhanced ALP activity and calcium deposition as well as higher osteogenic gene expression. e) Oil red O staining of the lipid droplet after the adipogenic differentiation of hMSCs. More lipid droplets were visualized in the H-group. f) Quantification of Oil red O staining. Hypoxia significantly enhanced the oil red ratio. g) Gene expression of adipogenic differentiation genes and stemness genes. Expression of adipogenic genes was enhanced in the hypoxia samples. (*p < 0.05, **p < 0.01)
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References

    1. Aguirre A, Planell JA, Engel E. Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis. Biochem Biophys Res Commun. 2010;400(2):284–291. - PubMed
    1. Akhurst RJ, Lehnert SA, Faissner A, Duffie E. TGF beta in murine morphogenetic processes: the early embryo and cardiogenesis. Development. 1990;108(4):645–656. - PubMed
    1. Armulik A, Abramsson A, Betsholtz C. Endothelial/pericyte interactions. Circ Res. 2005;97(6):512–523. - PubMed
    1. Au P, Tam J, Fukumura D, Jain RK. Bone marrow–derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood. 2008;111(9):4551–4558. - PMC - PubMed
    1. Barati D, Shariati SRP, Moeinzadeh S, Melero-Martin JM, Khademhosseini A, Jabbari E. Spatiotemporal release of BMP-2 and VEGF enhances osteogenic and vasculogenic differentiation of human mesenchymal stem cells and endothelial colony-forming cells co-encapsulated in a patterned hydrogel. J Controlled Release. 2016;223:126–136. - PMC - PubMed

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