Adipose-derived stem cells promote angiogenesis and tissue formation for in vivo tissue engineering

Abstract

Adult mesenchymal stem cells secrete a variety of angiogenic cytokines and growth factors, so we proposed that these paracrine mechanisms may be used to promote vascularization and growth for tissue engineering in vivo. We tested whether or not human adipose-derived stem cells (ASCs) promote tissue formation in rats. ASCs were evaluated in vitro for mRNA expression of angiogenic factors, including the vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8 (IL-8), and stromal cell-derived factor-1 (SDF-1) and proliferative activity on human microvascular endothelial cells. For in vivo analysis, CM-DiI-labeled ASCs were implanted with a rat cardiac extracellular matrix (ECM) extract-derived hydrogel into a chamber with a femoral arteriovenous loop in the groin of male nude rats for 7 days. Vascularization in newly generated tissue was estimated by histomorphometry after endothelial nitric oxide synthase (eNOS) immunostaining. ASCs expressed growth factor mRNA and produced an angiogenic activity in vitro. After implantation, ASCs survived, but remained suspended in the ECM and relatively few were incorporated into the newly formed tissue. The volume of newly generated tissue was significantly higher in chambers containing ASCs and it was enriched with vasculature when compared with the ECM alone. We conclude that human ASCs promote tissue growth and angiogenesis in the rat vascularized chamber, thereby showing promise for tissue-engineering applications for regenerative therapy.

Figures

FIG. 1.

mRNA expression of candidate angiogenic factors in adipose-derived stem cells (ASCs) on matrix substrates. Transcript levels of (a) interleukin-8 (IL-8) show dose-dependent increases in ASCs plated on increasing concentrations of cardiogel. Expression of (b) vascular endothelial growth factor (VEGF), (c) basic fibroblast growth factor (bFGF), and (d) stromal cell-derived factor-1 (SDF-1) was unchanged by treatment. Results are expressed as relative fold change in the mRNA level compared to plastic (mean±standard error of the mean (SEM)). *represents p<0.05 compared to plastic.

FIG. 2.

Endothelial cell proliferation in the medium conditioned by ASCs grown on tissue culture plastic, cardiogel, or fibronectin was greater than in the unconditioned (negative control) medium, and was at least that seen in the high serum (positive control) medium. * represents p<0.05 compared to the negative control.

FIG. 3.

Schematic of the in vivo study design. Four experimental groups were made: alginate only, cardiogel only, alginate with ASCs, and cardiogel with ASCs (n=6 each, N=24 total). Tissues were taken 7 days later for histological and morphometric assessment. Color images available online at www.liebertpub.com/tea

FIG. 4.

Photomicrographs of hematoxylin and eosin (HE) stain (upper panels) and CM-DiI fluorescence (lower panels) of the chamber without (left) and with (right) ASCs (arrows). Tissue was categorized into new tissue, gel, arterio-venous (AV) loop, and fibrin (middle panels) (scale bar=1 mm). Higher power micrographs (bottom panels) show the DiI fluorescence predominantly localized to the gel space (G) (F, fibrin; N, new tissue; A, AV loop; scale bar=175 μm). Color images available online at www.liebertpub.com/tea

FIG. 5.

Stereological analysis showing percentage volume of the constructs. In the groups with ASCs, percentage volume of the new tissue was significantly increased [p<0.05, analysis of variance (ANOVA)].

FIG. 6.

(a) Photomicrographs of newly formed tissue with endothelial nitric oxide synthase immunohistochemistry without (left) and with (right) ASCs (scale bar=50 μm). (b) Stereological analysis showing vascularization in the newly formed tissue (mean±SEM). In the groups with ASCs, percentage vascularization in newly formed tissue were significantly higher than without ASCs (p<0.05, ANOVA). Color images available online at www.liebertpub.com/tea