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(Circulation. 2006;114:I-193 – I-199.)
© 2006 American Heart Association, Inc.

Cell Transplantation and Tissue Engineering

Transforming Growth Factor-ß1 Modulates Extracellular Matrix Production, Proliferation, and Apoptosis of Endothelial Progenitor Cells in Tissue-Engineering Scaffolds

Virna L. Sales, MD; George C. Engelmayr, Jr, PhD; Bret A. Mettler, MD; John A. Johnson, Jr; Michael S. Sacks, PhD; John E. Mayer, Jr, MD

From the Department of Cardiac Surgery (V.L.S., B.A.M., J.A.J., J.E.M.), Children’s Hospital Boston, Harvard Medical School, Boston, Mass; and Engineered Tissue Mechanics Laboratory (G.C.E., M.S.S.), Department of Bioengineering and McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pa

Correspondence to John E. Mayer, Jr, MD, Department of Cardiac Surgery, Children’s Hospital Boston, 300 Longwood Ave, Boston, MA 02115. E-mail john.mayer{at}cardio.chboston.org

Background— Valvular endothelial cells and circulating endothelial progenitor cells (EPCs) can undergo apparent phenotypic change from endothelial to mesenchymal cell type. Here we investigated whether EPCs can promote extracellular matrix formation in tissue engineering scaffolds in response to transforming growth factor (TGF)-ß1.

Method and Results— Characterized ovine peripheral blood EPCs were seeded onto poly (glycolic acid)/poly (4-hydroxybutyrate) scaffolds for 5 days. After seeding at 2x10⁶ cells/cm², scaffolds were incubated for 5 days in a roller bottle, with or without the addition of TGF-ß1. After seeding at 15x10⁶ cells/cm², scaffolds were incubated for 10 days in a roller bottle with or without the addition of TGF-ß1 for the first 5 days. Using immunofluorescence and Western blotting, we demonstrated that EPCs initially exhibit an endothelial phenotype (ie, CD31⁺, von Willebrand factor⁺, and –smooth muscle actin (SMA)^–) and can undergo a phenotypic change toward mesenchymal transformation (ie, CD31⁺ and -SMA⁺) in response to TGF-ß1. Scanning electron microscopy and histology revealed enhanced tissue formation in EPC-TGF-ß1 scaffolds. In both the 10- and 15-day experiments, EPC-TGF-ß1 scaffolds exhibited a trend of increased DNA content compared with unstimulated EPC scaffolds. TGF-ß1–mediated endothelial to mesenchymal transformation correlated with enhanced expression of laminin and fibronectin within scaffolds evidenced by Western blotting. Strong expression of tropoelastin was observed in response to TGF-ß1 equal to that in the unstimulated EPC. In the 15-day experiments, TGF-ß1–stimulated scaffolds revealed dramatically enhanced collagen production (types I and III) and incorporated more 5-bromodeoxyuridine and TUNEL staining compared with unstimulated controls.

Conclusions— Stimulation of EPC-seeded tissue engineering scaffolds with TGF-ß1 in vitro resulted in a more organized cellular architecture with glycoprotein, collagen, and elastin synthesis, and thus noninvasively isolated EPCs coupled with the pleiotropic actions of TGF-ß1 could offer new strategies to guide tissue formation in engineered cardiac valves.

Key Words: cells • growth substances • valves • vessels