Material-based deployment enhances efficacy of endothelial progenitor cells

Abstract

Cell-based therapies are attractive for revascularizing and regenerating tissues and organs, but clinical trials of endothelial progenitor cell transplantation have not resulted in consistent benefit. We propose a different approach in which a material delivery system is used to create a depot of vascular progenitor cells in vivo that exit over time to repopulate the damaged tissue and participate in regeneration of a vascular network. Microenvironmental conditions sufficient to maintain the viability and outward migration of outgrowth endothelial cells (OECs) have been delineated, and a material incorporating these signals improved engraftment of transplanted cells in ischemic murine hindlimb musculature, and increased blood vessel densities from 260 to 670 vessels per mm(2), compared with direct cell injection. Further, material deployment dramatically improved the efficacy of these cells in salvaging ischemic murine limbs, whereas bolus OEC delivery was ineffective in preventing toe necrosis and foot loss. Finally, material deployment of a combination of OECs with another cell population commonly isolated from peripheral or cord blood, endothelial progenitor cells (EPCs) returned perfusion to normal levels in 40 days, and prevented toe and foot necrosis. Direct injection of an EPC/OEC combination was minimally effective in improving limb perfusion, and untreated limbs underwent autoamputation in 3 days. These results demonstrate that vascular progenitor cell utility is highly dependent on the mode of delivery, and suggest that one can create new vascular beds for a variety of applications with this material-controlled deployment of cells.

Fig. 1.

Proposed cell delivery approach, characterization of cell migration from macroporous alginate scaffolds. (A) Diagram of approach to present cell adhesion ligands (RGD-containing peptides) and local morphogens (VEGF) in the material to maintain cell viability, and to activate and induce cell migration out of scaffold. (B) Phase-contrast micrographs of OECs that have migrated out from scaffolds that contain no VEGF (blank), VEGF₁₂₁, or VEGF₁₆₅ and populated the surrounding tissue mimic (collagen gel) after 72 h. (C) Quantification of OECs populating the collagen matrix when VEGF₁₂₁ was incorporated into scaffolds (gray filled bar), compared with the presentation of VEGF₁₆₅ (open bar), or no VEGF (black filled bar). Values were normalized to the initial cell number placed in scaffolds. (D) Viability of the cells that migrated out from scaffolds with no VEGF (blank), VEGF₁₂₁ or VEGF₁₆₅ in the scaffolds. (E) Proliferation of OECs that had migrated out of scaffolds and were subsequently recovered and placed in culture on tissue culture dishes in the presence of VEGF in the medium. Control OECs that had never been placed in scaffolds were cultured in parallel, maintained in culture in the absence of VEGF stimulation (control) for comparison. Values in C–E represent mean and standard deviations (n = 6). Magnification 200× for all photomicrographs.

Fig. 2.

Analysis of angiogenesis in ischemic hindlimbs after OEC transplantation. (A) Implantation of blank scaffolds, bolus injection of OECs and VEGF (same quantities as placed in scaffolds), transplantation of OECs on scaffolds lacking VEGF (alginate scaffold OEC), and transplantation of OECs on scaffolds presenting VEGF₁₂₁ [alginate scaffold (VEGF) OEC]. Photomicrographs of tissue sections from ischemic hindlimbs of SCID mice at postoperative day 15, immunostained for the mouse endothelial cell marker CD-31 (B), and human CD-31 (C). (D) Quantification of the total blood vessel densities in hindlimb muscle tissue after 2 weeks with bolus injection of VEGF₁₂₁ and OECs (+ − +), scaffold delivery (no VEGF₁₂₁) of OECs (+ + −), or scaffold delivering OECs with VEGF₁₂₁ (+ + +) in SCID mice. (E) Hindlimbs subjected to surgery were also visually examined, and grouped as normal (displaying no discrepancy in color or limb integrity from nonischemic hindlimbs of the same animal), or presenting one necrotic toe, multiple necrotic toes, or a complete necrotic foot. Mean values are presented with standard deviations (n = 6) in both graphs. *, P < 0.05 between conditions.

Fig. 3.

Gross photographs and perfusion images of ischemic hindlimbs as a function of time postsurgery. (A) Limbs with no treatment (blank scaffold), demonstrated precocious and rapid limb necrosis (<3 days) (left-most column), and no perfusion images were obtained. For other conditions, hindlimbs were maintained over time, and perfusion images could be obtained. In all of these conditions, scaffolds presenting RGD ligands and VEGF₁₂₁ were used. The normal baseline (before) perfusion was immediately reduced after unilateral femoral artery ligation (after), and subsequent recovery was tracked as a function of time postsurgery. (B) Total blood vessel densities in hindlimb muscle tissue at six weeks postligation for the various experimental groups. (C) Quantification of hindlimb perfusion for the conditions, including bolus injection of EPC and OEC (inverted filled triangle), EPC transplantation with scaffolds (open square), OEC transplantation with scaffolds (open triangle), and EPC and OEC combined transplantation on scaffolds (filled circle) in SCID mice. (D) Quantification and distribution of hindlimb ischemia severity observed in different experimental groups over time. Mean values are presented with standard deviations (n = 6). *, statistically significant difference (P < 0.05), as compared with control (EPC and OEC bolus injection); #, statistically significant difference (P < 0.05) between conditions.