Abstract 1297: A Novel Nanotechnology-Based Cell Sheet Strategy for Therapeutic Neovascularization: Multilayered Mesenchymal Stem Cell Sheet with Magnetite Nanoparticle Improves Therapeutic Efficacy of Ischemic Neovascularization
Bone marrow cell therapy contributes to collateral formation through the secretion of angiogenic factors by progenitor cells and muscle cells per se. However, we experience some cases that are refractory to this therapy due to graft failure.
[Methods & Results] To develop a novel nanotechnology-based therapeutic system, we prepared “scaffold-less” multilayered 3-D cell sheets using magnetic force-based tissue engineering. Original magnetite cationic liposomes were added to the medium of human mesenchymal stem cells (hMSC, magnetite concentration, 20 pg/cell). hMSC with magnetite particles were replated onto the ultra-low attachment culture dish laid over the neodymium magnet (0.4 Tesla) and allowed to form multilayered 3-D sheets. No toxicity was observed after a 24-h incubation period and the apoptosis rates were similar to those of attached hMSCs (attached hMSCs: sheeted hMSCs: floated hMSCs=4.2±0.2*: 5.3±0.3*: 8.0±0.6 %, respectively, *P<0.05 vs floated MSCs, data±SD). Although the phosphorylation levels of focal adhesion kinase (FAK) in sheeted MSCs were reduced if compared with attached hMSCs (p-FAK/total FAK; 1.2±0.2 vs 0.72±0.2, P<0.05), cell to cell connection was confirmed by the positive immunostaining of connexin43 at 24-h. We, then, produced unilateral hindlimb ischemia in nude mice, which were injected with vehicle (PBS), or hMSCs with magnetite (2×106 cells into 6 to 8 ischemic sites), or covered with multilayered 3-D hMSC sheets (2×106 cels/2.0 cm2, n=9 each). Trace experiments with MRI and microscopy showed that neovascularization was enhanced within and around the hMSC sheet and a part of hMSCs merged with local tissues without inflammation. Laser Doppler and morphometric analyses showed that hMSC sheet placement significantly enhanced recovery of blood perfusion to the ischemic limb and increased capillary and muscle bundle density (MBD) to a greater degree than cell injection group (MBD, PBS : injection : sheet = 609±76* : 647±88* : 980±240/mm2, *P<0.05 vs sheets).
[Conclusions] hMSC sheets were successfully engrafted to the ischemic tissues and recovered blood perfusion. The present data demonstrates the potential therapeutic benefits of our nanotechnology-based cell sheet strategy for therapeutic neovascularization.