Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels

Abstract

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Figure 1. Overall experimental scheme of fabricating an hMSC-based scaffold-free TEBV.

Figure 2. Images of aligned cell sheets and resulting TEBV.

(A) SEM image of nanopatterned PDMS; (B,C) Phase images of aligned hMSCs grown on nanopatterned PDMS, (D) hMSC sheets wrapped around 1.3 mm diameter glass rod; (E,F) SEM images of circumferentially aligned hMSC in outer layer of TEBV; (G,H) Confocal images showing phalloidin staining (Red) and DAPI (Blue) of TEBV; (I–L) SEM images of various TEBV sections demonstrating that the cell sheets were well fused to create multiple layers of the vessel wall with confluent cell density throughout the vessel from the lumen to the exterior ((I): TEBV; (J): longitudinal section showing lumen; (K): longitudinal section showing the outer wall; and (L): cross-section)

Figure 3

Response of scaffold-free TEBVs to flow rate (A) and 1 μM phenylephrine (B). The flow rate was gradually increased from 0.5 to 4 mL/min in doubling increments and the vessel diameter increased as the flow rate increased (*p < 0.05). The decrease of vessel diameter and the amount of NO release in response to the drug was comparable to those of native vessels (**p > 0.05).

Figure 4. Confocal images showing immunostaining for PECAM, SMA, Type IV collagen, and laminin of TEBV cultured for 7 days in the perfusion chamber and native porcine femoral vein, respectively

(A).Immunofluorescence staining images show that the hMSCs align perpendicular to the direction of flow in the bioreactor, which is consistent with previous report that smooth muscle cells tend to orient in a direction perpendicular to blood flow (B). Monocyte adhesion assay shows the attachment of HL-60 s (arrows) to the endothelial cells and beside the aligned smooth muscle cells in the TEBV (C). Density of the attached HL-60 cells on the activated endothelium of TEBV is shown in (D). No attached HL-60 cells were observed if the TEBV was not endothelialized. Error bar indicates standard error.