Human TEVGs, constructed from hBMC-seeded scaffolds, transform into living blood vessels in SCID/bg mice. (A) Micro-computed tomography angiography at week 10 shows a patent TEVG functioning as an IVC venous conduit. Gross images of a human TEVG interposed into the IVC of the SCID/bg mouse at operative day 0 (B) and after 24 wk in vivo (C). (D) Gross image of native mouse IVC for comparison. Corresponding H&E images of a TEVG at day 0 (demonstrating hBMCs transplanted into the scaffold wall) (E), a TEVG at 24 wk (notice scaffold has degraded) (F), and native mouse IVC (G). Low-magnification (×100) photomicrographs of a TEVG at 10 wk postimplantation show scaffold materials still present, but the development of a confluent SMC (α-SMA, brown) layer (H) and EC [von Willebrand factor, brown] lining (I) throughout the inner lumen. By 24 wk, the scaffold material has degraded and the TEVG displays mature vessel architecture. (J) High-magnification (400×) photomicrograph demonstrates an organized EC-lined intima (von Willebrand factor, red) and SMC media (α-SMA, green). (K) Low-magnification (100×) Verhoeff–van Gieson stain shows scaffold replaced by a supportive adventitial layer composed of collagen (collagen, pink). (L) High-magnification (400×) Verhoeff–van Gieson stain demonstrates collagen fibrils but no elastin fibers (elastin, black; collagen, pink).

Seeded hBMCs do not directly contribute to the cellularity of the developing TEVG. Immunohistochemistry of the preimplant TEVG shows evidence of human leukocytes (hCD45) (A), human ECs (hCD31) (B), and human stem cells (hCD34) (C). Immunohistochemical analysis of hBMC-seeded scaffolds explanted at 1 wk in vivo detected only small numbers of retained human monocytes (hCD68) (D) and human ECs (hCD31) (E). No hCD34 was detectable. After 1 wk, no human antigen expression in the TEVG was detectable via immunohistochemistry. (F) Majority of cells in scaffold wall at 1 wk express F4/80, a marker for mouse monocytes. (G) α-SMA expression starts to be detected around the inner luminal lining by 1 wk. (H) Patchy endothelialization, indicated by positive von Willebrand factor expression, begins to form in the inner lumen by 3 wk. (I) RT-PCR of explanted hBMC-seeded scaffolds confirms no detectable human RNA expression after 1 wk in vivo. All photomicrographs are at a magnification of 400× (brown, positive expression). Data in graph are expressed as mean ± SD.

Seeded hBMCs increase early monocyte recruitment and secrete MCP-1. (A) Although seeded hBMCs are no longer present after 1 wk in vivo, scaffold cellularity rapidly increases during week 1 of development and then stabilizes until scaffold degradation. Seeded hBMCs significantly increase early cellularity by 1 wk. (B) Difference in cellularity between hBMC-seeded and unseeded scaffolds at week 1 is primarily attributable to a significant increase in infiltrating mouse monocytes (positive F4/80 expression). (C–E) Luminex assay shows that seeding onto PGA-P(CL/LA) scaffolds induces hBMCs to increase secretion of multiple cytokines associated with monocyte chemotaxis. MCP-1 was expressed in the highest concentration, with log fold differences over other cytokines tested. Interferon-inducible protein 10 and PDGF secretion by seeded hBMCs was notably decreased by exposure to scaffolds. GRO; MIP, macrophage inflammatory protein. (F) Confirmatory MCP-1-specific ELISA shows a significant increase in MCP-1 production when hBMCs were seeded onto scaffolds. Data in graphs are expressed as mean ± SD. *P < 0.05;** P < 0.001.

MCP-1-releasing microparticles mimic function of seeded hBMCs by increasing early monocyte recruitment to scaffolds. Microparticles were created to function as hBMC analogues capable of secreting MCP-1. Scanning electron microscopy images of MCP-1 microparticles similar in size distribution to hBMCs (1–20 μm) (A), cross-section of scaffold embedded with MCP-1 microparticles (B), and higher magnification of scaffold demonstrating individual PGA fibers with MCP-1 microparticles securely embedded into P(CL/LA) sealant (C). (D) Cumulative MCP-1 release profile of MCP-1 microparticle scaffolds (n = 5) shows they release ≈200 ng of MCP-1 over 72 h. (E) At 1 wk, MCP-1 microparticle scaffolds have significantly greater numbers of mouse monocytes (F4/80⁺ cells) than both hBMC-seeded and unseeded scaffolds. At 10 wk, all MCP-1 microparticle scaffolds are patent and demonstrate similar vascular architecture to hBMC-seeded scaffolds. Immunohistochemical staining shows an EC lining [von Willebrand factor (vWF) expression] along the inner lumen (F) and an SMC media layer (α-SMA expression) below the EC lining (G). (H) Gomori trichrome staining shows a supportive layer of collagen within the vascular neotissue and an external ring of degrading scaffold material infiltrated with monocytes and collagen. Photomicrographs of the hBMC-seeded scaffold at 10 wk stained with vWF (I), α-SMA (J), and Gomori trichrome (K), for comparison. All histologic photomicrographs are at magnification of 400×. Data in graphs are expressed as mean ± SD. *P < 0.05; **P < 0.001.

Proposed mechanism of vascular transformation of hBMC-seeded biodegradable scaffolds. (A) Early pulse of MCP-1, secreted from seeded hBMCs, enhances early monocyte recruitment to the scaffold. Infiltrating monocytes release multiple angiogenic cytokines and growth factors (i.e., VEGF), which recruit SMCs and ECs to the scaffold. Vascular cells potentially come from circulating progenitors and/or proliferation/migration of mature vascular cells in adjacent vessel segments. ECs and SMCs appropriately organize into a mature blood vessel structure on the luminal surface of the scaffold. As the scaffold degrades, monocytes migrate away, leaving behind a completely autologous neovessel. (B) Immunohistochemical VEGF staining of hBMC-seeded scaffolds at postimplantation weeks 1, 6, and 10 shows continued VEGF expression throughout TEVG development (brown, positive VEGF expression). Photomicrographs at a magnification of 400×.