Bone Marrow-Derived Macrophages Enhance Vessel Stability in Modular Engineered Tissues

Abstract

Macrophages play a central role in the host response to tissue engineered constructs and their resultant vascularization. With a diversity of phenotypes, it is important to control the macrophage response to maximize vessel formation. We use a bottom-up tissue fabrication system ("modular tissue engineering") consisting of mesenchymal stromal cells (MSC) embedded in injectable collagen gel modules that are coated with endothelial cells. Here we characterize the macrophage response to subcutaneously injected human umbilical vein endothelial cell (HUVEC)-coated modules with and without embedded human adipose-derived MSC or additional bone marrow-derived macrophages (BMDM) in SCID Beige mice. Clodronate depletion obviated vessel formation indicating that macrophages are essential to vessel formation. With MSC, macrophage infiltration (at day 3) was lessened relative to the HUVEC-only control, whereas they preferentially adopted a mixed M1/M2 phenotype, characterized by expression of both CD206 and MHCII, which was similar with or without MSC. Vessel numbers were higher with MSC as expected at day 7, but adding BMDM reduced the regression seen by day 14. Pretreating the BMDM with interleukin 4 (IL-4) or interferon γ(IFNγ) had no effect on vessel number or maturity (smooth muscle actin expression) or the inflammatory markers. Coculturing HUVEC with IFNγ-treated or untreated BMDM altered their expression of polarization markers, reducing the impact of the pretreatment. A similar phenomenon likely occurred in vivo reducing the expected impact of pretreatment. Although there is much to learn about the role of macrophages in module vascularization, the versatility of this bottom-up approach, afforded in part by enabling mixtures containing different components, is a useful means to alter the remodeling that occurs when constructs are deployed in vivo. This work demonstrates an effective method of enhancing the stability of a newly formed vasculature in modular engineered tissues in vivo through the counterintuitive implantation of allogeneic macrophages. Furthermore, we provide a detailed method for analyzing macrophage phenotype in vascularized explants and assess the role of added mesenchymal stromal cells (MSC) on that phenotype. Enhanced vascular stability may permit the development of increasingly large, clinically relevant tissues, fully permeated by blood vessels integrated with the host vasculature.