Angiogenesis is crucial for the success of most tissue engineering strategies. The natural inflammatory response is a major regulator of vascularization, through the activity of different types of macrophages and the cytokines they secrete. Macrophages exist on a spectrum of diverse phenotypes, from “classically activated” M1 to “alternatively activated” M2 macrophages. M2 macrophages, including the subsets M2a and M2c, are typically considered to promote angiogenesis and tissue regeneration, while M1 macrophages are considered to be anti-angiogenic, although these classifications are controversial. Here we show that in contrast to this traditional paradigm, primary human M1 macrophages secrete the highest levels of potent angiogenic stimulators including VEGF; M2a macrophages secrete the highest levels of PDGF-BB, a chemoattractant stabilizing pericytes, and also promote anastomosis of sprouting endothelial cells in vitro; and M2c macrophages secrete the highest levels of MMP9, an important protease involved in vascular remodeling. In a murine subcutaneous implantation model, porous collagen scaffolds were surrounded by a fibrous capsule, coincident with high expression of M2 macrophage markers, while scaffolds coated with the bacterial lipopolysaccharide were degraded by inflammatory macrophages, and glutaraldehyde-crosslinked scaffolds were infiltrated by substantial numbers of blood vessels accompanied by high levels of M1 and M2 macrophages. These results suggest that coordinated efforts by both M1 and M2 macrophages are required for angiogenesis and scaffold vascularization, which may explain some of the controversy over which phenotype is the angiogenic phenotype.

In normal tissue repair, macrophages exhibit a pro-inflammatory phenotype (M1) at early stages and a pro-healing phenotype (M2) at later stages. We have previously shown that M1 macrophages initiate angiogenesis while M2 macrophages promote vessel maturation. Therefore, we reasoned that scaffolds that promote sequential M1 and M2 polarization of infiltrating macrophages should result in enhanced angiogenesis and healing. To this end, we first analyzed the in vitro kinetics of macrophage phenotype switch using flow cytometry, gene expression, and cytokine secretion analysis. Then, we designed scaffolds for bone regeneration based on modifications of decellularized bone for a short release of interferon-gamma (IFNg) to promote the M1 phenotype, followed by a more sustained release of interleukin-4 (IL4) to promote the M2 phenotype. To achieve this sequential release profile, IFNg was physically adsorbed onto the scaffolds, while IL4 was attached via biotin-streptavidin binding. Interestingly, despite the strong interactions between biotin and streptavidin, release studies showed that biotinylated IL4 was released over 6 days. These scaffolds promoted sequential M1 and M2 polarization of primary human macrophages as measured by gene expression of ten M1 and M2 markers and secretion of four cytokines, although the overlapping phases of IFNg and IL4 release tempered polarization to some extent. Murine subcutaneous implantation model showed increased vascularization in scaffolds releasing IFNg compared to controls. This study demonstrates that scaffolds for tissue engineering can be designed to harness the angiogenic behavior of host macrophages towards scaffold vascularization.

Background The only definitive treatment for end-stage organ failure is orthotopic transplantation. Lung extracellular matrix (ECM) holds great potential as a scaffold for lung tissue engineering since it retains the complex architecture, biomechanics and topological specificity of the lung. Decellularization of human lungs rejected from transplantation could provide “ideal” biological scaffolds for lung tissue engineering, but the availability of such lungs remains limited. The present study was designed to determine whether porcine lung could serve as a suitable substitute of human lung to study tissue-engineering therapies. Methods Human and porcine lungs were procured, sliced into sheets, and decellularized using three different methods. Compositional, ultrastructural, and biomechanical changes to the ECM were characterized. The suitability of LECM for cellular re-population was evaluated by assessing the viability, growth, and metabolic activity of human lung fibroblasts (hMRC-5s), human small airway epithelial cells (hSAECs), and human adipose-derived mesenchymal stem cells (hMSCs) over a period of seven days. Results Decellularization using CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate, showed the best maintenance of both human and porcine LECM, with similar retention of ECM proteins, except for elastin. Human and porcine LECM supported the cultivation of pulmonary cells in a similar way, except that the human LECM was stiffer and resulted in higher metabolic activity of the cells than porcine LECM. Conclusions Porcine lungs can be decellularized using CHAPS to produce lung ECM scaffolds with properties resembling those of human lungs, for pulmonary tissue engineering. We propose that porcine lung ECM can be an excellent screening platform for the envisioned human tissue engineering applications of decellularized lungs.

NA Laryngoscope, 2018.

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