Sequential delivery of immunomodulatory cytokines to facilitate the M1-to-M2 transition of macrophages and enhance vascularization of bone scaffolds

Abstract

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.

In vitro response of macrophages to ceramic scaffolds used for bone regeneration

“…We also used protein secretion analysis instead of gene expression analysis to confirm that macrophages secrete the protein products of these genes at appreciable levels. While all proteins evaluated were secreted at the expected levels based on previous reports [20,21], varying baghdadite grain size between 2 and 4 mm had only a modest effect on macrophage behaviour (electronic supplementary material, figure S4). For example, macrophages cultured on larger grain baghdadite scaffolds secreted slightly higher levels of TNF at day 2 ( p , 0.05).…”

“…This multifaceted behaviour stems from the fact that macrophages rapidly shift their phenotype in response to changing environmental stimuli, which is critical for successful wound healing [18]. In the normal healing process of many tissues, including bone fractures, the macrophage population shifts from predominantly pro-inflammatory (M1) to predominantly anti-inflammatory (M2) over time [19], perhaps because of sequential actions of the two populations in promoting angiogenesis [20,21]. M1 macrophages can also be activated in vitro by pro-inflammatory signals such as lipopolysaccharide and interferon-gamma.…”

“…On day 6, cells were lysed directly in the well in buffer RLT and stored at 2208C overnight. All samples were cultured in medium containing 20 ng ml 21 MCSF.…”

In vitro chemotaxis and tissue remodeling assays quantitatively characterize foreign body reaction

“…In this study, differentiation to a moderate anti-inflammatory M2 macrophage subtype involved in long-term chronic tissue-remodeling and fibrosis was preferred (Franz et al, 2011). Thus, monocytes were incubated with M-CSF for six days in culture medium (Ambarus et al, 2012;Spiller et al, 2014;Vogel et al, 2014). Robustness of differentiation was confirmed by analyzing phenotypic clusters of differentiation in flow cytometry (Tab.…”

The roles of immune cells in bone healing; what we know, do not know and future perspectives

“…This role is mediated via M1 cells that produce the angiogenesis prompting factors, VEGF, IL-8, bFGF and RANTES. Additionally, M2 cells have been proposed to enhance the blood vessel fusion, vascular remodelling and regulation of M1 activity [145]. These findings had a great potential to be applied in the regenerative bone therapies to fabricate scaffolds that help to polarise macrophages and consequently supporting blood vessel formation during bone healing [145] together with promoting the proliferation and polarisation of bone progenitors cells to achieve a completed healing process.…”