Biomaterial-assisted macrophage cell therapy for regenerative medicine.

Although most tissue types are capable of some form of self-repair and regeneration, injuries that are larger than a critical threshold or those occurring in the setting of certain diseases can lead to impaired healing and ultimately loss of structure and function. The immune system plays an important role in tissue repair and must be considered in the design of therapies in regenerative medicine. In particular, macrophage cell therapy has emerged as a promising strategy that leverages the reparative roles of these cells. Macrophages are critical for successful tissue repair and accomplish diverse functions throughout all phases of the process by dramatically shifting in phenotypes in response to microenvironmental cues. Depending on their response to various stimuli, they may release growth factors, support angiogenesis, and facilitate extracellular matrix remodeling. However, this ability to rapidly shift phenotype is also problematic for macrophage cell therapy strategies, because adoptively transferred macrophages fail to maintain therapeutic phenotypes following their administration to sites of injury or inflammation. Biomaterials are a potential way to control macrophage phenotype in situ while also enhancing their retention at sites of injury. Cell delivery systems incorporated with appropriately designed immunomodulatory signals have potential to achieve tissue regeneration in intractable injuries where traditional therapies have failed. Here we explorecurrent challenges in macrophage cell therapy, especially retention and phenotype control, how biomaterials may overcome them, and opportunities for next generation strategies. Biomaterials will be an essential tool to advance macrophage cell therapy for widespread clinical applications.

Effects of Interleukin-4 (IL-4)-releasing microparticles and adoptive transfer of macrophages on immunomodulation and angiogenesis.

Macrophages are major regulators of angiogenesis in response to injury, but the mechanisms behind their diverse and phenotypically specific functions are still poorly understood. In particular, the effects of interleukin-4 (IL-4) on macrophage behavior have been well studied in vitro, but it remains unclear whether the release of IL-4 from biomaterials can be used to control macrophage phenotype and subsequent effects on angiogenesis in vivo. We used the murine hindlimb ischemia model to investigate the effects of IL-4-releasing poly(lactic-co-glycolic acid) microparticles co-delivered with IL-4-polarized macrophages on host macrophage phenotype and angiogenesis in vivo. We established a minimum dose of IL-4 required to modulate macrophage phenotype in vivo and evaluated effects on macrophage subpopulation diversity using multidimensional flow cytometry and pseudotime analysis. The delivery of IL-4-releasing microparticles did not affect the density or size of blood vessels as measured by immunohistochemical (IHC) analysis, but it did increase perfused tissue volume as measured by 3D microcomputed tomography (microCT). In contrast, the co-delivery of IL-4-releasing microparticles and exogenously IL-4-polarized macrophages increased the size of blood vessels as measured by IHC, but without effects on perfused tissue volume via microCT. These effects occurred in spite of low recovery of adoptively transferred macrophages at 4 days after administration. Spatial analysis of macrophage-blood vessel interactions via IHC showed that macrophages closely interacted with blood vessels, which was slightly influenced by treatment, and that blood vessel size was positively correlated with number of macrophages in close proximity. Altogether, these findings indicate that delivery of IL-4-releasing microparticles and exogenously IL-4-polarized macrophages can be beneficial for angiogenesis, but further mechanistic investigations are required.

Characterizing the Macrophage Response to Immunomodulatory Biomaterials Through Gene Set Analyses.

The primary regulators of the innate immune response to implanted biomaterials are macrophages, which change phenotype over time to regulate multiple phases of the tissue repair process. Immunomodulatory biomaterials that target macrophage phenotype are a promising approach for promoting tissue repair. Although expression of multiple markers has been widely used to characterize macrophage phenotype, the complexity of the macrophage response to biomaterials makes interpretation difficult. The aim of this study was to put forth an objective method to characterize macrophage phenotype with respect to specific biological processes or standard phenotypes of interest. We investigated the utility of gene set analyses to analyze macrophages as they respond to model biomaterials in comparison to "reference" M1 and M2a macrophage phenotypes. Primary human macrophages were seeded onto crosslinked collagen scaffolds with or without adsorption of the proinflammatory cytokine interferon-gamma (IFNg). Gene expression of a custom-curated panel of 48 genes, representing the M1 and M2a gene signatures as well as other genes important for angiogenesis and tissue repair, was quantified using NanoString on days 3, 5, and 8 of culture. A dataset of phenotype controls, consisting of M0, M1, and M2a macrophages, was used as a source of comparison and to validate the methods of characterization. Gene expression of M1 and M2a markers showed mixed upregulation and downregulation by macrophages seeded on collagen and IFNg-adsorbed collagen scaffolds, highlighting the need for more holistic analyses. Euclidean distance measurements to the reference phenotypes were unable to resolve differences between groups. In contrast, rotation gene set testing with and without gene weighting based on the genes' ability to differentiate between M1, M2a, and M0 controls, followed by gene set variation analysis, showed that collagen scaffolds inhibited the classic M1 phenotype without promoting a classic M2a phenotype, and that IFNg-adsorbed collagen scaffolds promoted the M1 phenotype and inhibited the M2a phenotype. In summary, this work demonstrates a powerful, objective methodology for characterizing the macrophage response to biomaterials in comparison to reference macrophage phenotypes. With the addition of more macrophage phenotypes with defined gene expression signatures, this method could prove beneficial for characterizing complex hybrid phenotypes.

Sequential drug delivery to modulate macrophage behavior and enhance implant integration.

Macrophages are major upstream regulators of the inflammatory response to implanted biomaterials. Sequential functions of distinct macrophage phenotypes are essential to the normal tissue repair process, which ideally results in vascularization and integration of implants. Improper timing of M1 or M2 macrophage activation results in dysfunctional healing in the form of chronic inflammation or fibrous encapsulation of the implant. Thus, biphasic drug delivery systems that modulate macrophage behavior are an appealing approach to promoting implant integration. In this review, we describe the timing and roles of macrophage phenotypes in healing, then highlight current drug delivery systems designed to sequentially modulate macrophage behavior.

Structural properties of starch-chitosan-gelatin foams and the impact of gelatin on MC3T3 mouse osteoblast cell viability.

BACKGROUND: This study examines the effects of adding gelatin to a starch-chitosan composite foam, focusing on the altered structural and biological properties. The compressive modulus of foams containing different gelatin concentrations was tested in dry, wet, and lyophilized states. MC3T3 mouse osteoblast cells were used to test the composite's ability to support cell growth. The stability of the foams in α-MEM culture media with and without cells was also examined. RESULTS: It was found that for dry foams, the compressive modulus increased with increasing gelatin content. For foams tested in wet and lyophilized states, the compressive modulus peaked at a gelatin concentration of 2.5% and 5%, respectively. The growth of MC3T3 mouse osteoblast cells was tested on the foams with different gelatin concentrations. The addition of gelatin had a positive effect on the cell growth and proliferation. CONCLUSION: The composite foam containing gelatin improved cell growth and is only dissolved by the growing cells at a rate influenced by the initial concentration of gelatin added to the foam.