Effects of a bioengineered allogeneic cellular construct on burn-related macrophage phenotype.

Bioengineered allogeneic cellularised constructs (BACC) exert pro-healing effects in burn wounds and skew macrophage phenotype towards a predominately reparative phenotype. However, whether BACC can modulate the phenotype of dysregulated macrophages, like those present in burn wounds, is not known. To better understand the macrophage modulatory characteristics of the BACC, primary human macrophages were polarised to the M2b phenotype, an immunosuppressive phenotype relevant to burn wounds, by simultaneously exposing macrophages to polystyrene plate-coated immunoglobulin G and the endotoxin lipopolysaccharide (LPS). The resulting macrophage phenotype upregulated both inflammatory and reparative genes, and increased secretion of the M2b marker CCL1 compared to five different in vitro macrophage phenotypes. M2b macrophages were cultured with the BACC in the presence or absence of LPS to mimic infection, which is a common occurrence in burn wounds. The BACC caused up-regulation of reparative gene sets and down-regulation of pro-inflammatory gene sets, even when LPS was present in the cell culture media. Co-cultures were maintained for 1, 3, or 5 days in the presence of LPS, and by day 1 both non-activated macrophages and M2b macrophages exhibited signs of endotoxin tolerance, as demonstrated by a reduced secretion of tumour necrosis factor α (TNFα) in response to fresh LPS stimulus. The BACC was not able to prevent endotoxin tolerance, but reparative genes were upregulated in macrophages chronically exposed to LPS. These results suggest that the BACC can promote a reparative phenotype in dysregulated macrophages relevant to the pathophysiology of burns.

Immunomodulatory Biomaterials for Tissue Repair.

All implanted biomaterials are targets of the host's immune system. While the host inflammatory response was once considered a detrimental force to be blunted or avoided, in recent years, it has become a powerful force to be leveraged to augment biomaterial-tissue integration and tissue repair. In this review, we will discuss the major immune cells that mediate the inflammatory response to biomaterials, with a focus on how biomaterials can be designed to modulate immune cell behavior to promote biomaterial-tissue integration. In particular, the intentional activation of monocytes and macrophages with controlled timing, and modulation of their interactions with other cell types involved in wound healing, have emerged as key strategies to improve biomaterial efficacy. To this end, careful design of biomaterial structure and controlled release of immunomodulators can be employed to manipulate macrophage phenotype for the maximization of the wound healing response with enhanced tissue integration and repair, as opposed to a typical foreign body response characterized by fibrous encapsulation and implant isolation. We discuss current challenges in the clinical translation of immunomodulatory biomaterials, such as limitations in the use of in vitro studies and animal models to model the human immune response. Finally, we describe future directions and opportunities for understanding and controlling the biomaterial-immune system interface, including the application of new imaging tools, new animal models, the discovery of new cellular targets, and novel techniques for in situ immune cell reprogramming.

Effects of a Bioengineered Allogeneic Cellularized Construct (BACC) on Primary Human Macrophage Phenotype.

The mechanisms behind the pro-healing effects of multicellular, bioengineered allogeneic cellularized constructs (BACC) are not known. Macrophages are key regulators of every phase of the wound healing process and the primary cells that mediate the response to biomaterials. It is hypothesized that cells within the BACC modulate macrophage behavior, which may contribute to the mechanism by which BACC promotes healing. To probe the influence of cells within the BACC compared to effects of the underlying collagen substrate, primary human macrophages are cultured in direct or indirect contact with BACC or with the same collagen substrate used in the BACC manufacturing. Macrophage phenotype is characterized over time via multiplex gene expression, protein secretion, multidimensional flow cytometry, and functional assays with fibroblasts and endothelial cells. The BACC causes macrophages to exhibit a predominately reparative phenotype over time compared to relevant collagen substrate controls, with multiple subpopulations expressing both pro-inflammatory and reparative markers. Conditioned media from macrophage-BACC co-cultures causes distinct effects on fibroblast and endothelial cell proliferation, migration, and network formation. Given the critical role of the reparative macrophage phenotype in wound healing, these results suggest that modulation of macrophage phenotype may be a critical part of the mechanisms behind BACC's pro-healing effects.

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.

Human Hair Follicle-Derived Mesenchymal Stromal Cells from the Lower Dermal Sheath as a Competitive Alternative for Immunomodulation.

Mesenchymal stromal cells (MSCs) have unique immunomodulatory capacities. We investigated hair follicle-derived MSCs (HF-MSCs) from the dermal sheath, which are advantageous as an alternative source because of their relatively painless and minimally risky extraction procedure. These cells expressed neural markers upon isolation and maintained stemness for a minimum of 10 passages. Furthermore, HF-MSCs showed responsiveness to pro-inflammatory environments by expressing type-II major histocompatibility complex antigens (MHC)-II to a lesser extent than adipose tissue-derived MSCs (AT-MSCs). HF-MSCs effectively inhibited the proliferation of peripheral blood mononuclear cells equivalently to AT-MSCs. Additionally, HF-MSCs promoted the induction of CD4+CD25+FOXP3+ regulatory T cells to the same extent as AT-MSCs. Finally, HF-MSCs, more so than AT-MSCs, skewed M0 and M1 macrophages towards M2 phenotypes, with upregulation of typical M2 markers CD163 and CD206 and downregulation of M1 markers such as CD64, CD86, and MHC-II. Thus, we conclude that HF-MSCs are a promising source for immunomodulation.