Staphylococcus aureus is the most common pathogen associated with bacterial infections in orthopedic procedures. Infections often lead to implant failure and subsequent removal, motivating the development of bifunctional materials that both promote repair and prevent failure due to infection. Lysostaphin is an anti-staphylococcal enzyme resulting in bacterial lysis and biofilm reduction. Lysostaphin use is limited by the lack of effective delivery methods to provide sustained, high doses of enzyme to infection sites. We engineered a BMP-2–loaded lysostaphin-delivering hydrogel that simultaneously prevents S. aureus infection and repairs nonhealing segmental bone defects in the murine radius. Lysostaphin-delivering hydrogels eradicated S. aureus infection and resulted in mechanically competent bone. Cytokine and immune cell profiling demonstrated that lysostaphin-delivering hydrogels restored the local inflammatory environment to that of a sterile injury. These results show that BMP-2–loaded lysostaphin-delivering hydrogel therapy effectively eliminates S. aureus infection while simultaneously regenerating functional bone resulting in defect healing.
Many goals in tissue engineering rely on modulating cellular localization and polarization of cell signaling, including the inhibition of inflammatory infiltrate, facilitation of inflammatory cell egress, and clearance of apoptotic cells. Omega-3 polyunsaturated fatty acid-derived resolvins are gaining increasing recognition for their essential roles in inhibition of neutrophil invasion into inflamed tissue and promotion of macrophage phagocytosis of cellular debris as well as their egress to the lymphatics. Biomaterial-based release of lipid mediators is a largely under-explored approach that provides a method to manipulate local lipid signaling gradients in vivo and direct the recruitment and/or polarization of anti-inflammatory cell subsets to suppress inflammatory signaling and enhance angiogenesis and tissue regeneration. The goal of this study was to encapsulate Aspirin-Triggered Resolvin D1 (AT-RvD1) into a degradable biomaterial in order to elucidate the effects of sustained, localized delivery in a model of sterile inflammation. Flow cytometric and imaging analysis at both 1 and 3 days after injury showed that localized AT-RvD1 delivery was able significantly increase the accumulation of anti-inflammatory monocytes and M2 macrophages while limiting the infiltration of neutrophils. Additionally, cytokine profiling and longitudinal vascular analysis revealed a shift towards a pro-angiogenic profile with increased concentrations of VEGF and SDF-1α, and increased arteriolar diameter and tortuosity. These results demonstrate the ability of locally-delivered AT-RvD1 to increase pro-regenerative immune subpopulations and promote vascular remodeling.
To gain insights into neutrophil heterogeneity dynamics in the context of sterile inflammation and wound healing, we performed a pseudotime analysis of single-cell flow cytometry data using the spanning-tree progression analysis of density-normalized events algorithm. This enables us to view neutrophil transitional subsets along a pseudotime trajectory and identify distinct VEGFR1, VEGFR2, and CXCR4 high-expressing pro-angiogenic neutrophils. While the proresolving lipid mediator aspirin-triggered resolvin D1 (AT-RvD1) has a known ability to limit neutrophil infiltration, our analysis uncovers a mode of action in which AT-RvD1 leads to inflammation resolution through the selective reprogramming toward a therapeutic neutrophil subset. This accumulation leads to enhanced vascular remodeling in the skinfold window chamber and a proregenerative shift in macrophage and dendritic cell phenotype, resulting in improved wound closure after skin transplantation. As the targeting of functional immune subsets becomes the key to regenerative immunotherapies, single-cell pseudotime analysis tools will be vital in this field.
Full thickness rotator cuff tears (RCT) and the associated muscle degeneration that results due to this injury presents a significant clinical burden. The prevention or recovery from this degeneration requires the synchronized behavior of many cells that participate in regeneration. Strategies that tune the inflammatory cascade that is initiated after injury serves as a powerful way to influence tissue repair. Here, we use the local, sustained delivery of the immunomodulatory small molecule FTY720 to examine whether the recruitment of pro-regenerative myeloid cells affects the healing outcome. We find that PLGA microparticles have an atrophic effect on the muscle that is ameliorated with the release of FTY720. However, the inability of FTY720 delivery to induce pro-regenerative monocyte and macrophage recruitment and our findings demonstrating enrichment of CD4+ T cells suggest that effects of this small molecule are context dependent and that the underlying mechanisms behind this RCT associated muscle degeneration require further studies.
Control of microvascular network growth is critical to treatment of ischemic tissue diseases and enhancing regenerative capacity of tissue engineering implants. Conventional therapeutic strategies for inducing angiogenesis aim to deliver one or more proangiogenic cytokines or to over-express known pro-angiogenic genes, but seldom address potential compensatory or cooperative effects between signals and the overarching signaling pathways that determine successful outcomes. An emerging grand challenge is harnessing the expanding knowledge base of angiogenic signaling pathways toward development of successful new therapies. We previously performed drug optimization studies by various substitutions of a 2-(2,6-dioxo-3-piperidyl)isoindole-1,3-dione scaffold to discover novel bioactive small molecules capable of inducing growth of microvascular networks, the most potent of which we termed phthalimide neovascularization factor 1 (PNF1, formerly known as SC-3–149). We then showed that PNF-1 regulates the transcription of signaling molecules that are associated with vascular initiation and maturation in a time-dependent manner through a novel pathway compendium analysis in which transcriptional regulatory networks of PNF-1-stimulated microvascular endothelial cells are overlaid with literature-derived angiogenic pathways. In this study, we generated three analogues (SC-3–143, SC-3–263, SC-3–13) through systematic transformations to PNF1 to evaluate the effects of electronic, steric, chiral, and hydrogen bonding changes on angiogenic signaling. We then expanded our compendium analysis toward these new compounds. Variables obtained from the compendium analysis were then used to construct a PLSR model to predict endothelial cell proliferation. Our combined approach suggests mechanisms of action involving suppression of VEGF pathways through TGF-β andNR3C1 network activation.
Full thickness rotator cuff tears (RCT) and the associated muscle degeneration that results due to this injury presents a significant clinical burden. The prevention or recovery from this degeneration requires the synchronized behavior of many cells that participate in regeneration. Strategies that tune the inflammatory cascade that is initiated after injury serves as a powerful way to influence tissue repair. Here, we use the local, sustained delivery of the immunomodulatory small molecule FTY720 to examine whether the recruitment of pro-regenerative myeloid cells affects the healing outcome. We find that PLGA microparticles have an atrophic effect on the muscle that is ameliorated with the release of FTY720. However, the inability of FTY720 delivery to induce pro-regenerative monocyte and macrophage recruitment and our findings demonstrating enrichment of CD4+ T cells suggest that effects of this small molecule are context dependent and that the underlying mechanisms behind this RCT associated muscle degeneration require further studies.
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