Highlights d Setdb2 regulates macrophage plasticity during normal and pathologic wound repair d Setdb2 expression in wound macrophages depends on IFN-I/ JAK/STAT1 signaling d This pathway is altered in chronic diabetic wounds because of failed surge of IFN-I d Elevated uric acid in diabetic wounds is driven by Setdb2 regulation of Xdh

The performance of indwelling medical devices that depend on an interface with soft tissue is plagued by complex, unpredictable foreign body responses. Such devices—including breast implants, biosensors, and drug delivery devices—are often subject to a collection of biological host responses, including fibrosis, which can impair device functionality. This work describes a milliscale dynamic soft reservoir (DSR) that actively modulates the biomechanics of the biotic-abiotic interface by altering strain, fluid flow, and cellular activity in the peri-implant tissue. We performed cyclical actuation of the DSR in a preclinical rodent model. Evaluation of the resulting host response showed a significant reduction in fibrous capsule thickness (P = 0.0005) in the actuated DSR compared with non-actuated controls, whereas the collagen density and orientation were not changed. We also show a significant reduction in myofibroblasts (P = 0.0036) in the actuated group and propose that actuation-mediated strain reduces differentiation and proliferation of myofibroblasts and therefore extracellular matrix production. Computational models quantified the effect of actuation on the reservoir and surrounding fluid. By adding a porous membrane and a therapy reservoir to the DSR, we demonstrate that, with actuation, we could (i) increase transport of a therapy analog and (ii) enhance pharmacokinetics and time to functional effect of an inotropic agent. The dynamic reservoirs presented here may act as a versatile tool to further understand, and ultimately to ameliorate, the host response to implantable biomaterials.

Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20 days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8 days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration.

Objective-Osteopontin (OPN) is a highly phosphorylated extracellular matrix glycoprotein that is involved in a diversity of biological processes. In the vascular wall, OPN is produced by monocytes/macrophages, endothelial cells, and smooth muscle cells, and it is thought to mediate adhesion, migration, and survival of these cell types. In this study, we hypothesized that OPN plays a critical role in recovery from limb ischemia. Methods and Results-We induced hind limb ischemia in wild-type and OPN Ϫ/Ϫ mice. OPN Ϫ/Ϫ mice exhibited significantly delayed recovery of ischemic foot perfusion as determined by LDPI, impaired collateral vessel formation as measured using micro-CT, and diminished functional capacity of the ischemic limb. In the aortic ring assay, normal endothelial cell sprouting was found in OPN Ϫ/Ϫ mice. However, OPN Ϫ/Ϫ peritoneal monocytes/macrophages were found to possess significantly reduced migration in response to chemoattraction. Conclusions-This

Control of inflammation is critical for the treatment of nonhealing wounds, but a delicate balance exists between early inflammation that is essential for normal tissue repair and the pathologic inflammation that can occur later in the repair process. This necessitates the development of novel therapies that can target inflammation at the appropriate time during repair. Here, we found that SIRT3 is essential for normal healing and regulates inflammation in wound macrophages after injury. Under prediabetic conditions, SIRT3 was decreased in wound macrophages and resulted in dysregulated inflammation. In addition, we found that FABP4 regulates SIRT3 in human blood monocytes, and inhibition of FABP4 in wound macrophages decreases inflammatory cytokine expression, making FABP4 a viable target for the regulation of excess inflammation and wound repair in diabetes. Using a series of ex vivo and in vivo studies with genetically engineered mouse models and diabetic human monocytes, we showed that FABP4 expression is epigenetically upregulated in diabetic wound macrophages and, in turn, diminishes SIRT3 expression, thereby promoting inflammation. These findings have significant implications for controlling inflammation and promoting tissue repair in diabetic wounds.

Macrophages are a primary immune cell involved in inflammation, and their cell plasticity allows for transition from an inflammatory to a reparative phenotype and is critical for normal tissue repair following injury. Evidence suggests that epigenetic alterations play a critical role in establishing macrophage phenotype and function during normal and pathologic wound repair. Here, we find in human and murine wound macrophages that cyclooxygenase 2/prostaglandin E 2 (COX-2/PGE 2 ) is elevated in diabetes and regulates downstream macrophage-mediated inflammation and host defense. Using single-cell RNA sequencing of human wound tissue, we identify increased NF-κB–mediated inflammation in diabetic wounds and show increased COX-2/PGE 2 in diabetic macrophages. Further, we identify that COX-2/PGE 2 production in wound macrophages requires epigenetic regulation of 2 key enzymes in the cytosolic phospholipase A 2 /COX-2/PGE 2 (cPLA 2 /COX-2/PGE 2 ) pathway. We demonstrate that TGF-β–induced miRNA29b increases COX-2/PGE 2 production via inhibition of DNA methyltransferase 3b–mediated hypermethylation of the Cox-2 promoter. Further, we find mixed-lineage leukemia 1 (MLL1) upregulates cPLA 2 expression and drives COX-2/PGE 2 . Inhibition of the COX-2/PGE 2 pathway genetically ( Cox2 fl/fl Lyz2 Cre+ ) or with a macrophage-specific nanotherapy targeting COX-2 in tissue macrophages reverses the inflammatory macrophage phenotype and improves diabetic tissue repair. Our results indicate the epigenetically regulated PGE 2 pathway controls wound macrophage function, and cell-targeted manipulation of this pathway is feasible to improve diabetic wound repair.

Introduction Critical limb ischemia (CLI) is a life and limb-threatening condition affecting 1–10% of the peripheral arterial disease (PAD) population. Traditional revascularization options are not possible for up to 50% of CLI patients, in which case, the use of cellular therapies, such as bone marrow derived mesenchymal stem cells (MSCs), hold great promise as an alternative revascularization therapy. However no randomized controlled phase 3 trials to date have demonstrated an improvement in limb salvage with cellular therapies. This may be due to either: poor cell quality (i.e. inability to generate a sufficient number of angiogenic MSCs) and/or the inadequate retention and viability of MSCs after delivery. Because concerns remain about the expansion and angiogenic potential of autologous MSCs in the CLI population, the objective of this study was to examine: the impact of our novel culture media supplement, pooled human platelet lysate (PL), in lieu of the standard fetal bovine serum (FBS), to improve the expansion potential of MSCs from CLI patients. We also characterized the in vitro angiogenic activity of MSCs from the tibia of amputated CLI limbs compared to MSCs from healthy donors. Methods MSCs from CLI patients were obtained from the tibia of patients undergoing major amputation; n=4 ischemic (ISC); n=4 ischemic and diabetic (ISC + DM). Healthy MSCs (n=4) were aspirated from the iliac crest of young and healthy donors. MSCs were successfully isolated and expanded in culture with PL or FBS. MSCs from passage 3–6 were used for phenotypic marker expression, adipogenic and osteogenic differentiation, and tested for their in vitro angiogenic activity on human microdermal endothelial cells (EC). In parallel MSCs were cultured to passage 11 for population doubling calculations. Results MSCs from both ischemic and healthy patients exhibited appropriate expression of cell surface markers and differentiation capacity. Population doublings were significantly greater for PL-stimulated compared to FBS-stimulated MSCs in all groups. The secretome of all MSCs had biologically active amounts of angiogens identified without consistent trends between groups. PL expansion did not adversely affect MSCs’ angiogenic activity compared to FBS, and both ISC and ISC + DM MSCs demonstrated similar angiogenic effects on ECs by healthy and ischemic MSCs. Conclusion PL promotes the rapid expansion of MSCs from CLI and healthy persons. Importantly, MSCs expanded from CLI patients demonstrate the desired angiogenic activity as compared to their healthy counterparts. We conclude that autologous MSCs from CLI patients can be sufficiently expanded with PL and be expected to deliver requisite angiogenic effects in vivo. We expect the improved expansion of both ISC and ISC + DM with PL to be helpful in improving the successful delivery of autologous MSCs to patients with CLI.

Fibrous capsule (FC) formation, secondary to the foreign body response (FBR), impedes molecular transport and is detrimental to the long-term efficacy of implantable drug delivery devices, especially when tunable, temporal control is necessary. We report the development of an implantable mechanotherapeutic drug delivery platform to mitigate and overcome this host immune response using two distinct, yet synergistic soft robotic strategies. Firstly, daily intermittent actuation (cycling at 1 Hz for 5 minutes every 12 hours) preserves long-term, rapid delivery of a model drug (insulin) over 8 weeks of implantation, by mediating local immunomodulation of the cellular FBR and inducing multiphasic temporal FC changes. Secondly, actuation-mediated rapid release of therapy can enhance mass transport and therapeutic effect with tunable, temporal control. In a step towards clinical translation, we utilise a minimally invasive percutaneous approach to implant a scaled-up device in a human cadaveric model. Our soft actuatable platform has potential clinical utility for a variety of indications where transport is affected by fibrosis, such as the management of type 1 diabetes.