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Different macrophage phenotypes play important roles in diverse biological processes and diseases. In this study, we have characterized the dielectrophoretic responses of human monocytes and macrophage phenotypes: nonactivated (M0), pro-inflammatory (M1), and pro-healing (M2a). Dielectrophoretic responses of cells change as a function of frequency of the applied electric field. We measured the crossover frequency at which cells transition from negative to positive dielectrophoresis (DEP) or vice versa using interdigitated electrodes. For these characterization experiments, we also developed a new low-conductivity media formulation that retained 100% of the initial viability for 1 h. Human THP1 monocytes showed a distinguishable DEP response from mature macrophages. M1 macrophages also showed a distinct DEP response compared to M0 and M2a macrophages. No clear distinction could be drawn between M0 and M2a. The median values of the crossover frequencies of monocytes, M0, M1, and M2a were 38, 21, 11, and 23 kHz, respectively. Membrane capacitances of these cells were calculated consequently, and the values were 0.0111, 0.0128, 0.0244, and 0.0117 F/m

Cyclic strain generated at the cell-material interface is critical for the engraftment of biomaterials. Mechanosensitive immune cells, macrophages regulate the host-material interaction immediately after implantation by priming the environment and remodeling ongoing regenerative processes. This study investigated the ability of mechanically active scaffolds to modulate macrophage function

BACKGROUND: Acellular dermal matrix (ADM) supported implant-based reconstruction remains the most commonly performed mode of reconstruction after breast cancer. Acellular dermal matrix clinical usage has reported benefits but requires rapid and efficient vascular and cellular incorporation into the recipient to have the best outcomes. Orderly transition from M1 to M2 macrophage phenotypic profile, coordinated in part by interleukin 4 (IL-4), is an important component of vascular stabilization and remodeling. Using the ADM substrate as a delivery device for immunomodulation of macrophage phenotype holds the potential to improve integration. METHODS: Interleukin 4 was adsorbed onto ADM samples and drug elution curves were measured. Next, experimental groups of 8 C57BL/6 mice had 5-mm ADM discs surgically placed in a dorsal window chamber with a vascularized skin flap on one side and a plastic cover slip on the other in a model of implant-based breast reconstruction. Group 1 consisted of IL-4 (5 μg) adsorbed into the ADM preoperatively and group 2 consisted of an untreated ADM control. Serial gross examinations were performed with histology at day 21 for markers of vascularization, mesenchymal cell infiltration, and macrophage lineage. RESULTS: Drug elution curves showed sustained IL-4 release for 10 days after adsorption. Serial gross examination showed similar rates of superficial vascular investment of the ADM beginning at the periphery by day 14 and increasing through day 21. Interleukin-4 treatment led to significantly increased CD31 staining of vascular endothelial cells within the ADM over the control group (P < 0.05) at 21 days. Although vimentin staining did not indicate a significant increase in fibroblasts overall, IL-4 did result in a significant increase in expression of α-smooth muscle actin. The expression of macrophage phenotype markers Arginase1 and iNOS present within the ADM were not significantly affected by IL-4 treatment at the day 21 time point. CONCLUSIONS: Acellular dermal matrix has the potential to be used for immunomodulatory cytokine delivery during the timeframe of healing. Using implanted ADM as a delivery vehicle to drive IL-4 mediated angiogenesis and vascular remodeling significantly enhanced vascularity within the ADM substrate.

Injectable hydrogels may be pre-formed through dynamic crosslinks, allowing for injection and subsequent retention in the tissue by shear-thinning and self-healing processes, respectively. These properties enable the site-specific delivery of encapsulated therapeutics; yet, the sustained release of small-molecule drugs and their cell-targeted delivery remains challenging due to their rapid diffusive release and non-specific cellular biodistribution. Herein, we develop an injectable hydrogel system composed of a macrophage-targeted nanoparticle (cyclodextrin nanoparticles, CDNPs) crosslinked by adamantane-modified hyaluronic acid (Ad-HA). The polymer-nanoparticle hydrogel uniquely leverages cyclodextrin's interaction with small molecule drugs to create a spatially discrete drug reservoir and with adamantane to yield dynamic, injectable hydrogels. Through an innovative two-step drug screening approach and examination of 45 immunomodulatory drugs with subsequent in-depth transcriptional profiling of both murine and human macrophages, we identify celastrol as a potent inhibitor of pro-inflammatory (M1-like) behavior that furthermore promotes a reparatory (M2-like) phenotype. Celastrol encapsulation within the polymer-nanoparticle hydrogels permitted shear-thinning injection and sustained release of drug-laden nanoparticles that targeted macrophages to modulate cell behavior for greater than two weeks

Biotin-avidin interactions have been explored for decades as a technique to functionalize biomaterials, as well as for in vivo targeting, but whether changes in these interactions can be leveraged for immunomodulation remain unknown. The goal of this study was to investigate how biotin density and avidin variant can be used to deliver the immunomodulatory cytokine, interleukin 4 (IL4), from a porous gelatin scaffold, Gelfoam, to primary human macrophages in vitro. Here, we demonstrate that the degree of scaffold biotinylation controlled the binding of two different avidin variants, streptavidin and CaptAvidin. Biotinylated scaffolds were also loaded with streptavidin and biotinylated IL4 under flow, suggesting a potential use for targeting this biomaterial in vivo. While biotin-avidin interactions did not appear to influence the protein release in this system, increasing degrees of biotinylation did lead to increased M2-like polarization of primary human macrophages over time in vitro, highlighting the capability to leverage biotin-avidin interactions to modulate the macrophage phenotype. These results demonstrate a versatile and modular strategy to impart immunomodulatory activity to biomaterials.

Tissue repair is largely regulated by diverse Mϕ populations whose functions are timing- and context-dependent. The early phase of healing is dominated by pro-inflammatory Mϕs, also known as M1, followed by the emergence of a distinct and diverse population that is collectively referred to as M2. The extent of the diversity of the M2 population is unknown. M2 Mϕs may originate directly from circulating monocytes or from phenotypic switching of pre-existing M1 Mϕs within the site of injury. The differences between these groups are poorly understood, but have major implications for understanding and treating pathologies characterized by deficient M2 activation, such as chronic wounds, which also exhibit diminished M1 Mϕ behavior. This study investigated the influence of prior M1 activation on human Mϕ polarization to an M2 phenotype in response to IL-4 treatment in vitro. Compared to unactivated (M0) Mϕs, M1 Mϕs up-regulated several receptors that promote the M2 phenotype, including the primary receptor for IL-4. M1 Mϕs also up-regulated M2 markers in response to lower doses of IL-4, including doses as low as 10 pg/mL, and accelerated STAT6 phosphorylation. However, M1 activation appeared to also change the Mϕ response to treatment with IL-4, generating an M2-like phenotype with a distinct gene and protein expression signature compared to M2 Mϕs prepared directly from M0 Mϕs. Functionally, compared to M0-derived M2 Mϕs, M1-derived M2 Mϕs demonstrated increased migratory response to SDF-1α, and conditioned media from these Mϕs promoted increased migration of endothelial cells in transwell assays, although other common Mϕ-associated functions such as phagocytosis were not affected by prior polarization state. In summary, M1 polarization appears to prime Mϕs to transition into a distinct M2 phenotype in response to IL4, which leads to increased expression of some genes and proteins and decreased expression of others, as well as functional differences. Together, these findings indicate the importance of prior M1 activation in regulating subsequent M2 behavior, and suggest that correcting M1 behavior may be a therapeutic target in dysfunctional M2 activation.

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.

Background: Impaired healing after rotator cuff repair is a major concern, with retear rates as high as 94%. A method to predict whether patients are likely to experience poor surgical outcomes would change clinical practice. While various patient factors, such as age and tear size, have been linked to poor functional outcomes, it is currently very challenging to predict outcomes before surgery. Purpose: To evaluate gene expression differences in tissue collected during surgery between patients who ultimately went on to have good outcomes and those who experienced a retear, in an effort to determine if surgical outcomes can be predicted. Study Design: Case-control study; Level of evidence, 3. Methods: Rotator cuff tissue was collected at the time of surgery from 140 patients. Patients were tracked for a minimum of 6 months to identify those with good or poor outcomes, using clinical functional scores and follow-up magnetic resonance imaging to confirm failure to heal or retear. Gene expression differences between 8 patients with poor outcomes and 28 patients with good outcomes were assessed using a multiplex gene expression analysis via NanoString and a custom-curated panel of 145 genes related to various stages of rotator cuff healing. Results: Although significant differences in the expression of individual genes were not observed, gene set enrichment analysis highlighted major differences in gene sets. Patients who had poor healing outcomes showed greater expression of gene sets related to extracellular matrix production ( P < .0001) and cellular biosynthetic pathways ( P < .001), while patients who had good healing outcomes showed greater expression of genes associated with the proinflammatory (M1) macrophage phenotype ( P < .05). Conclusion: These results suggest that a more proinflammatory, fibrotic environment before repair may play a role in poor healing outcome. With validation in a larger cohort, these results may ultimately lead to diagnostic methods to preoperatively predict those at risk for poor surgical outcomes.

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. IMPACT STATEMENT: Immunomodulatory biomaterials that target macrophage phenotype are a promising approach for promoting tissue repair. However, due to the complexity of macrophage behavior as they interact with biomaterials, there is a need for improved methods for their characterization. We demonstrate the utility of gene set analyses to characterize the macrophage response to biomaterials using the classic M1 and M2a phenotypes as a reference, and suggest that this method may be adopted as a useful method for characterizing macrophage phenotype in a way that is practical, thorough, objective, and tailorable to particular phenotypes of interest. This methodology allows for identification of hybrid phenotypes and incorporation of additional phenotypes as their set of markers is established. Due to the variety of applications, this methodology is likely to be useful for engineers, scientists, and clinicians.

Low-frequency, low-intensity ultrasound has been previously shown to promote healing of chronic wounds in humans, but mechanisms behind these effects are poorly understood. The purpose of this study was to evaluate gene expression differences in debrided human venous ulcer tissue from patients treated with low-frequency (20 kHz), low-intensity (100 mW/cm2) ultrasound compared to a sham treatment in an effort to better understand the potential biological mechanisms. Debrided venous ulcer tissue was collected from 32 subjects one week after sham treatment or low-frequency, low-intensity ultrasound treatment. Of these samples, 7 samples (3 ultrasound treated and 4 sham treated) yielded sufficient quality total RNA for analysis by ultra-high multiplexed PCR (Ampliseq) and expression of more than 24,000 genes was analyzed. 477 genes were found to be significantly different between the ultrasound and sham groups using cut-off values of p

Although different metabolic pathways have been associated with distinct macrophage phenotypes, the field of utilizing metabolites to modulate macrophage phenotype is in a nascent stage. In this report, we developed microparticles based on polymerization of alpha-ketoglutarate (a Krebs cycle metabolite), with or without encapsulation of spermine (a polyamine metabolite), to modulate cell phenotype that are critical for resolution of inflammation. Poly (alpha-ketoglutarate) microparticles encapsulated and released spermine (spermine (encap)paKG MPs) in vitro, which was accelerated in an acidic environment. When delivered to bone marrow-derived-macrophages, spermine (encap)paKG MPs induced a complex phenotypic profile outside of the typical M1/M2 paradigm, with distinct effects in the presence or absence of the pro-inflammatory stimulus lipopolysaccharide. Of particular interest was the increase in expression of CD163, which has been linked to anti-inflammatory responses in sepsis. Therefore, we systemically administered spermine (encap)paKG MPs to two different murine models of sepsis using acute or chronic injection of LPS. Macrophages and neutrophils in the liver and spleen of animals treated with spermine (encap)paKG MPs increased expression of CD163, concomitant with normalizing of glycolysis and oxidative phosphorylation, in both models. Overall, these results show that spermine (encap)paKG MPs modulate macrophage phenotype in vitro and in vivo, with potential applications in inflammation-associated diseases.

BackgroundPsoriatic arthritis (PsA) is a chronic inflammatory disease, which is subsumed together with axial spondyloarthritis (axial SpA) under the umbrella term spondyloarthritis, whose clinical presentations are very heterogeneous. Axial involvement (axial PsA) has been described to be present in 34% of PsA patients [1] and has been systematically investigated only retrospectively or in cross-sectional studies [2]. Although axial PsA seems to have similar characteristics to axial SpA, it is not clear whether axial PsA and axial SpA, are a spectrum of the same disease with different expression patterns or different diseases with great similarities.ObjectivesTo prospectively investigate the clinical and imaging morphology patterns in a well-defined cohort of patients with axial PsA from the German SPondyloarthritis Inception Cohort (GESPIC).MethodsProspective, longitudinal, observational study of patients with an imaging confirmed diagnosis of axial PsA. In addition to clinical and laboratory characterization, conventional radiographs and magnetic resonance imaging (MRI) - scans of the entire spine and sacroiliac joints (SIJs) are performed at the baseline visit and patients are followed up every 6 months according to a predefined protocol. After 2 years, additional imaging (X-ray and MRI) is performed for follow-up. In this interim analysis baseline data are presented.ResultsBetween August 2019 and December 2021, 85 axial PsA patients were included. The mean age was 45.2 ± 12.9 years with a proportion of 55.3% female patients. Peripheral involvement was present in 43 (50.6%) patients, HLA-B27 was positive in 39 (45.9%), and C-reactive protein was elevated (>5mg/l) in 27 (31.8%) patients. Inflammatory back pain (IBP) in the discretion of the treating rheumatologist was present in 64 (75.3%) patients (Table 1). The modified New York criteria were fulfilled in 44.9% (n=35). MRI of the SIJ showed active inflammatory changes in 44 (55%) and structural changes in 59 (73.8%) patients. MRI of the spine showed active inflammation in 60% (n=48). Exclusively active and/or structural changes of the spine without changes in the SIJ were seen in 18.8% (n=15) (Figure 1).Table 1.Characteristics of patients with axial PsAParametersAxial PsA (n=85)Age in years (mean ± SD)45.2 ± 12.9Female sex, n (%)47 (55.3%)Inflammatory back pain (IBP) present, n (%)64 (75.3%)Peripheral Involvement, n (%)43 (50.6%)Nail Involvement, n (%)39 (45.9%)PASI-Score (mean ± SD)3.3 ± 5.1BASDAI (mean ± SD), 0-104.9 ± 2.0BASFI (mean ± SD), 0-103.8 ± 2.5ASDAS-CRP (mean ± SD)2.8 ± 1.0DAPSA (mean ± SD)14.5 ± 9.2HLA-B 27 positive, n (%)39 (45.9%)CRP >5mg/l, n (%)27 (31.8%)ASDAS-CRP =Ankylosing Spondylitis Disease Activity Score - CRP, BASDAI = Bath Ankylosing Spondylitis Disease Activity Index, BASFI =Bath Ankylosing Spondylitis Functional Index, CRP = C-reactive protein, DAPSA = Disease Activity in PSoriatic Arthritis-Score, PASI = Psoriasis Area Severity Index, PsA = Psoriatic arthritis, SD = standard deviation.Figure 1.MRI-imaging patterns of axial PsA patients (n=80*). MRI= magnetic resonance imaging, SIJs= sacroiliac joints. *Full imaging data available for 80 patients only due to variable reasons.ConclusionIn the here presented interims analysis of the baseline data of our prospective cohort study of patients with an imaging-based diagnosis of axial PsA, it is shown that these patients are less frequently HLA-B27 positive and more frequently female when compared to previously described cohorts of “classical” axial SpA patients. Noteworthy, nearly 20% of the patients showed an isolated spinal involvement without active or structural changes in the SIJs.References[1]Gladman, D.D., et al., Psoriatic arthritis (PSA)--an analysis of 220 patients. Q J Med, 1987. 62(238): p. 127-41.[2]Feld, J., et al., Axial disease in psoriatic arthritis and ankylosing spondylitis: a critical comparison. Nat Rev Rheumatol, 2018. 14(6): p. 363-371.AcknowledgementsThe GESPIC-axial PsA cohort is partially supported by an independent research grant from Novartis.We would especially like to thank C. Höppner, C. Lorenz, and all referring rheumatologists for their tireless support.Disclosure of InterestsHenriette Käding: None declared, Susanne Lüders: None declared, Mikhail Protopopov: None declared, Judith Rademacher: None declared, Valeria Rios Rodriguez: None declared, Laura Spiller: None declared, Murat Torgutalp: None declared, Denis Poddubnyy: None declared, Fabian Proft Speakers bureau: AMGEN, AbbVie, BMS, Celgene, Janssen, MSD, Novartis, Pfizer, Roche, UCB.Consultant of: AbbVie, Celgene, Janssen, Novartis, UCB.Grant/research support from: Novartis, Lilly, UCB.

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.

Older adults suffer from weakened and delayed bone healing due to age-related alterations in bone cells and in the immune system. Given the interaction between the immune system and skeletal cells, therapies that address deficiencies in both the skeletal and the immune system are required to effectively treat bone injuries of older patients. The sequence of macrophage activation observed in healthy tissue repair involves a transition from a pro-inflammatory state followed by a pro-reparative state. In older patients, inflammation is slower to resolve and impedes healing. The goal of this study was to design a novel drug delivery system for temporal guidance of the polarization of macrophages using bone grafting materials. A biomimetic calcium phosphate coating (bCaP) physically and temporally separated the pro-inflammatory stimulus interferon-gamma (IFNγ) from the pro-reparative stimulus simvastatin (SIMV). Effective doses were identified using a human monocyte line (THP-1) and testing culminated with bone marrow macrophages obtained from old mice. Sequential M1-to-M2 activation was achieved with both cell types. These results suggest that this novel immunomodulatory drug delivery system holds potential for controlling macrophage activation in bones of older patients.

Tissue repair is largely regulated by diverse macrophage populations whose functions are timing- and context-dependent. The early phase of healing is dominated by pro-inflammatory macrophages, also known as M1, followed by the emergence of a distinct and diverse population that is collectively referred to as M2. The extent of the diversity of the M2 population is unknown. M2 macrophages may originate directly from circulating monocytes or from phenotypic switching of pre-existing M1 macrophages within the site of injury. The differences between these groups have not been investigated, but have major implications for understanding and treating pathologies characterized by deficient M2 activation, such as chronic wounds, which also exhibit diminished M1 macrophage behavior. This study investigated the influence of prior M1 activation on human macrophage polarization to an M2 phenotype in response to IL-4 treatment in vitro. Compared to unactivated (M0) macrophages, M1 macrophages upregulated several receptors that promote the M2 phenotype, including the primary receptor for IL-4. M1 activation also changed the macrophage response to treatment with IL-4, generating an M2-like phenotype with a distinct gene and protein expression signature compared to M2 macrophages prepared directly from M0 macrophages. Functionally, compared to M0-derived M2 macrophages, M1-derived M2 macrophages demonstrated increased migratory response to SDF-1α, and conditioned media from these macrophages promoted increased recruitment of endothelial cells in transwell assays. Together, these findings indicate the importance of prior M1 activation in regulating subsequent M2 behavior, and suggest that augmentation of M1 behavior may be a therapeutic target in dysfunctional tissue repair.Summary sentenceM1 macrophages that are switched to an M2 phenotype exhibit a distinct functional phenotype compared to M2 macrophages derived directly from unactivated (M0) macrophages.

Macrophages play an important role in tissue repair, regeneration, and the ability of biomaterials to mediate these processes. Macrophages are highly plastic cells that exhibit altered behavior in response to changes in the microenvironment. With the growing knowledge of the roles that different macrophage phenotypes play in specific pathologies and/or injuries, researchers are now focusing on designing biomaterials to actively control macrophage behavior and promote healing outcomes. In this review, we highlight a variety of biomaterial strategies for controlling macrophage phenotype in chronic wounds, tissue defects, and inflammatory conditions, although these strategies can be applied to many other applications. In particular, we highlight the different situations in which biomaterials should inhibit or promote M1 or M2 activation, or both, for therapeutic outcomes.

Host-biomaterial interactions are critical determinants of the success or failure of an implant. However, detailed understanding of this process is limited due to a lack of dynamic tools for in vivo analyses. Here we characterize host-biomaterial interactions in zebrafish (Danio rerio), which are optically translucent and genetically tractable. Histological and immunohistochemical analyses following polypropylene suture implantation into adult zebrafish showed prolonged elevation of immune cell recruitment and collagen deposition, resembling a foreign body response. Live in vivo analysis showed that adsorption of the immunomodulatory cytokine interleukin-10 to a polystyrene microparticle, microinjected into transgenic larval zebrafish, inhibited neutrophil recruitment after 24 h compared to control microparticles, with no change in macrophage recruitment. This study illustrates that zebrafish are useful to investigate host-biomaterial interactions and have potential for high-throughput analysis of novel immunomodulatory biomaterials.

Aberrant extracellular matrix (ECM) assembly surrounding implanted biomaterials is the hallmark of the foreign body response, in which implants become encapsulated in thick fibrous tissue that prevents their proper function. While macrophages are known regulators of fibroblast behavior, how their phenotype influences ECM assembly and the progression of the foreign body response is poorly understood. In this study, we usedin vitromodels with physiologically relevant macrophage phenotypes, as well as controlled release of macrophage-modulating cytokines from gelatin hydrogels implanted subcutaneouslyin vivoto investigate the role of macrophages in ECM assembly. Primary human macrophages were polarized to four distinct phenotypes, which have each been associated with fibrosis, including pro-inflammatory M1, pro-healing M2, and a hybrid M1/M2, generated by exposing macrophages to M1- and M2-promoting stimuli simultaneously. Additionally, macrophages were first polarized to M1 and then to M2 (M1→M2) to generate a phenotype typically observed during normal wound healing. Human dermal fibroblasts that were cultured in macrophage-conditioned media upregulated numerous genes involved in regulation of ECM assembly, especially in M2-conditioned media. Hybrid M1/M2 macrophage-conditioned media caused fibroblasts to produce a matrix with thicker and less aligned fibers, while M2 macrophage-conditioned media caused the formation of a more aligned matrix with thinner fibers. Gelatin methacrylate hydrogels containing interleukin-4 (IL4) and IL13-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles were designed to promote the M2 phenotype in a murine subcutaneousin vivomodel. NanoString multiplex gene expression analysis of hydrogel explants showed that hydrogels with and without drug caused markers of both M1 and M2 phenotypes to be highly expressed, but the release of IL4+IL13 promoted upregulation of M2 markers and genes associated with regulation of ECM assembly, such asCol5a1andCol6a1. Biochemical analysis and second harmonic generation microscopy showed that the release of IL4+IL13 increased total sulfated glycosaminoglycan content and decreased fibril alignment, which is typically associated with less fibrotic tissue. Together, these results show that hybrid M1/M2 macrophages regulate ECM assembly, and that shifting the balance towards M2 may promote architectural and compositional changes in ECM with enhanced potential for downstream remodeling.

Monocyte-derived macrophages play a critical role in directing wound pathology following injury. Depending on their phenotype, macrophages also promote tissue regeneration. However, the therapeutic administration of macrophages with a controlled phenotype is challenging because macrophages are highly plastic and quickly revert to a detrimental, inflammatory phenotype in response to the environment of a damaged tissue. To address this issue, we developed a novel strategy to modulate macrophage phenotype intracellularly through phagocytosis of drug-loaded microparticles. Poly(lactic-co-glycolic acid) microparticles loaded with the anti-inflammatory drug dexamethasone (Dex) were phagocytosed by monocytes and stored intracellularly for at least 5 days. After differentiation into macrophages, cell phenotype was characterized over time with high-throughput gene expression analysis via NanoString. We found that the microparticles modulated macrophage phenotype for up to 7 days after microparticle uptake, with decreases in inflammation-related genes at early timepoints and upregulation of homing- and phagocytosis-related genes at multiple timepoints in a manner similar to cells treated with continuous free Dex. These data suggest that intracellularly loading macrophages with Dex microparticles via phagocytosis could be a unique methodology to selectively modulate macrophage phenotype over time. This strategy would allow therapeutic administration of macrophages for the treatment of a number of inflammatory disease and disorders. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1213-1224, 2019.

BackgroundMagnetic resonance tomography (MRI) plays a key role in the early diagnosis of axial spondyloarthritis (axSpA). However, the detection of changes indicative of axSpA requires specific expertise, which poses a challenge to non-specialized centers. Deep learning (an advanced machine learning method) based on training an artificial neural network may facilitate and support diagnostics in clinical practice.ObjectivesTo create a reliable deep learning tool for the detection of active inflammatory and structural changes indicative of axSpA on MRI of sacroiliac joints.MethodsIn this study, MRIs of sacroiliac joints from 477 patients from four cohorts (GESPIC-AS, GESPIC-Crohn, GESPIC-Uveitis and OptiRef comprising 266 patients with and 211 without axSpA) were used to develop a deep learning framework (randomly divided into training, n=404, and validation, n=73, datasets). MRIs from the ASAS cohort (n=116) were used for independent testing (test dataset). Each examination in the training/validation dataset was evaluated for the presence of active inflammatory and structural changes indicative of SpA by six experienced, trained and calibrated readers and by seven expert readers in the test dataset. The presence of the changes was defined as the majority vote amongst readers. Discordant cases in the training/validation dataset underwent consensus reading. In addition, the test dataset was evaluated by three radiologists not specifically trained in SpA. Diagnostic performance was evaluated using the area under the receiver operating characteristic curve (AUC), accuracy, sensitivity and specificity.ResultsThe prevalence of positive imaging findings for active inflammatory/structural changes indicative of axSpA was 41%/51% in the training/validation dataset and 22%/22% in the test dataset. The model for the detection of active inflammatory changes showed an AUC of 0.91 (0.83 – 0.97) – Figure 1 – and an accuracy of 84% on the validation dataset; the corresponding sensitivity and specificity were 96% and 76%, respectively. Despite a substantially lower prevalence of active inflammatory changes in the test dataset, the model showed good generalization with an AUC of 0.91 (0.84−0.97) and an accuracy of 75%; the sensitivity and specificity were 88% and 71%, respectively. The model demonstrated a similar performance on the validation and test datasets for the detection of active inflammatory changes fulfilling the ASAS definition. The model for the detection of structural changes indicative of axSpA showed good performance on the validation dataset with an AUC of 0.90 (0.82-0.96) for the detection of structural changes and an overall accuracy of 85%. The associated sensitivity and specificity were 95% and 75%, respectively. The model showed reasonable generalization to new data with an AUC of 0.89 (0.81−0.96) and an accuracy of 79%; the sensitivity and specificity were 85% and 78%, respectively. Overall, the model performed close to the individual human experts - Figure 1.ConclusionThe developed framework allowed the detection of active inflammatory and structural changes indicative of axSpA on MRI. This approach may be used as an assistant tool in the diagnostic workflow.AcknowledgementsGESPIC-AS has been financially supported by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung - BMBF). GESPIC-Crohn has been supported by the Clinical Research Unit grant from the Berlin Institute of Health (BIH). GESPIC-Uveitis has been supported by a research grant from AbbVie. OptiRef has been supported by a research grant from Novartis. The Assessment of Spondyloarthritis International Society (ASAS) has supported the project with a research grant and provided access to the MRI images of the ASAS calssifiaction cohort.We want to thank colleagues who performed annotation of the images from the ASAS classification cohort: Pedro Machado, Mikkel Ostergaard, Suzanne Juhl Pedersen, Ulrich Weber. Further, we thank Torsten Karge for the development of the MRI reading interface for GESPIC and OptiRef images, Joel Paschke for development of the scoring interface for ASAS images.LCA is grateful for her participation in the BIH Charité–Junior Clinician and Clinician Scientist Program and KKB is grateful for his participation in the BIH Charité Digital Clinician Scientist Program all funded by the Charité–Universitätsmedizin Berlin and the Berlin Institute of Health. JR is grateful for her participation in the BIH Charité–Junior Clinician and Clinician Scientist Program.Disclosure of InterestsNone declared

The purpose of this clinical study was to assess, in a limited patient population, the potential for a novel advanced wound care treatment based on low intensity, low frequency ultrasound (LFLI-US) to affect wound closure rate in human diabetic foot ulcers (DFU) and to effect changes in the relative expression of pro-inflammatory and anti-inflammatory genes. The ratio of expression of these genes, termed the M1/M2 score because it was inspired by the transition of macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) phenotypes as wound healing progresses, was previously presented as a potential healing indicator for DFUs treated with the standard of care (Nassiri et al. 2015). We have also shown that noncavitational, nonthermal, low frequency (20 kHz), low intensity (spatial peak temporal peak intensity

Mounting evidence suggests that therapeutic cell and drug delivery strategies designed to actively harness the regenerative potential of the inflammatory response have great potential in regenerative medicine. In particular, macrophages have emerged as a primary target because of their critical roles in regulating multiple phases of tissue repair through their unique ability to rapidly shift phenotypes. Herein, we review macrophage-based therapies, focusing on the translational potential for cell delivery of ex vivo-activated macrophages and delivery of molecules and biomaterials to modulate accumulation and phenotype of endogenous macrophages. We also review current obstacles to progress in translating basic findings to therapeutic applications, including the need for improved understanding of context-dependent macrophage functions and the myriad factors that regulate macrophage phenotype; potential species-specific differences (e.g. humans versus mice); quality control issues; and the lack of standardized procedures and nomenclature for characterizing macrophages. Looking forward, the inherent plasticity of macrophages represents a daunting challenge for harnessing these cells in regenerative medicine therapies but also great opportunity for improving patient outcomes in a variety of pathological conditions.

Background In contrast to adults, the fetal response to myocardial infarction (MI) is regenerative, requiring the recruitment of cardiac progenitor cells to replace infarcted myocardium. Macrophage contribution to tissue repair depends on their phenotype: M1 are proinflammatory and initiate angiogenesis; M2a are profibrotic and contribute to blood vessels maturation; and M2c are proremodeling and proangiogenesis. The goal of the present study was to expand on this work by examining cardiac progenitor cells recruitment, and the role of macrophages in promoting angiogenesis and cardiac regeneration in the fetal heart after MI. Methods Fetal and adult sheep underwent MI and were sacrificed 3 or 30 days after MI. Some fetal hearts received stromal cell-derived factor-1α-inhibitor treatment. The microvasculature was evaluated by micro–computed tomography, gene expression was evaluated by real-time polymerase chain reaction, and vessels counts were evaluated by immunohistochemistry. Results Micro–computed tomography analysis showed restoration of microvasculature in fetal hearts after MI. Vascular endothelial growth factor-α increased, and the expression of tissue markers associated with the M1, M2a, and M2c macrophage phenotypes were elevated at day 3 after MI, but returned to baseline by 30 days after MI. In contrast, adult hearts after MI exhibited low vascular endothelial growth factor-α and persistent upregulation of all macrophage markers, consistent with prolonged inflammation, fibrosis, and remodeling. Inhibition of stromal cell-derived factor-1α in fetal infarcts prevented angiogenesis, decreased vascular endothelial growth factor-α, and was associated with a sustained increase in M1, M2a, and M2c markers after MI. Conclusions Changes in angiogenesis and macrophage phenotype-related gene expression after MI are important for the fetal regenerative response to MI and are mediated at least in part by cardiac progenitor cells recruitment.

Chronic wounds remain a major clinical challenge. Human cryopreserved viable amniotic membrane (hCVAM) is among the most successful therapies, but the mechanisms of action remain loosely defined. Because proper regulation of macrophage behavior is critical for wound healing with biomaterial therapies, we hypothesized that hCVAM would positively regulate macrophage behavior in vitro, and that soluble factors released from the hCVAM would be important for this effect. Primary human pro-inflammatory (M1) macrophages were seeded directly onto intact hCVAM or cultured in separation via transwell inserts (Soluble Factors) in the presence of pro-inflammatory stimuli (interferon-γ and lipopolysaccharide) to simulate the chronic wound environment. Macrophages were characterized after 1 and 6 days using multiplex gene expression analysis of 37 macrophage phenotype- and angiogenesis-related genes via NanoString™, and protein content from conditioned media collected at days 1, 3 and 6 was analyzed via enzyme linked immunosorbent assays. Gene expression analysis showed that Soluble Factors promoted significant upregulation of pro-inflammatory marker IL1B on day 1 yet downregulation of TNF on day 6 compared to the M1 macrophage control. In contrast, intact hCVAM, which includes both extracellular matrix, viable cells, and soluble factors, promoted downregulation of pro-inflammatory markers TNF, CCL5 and CCR7 on day 1 and endothelial receptor TIE1 on day 6, and upregulation of the anti-inflammatory marker IL10 on day 6 compared to the M1 Control. Other genes related to inflammation and angiogenesis (MMP9, VEGF, SPP1, TGFB1, etc.) were differentially regulated between the Soluble Factors and intact hCVAM groups at both time points, though they were not expressed at significantly different levels compared to the M1 Control. Interestingly, Soluble Factors promoted increased secretion of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), while direct contact with hCVAM inhibited secretion of TNF, relative to the M1 Control. Both Soluble Factors and intact hCVAM inhibited secretion of MMP9 and VEGF, pro-inflammatory proteins that are critical for angiogenesis and remodeling, compared to the M1 Control, with intact hCVAM having a stronger effect. In a simulated pro-inflammatory environment, intact hCVAM has distinct anti-inflammatory effects on primary human macrophages, and direct macrophage contact with intact hCVAM is required for these effects. These findings are important for the design of next generation immunomodulatory biomaterials for wound repair and regenerative medicine that may include living cells, soluble factors, or a controlled drug delivery system.

Monocyte-derived macrophages orchestrate tissue regeneration by homing to sites of injury, phagocytosing pathological debris, and stimulating other cell types to repair the tissue. Accordingly, monocytes have been investigated as a translational and potent source for cell therapy, but their utility has been hampered by their rapid acquisition of a pro-inflammatory phenotype in response to the inflammatory injury microenvironment. To overcome this problem, we designed a cell therapy strategy where monocytes are exogenously reprogrammed by intracellularly loading the cells with biodegradable microparticles containing an anti-inflammatory drug in order to modulate and maintain an anti-inflammatory phenotype over time. To test this concept, poly(lactic-co-glycolic) acid microparticles were loaded with the anti-inflammatory drug dexamethasone (Dex) and administered to primary human monocytes for four hours to facilitate phagocytic uptake. After removal of non-phagocytosed microparticles, microparticle-loaded monocytes differentiated into macrophages and stored the microparticles intracellularly for several weeks in vitro, releasing drug into the extracellular environment over time. Cells loaded with intracellular Dex microparticles showed decreased expression and secretion of inflammatory factors even in the presence of pro-inflammatory stimuli up to 7 days after microparticle uptake compared to untreated cells or cells loaded with blank microparticles, without interfering with phagocytosis of tissue debris. This study represents a new strategy for long-term maintenance of anti-inflammatory macrophage phenotype using a translational monocyte-based cell therapy strategy without the use of genetic modification. Because of the ubiquitous nature of monocyte-derived macrophage involvement in pathology and regeneration, this strategy holds potential as a treatment for a vast number of diseases and disorders. STATEMENT OF SIGNIFICANCE: We report a unique and translational strategy to overcome the challenges associated with monocyte- and macrophage-based cell therapies, in which the cells rapidly take on inflammatory phenotypes when administered to sites of injury. By intracellularly loading monocytes with drug-loaded microparticles prior to administration via phagocytosis, we were able to inhibit inflammation while preserving functional behaviors of human primary macrophages derived from those monocytes up to seven days later. To our knowledge, this study represents the first report of reprogramming macrophages to an anti-inflammatory phenotype without the use of genetic modification.

Monocyte-derived macrophages orchestrate tissue regeneration by homing to sites of injury, phagocytosing pathological debris, and stimulating other cell types to repair the tissue. Accordingly, monocytes have been investigated as a translational and potent source for cell therapy, but their utility has been hampered by their rapid acquisition of a pro-inflammatory phenotype in response to the inflammatory injury microenvironment. To overcome this problem, we designed a cell therapy strategy where we collect and exogenously reprogram monocytes by intracellularly loading the cells with biodegradable microparticles containing an anti-inflammatory drug in order to modulate and maintain an anti-inflammatory phenotype over time. To test this concept, poly(lactic-co-glycolic) acid microparticles were loaded with the anti-inflammatory drug dexamethasone (Dex) and administered to primary human monocytes for four hours to facilitate phagocytic uptake. After removal of non-phagocytosed microparticles, microparticle-loaded monocytes differentiated into macrophages and stored the microparticles intracellularly for several weeks in vitro, releasing drug into the extracellular environment over time. Cells loaded with intracellular Dex microparticles showed decreased expression and secretion of inflammatory factors even in the presence of pro-inflammatory stimuli up to 7 days after microparticle uptake compared to untreated cells or cells loaded with blank microparticles. This study represents a new strategy for long-term maintenance of anti-inflammatory macrophage phenotype using a translational monocyte-based cell therapy strategy without the use of genetic modification. Because of the ubiquitous nature of monocyte-derived macrophage involvement in pathology and regeneration, this strategy holds potential as a treatment for a vast number of diseases and disorders.

Although infiltrating macrophages influence many pathological processes after spinal cord injury (SCI), the intrinsic molecular mechanisms that regulate their function are poorly understood. A major hurdle has been dissecting macrophage-specific functions from those in other cell types as well as understanding how their functions change over time. Therefore, we used the RiboTag method to obtain macrophage-specific mRNA directly from the injured spinal cord in mice and performed RNA sequencing to investigate their transcriptional profile. Our data show that at 7 d after SCI, macrophages are best described as foam cells, with lipid catabolism representing the main biological process, and canonical nuclear receptor pathways as their potential mediators. Genetic deletion of a lipoprotein receptor, CD36, reduces macrophage lipid content and improves lesion size and locomotor recovery. Therefore, we report the first macrophage-specific transcriptional profile after SCI and highlight the lipid catabolic pathway as an important macrophage function that can be therapeutically targeted after SCI.SIGNIFICANCE STATEMENTThe intrinsic molecular mechanisms that regulate macrophage function after spinal cord injury (SCI) are poorly understood. We obtained macrophage-specific mRNA directly from the injured spinal cord and performed RNA sequencing to investigate their transcriptional profile. Our data show that at 7 d after SCI, macrophages are best described as foam cells, with lipid catabolism representing the main biological process and canonical nuclear receptor pathways as their potential mediators. Genetic deletion of a lipoprotein receptor, CD36, reduces macrophage lipid content and improves lesion size and locomotor recovery. Therefore, we report the first macrophage-specific transcriptional profile after SCI and highlight the lipid catabolic pathway as an important macrophage function that can be therapeutically targeted after SCI.

Macrophages become polarized by cues in their environment and this polarization causes a functional change in their behavior. Two main subsets of polarized macrophages have been described. M1, or “classically activated” macrophages, are pro-inflammatory and M2, or “alternatively activated” macrophages, are anti-inflammatory. In this study, we investigated the motility and force generation of primary human macrophages polarized down the M1 and M2 pathways using chemokinesis assays and traction force microscopy on polyacrylamide gels. We found that M1 macrophages are significantly less motile and M2 macrophages are significantly more motile than unactivated M0 macrophages. We also showed that M1 macrophages generate significantly less force than M0 or M2 macrophages. We further found that M0 and M2, but not M1, macrophage force generation is dependent on ROCK signaling, as identified using the chemical inhibitor Y27632. Finally, using the chemical inhibitor blebbistatin, we found that myosin contraction is required for force generation by M0, M1, and M2 macrophages. This study represents the first investigation of the changes in the mechanical motility mechanisms used by macrophages after polarization.

Chronic wounds remain a major burden to the global healthcare system. Myriad wound matrices are commercially available but their mechanisms of action are poorly understood. Recent studies have shown that macrophages are highly influenced by their microenvironment, but it is not known how different biomaterials affect this interaction. Here, it was hypothesized that human macrophages respond differently to changes in biomaterial properties in vitro with respect to phenotype, including pro-inflammatory M1, anti-inflammatory M2a, known for facilitating extracellular matrix deposition and proliferation, and M2c, which has recently been associated with tissue remodeling. Using multiple donors, it was found that collagen scaffolds cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) promoted the least inflammatory phenotype in primary human macrophages compared with scaffolds cross-linked with formaldehyde or glutaraldehyde. Importantly, gene expression analysis trends were largely conserved between donors, especially TNFa (M1), CCL22 (M2a), and MRC1 (M2a). Then the response of primary and THP1 monocyte-derived macrophages to four commercially available wound matrices were compared-Integra Dermal Regeneration Template (Integra), PriMatrix Dermal Repair Scaffold (PriMatrix), AlloMend Acellular Dermal Matrix (AlloMend), and Oasis Wound Matrix (Oasis). Gene expression trends were different between primary and THP1 monocyte-derived macrophages for all six genes analyzed in this study. Finally, the behavior of primary macrophages cultured onto the wound matrices over time was analyzed. Integra and Oasis caused down-regulation of M2a markers CCL22 and TIMP3. PriMatrix caused up-regulation of TNFa (M1) and CD163 (M2c) and down-regulation of CCL22 and TIMP3 (both M2a). AlloMend caused up-regulation in CD163 (M2c). Lastly, Oasis promoted the largest increase in the combinatorial M1/M2 score, defined as the sum of M1 genes divided by the sum of M2 genes. This preliminary study suggested that biomaterials influenced the wound microenvironment to affect macrophage phenotype.

In recent years, macrophage phenotype has emerged as an important determinant of the success or failure of implanted polymeric biomaterials. However, it is not well understood how changes in biomaterials properties affect the foreign body response or macrophage behavior. Because failed attempts at biomaterial degradation by macrophages have been linked to frustrated phagocytosis, a defining feature of the foreign body response, we hypothesized that increased hydrogel crosslinking density (and decreased degradability) would exacerbate the foreign body response. Gelatin hydrogels were crosslinked with glutaraldehyde (0.05%, 0.1%, and 0.3%) and implanted subcutaneously in C57BL/6 mice over the course of 3 weeks. Histological and immunohistochemical analysis was used to characterize fibrous capsule formation and the temporal and spatial distribution of macrophage phenotype markers. Interestingly, changes in hydrogel crosslinking did not affect the thickness of the fibrous capsule surrounding the hydrogels, expression of the pan-macrophage marker F480, expression of three macrophage phenotype markers (iNOS, Arg1, CD163), or expression of the myofibroblast marker aSMA. With respect to temporal changes, the level of expression of the M1 marker (iNOS) remained relatively constant throughout the study, while the M2 markers Arg1 and CD163 increased over time. Expression of these M2 markers was highly correlated with fibrous capsule thickness. Differences in spatial distribution of staining also were noted, with the strongest staining for iNOS at the hydrogel surface and increasing expression of the myofibroblast marker aSMA toward the outer edge of the fibrous capsule. These results confirm previous reports that macrophages in the foreign body response exhibit characteristics of both M1 and M2 phenotypes. Understanding the effects (or lack of effects) of biomaterial properties on the foreign body response and macrophage phenotype may aid in the rational design of biomaterials to integrate with surrounding tissue.

We previously demonstrated that octadecylamine-functionalized nanodiamond (ND-ODA) and dexamethasone (Dex)-adsorbed ND-ODA (ND-ODA–Dex) promoted anti-inflammatory and pro-regenerative behavior in human macrophages in vitro. In this study, we performed a pilot study to investigate if these immunomodulatory effects translate when used as a treatment for rheumatoid arthritis in mice. Following local injection in limbs of mice with collagen type II-induced arthritis, microcomputed tomography showed that mice treated with a low dose of ND-ODA and ND-ODA–Dex did not experience bone loss to the levels observed in non-treated arthritic controls. A low dose of ND-ODA and ND-ODA–Dex also reduced macrophage infiltration and expression of pro-inflammatory mediators iNOS and tumor necrosis factor-α compared to the arthritic control, while a high dose of ND-ODA increased expression of these markers. Overall, these results suggest that ND-ODA may be useful as an inherently immunomodulatory platform, and support the need for an in-depth study, especially with respect to the effects of dose.

Arterial stiffening is a consequence of aging and a cholesterol-independent risk factor for cardiovascular disease (CVD). Arterial stiffening and CVD show a sex bias, with men more susceptible than premenopausal women. How arterial stiffness and sex interact at a molecular level to confer risk of CVD is not well understood. Here, we used the sexual dimorphism in LDLR-null mice to show that the protective effect of female sex on atherosclerosis is linked to reduced aortic stiffness and reduced expression of matrix metalloproteinase-12 (MMP12) by lesional macrophages. Deletion of MMP12 in LDLR-null mice attenuated the male sex bias for both arterial stiffness and atherosclerosis, and these effects occurred despite high serum cholesterol. Mechanistically, we found that oxidized LDL stimulates secretion of MMP12 in human as well as mouse macrophages. Estrogen antagonizes this effect by downregulating MMP12 expression. Our data support cholesterol-independent causal relationships between estrogen, oxidized LDL–induced secretion of macrophage MMP12, and arterial stiffness that protect against atherosclerosis in females and emphasize that reduced MMP12 functionality can confer atheroprotection to males.

Chronic wounds are the result of disruptions in the body's usual process of healing. They are not only a source of significant pain and discomfort but also, more importantly, an unguarded port of entry for pathogens into the body. While our current understanding of this phenomenon is far from complete, findings in physiological patterns and advancements in wound healing technologies have helped develop wound management and healing solutions to this long-standing medical challenge. This review presents an overview of known wound healing mechanics, abnormalities that lead to chronic wounds, and a summary of established and new wound healing technologies. Various approaches to heal wounds are discussed, from dermal replacements to advanced biomaterial-based treatments, from cell-, synthetic-, and composite-based approaches to preclinical approaches, which make developing such products possible. While tested breakthrough products are described, the authors focused more on recently developed innovations, which are at varying stages of maturity. The review concludes with a note on future perspectives and opinions on where the field and industry are headed and where they should be. Impact Statement Wound healing is an important area of research and clinical practice, and has captured the attention of tissue engineers since the nascent beginnings of the discipline. Tissue-engineered skin was the first FDA-approved product, achieved in 1996. Despite this success, and the passage of time, healing wounds, particularly chronic wounds, remains a vexing challenge. This comprehensive review article will provide readers with a synopsis of current issues, research approaches, animal models, technologies, and products that span the continuum from early development to clinical studies, in the hope of fueling new interests and ideas to overcome this long-standing medical challenge.

Diabetic foot ulcers (DFUs) are a major clinical problem exacerbated by prolonged bacterial infection. Macrophages, the primary innate immune cells, are multifunctional cells that regulate diverse processes throughout multiple phases of wound healing. To better understand the influence of microbial species on macrophage behavior, we cultured primary human monocyte-derived macrophages from four donors for 24 hours in media conditioned by bacteria and fungi (Pseudomonas aeruginosa, Corynebacterium amycolatum, Corynebacterium striatum, Staphylococcus aureus, Staphylococcus simulans, and Candida albicans) isolated from the DFUs of six patients. The effects of these microbe-derived signals on macrophage behavior were assessed by measuring the gene expression of a panel of 25 genes related to macrophage phenotype, angiogenesis, bacterial recognition, and cell survival, as well as secretion of two inflammatory cytokines using NanoString multiplex analysis. Principal component analysis showed that macrophage gene expression and protein secretion were affected by both microbial species as well as human donor. S. simulans and C. albicans caused up-regulation of genes associated with a proinflammatory (M1) phenotype, and P. aeruginosa caused an increase in the secretion of the proinflammatory cytokine and M1 marker tumor necrosis factor-alpha (TNFα). Together, these results suggest that macrophages respond to secreted factors from microbes by up-regulating inflammatory markers, and that the effects are strongly dependent on the monocyte donor. Ultimately, increased understanding of macrophage-microbe interactions will lead to the development of more targeted therapies for DFU healing.

Tissue engineering and regenerative medicine, facilitated by biomaterial-based therapies, hold promise for the repair, replacement, or regeneration of damaged tissue. The success or failure of all implanted biomaterials, ranging from stainless steel total joint replacements to naturally or synthetically derived skin grafts, is predominantly mediated by macrophages, the primary cell of the innate immune system. In an effort to better assess safety and efficacy of novel biomaterials, evaluating and understanding macrophage-biomaterial interactions is a necessary first step. Here, we describe the culture of macrophages on 3D biomaterials, such as decellularized human cortical bone or commercially available wound matrices, and subsequent analysis using gene expression and protein secretion to help understand how biomaterial properties may influence macrophage phenotype in vitro.