Interest in human mesenchymal stromal cells (MSCs) as an immune therapeutic has been on the increase over the past two decades with preclinical research yielding promising results, but so far only a singular MSC-based immunotherapy has been approved by the U.S. Food and Drug Administration (FDA). A major barrier for MSC clinical translation is the lack of reliable potency metrics and sufficient understanding of the mechanisms of action, as reported by the National Cell Manufacturing Consortium (NCMC). We integrated high-content morphological profiling and targeted sphingolipid analysis across MSC donors exhibiting a range of IFN-γ induced indoleamine-2,3-deoxygenase (IDO) activities to investigate the role of cell membrane components in MSC immunomodulatory function. High- and low IDO potency cells have distinct morphological signatures that are also reflected in the sphingolipid (SL) activity, with low IDO potency cell lines having low sphingomyelinase activity and high IDO potency cell lines having high sphingomyelinase activity. Perturbation of SL metabolism through the addition of exogenous neutral sphingomyelinase not only shifted morphological signatures to mimic those of high potency, but also significantly increased IDO activity within both high and low IDO potency donors. Additionally, these perturbations resulted in increased extracellular vesicle production with rich SL content and a stark reduction of TNF-α following EV treatment of activated macrophages. Together, these findings link morphology features from potent MSCs to sphingolipid remodeling and suggest a tractable strategy to tune MSC immunomodulatory potency.

Mesenchymal stromal cells (MSCs) show great promise as a clinical treatment for a variety of diseases, but their susceptibility to senescence during culture reduces the therapeutic potential and limits cell expansion. In this study, we explored how MSC lipid metabolism is altered in culture over time using ultrahigh-performance liquid chromatography mass spectrometry. The proportion of cells with senescence-associated β-galactosidase (SA-β-gal) activity was evaluated during 12 days of culture expansion of MSCs from two human donors. Lipid profiles were evaluated in parallel using exact mass and tandem mass spectrometry spectral database matching to generate 237 unique lipid annotations. Lipid abundance generally increased across most lipid classes over serial culture; however, many changes were heterogeneous between donors. Despite donor differences, 12 lipids, including 4 triglycerides (TG), provided discrimination between cultures with less than 10% SA-β-gal+, those with 10-20% SA-β-gal+, and greater than 20% SA-β-gal+ senescence proportion regardless of donor. More specifically, TG composed of long-chain, highly unsaturated fatty acids was strongly associated with higher MSC senescence. These changes in bulk lipid profiles may inform future strategies to monitor early culture senescence during the expansion of MSCs.

Understanding the immune response to hydrogel implantation is critical for the design of immunomodulatory biomaterials. To study the progression of inflammation around poly(ethylene glycol) hydrogels presenting Arg-Gly-Asp (RGD) peptides and vascular endothelial growth factor, we used temporal analysis of high-dimensional flow cytometry data paired with intravital imaging, immunohistochemistry, and multiplexed proteomic profiling. RGD-presenting hydrogels created a reparative microenvironment promoting CD206 + cellular infiltration and revascularization in wounded dorsal skin tissue. Unbiased clustering algorithms (SPADE) revealed significant phenotypic transition shifts as a function of the cell-adhesion hydrogel properties. SPADE identified an intermediate macrophage subset functionally regulating in vivo cytokine secretion that was preferentially recruited for RGD-presenting hydrogels, whereas dendritic cell subsets were preferentially recruited to RDG-presenting hydrogels. Last, RGD-presenting hydrogels controlled macrophage functional cytokine secretion to direct polarization and vascularization. Our studies show that unbiased clustering of single-cell data provides unbiased insights into the underlying immune response to engineered materials.

Cell therapies are expected to increase over the next decade owing to increasing demand for clinical applications. Mesenchymal stromal cells (MSCs) have been explored to treat a number of diseases, with some successes in early clinical trials. Despite early successes, poor MSC characterization results in lessened therapeutic capacity once in vivo. Here, we characterized MSCs derived from bone marrow (BM), adipose tissue and umbilical cord tissue for sphingolipids (SLs), a class of bioactive lipids, using liquid chromatography/tandem mass spectrometry. We found that ceramide levels differed based on the donor's sex in BM-MSCs. We detected fatty acyl chain variants in MSCs from all three sources. Linear discriminant analysis revealed that MSCs separated based on tissue source. Principal component analysis showed that interferon-γ-primed and unstimulated MSCs separated according to their SL signature. Lastly, we detected higher ceramide levels in low indoleamine 2,3-dioxygenase MSCs, indicating that sphingomyelinase or ceramidase enzymatic activity may be involved in their immune potency.

Volumetric muscle loss (VML) injuries after extremity trauma results in an important clinical challenge often associated with impaired healing, significant fibrosis, and long-term pain and functional deficits. While acute muscle injuries typically display a remarkable capacity for regeneration, critically sized VML defects present a dysregulated immune microenvironment which overwhelms innate repair mechanisms leading to chronic inflammation and pro-fibrotic signaling. In this series of studies, we developed an immunomodulatory biomaterial therapy to locally modulate the sphingosine-1-phosphate (S1P) signaling axis and resolve the persistent pro-inflammatory injury niche plaguing a critically sized VML defect. Multiparameter pseudo-temporal 2D projections of single cell cytometry data revealed subtle distinctions in the altered dynamics of specific immune subpopulations infiltrating the defect that were critical to muscle regeneration. We show that S1P receptor modulation via nanofiber delivery of Fingolimod (FTY720) was characterized by increased numbers of pro-regenerative immune subsets and coincided with an enriched pool of muscle stem cells (MuSCs) within the injured tissue. This FTY720-induced priming of the local injury milieu resulted in increased myofiber diameter and alignment across the defect space followed by enhanced revascularization and reinnervation of the injured muscle. These findings indicate that localized modulation of S1P receptor signaling via nanofiber scaffolds, which resemble the native extracellular matrix ablated upon injury, provides great potential as an immunotherapy for bolstering endogenous mechanisms of regeneration following VML injury.

Abstract Background Human Mesenchymal stromal cells (hMSCs) from various tissue sources are widely investigated in clinical trials. These MSCs are often administered to patients immediately after thawing the cryopreserved product (out-of-thaw), yet little is known about the single-cell transcriptomic landscape and tissue-specific differences of out-of-thaw human MSCs. Methods 13 hMSC samples derived from 10 “healthy” donors were used to assess donor variability and tissue-of-origin differences in single-cell gene expression profiles. hMSCs derived and expanded from the bone marrow (BM) or cord tissue (CT) underwent controlled-rate freezing for 24 h. Cells were then transferred to the vapor phase of liquid nitrogen for cryopreservation. hMSCs cryopreserved for at least one week, were characterized immediately after thawing using a droplet-based single-cell RNA sequencing method. Data analysis was performed with SC3 and SEURAT pipelines followed by gene ontology analysis. Results scRNA-seq analysis of the hMSCs revealed two major clusters of donor profiles, which differ in immune-signaling, cell surface properties, abundance of cell-cycle related transcripts, and metabolic pathways of interest. Within-sample transcriptomic heterogeneity is low. We identified numerous differentially expressed genes (DEGs) that are associated with various cellular functions, such as cytokine signaling, cell proliferation, cell adhesion, cholesterol/steroid biosynthesis, and regulation of apoptosis. Gene-set enrichment analyses indicated different functional pathways in BM vs. CT hMSCs. In addition, MSC-batches showed significant variations in cell cycle status, suggesting different proliferative vs. immunomodulatory potential. Several potential transcript-markers for tissue source differences were identified for further investigation in future studies. In functional assays, both BM and CT MSCs suppressed macrophage TNFα secretion upon interferon stimulation. However, differences between donors, tissue-of-origin, and cell cycle are evident in both TNF suppression and cytokine secretion. Conclusions This study shows that donor differences in hMSC transcriptome are minor relative to the intrinsic differences in tissue-of-origin. hMSCs with different transcriptomic profiles showed potential differences in functional characteristics. These findings contribute to our understanding of tissue origin-based differences in out-of-thaw therapeutic hMSC products and assist in the identification of cells with immune-regulatory or survival potential from a heterogeneous MSC population. Our results form the basis of future studies in correlating single-cell transcriptomic markers with immunomodulatory functions.

SUMMARYThroughout life, skeletal muscle, the arbiter of voluntary movements, is maintained by a population of skeletal muscle-dedicated stem cells, called muscle satellite cells (MuSCs). Similar to other adult stem cells, the function of MuSCs is tightly coordinated by the cellular and acellular components of their microenvironment, or the niche. While the processes that control the coupling of neurotransmission and muscle contraction have been well characterized, little is known on the reciprocal crosstalk between neural cells and MuSCs within the muscle microenvironment. Here, we report that mild peripheral nerve injury enhances MuSC myogenic function and muscle regeneration by synergistically augmenting MuSC mitochondrial bioenergetics and upregulating anabolic protein synthesis pathways. We also demonstrate that chronic disruption or degeneration of neuromuscular synapses, such as in muscular dystrophy and biological aging, abolishes MuSC and motor neuron interactions, causing significant deficits in muscle regeneration following injury. These results underscore the importance of neuromuscular junction and neural network as an essential niche of MuSCs. Determining the significance of MuSC-nerve interactions and their functional outcomes, as well as the possibility of modulating these connections, have important implications for our understanding of neuromuscular disease pathology and development of therapeutic interventions.HighlightsMild peripheral nerve injury increases muscle stem cell bioavailability of healthy muscle.Nerve perturbation stimulates myogenesis by enhancing protein synthesis and mitochondrial metabolism in young, healthy muscle.Synergistic crosstalk within neuromuscular niche boosts muscle regeneration in young, healthy muscle.Positive influences from the neural network on muscle stem cells are abolished in pathological denervation manifested in dystrophic and aging muscle.

Current cell culture surfaces used for the expansion and production of mesenchymal stromal cells (MSCs) are not optimized for the production of highly secretory and nonsenescent cells. In this study, we used poly (ethylene glycol) hydrogel substrates with tunable mechanical and biochemical properties to screen the effect of culture surfaces on pro-regenerative secretome by multiplex enzyme-linked immunosorbent assay, proliferation by PicoGreen DNA analysis, and senescence by senescence-associated β-galactosidase activity. We demonstrate that MSCs cultured on 30 kPa hydrogels, regardless of biochemical functionalization, broadly enhanced the secretion of immunomodulatory and regenerative factors versus stiffer 100 kPa or tissue culture plastic surfaces, but did not support robust proliferation. In contrast, culture on 100 kPa hydrogel surfaces promoted proliferation at a similar level and did not substantially alter the amount of secreted factors as compared with tissue culture plastic. Culture on integrin-engaging, cadherin-engaging, and hyaluronic acid-containing 30 kPa substrates enhanced MSC-conditioned media (CM) angiogenic activity in a human umbilical vein endothelial cell tube formation assay and human THP-1 monocyte chemoattraction in a transwell assay. However, 30 kPa substrate culture did not impact the myogenic activity of MSC CM in a C2C12 myoblast tube formation assay. Culture on selected 100 kPa surfaces enhanced CM angiogenic activity and monocyte chemotaxis, but not myogenic activity. Serial culture on 100 kPa RGD hydrogel surfaces significantly reduced senescence in MSCs versus tissue culture plastic, while maintaining the capacity of the cells to enhance their secretome in response to 30 kPa surfaces. Thus, hydrogel substrates that exhibit stiffness orders of magnitude lower than standard tissue culture plastic can serve as novel surfaces for the production of MSCs with an improved therapeutic secretory capacity and reduced senescence. Impact statement The success of mesenchymal stromal cell (MSC)-based therapies is dependent on the manufacture of a large number of cells with high therapeutic potency. Among the culture surfaces tested in this study, we demonstrate that substrate stiffness rather than biochemical functionalization predominantly guides changes in MSC proliferation and secretory capacity. We have identified substrate parameters to support MSC proliferation, enhance secretion of paracrine factors, and to reduce replicative senescence. By maximizing secretory capacity and reducing senescence through the choice of hydrogel culture materials, these findings have great potential to improve the large-scale production of therapeutic MSCs.

Regeneration of skeletal muscle after volumetric injury is thought to be impaired by a dysregulated immune microenvironment that hinders endogenous repair mechanisms. Such defects result in fatty infiltration, tissue scarring, chronic inflammation, and debilitating functional deficits. Here, we evaluated the key cellular processes driving dysregulation in the injury niche through localized modulation of sphingosine-1-phosphate (S1P) receptor signaling. We employ dimensionality reduction and pseudotime analysis on single cell cytometry data to reveal heterogeneous immune cell subsets infiltrating preclinical muscle defects due to S1P receptor inhibition. We show that global knockout of S1P receptor 3 (S1PR3) is marked by an increase of muscle stem cells within injured tissue, a reduction in classically activated relative to alternatively activated macrophages, and increased bridging of regenerating myofibers across the defect. We found that local S1PR3 antagonism via nanofiber delivery of VPC01091 replicated key features of pseudotime immune cell recruitment dynamics and enhanced regeneration characteristic of global S1PR3 knockout. Our results indicate that local S1P receptor modulation may provide an effective immunotherapy for promoting a proreparative environment leading to improved regeneration following muscle injury.

Inflammation after traumatic injury or surgical intervention is both a protective tissue response leading to regeneration and a potential cause of wound complications. One potentially successful strategy to harness to proregenerative roles of host inflammation is the localized delivery of bioactive materials to induce immune suppressive cellular responses by cells responding to injury. In this study, we designed a fully synthetic poly (ethylene) glycol (PEG)-based hydrogel to release the specialized pro-resolving lipid mediator aspirin-triggered resolvin-D1 (AT-RvD1) and recombinant human interleukin 10 (IL-10). We utilized a unique side-by-side internally controlled implant design wherein bioactive hydrogels were implanted adjacent to control hydrogels devoid of immune modulatory factors in the dorsal skinfold window chamber. We also explored singleimmune cell data with unsupervised approaches such as SPADE. First, we show that RGD-presenting hydrogel delivery results in enhanced immune cell recruitment to the site of injury. We then use intra-vital imaging to assess cellular recruitment and microvascular remodeling to show an increase in the caliber and density of local microvessels. Finally, we show that the recruitment and re-education of mononuclear phagocytes by combined delivery IL-10 and AT-RvD1 localizes immune suppressive subsets to the hydrogel, including CD206(+) macrophages (M2a/c) and IL-10 expressing dendritic cells in the context of chronic inflammation following surgical tissue disruption. These data demonstrate the potential of combined delivery on the recruitment of regenerative cell subsets involved in wound healing complications.

Purpose - Wound care following surgical procedures involves careful management, reducing risk of infection, and maximizing tissue oxygenation. Even with new strides in biological and synthetic products, achieving adequate wound care remains an important topic of interest. In this study, we develop an injectable hydrogel to promote wound resolution while increasing polarization of immune subsets towards healing phenotypes. These poly(ethylene glycol)-maleimide (PEG-MAL) hydrogels are capable of locally delivering therapeutic doses of the specialized pro-resolving mediator aspirin-triggered resolvin D1 (AT-RvD1) and the immunomodulatory cytokine interleukin 10 (IL-10) to enrich the injured tissue niche. To objectively uncover cellular heterogeneity from flow cytometry data, we utilized Spanning-tree Progression Analysis of Density-normalized Events (SPADE), a computational dimensionality reduction technique. This method highlighted established responses to wound healing, and, more notably, revealed novel aspects of the wound healing cascade in response to immunomodulatory treatment. Methods: The murine dorsal skinfold window chamber model was used to monitor vascularization and wound healing in response to hydrogel treatment. Each animal received an unloaded control hydrogel on the caudal side of the window chamber, and a loaded hydrogel rostrally. Loaded gels contained either IL-10, AT-RvD1, or both. At days 1, 3, and 7, dorsal tissue was excised and digested for flow cytometry. SPADE analysis was performed on manually gated single cell events to identify proportions of cells from the innate and adaptive immune system. Results: We show that AT-RvD1 and IL-10 alone are able to modulate the recruitment of various pro-inflammatory and pro-regenerative immune cells, but dual delivery of these factors enhances the recruitment of pro-regenerative immune cells, including M2 macrophages and tolerogenic dendritic cells, suggesting a synergistic interplay. Moreover, novel computational methods revealed the recruitment of rare immune cell subtypes, particularly from the adaptive immune system, to the site of injury following immunomodulatory hydrogel treatment. These findings suggest a promising method to target pro-regenerative cells from different branches of the immune system, and this treatment has the potential to enhance tissue regeneration and prevent wound healing complications after skin tissue injury. Conclusions: Biomaterial implants to deliver cells or molecules capable of recruiting and promoting the host immune response after injury can be instrumental to the restoration of tissue homeostasis and the promotion of wound healing. This dual-delivery system has the potential to improve therapeutic healing outcomes via synergy of cellular recruitment and polarization processes. Moreover, dimensionality reduction techniques, such as SPADE, provide novel and objective analytical approaches for analyzing high-dimensional data. We applied SPADE to mouse flow cytometry data and demonstrated that SPADE can be used to identify functional changes in response to treatment. Interestingly, SPADE enabled the identification of unexpected immune cell populations. This allows for the future development of immunomodulatory treatments that tailor the immune response and enhance the process of healing.

IMPACT STATEMENT:The goal of this study was to determine the threshold for a critically sized, nonhealing muscle defect by characterizing key components in the balance between fibrosis and regeneration as a function of injury size in the mouse quadriceps. There is currently limited understanding of what leads to a critically sized muscle defect and which muscle regenerative components are functionally impaired. With the substantial increase in preclinical VML models as testbeds for tissue engineering therapeutics, defining the critical threshold for VML injuries will be instrumental in characterizing therapeutic efficacy and potential for subsequent translation.

Mesenchymal stem and progenitor cells (MSCs), which normally reside in the bone marrow, are critical to bone health and can be recruited to sites of traumatic bone injury, contributing to new bone formation. The ability to control the trafficking of MSCs provides therapeutic potential for improving traumatic bone healing and therapy for genetic bone diseases such as hypophosphatasia.

Skeletal muscle has a remarkable regenerative capacity; however, after volumetric muscle loss (VML) due to traumatic injury or surgery this regenerative response is significantly diminished, causing chronic functional deficits. The critical defect size at which the muscle will not functionally recover has not yet been established and subsequently, the relative contribution of crucial muscle components, including muscle stem cells and the muscle stem cell niche, are unknown. In this study, we created VML injuries of 2, 3, or 4 mm diameter, full-thickness defects in the mouse quadriceps. The 2, 3, and 4 mm injuries resulted in a defect of 5, 15, or 30% of the quadriceps mass, respectively. At 14 and 28 days after injury, histological analyses revealed injury size-dependent differences in myofiber morphology and fibrosis; the number of small myofibers increased with increasing injury size. The results showed that the 3 mm injury was at a threshold point, as myofibers were unable to bridge the defect, there was persistent fibrosis and inflammation, and significantly increased number of myofibers with centrally located nuclei. We then further investigated the 3 mm VML for nerve and vascular regeneration. These injured muscles were accompanied by a drastic increase in denervated neuromuscular junctions (NMJ), while assessment of angiogenesis via micro-CT analysis revealed a significant increase in vascular volume primarily from small diameter vessels after VML injury. Collectively, these data indicate that the spatial and temporal control of the fibrotic and neuromotor response are critical to regeneration and could be potential therapeutic targets, as they are the most dysregulated components of the muscle stem cell niche after VML.

The immune response to biomaterial implants critically regulates functional outcomes such as vascularization, transplant integration/survival, and fibrosis. To create "immunologically smart" materials, the host-material response may be engineered to optimize the recruitment of pro-regenerative leukocyte subsets which mature into corresponding wound-healing macrophages. We have recently identified a unique feature of pro-regenerative Ly6Clow monocytes that is a higher expression of both the bioactive lipid receptor sphingosine-1-phosphate receptor 3 (S1PR3) and the stromal derived factor-1α (SDF-1α) receptor CXCR4. Therefore, we designed a bifunctional hydrogel to harnesses a mechanistic synergy between these signaling axes to enhance the recruitment of endogenous pro-regenerative monocytes. To overcome the challenge of codelivering two physiochemically distinct molecules-a large hydrophilic protein and hydrophobic small molecule-we engineered a dual affinity hydrogel that exploits the growth factor affinity of a heparin derivative (Hep-N) and lipid chaperone activity of albumin. The sphingosine analog FTY720 and SDF-1α are successfully loaded and coreleased from the Hep-N-functionalized PEG-DA hydrogels while maintaining bioactivity. Placement of these hydrogels into a murine partial thickness skin wound demonstrates that corelease of FTY720 and SDF-1α yields superior recruitment of myeloid cells to the implant interface compared to either factor alone. Although in vivo delivery of FTY720 or SDF-1α individually promotes the enhanced recruitment of Ly-6Clow anti-inflammatory monocytes, codelivery enhances the early accumulation and persistence of the differentiated wound healing CD206+ macrophages in the tissue surrounding the gel. Co-delivery similarly promoted the synergistic expansion of vasculature adjacent to the implant, a key step in tissue healing. Taken together, these findings suggest that the combination of chemotactic molecules may provide additional maturation signals to the infiltrating leukocytes to facilitate macrophage transition and vascular network expansion, thus, ultimately, potentiating tissue repair. The coupling of multiple pro-regenerative biological cues provides a foundation for more fine-tuned immunoregenerative modulation to facilitate tissue repair.

Successful tissue repair requires the activities of myeloid cells such as monocytes and macrophages that guide the progression of inflammation and healing outcome. Immunoregenerative materials leverage the function of endogenous immune cells to orchestrate complex mechanisms of repair; however, a deeper understanding of innate immune cell function in inflamed tissues and their subsequent interactions with implanted materials is necessary to guide the design of these materials. Blood monocytes exist in two primary subpopulations, characterized as classical inflammatory or non-classical. While classical monocytes extravasate into inflamed tissue and give rise to macrophages or dendritic cells, the recruitment kinetics and functional role of non-classical monocytes remains unclear. Here, we demonstrate that circulating non-classical monocytes are directly recruited to polymer films within skin injuries, where they home to a perivascular niche and generate alternatively activated, wound healing macrophages. Selective labeling of blood monocyte subsets indicates that non-classical monocytes are biased progenitors of alternatively activated macrophages. On-site delivery of the immunomodulatory small molecule FTY720 recruits S1PR3-expressing non-classical monocytes that support vascular remodeling after injury. These results elucidate a previously unknown role for blood-derived non-classical monocytes as contributors to alternatively activated macrophages, highlighting them as key regulators of inflammatory response and regenerative outcome.

Hematopoietic stem and progenitor cells (HSPCs) egress from bone marrow ( BM) during homeostasis and at increased rates during stress; however, the mechanisms regulating their trafficking remain incompletely understood. Here we describe a novel role for lipid receptor, sphingosine-1-phosphate receptor 3 (S1PR3), in HSPC residence within the BM niche. HSPCs expressed increased levels of S1PR3 compared to differentiated BM cells. Pharmacological antagonism or knockout ( KO) of S1PR3 mobilized HSPCs into blood circulation, suggesting that S1PR3 influences niche localization. S1PR3 antagonism suppressed BM and plasma SDF-1, enabling HSPCs to migrate toward S1P-rich plasma. Mobilization synergized with AMD3100-mediated antagonism of CXCR4, which tethers HSPCs in the niche, and recovered homing deficits of AMD3100-treated grafts. S1PR3 antagonism combined with AMD3100 improved re-engraftment and survival in lethally irradiated recipients. Our studies indicate that S1PR3 and CXCR4 signaling cooperate to maintain HSPCs within the niche under homeostasis. These results highlight an important role for S1PR3 in HSPC niche occupancy and trafficking that can be harnessed for both rapid clinical stem cell mobilization and re-engraftment strategies, as well as the opportunity to design novel therapeutics for control of recruitment, homing, and localization through bioactive lipid signaling.

Event Abstract Back to Event Monocyte recruitment to polymer implants regulates the in situ generation of macrophages and vascular remodeling Claire Segar1, Cheryl M. Lau1, Jack R. Krieger1, Molly E. Ogle1, Brett J. Jordan1 and Edward A. Botchwey1 1 Georgia Institute of Technology and Emory University, Biomedical Engineering, United States Introduction: Critical roles for the recruitment of inflammatory cells have been described for a broad spectrum of therapeutic applications, including skeletal muscle repair and limb regeneration, as well as generic host responses to biomedical implants. Biphasic recruitment kinetics of Ly6Chi (“pro-inflammatory”) monocytes from the blood is a hallmark of acute inflammation and is followed temporally by recruitment of Ly6Clo (“anti-inflammatory”) monocytes that facilitate inflammation resolution and re-vascularization of damaged tissue. While macrophages have been widely appreciated for their functions in mediating host response to implanted materials, a role for their monocyte precursors has remained largely unexplored. Using the murine dorsal skinfold window chamber model to monitor inflammatory processes in situ, we designed a series of experiments to explore the role that circulating monocytes play in wound healing. We hypothesized that enhancing recruitment of Ly6Clo monocytes from blood would increase the generation of pro-regenerative “M2” macrophages and improve vascular remodeling. Methods and Materials: To monitor monocyte recruitment and vascular remodeling in real-time, we performed dorsal skinfold window chambers in mice, which entails split thickness skin wounding. Poly(lactic-co-glycolic acid) (PLGA) thin films were made by using a solvent-casting technique with or without the small molecule FTY720 and subsequently implanted in window chambers. Selective labeling of Ly6Clo monocytes was performed by intravenous injection of fluorescently-labeled latex beads 1 day prior to surgery. In separate studies, labeling of Ly6Chi monocytes was performed by intravenous administration of clodronate liposomes (ClodLip) 2 days prior to surgery, followed by latex beads 16 hours later. Intravital microscopy and whole mount immunohistochemistry was used to investigate longitudinal changes to the vasculature and monocyte/macrophage-vessel interactions, respectively. Monocyte subsets were immunophenotyped and quantified using flow cytometry analysis of blood and collagenase-digested tissue. Results and Discussion: Circulating Ly6Clo monocytes labeled with latex beads were seen in inflamed tissue surround unloaded PLGA implants by 3 days post-surgery (Fig. 1A) and preferentially became CD206+ M2-like macrophages (Fig. 1B). Conversely, Ly6Chi monocytes labeled via sequential administration of ClodLip and latex beads similarly homed to inflamed tissue (Fig. 1C), but displayed no preference for differentiating into CD206+ macrophages (Fig. 1D). Selective depletion of blood Ly6Clo monocytes with ClodLip (Fig. 1E) severely impaired the in situ generation of CD206+ macrophages (Fig. 1F). Consequently, blood-derived Ly6Clo monocytes are biased progenitors of CD206+ wound macrophages that efficiently undergo differentiation post-extravasation. On site delivery of the immunomodulatory small molecule FTY720 increased the frequency of recruited Ly6Clo monocytes (data not shown) and CD206+F4/80+ macrophages 3 days post-surgery (Fig. 2A). In loss-of-function studies employing ClodLip, we observed that FTY720 no longer increased the frequency of M2 macrophages (Fig. 2B), indicating that circulating Ly6Clo monocytes are required. To explore the function of CD206+ macrophages, we probed the distribution of CD68+CD206+ cells in explanted whole tissue samples. FTY720 increased the frequency of CD68+CD206+ macrophages and positioned these cells around remodeling blood vessels (Fig. 2C). Moreover, FTY720-mediated regulation of monocyte fate corresponded with expansion of arterioles and angiogenic sprouting (Fig. 2D). Conclusions: We have demonstrated that circulating Ly6Clo monocytes are robustly recruited to inflamed peri-implant tissue following injury and efficiently undergo differentiation into pro-regenerative macrophages. Recruitment of Ly6Clo monocytes by biomaterial-released FTY720 is a promising strategy for tuning the innate immune response to improve vascularization and wound healing. National Institutes of Health grants R01AR056445-01A2 and R01DE019935-01 to Dr. Botchwey; National Science Foundation Graduate Research Fellowship to Claire Segar under Grant No. No. DGE-1148903; American Heart Association Pre-doctoral Fellowship 15PRE25090024 to Claire Segar Keywords: blood vessel, cell fate, in vivo tissue engineering Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: General Session Oral Topic: Role of biomaterials in inflammation Citation: Segar C, Lau CM, Krieger JR, Ogle ME, Jordan BJ and Botchwey EA (2016). Monocyte recruitment to polymer implants regulates the in situ generation of macrophages and vascular remodeling. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00159 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Claire Segar Cheryl M Lau Jack R Krieger Molly E Ogle Brett J Jordan Edward A Botchwey Google Claire Segar Cheryl M Lau Jack R Krieger Molly E Ogle Brett J Jordan Edward A Botchwey Google Scholar Claire Segar Cheryl M Lau Jack R Krieger Molly E Ogle Brett J Jordan Edward A Botchwey PubMed Claire Segar Cheryl M Lau Jack R Krieger Molly E Ogle Brett J Jordan Edward A Botchwey Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.