Dapansutrile OLT1177 suppresses foreign body response inflammation while preserving vascularisation of implanted materials.

Mitigating inflammation associated with the foreign body response (FBR) remains a significant challenge in enhancing the performance of implantable medical devices. Current anti-inflammatory approaches aim to suppress implant fibrosis, the major outcome of the FBR, but also inadvertently inhibit beneficial immune signalling necessary for tissue healing and vascularization. In a previous study, we demonstrated the feasibility of 'selective' immunosuppression targeting the NLRP3 inflammasome using the small molecule inhibitor MCC950, leading to reduced implant fibrosis without compromising healing and leading to enhanced vascularization. However, the clinical potential of MCC950 is severely limited due to its failure to pass Phase I clinical safety trials. This has triggered substantial efforts to develop safer analogues of NLRP3 inhibitors. Dapansutrile (OLT1177) is emerging as a leading candidate amongst current NLRP3 inhibitors, demonstrating both safety and effectiveness in a growing number of clinical indications and Phase 2 trials. While the anti-inflammatory effects of OLT1177 have been shown, validation of these effects in the context of implanted materials and the FBR have not yet been demonstrated. In this study, we show OLT1177 possesses beneficial effects on key cell types which drive FBR outcomes, including macrophages, fibroblasts, and smooth muscle cells. Evaluation of OLT1177 in a 28 day subcutaneous implantation model showed OLT1177 reduced fibrotic capsule formation while promoting implant vascularization. Mechanistic studies revealed that this occurred through activation of early pro-angiogenic markers while suppressing late-stage anti-angiogenic markers. These findings establish OLT1177 as a promising therapeutic approach for mitigating implant fibrosis while supporting vascularisation, suggesting a highly promising selective immunosuppressive strategy for the FBR warranting further research to explore its optimal integration into medical materials and devices.

On-Demand Bioactivation of Inert Materials With Plasma-Polymerized Nanoparticles.

Conventional gas plasma treatments are crucial for functionalizing materials in biomedical applications, but have limitations hindering their broader use. These methods require exposure to reactive media under vacuum conditions, rendering them unsuitable for substrates that demand aqueous environments, such as proteins and hydrogels. In addition, complex geometries are difficult to treat, necessitating extensive customization for each material and shape. To address these constraints, an innovative approach employing plasma polymer nanoparticles (PPN) as a versatile functionalization tool is proposed. PPN share similarities with traditional plasma polymer coatings (PPC) but offer unique advantages: compatibility with aqueous systems, the ability to modify complex geometries, and availability as off-the-shelf products. Robust immobilization of PPN on various substrates, including synthetic polymers, proteins, and complex hydrogel structures is demonstrated in this study. This results in substantial improvements in surface hydrophilicity. Materials functionalization with arginylglycylaspartic acid (RGD)-loaded PPN significantly enhances cell attachment, spreading, and substrate coverage on inert scaffolds compared to passive RGD coatings. Improved adhesion to complex geometries and subsequent differentiation following growth factor exposure is also demonstrated. This research introduces a novel substrate functionalization approach that mimics the outcomes of plasma coating technology but vastly expands its applicability, promising advancements in biomedical materials and devices.

Selective Immunosuppression Targeting the NLRP3 Inflammasome Mitigates the Foreign Body Response to Implanted Biomaterials While Preserving Angiogenesis.

Medical devices are a mainstay of the healthcare industry, providing clinicians with innovative tools to diagnose, monitor, and treat a range of medical conditions. For implantable devices, it is widely regarded that chronic inflammation during the foreign body response (FBR) is detrimental to device performance, but also required for tissue regeneration and host integration. Current strategies to mitigate the FBR rely on broad acting anti-inflammatory drugs, most commonly, dexamethasone (DEX), which can inhibit angiogenesis and compromise long-term device function. This study challenges prevailing assumptions by suggesting that FBR inflammation is multifaceted, and selectively targeting its individual pathways can stop implant fibrosis while preserving beneficial repair pathways linked to improved device performance. MCC950, an anti-inflammatory drug that selectively inhibits the NLRP3 inflammasome, targets pathological inflammation without compromising global immune function. The effects of MCC950 and DEX on the FBR are compared using implanted polycaprolactone (PCL) scaffolds. The results demonstrate that both DEX and MCC950 halt immune cell recruitment and cytokine release, leading to reduced FBR. However, MCC950 achieves this while supporting capillary growth and enhancing tissue angiogenesis. These findings support selective immunosuppression approaches as a potential future direction for treating the FBR and enhancing the longevity and safety of implantable devices.

Plasma polymerized nanoparticles are a safe platform for direct delivery of growth factor therapy to the injured heart.

Introduction: Heart failure due to myocardial infarction is a progressive and debilitating condition, affecting millions worldwide. Novel treatment strategies are desperately needed to minimise cardiomyocyte damage after myocardial infarction and to promote repair and regeneration of the injured heart muscle. Plasma polymerized nanoparticles (PPN) are a new class of nanocarriers which allow for a facile, one-step functionalization with molecular cargo. Methods: Here, we conjugated platelet-derived growth factor AB (PDGF-AB) to PPN, engineering a stable nano-formulation, as demonstrated by optimal hydrodynamic parameters, including hydrodynamic size distribution, polydisperse index (PDI) and zeta potential, and further demonstrated safety and bioactivity in vitro and in vivo. We delivered PPN-PDGF-AB to human cardiac cells and directly to the injured rodent heart. Results: We found no evidence of cytotoxicity after delivery of PPN or PPN-PDGFAB to cardiomyocytes in vitro, as determined through viability and mitochondrial membrane potential assays. We then measured contractile amplitude of human stem cell derived cardiomyocytes and found no detrimental effect of PPN on cardiomyocyte contractility. We also confirmed that PDGF-AB remains functional when bound to PPN, with PDGF receptor alpha positive human coronary artery vascular smooth muscle cells and cardiac fibroblasts demonstrating migratory and phenotypic responses to PPN-PDGF-AB in the same manner as to unbound PDGF-AB. In our rodent model of PPN-PDGF-AB treatment after myocardial infarction, we found a modest improvement in cardiac function in PPN-PDGF-AB treated hearts compared to those treated with PPN, although this was not accompanied by changes in infarct scar size, scar composition, or border zone vessel density. Discussion: These results demonstrate safety and feasibility of the PPN platform for delivery of therapeutics directly to the myocardium. Future work will optimize PPN-PDGF-AB formulations for systemic delivery, including effective dosage and timing to enhance efficacy and bioavailability, and ultimately improve the therapeutic benefits of PDGF-AB in the treatment of heart failure cause by myocardial infarction.

Selective NLRP3 Inflammasome Inhibitor MCC950 Suppresses Inflammation and Facilitates Healing in Vascular Materials.

Minimally invasive interventions using drug-eluting stents or balloons are a first-line treatment for certain occlusive cardiovascular diseases, but the major long-term cause of failure is neointimal hyperplasia (NIH). The drugs eluted from these devices are non-specific anti-proliferative drugs, such as paclitaxel (PTX) or sirolimus (SMS), which do not address the underlying inflammation. MCC950 is a selective inhibitor of the NLRP3-inflammasome, which drives sterile inflammation commonly observed in NIH. Additionally, in contrast to broad-spectrum anti-inflammatory drugs, MCC950 does not compromise global immune function due this selective activity. In this study, MCC950 is found to not impact the viability, integrity, or function of human coronary endothelial cells, in contrast to the non-specific anti-proliferative effects of PTX and SMS. Using an in vitro model of NLRP3-mediated inflammation in murine macrophages, MCC950 reduced IL-1ß expression, which is a key driver of NIH. In an in vivo mouse model of NIH in vascular grafts, MCC950 significantly enhanced re-endothelialization and reduced NIH compared to PTX or SMS. These findings show the effectiveness of a targeted anti-inflammatory drug-elution strategy with significant implications for cardiovascular device intervention.

Engineering Vascular Bioreactor Systems to Closely Mimic Physiological Forces In Vitro.

In vitro models of the vasculature play an important role in biomedical discovery research, with diverse applications in vascular biology, drug discovery, and tissue engineering. These models aim to replicate the conditions of the human vasculature including physical geometry, employing appropriate vascular cells exposed to physiological forces. However, vessel biology is complex, with multiple relevant cell types, precise three-dimensional (3D) architectural arrangement, an array of biological cues and pressure, flow rate, and shear stress stimulation that are difficult to replicate outside of the body. Vessel bioreactors typically comprise core modules, common to most systems: a 3D tubular scaffold to support cells, media and nutrient exchange for cell viability, a pumping module, and sensor arrays for monitoring. In our comprehensive review of the literature, foundational elements such as maintenance of cell viability, nutrient exchange with flow, use of 3D scaffolds, and basic sensing capabilities are well established. However, most bioreactor systems fail to adequately replicate combinations of physiologically relevant stimuli-including pressure, shear stress, and flow rate-independently, as system input parameters. At the root of this deficiency is the field's reliance on simple pumping systems designed for other applications, making it necessary to add resistors and compliance chambers to even approach human vascular conditions. As vascular biology research rapidly progressed it became increasingly clear that combinations of physical forces strongly influence cell phenotype, gene expression, and in turn can be drivers of pathology. We highlight the need for renewed innovation in vascular bioreactor development with a focus on the importance of providing appropriate physiological forces in the same system. Impact statement In vitro systems modeling aspects of the human vasculature are increasingly important in tissue engineering and biomedical research. Current systems maintain basic cell viability and facilitate nutrient exchange but poorly replicate physiological forces, reliant on simplistic pumping systems. Our review highlights the need to more accurately mimic arterial pressure, flow rate, and shear stress in the same system. Innovation in this area would improve in vitro modeling of the vasculature, significantly impacting tissue engineering and vascular biology in this area.

Synthetic vascular graft with spatially distinct architecture for rapid biomimetic cell organisation in a perfusion bioreactor.

Access to lab-grown fully functional blood vessels would provide an invaluable resource to vascular medicine. The complex architecture and cellular makeup of native vessels, however, makes this extremely challenging to reproducein vitro. Bioreactor systems have helped advanced research in this area by replicating many of the physiological conditions necessary for full-scale tissue growth outside of the body. A key element underpinning these technologies are 3D vascular graft templates which serve as temporary scaffolds to direct cell growth into similar cellular architectures observed in native vessels. Grafts further engineered with appropriate physical cues to accommodate the multiple cell types that reside within native vessels may help improve the production efficiency and physiological accuracy of bioreactor-grown vessel substitutes. Here, we engineered two distinct scaffold architectures into an electrospun vascular graft aiming to encourage the spatial organisation of human vascular endothelial cells (hCAECs) in a continuous luminal monolayer, co-cultured with human fibroblasts (hFBs) populating the graft wall. Using an electrospun composite of polycaprolactone and gelatin, we evaluated physical parameters including fibre diameter, fibre alignment, and porosity, that best mimicked the spatial composition and growth of hCAECs and hFBs in native vessels. Upon identifying the optimal scaffold architectures for each cell type, we constructed a custom-designed mandrel that combined these distinct architectures into a single vascular graft during a single electrospinning processing run. When connected to a perfusion bioreactor system, the dual architecture graft spatially oriented hCAECs and hFBs into the graft wall and lumen, respectively, directly from circulation. This biomimetic cell organisation was consistent with positive graft remodelling with significant collagen deposition in the graft wall. These findings demonstrate the influence of architectural cues to direct cell growth within vascular graft templates and the future potential of these approaches to more accurately and efficiency produce blood vessel substitutes in bioreactor systems.

Macrophage Polarization as a Novel Therapeutic Target for Endovascular Intervention in Peripheral Artery Disease.

Peripheral artery disease (PAD) has a significant impact on human health, affecting 200 million people globally. Advanced PAD severely diminishes quality of life, affecting mobility, and in its most severe form leads to limb amputation and death. Treatment of PAD is among the least effective of all endovascular procedures in terms of long-term efficacy. Chronic inflammation is a key driver of PAD; however, stents and coated balloons eluting antiproliferative drugs are most commonly used. As a result, neither stents nor coated balloons produce durable clinical outcomes in the superficial femoral artery, and both have recently been associated with significantly increased mortality. This review summarizes the most common clinical approaches and limitations to treating PAD and highlights the necessity to address the underlying causes of inflammation, identifying macrophages as a novel therapeutic target in the next generation of endovascular PAD intervention.

Bioengineering silk into blood vessels.

The rising incidence of cardiovascular disease has increased the demand for small diameter (<6 mm) synthetic vascular grafts for use in bypass surgery. Clinically available synthetic grafts (polyethylene terephthalate and expanded polytetrafluorethylene) are incredibly strong, but also highly hydrophobic and inelastic, leading to high rates of failure when used for small diameter bypass. The poor clinical outcomes of commercial synthetic grafts in this setting have driven significant research in search of new materials that retain favourable mechanical properties but offer improved biocompatibility. Over the last several decades, silk fibroin derived from Bombyx mori silkworms has emerged as a promising biomaterial for use in vascular applications. Progress has been driven by advances in silk manufacturing practices which have allowed unprecedented control over silk strength, architecture, and the ensuing biological response. Silk can now be manufactured to mimic the mechanical properties of native arteries, rapidly recover the native endothelial cell layer lining vessels, and direct positive vascular remodelling through the regulation of local inflammatory responses. This review summarises the advances in silk purification, processing and functionalisation which have allowed the production of robust vascular grafts with promise for future clinical application.

Silk Fibroin Scaffold Architecture Regulates Inflammatory Responses and Engraftment of Bone Marrow-Mononuclear Cells.

Despite being one of the most clinically trialed cell therapies, bone marrow-mononuclear cell (BM-MNC) infusion has largely failed to fulfill its clinical promise. Implanting biomimetic scaffolds at sites of injury prior to BM-MNC infusion is a promising approach to enhance BM-MNC engraftment and therapeutic function. Here, it is demonstrated that scaffold architecture can be leveraged to regulate the immune responses that drive BM-MNC engraftment. Silk scaffolds with thin fibers and low porosity (LP) impairs immune activation in vitro compared with thicker fiber, high porosity (HP) scaffolds. Using the authors' established in vivo bioluminescent BM-MNC tracking model, they showed that BM-MNCs home to and engraft in greater numbers in HP scaffolds over 14 days. Histological analysis reveals thicker fibrous capsule formation, with enhanced collagen deposition in HP compared to LP scaffolds consistent with substantially more native CD68+ macrophages and CD4+ T cells, driven by their elevated pro-inflammatory M1 and Th1 phenotypes, respectively. These results suggest that implant architecture impacts local inflammation that drives differential engraftment and remodeling behavior of infused BM-MNC. These findings inform the future design of biomimetic scaffolds that may better enhance the clinical effectiveness of BM-MNC infusion therapy.

Comprehensive Evaluation of the Toxicity and Biosafety of Plasma Polymerized Nanoparticles.

The rapid growth of nanoparticle-based therapeutics has underpinned significant developments in nanomedicine, which aim to overcome the limitations imposed by conventional therapies. Establishing the safety of new nanoparticle formulations is the first important step on the pathway to clinical translation. We have recently shown that plasma-polymerized nanoparticles (PPNs) are highly efficient nanocarriers and a viable, cost-effective alternative to conventional chemically synthesized nanoparticles. Here, we present the first comprehensive toxicity and biosafety study of PPNs using both established in vitro cell models and in vivo models. Overall, we show that PPNs were extremely well tolerated by all the cell types tested, significantly outperforming commercially available lipid-based nanoparticles (lipofectamine) used at the manufacturer's recommended dosage. Supporting the in vitro data, the systemic toxicity of PPNs was negligible in BALB/c mice following acute and repeated tail-vein intravenous injections. PPNs were remarkably well tolerated in mice without any evidence of behavioral changes, weight loss, significant changes to the hematological profile, or signs of histological damage in tissues. PPNs were tolerated at extremely high doses without animal mortality observed at 6000 mg/kg and 48,000 mg/kg for acute and repeated-injection regimens, respectively. Our findings demonstrate the safety of PPNs in biological systems, adding to their future potential in biomedical applications.

Bioactivation of Encapsulation Membranes Reduces Fibrosis and Enhances Cell Survival.

Encapsulation devices are an emerging barrier technology designed to prevent the immunorejection of replacement cells in regenerative therapies for intractable diseases. However, traditional polymers used in current devices are poor substrates for cell attachment and induce fibrosis upon implantation, impacting long-term therapeutic cell viability. Bioactivation of polymer surfaces improves local host responses to materials, and here we make the first step toward demonstrating the utility of this approach to improve cell survival within encapsulation implants. Using therapeutic islet cells as an exemplar cell therapy, we show that internal surface coatings improve islet cell attachment and viability, while distinct external coatings modulate local foreign body responses. Using plasma surface functionalization (plasma immersion ion implantation (PIII)), we employ hollow fiber semiporous poly(ether sulfone) (PES) encapsulation membranes and coat the internal surfaces with the extracellular matrix protein fibronectin (FN) to enhance islet cell attachment. Separately, the external fiber surface is coated with the anti-inflammatory cytokine interleukin-4 (IL-4) to polarize local macrophages to an M2 (anti-inflammatory) phenotype, muting the fibrotic response. To demonstrate the power of our approach, bioluminescent murine islet cells were loaded into dual FN/IL-4-coated fibers and evaluated in a mouse back model for 14 days. Dual FN/IL-4 fibers showed striking reductions in immune cell accumulation and elevated levels of the M2 macrophage phenotype, consistent with the suppression of fibrotic encapsulation and enhanced angiogenesis. These changes led to markedly enhanced islet cell survival and importantly to functional integration of the implant with the host vasculature. Dual FN/IL-4 surface coatings drive multifaceted improvements in islet cell survival and function, with significant implications for improving clinical translation of therapeutic cell-containing macroencapsulation implants.

Evaluation of synthetic vascular grafts in a mouse carotid grafting model.

Current animal models for the evaluation of synthetic grafts are lacking many of the molecular tools and transgenic studies available to other branches of biology. A mouse model of vascular grafting would allow for the study of molecular mechanisms of graft failure, including in the context of clinically relevant disease states. In this study, we comprehensively characterise a sutureless grafting model which facilitates the evaluation of synthetic grafts in the mouse carotid artery. Using conduits electrospun from polycaprolactone (PCL) we show the gradual development of a significant neointima within 28 days, found to be greatest at the anastomoses. Histological analysis showed temporal increases in smooth muscle cell and collagen content within the neointima, demonstrating its maturation. Endothelialisation of the PCL grafts, assessed by scanning electron microscopy (SEM) analysis and CD31 staining, was near complete within 28 days, together replicating two critical aspects of graft performance. To further demonstrate the potential of this mouse model, we used longitudinal non-invasive tracking of bone-marrow mononuclear cells from a transgenic mouse strain with a dual reporter construct encoding both luciferase and green fluorescent protein (GFP). This enabled characterisation of mononuclear cell homing and engraftment to PCL using bioluminescence imaging and histological staining over time (7, 14 and 28 days). We observed peak luminescence at 7 days post-graft implantation that persisted until sacrifice at 28 days. Collectively, we have established and characterised a high-throughput model of grafting that allows for the evaluation of key clinical drivers of graft performance.

Mechanically Robust Plasma-Activated Interfaces Optimized for Vascular Stent Applications.

The long-term performance of many medical implants is limited by the use of inherently incompatible and bioinert materials. Metallic alloys, ceramics, and polymers commonly used in cardiovascular devices encourage clot formation and fail to promote the appropriate molecular signaling required for complete implant integration. Surface coating strategies have been proposed for these materials, but coronary stents are particularly problematic as the large surface deformations they experience in deployment require a mechanically robust coating interface. Here, we demonstrate a single-step ion-assisted plasma deposition process to tailor plasma-activated interfaces to meet current clinical demands for vascular implants. Using a process control-feedback strategy which predicts crucial coating growth mechanisms by adopting a suitable macroscopic plasma description in combination with noninvasive plasma diagnostics, we describe the optimal conditions to generate highly reproducible, industry-scalable stent coatings. These interfaces are mechanically robust, resisting delamination even upon plastic deformation of the underlying material, and were developed in consideration of the need for hemocompatibility and the capacity for biomolecule immobilization. Our optimized coating conditions combine the best mechanical properties with strong covalent attachment capacity and excellent blood compatibility in initial testing with plasma and whole blood, demonstrating the potential for improved vascular stent coatings.

Blended Polyurethane and Tropoelastin as a Novel Class of Biologically Interactive Elastomer.

Polyurethanes are versatile elastomers but suffer from biological limitations such as poor control over cell attachment and the associated disadvantages of increased fibrosis. We address this problem by presenting a novel strategy that retains elasticity while modulating biological performance. We describe a new biomaterial that comprises a blend of synthetic and natural elastomers: the biostable polyurethane Elast-Eon and the recombinant human tropoelastin protein. We demonstrate that the hybrid constructs yield a class of coblended elastomers with unique physical properties. Hybrid constructs displayed higher elasticity and linear stress-strain responses over more than threefold strain. The hybrid materials showed increased overall porosity and swelling in comparison to polyurethane alone, facilitating enhanced cellular interactions. In vitro, human dermal fibroblasts showed enhanced proliferation, while in vivo, following subcutaneous implantation in mice, hybrid scaffolds displayed a reduced fibrotic response and tunable degradation rate. To our knowledge, this is the first example of a blend of synthetic and natural elastomers and is a promising approach for generating tailored bioactive scaffolds for tissue repair.

Immobilization of bioactive plasmin reduces the thrombogenicity of metal surfaces.

Components of many vascular prostheses including endovascular stents, heart valves and ventricular assist devices are made using metal alloys. In these blood contacting applications, metallic devices promote blood clotting, which is managed clinically by profound platelet suppression and/or anticoagulation. Here it is proposed that the localized immobilization of bioactive plasmin, a critical mediator of blood clot stability, may attenuate metallic prosthesis-induced thrombus formation. Previously described approaches to covalently immobilize biomolecules on implantable materials have relied on complex chemical linker chemistry, increasing the possibility of toxic side effects and reducing bioactivity. We utilize a plasma deposited thin film platform to covalently immobilize biologically active plasmin on stainless steel substrates, including stents. A range of in vitro whole blood assays demonstrate striking reductions in thrombus formation. This approach has profound potential to improve the efficacy of a wide range of metallic vascular implants.

Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin.

The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs) are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE) and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVß3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVß3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVß3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.