Design of an Ultralow Molecular Weight Heparin That Resists Heparanase Biodegradation.

Heparanase, an endo-ß-d-glucuronidase produced by a variety of cells and tissues, cleaves the glycosidic linkage between glucuronic acid (GlcA) and a 3-O- or 6-O-sulfated glucosamine, typified by the disaccharide -[GlcA-GlcNS3S6S]-, which is found within the antithrombin-binding domain of heparan sulfate or heparin. As such, all current forms of heparin are susceptible to degradation by heparanase with neutralization of anticoagulant properties. Here, we have designed a heparanase-resistant, ultralow molecular weight heparin as the structural analogue of fondaparinux that does not contain an internal GlcA residue but otherwise displays potent anticoagulant activity. This heparin oligosaccharide was synthesized following a chemoenzymatic scheme and displays nanomolar anti-FXa activity yet is resistant to heparanase digestion. Inhibition of thrombus formation was further demonstrated after subcutaneous administration of this compound in a murine model of venous thrombosis. Thrombus inhibition was comparable to that observed for enoxaparin with a similar effect on bleeding time.

Sulfated poly-amido-saccharides (sulPASs) are anticoagulants in vitro and in vivo.

Anticoagulant therapeutics are a mainstay of modern surgery and of clotting disorder management such as venous thrombosis, yet performance and supply limitations exist for the most widely used agent - heparin. Herein we report the first synthesis, characterization, and performance of sulfated poly-amido-saccharides (sulPASs) as heparin mimetics. sulPASs inhibit the intrinsic pathway of coagulation, specifically FXa and FXIa, as revealed by ex vivo human plasma clotting assays and serine protease inhibition assays. sulPASs activity positively correlates with molecular weight and degree of sulfation. Importantly, sulPASs are not degraded by heparanases and are non-hemolytic. In addition, their activity is reversed by protamine sulfate, unlike small molecule anticoagulants. In an in vivo murine model, sulPASs extend clotting time in a dose dependent manner with bleeding risk comparable to heparin. These findings support continued development of synthetic anticoagulants to address the clinical risks and shortages associated with heparin.

A rechargeable anti-thrombotic coating for blood-contacting devices.

Despite the potential of anti-thrombogenic coatings, including heparinized surfaces, to improve the performance of blood-contacting devices, the inevitable deterioration of bioactivity remains an important factor in device failure and related thrombotic complications. As a consequence, the ability to restore the bioactivity of a surface coating after implantation of a blood-contacting device provides a potentially important strategy to enhance its clinical performance. Here, we report the regeneration of a multicomponent anti-thrombogenic coating through use of an evolved sortase A to mediate reversible transpeptidation. Both recombinant thrombomodulin and a chemoenzymatically synthesized ultra-low molecular weight heparin were repeatedly and selectively immobilized or removed in a sequential, alternating, or simultaneous manner. The generation of activated protein C (aPC) and inhibition of activated factor X (FXa) was consistent with the molecular composition of the surface. The fabrication of a rechargeable anti-thrombogenic surface was demonstrated on an expanded polytetrafluoroethylene (ePTFE) vascular graft with reconstitution of the surface bound coating 4 weeks after in vivo implantation in a rat model.

A PSGL-1 glycomimetic reduces thrombus burden without affecting hemostasis.

Events mediated by the P-selectin/PSGL-1 pathway play a critical role in the initiation and propagation of venous thrombosis by facilitating the accumulation of leukocytes and platelets within the growing thrombus. Activated platelets and endothelium express P-selectin, which binds P-selectin glycoprotein ligand-1 (PSGL-1) that is expressed on the surface of all leukocytes. We developed a pegylated glycomimetic of the N terminus of PSGL-1, PEG40-GSnP-6 (P-G6), which proved to be a highly potent P-selectin inhibitor with a favorable pharmacokinetic profile for clinical translation. P-G6 inhibits human and mouse platelet-monocyte and platelet-neutrophil aggregation in vitro and blocks microcirculatory platelet-leukocyte interactions in vivo. Administration of P-G6 reduces thrombus formation in a nonocclusive model of deep vein thrombosis with a commensurate reduction in leukocyte accumulation, but without disruption of hemostasis. P-G6 potently inhibits the P-selectin/PSGL-1 pathway and represents a promising drug candidate for the prevention of venous thrombosis without increased bleeding risk.

Standardized User-Independent Confocal Microscopy Image Acquisition and Analysis for Thickness Measurements of Microscale Collagen Scaffolds.

The ability to accurately and precisely measure the thickness of biomaterial constructs is critical for characterizing both specific dimensional features and related mechanical properties. However, in the absence of a standardized approach for thickness measurements, a variety of imaging modalities have been employed, which have been associated with varying limits of accuracy, particularly for ultrathin hydrated structures. Electron microscopy (EM), a commonly used modality, yields thickness values for extensively processed and nonhydrated constructs, potentially resulting in overestimated mechanical properties, including elastic modulus and ultimate tensile strength. Confocal laser scanning microscopy (CLSM) has often been used as a nondestructive imaging alternative. However, published CLSM-derived image analysis protocols use arbitrary signal intensity cutoffs and provide minimal information regarding thickness variability across imaged surfaces. To address the aforementioned limitations, we present a standardized, user-independent CLSM image acquisition and analysis approach developed as a custom ImageJ macro and validated with collagen-based scaffolds. In the process, we also quantify thickness discrepancies in collagen-based scaffolds between CLSM and EM techniques, further illustrating the need for improved strategies. Employing the same image acquisition protocol, we also demonstrate that this approach can be used to estimate the surface roughness of the same scaffolds without the use of specialized instrumentation.

Resident and elicited murine macrophages differ in expression of their glycomes and glycan-binding proteins.

The pleiotropic functions of macrophages in immune defense, tissue repair, and maintenance of tissue homeostasis are supported by the heterogeneity in macrophage sub-populations that differ both in ontogeny and polarization. Although glycans and glycan-binding proteins (GBPs) are integral to macrophage function and may contribute to macrophage diversity, little is known about the factors governing their expression. Here, we provide a resource for characterizing the N-/O-glycomes of various murine peritoneal macrophage sub-populations, demonstrating that glycosylation primarily reflects developmental origin and, to a lesser degree, cellular polarization. Furthermore, comparative analysis of GBP-coding genes in resident and elicited macrophages indicated that GBP expression is consistent with specialized macrophage functions and correlates with specific types of displayed glycans. An integrated, semi-quantitative approach was used to confirm distinct expression patterns of glycans and their binding proteins across different macrophages. The data suggest that regulation of glycan-protein complexes may be central to macrophage residence and recruitment.

Continuous Formation of Ultrathin, Strong Collagen Sheets with Tunable Anisotropy and Compaction.

The multiscale organization of protein-based fibrillar materials is a hallmark of many organs, but the recapitulation of hierarchal structures down to fibrillar scales, which is a requirement for withstanding physiological loading forces, has been challenging. We present a microfluidic strategy for the continuous, large-scale formation of strong, handleable, free-standing, multicentimeter-wide collagen sheets of unprecedented thinness through the application of hydrodynamic focusing with the simultaneous imposition of strain. Sheets as thin as 1.9 µm displayed tensile strengths of 0.5-2.7 MPa, Young's moduli of 3-36 MPa, and modulated the diffusion of molecules as a function of collagen nanoscale structure. Smooth muscle cells cultured on engineered sheets oriented in the direction of aligned collagen fibrils and generated coordinated vasomotor responses. The described biofabrication approach enables rapid formation of ultrathin collagen sheets that withstand physiologically relevant loads for applications in tissue engineering and regenerative medicine, as well as in organ-on-chip and biohybrid devices.

Modulation of lymphocyte-mediated tissue repair by rational design of heterocyclic aryl hydrocarbon receptor agonists.

Aryl hydrocarbon receptor (AHR) is an essential regulator of gut immunity and a promising therapeutic target for inflammatory bowel disease (IBD). Current AHR agonists are inadequate for clinical translation due to low activity, inadequate pharmacokinetics, or toxicity. We synthesized a structurally diverse library and used integrated computational and experimental studies to discover mechanisms governing ligand-receptor interaction and to design potent drug leads PY109 and PY108, which display physiochemical drug-likeness properties, desirable pharmacokinetic profiles, and low toxicity. In a murine model of dextran sulfate sodium-induced colitis, orally administered compounds increase interleukin-22 (IL-22) production and accelerate mucosal healing by modulating mucosal adaptive and innate lymphoid cells. AHR and IL-22 pathway induction was confirmed using RNA sequencing and characterization of the lymphocyte protein-protein interaction network. Significant induction of IL-22 was also observed using human T cells from patients with IBD. Our findings support rationally designed AHR agonists for IBD therapy.

Evaluation of a bioengineered construct for tissue engineering applications.

Effective biomaterial options for tissue repair and regeneration are limited. Current biologic meshes are derived from different tissue sources and are generally sold as decellularized tissues. This work evaluated two collagen based bioengineered constructs and a commercial product in a model of abdominal full thickness defect repair. To prepare the bioengineered construct, collagen type 1 from porcine skin was isolated using an acid solubilization method. After purification, the collagen was formed into collagen sheets that were physically bonded to form a mechanically robust construct that was subsequently laser micropatterned with pores as a means to promote tissue integration (collagen only construct). A second engineered construct consisted of the aforementioned collagen construct embedded in an RGD-functionalized alginate gel that serves as a bioactive interface (collagen-alginate construct). The commercial product is a biologic mesh derived from bovine pericardium (Veritas® ). We observed enhanced vascularization in the midportion of the engineered collagen-alginate construct 2 weeks after implantation. Overall, the performance of the bioengineered constructs was similar to that of the commercial product with comparable integration strength at 8 weeks. Bioengineered constructs derived from monomeric collagen demonstrate promise for a variety of load bearing applications in tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2345-2354, 2018.

An immobilized liquid interface prevents device associated bacterial infection in vivo.

Virtually all biomaterials are susceptible to biofilm formation and, as a consequence, device-associated infection. The concept of an immobilized liquid surface, termed slippery liquid-infused porous surfaces (SLIPS), represents a new framework for creating a stable, dynamic, omniphobic surface that displays ultralow adhesion and limits bacterial biofilm formation. A widely used biomaterial in clinical care, expanded polytetrafluoroethylene (ePTFE), infused with various perfluorocarbon liquids generated SLIPS surfaces that exhibited a 99% reduction in S. aureus adhesion with preservation of macrophage viability, phagocytosis, and bactericidal function. Notably, SLIPS modification of ePTFE prevents device infection after S. aureus challenge in vivo, while eliciting a significantly attenuated innate immune response. SLIPS-modified implants also decrease macrophage inflammatory cytokine expression in vitro, which likely contributed to the presence of a thinner fibrous capsule in the absence of bacterial challenge. SLIPS is an easily implementable technology that provides a promising approach to substantially reduce the risk of device infection and associated patient morbidity, as well as health care costs.

Targeting P-selectin glycoprotein ligand-1/P-selectin interactions as a novel therapy for metabolic syndrome.

Obesity-induced insulin resistance and metabolic syndrome continue to pose an important public health challenge worldwide as they significantly increase the risk of type 2 diabetes and atherosclerotic cardiovascular disease. Advances in the pathophysiologic understanding of this process has identified that chronic inflammation plays a pivotal role. In this regard, given that both animal models and human studies have demonstrated that the interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin is not only critical for normal immune response but also is upregulated in the setting of metabolic syndrome, PSGL-1/P-selectin interactions provide a novel target for preventing and treating resultant disease. Current approaches of interfering with PSGL-1/P-selectin interactions include targeted antibodies, recombinant immunoglobulins that competitively bind P-selectin, and synthetic molecular therapies. Experimental models as well as clinical trials assessing the role of these modalities in a variety of diseases have continued to contribute to the understanding of PSGL-1/P-selectin interactions and have demonstrated the difficulty in creating clinically relevant therapeutics. Most recently, however, computational simulations have further enhanced our understanding of the structural features of PSGL-1 and related glycomimetics, which are responsible for high-affinity selectin interactions. Leveraging these insights for the design of next generation agents has thus led to development of a promising synthetic method for generating PSGL-1 glycosulfopeptide mimetics for the treatment of metabolic syndrome.

Efferocytosis as a regulator of macrophage chemokine receptor expression and polarization.

Efferocytosis has been suggested to promote macrophage resolution programs that are dependent on motility and emigration, however, few studies have addressed directed migration in resolving macrophages. In this report, we hypothesized that efferocytosis would induce differential chemokine receptor expression. Polarized macrophage populations, including macrophages actively engaged in efferocytosis, were characterized by PCR array and traditional transwell motility assays. We identified specific up-regulation of chemokine receptor CXCR4 on both mouse and human macrophages and characterized in vivo expression of CXCR4 in a resolving model of murine peritonitis. Using adoptive transfer and AMD3100 blocking, we confirmed a role for CXCR4 in macrophage egress to draining lymphatics. Collectively these data provide an important mechanistic link between efferocytosis and macrophage emigration.

In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A.

Surface immobilization of bioactive molecules is a central paradigm in the design of implantable devices and biosensors with improved clinical performance capabilities. However, in vivo degradation or denaturation of surface constituents often limits the long-term performance of bioactive films. Here we demonstrate the capacity to repeatedly regenerate a covalently immobilized monomolecular thin film of bioactive molecules through a two-step stripping and recharging cycle. Reversible transpeptidation by a laboratory evolved Staphylococcus aureus sortase A (eSrtA) enabled the rapid immobilization of an anti-thrombogenic film in the presence of whole blood and permitted multiple cycles of film regeneration in vitro that preserved its biological activity. Moreover, eSrtA transpeptidation facilitated surface re-engineering of medical devices in situ after in vivo implantation through removal and restoration film constituents. These studies establish a rapid, orthogonal and reversible biochemical scheme to regenerate selective molecular constituents with the potential to extend the lifetime of bioactive films.

Rapid homogeneous endothelialization of high aspect ratio microvascular networks.

Microvascularization of an engineered tissue construct is necessary to ensure the nourishment and viability of the hosted cells. Microvascular constructs can be created by seeding the luminal surfaces of microfluidic channel arrays with endothelial cells. However, in a conventional flow-based system, the uniformity of endothelialization of such an engineered microvascular network is constrained by mass transfer of the cells through high length-to-diameter (L/D) aspect ratio microchannels. Moreover, given the inherent limitations of the initial seeding process to generate a uniform cell coating, the large surface-area-to-volume ratio of microfluidic systems demands long culture periods for the formation of confluent cellular microconduits. In this report, we describe the design of polydimethylsiloxane (PDMS) and poly(glycerol sebacate) (PGS) microvascular constructs with reentrant microchannels that facilitates rapid, spatially homogeneous endothelial cell seeding of a high L/D (2 cm/35 µm; > 550:1) aspect ratio microchannels. MEMS technology was employed for the fabrication of a monolithic, elastomeric, reentrant microvascular construct. Isotropic etching and PDMS micromolding yielded a near-cylindrical microvascular channel array. A 'stretch - seed - seal' operation was implemented for uniform incorporation of endothelial cells along the entire microvascular area of the construct yielding endothelialized microvascular networks in less than 24 h. The feasibility of this endothelialization strategy and the uniformity of cellularization were established using confocal microscope imaging.

Engineered composite fascia for stem cell therapy in tissue repair applications.

A critical challenge in tissue regeneration is to develop constructs that effectively integrate with the host tissue. Here, we describe a composite, laser micromachined, collagen-alginate construct containing human mesenchymal stem cells (hMSCs) for tissue repair applications. Collagen type I was fashioned into laminated collagen sheets to form a mechanically robust fascia that was subsequently laser micropatterned with pores of defined dimension and spatial distribution as a means to modulate mechanical behavior and promote tissue integration. Significantly, laser micromachined patterned constructs displayed both substantially greater compliance and suture retention strength than non-patterned constructs. hMSCs were loaded in an RGD-functionalized alginate gel modified to degrade in vivo. Over a 7 day observation period in vitro, high cell viability was observed with constant levels of VEGF, PDGF-ß and MCP-1 protein expression. In a full thickness abdominal wall defect model, the composite construct prevented hernia recurrence in Wistar rats over an 8-week period with de novo tissue and vascular network formation and the absence of adhesions to underlying abdominal viscera. As compared to acellular constructs, constructs containing hMSCs displayed greater integration strength (cell seeded: 0.92 ± 0.19 N/mm vs. acellular: 0.59 ± 0.25 N/mm, p=0.01), increased vascularization (cell seeded: 2.7-2.1/hpf vs. acellular: 1.7-2.1/hpf, p<0.03), and increased infiltration of macrophages (cell seeded: 2021-3630 µm(2)/hpf vs. acellular: 1570-2530 µm(2)/hpf, p<0.05). A decrease in the ratio of M1 macrophages to total macrophages was also observed in hMSC-populated samples. Laser micromachined collagen-alginate composites containing hMSCs can be used to bridge soft tissue defects with the capacity for enhanced tissue repair and integration. STATEMENT OF SIGNIFICANCE: Effective restoration of large soft tissue defects caused by trauma or treatment complications represents a critical challenge in the clinic. In this study, a novel composite construct was engineered and evaluated for stem cell delivery and tissue repair. Laser micromachining was used to fabricate patterned, microporous constructs designed with pores of defined size and distribution as a means to tune mechanical responses, accommodate and protect incorporated cells, and enhance tissue integration. The construct was embedded within an engineered alginate gel containing hMSCs. Upon repair of a full thickness abdominal wall defect in a rat model, the composite construct modulated host innate immunity towards a reparative phenotypic response, promoted neovascularization and associated matrix production, and increased the strength of tissue integration.

Syndecan-1 modulates the motility and resolution responses of macrophages.

OBJECTIVE: Syndecan-1 (Sdc-1) is a member of a family of cell surface proteoglycans, which has been reported to participate in the regulation of events relevant to tissue repair and chronic injury responses, including cell-substrate interactions, matrix remodeling, and cell migration. In this study, we report the functional significance of Sdc-1 in polarized macrophage populations and its role in adhesion and motility events relevant to resolution of the inflammatory program. APPROACH AND RESULTS: Macrophage Sdc-1 expression is associated with differentiated M2 macrophages with high intrinsic motility, and Sdc-1 deficiency is characterized by impaired migration and enhanced adhesion. Leukocyte infiltration and emigration were examined in a thioglycollate-induced model of peritonitis in Sdc-1(+/+) and Sdc-1(-/-) mice. Although the infiltration of inflammatory cells was similar in both cohorts, a significant delay in the lymphatic clearance of Sdc-1(-/-) macrophages was observed. Moreover, we observed enhanced inflammation and greater burden of atherosclerotic plaques in ApoE(-/-)Sdc-1(-/-) mice maintained on a Western diet. CONCLUSIONS: These results demonstrate that defective motility in Sdc-1(-/-) macrophages promotes a persistent inflammatory state with relevance to the pathogenesis of atherosclerosis.

Microablation of collagen-based substrates for soft tissue engineering.

Noting the abundance and importance of collagen as a biomaterial, we have developed a facile method for the production of a dense fibrillar extracellular matrix mimicking collagen-elastin hybrids with tunable mechanical properties. Through the use of excimer-laser technology, we have optimized conditions for the ablation of collagen lamellae without denaturation of protein, maintenance of fibrillar ultrastructure and preservation of native D-periodicity. Strengths of collagen-elastin hybrids ranged from 0.6 to 13 MPa, elongation at break from 9 to 70% and stiffness from 2.9 to 94 MPa, allowing for the design of a wide variety of tissue specific scaffolds. Further, large (centimeter scale) lamellae can be fabricated and embedded with recombinant elastin to generate collagen-elastin hybrids. Exposed collagen in hybrids act as cell adhesive sites for rat mesenchymal stem cells that conform to ablate waveforms. The ability to modulate these features allows for the generation of a class of biopolymers that can architecturally and physiologically replicate native tissue.

Immobilization of actively thromboresistant assemblies on sterile blood-contacting surfaces.

Rapid one-step modification of thrombomodulin with alkylamine derivatives such as azide, biotin, and PEG is achieved using an evolved sortase (eSrtA) mutant. The feasibility of a point-of-care scheme is demonstrated herein to site-specifically immobilize azido-thrombomodulin on sterilized commercial ePTFE vascular grafts, which exhibit superior thromboresistance compared with commercial heparin-coated grafts in a primate model of acute graft thrombosis.

Collagen-Based Substrates with Tunable Strength for Soft Tissue Engineering.

Through the use of mechanical reinforcement of collagen matrices, mechanically strong and compliant 3D tissue mimetic scaffolds can be generated that act as scaffolds for soft tissue engineering. Collagen has been widely used for the development of materials for repair, augmentation or replacement of damaged or diseased tissue. Herein we describe a facile method for the layer-by-layer fabrication of robust planar collagen fiber constructs. Collagen gels cast in a phosphate buffer were dried to form dense collagen mats. Subsequent gels were layered and dried atop mats to create multilayer constructs possessing a range of tunable strengths (0.5 - 11 MPa) and stiffness (1 - 115 MPa). Depending on processing conditions and crosslinking of constructs, strain to failure ranged between 9 to 48%. Collagen mats were constructed into hernia patches that prevented hernia recurrence in Wistar rats.