A platform to reproducibly evaluate human colon permeability and damage.

The intestinal epithelium comprises diverse cell types and executes many specialized functions as the primary interface between luminal contents and internal organs. A key function provided by the epithelium is maintenance of a barrier that protects the individual from pathogens, irritating luminal contents, and the microbiota. Disruption of this barrier can lead to inflammatory disease within the intestinal mucosa, and, in more severe cases, to sepsis. Animal models to study intestinal permeability are costly and not entirely predictive of human biology. Here we present a model of human colon barrier function that integrates primary human colon stem cells into Draper's PREDICT96 microfluidic organ-on-chip platform to yield a high-throughput system appropriate to predict damage and healing of the human colon epithelial barrier. We have demonstrated pharmacologically induced barrier damage measured by both a high throughput molecular permeability assay and transepithelial resistance. Using these assays, we developed an Inflammatory Bowel Disease-relevant model through cytokine induced damage that can support studies of disease mechanisms and putative therapeutics.

Mesenchymal stromal cell-derived extracellular vesicles reduce lung inflammation and damage in nonclinical acute lung injury: Implications for COVID-19.

Mesenchymal stem cell derived extracellular vesicles (MSC-EVs) are bioactive particles that evoke beneficial responses in recipient cells. We identified a role for MSC-EV in immune modulation and cellular salvage in a model of SARS-CoV-2 induced acute lung injury (ALI) using pulmonary epithelial cells and exposure to cytokines or the SARS-CoV-2 receptor binding domain (RBD). Whereas RBD or cytokine exposure caused a pro-inflammatory cellular environment and injurious signaling, impairing alveolar-capillary barrier function, and inducing cell death, MSC-EVs reduced inflammation and reestablished target cell health. Importantly, MSC-EV treatment increased active ACE2 surface protein compared to RBD injury, identifying a previously unknown role for MSC-EV treatment in COVID-19 signaling and pathogenesis. The beneficial effect of MSC-EV treatment was confirmed in an LPS-induced rat model of ALI wherein MSC-EVs reduced pro-inflammatory cytokine secretion and respiratory dysfunction associated with disease. MSC-EV administration was dose-responsive, demonstrating a large effective dose range for clinical translation. These data provide direct evidence of an MSC-EV-mediated improvement in ALI and contribute new insights into the therapeutic potential of MSC-EVs in COVID-19 or similar pathologies of respiratory distress.

Spontaneous Coronary Artery Dissection in a Postpartum Female: Case Report and Simplified Algorithm for its Diagnosis and Management.

Spontaneous coronary artery dissection is a rare, life-threatening cause of acute myocardial infarction that must always be considered in the differential diagnosis, particularly in young, healthy women with a paucity of typical risk factors for heart disease. We present a case of a 39-year-old White woman, three months postpartum, presenting with severe epigastric chest pain radiating to her neck. Subsequent workup using coronary angiography revealed spontaneous dissection of the distal left anterior descending artery. The patient was successfully managed by conservative treatment using low dose aspirin, metoprolol, and captopril, highlighting that hemodynamically stable patients with lesions in distal branches of coronary arteries and single-vessel disease can be successfully managed in a conservative fashion, without the need for surgical or percutaneous revascularization.

Macrophages modulate adult zebrafish tail fin regeneration.

Neutrophils and macrophages, as key mediators of inflammation, have defined functionally important roles in mammalian tissue repair. Although recent evidence suggests that similar cells exist in zebrafish and also migrate to sites of injury in larvae, whether these cells are functionally important for wound healing or regeneration in adult zebrafish is unknown. To begin to address these questions, we first tracked neutrophils (lyzC(+), mpo(+)) and macrophages (mpeg1(+)) in adult zebrafish following amputation of the tail fin, and detailed a migratory timecourse that revealed conserved elements of the inflammatory cell response with mammals. Next, we used transgenic zebrafish in which we could selectively ablate macrophages, which allowed us to investigate whether macrophages were required for tail fin regeneration. We identified stage-dependent functional roles of macrophages in mediating fin tissue outgrowth and bony ray patterning, in part through modulating levels of blastema proliferation. Moreover, we also sought to detail molecular regulators of inflammation in adult zebrafish and identified Wnt/ß-catenin as a signaling pathway that regulates the injury microenvironment, inflammatory cell migration and macrophage phenotype. These results provide a cellular and molecular link between components of the inflammation response and regeneration in adult zebrafish.

Botulinum toxin induces muscle paralysis and inhibits bone regeneration in zebrafish.

Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogenesis in diverse conditions of bone formation (eg, development, growth, and healing), yet the mechanisms of neuromuscular-bone crosstalk underlying these deficits have yet to be identified. Motivated by the emerging utility of zebrafish (Danio rerio) as a rapid, genetically tractable, and optically transparent model for human pathologies (as well as the potential to interrogate neuromuscular-mediated bone disorders in a simple model that bridges in vitro and more complex in vivo model systems), in this study, we developed a model of BTx-induced muscle paralysis in adult zebrafish, and we examined its effects on intramembranous ossification during tail fin regeneration. BTx administration induced rapid muscle paralysis in adult zebrafish in a manner that was dose-dependent, transient, and focal, mirroring the paralytic phenotype observed in animal and human studies. During fin regeneration, BTx impaired continued bone ray outgrowth, morphology, and patterning, indicating defects in early osteogenesis. Further, BTx significantly decreased mineralizing activity and crystalline mineral accumulation, suggesting delayed late-stage osteoblast differentiation and/or altered secondary bone apposition. Bone ray transection proximal to the amputation site focally inhibited bone outgrowth in the affected ray, implicating intra- and/or inter-ray nerves in this process. Taken together, these studies demonstrate the potential to interrogate pathological features of BTx-induced osteoanabolic dysfunction in the regenerating zebrafish fin, define the technological toolbox for detecting bone growth and mineralization deficits in this process, and suggest that pathways mediating neuromuscular regulation of osteogenesis may be conserved beyond established mammalian models of bone anabolic disorders.

Nanofiber-based in vitro system for high myogenic differentiation of human embryonic stem cells.

Myogenic progenitor cells derived from human embryonic stem cells (hESCs) can provide unlimited sources of cells in muscle regeneration but their clinical uses are largely hindered by the lack of efficient methods to induce differentiation of stem cells into myogenic cells. We present a novel approach to effectively enhance myogenic differentiation of human embryonic stem cells using aligned chitosan-polycaprolactone (C-PCL) nanofibers constructed to resemble the microenvironment of the native muscle extracellular matrix (ECM) in concert with Wnt3a protein. The myogenic differentiation was assessed by cell morphology, gene activities, and protein expression. hESCs grown on C-PCL uniaxially aligned nanofibers in media containing Wnt3a displayed an elongated morphology uniformly aligned in the direction of fiber orientation, with increased expressions of marker genes and proteins associated with myogenic differentiation as compared to control substrates. The combination of Wnt3a signaling and aligned C-PCL nanofibers resulted in high percentages of myogenic-protein expressing cells over total treated hESCs (83% My5, 91% Myf6, 83% myogenin, and 63% MHC) after 2 days of cell culture. Significantly, this unprecedented high-level and fast myogenic differentiation of hESC was demonstrated in a culture medium containing no feeder cells. This study suggests that chitosan-based aligned nanofibers combined with Wnt3a can potentially act as a model system for embryonic myogenesis and muscle regeneration.

Parkinson's disease-associated DJ-1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction.

Mitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson's disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H(2) O(2)) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.

Multivalent integrin-specific ligands enhance tissue healing and biomaterial integration.

Engineered biointerfaces covered with biomimetic motifs, including short bioadhesive ligands, are a promising material-based strategy for tissue repair in regenerative medicine. Potentially useful coating molecules are ligands for the integrins, major extracellular matrix receptors that require both ligand binding and nanoscale clustering for maximal signaling efficiency. We prepared coatings consisting of well-defined multimer constructs with a precise number of recombinant fragments of fibronectin (monomer, dimer, tetramer, and pentamer) to assess how nanoscale ligand clustering affects integrin binding, stem cell responses, tissue healing, and biomaterial integration. Clinical-grade titanium was grafted with polymer brushes that presented monomers, dimers, trimers, or pentamers of the alpha(5)beta(1) integrin-specific fibronectin III (7 to 10) domain (FNIII(7-10)). Coatings consisting of trimers and pentamers enhanced integrin-mediated adhesion in vitro, osteogenic signaling, and differentiation in human mesenchymal stem cells more than did surfaces presenting monomers and dimers. Furthermore, ligand clustering promoted bone formation and functional integration of the implant into bone in rat tibiae. This study establishes that a material-based strategy in which implants are coated with clustered bioadhesive ligands can promote robust implant-tissue integration.

Molecular assembly and biological activity of a recombinant fragment of fibronectin (FNIII(7-10)) on poly(ethyl acrylate).

Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some particular chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network on the material surface. We have investigated the organization of a recombinant fragment of fibronectin (FNIII(7-10)) upon adsorption on this particular chemistry, PEA. Atomic force microscopy (AFM) was used to identify individual molecules of the fragment after adsorption, as well as the evolution of the distribution of adsorbed molecules on the surface of the material as the concentration of the adsorbing solution increased. Globular molecules that turn into small aggregates were found as a function of solution concentration. Above a threshold concentration of the adsorbing solution (50 microg/mL) an interconnected network of the FNIII(7-10) fragment is obtained on the material surface. The bioavailability of specific cell adhesion domains, including RGD, within the molecules was higher on PEA than on the control glass. The biological activity of the fragment was further investigated by evaluating focal adhesion formation and actin cytoskeleton for MC3T3-E1 osteoblast-like cells. Well-developed focal adhesion complexes and insertions of actin stress fibers were found on PEA in a similar way as it happens in the control SAM-OH. Moreover, increasing the hydrophilicity of the surface by incorporating -OH groups led to globular molecules of the fragment homogeneously distributed throughout the surface; and the cell-material interaction is reduced as depicted by the lack of well-developed focal plaques and actin cytoskeleton.

Human mesenchymal stem cell differentiation on self-assembled monolayers presenting different surface chemistries.

Human mesenchymal stem cells (hMSCs) have tremendous potential as a cell source for regenerative medicine due to their capacity for differentiation into a wide range of connective tissue cell types. Although significant progress has been made in the identification of defined growth factor conditions to induce lineage commitment, the effect of underlying biomaterial properties on functional differentiation is far less understood. Here we conduct a systematic assessment of the role for surface chemistry on cell growth, morphology, gene expression and function during hMSC commitment along osteogenic, chondrogenic and adipogenic lineages. Using self-assembled monolayers of omega-functionalized alkanethiols on gold as model substrates, we demonstrate that biomaterial surface chemistry differentially modulates hMSC differentiation in a lineage-dependent manner. These results highlight the importance of initial biomaterial surface chemistry on long-term functional differentiation of adult stem cells, and suggest that surface properties are a critical parameter that must be considered in the design of biomaterials for stem cell-based regenerative medicine strategies.

Dendritic cell responses to self-assembled monolayers of defined chemistries.

Biomaterial contact triggers dendritic cell (DC) maturation, to an extent depending on the biomaterial, ultimately enhancing an immune response toward associated antigens, implying a role for biomaterials as adjuvants. Self-assembled monolayers (SAM) of alkanethiols on titanium/gold-coated surfaces presenting different chemistries were used to study effects of biomaterial surface chemistry on DC maturation. Although DCs treated with OH, COOH, or NH(2) SAMs showed modest maturation, those treated with CH(3) SAMs were least mature, all based on cytospins, allostimulatory capacity, or maturation marker expression. Surprisingly, DCs treated with CH(3) SAMs secreted highest levels of proinflammatory tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6) but were least mature. Secretion of anti-inflammatory mediators by DCs treated with CH(3) SAMs was not responsible for mitigating DC maturation under these conditions. Interestingly, elevated levels of apoptotic markers were measured associated with DCs and T cells upon CH(3) SAMs contact. Since phagocytosis of apoptotic DCs has strong immunosuppressive effects on DCs, more apoptotic DCs on CH(3) SAMs may account for lower DC maturation. Finally, higher expression of cytotoxic T lymphocyte associated antigen receptor-4 (CTLA-4) on T cells may imply a mechanism of T cell inhibition on CH(3) SAMs.

Saccharide polymer brushes to control protein and cell adhesion to titanium.

Attaining control over the surface chemistry of titanium is critical to its use in medical implants, especially to address complications such as infection and loosening of implants over time, which still present significant challenges. The surface-initiated atom transfer radical polymerization (SI-ATRP) of a saccharide-substituted methacrylate, 2-gluconamidoethyl methacrylate (GAMA), affords dense polymer brushes that resist protein adsorption and cell adhesion. We further tailored the nature of the surfaces by covalent attachment of an adhesion peptide to afford control over cell adhesion. Whereas unmodified poly(GAMA) brushes prevent cell adhesion, brushes with a tethered GFOGER-containing peptide sequence promote the deposition of confluent well-spread cells. The presentation of adhesion proteins on a robust bioresistive background in this fashion constitutes a versatile approach to the development of new biomaterials.

Simple application of fibronectin-mimetic coating enhances osseointegration of titanium implants.

Integrin-mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device integration and performance. Because of the central role of integrin-fibronectin (FN) interactions in osteoblastic function and bone formation, we evaluated the ability of FN-inspired biomolecular coatings to promote osteoblastic differentiation and implant osseointegration. Notably, these biomolecular coatings relied on physical adsorption of FN-based ligands onto biomedical-grade titanium as a simple, clinically translatable strategy to functionalize medical implants. Surfaces coated with a recombinant fragment of FN spanning the central cell binding domain enhanced osteoblastic differentiation and mineralization in bone marrow stromal cell cultures and increased implant osseointegration in a rat cortical bone model compared to passively adsorbed arginine-glycine-aspartic acid peptides, serum proteins and full-length FN. Differences in biological responses correlated with integrin binding specificity and signalling among surface coatings. This work validates a simple, clinically translatable, surface biofunctionalization strategy to enhance biomedical device integration.

Micropatterned surfaces with controlled ligand tethering.

Microcontact printing (micro-CP) is a facile, cost-effective, and versatile soft-lithography technique to create two-dimensional patterns of domains with distinct functionalities that provides a robust platform to generate micropatterned biotechnological arrays and cell culture substrates. Current micro-CP approaches rely on nonspecific immobilization of biological ligands, either by direct printing or adsorption from solution, onto micropatterned domains surrounded by a nonfouling background. This technique is limited by insufficient control over ligand density. We present a modified micro-CP protocol involving stamping mixed ratios of carboxyl- and tri(ethylene glycol)-terminated alkanethiols that provides for precise covalent tethering of single or multiple ligands to prescribed micropatterns via standard peptide chemistry. Processing parameters were optimized to identify conditions that control relevant endpoint pattern characteristics. This technique provides a facile method to generate micropatterned arrays with tailorable and controlled presentation of biological ligands for biotechnological applications and analyses of cell-material interactions.

Polymer brushes and self-assembled monolayers: Versatile platforms to control cell adhesion to biomaterials (Review).

This review focuses on the surface modification of substrates with self-assembled monolayers (SAMs) and polymer brushes to tailor interactions with biological systems and to thereby enhance their performance in bioapplications. Surface modification of biomedical implants promotes improved biocompatibility and enhanced implant integration with the host. While SAMs of alkanethiols on gold substrates successfully prevent nonspecific protein adsorption in vitro and can further be modified to tether ligands to control in vitro cell adhesion, extracellular matrix assembly, and cellular differentiation, this model system suffers from lack of stability in vivo. To overcome this limitation, highly tuned polymer brushes have been used as more robust coatings on a greater variety of biologically relevant substrates, including titanium, the current orthopedic clinical standard. In order to improve implant-bone integration, the authors modified titanium implants with a robust SAM on which surface-initiated atom transfer radical polymerization was performed, yielding oligo(ethylene glycol) methacrylate brushes. These brushes afforded the ability to tether bioactive ligands, which effectively promoted bone cell differentiation in vitro and supported significantly better in vivo functional implant integration.

Mixed extracellular matrix ligands synergistically modulate integrin adhesion and signaling.

Cell adhesion to extracellular matrix (ECM) components through cell-surface integrin receptors is essential to the formation, maintenance and repair of numerous tissues, and therefore represents a central theme in the design of bioactive materials that successfully interface with the body. While the adhesive responses associated with a single ligand have been extensively analyzed, the effects of multiple integrin subtypes binding to multivalent ECM signals remain poorly understood. In the present study, we generated a high throughput platform of non-adhesive surfaces presenting well-defined, independent densities of two integrin-specific engineered ligands for the type I collagen (COL-I) receptor alpha(2)beta(1) and the fibronectin (FN) receptor alpha(5)beta(1) to evaluate the effects of integrin cross-talk on adhesive responses. Engineered surfaces displayed ligand density-dependent adhesive effects, and mixed ligand surfaces significantly enhanced cell adhesion strength and focal adhesion assembly compared to single FN and COL-I ligand surfaces. Moreover, surfaces presenting mixed COL-I/FN ligands synergistically enhanced FAK activation compared to the single ligand substrates. The enhanced adhesive activities of the mixed ligand surfaces also promoted elevated proliferation rates. Our results demonstrate interplay between multivalent ECM ligands in adhesive responses and downstream cellular signaling.

Synthesis and modification of functional poly(lactide) copolymers: toward biofunctional materials.

A polylactide copolymer with pendant benzyloxy groups has been synthesized by the copolymerization of a benzyl-ether substituted monomer with lactide. Debenzylation of the polymer to provide pendant hydroxyl groups followed by modification with succinic anhydride affords the corresponding carboxylic acid functionalized copolymer that is amenable to standard carbodiimide coupling conditions to attach amine-containing biological molecules. An amino-substituted biotin derivative was coupled to the carboxyl functional groups of copolymer films as proof-of-concept. In a demonstration of the function of these new materials, an RGD-containing peptide sequence was tethered to copolymer films at various densities and was shown to enhance the adhesion of epithelial cells. This strategy provides the opportunity for the attachment of a variety of ligands, allowing for the fabrication of a versatile class of biodegradable, biocompatible materials.

The effect of integrin-specific bioactive coatings on tissue healing and implant osseointegration.

Implant osseointegration, defined as bone apposition and functional fixation, is a requisite for clinical success in orthopaedic and dental applications, many of which are restricted by implant loosening. Modification of implants to present bioactive motifs such as the RGD cell-adhesive sequence from fibronectin (FN) represents a promising approach in regenerative medicine. However, these biomimetic strategies have yielded only marginal enhancements in tissue healing in vivo. In this study, clinical-grade titanium implants were grafted with a non-fouling oligo(ethylene glycol)-substituted polymer coating functionalized with controlled densities of ligands of varying specificity for target integrin receptors. Biomaterials presenting the alpha5beta1-integrin-specific FN fragment FNIII 7-10 enhanced osteoblastic differentiation in bone marrow stromal cells compared to unmodified titanium and RGD-presenting surfaces. Importantly, FNIII 7-10-functionalized titanium significantly improved functional implant osseointegration compared to RGD-functionalized and unmodified titanium in vivo. This study demonstrates that bioactive coatings that promote integrin binding specificity regulate marrow-derived progenitor osteoblastic differentiation and enhance healing responses and functional integration of biomedical implants. This work identifies an innovative strategy for the rational design of biomaterials for regenerative medicine.

Biomolecular surface coating to enhance orthopaedic tissue healing and integration.

Implant osseointegration is a prerequisite for clinical success in orthopaedic and dental applications, many of which are restricted by loosening. Biomaterial surface modification approaches, including calcium-phosphate ceramic coatings and macro/microporosity, have had limited success in promoting integration. To improve osseointegration, titanium surfaces were coated with the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) collagen-mimetic peptide, selectively promoting alpha2beta1 integrin binding, a crucial event for osteoblastic differentiation. Titanium surfaces presenting GFOGER triggered osteoblastic differentiation and mineral deposition in bone marrow stromal cells, leading to enhanced osteoblastic function compared to unmodified titanium. Furthermore, this integrin-targeted coating significantly improved in vivo peri-implant bone regeneration and osseointegration, as characterized by bone-implant contact and mechanical fixation, compared to untreated titanium in a rat cortical bone-implant model. GFOGER-modified implants also significantly enhanced osseointegration compared to surfaces modified with full-length type I collagen, highlighting the importance of presenting specific biofunctional domains within the native ligand. In addition, this biomimetic implant coating is generated using a simple, single-step procedure that readily translates to a clinical environment with minimal processing and cytotoxicity concerns. Therefore, this study establishes a biologically active and clinically relevant implant-coating strategy that enhances bone repair and orthopaedic implant integration.

Integrin specificity and enhanced cellular activities associated with surfaces presenting a recombinant fibronectin fragment compared to RGD supports.

Biomimetic strategies focusing on presenting short bioadhesive oligopeptides, including the arginine-glycine-aspartic acid (RGD) motif present in numerous adhesive proteins, on a non-fouling support have emerged as promising approaches to improve cellular activities and healing responses. Nevertheless, these bio-inspired strategies are limited by low activity of the oligopeptides compared to the native ligand due to the absence of complementary or modulatory domains. In the present analysis, we generated well-defined biointerfaces presenting RGD-based ligands of increasing complexity to directly compare their biological activities in terms of cell adhesion strength, integrin binding and signaling. Mixed self-assembled monolayers of alkanethiols on gold were optimized to engineer robust supports that present anchoring groups for ligand tethering within a non-fouling, protein adsorption-resistant background. Controlled bioadhesive interfaces were generated by tethering adhesive ligands via standard peptide chemistry. On a molar basis, biointerfaces functionalized with the FNIII7-10 recombinant fragment presenting the RGD and PHSRN adhesive motifs in the correct structural context exhibited significantly higher adhesion strength, FAK activation, and cell proliferation rate than supports presenting RGD ligand or RGD-PHSRN, an oligopeptide presenting these two sites separated by a polyglycine linker. Moreover, FNIII7-10-functionalized surfaces displayed specificity for alpha5beta1 integrin, while cell adhesion to supports presenting RGD or RGD-PHSRN was primarily mediated by alphavbeta3 integrin. These results are significant to the rational engineering of bioactive materials that convey integrin binding specificity for directed cellular and tissue responses in biomedical and biotechnological applications.