Riboflavin-UVA crosslinking of amniotic membranes and its influence on the culture of adipose-derived stem cells.

The human amniotic membrane (hAM) is a collagen-based extracellular matrix whose applications are restricted by its moderate mechanical properties and rapid biodegradation. In this work, we investigate the use of riboflavin, a water-soluble vitamin, to crosslink and strengthen the human amniotic membrane under UVA light. The effect of riboflavin-UVA crosslinking on hAM properties were determined via infrared spectroscopy, uniaxial tensile testing, proteolytic degradation, permeability testing, SEM, and quantification of free (un-crosslinked) amine groups. Samples crosslinked with glutaraldehyde, a common and effective yet cytotoxic crosslinking agent, were used as controls. Improved hAM mechanical properties must not come at the expense of reduced cellular proliferation and induction capabilities. In this study, we assessed the viability, proliferation, immunophenotype, and multilineage differentiation ability of human adipose-derived stem cells seeded on riboflavin-UVA crosslinked membranes. Overall, hAM crosslinked with riboflavin-UVA benefited from a stable three-fold increase in mechanical properties (comparable to the increase seen with glutaraldehyde crosslinked membranes) and improved biodegradation, all while retaining their biocompatibility and abilities to support the cultivation and differentiation of adipose-derived stem cells. Together, these results suggest that riboflavin-UVA crosslinking is an effective strategy to enhance the hAM for tissue engineering and regenerative medicine applications establishing it as an attractive and tuneable biomaterial.

Oscillatory shear potentiates latent TGF-ß1 activation more than steady shear as demonstrated by a novel force generator.

Cardiovascular mechanical stresses trigger physiological and pathological cellular reactions including secretion of Transforming Growth Factor ß1 ubiquitously in a latent form (LTGF-ß1). While complex shear stresses can activate LTGF-ß1, the mechanisms underlying LTGF-ß1 activation remain unclear. We hypothesized that different types of shear stress differentially activate LTGF-ß1. We designed a custom-built cone-and-plate device to generate steady shear (SS) forces, which are physiologic, or oscillatory shear (OSS) forces characteristic of pathologic states, by abruptly changing rotation directions. We then measured LTGF-ß1 activation in platelet releasates. We modeled and measured flow profile changes between SS and OSS by computational fluid dynamics (CFD) simulations. We found a spike in shear rate during abrupt changes in rotation direction. OSS activated TGF-ß1 levels significantly more than SS at all shear rates. OSS altered oxidation of free thiols to form more high molecular weight protein complex(es) than SS, a potential mechanism of shear-dependent LTGF-ß1 activation. Increasing viscosity in platelet releasates produced higher shear stress and higher LTGF-ß1 activation. OSS-generated active TGF-ß1 stimulated higher pSmad2 signaling and endothelial to mesenchymal transition (EndoMT)-related genes PAI-1, collagen, and periostin expression in endothelial cells. Overall, our data suggest variable TGF-ß1 activation and signaling occurs with competing blood flow patterns in the vasculature to generate complex shear stress, which activates higher levels of TGF-ß1 to drive vascular remodeling.

Human Amniotic Membrane: A Versatile Scaffold for Tissue Engineering.

The human amniotic membrane (hAM) is a collagen-based extracellular matrix derived from the human placenta. It is a readily available, inexpensive, and naturally biocompatible material. Over the past decade, the development of tissue engineering and regenerative medicine, along with new decellularization protocols, has recast this simple biomaterial as a tunable matrix for cellularized tissue engineered constructs. Thanks to its biocompatibility, decellularized hAM is now commonly used in a broad range of medical fields. New preparation techniques and composite scaffold strategies have also emerged as ways to tune the properties of this scaffold. The current state of understanding about the hAM as a biomaterial is summarized in this review. We examine the processing techniques available for the hAM, addressing their effect on the mechanical properties, biodegradation, and cellular response of processed scaffolds. The latest in vitro applications, in vivo studies, clinical trials, and commercially available products based on the hAM are reported, organized by medical field. We also look at the possible alterations to the hAM to tune its properties, either through composite materials incorporating decellularized hAM, chemical cross-linking, or innovative layering and tissue preparation strategies. Overall, this review compiles the current literature about the myriad capabilities of the human amniotic membrane, providing a much-needed update on this biomaterial.

Properties of porcine adipose-derived stem cells and their applications in preclinical models.

Adipose-derived stem cells represent a reliable adult stem cell source thanks to their abundance, straightforward isolation, and broad differentiation abilities. Consequently, human adipose-derived stem cells (hASCs) have been used in vitro for several innovative cellular therapy and regenerative medicine applications. However, the translation of a novel technology from the laboratory to the clinic requires first to evaluate its safety, feasibility, and potential efficacy through preclinical studies in animals. The anatomy and physiology of pigs and humans are very similar, establishing pigs as an attractive and popular large animal model for preclinical studies. Knowledge of the properties of porcine adipose-derived stem cells (pASCs) used in preclinical studies is critical for their success. While hASCs have been extensively studied this past decade, only a handful of reports relate to pASCs. The aim of this concise review is to summarize the current findings about the isolation of pASCs, their culture, proliferation, and immunophenotype. The differentiation abilities of pASCs and their applications in porcine preclinical models will also be reported.

Designing and evaluating a STEM teacher learning opportunity in the research university.

This study examines the design and evaluation strategies for a year-long teacher learning and development experience, including their effectiveness, efficiency and recommendations for strategic redesign. Design characteristics include programmatic features and outcomes: cognitive, affective and motivational processes; interpersonal and social development; and performance activities. Program participants were secondary math and science teachers, partnered with engineering faculty mentors, in a research university-based education and support program. Data from multiple sources demonstrated strengths and weaknesses in design of the program's learning environment, including: face-to-face and via digital tools; on-site and distance community interactions; and strategic evaluation tools and systems. Implications are considered for the strategic design and evaluation of similar grant-funded research experiences intended to support teacher learning, development and transfer.

Single-walled carbon nanotubes do not pierce aqueous phospholipid bilayers at low salt concentration.

Because of their unique physical, chemical, and electrical properties, carbon nanotubes are an attractive material for many potential applications. Their interactions with biological entities are, however, not yet completely understood. To fill this knowledge gap, we present experimental results for aqueous systems containing single-walled carbon nanotubes and phospholipid membranes, prepared in the form of liposomes. Our results suggest that dispersed single-walled carbon nanotubes, instead of piercing the liposome membranes, adsorb on them at low ionic strength. Transmission electron microscopy and dye-leakage experiments show that the liposomes remain for the most part intact in the presence of the nanotubes. Further, the liposomes are found to stabilize carbon nanotube dispersions when the surfactant sodium dodecylbenezenesulfonate is present at low concentrations. Quantifying the interactions between carbon nanotubes and phospholipid membranes could not only shed light on potential nanotubes cytotoxicity but also open up new research venues for their use in controlled drug delivery and/or gene and cancer therapy.

Design considerations for a microfluidic device to quantify the platelet adhesion to collagen at physiological shear rates.

Accurate assessment of blood platelet function is essential in understanding thrombus formation which plays a central role in cardiovascular disease. Parallel plate flow chambers have been widely used as they allow for platelet adhesion on a collagen surface at physiologically relevant fluid mechanical forces. Standard parallel plate flow chambers typically need several milliliters of blood, which is substantially more than can be obtained from small animals. We designed, fabricated, and assessed the functionality of a microfluidic channel with a width of 500 microm and a height of 50 microm in which a wall shear rate of 1000 s(-1) can be achieved with a flow rate of 15 microL/min. The velocity distribution in the microchannel predicted from the equations of motion was compared to experimentally measured velocities of fluorescent beads. This analysis showed that the motion of beads was quite similar to the predicted motion. Adhesion of platelets from whole blood at a shear rate of 1000 s(-1) onto a collagen surface using the microfluidic flow channel was qualitatively similar to platelet adhesion observed with a standard sized parallel plate flow chamber. After 5 min flow the surface coverage of platelets in the microfluidic device was about 55% while in a traditional size flow chamber the surface coverage was about 75%. This suggests that the microfluidic flow chamber can be used to quantify platelet adhesion for system where only very small amounts of blood are available.

Modification of single walled carbon nanotube surface chemistry to improve aqueous solubility and enhance cellular interactions.

Single walled carbon nanotubes (SWNTs) continue to demonstrate the potential of nanoscaled materials in a wide range of applications. The ability to modulate the mechanical or electrical properties of a material by varying the SWNT component may result in diverse "application tunable" materials. Similarly, biomaterials used in tissue engineering applications may benefit from these characteristics by varying electrical and mechanical properties to enhance or direct tissue specific regeneration. The interactions between SWNTs and cellular systems need to be optimized to integrate these highly hydrophobic nanoparticles within an aqueous environment while maintaining their unique properties. We assessed solubility, conductance, and cellular interactions between four different SWNT preparations (unrefined, refined, and SWNT with either albumin or human plasma adsorbed). Initial interactions between cells and SWNTs were assessed within a 3D environment using a red blood cell lysis model, with longer-term interactions assessing the effects on PC12 and 3T3 fibroblast function when cultured on SWNT-collagen composite hydrogels. After SWNT purification, the lytic effect on red blood cells (RBCs) is significantly reduced from 11% to 0.7%, indicating manufacturing contaminants play a significant role in undesirable cell interactions. Nanotubes with either human plasma or albumin physisorbed onto the nanotube surface were significantly more hydrophilic than either unrefined or refined preparations and displayed improved RBC interactions. Despite improved dispersion, purification, and adsorption of either plasma or albumin, SWNTs caused a significant reduction in conductance. Although the molecular interactions occurring at the cell membrane remain unclear, these investigations have identified two main factors contributing to membrane failure: manufacturing impurities and to a lesser extend the material's innate hydrophobicity. Although purification is a critical step to remove toxic manufacturing contaminants, care must be taken to ensure improved aqueous dispersion does not compromise desirable mechanical and electrical attributes.

The synergy site of fibronectin is required for strong interaction with the platelet integrin alphaIIbbeta3.

Integrins are a class of cell adhesion molecules that bind to ligands containing the RGD peptide sequence. There is increasing evidence that peptide sites other than the RGD site are required for optimal binding of integrins with their ligands. We have examined the sites on the protein fibronectin that are needed for optimal binding to the platelet integrin alphaIIbbeta3 using a strategy of site directed mutagenesis. Single amino acids near the RGD site or near the synergy site of fibronectin were mutated and the resultant proteins were expressed in a bacterial expression system. The purified protein was coated onto glass cover slips. Platelets, expressing alphaIIbbeta3 were perfused over the surface at physiologically relevant shear rates and the extent of adhesion was quantified. We found that the single amino acid substitution of the aspartic acid in the RGD sequence, D1495A, completely abolished adhesion. Surprisingly, the mutants R1445A and R1448Q that are near the RGD site also abolished adhesion of platelets under flow. Additionally, the synergy site mutants R1371A, R1374Q, or R1379A displayed only minimal adhesion of platelets. These results show that the binding site for alphaIIbbeta3 on fibronectin extends over a considerable distance from the RGD site and that these distant sites are required for optimal attachment of cells in the presence of physiologically relevant shear stress.

Chemical modification of SWNT alters in vitro cell-SWNT interactions.

Single-walled carbon nanotubes (SWNT) have been the focus of considerable attention as a material with extraordinary mechanical and electrical properties. SWNT have been proposed in a number of biomedical applications, including neural, bone, and dental tissue engineering. In these applications, it is clear that surrounding tissues will come into surface contact with SWNT composites, and compatibility between SWNT and host cells must be addressed. This investigation describes the gross physical and chemical effects of different SWNT preparations on in vitro cell viability and metabolic activity. Three different SWNT preparations were analyzed: as purchased (AP-NT), purified (PUR-NT), and functionalized with glucosamine (GA-NT), over concentrations of 0.001-1.0% (wt/vol). With the exception of the lowest SWNT concentrations, increasing concentrations of SWNT resulted in a decrease of cell viability, which was dependent on SWNT preparation. The metabolic activity of 3T3 cells was also dependent on SWNT preparation and concentration. These investigations have shown that these SWNT preparations have significant effects on in vitro cellular function that cannot be attributed to one factor alone, but are more likely the result of several unfavorable interactions. Effects, such as destabilizing the cell membrane, soluble toxic contaminants, and limitations in mass transfer as the SWNT coalesce into sheets, may all play a role in these interactions. Using comprehensive purification processes and modifying the NT-surface chemistry to introduce functional groups or reduce hydrophobicity or both, these interactions can be significantly improved.

Platelet-derived NO slows thrombus growth on a collagen type III surface.

Nitric oxide (NO) is a free radical that plays an important role in modulating platelet adhesion and aggregation. Platelets are a source of vascular NO, but since erythrocytes avidly scavenge NO, the functional significance of platelet-derived NO is not clear. Our purpose was to determine if NO from platelets affects platelet thrombus formation in the presence of anticoagulated whole blood in an in vitro parallel plate flow system. We studied platelet adhesion and aggregation on a collagen type III surface in the presence of physiologically relevant fluid mechanical shear stress. We found that certain receptor mediated agonists (insulin and isoproterenol) caused a concentration dependent reduction in thrombus formation at a shear rate of 1000 s-1. This effect was mediated by NO since it was abolished in the presence of the NO inhibitor L-nitro-arginine-methyl-ester (L-NAME). As expected, at venous levels of shear rate (100 s-1) neither of the agonists had any effect on thrombus formation since platelet adhesion does not depend on activation at these low levels of shear. Interestingly, at a shear rate of 2000 s-1 the addition of L-NAME caused an increase in platelet coverage suggesting that shear, by itself, induces NO production by platelets. This is the first demonstration of shear stress causing platelets to produce an inhibitor of platelet activation. These results demonstrate that the development of a platelet thrombus is regulated in a complex way and that platelets produce functionally significant amounts of NO even in the presence of whole blood.

Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow.

Leukocytes roll on selectins at nearly constant velocities over a wide range of wall shear stresses. Ligand-coupled microspheres roll faster on selectins and detach quickly as wall shear stress is increased. To examine whether the superior performance of leukocytes reflects molecular features of native ligands or cellular properties that favor selectin-mediated rolling, we coupled structurally defined selectin ligands to microspheres or K562 cells and compared their rolling on P-selectin. Microspheres bearing soluble P-selectin glycoprotein ligand (sPSGL)-1 or 2-glycosulfopeptide (GSP)-6, a GSP modeled after the NH2-terminal P-selectin-binding region of PSGL-1, rolled equivalently but unstably on P-selectin. K562 cells displaying randomly coupled 2-GSP-6 also rolled unstably. In contrast, K562 cells bearing randomly coupled sPSGL-1 or 2-GSP-6 targeted to a membrane-distal region of the presumed glycocalyx rolled more like leukocytes: rolling steps were more uniform and shear resistant, and rolling velocities tended to plateau as wall shear stress was increased. K562 cells treated with paraformaldehyde or methyl-beta-cyclodextrin before ligand coupling were less deformable and rolled unstably like microspheres. Cells treated with cytochalasin D were more deformable, further resisted detachment, and rolled slowly despite increases in wall shear stress. Thus, stable, shear-resistant rolling requires cellular properties that optimize selectin-ligand interactions.