Decreased tissue factor expression with increased CD11b upregulation on elastin-based biomaterial coatings.

Elastin-like polypeptide (ELP) coatings have been shown to have non-thrombogenic properties both in vitro and in vivo. In this work, we expand our understanding of this phenomenon by investigating the interaction of these coatings with leukocytes. Citrated whole blood was exposed to a shear rate of 300 s-1 for 2 hours at 37 °C on ELP1- and ELP4-coated polyethylene terephthalate (Mylar™) surfaces in a cone and plate device. Scanning electron microscopy and flow cytometry were used to measure leukocyte activation and platelet-leukocyte aggregation in response to the ELP1 and ELP4 coatings on the surface and in the bulk, respectively. Surface analysis showed little leukocyte activity on the surface of uncoated positive controls. Both the tissue factor (TF) expression (indicative of leukocyte activation) and CD61 expression (indicative of platelet-leukocyte aggregates), in the bulk were decreased by 40% and 20%, respectively, with the ELP coating of Mylar™, while a two- to three-fold increase in CD11b upregulation (indicative of leukocyte activation) for ELP1 and ELP4 was determined. Two of three bulk markers indicated that ELP-coated Mylar™ decreased the leukocyte response compared to the uncoated Mylar™, while the third, CD11b, indicated an increase in leukocyte response to the ELP coatings.

Platelet Adhesion and Fibrinogen Accretion on a Family of Elastin-Like Polypeptides.

Previous work in our laboratory showed the potential of using a human recombinant elastin-like polypeptide (ELP) as a thromboresistant coating. In this work we investigate the use of three particular ELPs (ELP1, ELP2 and ELP4), that differ by molecular weight and number of repeating hydrophobic and cross-linking domains, as coatings to improve blood-contacting properties. All three ELPs were passively adsorbed on Mylar surfaces. Differences in water contact angle and surface concentration were found among the three ELP coatings, with the shortest polypeptide, ELP1, being the most hydrophilic and abundant on the surface (55°, 0.76 µg/cm(2)), followed by ELP2 (55°, 0.35 µg/cm(2)) and ELP4, the longest of the three (66°, 0.25 µg/cm(2)), respectively. The blood interactions of the ELP coatings were investigated by measuring fibrinogen adsorption and platelet adhesion in whole blood under laminar flow in a cone and plate viscometer configuration. In general, platelet adhesion to the ELP-coated surfaces was found to correlate with fibrinogen adsorption. Decreases in fibrinogen accretion and platelet adhesion were observed for ELP-coated compared to uncoated surfaces. The magnitude of the decreases was found to depend on the ELP sequence length, with ELP4 exhibiting the lowest levels of fibrinogen adsorption and platelet adhesion at 43 ± 24 ng/cm(2) and 113 ± 77 platelets/mm(2), respectively.

Adipose tissue engineering in vivo with adipose-derived stem cells on naturally derived scaffolds.

Placental decellular matrix (PDM) and PDM combined with cross-linked hyaluronan (XLHA) scaffolds, seeded with primary human adipose-derived stem cells (ASC), were investigated in a subcutaneous athymic mouse model. The in vivo response at 3 and 8 weeks was characterized using histological and immunohistochemical staining. Fibrous capsule formation was assessed and the relative number of adipocytes in each scaffold was quantified. Undifferentiated ASC were localized using immunostaining for human vimentin. Unilocular and multilocular adipocytes were identified by intracellular lipid accumulation. Staining for murine CD31 assessed implant vascularization. Both scaffolds macroscopically maintained their three-dimensional volume and supported mature adipocyte populations in vivo. There was evidence of implant integration and a host contribution to the adipogenic response. The results suggested that incorporating the XLHA had a positive effect in terms of angiogenesis and adipogenesis. Overall, the PDM and PDM with XLHA scaffolds showed great promise for adipose tissue regeneration.

Polyurethane films seeded with embryonic stem cell-derived cardiomyocytes for use in cardiac tissue engineering applications.

Cardiomyocytes are terminally differentiated cells and therefore unable to regenerate heart tissue after infarction. The successful engraftment of various cell types resulting in improved cardiac function has been reported, however methods for improving the delivery of donor cells to the infarct site still need to be developed. The use of bioengineered cardiac grafts has been suggested to replace infarcted myocardium and enhance cardiac function. In this study, we cultured embryonic stem (ES) cell-derived cardiomyocytes on thin polyurethane (PU) films. The films were coated with gelatin, laminin or collagen IV in order to encourage cell adhesion. Constructs were examined for 30 days after seeding. Cells cultured on laminin and collagen IV, exhibited preferential attachment, as assessed by cellular counts, and viability assays. These surfaces also resulted in a greater number of contracting films compared to controls. A degradable elastomer seeded with embryonic stem cell-derived cardiomyocytes may hold potential for the repair of damaged heart tissue.

Elastomeric biodegradable polyurethane blends for soft tissue applications.

Four biodegradable polyurethane blends were made from segmented polyurethanes that contain amino acid-based chain extender and diisocyanate groups. The soft segments of these parent polyurethanes were either polyethylene oxide (PEO) or polycaprolactone (PCL) diols. The blends were developed to investigate the effect of varying soft segment compositions on the overall morphological, mechanical, and degradative properties of the materials, with a view to producing a family of materials with a wide range of properties. The highly hydrophilic PEO material was incorporated to increase the blend's susceptibility to degradation, while the PCL polyurethane was selected to provide higher moduli and percent elongations (strains) than the PEO parent materials can achieve. All four blends were determined to be semi-crystalline, elastomeric materials that possess similarly shaped stress-strain curves to that of the PCL-based parent polyurethane. As the percent composition of PEO polyurethane within the blend increased, the material became weaker and less extensible. The blends demonstrated rapid initial degradation in buffer followed by significantly slower, prolonged degradation, likely corresponding to an initial loss of primarily PEO-containing polymer, followed by the slower degradation of the PCL polyurethane. All four blends were successfully formed into three-dimensional porous scaffolds utilizing solvent casting/particulate leaching methods. Since these new blends possess a range of mechanical and degradation properties and can be shaped into three-dimensional objects, these materials may hold potential for use in soft tissue engineering scaffold applications.

22 week assessment of bladder acellular matrix as a bladder augmentation material in a porcine model.

Previous studies on the reconstruction of porcine bladder using bladder acellular matrix allograft (BAMA) have indicated positive preliminary results with respect to graft shrinkage and cellular repopulation. The current study was conducted to investigate the feasibility of using BAMA in a similar model of bladder reconstruction out to longer time frames (22 weeks). At predetermined time points, the macroscopic, histological and mechanical properties of explanted native and BAMA tissues were evaluated and compared. Macroscopically, contracture of the BAMA was observed. The peripheral regions of the grafts experienced extensive cellular repopulation. Towards the centre however, all grafts were consistently devoid of organized smooth muscle bundles and a well-developed urothelium. An alteration in both the amount and organization of collagen was also observed within this region. Significant differences (p < 0.05) in the rupture strain and the elastic modulus of the BAMA compared to native bladder tissue appear to correlate with macroscopic graft contracture as well as the fibroproliferative tissue response of the matrix.

Self-aggregation characteristics of recombinantly expressed human elastin polypeptides.

Elastin is an extracellular matrix protein found in tissues requiring extensibility and elastic recoil. Monomeric elastin has the ability to aggregate into fibrillar structures in vitro, and has been suggested to participate in the organization of its own assembly into a polymeric matrix in vivo. Although hydrophobic sequences in elastin have been suggested to be involved in this process of self-organization, the contributions of specific hydrophobic and crosslinking domains to the propensity of elastin to self-assemble have received less attention. We have used a series of defined, recombinant human elastin polypeptides to investigate the factors contributing to elastin self-assembly. In general, coacervation temperature of these polypeptides, used as a measure of their propensity to self-assemble, was influenced both by salt concentration and polypeptide concentration. In addition, hydrophobic domains appeared to be essential for the ability of these polypeptides to self-assemble. However, neither overall molecular mass, number of hydrophobic domains nor general hydropathy of the polypeptides provided a complete explanation for differences in coacervation temperature, suggesting that the specific nature of the sequences of these hydrophobic domains are an important determinant of the ability of elastin polypeptides to self-assemble.

In vitro degradation and erosion of degradable, segmented polyurethanes containing an amino acid-based chain extender.

In vitro degradation and erosion of novel, degradable segmented polyurethanes containing a phenylalanine diester chain extender were investigated by exposing the polymers to buffer. chymotrypsin, and trypsin solutions for up to 28 days. Polyurethane degradation and erosion were monitored by gravimetry, scanning electron microscopy (SEM), and gel permeation chromatography (GPC) and compared to a control polyurethane. Polyurethanes were synthesized using two different soft segments (polycaprolactone diol and polyethylene oxide) of variable molecular weight. Inclusion of the phenylalanine-based chain extender resulted in an increased susceptibility to enzyme-mediated, but not buffer-mediated, erosion in comparison to the control polyurethane. SEM analysis indicated that enzyme-mediated erosion proceeded via a surface-limited mechanism resulting in a progressive removal of material from the surface inwards with time. The magnitude of degradation and erosion was highly variable and was dependent on soft segment type and molecular weight. The range of degradation rates, as well as physicochemical properties, makes these polyurethanes potentially useful for a wide range of biomedical applications.

Plasmin degradation of fibrin coatings on synthetic polymer substrates.

The goal of this research was to evaluate the in vitro stability of fibrin coatings on polymeric materials in the presence of plasmin. Factor XIIIa-crosslinked and noncrosslinked fibrin layers were coated on three different polyurethane substrates: Corethane, Tegaderm, and a biodegradable polyurethane, PCL/HDI/Phe. Degradation assays indicated that crosslinking the fibrin coatings enhanced the stability of the coatings on both Tegaderm and PCL/HDI/Phe; however, the persistence of the coating on the woven Corethane was not influenced by crosslinking. Degradation assay results also showed that the fibrin coating on the Corethane was significantly less stable than the fibrin coatings on the Tegaderm and PCL/HDI/Phe films. The chromogenic substrate assay data showed crosslinking did not affect the specific plasmin activity on the coatings; therefore, the increased stability resulting from crosslinking was not achieved through a reduction of fibrinolysis. The plasmin activity on the coated Corethane samples was much greater than that on either of the coated flat wound dressing materials. The large surface area of Corethane, a porous woven vascular graft material, may have had a direct influence on the fibrinolysis of its coatings by providing a large number of tissue-type plasminogen activator (tPA) binding sites. A thin, crosslinked, fibrin-coated polyurethane provides a theoretically attractive biomaterial for use in a wound dressing application and should be subject to ongoing research.

Tumor necrosis factor (TNFalpha) production by rat peritoneal macrophages is not polyacrylate surface-chemistry dependent.

Polyacrylate films in the absence of added endotoxin caused rat peritoneal macrophages to secrete a small amount of TNFalpha. There was little difference, if any, among the materials, which included various co- or ter-polymers of hydroxyethyl methacrylate, dimethylaminoethyl methacrylate, methacrylic acid, methyl methacrylate, and butyl methacrylate. The materials were surface characterized and endotoxin cleaned prior to testing. Equivalent endotoxin levels associated with the material were <0.03 EU/mL for all materials but two; for polyHEMA, the most contaminated material, it was 0.23 EU/mL. Films of the materials were incubated with freshly isolated rat peritoneal macrophages for 6 to 24 h before the TNFalpha levels in the supernatant were analyzed for biological activity, using L929 cells as a target. When endotoxin was added, far greater quantities of TNFalpha were generated at 24 h compared to 6 h, but still there was little effect with regard to material chemistry. Such an in vitro assay proved not to be useful for the screening of potential microencapsulation materials for peritoneal biocompatibility.

Synthesis and characterization of degradable polyurethane elastomers containing and amino acid-based chain extender.

Degradable polyurethane elastomers were synthesized using a diester chain extender. The chain extender was synthesized by a diesterification reaction between L-phenylalanine and 1,4-cyclohexane dimethanol to yield a diester, diamine. Soft segment chemistry (polycaprolactone diol, PCL and polyethylene oxide, PEO) and molecular weight were varied and the impact on polyurethane physicochemical and degradation characteristics was evaluated. It was found that the PEO containing polyurethanes absorbed large amounts of water while the PCL containing ones did not, indicating a large difference in bulk hydrophilicity. The rate of water vapor permeance (WVP) through the polyurethane films generally followed the water absorption trends. However, soft segment crystallinity, noted by DSC, for the PCL containing polyurethanes served to reduce WVP values with increasing PCL molecular weight. Polyurethane surface characterization was carried out by water contact angles and XPS. The PEO containing polyurethanes exhibited low contact angles in comparison with the PCL ones. In addition, angle-resolved XPS demonstrated soft segment surface enrichment in all cases typical for phase segregated materials. Significant variation in the physicochemical properties of the experimental polyurethanes was observed indicating potential use in a variety of biomaterials applications. An in vitro degradation study was carried out by incubating the polymers in 0.1 M TBS at 37 degrees C, pH 8.0 for up to 56 days. Degradation was followed by measuring mass loss, change in molecular weight by GPC and surface alteration by scanning electron microscopy. The polyurethane containing PEO was found to exhibit substantial mass and molecular weight loss over 56 days resulting in a porous material of little strength. In contrast, the PCL containing polyurethane displayed modest mass and molecular weight loss after 56 days. This polyurethane retained its strength and displayed little surface alteration after 56 days in buffer. It was hypothesized that differences in polyurethane hydrophilicity as well as initial molecular weight may have been responsible for the dramatic difference in degradation rate observed here.

Protein and platelet interactions with thermally denatured fibrinogen and cross-linked fibrin coated surfaces.

In this work the hypothesis that a mature, cross-linked fibrin clot, pre-formed on a biomaterial, may be relatively nonthrombogenic was investigated. A cross-linked fibrin layer was formed on polyethylene which had been precoated with thermally denatured fibrinogen. Plasma protein adsorption and platelet interactions with the cross-linked fibrin and denatured fibrinogen surfaces were investigated. The adsorption of albumin, fibrinogen, and fibronectin from plasma was measured. For all three proteins, the cross-linked fibrin surface exhibited much higher levels of adsorption than either the thermally denatured fibrinogen or the polyethylene surface. Vroman peaks were observed for fibrinogen and fibronectin on polyethylene but not on the cross-linked fibrin and thermally denatured fibrinogen materials. In dilute plasma the thermally denatured fibrinogen surface showed considerable resistance to protein adsorption. However, at plasma concentrations greater than about 5% normal, this protein resistance was apparently lost. Platelet interactions (adhesion and release of granule constituents from adherent platelets) using suspensions of washed platelets in the presence of red cells were investigated at shear rates of 50, 300, and 525 s(-1) using a cone and plate apparatus. The levels of platelet adhesion on the different surfaces were in the order: adsorbed fibrinogen > cross-linked fibrin > thermally denatured fibrinogen = polyethylene. Platelets on the cross-linked fibrin surface also showed high levels of release indicating significant platelet activation. Scanning electron microscopic observations were in agreement with the platelet adhesion and release data, showing only a few (but well-spread) adherent platelets on the cross-linked fibrin surface.

Tissue engineering.

Wound care has become one of the first fields to see the benefit of a new technology: tissue engineering. Tissue engineering involves the development of new materials or devices capable of specific interactions with biological tissues. In wound care, these materials may be based entirely on naturally occurring tissues and cells, or may be materials that combine synthetics, usually polymers, with biological layers. Both wound dressings and skin substitutes are available. The complexity of the materials depends on the end uses. Generally, synthetics made from polymeric materials such as Tegaderm and Opsite are used as wound dressings over relatively simple and shallow wounds or as coverings over more complex dressings. Their function is one of protection from water loss, drying, and mechanical injury. More complex dressings vary from dermal replacements made of reconstituted collagen and chondroitan sulfate backed by a polymer layer such as Integra(R) to the complex Apligraftrade mark that contains collagen and seeded cells. This last is designed as a complete skin replacement or skin substitute. Ultimately, engineered skin will contain all of the components necessary to modulate healing and provide the desired response: a wound closed with limited scar tissue that retains all of the characteristics of natural skin.

Lysis of surface-localized fibrin clots by adsorbed plasminogen in the presence of tissue plasminogen activator.

The ability of plasminogen adsorbed from buffer onto sulphonated silica glass or lysine-derivatized silica glass to lyse fibrin I clots has been investigated. Clots were formed around the test surface by adding reptilase to fibrinogen solutions in which the surfaces were immersed. Tissue plasminogen activator (t-PA) was then added and the extent of clot lysis was determined by measuring the levels of the specific plasmin cleavage product of fibrinogen, B beta 1-42 peptide. The data indicate that in the presence of t-PA, B beta 1-42 generation per mole of bound plasminogen on the lysinized material is approximately two-fold higher than on the sulphonated material. It is concluded that a preformed clot may be lysed by adsorbed plasminogen in the presence of t-PA, and that clot lysis is significantly enhanced when the plasminogen is adsorbed via its lysine binding sites.

Interactions of plasminogen and fibrinogen with model silica glass surfaces: adsorption from plasma and enzymatic activity studies.

The adsorption of fibrinogen and plasminogen from plasma to silica glass, sulfonated silica glass, and lysine-derivatized silica glass has been investigated. The data indicate that the sulfonated material has a high affinity for both fibrinogen and plasminogen, but that the ratio of plasminogen to fibrinogen is greater on the lysine-derivatized surface. The adsorption data also suggest plasminogen as a possible contributor to the fibrinogen Vroman effect, whereby initially absorbed fibrinogen is displaced from the surface. The plasmin activity of plasminogen adsorbed to the lysine-derivatized silica glass and its sulfonated precursor was assessed by both a chromogenic substrate assay and a radioimmunoassay for the plasmin cleavage product of fibrinogen, the B beta 1-42 peptide. The data indicate that 1) the adsorbed plasminogen is not inherently plasmin-like; 2) the enzymatic activity associated with the bound plasminogen is significantly enhanced on both surfaces in the presence of activator; and 3) in the presence of activator, the plasmin activity per mole of bound plasminogen on the lysinized material is approximately a factor of two greater than on the sulfonated material based on the chromogenic substrate assay, and a factor of four greater based on the B beta 1-42 radioimmunoassay. The lysinized material thus exhibits several properties that are different from its sulfonated precursor. It adsorbs more plasminogen relative to fibrinogen after the Vroman peak, and this adsorbed plasminogen appears to be in a conformation that is more readily activated to plasmin. Once activated, the surface bound plasmin shows enhanced ability to cleave either a low molecular weight chromogenic substrate or a macromolecular substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption of plasminogen from plasma to lysine-derivatized polyurethane surfaces.

The adsorption of plasminogen, the principal protein of the fibrinolytic pathway in blood, to a number of solid surfaces from plasma was investigated. This study forms part of a larger project to develop a fibrinolytic surface for blood-contacting applications. Polyurethanes incorporating lysine residues were developed in an attempt to promote selective adsorption of plasminogen from plasma through lysine-binding sites in the plasminogen molecule. The adsorption of plasminogen to these surfaces as well as to glass, 'conventional' polyurethanes and precursor sulphonated polyurethanes was investigated. Adsorption from citrated human plasma diluted with isotonic Tris buffer (pH 7.4) was measured under static conditions at room temperature using radioiodinated plasminogen. The following trends were observed. (1) Adsorption increases monotonically with increasing plasma concentration and there is no suggestion of transient adsorption (Vroman effect) on any of the surfaces studied. (2) Sulphonate groups appear to have a strong effect on plasminogen adsorption as was found previously for adsorption from buffer. (3) The lysine-derivatized material having the highest lysine content may show a slight increase in plasminogen binding affinity compared to its sulphonated precursor.