Perspective on hemocompatibility testing.
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Biocompatible Materials↗
Biomedical subjects
M V Sefton
Publications and source records attributed to M V Sefton.
Does surface chemistry affect thrombogenicity of surface modified polymers?
With some exceptions, surface chemistry had little effect on platelet and leukocyte activation, and cell deposition, by scanning electron microscopy after blood exposure and clotting times among a group of 12 unmodified and plasma modified tubings. All materials activated platelets and leukocytes to detectable levels, although some materials increased the value of one activation parameter but not another. Unmodified materials [polyethylene (PE), Pellethane (PEU), latex, nylon, and Silastic] and modified materials (H(2)O plasma treated PE and PEU, CF(4) plasma treated PE, fluorinated PEU, NH(4) plasma treated PEU, polyethylene imine treated PEU, and heparin treated PEU) were characterised by XPS and contact angle. The objective of this project was to define a series of assays for the evaluation of hemocompatibility of cardiovascular devices with a view to clarify the specific requirements of ISO-10993-4, and to define an appropriate screening program for new blood contacting biomaterials. PE, PE--CF(4), PE--H(2)0, PEU--F, latex, and PEU-heparin were the exceptions to the general observations, although each behaved differently. PE proved to be least reactive, whereas PE-CF(4) was most reactive by several assays. Platelet microparticle formation (determined by flow cytometry), PTT, postblood exposure SEM, total SC5b-9, C3a, and platelet and leukocyte loss (cell counts) were able to distinguish differences among these materials, and often, but not always, showed expected correlations.
Biocompatible Materials↗
Leukocyte activation and leukocyte procoagulant activities after blood contact with polystyrene and polyethylene glycol-immobilized polystyrene beads.
Beads (45 microm) of polystyrene (PS) and polyethylene glycol modified PS (TentaGel) with an amino or hydroxyl terminal group were incubated with blood to assess the effect of surface area and material chemistry on leukocyte activation. After a 2-hour incubation, blood contact with beads activated leukocytes in the bulk (tissue factor expression, CD11b up-regulation, and association with platelets) independently of material surface chemistry. On the other hand, activation of adherent leukocytes was material dependent. After blood contact with PS, polyethylene glycol-immobilized PS (PS-PEG) and PS-PEG-NH2 beads, CD11b up-regulation in the bulk, platelet-leukocyte aggregates, and leukocyte adhesion were all dependent on surface area, whereas tissue factor (TF) expression was not. Material-induced leukocyte activation in the bulk was also independent of the beads' capacity to activate platelets. However, monocyte adhesion and TF expression on beads appeared to be related to the presence of platelets on the surface. Material-induced TF expression was able to initiate the extrinsic pathway of coagulation, resulting in significant fibrin formation. Although not all of our markers of leukocyte activation varied with material area or chemistry, it was clear that these materials activated leukocytes in a way that resulted in increased procoagulant activity. During blood-material interactions, material-induced leukocyte activation may then contribute to thrombogenesis.
Blood Coagulation↗
Making microencapsulation work: conformal coating, immobilization gels and in vivo performance.
Microencapsulation of cells as a means of insulin or other protein delivery (for example, for gene therapy) has not yet realized its potential. Three aspects of this problem are illustrated with reference to the use of poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA-MMA). Conformal coating was used to coat cell aggregates with a very thin layer of a water-insoluble HEMA-MMA membrane that conforms to the shape of the aggregate, and minimizes the polymer's contribution to the total transplant volume. Cell aggregates were coated at a liquid-liquid interface of a discontinuous density gradient composed of both aqueous and organic liquids. Aggregates of HepG2 cells were coated and remained viable. Immobilization matrices were co-encapsulated in order to control cell phenotype. Ultralow gelling temperature agarose promoted the proliferation of HEK293 cells, while the viability of transfected C2C12 cells was improved in microcapsules that contained Matrigel. Rat or human hepatoma cells in HEMA-MMA microcapsules lost viability within a week after implantation into an omental pouch in Wistar rats. The loss of viability was attributed to the tissue reaction, although it is not clear if the cells lost their viability in vivo leading to the aggressive tissue reaction or if the latter caused the cells to starve or otherwise die. On the other hand, intraperitoneal implantation of microcapsules containing L929 cells in 'syngeneic' C3H mice in a high-strength agarose gel resulted in maintenance of viability of approximately 50% of the encapsulated cells. While progress is being made on several fronts, this type of tissue engineering construct is still several years away from routine use in humans.
Animals↗
Complement activation by PVA as measured by ELIFA (enzyme-linked immunoflow assay) for SC5b-9.
Polyvinyl alcohol (PVA) coated onto polyethylene (PE) tubes exposed to human serum for 1 hour at 37 degrees C resulted in the production of 1.03 +/- 0.04 microg/cm2 of the soluble form of the terminal membrane attack complex, SC5b-9. This was approximately 20 x that produced by the polyethylene. About one quarter of this total was found associated with the surface of PVA. SC5b-9 concentrations were determined by enzyme-linked immunoflow assay (ELIFA) a variant on ELISA that involved drawing the test sample, the antibodies and the chromogenic reagent through a nitrocellulose membrane filter. ELIFA enabled analysis of protein concentrations in the presence of SDS, so that SDS (0.05%) was used to desorb adsorbed SC5b-9 prior to analysis together with SC5b-9 in the bulk to get a more complete picture of PVA-associated complement activation.
Biocompatible Materials↗
Effect of an immobilization matrix and capsule membrane permeability on the viability of encapsulated HEK cells.
The effect of inclusion of an immobilization matrix and the capsule membrane permeability on the viability, metabolic activity, and proliferation of encapsulated HEK cells was investigated in vitro. In the absence of a matrix, a particular transfected HEK cell line formed a single aggregate in the core of the poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA-MMA) capsule, and the number of live cells decreased significantly with the passage of time. In contrast, co-encapsulation with a 1% (w/v) ultralow gelling temperature agarose matrix promoted the proliferation of the encapsulated cells. The initial number of approximately 200 live cells/capsule doubled 14 d after encapsulation and reached a plateau of approximately 500 live cells/capsule 28 d after encapsulation. The agarose matrix provided uniform distribution of the cells within the capsule core giving rise to multiple aggregates upon proliferation. Reduction of the polymer solution concentration, and hence the increase of the permeability of the capsule membrane, did not have an effect on the extent or rate of proliferation of cells co-encapsulated with agarose, and did not improve the viability of cells that were encapsulated without a matrix. These cells (transfected with the cDNA for human hepatic lipase) served as a model as part of a program evaluating the use of encapsulated cells for gene therapy.
Biocompatible Materials↗
Microencapsulation of normal and transfected L929 fibroblasts in a HEMA-MMA copolymer.
Mouse L929 fibroblasts transfected to express a secreted form of human alkaline phosphatase (SEAP) were encapsulated in approximately 400-microm poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA-MMA) microcapsules as a baseline for the use of genetically engineered cells in encapsulation therapy. Although incubation of microcapsules with serum-containing medium resulted in maintaining the number of live encapsulated cells with the passage of time, incubation in a serum-free medium resulted in a three-fold proliferation of the encapsulated cells within a 3-week observation period. Similar to the results for incubation with serum-containing medium, co-encapsulation with a bovine dermal type I collagen, i.e., the inclusion of a matrix in the core of the capsules, resulted in maintenance of the initial number of live cells with the passage of time. SEAP measurements indicated that the transfected cells not only continued to express the transgene product after encapsulation, but also adapted to the capsule microenvironment to secrete SEAP at progressively larger amounts with the passage of time. However, SEAP expression only occurred when the transfected cells (encapsulated or non-encapsulated) were cultivated in serum-containing medium.
Alkaline Phosphatase↗
Viability of HEMA-MMA microencapsulated model hepatoma cells in rats and the host response.
Small diameter hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA; 75% HEMA) microcapsules containing an aggregate of viable rat hepatoma H4IIEC3 cells, after implantation into an omental pouch in Wistar rats, contained viable cells at 7 days but not 14 days. A similar transplantation of microencapsulated aggregates of human hepatoma HepG2 cells did not result in viable cells even at 7 days. The loss of viability was attributed to the tissue reaction, because both encapsulated cell types remained viable in vitro. However, it is not clear if the cells lost their viability in vivo, leading to the aggressive tissue reaction or if the latter caused the cells to starve or otherwise die. The tissue reactions to microcapsules containing rat or human hepatoma cells at day 1 was one cell layer thick and avascular. At later times, tissue reactions were comprised of three regions: macrophages, fibroblasts, and some foreign body giant cells apposed to the polymer membrane, a dense region of fibroblasts and collagen, and a region of vascularized granulation tissue. Prompt vascularization of the tissue reactions occurred after 4 days and was maintained for up to 14 days. Even at 14 days, immune cells were observed, suggesting a continued immune response toward antigens shed from the encapsulated cells.
Animals↗
Hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymers for cell microencapsulation: effect of HEMA purity.
Thermoplastic copolymers of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) (molar ratio: 75/25 HEMA-MMA) were synthesized using HEMA containing different amounts of ethylene glycol dimethacrylate (EGDMA) to investigate their suitability for cell microencapsulation. Pure HEMA (0.0% EGDMA) was obtained with preparative chromatography to prepare a linear copolymer. Microcapsules (with a diameter of 300-400 microm) were readily made with the copolymers by interfacial precipitation. Smaller and more transparent capsules were obtained using the copolymer prepared from purer HEMA. Chinese hamster ovary (CHO) fibroblasts, as model cells, were microencapsulated in the linear copolymer. The CHO cells survived the microencapsulation process and the metabolic activity of the encapsulated cells increased within the 14 days observation period.
Animals↗
What really is blood compatibility?
The criteria for nonthrombogenicity are classically defined as long clotting times and minimal platelet deposition. The inability to point to unequivocal progress in the development of truly nonthrombogenic materials, highlights the inadequacy if not actually invalidity of these criteria. Our approach is to define nonthrombogenicity in terms of: (1) a thrombin production rate constant, kp < 10(-4) cm s(-1); (2) low platelet consumption and low degree of platelet activation (e.g., microparticle formation); (3) perhaps some platelet spreading; and (4) low complement and leukocyte activation. Only when the target becomes clear, will it be possible to identify clear strategies for producing the materials we need.
Animals↗
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.
Animals↗
Conformal coating of small particles and cell aggregates at a liquid-liquid interface.
Polymer encapsulation of allogeneic or xenogeneic tissue is under active investigation as a means of isolating transplanted cells, such as pancreatic islets, from the immune system. We report here a method for coating small particles and cell aggregates with a very thin water insoluble hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) membrane that conforms to the shape of the aggregate, and minimizes the polymer's contribution to the total transplant volume. Cell aggregates were coated at a liquid-liquid interface of a discontinuous density gradient composed of both aqueous and organic liquids. By increasing the viscosity difference and decreasing the density difference between the two liquids of the coating interface, coatings from approximately 1 to 15 microns thick were formed. Aggregates of HepG2 cells and pancreatic islets were coated and remained viable.
Biocompatible Materials↗
Flow cytometric study of in vitro neutrophil activation by biomaterials.
Neutrophil activation for adherent and nonadherent cells, as measured by flow cytometry, was not strongly dependent on material surface chemistry. We had hypothesized that material-induced neutrophil activation was an important parameter associated with material failure. All materials tested [cellophane, an acrylonitrile copolymer (AN69), Pellethane, nylon, polyethylene terephthalate, low density polyethylene, and polydimethylsiloxane] activated isolated human neutrophils, which were resuspended in plasma or serum, to similar extents based on L-selectin shedding, CD11b upregulation, and stimulation of the oxidative burst after 30-min exposure. Inhibition of complement activation by sCR1 unexpectedly had little effect if any on nonadherent neutrophils. However, neutrophil adhesion, but not the level of activation of the adherent cells, was strongly dependent on complement activation. Pretreatment with albumin did not inhibit adhesion or reduce neutrophil activation, but plasma pretreatment resulted in increased activation for nonadherent and adherent cells. More adhesion and a higher level of activation of adherent cells was observed following pretreatment with fibrinogen, a ligand of CD11b. Taken together these results suggest that upon contact with a material, neutrophil activation may occur though mechanisms that are not mediated by complement. For example, the presence of plasma proteins such as fibrinogen at the interface may trigger activation and the release of other activating agents. Although the material differences are small, the extent of activation may be significant and warrant further study of the mechanism and consequences of that activation.
Acrylic Resins↗
Characterization of transient platelet contacts on a polyvinyl alcohol hydrogel by video microscopy.
Acridine orange labelled, washed human platelets were counted and tracked on polyvinyl alcohol (PVA), heparin-PVA and polyethylene (PE)-coated coverslips with a view to understand why transient contact on the PVA hydrogels lead to elevated platelet activation and consumption relative to polyethylene. Over the 4 min of initial contact that was studied, platelet adhesion was higher on PE than on PVA or heparin-PVA at both 40 and 200 s(-1), as expected, regardless of whether the surfaces were pre-treated with albumin or fibrinogen. Not all platelets appearing to make contact with the surface, actually attached. For example, less than 2% of the platelets contacting albumin pre-treated PVA (at 40 s(-1)) remained adherent at the end of the initial 60 s observation time, while the corresponding number for PE was greater than 9%. A greater fraction of the platelets remained adherent at the higher shear rate or with fibrinogen pre-treatment, but the difference between PVA and PE remained similar: for example, with fibrinogen pre-treatment at 200 s(-1), approximately 25% of the platelet contacts resulted in adhesion on PVA while 66% did so on PE. While net platelet adhesion was less for the hydrogels, than for PE, the total number of contacts (adherents + non-adherents) were more comparable and unexpectedly higher for albumin pre-treatment than for fibrinogen. Net platelet adhesion is but one component of the total platelet interaction with a material surface. Fluorescent video microscopy has been shown to be a useful, albeit not unequivocal, method for assessing the platelets that make contact with but do not adhere to a surface. reserved
Biocompatible Materials↗
Surface grafting of poly(ethylene glycol) onto poly(acrylamide-co-vinyl amine) cross-linked films under mild conditions.
Poly(ethylene glycol) (PEG) was grafted onto poly(acrylamide-co-vinyl amine) (poly(AM-co-VA)) film using tresylated PEG (TPEG) at 37 degrees C in aqueous buffers (pH 7.4) with a view to surface-modifying microencapsulated mammalian cells. Poly(AM-co-VA) film was synthesized by Hofmann degradation of a cross-linked poly(acrylamide) film. Conversion to vinyl amine on the surface of the film was approximately 50%, but bulk conversion was not observed; surface specificity was thought to be the result of cleavage of aminated polymer chains at the surface due to chain scission. Reaction between primary amine and TPEG gave a graft yield of 2 mol% (based on XPS) with respect to available surface amine groups, equivalent to 54 mol% ethylene oxide based on monomer units. Physical adsorption of non-activated polymer was done under identical conditions as a control and the difference in oxygen content was significant compared to TPEG. The type of buffer agent and buffer concentration did not influence graft yields. This graft reaction, which was completed in as little as 2 h was considered to be mild enough to be used for a surface modification of microcapsules containing cells without affecting their viability. Such a surface modification technique may prove to be a useful means of enhancing the biocompatibility of microcapsules (or any tissue engineering construct) even after cell encapsulation or seeding.
Acrylic Resins↗
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.
Biocompatible Materials↗
Selected aspects of the microencapsulation of mammalian cells in HEMA-MMA.
Microencapsulation of live mammalian cells is one means of creating hybrid artificial organs, like an artificial pancreas or an artificial liver. In addition to creating and developing the methodologies for enclosing cells within the appropriate semipermeable and biocompatible membranes, novel techniques are needed to assess the various features of the resulting capsules. The small size of a capsule or its heterogeneity can lead to additional complexities that go beyond the problem of examining cell behavior in the presence of biomaterials. These problems are illustrated here by comparison of protein release by microencapsulated HepG2 cells within large and small HEMA-MMA (hydroxyethyl methacrylate-methyl methacrylate) capsules, by assessment of the effect of processing conditions on HEMA-MMA microcapsule permeability to horseradish peroxidase at the individual capsule level, and by a confocal microscopy technique for assessing intracapsule cell viability.
Animals↗
Flow cytometric analysis of material-induced platelet activation in a canine model: elevated microparticle levels and reduced platelet life span.
Assessment of material-induced platelet activation is important given that it is thought to be a major mechanism of biomaterials thrombogenicity. We monitored, by flow cytometry, platelet microparticle (MP) levels in the circulation during the connection of polyvinyl alcohol (PVA) hydrogel and polyethylene (PE) test segments (3.18 mm ID, 20 and 50 cm L) to our chronically shunted beagle dogs. We report that circulating microparticle levels were dependent on test segment material, length, and time. The connection of 50-cm lengths of PVA hydrogel test segments led to MP levels two to three times greater than background at 48 h, while the connection of polyethylene test segments did not lead to elevated microparticle levels. MP levels were near background 24 h after removal of the PVA test segment. To determine platelet life span during the connection of test segments, platelets were labeled in vivo with biotin and their disappearance monitored flow cytometrically. While platelet life span for shunted dogs (no test segment) was 4.7 +/- 0.2 days, the connection of PVA hydrogel test segments led to a platelet life span of < 2 days.
Animals↗