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Michael V Sefton

Publications and source records attributed to Michael V Sefton.

17 recordsLinked to original sources
PubMed2007

Tissue factor and thrombomodulin expression on endothelial cell-seeded collagen modules for tissue engineering.

The creation of functional tissue engineering constructs to repair or replace diseased tissues requires a well-formed vasculature network within the construct and the endothelial cells lining that vascular bed must display a nonthrombogenic phenotype. A new approach to tissue engineering involves the assembly of smaller components (modules fabricated at the hundred micron scale) into larger constructs. The modules, collagen gel containing the particular tissue cell of interest, are covered with endothelial cells prior to assembly so that the interconnected channels that are formed are lined with endothelial cells, creating a mimic of a vascular network. Here, we confirmed (using confocal microscopy primarily) that the human umbilical vein endothelial cells, seeded on collagen gel modules without a second embedded cell and without flow, bore the molecular markers of low thrombogenicity. Two days, after seeding on the modules, endothelial cells displayed the typical cobblestone morphology, formed tight cell-cell junctions and covered the whole module surface. Immunofluorescence staining showed that at both 2 days and 7 days after seeding, only a few cells expressed tissue factor while this number was dramatically increased after TNFalpha stimulation. On the other hand, thrombomodulin was expressed by the majority of seeded cells and expression was reduced after TNFalpha stimulation.

Collagen↗
PubMed2006

Suppressed splenocyte proliferation following a xenogeneic skin graft due to implanted biomaterials.

BACKGROUND: Immune system responses to antigens in the context of biomaterials are poorly understood. Biomaterial implantation results in an inflammatory reaction, which is anticipated to alter the adaptive immune response, in the case presented here, to a skin xenograft. Our earlier work showed unexpectedly low splenocyte proliferation following a xenogeneic cell implant in tandem with a biomaterial in the form of a microcapsule. Here we explore whether that effect was due to the cells or the biomaterial, and attempt to dissect the mechanism of immune deviation. METHODS: We assessed the immune response of Balb/c mice to hamster skin grafts accompanied by one of three implants: encapsulated xenogeneic cells; free cells accompanied by the same encapsulation biomaterials; and the encapsulation biomaterials without cells. Cells were encapsulated in a hydroxyethyl methacrylate-methyl methacrylate copolymer then embedded in an agarose gel. Splenocyte proliferation upon re-challenge in vitro, antibody titer in serum, and Th1/2 polarization (by cytokines in splenocyte challenge supernatants and antibody isotypes in serum) were measured. RESULTS: All skin grafts with encapsulation materials (even without cells) suppressed subsequent splenocyte proliferation at 10 days postimplant, although this effect disappeared by two months. In contrast, the antibody response was equal to or greater than that for a skin graft alone. Th1/2 polarization could not explain these observations because it did not correlate with the suppression of splenocyte proliferation. CONCLUSIONS: Implanted biomaterials caused nonspecific, transient suppression of splenocyte responses to hamster cells following hamster skin grafts, which is potentially important in the context of tissue engineering.

Animals↗
PubMed2006

Vascularized organoid engineered by modular assembly enables blood perfusion.

Tissue engineering is one approach to address the donor-organ shortage, but to attain clinically significant viable cell densities in thick tissues, laboratory-constructed tissues must have an internal vascular supply. We have adopted a biomimetic approach and assembled microscale modular components, consisting of submillimeter-sized collagen gel rods seeded with endothelial cells (ECs) into a (micro)vascularized tissue; in some prototypes the gel contained HepG2 cells to illustrate the possibilities. The EC-covered modules then were assembled into a larger tube and perfused with medium or whole blood. The interstitial spaces among the modules formed interconnected channels that enabled this perfusion. Viable cell densities were high, within an order of magnitude of cell densities within tissues, and the percolating nature of the flow through the construct was evident in microcomputed tomography and Doppler ultrasound measurements. Most importantly, the ECs retained their nonthrombogenic phenotype and delayed clotting times and inhibited the loss of platelets associated with perfusion of whole blood through the construct. Unlike the conventional scaffold and cell-seeding paradigm of other tissue-engineering approaches, this modular construct has the potential to be scalable, uniform, and perfusable with whole blood, circumventing the limitations of other approaches.

Animals↗
PubMed2006

Methylation of poloxamine for enhanced cell adhesion.

Aiming at producing a synthetic collagen-mimetic material that is stiffer than collagen but that like collagen allows both cell encapsulation and cell growth on the surface, a positively charged poloxamine hydrogel was prepared by methylating the tertiary amine groups of a four-arm poly(ethylene oxide)-poly(propylene oxide) block copolymer derivative (Tetronic 1107). This derivative was subsequently reacted with methacryloyl isocyanate, rendering positively charged materials that are further cross-linkable by a photointiated free radical polymerization. Different hydrogels containing methylated poloxamine methacrylate concentrations between 6% and 18% were produced and characterized by means of water uptake and viscoelastic properties. A sharp increase in water content was observed in distilled water during the first week; some of the gels showed water uptakes as high as 2 times the initial wet weight. In PBS, this effect was less prominent due to the decrease in the osmotic gradient. Also, a gradual increase of both the storage modulus (G') and the loss modulus (G") resulted from increasing the polymer concentration: for example, G' values ranged between 70 and 23000 Pa for 6% and 18% methylated poloxamine methacrylate hydrogels (at 1 Hz, 100 Pa of oscillatory stress). HepG2 cells embedded in different compositions and exposed to UV light displayed good viability levels after the cross-linking, unlike a previously reported attempt at creating a synthetic collagen-mimetic material. A well-spread endothelial cell morphology was apparent on methylated poloxamine films after preincubation in serum-containing medium, while on unmodified poloxamine methacrylate hydrogels cells attached poorly. However, EC did not attach well to the same material when fabricated not as films but as cylindrical modules as needed for the modular construct for which this material was intended. Thus, for this apparently more challenging geometry, it was necessary to combine collagen with the methylated poloxamine to have good attachment of EC on the surface of modules as well as films. While the challenge of creating a synthetic alternative to collagen as a stiffer cell-compatible substrate remains, methylated poloxamine displays many of the attributes that make it a useful material for tissue engineering.

Cell Adhesion↗
PubMed2005

Surface study of collagen/poloxamine hydrogels by a 'deep freezing' ToF-SIMS approach.

In order to determine the presence of collagen molecules at the surface of a collagen-modified poloxamine hydrogel (a semi-interpenetrating network), the surface composition was studied using Time-of-Flight Secondary Ion Mass Spectra (ToF-SIMS). Collagen was added to the poloxamine hydrogel (poloxamine is a commercially available four-arm poly(ethylene oxide)/poly(propylene oxide) block copolymer, PEO/PPO) to promote the attachment of endothelial or liver cells. X-ray photoelectron spectroscopy (XPS) of dry samples showed a sharp increase in the N content from 0.6% in a pure poloxamine hydrogel to 8.8% in the collagen-containing material. Afterwards, the surface was studied by a 'deep freezing' ToF-SIMS approach under progressive heating from -120 to -60 degrees C. The positive spectrum of collagen/poloxamine at -65 degrees C displayed distinct signals corresponding to different amino acid fragments such as CH4N+ (30 m/z, Gly), C3HN2+ (43 m/z, Arg), C2H6N+ (44 m/z, Ala) and C4H5N2+(81m/z, His) and others corresponding to the PEO and PPO blocks of poloxamine. In addition, the negative spectrum showed peaks at 26 m/z (CN-), 32 m/z (S-) and 42 m/z (CNO-) characteristic of fragments of the collagen molecule. Imaging experiments indicated the homogeneous distribution of the collagen on the surface. These results supported the use of ToF-SIMS for the surface characterization of hydrated hydrogels and confirmed the collagen presence as the means whereby cells attach to the modified poloxamine matrix.

Animals↗
PubMed2005

Poloxamine hydrogels with a quaternary ammonium modification to improve cell attachment.

Four-arm poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO) block copolymer (poloxamine, Tetronic 1107) hydrogels were modified with positively charged groups with the aim of overcoming the low cell adhesion properties of these PEO-rich systems. Different matrices containing poloxamine-methacrylate (6-12%) and a quaternary ammonium methacrylate ([2-(methacryloyloxy)ethyl]-trimethylammonium chloride [MAETAC], 0-0.48 M) were produced by a photo-initiated free radical copolymerization. A sharp increase in water content for MAETAC-containing gels was seen during the first 2 days of incubation in distilled water; some of the gels showed water uptakes as high as 12 times the initial wet weight. In phosphate-buffered saline (PBS), this effect was less pronounced because of the decrease in the osmotic gradient. In addition, a gradual increase of both the storage and the loss modulus of the gels resulted from increasing the MAETAC content [e.g., G' values increased from 13,500 Pa for 12% poloxamine-methacrylate gels without MAETAC to 151,000 Pa for 0.48M MAETAC contents (at 1 Hz, 100 Pa of oscillatory stress)]. Although on pure poloxamine-methacrylate gels HepG2 and HUVEC cells attached poorly, on MAETAC-containing specimens a well-spread morphology and confluent monolayers were obtained, at least after preincubation in serum containing medium. Although not having all the cell compatibility of collagen, these fully synthetic quaternary ammonium-modified PEO-rich gels may have some utility in tissue-engineering applications where stiff gels and cell attachment after gelation are desired.

Biocompatible Materials↗
PubMed2005

Endotoxin: the uninvited guest.

In the laboratory environment where biomaterials are synthesized and their biocompatibility assessed, we find that endotoxin contamination is hard to avoid and must not be ignored. In those relatively few cases where endotoxin was known to be present, it has been clearly shown that endotoxin can significantly affect the biological response observed and hence confound any effect of the material. This short review explains what endotoxin is, how to test for it and remove it and what its effect on the biological response to biomaterials is. We advocate routine testing of endotoxin on biomaterials and of reagents used in experimental evaluation of biomaterials and this should be the responsibility of every scientist to ensure the validity of any biomaterial study.

Biocompatible Materials↗
PubMed2005

Semi-synthetic collagen/poloxamine matrices for tissue engineering.

Collagen-containing poloxamine hydrogels were produced with the aim of overcoming the low stiffness displayed by collagen gels that are not otherwise chemically crosslinked. Matrices were obtained by functionalization of a four-arm PEO-PPO block copolymer (poloxamine, Tetronic) with methcrylate groups and subsequent free radical polymerization of water solutions of the modified polymer in the presence of collagen. The resulting matrices had a sharp increase in stiffness, when compared to pure collagen gels. For example, whereas collagen had a storage modulus (G') around 70 Pa and a loss modulus (G'') of 10 Pa, a crosslinked collagen/poloxamine system containing 8.3% crosslinked poloxamine had G' and G'' values of 7400 and 1000 Pa, respectively. HepG2 cells were seeded within the gels before the crosslinking and the viability levels estimated by AlamarBlue assay were between 65% and 91% for systems containing 0.04-0.09 wt% photoinitiator. HepG2 and endothelial cells also adhered to and spread on the surface of the collagen-containing specimens, suggesting their potential utility in tissue engineering.

Animals↗
PubMed2004

In vivo recognition by the host adaptive immune system of microencapsulated xenogeneic cells.

BACKGROUND: Microencapsulation is under consideration as a means of enabling pancreatic islet transplantation. To understand better the ongoing destructive host response, we examined whether the adaptive immune system of the recipient recognized polymer-encapsulated xenogeneic cells implanted intraperitoneally. METHODS: Balb/c mice were implanted with xenogeneic Chinese hamster ovary cells, inside and outside poly(hydroxyethyl methacrylate-methyl methacrylate) microcapsules, and responses were compared with xenografted Chinese hamster skin (positive control). Capsules were localized within an agarose rod. Splenocyte proliferation upon rechallenge in vitro, antibody titer in serum, and Th1/2 polarization (assessed by interleukin-4 and interferon-gamma in supernatants of antigen-challenged splenocytes and immunoglobulin [Ig]G1 and IgG2a antibody isotypes in serum) were measured. RESULTS: Encapsulation did not prevent a strong recipient antibody response. Splenocyte proliferation in vitro did not differ after priming by implanted cells, inside or outside capsules. Thus, the capsule membrane did not prevent indirect recognition of shed antigens. However, after 10 days of implantation, proliferation was lower than that induced by skin grafts, although this difference disappeared by 2 months. This transient T-cell suppression was unexpected because encapsulated cell viability was already compromised by 10 days. The influence of Th1/2 bias did not explain the observed suppression. Cells inside capsules elicited a consistent Th2 response, whereas cells outside capsules elicited a mixed response, and skin xenografts showed an initial Th2 response that became mixed by 2 months. CONCLUSIONS: Encapsulation does not prevent host immune responses, but the inflammatory response to the implanted biomaterials or xenogeneic cells may be responsible both for encapsulated cell death and transient T-cell suppression.

Animals↗
PubMed2004

Expression of matrix metalloproteinase-2 and -9 in exudates associated with polydimethyl siloxane and gelatin tubes implanted in mice.

The expression of matrix metalloproteinases (MMPs), MMP-2 and -9, during the inflammatory response to two implanted biomaterials was determined. To illustrate this phenomenon, polydimethyl siloxane (PDMS), weakly crosslinked gelatin, and highly crosslinked gelatin tubes were implanted subcutaneously in mice for up to 3 weeks. Latent MMP-2 (pro-MMP-2) was seen in exudates throughout the experimental period and did not show any apparent changes in time or among the three biomaterials. The expression of MMP-9, however, showed a very different pattern. At days 4 and 8, the predominant form of MMP-9 is pro-MMP-9 (the latent form) and its level is much higher for the PDMS tube than either of the gelatin tubes. The active form of MMP-9, initially low, rose progressively over the 3-week period and reached its peak at week 2 for PDMS and highly crosslinked gelatin and at week 3 for the more degradable, weakly crosslinked gelatin. Data from the histological and cytological analysis also showed that the PDMS tube induced a much stronger cellular response than gelatin tubes, although endotoxin contamination precludes a conclusion related to biomaterial effects. We concluded that MMP-9 is a useful marker of the host inflammatory response to the implanted biomaterials and it may be useful in the assessment of biomaterial biocompatibility. Perhaps more importantly, the consequences of MMPs on matrix remodeling may become of concern in biomaterials used for tissue engineering.

Absorbable Implants↗
PubMed2004

Poly(methylidene malonate 2.1.2) nanoparticles: a biocompatible polymer that enhances peri-adventitial adenoviral gene delivery.

Vascular gene therapy is currently limited by low and transient levels of gene transfection. The objectives of this study were to determine whether peri-adventitial delivery of adenovirus coupled to nanoparticles could improve transfection efficiency and duration. Adenovirus was absorbed to the surface of nanoparticles that were made from poly(methylidene malonate)2.1.2 (PMM2.1.2). These complexes were found to have good adhesive properties to both cultured vascular smooth muscle cells and to the luminal and adventitial layers of excised rabbit carotid arteries. Adenovirus encoding to beta-galactosidase coupled to PMM2.1.2 nanoparticles or adenovirus alone were delivered locally to the adventitia of rabbit carotid arteries. Transfection rate was assessed histologically by the percentage of beta-galactosidase positive cells in the vessel wall at 1 and at 2 weeks. There was significantly higher transfection rate when adenovirus was complexed with nanoparticles as compared to free adenovirus (At 1 week: 10+/-3.9% beta-gal positive cells vs. 2.4+/-0.3% and at 2 weeks: 4.3+/-4.1% vs. 0%, P<0.005 for all). This difference was present in both the medial and adventitial layers. In conclusion, adenoviral mediated gene therapy was significantly enhanced by adsorbing the virus to PMM2.1.2 nanoparticles. This delivery method may be a good therapeutic strategy for the treatment of various vascular diseases.

Adenoviridae↗
PubMed2004

Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelets and leukocytes.

Our failure to produce truly non-thrombogenic materials may reflect a failure to fully understand the mechanisms of biomaterial-associated thrombosis. The community has focused on minimizing coagulation or minimizing platelet adhesion and activation. We have infrequently considered the interactions between the two although we are generally familiar with these interactions. However, we have rarely considered in the context of biomaterial-associated thrombosis the other major players in blood: complement and leukocytes. Biomaterials are known agonists of complement and leukocyte activation, but this is frequently studied only in the context of inflammation. For us, thrombosis is a special case of inflammation. Here we summarize current perspectives on all four of these components in thrombosis and with biomaterials and cardiovascular devices. We also briefly highlight a few features of biomaterial-associated thrombosis that are not often considered in the biomaterials literature: The importance of tissue factor and the extrinsic coagulation system. Complement activation as a prelude to platelet activation and its role in thrombosis. The role of leukocytes in thrombin formation. The differing time scales of these contributions.

Biocompatible Materials↗
PubMed2003

Reaction of poly(acrylamide-co-vinylamine) with tresyl-PEG in the presence of PC12 cells.

Tresylation of an amine containing polymer film in the presence of PC12 cells did not result in a significant loss of cell viability, at least as assessed by trypan blue exclusion or MTT assay. PC12 cells were cultured atop reactive poly(acrylamide-co-vinyl amine) films or tissue culture polystyrene and exposed for 2 h to tresylated polyethylene glycol (TPEG) or unreactive hydrolyzed TPEG in 0.1M TES (N-tris hydroxymethyl-2-aminoethane sulfonic acid). The loss in trypan blue viability was limited ( approximately 80% retained), provided the TPEG concentration was 10 micromol/g or less. Similarly when microencapsulated PC12 cells (in a non-reactive polyacrylate hydrogel) were exposed to TPEG (10 micromol/g in 0.1M TES) the loss of MTT activity was small. The loss of vaibility was attributed to the toxicity of the tresyl leaving group and not the reaction itself. Thus, it may be possible to surface modify cell containing microcapsules, at least under limited conditions, in order to improve their biocompatibility without compromising the viability of the enclosed cells. This should lead to the development of new (reactive) polymers for microencapsulation since biocompatibility need not be a design consideration in the first instance.

Amines↗
PubMedDate not supplied

Conformal coating of mammalian cells immobilized onto magnetically driven beads.

A novel cell bead system, comprising a magnetic core, a spherical annulus of agarose-immobilized cells, all conformally coated within a synthetic polymer, is proposed as a means of immunoisolating mammalian cells in a system that provides a balance between low total implant volume, retrievability, and diffusion limitations. A successful immunoisolation system could be used to transplant cells without eliciting an inappropriate host response. Chinese hamster ovary (CHO) cells were immobilized at the periphery of large (approximately 2 mm) agarose beads containing inert magnetic cores (< or = 1 mm) and coated in a hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer by interfacial precipitation. The beads were coated in liquid gradients containing polyethylene glycol 200 (PEG) or bromooctane. Although many cells were adversely affected by the coating process, the cells that did survive (30-50% of those loaded into the beads) remained viable for a period of at least 2 weeks. This viability was much higher than achieved previously because of a number of factors, such as the aqueous agarose, the hydrophobic bromooctane intermediate layer, and faster coating times that minimize the exposure of the cells to organic solvents. Also, a mathematical model was used to describe oxygen transport within the annular agarose beads. These results provide evidence that the proposed geometry and the fabrication approach may be useful for a variety of applications that involve cell encapsulation.

Animals↗
PubMedDate not supplied

Collagen/poloxamine hydrogels: cytocompatibility of embedded HepG2 cells and surface-attached endothelial cells.

The effects of cross-linked poloxamine hydrogels on the cellular function of embedded HepG2 cells and surface-attached endothelial cells were assessed. HepG2 cells embedded within collagen/poloxamine-methacrylate gel survived photo-cross-linking (MTT viability, 78%). There was a gradual increase in cell number during the first week. The cumulative secretion of alpha1-antitrypsin by HepG2 cells showed an almost linear profile. However, lower levels for the collagen/poloxamine-methacrylate matrix were observed when compared with collagen. Endothelial cells attached poorly to poloxamine gels without collagen (alamarBlue reduction ranged from 36 to 63%) and did not spread well. The addition of collagen led to spread cells and alamarBlue reduction levels of 75-93% (24 h after seeding). On day 5, some detachment was noted through analysis of vascular endothelial cadherin staining. Finally, the collagen-containing matrix was used to prepare cylindrical modules containing HepG2 cells to show the utility of this material in modular tissue constructs. A fluorescent cytoplasmic tracer, Vybrant CFDA SE, showed that embedded cells remained viable for more than 2 months, confirming the good cytocompatibility of collagen/poloxamine-methacrylate in the form of modules. The suitability of these modules for preparing uniform, scaleable, and vascularized constructs remains to be demonstrated.

Animals↗