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This paper describes the synthesis and characterization of an injectable methacrylate functionalized urethane-based photopolymerizable prepolymer to form biodegradable hydrogels. The tetramethacrylate prepolymer was based on the reaction between two synthesized compounds, diisocyanato poly(ethylene glycol) and monohydroxy dimethacrylate poly(epsilon-caprolactone) triol. The final prepolymer was hydrated with phosphate-buffered saline (pH 7.4) to yield a biocompatible hydrogel containing up to 86% water. The methacrylate functionalized prepolymer was polymerized using blue light (450 nm) with an initiator, camphorquinone and a photosensitizer, N,N-dimethylaminoethyl methacrylate. The polymer was stable in vitro in culture media over the 28 days tested (1.9% mass loss); in the presence of lipase, around 56% mass loss occurred over the 28 days in vitro. Very little degradation occurred in vivo in rats over the same time period. The polymer was well tolerated with very little capsule formation and a moderate host tissue response. Human chondrocytes, seeded onto Cultispher-S beads, were viable in the tetramethacrylate prepolymer and remained viable during and after polymerization. Chondrocyte-bead-polymer constructs were maintained in static and spinner culture for 8 weeks. During this time, cells remained viable, proliferated and migrated from the beads through the polymer towards the edge of the polymer. New extracellular matrix (ECM) was visualized with Masson's trichrome (collagen) and Alcian blue (glycosaminoglycan) staining. Further, the composition of the ECM was typical for articular cartilage with prominent collagen type II and type VI and moderate keratin sulphate, particularly for tissue constructs cultured under dynamic conditions., (2010. Published by Elsevier Ltd. All rights reserved.)

For tissue engineering and cell therapy applications, expansion of cells such as chondrocytes on beads in spinner culture can provide advantages compared with monolayer culture. The use of resorbable beads that can be included as an integral part of the construct provides the advantage of minimizing the extent of cell handling and eliminating a final trypsin treatment to detach cells from the bead. In this study, we have made various types of beads based on native collagen and denatured collagen (gelatin). The beads have been stabilized by different extents of glutaraldehyde cross-linking, and characterized by a combination of chemical analysis, thermal stability, and microscopy. In vitro examination in the presence and absence of chondrocytes showed that stability increased with the extent of crosslinking and could also be influenced by the manner of fabrication. On the basis of the in vitro stability studies, gelatin beads of a defined stability were shown to resorb over time in subcutaneous implants in nude mice compared with more stable demineralized bone particle (DMB) carriers. These data indicate that for direct use in tissue engineering or cell therapy applications, where resorbable beads can be used for cell expansion and then direct delivery of cells, it is possible to design suitable carrier beads with a range of stabilities that match the implant requirements., ((c) 2009 Wiley Periodicals, Inc.)

We recently reported the generation of a highly elastic, crosslinked protein biomaterial via a rapid photochemical process using visible light illumination. In light of these findings, we predicted that other unmodified, tyrosine-rich, self-associating proteins might also be susceptible to this covalent crosslinking method. Here we show that unmodified native fibrinogen can also be photochemically crosslinked into an elastic hydrogel biomaterial through the rapid formation of intermolecular dityrosine. Photochemically crosslinked fibrinogen forms tissue sealant bonds at least 5-fold stronger than commercial fibrin glue and is capable of producing maximum bond strength within 20s. In vitro studies showed that components of the photochemical crosslinking reaction are non-toxic to cells. This material will find useful application in various surgical procedures where rapid curing for high strength tissue sealing is required.

A method for the preparation of bioresorbable collagen beads with an open porous structure is presented. These beads were prepared from collagen-alginate composite beads by removal of the alginate component. These collagen beads were suitable for rapid proliferation of chondrocytes in a dynamic, spinner culture system. Histology and immuno-histology showed that biochemical markers of chondrocytes are present during this cell proliferation, indicating that there was control of phenotype and that cell de-differentiation had not occurred. Unlike other 3-D scaffolds that have been used, the cells were amplified from very low cell densities and were able to proliferate freely without loss of phenotype.

Articular cartilage tissue engineering procedures require the transplantation of chondrocytes that have been expanded in vitro. The expansion is carried out for a considerable time and can lead to a modulation of cell phenotype. However, microcarrier cultures have been shown to allow cell expansion while maintaining the phenotype. Here, we have used the biodegradable polyester poly(lactide-co-glycolide) (PLGA) in the form of microspheres and irregular shaped microparticles with a diameter between 47 and 210 microm. Surface modification of particles was carried out by ammonia plasma treatment and subsequent adsorption of collagen. Alternatively, particles were modified by partial hydrolysis and subsequent immobilization of an amine-terminated dendrimer. Each surface modification step was characterized by X-ray photoelectron spectroscopy. The effectiveness of the surface modification procedures was demonstrated by in vitro cell culture experiments using sheep articular cartilage chondrocytes. A significant influence of both the particle shape and the surface chemistry on the proliferation rate was observed while the phenotype was maintained independent of the surface chemistry or particle shape. Chondrocytes cultured on PLGA microspheres were further assessed for cartilage tissue formation in collagen type I gels in nude mice. The tissue that were formed showed the appearance of a hyaline-like cartilage and the presence of the microspheres substantially reduced the degree of collagen gel contraction over 1-2 months.

A recombinant hydroxylated fragment of human type III collagen has been produced in Saccharomyces cerevisiae by coordinated coexpression of a collagen gene fragment together with both the alpha- and beta-subunit genes for prolyl-4-hydroxylase (EC 1.14.11.2). The collagen fragment consisted of 255 residues of the helical domain and the complete C-telopeptide and C-propeptide domains. It was inserted under the control of the ethanol-inducible ADH2 promoter in a multicopy, TRP1-selectable, yeast expression vector, YEpFlag1. The prolyihydroxylase subunit genes were cloned on either side of a bidirectional galactose-inducible promoter in a low-copy minichromosome yeast expression vector, pYEUra3, which is URA3 selectable. Coordinated expression of the three different gene products after cotransformation into S. cerevisiae was detected by immunoblotting. Amino acid analysis of an immunoreactive collagen fraction demonstrated the presence of hydroxyproline, while the presence of a triple-helical domain in the collagen fragment was demonstrated by its resistance to pepsin proteolysis.

We developed a panel of highly specific monoclonal antibodies (MAb) to either human or dog collagen Type VI. Various degrees of species crossreactivities were found with ELISA and immunohistology. Because of these differentiating species specificities, which allowed distinction between the original donor collagen and newly formed host collagen, the MAb proved to be valuable tools in examination of explanted samples of an ovine composite vascular prosthesis retrieved from a canine model. With an MAb that reacts with dog but not sheep collagen Type VI, newly synthesized pockets of collagen Type VI could readily be detected within the prosthesis as early as 3 months after implantation. These areas were associated with regions of cell infiltration, presumably derived from the host. This association was also apparent in the newly formed intimal region of the prosthesis where only host cells were found. Another of the MAb, which was positive against human but not sheep collagen, was also used to demonstrate marked deposition of host collagen Type VI in a retrieved human sample of the prosthesis. In this case the antibody was able to detect collagen on a formalin-fixed tissue, which would broaden the scope of its use in clinical and pathological situations. Use of these novel antibody probes provides a rapid marker for new tissue augmentation of implanted biological devices which would be an indicator of the long-term performance of a prosthesis.

Whilst monoclonal antibodies (Mab) to potyviruses have been generated, it has not been possible to produce molecules with high specificity or broad reactivity to defined conserved amino acid sequences. In the current study, peptide-mediated electrofusion was used to select for high efficiency antibody-secreting hybridomas after mice were immunized with highly immunogenic viral coat protein. Mice were immunized with coat protein from either one potyvirus (potato virus Y, PVY-D) or a mixture of five distinct potyviruses. Two well-defined peptides were used for selective electrofusions. Peptide-1 was selected from the highly specific N terminal region of PVY-D and peptide-2 from the highly conserved N terminal/core junction region of Johnson grass mosaic virus (JGMV). Conventional PEG-mediated fusions using mice immunized with these peptides did not result in hybridoma formation. On the other hand, electrofusions using biotin-streptavidin to bridge peptide-specific B cells to myeloma cells produced hybridomas secreting antibodies either highly specific to PVY-D or cross-reactive with all potyviruses, depending on the peptide used.

The antigenic response to the helical domain of collagens is normally very low, with the nature of the epitopes recognized by antibodies being dependent on the species of origin. Thus, in certain species, for example rabbit, sequential determinants on single alpha-chains are found, whereas in other species such as mouse, conformational epitopes are predominant. A variety of techniques for identification of epitopes, including rotary shadowing, examination of specific fragments and chemical modification reactions are discussed. The application of these techniques is illustrated using a range of monoclonal antibodies to interstitial collagens. These antibodies show that epitopes are distributed over the length of the collagen molecule.

Samples of an ovine collagen-polyester composite device suitable for peripheral revascularization, the Omniflow Vascular Prosthesis, have been retrieved for morphological and immunohistological analyses during and up to 4 years of implantation in a dog aortoiliac by-pass model. At the various sample times, the prosthesis explants were shown to retain their structural integrity, with no aneurysm formation and with little thrombus accumulation. Immunohistological studies on samples of the prosthesis showed that the original ovine collagen was still present after 4 years, and that there had been augmentation by the deposition of new, host-derived connective tissue.