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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.)

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.

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.