Interaction of preservation methods and radiation sterilization in human skin processing, with particular insight on the impact of the final water content and collagen disruption. Part I: process validation, water activity and collagen changes in tissues cryopreserved or processed using 50, 85 or 98% glycerol solutions.

Current regulatory requirements demand an in-depth understanding and validation of protocols used in tissue banking. The aim of this work was to characterize the quality of split thickness skin allografts cryopreserved or manufactured using highly concentrated solutions of glycerol (50, 85 or 98%), where tissue water activity (aw), histology and birefringence changes were chosen as parameters. Consistent aw outcomes validated the proposed processing protocols. While no significant changes in tissue quality were observed under bright-field microscopy or in collagen birefringence, in-process findings can be harnessed to fine-tune and optimize manufacturing outcomes in particular when further radiation sterilization is considered. Furthermore, exposing the tissues to 85% glycerol seems to derive the most efficient outcomes as far as aw and control of microbiological growth.

Non-animal collagens as new options for cosmetic formulation.

OBJECTIVE: To examine the potential of non-animal collagens as a new option for cosmetic applications. METHODS: Non-animal collagens from three species, Streptococcus pyogenes, Solibacter usitatus and Methylobacterium sp 4-46, have been expressed as recombinant proteins in Escherichia coli using a cold-shock, pCold, expression system. The proteins were purified using either metal affinity chromatography or a simple process based on precipitation and proteolytic digestion of impurities, which is suitable for large-scale production. Samples were examined using a range of analytical procedures. RESULTS: Analyses by gel electrophoresis and mass spectrometry were used to examine the purity and integrity of the products. Circular dichroism spectroscopy showed stabilities around 38°C, and calculated pI values were from 5.4 to 8.6. UV-visible light spectroscopy showed the clarity of collagen solutions. The collagens were soluble at low ionic strength between pH 5 and pH 8, but were less soluble under more acidic conditions. At lower pH, the insoluble material was well dispersed and did not form the fibrous associations and aggregates found with animal collagens. The materials were shown to be non-cytotoxic to cells in culture. CONCLUSIONS: These novel, non-animal collagens may be potential alternatives to animal collagens for inclusion in cosmetic formulations.

Impact of heparan sulfate chains and sulfur-mediated bonds on the mechanical properties of bovine lens capsule.

We assessed the importance of glycosaminoglycans and sulfur-mediated bonds for the mechanical properties of lens capsules by comparing the stress-strain responses from control and treated pairs of bovine source. No significant change in mechanical properties was observed upon reduction of disulfide bonds. However, removal of glycosaminoglycan chains resulted in a significantly stiffer lens capsule, whereas high concentrations of reducing agent, which is expected to reduce the recently reported sulfilimine bond of collagen IV, resulted in a significantly less stiff lens capsule. A comparison of the diffraction patterns of the control and strongly reduced lens capsules indicated structural rearrangements on a nanometer scale.

TLiSA1, a human T lineage-specific activation antigen involved in the differentiation of cytotoxic T lymphocytes and anomalous killer cells from their precursors.

The characteristics of a novel T lineage-specific activation antigen, termed TLiSA1, are described. The antigen was detected with a mouse monoclonal antibody, LeoA1, that was raised against activated human T cells generated in mixed lymphocyte culture (MLC). The antigen became strongly expressed on T cells 48-72 h after stimulation with phytohemagglutinin, and retained expression on MLC-activated T cells after 10 d of culture. The antigen was absent from a range of human T, B, myeloid, fibroblast, and tumour cell lines, but was present on the surface of the interleukin 2 (IL-2)-dependent gibbon cell line MLA-144. Analysis of the antigen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from activated human T cells demonstrated a broad band in the region of 70 kD, whereas precipitates obtained from MLA-144 revealed a single narrow band of 95 kD. The molecule was expressed with a maximum density of 66,000 copies per cell on the surface of MLC-activated T cell blasts, as assessed by Scatchard analysis. TLiSA1 was distinguished from the IL-2 receptor bound by the anti-Tac monoclonal antibody by demonstrating that the antigens did not comodulate or coprecipitate, and by constructing an IL-2-independent human T X T hybrid that expressed the TLiSA1 but not the Tac antigen. MLC with B lymphoblasts was used to generate cytotoxic T lymphocytes (CTL) specific for the stimulating cell, and anomalous killer (AK) cells able to kill melanoma target cells. The presence of LeoA1 or F(ab')2 fragments of the antibody from the beginning of coculture did not affect proliferation in these cultures, but did inhibit the induction of both CTL and AK cells from their precursors. This inhibition of differentiation by LeoA1 was confirmed under conditions of limiting dilution, where it was shown that the antibody reduced the frequency of CTL produced, and greatly (fourfold) reduced the frequency of AK cells generated from their precursors. We discuss the possibility that human CTL may express a differentiation factor receptor that is distinct from the receptor for IL-2.

Synergism between membrane gangliosides and Arg-Gly-Asp-directed glycoprotein receptors in attachment to matrix proteins by melanoma cells.

The identification of specific cell surface glycoprotein receptors for Arg-Gly-Asp-containing extracellular matrix proteins such as fibronectin has focused attention on the role of gangliosides in this process. Is their involvement dependent or independent of the protein receptors? In attachment assays with cells from a human melanoma cell line, titration experiments with an antibody (Mel 3) with specificity for the disialogangliosides GD2 and GD3, used together with a synthetic peptide containing the cell binding sequence Arg-Gly-Asp, show that their joint effect is synergistic. Both the Mel 3 antibody and the synthetic peptide individually cause rapid detachment of melanoma cells from fibronectin substrate but, when used together, much smaller concentrations of both are required to achieve the same effect. The Mel 3 antibody was not nonspecifically reducing receptor binding to the Arg-Gly-Asp sequence since, in binding assays with radiolabeled peptide performed with cells in suspension, very little peptide is bound by the melanoma cells under these conditions but addition of Mel 3, an antibody of IgM isotype, causes a two- to threefold increase in specific binding. The simplest interpretation of these data is that the Mel 3 antibody is causing sufficient clustering of membrane gangliosides in local areas and producing a favorably charged environment to facilitate peptide binding by specific glycoprotein receptors.

Composite mesh design for delivery of autologous mesenchymal stem cells influences mesh integration, exposure and biocompatibility in an ovine model of pelvic organ prolapse.

The widespread use of synthetic transvaginal polypropylene mesh for treating Pelvic Organ Prolapse (POP) has been curtailed due to serious adverse effects highlighted in 2008 and 2011 FDA warnings and subsequent legal action. We are developing new synthetic mesh to deliver endometrial mesenchymal stem cells (eMSC) to improve mesh biocompatibility and restore strength to prolapsed vaginal tissue. Here we evaluated knitted polyamide (PA) mesh in an ovine multiparous model using transvaginal implantation and matched for the degree of POP. Polyamide mesh dip-coated in gelatin and stabilised with 0.5% glutaraldehyde (PA/G) were used either alone or seeded with autologous ovine eMSC (eMSC/PA/G), which resulted in substantial mesh folding, poor tissue integration and 42% mesh exposure in the ovine model. In contrast, a two-step insertion protocol, whereby the uncoated PA mesh was inserted transvaginally followed by application of autologous eMSC in a gelatin hydrogel onto the mesh and crosslinked with blue light (PA + eMSC/G), integrated well with little folding and no mesh exposure. The autologous ovine eMSC survived 30 days in vivo but had no effect on mesh integration. The stiff PA/G constructs provoked greater myofibroblast and inflammatory responses in the vaginal wall, disrupted the muscularis layer and reduced elastin fibres compared to PA + eMSC/G constructs. This study identified the superiority of a two-step protocol for implanting synthetic mesh in cellular compatible composite constructs and simpler surgical application, providing additional translational value.

Use of biodegradable urethane-based adhesives to appose meniscal defect edges in an ovine model: a preliminary study.

OBJECTIVE: To evaluate the biological response to two urethane-based adhesives used to repair full thickness meniscal wounds created in the partially vascularised (red-white) zone. DESIGN: An ovine bilateral meniscal defect model was used to evaluate the initial biological response of the meniscal cartilage and synovium over a 1-month period. A 10-mm full-thickness defect was created in the medial meniscus of each femorotibial joint. The defects were either left untreated or repaired using the urethane-based adhesives. Synovial fluid, synovial membrane and the meniscal cartilages were retrieved at necropsy for cytological and histological assessment. RESULTS: The ovine model proved to be a suitable system for examining meniscal repair. Untreated defects showed no tissue apposition or cellular healing response, whereas all eight defects repaired with the two urethane-based adhesive formulations showed signs of repair and tissue regeneration with indications of cell infiltration and new collagen deposition in and around the polymer. No adverse cellular response to the adhesives was observed in the meniscal defect or in the synovial membrane and fluid. CONCLUSION: Trauma to the knee commonly results in tears to the meniscal cartilage, with the majority of these occurring in the partially vascularised (red-white) or non-vascularised (white) zones of the meniscus. Repair, and subsequent healing, of these tears is poor because of the reduced vascularity and limited surgical access. The present data indicate that an ovine model is a suitable system for examining meniscal repair, and that development of urethane-based adhesives offers a strategy that may be clinically effective for the treatment of these injuries.

Endometrial Mesenchymal Stem/Stromal Cells Modulate the Macrophage Response to Implanted Polyamide/Gelatin Composite Mesh in Immunocompromised and Immunocompetent Mice.

The immunomodulatory properties of human endometrial mesenchymal stem cells (eMSC) have not been well characterised. Initial studies showed that eMSC modulated the chronic inflammatory response to a non-degradable polyamide/gelatin mesh in a xenogeneic rat skin wound repair model, but the mechanism remains unclear. In this study, we investigated the immunomodulatory effect of eMSC on the macrophage response to polyamide/gelatin composite mesh in an abdominal subcutaneous wound repair model in C57BL6 immunocompetent and NSG (NOD-Scid-IL2Rgamma null ) immunocompromised mice to determine whether responses differed in the absence of an adaptive immune system and NK cells. mCherry lentivirus-labelled eMSC persisted longer in NSG mice, inducing longer term paracrine effects. Inclusion of eMSC in the mesh reduced inflammatory cytokine (Il-1ß, Tnfα) secretion, and in C57BL6 mice reduced CCR7+ M1 macrophages surrounding the mesh on day 3 and increased M2 macrophage marker mRNA (Arg1, Mrc1, Il10) expression at days 3 and 7. In NSG mice, these effects were delayed and only observed at days 7 and 30 in comparison with controls implanted with mesh alone. These results show that the differences in the immune status in the two animals directly affect the survival of xenogeneic eMSC which leads to differences in the short-term and long-term macrophage responses to implanted meshes.

Synthetic biodegradable microparticles for articular cartilage tissue engineering.

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.

Enhanced natural killer sensitivity with concomitant clonal selection for cells bearing homogeneously staining regions in the human melanoma cell line MeWo upon induction of differentiation with theophylline.

Culture of the human melanoma cell line MeWo in the presence of 1 mM theophylline was associated with an increase in susceptibility to natural killer (NK)-mediated cytolysis. The phenomenon was detected as early as 72 hours after initiation of theophylline treatment, reaching maximum values at 3-4 weeks and remaining stable for longer than 3 months of testing, provided the cells were maintained in the presence of theophylline. The alteration in target sensitivity was selective for NK-mediated cytolysis, since other mechanisms of cell-mediated cytolysis, including antibody-dependent cell-mediated cytotoxicity and monocyte-mediated and lectin-induced cytolysis, were comparable between untreated and treated cells. The enhanced susceptibility of theophylline-treated cultures to NK lysis, as compared to NK lysis susceptibility of untreated MeWo cells, was not significantly changed by pretreatment of effector lymphocytes with interferon. Evidence for differentiation in theophylline-treated cultures was obtained. In addition, however, cytofluorometric and karyologic analysis revealed the existence of two subpopulations of differing ploidy in the MeWo line. The hypodiploid, NK-sensitive subpopulation, bearing homogeneously staining regions on two chromosomes, could be selected by growth in theophylline. Therefore, selection of subpopulations in heterogeneous tumor cell lines by chemical inducers suggests an alternative and novel mechanism for enhancement of NK sensitivity.

Fluctuations in the expression of a glycolipid antigen associated with differentiation of melanoma cells monitored by a monoclonal antibody, Leo Mel 3.

A monoclonal antibody, Leo Mel 3, raised against a melanoma cell line (LiBr), binds to a carbohydrate determinant of cell surface gangliosides, the simplest of which is GD3. This monoclonal antibody was screened for by its capacity to block the recognition and lysis of the melanoma cells by cytotoxic T-lymphocytes with anomalous killer cell function, illustrating a novel approach for identifying monoclonal antibody to biologically relevant tumor-associated antigens. Leo Mel 3 reacted selectively with melanoma cells by indirect immunofluorescent and immunoperoxidase staining; it reacted with tissue from all primary and metastatic melanoma tested, and it bound to cells from all but one of six cultured melanoma cell lines. Leo Mel 3 did not react with a variety of carcinomas, lymphomas, leukemias, and other neuroectodermal tumors, nor with adult or fetal tissues, except fetal liver. Very weak staining of cutaneous basal melanocytes was noted in a minority of skin sections, and 50 to 80% of melanocytes in four of seven benign nevi showed weak to moderate reactivity. The antibody was relatively specific for human adherent melanoma cells, since it did not bind to the adherent murine B16 melanoma line nor to a nonadherent human melanoma cell line (PMC-22). Expression of the Leo Mel 3-defined antigen was unrelated to changes in cell cycle. When cells from an adherent melanoma cell line were detached and maintained briefly in suspension culture, the cells became markedly less reactive with Leo Mel 3 and, after readherence to plastic, they rapidly reexpressed higher levels of the ganglioside antigen; since Leo Mel 3 prevented attachment and growth of melanoma cells in vitro, a functional role for the ganglioside is suggested in cell adhesion and metastasis. Differentiation of melanoma cells with dimethyl sulfoxide, retinoic acid, and theophylline resulted in a marked and selective increase in the amount of Leo Mel 3-defined antigen, together with an increase in the target cell binding ability of these cells, assessed by cold target competition assays using anomalous killer cells.

The effect of sequence variations and structure on the cytolytic activity of melittin peptides.

The importance of various amino acid residues in melittin for cytolytic function against mammalian cells was assessed by use of a monoclonal antibody to the C-terminal region, synthesis of peptide analogues and chemical modification of specific residues. A monoclonal anti-melittin antibody directed to the basic C-terminal region inhibited cytolytic activity. Consistent with this, deletion of one of the two Lys Arg sequences at the C terminal end of the peptide reduced cytolysis 8-fold, although significant activity was still present. A similar reduction in activity was also found with a synthetic analogue which had the reverse sequence to melittin. In contrast, when the last 6 residues of the C-terminal region were transferred to the N-terminus, a peptide with markedly reduced activity was obtained. Chemical modification of lysine and arginine residues of melittin indicated that lysine was only minimally important for functional activity compared with arginine which was essential. In particular, our results demonstrate that substitution of serine for lysine 7 has no significant effect on the activity of the peptide and suggest that this residue is important only in maintaining the amphipathic helix of the peptide.

Temporal changes in the biomechanical properties of endometrial mesenchymal stem cell seeded scaffolds in a rat model.

Use of synthetic clinical meshes in pelvic organ prolapse (POP) repair can lead to poor mechanical compliance in vivo, as a result of a foreign body reaction leading to excessive scar tissue formation. Seeding mesh with mesenchymal stem cells (MSCs) prior to implantation may reduce the foreign body reaction and lead to improved biomechanical properties of the mesh-tissue complex. This study investigates the influence of seeding human endometrial mesenchymal stem cells (eMSCs) on novel gelatin-coated polyamide scaffolds, to identify differences in scaffold/tissue biomechanical properties and new tissue growth following up to 90 days' implantation, in a subcutaneous rat model of wound repair. Scaffolds were subcutaneously implanted, either with or without eMSCs, in immunocompromised rats and following 7, 30, 60 and 90 days were removed and assessed for their biomechanical properties using uniaxial tensile testing. Following 7, 30 and 90 days' implantation scaffolds were assessed for tissue ingrowth and organization using histological staining and scanning electron microscopy. The eMSCs were associated with altered collagen growth and organization around the mesh filaments of the scaffold, affecting the physiologically relevant tensile properties of the scaffold-tissue complex, in the toe region of the load-elongation curve. Scaffolds seeded with eMSCs were significantly less stiff on initial stretching than scaffolds implanted without eMSCs. Collagen growth and organization were enhanced in the long-term in eMSC-seeded scaffolds, with improved fascicle formation and crimp configuration. Results suggest that neo-tissue formation and remodelling may be enhanced through seeding scaffolds with eMSCs.

The use of an electroporation apparatus for the production of murine hybridomas.

Antigen-directed electrofusion was carried out using biotin-streptavidin to bridge antigen-specific splenocytes to myeloma cells. Electrofusion was performed using a commercial electroporation apparatus. Electrofusion conditions were optimized by measuring the survival of myeloma cells after a range of electrical pulse conditions. The procedure was tested with antigens of high and low immunogenicity. The yields of hybridomas secreting antibodies specific for both antigens were considerably increased by the use of the electrofusion procedure.

The use of peptide-mediated electrofusion to select monoclonal antibodies directed against specific and homologous regions of the potyvirus coat protein.

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.

Monoclonal antibodies to type V collagen for immunohistological examination of new tissue deposition associated with biomaterial implants.

We developed a panel of highly specific monoclonal antibodies (MAb) against dog Type V collagen. Each antibody showed differential reactivities towards Type V collagen from other species. All the antibodies were highly reactive in conventional ELISA, as well as with electroblots of collagen after polyacrylamide gel electrophoresis using non-denaturing conditions. The MAb were shown to be suitable for the immunohistological detection of Type V collagen in tissue sections, although this normally required pre-treatment of sections with 50 mM acetic acid. In particular, the antibodies were shown to be useful for examining samples of a collagen-based biomaterial, a vascular prosthesis, after explant from evaluation in an animal model. This showed that Type V collagen was most prominent in regions of new tissue formation within the neointima, close to the inner surface of the prosthesis. The broad spectrum of differential reactivities allows the antibodies to be used for a wide range of experimental models. These MAb therefore provide a novel approach for the evaluation of biomaterial performance, particularly for collagen-based implants.

Monoclonal antibodies to type VI collagen demonstrate new tissue augmentation of a collagen-based biomaterial implant.

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.

A novel host-protective antigen from Trichostrongylus colubriformis.

The detergent-soluble fraction from Trichostrongylus colubriformis third-stage larvae contained a simple set of antigens, one of which (molecular weight 41,000) induced 43-51% protection in guinea pigs following immunization. Isolation and partial amino acid sequence analysis of this protective antigen showed it was parasite tropomyosin.

Production of recombinant hydroxylated human type III collagen fragment in Saccharomyces cerevisiae.

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.

Selection of polyclonal antibodies to the N terminus of bean yellow mosaic potyvirus coat protein by induction of tolerance with monoclonal antibody.

Polyclonal antisera to potyviruses contain virus-specific as well as cross-reacting antibodies. The virus-specific antibodies are directed to the surface-located, N-terminal region of the coat protein, whereas cross-reacting antibodies are produced against multiple epitopes within the core region of the coat protein (minus N and C termini), which displays extensive sequence homology among distinct potyviruses. In the present study, immunological tolerance was induced in mice against the cross-reactive central core region of bean yellow mosaic virus (BYMV) using a rat monoclonal antibody (mAb) to the L3T4 molecule (the mouse equivalent of CD4). Generation of specific antisera reactive to the N terminus of BYMV was attained in tolerized mice by secondary immunization with whole viral coat protein from BYMV. This approach appears to be ideally suited to potyviruses where a two-third of the coat protein molecule contains immunogenic epitopes which can result in cross-reacting antibodies.