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Bacterial systems, especially using Escherichia coli, provide a useful system for recombinant production of collagen and collagen-like proteins. These systems provide high yields of products but have lacked the ability to introduce hydroxyproline when it is needed for stability and function. Recent progress, in the last 30 months, of animal-based recombinant proteins has focussed on further development of collagen peptide materials and their applications. The proposal of new approaches for incorporation of hydroxyproline may enhance the utility of these products and enable production of new materials. Bacterial, collagen-like constructs have also continued to be developed for use in understanding collagen structure and functions, while progress has also been made on their application as new biomedical materials.

Rare perivascular mesenchymal stromal cells (MSCs) with therapeutic properties have been identified in many tissues. Their rarity necessitates extensive in vitro expansion, resulting in spontaneous differentiation, cellular senescence and apoptosis, producing therapeutic products with variable quality and decreased potency. We previously demonstrated that A83-01, a transforming growth factor beta (TGF-&beta, ) receptor inhibitor, maintained clonogenicity and promoted the potency of culture-expanded premenopausal endometrial MSCs using functional assays and whole-transcriptome sequencing. Here, we compared the effects of A83-01 on MSCs derived from postmenopausal endometrium, menstrual blood, placenta decidua-basalis, bone marrow and adipose tissue. Sushi-domain-containing-2 (SUSD2+) and CD34+CD31&minus, CD45&minus, MSCs were isolated. Expanded MSCs were cultured with or without A83-01 for 7 days and assessed for MSC properties. SUSD2 identified perivascular cells in the placental decidua-basalis, and their maternal origin was validated. A83-01 promoted MSC proliferation from all sources except bone marrow and only increased SUSD2 expression and prevented apoptosis in MSCs from endometrial-derived tissues. A83-01 only improved the cloning efficiency of postmenopausal endometrial MSCs (eMSCs), and expanded adipose tissue MSCs (adMSCs) underwent significant senescence, which was mitigated by A83-01. MSCs derived from bone marrow (bmMSCs) were highly apoptotic, but A83-01 was without effect. A83-01 maintained the function and phenotype in MSCs cultured from endometrial, but not other, tissues. Our results also demonstrated that cellular SUSD2 expression directly correlates with the functional phenotype.

Endometrial mesenchymal stem/stromal cells (eMSCs) exhibit excellent regenerative capacity in the endometrial lining of the uterus following menstruation and high proliferative capacity in vitro. Bioprinting eMSCs onto a mesh could be a potential therapy for Pelvic Organ Prolapse (POP). This study reports a novel strategy targeting vaginal repair using bioprinting of eMSCs encapsulated in a hydrogel and3D melt electrospun mesh to generate a tissue engineering construct. Following a CAD, 3D printed poly e-caprolactone (PCL) meshes were fabricated using melt electrospinning (MES) at different temperatures using a GMP clinical grade GESIM Bioscaffolder. Electron and atomic force microscopies revealed that MES meshes fabricated at 100°C and with a speed 20 mm/sec had the largest open pore diameter (47.2 ± 11.4 μm) and the lowest strand thickness (121.4 ± 46 μm) that promoted optimal eMSC attachment. An Aloe Vera-Sodium Alginate (AV-ALG) composite based hydrogel was optimised to a 1:1 mixture (1%AV-1%ALG) and eMSCs, purified from human endometrial biopsies, were then bioprinted in this hydrogel onto the MES printed meshes. Acute in vivo foreign body response assessment in NSG mice revealed that eMSC printed on MES constructs promoted tissue integration, eMSC retention and an anti-inflammatory M2 macrophage phenotype characterised by F4/80+CD206+ colocalization. Our results address an unmet medical need highlighting the potential of alternative 3D bioprinted eMSC-MES meshes as a novel approach to overcome the current challenges with non-degradable knitted meshes in POP treatment.

Recombinant bacterial collagens provide a new opportunity for safe biomedical materials. They are readily expressed in Escherichia coli in good yield and can be readily purified by simple approaches. However, recombinant proteins are limited in that direct secondary modification during expression is generally not easily achieved. Thus, inclusion of unusual amino acids, cyclic peptides, sugars, lipids, and other complex functions generally needs to be achieved chemically after synthesis and extraction. In the present study, we have illustrated that bacterial collagens that have had their sequences modified to include cysteine residue(s), which are not normally present in bacterial collagen-like sequences, enable a range of specific chemical modification reactions to be produced. Various model reactions were shown to be effective for modifying the collagens. The ability to include alkyne (or azide) functions allows the extensive range of substitutions that are available via "click" chemistry to be accessed. When bifunctional reagents were used, some crosslinking occurred to give higher molecular weight polymeric proteins, but gels were not formed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 806-813, 2017.

A range of non-animal collagens has been described, derived from bacterial species, which form stable triple-helical structures without the need for secondary modification to include hydroxyproline in the sequence. The non-animal collagens studied to date are typically smaller than animal interstitial collagens, around one quarter the length and do not pack into large fibrillar aggregates like those that are formed by the major animal interstitial collagens. A consequence of this for biomedical products is that fabricated items, such as collagen sponges, are not as mechanically and dimensionally stable as those of animal collagens. In the present study, we examined the production of larger, polymeric forms of non-animal collagens through introduction of tyrosine and cysteine residues that can form selective crosslinks through oxidation. These modifications allow the formation of larger aggregates of the non-animal collagens. When Tyr residues were incorporated, gels were obtained. And with Cys soluble aggregates were formed. These materials can be formed into sponges that are more stable than those formed without these modifications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2369-2376, 2016.

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-1β, 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.

Regenerative medicine strategies for restoring articular cartilage face significant challenges to recreate the complex and dynamic biochemical and biomechanical functions of native tissues. As an approach to recapitulate the complexity of the extracellular matrix, collagen-mimetic proteins offer a modular template to incorporate bioactive and biodegradable moieties into a single construct. We modified a Streptococcal collagen-like 2 protein with hyaluronic acid (HA) or chondroitin sulfate (CS)-binding peptides and then cross-linked with a matrix metalloproteinase 7 (MMP7)-sensitive peptide to form biodegradable hydrogels. Human mesenchymal stem cells (hMSCs) encapsulated in these hydrogels exhibited improved viability and significantly enhanced chondrogenic differentiation compared to controls that were not functionalized with glycosaminoglycan-binding peptides. Hydrogels functionalized with CS-binding peptides also led to significantly higher MMP7 gene expression and activity while the HA-binding peptides significantly increased chondrogenic differentiation of the hMSCs. Our results highlight the potential of this novel biomaterial to modulate cell-mediated processes and create functional tissue engineered constructs for regenerative medicine applications.

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.

Cuvierian tubules are expelled as a defence mechanism against predators by various species within the family Holothuridae. When the tubules are expelled, they become sticky almost immediately and ensnare the predator. The mechanism of this rapid adhesion is not clear, but proteins on the surface of the expelled tubules are widely believed to be involved. This study has examined such proteins from Holothuria dofleinii, sourced from adhesive prints left on glass after the removal of adhered tubules. Gel electrophoresis showed that seven strongly staining protein bands were consistently present in all samples, with molecular masses ranging from 89 to 17 kDa. N-terminal sequence data was obtained from two bands, while others seemed blocked. Tandem mass spectrometry-based sequencing of tryptic peptides derived from individual protein bands indicated that the proteins were unlikely to be homopolymers. PCR primers designed using the peptide sequences enabled us to amplify, clone and sequence cDNA segments relating to four gel bands; for each, the predicted translation product contained other peptide sequences observed for that band that had not been used in primer design. Database searches using the peptide and cDNA-encoded sequences suggest that two of the seven proteins are novel and one is a C-type lectin, while-surprisingly-at least three of the other four are closely related to enzymes associated with the pentose phosphate cycle and glycolysis. We discuss precedents in which lectins and metabolic enzymes are involved in attachment and adhesion phenomena.

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.

The aim of this study was to develop a 2-N, 6-O-sulfated chitosan (26SCS) modified electrospun fibrous PCL scaffold for bone morphogenetic protein-2 (BMP-2) delivery to improve osteoinduction. The PCL scaffold was modified by an aminolysis reaction using ethylenediamine (ED) and 26SCS was immobilized via electrostatic interactions (PCL-N-S). Scaffolds were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. In vitro BMP-2 adsorption and release kinetics indicated that modified PCL-N-S scaffolds showed higher levels of binding of BMP-2 (about 30-100 times), moderative burst release (about one third), and prolonged releasing time compared to the unmodified PCL scaffold. The bioactivity of released BMP-2 determined by alkaline phosphatase (ALP) activity assay was maintained and improved 8-12 times with increasing concentration of immobilized 26SCS on the scaffolds. In vitro studies demonstrated that bone marrow mesenchymal stem cells (BMSCs) attached more readily to the PCL-N-S scaffolds with increased spreading. In conclusion, 26SCS modified PCL scaffolds can be a potent system for the sustained and bioactive delivery of BMP-2.

Pelvic Organ Prolapse (POP) is the herniation of pelvic organs into the vagina. Despite broad acceptance of mesh use in POP surgical repair, the complication rate is unacceptable. We hypothesized that collagen-containing polypropylene (PP) mesh types could modulate mesh-tissue integration and reduce long-term inflammation, thereby reducing mesh-associated complications. This study compared the long-term tissue response to an unmodified PP mesh and two collagen containing meshes in an ovine model which has similar pelvic anatomy and vaginal size to human. Three commercially available macroporous PP meshes, uncoated PP mesh (Avaulta Solo) (PP), the same textile PP mesh layered with a sheet of cross-linked porcine acellular matrix (Avaulta Plus) (PP-ACM) and a different yet also macroporous PP (Sofradim) mesh coated with solubilized atelocollagen (Ugytex) (PP-sCOL) were implanted in the ovine vagina and tissue explanted after 60 and 180days. The macrophage phenotype and response to implanted meshes, and vascularity were quantified by immunostaining and morphometry. We quantified changes in extracellular matrix composition biochemically and collagen organisation and percentage area around the interface of the mesh implants by Sirius Red birefringence and morphometry. PP-ACM induced a more sustained inflammatory response, indicated by similar CD45(+) leukocytes but reduced CD163(+) M2 macrophages at 60days (P0.05). PP-sCOL increased Von Willebrand Factor (vWF)-immunoreactive vessel profiles after 60days. At the micro-molecular level, collagen birefringence quantification revealed significantly fewer mature collagen fibrils (red, thick fibrils) at the mesh-tissue interface than control tissue for all mesh types (P0.001) but still significantly greater than the proportion of immature (green thin fibrils) at 60days (P0.05). The proportion of mature collagen fibrils increased with time around the mesh filaments, particularly those containing collagen. The total collagen percent area at the mesh interface was greatest around the PP-ACM mesh at 60days (P0.05). By 180days the total mature and immature collagen fibres at the interface of the mesh filaments resembled that of native tissue. In particular, these results suggest that both meshes containing collagen evoke different types of tissue responses at different times during the healing response yet both ultimately lead to physiological tissue formation approaching that of normal tissue.Pelvic organ prolapse (POP) is the descent of the pelvic organs to the vagina. POP affects more than 25% of all women and the lifetime risk of undergoing POP surgery is 19%. Although synthetic polypropylene (PP) meshes have improved the outcome of the surgical treatment for POP, there was an unacceptable rate of adverse events including mesh exposure and contracture. It is hypothesized that coating the PP meshes with collagen would provide a protective effect by preventing severe mesh adhesions to the wound, resulting in a better controlled initial inflammatory response, and diminished risk of exposure. In this study we assessed the effect of two collagen-containing PP meshes on the long-term vaginal tissue response using new techniques to quantify these tissue responses.

Polyetheretherketone (PEEK) is a high performance polymer, with high melting temperature and high resistance to wear. PEEK biomedical devices are typically manufactured to produce nonflexible structures. In this study, we fabricated flexible PEEK scaffolds from multifilament and monofilament yarns, using weaving technologies. Scaffolds were compared for structural and mechanical properties, and assessed for in vitro biological response to L929 mouse fibroblast cells. PEEK scaffolds were found to support fibroblast cell attachment and proliferation, with similar cell numbers to a polyethylene terephthalate scaffold. The large pores (261–280 μm) of the monofilament scaffold prevented pore coverage by cells, confining cells to filaments, whereas the smaller pores (81–100 μm) of the multifilament scaffold permitted partial pore coverage. Poor cell adhesion, due to large filament curvature angles, created a checkered pattern on the woven surface, a previously undocumented phenomenon. The multifilament scaffold was found to be lighter, thinner, and less porous, with better mechanical properties (load at break: 657 N, elastic recovery: 66%, burst strength: 492 N) than the monofilament scaffold (load at break: 534 N, elastic recovery: 30%, burst strength: 401 N). Results indicate that flexible PEEK woven structures may find application as tissue engineering scaffolds, particularly for engineering soft tissues. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3326–3331, 2012.

Keratins extracted from various "hard tissues" such as wool, hair, and nails are increasingly being investigated as a source of abundant, biocompatible materials. In this study we explored a recent photochemical method to crosslink solubilized wool keratoses, with the aim of producing a mechanically favorable biomaterial. Wool proteins were isolated by oxidizing the disulfides and extracting the resulting soluble keratoses. The α- and γ-keratose fractions were analyzed by liquid chromatography-mass spectrometry to identify their constituent proteins. Hydrogels were produced by covalent crosslinking of the α-keratoses via a photo-oxidative process catalyzed by blue light, a ruthenium complex, and persulfate. The presence of dityrosine crosslinks was demonstrated by high performance liquid chromatography and mass spectrometry analyses. The crosslinked α-keratose material had moderate tensile strength and elasticity, and high adhesive strength. The material displayed modest shrinking after crosslinking, however the shrinking could be prevented by crosslinking in the presence of 2.5% glycerol, resulting in gels that did not shrink or swell. Small solutes such as Tris and glycerol influenced the crosslink density and elastic modulus of the crosslinked material. The α-keratose was able to support adhesion and growth of NIH/3T3 fibroblasts in vitro. The fabrication of mechanically stable keratin biomaterials by this facile photo-crosslinking method may be useful for various tissue engineering applications.

Molecules that mimic the cytokine thrombopoietin that act by an atypical mechanism of binding to a receptor transmembrane (TM) domain are widely understood to require zinc for their biological activity. We investigated potent thrombopoietin mimetics from three chemical classes including the recently registered drug Eltrombopag, which operate via this novel mechanism, to determine whether zinc is essential for inducing cell proliferation. Using addition of zinc and a potent metal chelator, we show that the existing paradigm is incorrect and the compounds exhibit excellent thrombopoietin-mimetic activity even in the presence of high concentrations of EDTA. The implications of these findings for the mechanism of action are discussed.

In this study we have prepared a tubular knitted scaffold from a 9 ply multiwalled carbon nanotube (MWCNT) yarn and a composite scaffold, formed by electrospinning poly(lactic-co-glycolic acid) (PLGA) nanofibres onto the knitted scaffold. Both structures were assessed for in vitro biocompatibility with NR6 mouse fibroblast cells for up to 22 days and their suitability as tissue engineering scaffolds considered. The MWCNT yarn was found to support cell growth throughout the culture period, with fibroblasts attaching to, and proliferating on, the yarn surface. The knitted tubular scaffold contained large pores that inhibited cell spanning, leading to the formation of cell clusters on the yarn, and an uneven cell distribution on the scaffold surface. The smaller pores, created through electrospinning, were found to promote cell spanning, leading to a uniform distribution of cells on the composite scaffold surface. Evaluation of the electrical and mechanical properties of the knitted scaffold determined resistance levels of 0.9 kOmega/cm, with a breaking load and extension to break approaching 0.7N and 8%, respectively. The PLGA/MWCNT composite scaffold presented in this work not only supports cell growth, but also has the potential to utilize the full range of electrical and mechanical properties that carbon nanotubes have to offer.

SynopsisObjective 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.

When provoked, Notaden bennetti frogs secrete an exudate which rapidly forms a tacky elastic solid ("frog glue"). This protein-based material acts as a promiscuous pressure-sensitive adhesive that functions even in wet conditions. We conducted macroscopic tests in air to assess the tensile strength of moist glue (up to 78 +/- 8 kPa) and the shear strength of dry glue (1.7 +/- 0.3 MPa). We also performed nanomechanical measurements in water to determine the adhesion (1.9-7.2 nN or greater), resilience (43-56%), and elastic modulus (170-1035 kPa) of solid glue collected in different ways. Dry glue contains little carbohydrate and consists mainly of protein. The protein complement is rich in Gly (15.8 mol %), Pro (8.8 mol %), and Glu/Gln (14.1 mol %); it also contains some 4-hydroxyproline (4.6 mol %) but no 5-hydroxylysine or 3,4-dihydroxyphenylalanine (L-Dopa). Denaturing gel electrophoresis of the glue reveals a characteristic pattern of proteins spanning 13-400 kDa. The largest protein (Nb-1R, apparent molecular mass 350-500 kDa) is also the most abundant, and this protein appears to be the key structural component. The solid glue can be dissolved in dilute acids; raising the ionic strength causes the glue components to self-assemble spontaneously into a solid which resembles the starting material. We describe scattering studies on dissolved and solid glue and provide microscopy images of glue surfaces and sections, revealing a porous interior that is consistent with the high water content (85-90 wt %) of moist glue. In addition to compositional similarities with other biological adhesives and well-known elastomeric proteins, the circular dichroism spectrum of dissolved glue is almost identical to that for soluble elastin and electron and scanning probe microscopy images invite comparison with silk fibroins. Covalent cross-linking does not seem to be necessary for the glue to set.

A novel bone graft substitute comprising a porous, collagenous scaffold was biomimetically coated with hydroxyapatite using a simulated body fluid solution chemistry method. The scaffold had a porosity of approximately 85%, with pore sizes between 30 μm and 100 μm. Glutaraldehyde vapor was used to stabilize the collagenous scaffold, giving a significantly increased thermal stability over an unstabilized scaffold, as shown by differential scanning calorimetry. A thin layer (

Synthetic peptides were constructed with the sequence of the first 20 residues of melittin and terminating with a range of different amino acid amides. These were found to have haemolytic and cytolytic activity similar to that of melittin, provided that certain charge constraints were observed. The nature of the 21st residue was not critical except when the residue introduced a negative charge. The presence of at least two positive charges in the molecule was found to be essential for activity. One of these charges could be the amino-terminal amine. Peptides could be inactivated by the addition of a non-acidic presequence which was acetylated at the N-terminus. Introducing a protease cleavable sequence into an N-terminal extension of the peptides produced analogues with low haemolytic activity that could be activated by proteolytic action. A peptide with extra positive charges introduced on the hydrophilic face of the helix possessed a haemolytic activity that was greater than that of melittin.

D-Pro14 melittin was synthesized to investigate the effect of increasing the angle of the bend in the hinge region between the helical segments of the molecule. Structural analysis by nuclear magnetic resonance indicated that, in methanol, the molecule consisted of two helices separated at Pro14, as in melittin. However, the two helices in D-Pro14 melittin were laterally displaced relative to each other by approximately 7 A, and in addition, there was a small rotation of the carboxyl-terminal helix relative to the amino-terminal helix around the long axis of the molecule. The peptide had less than 5% of the cytolytic activity of melittin. Modification of Arg22 with the 2,2,5,7,8-pentamethyl-chroman-6-sulphonyl (pmc) group restored hemolytic activity to close to that of unmodified melittin. Replacement of Arg22 with Phe was less effective in restoring hemolytic activity. Electron-paramagnetic resonance studies suggest that there is a positive correlation between hemolytic activity of the peptides and interaction with phospholipid bilayers.

The collagen like domain Scl2 from Streptococcus pyogenes has been proposed as a potential biomedical material. It is non-cytotoxic and non-immunogenic and can be prepared in good yield in fermentation. The Scl2 collagen domain is about a quarter of the length, 234 residues, of the main collagen type, mammalian type I collagen (1014 residues) that is currently used in biomedical devices. In the present study we have made constructs comprising 1 to 4 copies of the Scl2 collagen domain, plus these same constructs with a CysCys sequence at the C-terminal, analogous to that found in mammalian type III collagens. The yields of these constructs were examined from 2 L fermentation studies. The yields of both series declined with increasing size. Circular dichroism showed that the addition of further collagen domains did not lead to a change in the melting temperature compared to the monomer domain. Addition of the CysCys sequence led to a small additional stabilization of about 2-3°C for the monomer construct when the folding (V) domain was present.

OBJECTIVE: There are increasing numbers of reports describing human vaginal tissue composition in women with and without pelvic organ prolapse with conflicting results. The aim of this study was to compare ovine and human posterior vaginal tissue in terms of histological and biochemical tissue composition and to assess passive biomechanical properties of ovine vagina to further characterise this animal model for pelvic organ prolapse research. STUDY DESIGN: Vaginal tissue was collected from ovariectomised sheep (n = 6) and from postmenopausal women (n = 7) from the proximal, middle and distal thirds. Tissue histology was analyzed using Masson's Trichrome staining; total collagen was quantified by hydroxyproline assays, collagen III/I+III ratios by delayed reduction SDS PAGE, glycosaminoglycans by dimethylmethylene blue assay, and elastic tissue associated proteins (ETAP) by amino acid analysis. Young's modulus, maximum stress/strain, and permanent strain following cyclic loading were determined in ovine vagina. RESULTS: Both sheep and human vaginal tissue showed comparable tissue composition. Ovine vaginal tissue showed significantly higher total collagen and glycosaminoglycan values (p

Mammalian collagen has been widely used as a biomedical material. Nevertheless, there are still concerns about the variability between preparations, particularly with the possibility that the products may transmit animal-based diseases. Many groups have examined the possible application of bioengineered mammalian collagens. However, translating laboratory studies into large-scale manufacturing has often proved difficult, although certain yeast and plant systems seem effective. Production of full-length mammalian collagens, with the required secondary modification to give proline hydroxylation, has proved difficult in E. coli. However, recently, a new group of collagens, which have the characteristic triple helical structure of collagen, has been identified in bacteria. These proteins are stable without the need for hydroxyproline and are able to be produced and purified from E. coli in high yield. Initial studies indicate that they would be suitable for biomedical applications.

ObjectiveTo undertake a comprehensive analysis of the biochemical tissue composition and passive biomechanical properties of ovine vagina and relate this to the histo-architecture at different reproductive stages as part of the establishment of a large preclinical animal model for evaluating regenerative medicine approaches for surgical treatment of pelvic organ prolapse.MethodsVaginal tissue was collected from virgin (n = 3), parous (n = 6) and pregnant sheep (n = 6; mean gestation; 132 d; term = 145 d). Tissue histology was analyzed using H+E and Masson's Trichrome staining. Biochemical analysis of the extracellular matrix proteins used a hydroxyproline assay to quantify total collagen, SDS PAGE to measure collagen III/I+III ratios, dimethylmethylene blue to quantify glycosaminoglycans and amino acid analysis to quantify elastin. Uniaxial tensiometry was used to determine the Young's modulus, maximum stress and strain, and permanent strain following cyclic loading.ResultsVaginal tissue of virgin sheep had the lowest total collagen content and permanent strain. Parous tissue had the highest total collagen and lowest elastin content with concomitant high maximum stress. In contrast, pregnant sheep had the highest elastin and lowest collagen contents, and thickest smooth muscle layer, which was associated with low maximum stress and poor dimensional recovery following repetitive loading.ConclusionPregnant ovine vagina was the most extensible, but the weakest tissue, whereas parous and virgin tissues were strong and elastic. Pregnancy had the greatest impact on tissue composition and biomechanical properties, compatible with significant tissue remodeling as demonstrated in other species. Biochemical changes in tissue protein composition coincide with these altered biomechanical properties.

Recently, a different class of collagen-like molecules has been identified in numerous bacteria. Initial studies have shown that these collagens are readily produced in Escherichia coli and they have been isolated and purified by various small-scale chromatography approaches. These collagens are non-cytotoxic, are non-immunogenic, and can be produced in much higher yields than mammalian collagens, making them potential new collagens for biomedical materials. One of the major drawbacks with large-scale fermentation of collagens has been appropriate scalable down-stream processing technologies. Like other collagens, the triple helical domains of bacterial collagens are particularly resistant to proteolysis. The present study describes the development and optimization of a simple, scalable procedure using a combination of acid precipitation of the E. coli host proteins, followed by proteolysis of residual host proteins to produce purified collagens in large scale without the use of chromatographic methods.

The lipid binding behaviour of the antimicrobial peptides magainin 1, melittin and the C-terminally truncated analogue of melittin (21Q) was studied with a hybrid bilayer membrane system using surface plasmon resonance. In particular, the hydrophobic association chip was used which is composed of long chain alkanethiol molecules upon which liposomes adsorb spontaneously to create a hybrid bilayer membrane surface. Multiple sets of sensorgrams with different peptide concentrations were generated. Linearisation analysis and curve fitting using numerical integration analysis were performed to derive estimates for the association (ka) and dissociation (kd) rate constants. The results demonstrated that magainin 1 preferentially interacted with negatively charged dimyristoyl-L-K-phosphatidyl-DL-glycerol (DMPG), while melittin interacted with both zwitterionic dimyristoyl-L-K-phosphatidylcholine and anionic DMPG. In contrast, the C-terminally truncated melittin analogue, 21Q, exhibited lower binding affinity for both lipids, showing that the positively charged Cterminus of melittin greatly influences its membrane binding properties. Furthermore the results also demonstrated that these antimicrobial peptides bind to the lipids initially via electrostatic interactions which then enhances the subsequent hydrophobic binding. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high K-helicity was associated with high binding affinity. Overall, the results demonstrated that biosensor technology provides a new experimental approach to the study of peptide-membrane interactions through the rapid determination of the binding affinity of bioactive peptides for phospholipids. fl 2001 Elsevier Science B.V. All rights reserved.

Mandrel-grown, mesh-reinforced vascular pros- theses require adequate tissue coverage of the mesh for ef- fective clinical function, particularly in low blood flow situ- ations. Development of the ovine collagen-based Omni- flow™ vascular prosthesis has shown that the extent of this tissue cover is dependent on the interactions of the mandrel and the mesh with the sheep host. In the present study, the effects of chemical changes to the mesh have been examined. These data indicate that certain treatments of the mesh, par- ticularly collagen or heparin, lead to increased tissue cover- age while the number of sheep cells present and the ultra- structure of the resulting vessel remain unchanged. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 47, 309-315, 1999.

The interaction of melittin and a truncated analogue of melittin with an immobilised phosphatidylcholine monolayer has been studied using dynamic elution techniques. The melittin analogue (21Q analogue) had five amino acids omitted from the C-terminal region of melittin. The influence of temperature and methanol concentration on the binding affinity of the two peptides was determined and compared to the binding behaviour of two control moleculesN-acetyltryptophanamide and diphenylalanine. Both peptides exhibited non-linear dependence of affinity on % methanol at different temperatures, whileN-acetyltryptophanamide and diphenylalanine exhibited linear behaviour. In addition, both melittin and the 21Q analogue exhibited significant band broadening under a range of experimental conditions, which was not evident forN-acetyltryptophanamide and diphenylalanine. As melittin is known to adopt a significant degree of α-helical conformation in the presence of lipids, the results suggest that melittin and the 21Q analogue adopt different conformations and orientations upon binding to the immobilised phosphatidylcholine surface. Overall, the results of this study demonstrate that the immobilised lipid monolayer provides a powerful system to rapidly assess the affinity of peptides for different lipid surfaces.

Nine fatty acid-peptide hybrid molecules were constructed using the general formula CH3(CH2)nCO-Phe Asp Cys-amide and tested for their ability to inhibit cell lysis induced by the membrane-active peptide melittin. All of these molecules, where n = 4-14, inhibited the action of melittin to some extent, but the longer carbon chains were most effective. Several potential inhibitors were also constructed with conservative substitutions in the peptide portion of the molecule. All were effective to varying degrees. We concluded that in the hexapeptide inhibitor published by Blondelle et al. (1993), the role of the first three residues is only to provide hydrophobic interaction with the melittin and has no particular amino acid sequence specificity. Some of these inhibitors were found to inhibit the lytic activity of a melittin analogue which had only superficial sequence similarity to melittin and also a truncated form of melittin, indicating the generality of the action of the inhibitors.

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.

A library of eight conformation-dependent monoclonal antibodies that react with distinct epitopes on native human type III collagen has been examined for the ability of these antibodies to inhibit platelet aggregation induced by this collagen. Six of these antibodies had no effects; one, 1E7-D7/Col3, delayed the onset and slowed the rate of platelet aggregation, while another, 2G8-B1/Col3, completely inhibited aggregation. In order to identify the epitope recognized by this inhibitory antibody, a series of peptides that could fold to form triple-helical fragments was examined. Each peptide included six Gly-Xaa-Yaa triplets from the human type III collagen sequence, where Xaa and Yaa represent the particular amino acids in the sequence, and a C-terminal (Gly-Pro-Hyp)4 sequence to enhance triple-helical stability. Using these peptides we have identified the epitope as a nine-amino-acid sequence, GLAGAOGLR (where O is the one-letter code for 4-hydroxyproline), starting at position 520 in the human type III collagen helical domain. This sequence is proximal to the site proposed for the interaction of type III collagen with α2β1-integrin of platelets.

A synthetic peptide with the sequence of the first 20 residues of melittin and terminating with an additional cysteine amide was found to have cytolytic activity similar to that of melittin. It was apparent from MS data that the cysteine-terminating peptides had formed disulphide dimers. A peptide in which the thiol was blocked by iodoacetate showed no activity, whereas the same peptide blocked by acetamidomethyl showed activity marginally less haemolytic than that of melittin. Cytolytic activity of melittin analogues comprising the full 26 residues could be obtained with wide sequence permutations providing that a general amphipathic helical structure was preserved. In contrast, the activity of the dimers was dependent not only on retention of an amphipathic helix but also on certain individual residues and a free positive charge. A free N-terminus was essential for haemolytic activity. In addition, a lysine or arginine residue at position 7 and a proline at position 14 were found to be necessary for activity, although it was apparent that additional residues are important for retention of the full lytic potential.

The sequence of peptides necessary to inhibit melittin-induced lysis was studied using 13 peptide analogues of the inhibitor Ac-IVIFDC-NH2. Although this inhibitor is a disulfide-linked dimer, inhibition was equally effective if the thiol SH was blocked or replaced by methionine or lysine. The substitution of phenylalanine with other aromatic residues preserved activity, as did the replacement of aspartic acid by asparagine. The results suggest that the cytolytic activity of melittin can be inhibited by a short peptide of four hydrophobic residues followed by two other nonspecific residues. Fluorescence studies showed that the inhibitor caused a blue shift in the Trp emission spectrum. A spin label attached to the N-terminus of the inhibitor significantly quenched the fluorescence. These data confirmed the involvement of Trp 19 with the inhibitor, also predicted by molecular modeling of the probable binding site. Density gradient studies with large unilamellar vesicles indicated that the inhibitor prevented melittin from reacting with the lipid bilayer.

A range of alternative crosslinking conditions based on glutaraldehyde were examined for their effectiveness for stabilizing collagen-based materials using test samples of a collagen-polymer composite tube. Stabilization of collagen was performed with various concentrations of glutaraldehyde at acid pH, in the absence or presence of 0.7 m NaCl to control collagen swelling. For each condition, some samples were further treated at neutral pH. These test samples were compared with samples treated with glutaraldehyde at neutral pH and with samples of Omniflow vascular prosthesis. The effectiveness of the stabilization was examined by amino acid analysis, to assess the extent of modification, isometric tension analysis, to evaluate the extent of crosslinking, compliance and accelerated fatigue testing, to evaluate mechanical properties, and by a rat subcutaneous model to evaluate tissue response and propensity to calcification. The data indicated that effective crosslinking could be achieved at low pH and that this can be increased slightly by the presence of NaCl. At low pH, the extent of calcification was low compared to samples treated at pH 7. Subsequent treatment at pH 7 of samples given an initial low pH glutaraldehyde (GA) treatment generally did not alter shrinkage temperatures although the extent of lysine modification and calcification did increase. In general, a more inflammatory response was observed in samples tanned at low pH, although this was not as severe as responses to untreated tissue implants. The Omniflow vascular prosthesis showed excellent chemical and mechanical properties and did not show any calcification.

A method that allows stratigraphic analysis of the collagen in the connective tissue which forms around a biomaterial implant is described. Collagen was analysed by densitometry of pepsin soluble a-chains and by hydroxyproline analysis. The tissue formed around a silicone tubing implant was used as a model system for quantitation of collagen types. The amount of collagen was lowest close to the material surface, while this same region had the highest content of type III compared to type I collagen. Type V collagen, which was the only other collagen detected, was present at low levels throughout. Immunohistological examination of the same tissue with highly specific monoclonal antibodies also showed types I and III collagens. The relative staining of these collagens indicated that even if the affinities of antibodies and their effectiveness in penetrating tissue could be taken into account, any quantitative estimates of collagens from immunohistology alone must be treated with great caution.

Photocrosslinking, using 2 mM Ru(II)(bpy)(3)Cl(2) and various concentrations of sodium persulfate with irradiation by blue light, ∼455 nm, has been shown to be a rapid and effective method for crosslinking various tissues: tendon, amnion membrane, pericardium, and heart valve leaflet. The presence of new crosslinking was demonstrated by the increase in the shrinkage temperature of these tissues. In all the cases, increase in the shrinkage temperatures were seen, although at higher sodium persulfate concentrations, for example, 100 mM, both with and without the Ru(II)(bpy)(3)Cl(2) catalyst, some degradation of the collagenous tissues was found. The effectiveness of this photocrosslinking method when used with tissues was also shown through the increase in the break strength of tissues after crosslinking, and by the reduction of protein that could be extracted by urea. In solution studies, dityrosine has been shown to be formed during photocrosslinking. With tissues, Western blotting showed the presence of new dityrosine crosslinked proteins.

Tissue engineering and cell therapy represent significant emerging technologies in medicine. Tissue engineering is‚ "the persuasion of the body to heal itself, through the delivery to the appropriate sites of molecular signals, cells and supporting structures" (Williams, 1999). Various approaches have been described which are labelled tissue engineering, which form a continuum from complex regeneration of scaffold-based tissues to more simple cell-based therapies, where these supporting structures are usually absent or are present in the form of gels simply to aid cell delivery or encapsulation. In tissue engineering the implant to be delivered may frequently be a near mature functional product or a cell-seeded tissue scaffold, both derived in a bioreactor (Haycock, 2011). For cell-based therapies, a bioreactor is not necessary but may be used to amplify the cell numbers for the therapy. Indeed, a key element for both approaches is that they normally require significant expansion of the replacement cells, which have typically been derived in low numbers from small biopsy samples (Brittberg et al., 1994; Chiang et al., 2005; Thissen et al., 2005). A consequence of this expansion is that there can be a significant loss in the quality of cells available for the procedure. In many cases, monolayer cell culture has been the method of choice for cell expansion (Brittberg, 1999; Brittberg et al., 1994; Henderson et al., 2007). However, while this method is well established and simple it has problems. One of these is that certain cells may dedifferentiate and lose their phenotype and biochemical characteristics (Arterburn et al., 1995; Mallein-Gerin et al., 1991). For example, in extended culture, chondrocytes lose their rounded morphology (Fig. 1A) and develop an extended, fibroblast like appearance (Fig. 1B). Also, accompanying this morphological change, matrix biosynthesis switches from type II collagen to type I collagen (Aulthouse et al., 1989; Lefebvre et al., 1990; von der Mark et al., 1977). It is clear that the collagen produced changes from type II collagen (Fig. 2A), which is the characteristic collagen of articular, hyaline cartilage (Mayne, 1989; Miller & Matukas, 1969) to type I collagen (Fig. 2B), which is a mechanically-inferior fibrocartilage (Chiang et al., 2005; Wegener et al., 2009). Type I collagen is the principal collagen of many tissues

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.

In this study we present a novel approach for predicting tissue growth within the pores of fibrous tissue engineering scaffolds. Thin nonwoven polyethylene terephthalate scaffolds were prepared to characterize tissue growth within scaffold pores, by mouse NR6 fibroblast cells. On the basis of measurements of tissue lengths at fiber crossovers and along fiber segments, mathematical models were determined during the proliferative phase of cell growth. Tissue growth at fiber crossovers decreased with increasing interfiber angle, with exponential relationships determined on day 6 and 10 of culture. Analysis of tissue growth along fiber segments determined two growth profiles, one with enhanced growth as a result of increased tissue lengths near the fiber crossover, achieved in the latter stage of culture. Derived mathematical models were used in the development of a software program to visualize predicted tissue growth within a pore. This study identifies key pore parameters that contribute toward tissue growth, and suggests models for predicting this growth, based on fibroblast cells. Such models may be used in aiding scaffold design, for optimum pore infiltration during the tissue engineering process.

A range of bacteria have been shown to contain collagen-like sequences that form triple-helical structures. Some of these proteins have been shown to form triple-helical motifs that are stable around body temperature without the inclusion of hydroxyproline or other secondary modifications to the protein sequence. This makes these collagen-like proteins particularly suitable for recombinant production as only a single gene product and no additional enzyme needs to be expressed. In the present study, we have examined the cytotoxicity and immunogenicity of the collagen-like domain from Streptococcus pyogenes Scl2 protein. These data show that the purified, recombinant collagen-like protein is not cytotoxic to fibroblasts and does not elicit an immune response in SJL/J and Arc mice. The freeze dried protein can be stabilised by glutaraldehyde cross-linking giving a material that is stable at >37 degrees C and which supports cell attachment while not causing loss of viability. These data suggest that bacterial collagen-like proteins, which can be modified to include specific functional domains, could be a useful material for medical applications and as a scaffold for tissue engineering.

Here we report the use of a facile photochemical crosslinking method to fabricate stable polymer matrices from unmodified gelatin and fibrinogen. Gels were produced by covalent crosslinking of the proteins in a rapid photo-oxidative process, catalysed by a ruthenium metal complex and irradiation with visible light. For generation of macroporous, spongy matrices, the proteins and crosslinking reagents were mixed with catalase and hydrogen peroxide to achieve a foaming reaction, producing a stable, foamed matrix that was subsequently photo-crosslinked. C2C12 cells were either seeded onto the matrices after photo-curing or embedded in the protein matrix prior to foaming and crosslinking. Cells seeded onto scaffolds post-curing showed high cell viability and rapid proliferation in vitro. For cells embedded in the matrix prior to crosslinking there was some loss of initial viability, but surviving cells were able to proliferate after a period of in vitro cultivation. The matrices were shown to be biocompatible when implanted into nude mice, with evidence of proliferation and differentiation of cells seeded into the scaffolds. The results are promising for further development of tissue-engineering scaffolds based on this ruthenium-catalysed photo-crosslinking method.

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.

When provoked, Notaden bennetti frogs secrete a proteinaceous exudate, which rapidly forms a tacky and elastic glue. This material has potential in biomedical applications. Cultured cells attached and proliferated well on glue-coated tissue culture polystyrene, but migrated somewhat slower than on uncoated surfaces. In organ culture, dissolved glue successfully adhered collagen-coated perfluoropolyether lenses to debrided bovine corneas and supported epithelial regrowth. Small pellets of glue implanted subcutaneously into mice were resorbed by surrounding tissues, and all of the animals made a full recovery. An initial but transient skin necrosis at the implant site was probably caused by some of the potentially toxic metabolites present in the frog secretion; these include sterols and carotenoids, as well as fatty alcohols, aldehydes, ketones, acids, and aromatic compounds. Removal of the carotenoid pigments did not significantly alter the glue's material properties. In contrast, peroxidase treatment of dissolved glue introduced unnatural crosslinks between molecules of the major protein (Nb-1R) and resulted in the formation of a soft hydrogel, which was very different to the original material. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010

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.

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.

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.

Eight monoclonal antibodies have been produced against human pepsin-soluble type III collagen. All antibodies were shown to be highly specific for type III collagen and did not cross-react with a range of other collagen types or connective-tissue proteins. Examination of type III collagen from other species showed that these antibodies had a wide range of species specificities, indicating that several distinct epitopes were being recognized. The location of the epitopes was investigated by using reactivity of the antibodies to CNBr fragments and to sequential fragments formed by tryptic digestion of renaturing type III collagen. These data also indicated that several distinct epitopes were recognized and that they were located over the length of the type III collagen.