Jacinta F. White

The in vivo performance of an enzyme-assisted self-assembled peptide/protein hydrogel

We demonstrate the distribution of the important extracellular matrix protein laminin in a novel biomaterial consisting of a hydrogel underpinned by nanofibrillar networks. These are formed by the immobilised enzyme mediated self-assembly of fmoc-L(3) (9-fluorenylmethoxycarbonyl-tri-leucine). The peptide assembly yields nanofibrils formed of β-sheets that are locked together via π-stacking interactions. This ordering allows the localisation of the peptide sidechains on the surface, creating a hydrophobic environment. This induces the formation of bundles of these nanofibrils producing a clear hydrogel. This mechanism enables the three dimensional distribution of laminin throughout the network via supramolecular interactions. These forces favour the formation and improve the order of the network itself, as observed by spectroscopic and mechanical testing. In order to test the stability and suitability of this class of material for in vivo applications, we utilise microinjection to deliver the biomaterial under fine spatial control into a dystrophic zebrafish model organism, which lacks laminin as a result of a genetic mutation. Using confocal and transmission electron microscopy, we confirm that the biomaterial remains stable structurally, and is confined spatially to the site of injection.

Expression and analysis of the NS2 protein of influenza A virus

SummaryInfluenza NS2 protein was expressed inSaccharomyces cerevisiae using a copper-inducible promoter. The protein produced had a molecular weight of 13 kDa, was reactive with anti-NS2 antiserum and was localised to the yeast cell nucleus. Two-hybrid analysis identified a direct protein-protein interaction between NS2 and the M2 protein of the virus, involving the C-terminal 163 residues of M1. A filter-binding assay localised the M1 binding region to the C-terminal 70 amino acids of NS2.

In vivo evaluation of a collagenous membrane as an absorbable adhesion barrier

An absorbable membrane made from purified, pepsin-soluble collagen was compared to Interceed, an absorbable cellulose-based product, and to a control group for effectiveness in inhibiting the formation of adhesions between peritoneal surface injuries in adult rats. An adhesion scoring system was used to evaluate and compare the performance of the test materials with the control group in regard to the extent, tenacity, and type of any adhesions evident at 28 days following surgery. The collagen group performed significantly better (p < 0.05) than either the Interceed or control groups, showing fewer, less extensive adhesions. The collagen membranes resulted in either no or weak adhesions between the body wall and caecum. Adhesions in the Interceed group were quite variable and characterized by a marked peritoneal reaction in the caecal and body walls adjacent to adhesions. Control samples were characterized by close, dense fibrotic adhesions between the caecum and body wall. Both of the test materials showed some deficiencies in respect to their physical and handling properties that could be further improved for this indication.

Extracellular addition of a domain of HIV‐1 Vpr containing the amino acid sequence motif H(S/F)RIG causes cell membrane permeabilization and death

Vpr is a virion‐associated protein of human immuno‐deficiency virus type 1 (HIV‐1) whose function in acquired immune deficiency syndrome (AIDS) has been uncertain. We previously employed yeast as a model to examine the effects of Vpr on basic cellular functions; intracellular Vpr was shown to cause cell‐growth arrest and structural defects, and these effects were caused by a region of Vpr containing the sequence HFRIGCRHSRIG. Here we show that peptides containing the H(S/F)RIG amino acid sequence motif cause death when added externally to a variety of yeast including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Candida albicans and Schizosaccharomyces pombe. Such peptides rapldly entered the cell from the time of addition, resulting in cell death. Elevated levels of ions, particularly magnesium and calcium ions, abrogated the cytotoxic effect by preventing the Vpr peptides from entering the cells. Extracellular Vpr found in the serum, or breakdown products of extracellular Vpr, may have similar effects to the Vpr peptides described here and could explain the death of uninfected by‐stander cells during AIDS.

A preclinical evaluation of alternative synthetic biomaterials for fascial defect repair using a rat abdominal hernia model.

Introduction Fascial defects are a common problem in the abdominal wall and in the vagina leading to hernia or pelvic organ prolapse that requires mesh enhancement to reduce operation failure. However, the long-term outcome of synthetic mesh surgery may be unsatisfactory due to post-surgical complications. We hypothesized that mesh fabricated from alternative synthetic polymers may evoke a different tissue response, and provide more appropriate mechanical properties for hernia repair. Our aim was to compare the in vivo biocompatibility of new synthetic meshes with a commercial mesh. Methods We have fabricated 3 new warp-knitted synthetic meshes from different polymers with different tensile properties polyetheretherketone (PEEK), polyamide (PA) and a composite, gelatin coated PA (PA+G). The rat abdominal hernia model was used to implant the meshes (25×35 mm, n = 24/ group). After 7, 30, 60, 90 days tissues were explanted for immunohistochemical assessment of foreign body reaction and tissue integration, using CD31, CD45, CD68, alpha-SMA antibodies. The images were analysed using an image analysis software program. Biomechanical properties were uniaxially evaluated using an Instron Tensile® Tester. Results This study showed that the new meshes induced complex differences in the type of foreign body reaction over the time course of implantation. The PA, and particularly the composite PA+G meshes, evoked a milder early inflammatory response, and macrophages were apparent throughout the time course. Our meshes led to better tissue integration and new collagen deposition, particularly with the PA+G meshes, as well as greater and sustained neovascularisation compared with the PP meshes. Conclusion PA, PA+G and PEEK appear to be well tolerated and are biocompatible, evoking an overlapping and different host tissue response with time that might convey mechanical variations in the healing tissue. These new meshes comprising different polymers may provide an alternative option for future treatment of fascial defects.

Biodegradable and injectable cure-on-demand polyurethane scaffolds for regeneration of articular cartilage.

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 Massons 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.

Characterisation of clinical and newly fabricated meshes for pelvic organ prolapse repair.

Clinical meshes used in pelvic organ prolapse (POP) repair are predominantly manufactured from monofilament polypropylene (PP). Complications from the use of these meshes in transvaginal kits, including mesh exposure and pain, have prompted two public health notifications by the FDA. The aim of this study was to compare several clinical PP POP meshes to new fabricated POP meshes, knitted from alternative polymers, for their mechanical properties using standard and clinically relevant multi-axial testing methods. Five new meshes were warp knitted to different architectures and weights from polyamide and polyetheretherketone monofilaments. A composite mesh of a polyamide mesh incorporating a gelatin layer was also fabricated to enable the potential delivery of cells on these meshes. Meshes were assessed for their structural characteristics and mechanical properties, using uniaxial stiffness, permanent strain, bending rigidity and multi-axial burst strength methods. Results were compared to three clinical urogynaecological polypropylene meshes: Polyform®, Gynemesh(TM)PS, and IntePro®. New fabricated meshes were uniaxially less stiff (less than 0.24 N/mm and 1.20 N/mm in toe and linear regions, respectively) than the Gynemesh (0.48 N/mm and 2.08 N/mm in toe and linear regions, respectively) and IntePro (0.57 N/mm in toe region) clinical meshes, with the gelatin coated PA mesh exhibiting lower permanent strain than Polyform clinical mesh (8.1% vs. 23.5%). New meshes had lower burst stiffness than Polyform (less than 16.9 N/mm for new meshes and 26.6N/mm for Polyform). Within the new mesh prototypes, the PA meshes, either uncoated (4.7-5.7 μN m) or with gelatin coating (16.7 μN m) possessed lower bending rigidity than both Polyform and Gynemesh (46.2 μN m and 36.4 μN m, respectively). The new fabricated mesh designs were of similar architecture, but with some improved mechanical properties, compared to clinical POP meshes. Multi-axial analysis of new and clinical mesh designs provides greater discriminatory power in analysing mesh mechanical properties for clinical applications.

ORGANIZATION OF FIBRILLAR COLLAGEN IN THE HUMAN AND BOVINE CORNEA : COLLAGEN TYPES V AND III

The localization and fibrillar organization of collagen types V and III in the human and bovine corneal stromas were studied. In the chicken cornea, type V co-assembles with type I collagen as heterotypic fibrils and this interaction is involved in the regulation of fibril diameter necessary for corneal transparency. To determine whether this is a regulatory mechanism common to the corneas of different species the human and bovine corneal stroma were studied. Collagen type V was found in the epithelium and Bowmans membrane in the untreated adult human and bovine cornea using immunofluorescence microscopy. In the absence of any treatment, there was no type V reactivity within the stroma. However, type V collagen was detected homogeneously throughout the corneal stroma after treatments that partially disrupt fibril structure. The reactivity was strongest in the cornea, weaker in the limbus and weakest in the sclera. Fetal corneas showed similar reactivity for type V collagen, but unlike the adult, the stroma was slightly reactive. Immunoelectron microscopy demonstrated that type V collagen was associated with disrupted, but not with intact, fibrils in both human and bovine corneal stroma. Type III collagen reactivity was not detected in the cornea, but was present subepithelially in the limbus and in the scleral stroma. These data indicate that type V collagen is a component of striated collagen fibrils throughout the human and bovine corneal stromas. The interaction of type I and V collagen as heterotypic fibrils masks the helical epitope recognized by the monoclonal antibody against type V collagen. The heterotypic interactions of collagen type V indicate a role in the regulation of fibril diameter analogous to that described in the avian cornea.

Influence of Reproductive Status on Tissue Composition and Biomechanical Properties of Ovine Vagina

Objective To 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. Methods Vaginal 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 Massons 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 Youngs modulus, maximum stress and strain, and permanent strain following cyclic loading. Results Vaginal 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. Conclusion Pregnant 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.

Evaluation of a collagen-based biosynthetic material for the repair of abdominal wall defects

A collagen tissue polymer composite manufactured in sheep and prepared in two different forms (wet and dry) was compared to polypropylene mesh and to a control group for effectiveness in the repair of an abdominal wall defect in a rabbit model. The wet and dry patches were shown to differ significantly in their pore size. The wet material was shown to retain its natural porosity and promoted neovascularization, tissue integration, cellular infiltration, and neomatrix formation compared to the dry collagen-polymer patch. This material was superior to the polypropylene mesh implant, which was associated with significant adhesions. The appearance of type VI collagen was the earliest sign of new cell infiltration and neomatrix formation within the implant. New deposition of type VI collagen was apparent throughout the thickness of the implant within 4 weeks, followed by type III collagen accumulation. Decreased porosity of the collagen component in the dry patches resulted in a totally nonintegrated implant. This induced a foreign-body capsule with minimal cellular tissue infiltration and no deposition of collagen types VI and III within the implant.