Veronica Glattauer

Collagens as biomaterials

This paper reviews the structure, function and applications of collagens as biomaterials. The various formats for collagens, either as tissue-based devices or as reconstituted soluble collagens are discussed. The major emphasis is on the new technologies that are emerging that will lead to new and improved collagen-based medical devices. In particular, the development of recombinant collagens, especially using microorganism systems, is allowing the development of safe and reproducible collagen products. These systems also allow for the development of novel, non-natural structures, for example collagen like structures containing repeats of key functional domains or as chimeric structures where a collagen domain is covalently linked to another biologically active component.

Self-assembly of ciprofloxacin and a tripeptide into an antimicrobial nanostructured hydrogel

This work reports the self-assembly of a sparingly soluble antibiotic (ciprofloxacin) and a hydrophobic tripeptide ((D)Leu-Phe-Phe) into supramolecular nanostructures that yield a macroscopic hydrogel at physiological pH. Drug incorporation results in modified morphology and rheological properties of the self-assembled hydrogel. These changes can be correlated with intermolecular interactions between the drug and the peptide, as confirmed by spectroscopic analysis (fluorescence, circular dichroism, IR). The drug appears bound within the hydrogel by non-covalent interactions, and retains its activity over a prolonged release timescale. Antimicrobial activity of the ciprofloxacin-peptide self-assembled hydrogel was evaluated against Staphylococcus aureus, Escherichia coli, and a clinical strain of Klebsiella pneumoniae. Interestingly, the peptide hydrogel alone exhibited a mild anti-bacterial activity against Gram-negative bacteria. While toxic to bacteria, no major cytotoxicity was seen in haemolysis assays of human red blood cells or in mouse fibroblast cell cultures. This new approach of drug incorporation into the nanostructure of a simple tripeptide hydrogel by self-assembly may have important applications for cost-effective wound dressings and novel antimicrobial formulations.

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.

Bone regeneration using photocrosslinked hydrogel incorporating rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles

Although rhBMP-2 has excellent ability to accelerate the repair of normal bone defects, limitations of its application exist in the high cost and potential side effects. This study aimed to develop a composite photopolymerisable hydrogel incorporating rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (PH/rhBMP-2/NPs) as the bone substitute to realize segmental bone defect repair at a low growth factor dose. Firstly rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (rhBMP-2/NPs) were prepared and characterized by DLS and TEM. Composite materials, PH/rhBMP-2/NPs were developed and investigated by SEM-EDS as well as a series of physical characterizations. Using hMSCs as an in vitro cell model, composite photopolymerisable hydrogels incorporating NPs (PH/NPs) showed good cell viability, cell adhesion and time dependent cell ingrowth. In vitro release kinetics of rhBMP-2 showed a significantly lower initial burst release from the composite system compared with the growth factor-loaded particles alone or encapsulated directly within the hydrogel, followed by a slow release over time. The bioactivity of released rhBMP-2 was validated by alkaline phosphatase (ALP) activity as well as a mineralization assay. In in vivo studies, the PH/rhBMP-2/NPs induced ectopic bone formation in the mouse thigh. In addition, we further investigated the in vivo effects of rhBMP-2-loaded scaffolds in a rabbit radius critical defect by three dimensional micro-computed tomographic (μCT) imaging, histological analysis, and biomechanical measurements. Animals implanted with the composite hydrogel containing rhBMP-2-loaded nanoparticles underwent gradual resorption with more pronounced replacement by new bone and induced reunion of the bone marrow cavity at 12 weeks, compared with animals implanted with hydrogel encapsulated growth factors alone. These data provided strong evidence that the composite PH/rhBMP-2/NPs are a promising substitute for bone tissue engineering.

A Streptococcus pyogenes derived collagen-like protein as a non-cytotoxic and non-immunogenic cross-linkable biomaterial.

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.

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.

Collagen-based layer-by-layer coating on electrospun polymer scaffolds

Preparation of microfibre constructs of collagen by electrospinning has been problematic due to the instability of collagen in volatile solvents, such as 1,1,1,3,3,3-hexafluoro-2-propanol, so that electrospinning leads to a substantial amount of gelatin fibres. In the present study we have demonstrated the production of collagen-based microfibre constructs by use of a layer-by-layer coating process onto a preformed synthetic polymer microfibre base. Soluble native collagen, which has a basic isoelectric point, has been used with modified triple-helical collagens that have acidic isoelectric points. These modified collagens have been prepared as deamidated, succinylated, maleylated and citraconylated derivatives. Together, the acidic and basic collagens have successfully coated polyacrylonitrile and poly(DL-lactide-co-glycolide) fibres, as shown by spectroscopy and microscopy. These coatings allow good cell attachment and spreading on the fibres. The native, triple helical form of the collagen has been confirmed through use of a conformation dependent monoclonal antibody.

MR three-dimensional molecular imaging of intramural biomarkers with targeted nanoparticles.

In this study, porcine carotid arteries were subjected to balloon overstretch injury followed by local delivery of paramagnetic nanoparticles targeted to alphavbeta3-integrin expressed by smooth muscle cells or collagen III within the extracellular matrix. Carotid T1-weighted angiography and vascular imaging was performed at 1.5T. While MR angiograms were indistinguishable between control and targeted vessel segments, alphavbeta3-integrin-and collagen Ill-targeted nanoparticles spatially delineated patterns and volumes of stretch injury. In conclusion, MR molecular imaging with alphavbeta3-integrin or collagen Ill-targeted nanoparticles enables the non-invasive, three-dimensional characterization of arterial pathology unanticipated from routine angiography.

Micropatterning of Polymer Brushes: Grafting from Dewetting Polymer Films for Biological Applications

In this novel platform, a micropatterned polymer brush was obtained by grafting poly(poly(ethylene glycol) methyl ether methacrylate) (poly(PEGMA)) from a thin macroinitiator film using atom transfer radical polymerization (ATRP). A pattern of holes was formed in the macroinitiator film by taking advantage of its spontaneous dewetting above the glass transition temperature from a bottom polystyrene film, driven by unfavorable intermolecular forces. Patterning by dewetting can be achieved at length-scales from a few hundred nanometers to several tens of micrometers, by simply thermally annealing the bilayer above the glass transition temperature of the polymer. This approach is substrate-independent, as polymer films can be cast onto surfaces of different size, shape, or material. As a demonstration of its potential, proteins, and individual cells were attached on targeted bioadhesive polystyrene areas of the micropatterns within poly(PEGMA) protein-repellent brushes. We anticipate this approach will be suitable for the patterning of brushes, especially for biomedical applications such as in the study of single cells and of cell cocultures.

Evaluation of the immunogenicity and cell compatibility of avian collagen for biomedical applications.

There have been concerns regarding the suitability of bovine collagen as a biomaterial since the emergence of bovine spongiform encephalopathy. Consequently, collagens from other species may be used if they can meet appropriate standards, including negligible or lack of immunogenicity. In this study, the potential immunogenicity of both monomeric and pepsin-solubilized chicken collagens have been compared with a commercial, pepsin-solubilized bovine collagen that is approved for biomedical implantation. All collagens were poor immunogens compared with ovalbumin. No IgE responses were detected in sera of three strains of mice, and no hypersensitivity reactions were found in guinea pigs in maximization and Buehler tests. IgG(1) antibodies were found although the titre was substantially lower than against ovalbumin. All responses in mice and rabbits were found only when immunizations were performed with adjuvant, and after multiple injections over a long period of time. The response from the monomeric chicken collagen was less than for pepsin-solubilized collagens. Collagen sponges prepared from the two chicken collagen preparations both supported the attachment and growth of mouse fibroblasts. These data indicate that chicken collagen, particularly when monomeric, may be useful in certain biomedical applications.