The diazirine-mediated photo-crosslinking of collagen improves biomaterial mechanical properties and cellular interactions.

In tissue engineering, crosslinking with carbodiimides such as EDC is omnipresent to improve the mechanical properties of biomaterials. However, in collagen biomaterials, EDC reacts with glutamate or aspartate residues, inactivating the binding sites for cellular receptors and rendering collagen inert to many cell types. In this work, we have developed a crosslinking method that ameliorates the rigidity, stability, and degradation rate of collagen biomaterials, whilst retaining key interactions between cells and the native collagen sequence. Our approach relies on the UV-triggered reaction of diazirine groups grafted on lysines, leaving critical amino acid residues intact. Notably, GxxGER recognition motifs for collagen-binding integrins, ablated by EDC crosslinking, were left unreacted, enabling cell attachment, spreading, and colonization on films and porous scaffolds. In addition, our procedure conserves the architecture of biomaterials, improves their resistance to collagenase and cellular contraction, and yields material stiffness akin to that obtained with EDC. Importantly, diazirine-crosslinked collagen can host mesenchymal stem cells, highlighting its strong potential as a substrate for tissue repair. We have therefore established a new crosslinking strategy to modulate the mechanical features of collagen porous scaffolds without altering its biological properties, thereby offering an advantageous alternative to carbodiimide treatment. STATEMENT OF SIGNIFICANCE: This article describes an approach to improve the mechanical properties of collagen porous scaffolds, without impacting collagen's natural interactions with cells. This is significant because collagen crosslinking is overwhelmingly performed using carbodiimides, which results in a critical loss of cellular affinity. By contrast, our method leaves key cellular binding sites in the collagen sequence intact, enabling cell-biomaterial interactions. It relies on the fast, UV-triggered reaction of diazirine with collagen, and does not produce toxic by-products. It also supports the culture of mesenchymal stem cells, a pivotal cell type in a wide range of tissue repair applications. Overall, our approach offers an attractive option for the crosslinking of collagen, a prominent material in the growing field of tissue engineering.

Tailoring the biofunctionality of collagen biomaterials via tropoelastin incorporation and EDC-crosslinking.

Recreating the cell niche of virtually all tissues requires composite materials fabricated from multiple extracellular matrix (ECM) macromolecules. Due to their wide tissue distribution, physical attributes and purity, collagen, and more recently, tropoelastin, represent two appealing ECM components for biomaterials development. Here we blend tropoelastin and collagen, harnessing the cell-modulatory properties of each biomolecule. Tropoelastin was stably co-blended into collagen biomaterials and was retained after EDC-crosslinking. We found that human dermal fibroblasts (HDF), rat glial cells (Rugli) and HT1080 fibrosarcoma cells ligate to tropoelastin via EDTA-sensitive and EDTA-insensitive receptors or do not ligate with tropoelastin, respectively. These differing elastin-binding properties allowed us to probe the cellular response to the tropoelastin-collagen composites assigning specific bioactivity to the collagen and tropoelastin component of the composite material. Tropoelastin addition to collagen increased total Rugli cell adhesion, spreading and proliferation. This persisted with EDC-crosslinking of the tropoelastin-collagen composite. Tropoelastin addition did not affect total HDF and HT1080 cell adhesion; however, it increased the contribution of cation-independent adhesion, without affecting the cell morphology or, for HT1080 cells, proliferation. Instead, EDC-crosslinking dictated the HDF and HT1080 cellular response. These data show that a tropoelastin component dominates the response of cells that possess non-integrin based tropoelastin receptors. EDC modification of the collagen component directs cell function when non-integrin tropoelastin receptors are not crucial for cell activity. Using this approach, we have assigned the biological contribution of each component of tropoelastin-collagen composites, allowing informed biomaterial design for directed cell function via more physiologically relevant mechanisms. STATEMENT OF SIGNIFICANCE: Biomaterials fabricated from multiple extracellular matrix (ECM) macromolecules are required to fully recreate the native tissue niche where each ECM macromolecule engages with a specific repertoire of cell-surface receptors. Here we investigate combining tropoelastin with collagen as they interact with cells via different receptors. We identified specific cell lines, which associate with tropoelastin via distinct classes of cell-surface receptor. These showed that tropoelastin, when combined with collagen, altered the cell behaviour in a receptor-usage dependent manner. Integrin-mediated tropoelastin interactions influenced cell proliferation and non-integrin receptors influenced cell spreading and proliferation. These data shed light on the interplay between biomaterial macromolecular composition, cell surface receptors and cell behaviour, advancing bespoke materials design and providing functionality to specific cell populations.

Modulating hESC-derived cardiomyocyte and endothelial cell function with triple-helical peptides for heart tissue engineering.

In this study, we investigated the role of cardiomyocyte (CM) and endothelial cell (EC) specific interactions with collagen in the assembly of an operational myocardium in vitro. Engineered cardiac patches represent valuable tools for myocardial repair following infarction and are generally constituted of a suitable biomaterial populated by CMs and supportive cell types. Among those, ECs are required for tissue vascularization and positively modulate CM function. To direct the function of human embryonic stem cell (hESC)-derived CM and EC seeded on biomaterials, we replicated cell-collagen interactions, which regulate cellular behaviour in the native myocardium, using triple-helical peptides (THPs) that are ligands for collagen-binding proteins. THPs enhanced proliferation and activity of CMs and ECs separately and in co-culture, drove CM maturation and enabled coordinated cellular contraction on collagen films. These results highlight the importance of collagen interactions on cellular response and establish THP-functionalized biomaterials as novel tools to produce engineered cardiac tissues.

Zinc sulfide nanoparticle-decorated fibre mesh to enable localized H2S-amplified chemotherapy.

For the first time, we report the design and fabrication of a ZnS nanoparticle-decorated silica fibre mesh (ZnS@SiO2) for localized H2S-amplified chemotherapy. With incorporation of DOX, implanted ZnS@SiO2 fibres enable sufficient on-site drug dosage and intracellular H2S content, inducing significant in vitro and in vivo tumour inhibition.

Impact of UV- and carbodiimide-based crosslinking on the integrin-binding properties of collagen-based materials.

Collagen constructs are widely used for tissue engineering. These are frequently chemically crosslinked, using EDC, to improve their stability and tailor their physical properties. Although generally biocompatible, chemical crosslinking can modify crucial amino acid side chains, such as glutamic acid, that are involved in integrin-mediated cell adhesion. Instead UV crosslinking modifies aromatic side chains. Here we elucidate the impact that EDC, in combination with UV, exerts on the activity of integrin-binding motifs. By employing a model cell line that exclusively utilises integrin α2ß1, we found that whilst EDC crosslinking modulated cell binding, from cation-dependent to cation-independent, UV-mediated crosslinking preserved native-like cell binding, proliferation and surface colonisation. Similar results were observed using a purified recombinant I-domain from integrin α1. Conversely, binding of the I-domain from integrin α2 was sensitive to UV, particularly at low EDC concentrations. Therefore, from this in vitro study, it appears that UV can be used to augment EDC whist retaining a specific subset of integrin-binding motifs in the native collagen molecule. These findings, delineating the EDC- and UV-susceptibility of cell-binding motifs, permit controlled cell adhesion to collagen-based materials through specific integrin ligation in vitro. However, in vivo, further consideration of the potential response to UV wavelength and dose is required in the light of literature reports that UV initiated collagen scission may lead to an adverse inflammatory response. STATEMENT OF SIGNIFICANCE: Recently, there has been rapid growth in the use of extracellular matrix-derived molecules, and in particular collagen, to fabricate biomaterials that replicate the cellular micro-environment. Often chemical or physical crosslinkers are required to enhance the biophysical properties of these materials. Despite extensive use of these crosslinkers, the cell-biological consequences have not been ascertained. To address this, we have investigated the integrin-binding properties of collagen after chemically crosslinking with EDC and physically crosslinking with UV-irradiation. We have established that whilst EDC crosslinking abates all of the integrin binding sites in collagen, UV selectively inhibits interaction with integrin-α2 but not -α1. By providing a mechanistic model for this behaviour, we have, for the first time, defined a series of crosslinking parameters to systematically control the interaction of collagen-based materials with defined cellular receptors.

Cellular response to collagen-elastin composite materials.

Collagen is used extensively in tissue engineering due to its biocompatibility, near-universal tissue distribution, low cost and purity. However, native tissues are composites that include diverse extracellular matrix components, which influence strongly their mechanical and biological properties. Here, we provide important new findings on the differential regulation, by collagen and elastin, of the bio-response to the composite material. Soluble and insoluble elastin had differing effects on the stiffness and failure strength of the composite films. We established that Rugli cells bind elastin via EDTA-sensitive receptors, whilst HT1080 cells do not. These cells allowed us to probe the contribution of collagen alone (HT1080) and collagen plus elastin (Rugli) to the cellular response. In the presence of elastin, Rugli cell attachment, spreading and proliferation increased, presumably through elastin-binding receptors. By comparison, the attachment and spreading of HT1080 cells was modified by elastin inclusion, but without affecting their proliferation, indicating indirect modulation by elastin of the response of cells to collagen. These new insights highlight that access to elastin dominates the cellular response when elastin-binding receptors are present. In the absence of these receptors, modification of the collagen component and/or physical properties dictate the cellular response. Therefore, we can attribute the contribution of each constituent on the ultimate bioactivity of heterogeneous collagen-composite materials, permitting informed, systematic biomaterials design. STATEMENT OF SIGNIFICANCE: In recent years there has been a desire to replicate the complex extracellular matrix composition of tissues more closely, necessitating the need for composite protein-based materials. In this case both the physical and biochemical properties are altered with the addition of each component, with potential consequences on the cell. To date, the different contributions of each component have not been deconvolved, and instead the cell response to the scaffold as a whole has been observed. Instead, here, we have used specific cell lines, that are sensitive to specific components of an elastin-collagen composite, to resolve the bio-activity of each protein. This has shown that elastin-induced alteration of the collagen component can modulate early stage cell behaviour. By comparison the elastin component directly alters the cell response over the short and long term, but only where appropriate receptors are present on the cell. Due to the widespread use of collagen and elastin, we feel that this data permits, for the first time, the ability to systematically design collagen-composite materials to promote desired cell behaviour with associated advantages for biomaterials fabrication.

Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF.

Collagen-based scaffolds may require chemical crosslinking to achieve mechanical properties suitable for tissue engineering. Carbodiimide treatment, often used for this purpose, consumes amino acid side chains required for receptor recognition, thus reducing cell-collagen interaction. Here, we restore recognition and function of both von Willebrand Factor (VWF) and Discoidin Domain Receptor 2 (DDR2) to crosslinked collagen films by derivatisation with a specific triple-helical peptide (THP), an approach previously applied to integrin-mediated cellular adhesion. The THP contained the collagen III-derived active sequence, GPRGQOGVNleGFO, conjugated to a photoreactive moiety, diazirine, allowing UV-dependent covalent coupling to collagen films. Crosslinking of collagen films attenuated the binding of recombinant VWF A3 domain and of DDR2 (as the GST and Fc fusions, respectively), and coupling of the specific THP restored their attachment. These derivatised films supported activation of DDR2 expressed in either COS-7 or HEK293 cells, reflected by phosphorylation of tyrosine 740, and VWF-mediated platelet deposition from flowing blood was restored. Further, such films were able to increase low-density lipoprotein uptake in vascular endothelial cells, a marker for endothelial phenotype. Thus, covalent linkage of specific THPs to crosslinked collagen films i) restores their cognate protein binding, ii) triggers the corresponding cellular responses, and iii) demonstrates the broad applicability of the approach to a range of receptors for applications in regenerative medicine.

Structurally graduated collagen scaffolds applied to the ex vivo generation of platelets from human pluripotent stem cell-derived megakaryocytes: Enhancing production and purity.

Platelet transfusions are a key treatment option for a range of life threatening conditions including cancer, chemotherapy and surgery. Efficient ex vivo systems to generate donor independent platelets in clinically relevant numbers could provide a useful substitute. Large quantities of megakaryocytes (MKs) can be produced from human pluripotent stem cells, but in 2D culture the ratio of platelets harvested from MK cells has been limited and restricts production rate. The development of biomaterial cell supports that replicate vital hematopoietic micro-environment cues are one strategy that may increase in vitro platelet production rates from iPS derived Megakaryocyte cells. In this paper, we present the results obtained generating, simulating and using a novel structurally-graded collagen scaffold within a flow bioreactor system seeded with programmed stem cells. Theoretical analysis of porosity using micro-computed tomography analysis and synthetic micro-particle filtration provided a predictive tool to tailor cell distribution throughout the material. When used with MK programmed stem cells the graded scaffolds influenced cell location while maintaining the ability to continuously release metabolically active CD41 + CD42 + functional platelets. This scaffold design and novel fabrication technique offers a significant advance in understanding the influence of scaffold architectures on cell seeding, retention and platelet production.

Gold nanorod-assembled ZnGa2O4:Cr nanofibers for LED-amplified gene silencing in cancer cells.

Nanoparticles are now commonly used as non-viral gene vectors for RNA interference (RNAi) in cancer therapy but suffer from low targeting efficiency in situ. Meanwhile, localized drug delivery systems do not offer the effective capability for intracellular gene transportation. We describe here the design and synthesis of a localized therapeutic system, consisting of gold nanorods (Au NRs) loaded with hTERT siRNA assembled on the surface of ZnGa2O4:Cr (ZGOC) nanofibers. This composite system offers the potential for a LED-induced mild photothermal effect which enhances the phagocytosis of Au NRs carrying siRNA and the subsequent release of siRNA in the cytoplasm. Both phenomena amplify the gene silencing effect and consequently offer the potential for a superior therapeutic outcome.

Upconversion nanocrystal 'armoured' silica fibres with superior photoluminescence for miRNA detection.

We have fabricated a flexible membrane, consisting of SiO2 nanofibres armoured with upconversion nanoparticles, exhibiting intense photoluminescence. These assemblies were subsequently grafted with molecular beacons to produce a biosensor suitable for the detection of specific microRNA and with applications in early cancer detection and point-of-care diagnosis.

3D imaging of cells in scaffolds: direct labelling for micro CT.

The development of in-vitro techniques to characterise the behaviour of cells in biomedical scaffolds is a rapidly developing field. However, until now it has not been possible to visualise, directly in 3D, the extent of cell migration using a desktop X-ray microCT. This paper describes a new technique based on cell labelling with a radio opacifier (barium sulphate), which permits cell tracking without the need for destructive sample preparation. The ability to track cells is highlighted via a comparison of cell migration through demonstrator lyophilised collagen scaffolds with contrasting pore size and interconnectivity. The results demonstrate the ease with which the technique can be used to characterise the effects of scaffold architecture on cell infiltration.

Selecting the correct cellular model for assessing of the biological response of collagen-based biomaterials.

Accurate evaluation of the biological performance of biomaterials requires the correct assessment of their native-like cell ligation properties. However, cell attachment studies often overlook the details of the substrate-cell binding mechanisms, be they integrin-mediated or non-specific, and ignore the class- and species-specificities of the cell adhesion receptor involved. In this work we have used different collagen (Col) substrates (fibrillar collagens I, II and III and network-forming Col IV), containing different affinity cell-recognition motifs, to establish the influence of the receptor identity and species-specificity on collagen-cell interactive properties. Receptor expression was varied by using cells of different origin, or transfecting collagen-binding integrins into integrin-null cells. These include mouse C2C12 myoblasts transfected with human α1, α2, α10 or α11; human fibrosarcoma HT1080 cells which constitutively express only human α2ß1, and rat glioma Rugli cells, with only rat α1ß1. Using these lines, the nature of integrin binding sites was studied in order to delineate the bioactivity of different collagen substrates. Integrin ligation was studied on collagen coatings alongside synthetic (GFOGER/GLOGEN) and Toolkit (Col II-28/Col III-7) triple-helical peptides to evaluate (1) their affinity towards different integrins and (2) to confirm the activity of the inserted integrin in the transfected cells. Thin films of dermal and tendon Col I were used to evaluate the influence of the carbodiimide (EDC)-based treatment on the cellular response on Col of different origin. The results showed that the binding properties of transfected C2C12 cells to collagens depend on the identity of inserted integrin. Similar ligation characteristics were observed using α1+ and α10+ cells, but these were distinct from the similar binding features of α2+ and α11+ cells. Recombinant human and rat-α1 I domain binding to collagens and peptides correlated with the cell adhesion results, showing receptor class- and species-specificities. The understanding of the physiologically relevant cell anchorage characteristics of bio-constructs may assist in the selection of (1) the optimum collagen source for cellular supports and (2) the correct cellular model for their biological assessment. This, in turn, may allow reliable prediction of the biological performance of bio-scaffolds in vivo for specific TE applications. STATEMENT OF SIGNIFICANCE: Integrins play a vital role in cellular responses to environmental cues during early-stage cell-substrate interaction. We describe physiologically relevant cell anchorage to collagen substrates that present different affinity cell-recognition motifs, to provide experimental tools to assist in understanding integrin binding. Using different cell types and recombinant integrin α1-I-domains, we found that cellular response was highly dependent on collagen type, origin and EDC-crosslinking status, as well as on the integrin class and species of origin. This comprehensive study establishes selectivity amongst the four collagen-binding integrins and species-specific properties that together may influence choice of cell type and receptor in different experimental settings. This work offers key guidance in selecting of the correct cellular model for the biological testing of collagen-based biomaterials.

Production of a fluorescence resonance energy transfer (FRET) biosensor membrane for microRNA detection.

MicroRNAs (miRNAs) play a key role in regulating gene expression but can be associated with abnormalities linked to carcinogenesis and tumor progression. Hence there is increasing interest in developing methods to detect these non-coding RNA molecules in the human circulation system. Here, a novel FRET miRNA-195 targeting biosensor, based on silica nanofibers incorporated with rare earth-doped calcium fluoride particles (CaF2:Yb,Ho@SiO2) and gold nanoparticles (AuNPs), is reported. The formation of a sandwich structure, as a result of co-hybridization of the target miRNA which is captured by oligonucleotides conjugated at the surface of CaF2:Yb,Ho@SiO2 fibers and AuNPs, brings the nanofibers and AuNPs in close proximity and triggers the FRET effect. The intensity ratio of green to red emission, I541/I650, was found to decrease linearly upon increasing the concentration of the target miRNA and this can be utilized as a standard curve for quantitative determination of miRNA concentration. This assay offers a simple and convenient method for miRNA quantification, with the potential for rapid and early clinical diagnosis of diseases such as breast cancer.

Luminescent CaTiO3:Yb,Er nanofibers co-conjugated with Rose Bengal and gold nanorods for potential synergistic photodynamic/photothermal therapy.

Photodynamic therapy (PDT) and photothermal therapy (PTT) have been explored widely for application in cancer treatment. In this work, we describe the synthesis of CaTiO3:Yb,Er (CTO) nanofibers co-conjugated with Rose Bengal (RB) and gold nanorods (AuNRs), which offer the potential for combined upconversion photoluminescence (UCPL) and enhanced, synergistic PDT and PTT. Based on this delivery platform, RB and AuNRs served as the PDT and PTT agents, respectively. RB and AuNRs have strong and well-matched absorption with the green and red emissions of UCPL CTO nanofibers respectively, hence a single 980 nm continuous wave laser with deep tissue penetration can be employed to allow PDT and PTT to occur simultaneously. The nanocomposite can effectively convert the near-infrared (NIR) radiation from the laser into a combination of targeted hyperthermia and generation of reactive oxygen species (ROS). In comparison with PDT or PTT alone, the combined PDT/PTT treatment showed significantly enhanced suppression of the viability of Hep G2 cells in vitro, demonstrating its potential for use in oncology.

The Mechanics of Brow-Suspension Ptosis Repair: A Comparative Study of Fox Pentagon and Crawford Triangle Techniques.

PURPOSE: To perform quantitative analysis of the most commonly used brow-suspension configurations. METHODS: The inflection positions for Fox pentagon and Crawford triangle configurations were marked on 49 healthy volunteers (male and female) and photographs taken in 3 states: "normal," "closed," and "raised." The skin marks were measured vectorially with respect to the medial canthus, and displacement changes were evaluated for "normal-to-closed" ("blinking") and from "closed-to-raised" ("eye-opening") states. The distance between a pair of inflection marks, representing the approximate path of sling configurations, were also measured and analyzed in relation to the mechanical properties of a variety of synthetic brow-suspension materials. RESULTS: "Blinking" resulted in the greatest displacement in the medial eyelid incision, resulting in the greatest strain on the line connecting the medial eyelid and medial brow inflections. No significant differences in the strains for individual lines were found between the Fox and Crawford techniques, although the former shows a significantly lower overall strain in the whole loop than the latter. The displacements of some inflections and of the strains of a few lines differed significantly in men and women. CONCLUSIONS: Within the scope of this study, the blinking action was shown to result in the maximum strain of ~40%, which lies within the elastic region of stress-strain curves for some commonly used synthetic brow-suspension materials. No one method was statistically superior, although the Fox pentagon gave a significantly lower overall strain when the sling material was assumed to move somewhat around the inflections within a closed loop.

Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry.

Studies of cell attachment to collagen-based materials often ignore details of the binding mechanisms-be they integrin-mediated or non-specific. In this work, we have used collagen and gelatin-based substrates with different dimensional characteristics (monolayers, thin films and porous scaffolds) in order to establish the influence of composition, crosslinking (using carbodiimide) treatment and 2D or 3D architecture on integrin-mediated cell adhesion. By varying receptor expression, using cells with collagen-binding integrins (HT1080 and C2C12 L3 cell lines, expressing α2ß1, and Rugli expressing α1ß1) and a parent cell line C2C12 with gelatin-binding receptors (αvß3 and α5ß1), the nature of integrin binding sites was studied in order to explain the bioactivity of different protein formulations. We have shown that alteration of the chemical identity, conformation and availability of free binding motifs (GxOGER and RGD), resulting from addition of gelatin to collagen and crosslinking, have a profound effect on the ability of cells to adhere to these formulations. Carbodiimide crosslinking ablates integrin-dependent cell activity on both two-dimensional and three-dimensional architectures while the three-dimensional scaffold structure also leads to a high level of non-specific interactions remaining on three-dimensional samples even after a rigorous washing regime. This phenomenon, promoted by crosslinking, and attributed to cell entrapment, should be considered in any assessment of the biological activity of three-dimensional substrates. Spreading data confirm the importance of integrin-mediated cell engagement for further cell activity on collagen-based compositions. In this work, we provide a simple, but effective, means of deconvoluting the effects of chemistry and dimensional characteristics of a substrate, on the cell activity of protein-derived materials, which should assist in tailoring their biological properties for specific tissue engineering applications.

The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen.

Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2ß1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2ß1 and α1ß1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons.

Optimisation of UV irradiation as a binding site conserving method for crosslinking collagen-based scaffolds.

Short wavelength (λ = 254 nm) UV irradiation was evaluated over a range of intensities (0.06 to 0.96 J/cm(2)) as a means of cross-linking collagen- and gelatin-based scaffolds, to tailor their material characteristics whilst retaining biological functionality. Zero-link carbodiimide treatments are commonly applied to collagen-based materials, forming cross-links from carboxylate anions (for example the acidic E of GFOGER) that are an essential part of integrin binding sites on collagen. Cross-linking these amino acids therefore disrupts the bioactivity of collagen. In contrast, UV irradiation forms bonds from less important aromatic tyrosine and phenylalanine residues. We therefore hypothesised that UV cross-linking would not compromise collagen cell reactivity. Here, highly porous (~99 %) isotropic, collagen-based scaffolds were produced via ice-templating. A series of scaffolds (pore diameters ranging from 130-260 µm) with ascending stability in water was made from gelatin, two different sources of collagen I, or blends of these materials. Glucose, known to aid UV crosslinking of collagen, was added to some lower-stability formulations. These scaffolds were exposed to different doses of UV irradiation, and the scaffold morphology, dissolution stability in water, resistance to compression and cell reactivity was assessed. Stabilisation in aqueous media varied with both the nature of the collagen-based material employed and the UV intensity. Scaffolds made from the most stable materials showed the greatest stability after irradiation, although the levels of cross-linking in all cases were relatively low. Scaffolds made from pure collagen from the two different sources showed different optimum levels of irradiation, suggesting altered balance between stabilisation from cross-linking and destabilisation from denaturation. The introduction of glucose into the scaffold enhanced the efficacy of UV cross-linking. Finally, as hypothesized, cell attachment, spreading and proliferation on collagen materials were unaffected by UV cross-linking. UV irradiation may therefore be used to provide relatively low level cross-linking of collagen without loss of biological functionality.

Microstructure and mechanical properties of synthetic brow-suspension materials.

Levator palpebrae superioris (LPS) is a muscle responsible for lifting the upper eyelid and its malfunction leads to a condition called "ptosis", resulting in disfigurement and visual impairment. Severe ptosis is generally treated with "brow-suspension" surgery, whereby the eyelid is cross-connected to the mobile tissues above the eyebrow using a cord-like material, either natural (e.g. fascia lata harvested from the patient) or a synthetic cord. Synthetic brow-suspension materials are widely used, due to not requiring the harvesting of fascia lata that can be associated with pain and donor-site complications. The mechanical properties of some commonly-used synthetic brow-suspension materials were investigated--namely, monofilament polypropylene (Prolene®), sheathed braided polyamide (Supramid Extra® II), silicone frontalis suspension rod (Visitec® Seiff frontalis suspension set), woven polyester (Mersilene® mesh), and expanded polytetrafluoroethylene (Ptose-Up). Each material underwent a single tensile loading to the failure of the material, at three different displacement rates (1, 750 and 1500 mm/min). All the materials exhibited elastic-plastic tensile stress-strain behaviour with considerable differences in elastic modulus, ultimate tensile strength, elastic limit and work of fracture. The results suggest that, as compared to other materials, the silicone brow-suspension rod (Visitec® SFSS) might be the most suitable, providing relatively long-lasting stability and desirable performance. These findings, together with other factors such as commercial availability, cost and clinical outcomes, will provide clinicians with a more rational basis for selection of brow-suspension materials.

Collagen fibre implant for tendon and ligament biological augmentation. In vivo study in an ovine model.

PURPOSE: Although most in vitro studies indicate that collagen is a suitable biomaterial for tendon and ligament tissue engineering, in vivo studies of implanted collagen for regeneration of these tissues are still lacking. The objectives of this study were the following: (1) to investigate the regeneration of the central third of the ovine patellar tendon using implants made of an open array of collagen fibres (reconstituted, extruded bovine collagen); and (2) to compare two collagen crosslinking chemistries: carbodiimide and carbodiimide associated with ethyleneglycoldiglycidylether. METHODS: Forty-eight Welsh Mountain sheep were operated on their right hind leg. The central third of patellar tendon was removed and substituted with carbodiimide (n = 16) and carbodiimide-ethyleneglycoldiglycidylether-crosslinked implants (n = 16). In the control group the defect was left empty (n = 16). The central third of contralateral unoperated tendons was used as positive controls. Half of the sheep in each group were killed at 3- and 6-month time points. After proper dissection, tendon sub-units (medial, central and lateral) were tested to failure (n = 6 for each group), whilst 2 non-dissected samples were used for histology. RESULTS: Both the implants had significantly lower stress to failure and modulus with respect to native tendon at both 3- and at 6-month time points. The implants did not statistically differ in stress to failure, whilst carbodiimide-crosslinked implants had significantly higher modulus than carbodiimide-ethyleneglycoldiglycidylether-crosslinked implants both at 3 and at 6 months. Histology showed carbodiimide-crosslinked implants to have a better integration with the native tendon than carbodiimide-ethyleneglycoldiglycidylether-crosslinked implants. Carbodiimide-crosslinked implants appeared partially resorbed and showed increased tissue ingrowth with respect to carbodiimide-ethyleneglycoldiglycidylether-crosslinked implants. CONCLUSIONS: To deliver collagen implants as an open array of fibres allows optimal tendon-implant integration and good ingrowth of regenerated tissue. In the present study the resorption rate of both the examined implants was too low due to the high level of crosslinking. This led to only minor substitution of the implant with regenerated tissue, which in turn produced a low-strength implanted region. Further studies are needed to find the right balance between strength and resorption rate of collagen fibres.