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.

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.

Plasma processing of PDMS based spinal implants for covalent protein immobilization, cell attachment and spreading.

PDMS is widely used for prosthetic device manufacture. Conventional ion implantation is not a suitable treatment to enhance the biocompatibility of poly dimethyl siloxane (PDMS) due to its propensity to generate a brittle silicon oxide surface layer which cracks and delaminates. To overcome this limitation, we have developed new plasma based processes to balance the etching of carbon with implantation of carbon from the plasma source. When this carbon was implanted from the plasma phase it resulted in a surface that was structurally similar and intermixed with the underlying PDMS material and not susceptible to delamination. The enrichment in surface carbon allowed the formation of carbon based radicals that are not present in conventional plasma ion immersion implantation (PIII) treated PDMS. This imparts the PDMS surfaces with covalent protein binding capacity that is not observed on PIII treated PDMS. The change in surface energy preserved the function of bound biomolecules and enhanced the attachment of MG63 osteosarcoma cells compared to the native surface. The attached cells, an osteoblast interaction model, showed increased spreading on the treated over untreated surfaces. The carbon-dependency for these beneficial covalent protein and cell linkage properties was tested by incorporating carbon from a different source. To this end, a second surface was produced where carbon etching was balanced against implantation from a thin carbon-based polymer coating. This had similar protein and cell-binding properties to the surfaces generated with carbon inclusion in the plasma phase, thus highlighting the importance of balancing carbon etching and deposition. Additionally, the two effects of protein linkage and bioactivity could be combined where the cell response was further enhanced by covalently tethering a biomolecule coating, as exemplified here with the cell adhesive protein tropoelastin. Providing a balanced carbon source in the plasma phase is applicable to prosthetic device fabrication as illustrated using a 3-dimensional PDMS balloon prosthesis for spinal implant applications. Consequently, this study lays the groundwork for effective treatments of PDMS to selectively recruit cells to implantable PDMS fabricated biodevices.

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.

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.

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.

Cell patterning via linker-free protein functionalization of an organic conducting polymer (polypyrrole) electrode.

The interaction of proteins and cells with polymers is critical to their use in scientific and medical applications. In this study, plasma immersion ion implantation (PIII) was used to modify the surface of the conducting polymer, polypyrrole, which possesses electrical properties. PIII treatment enabled persistent, covalent binding of the cell adhesive protein, tropoelastin, without employing chemical linking molecules. In contrast tropoelastin was readily eluted from the untreated surface. Through this differential persistence of binding, surface bound tropoelastin supported cell adhesion and spreading on the PIII treated but not the untreated polypyrrole surface. The application of a steel shadow mask during PIII treatment allowed for spatial definition of tropoelastin exclusively to PIII treated regions. The general applicability of this approach to other extracellular matrix proteins was illustrated using collagen I, which displayed similar results to tropoelastin but required extended washing conditions. This approach allowed fine patterning of cell adhesion and spreading to tropoelastin and collagen, specifically on PIII treated polypyrrole regions. We therefore present a methodology to alter the functionality of polypyrrole surfaces, generating surfaces that can spatially control cellular interactions through protein functionalization with the potential for electrical stimulation.

Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells.

Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture.

Integrin alpha5beta1 and ADAM-17 interact in vitro and co-localize in migrating HeLa cells.

Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.

Cell adhesion to fibrillin-1 molecules and microfibrils is mediated by alpha 5 beta 1 and alpha v beta 3 integrins.

Fibrillins are the major glycoprotein components of microfibrils that form a template for tropoelastin during elastic fibrillogenesis. We have examined cell adhesion to assembled purified microfibrils, and its molecular basis. Human dermal fibroblasts exhibited Arg-Gly-Asp and cation-dependent adhesion to microfibrils and recombinant fibrillin-1 protein fragments. Strong integrin alpha 5 beta 1 interactions with fibrillin ligands were identified, but integrin alpha v beta 3 also contributed to cell adhesion. Fluorescence-activated cell sorting analysis confirmed the presence of abundant alpha 5 beta 1 and some alpha v beta 3 receptors on these cells. Adhesion to microfibrils and to Arg-Gly-Asp containing fibrillin-1 protein fragments induced signaling events that led to cell spreading, altered cytoskeletal organization, and enhanced extracellular fibrillin-1 deposition. Differences in cell shape when plated on fibrillin or fibronectin implied substrate-specific alpha 5 beta 1-mediated cellular responses. An Arg-Gly-Asp-independent cell adhesion sequence was also identified within fibrillin-1. Adhesion and spreading of smooth muscle cells on fibrillin ligands was enhanced by antibody-induced beta1 integrin activation. A375-SM melanoma cells bound Arg-Gly-Asp-containing fibrillin-1 protein fragments mainly through alpha v beta 3, whereas HT1080 cells used mainly alpha 5 beta 1. This study has shown that fibrillin microfibrils mediate cell adhesion, that alpha 5 beta 1 and alpha v beta 3 are both important but cell-specific fibrillin-1 receptors, and that cellular interactions with fibrillin-1 influence cell behavior.