Accelerated degradation testing impacts the degradation processes in 3D printed amorphous PLLA.

Additive manufacturing and electrospinning are widely used to create degradable biomedical components. This work presents important new data showing that the temperature used in accelerated tests has a significant impact on the degradation process in amorphous 3D printed poly-l-lactic acid (PLLA) fibres. Samples (c. 100 µ m diameter) were degraded in a fluid environment at 37 ° C, 50 ° C and 80 ° C over a period of 6 months. Our findings suggest that across all three fluid temperatures, the fibres underwent bulk homogeneous degradation. A three-stage degradation process was identified by measuring changes in fluid pH, PLLA fibre mass, molecular weight and polydispersity index. At 37 ° C, the fibres remained amorphous but, at elevated temperatures, the PLLA crystallised. A short-term hydration study revealed a reduction in glass transition (Tg), allowing the fibres to crystallise, even at temperatures below the dry Tg. The findings suggest that degradation testing of amorphous PLLA fibres at elevated temperatures changes the degradation pathway which, in turn, affects the sample crystallinity and microstructure. The implication is that, although higher temperatures might be suitable for testing bulk material, predictive testing of the degradation of amorphous PLLA fibres (such as those produced via 3D printing or electrospinning) should be conducted at 37 ° C.

Characterizing collagen scaffold compliance with native myocardial strains using an ex-vivo cardiac model: The physio-mechanical influence of scaffold architecture and attachment method.

Applied to the epicardium in-vivo, regenerative cardiac patches support the ventricular wall, reduce wall stresses, encourage ventricular wall thickening, and improve ventricular function. Scaffold engraftment, however, remains a challenge. After implantation, scaffolds are subject to the complex, time-varying, biomechanical environment of the myocardium. The mechanical capacity of engineered tissue to biomimetically deform and simultaneously support the damaged native tissue is crucial for its efficacy. To date, however, the biomechanical response of engineered tissue applied directly to live myocardium has not been characterized. In this paper, we utilize optical imaging of a Langendorff ex-vivo cardiac model to characterize the native deformation of the epicardium as well as that of attached engineered scaffolds. We utilize digital image correlation, linear strain, and 2D principal strain analysis to assess the mechanical compliance of acellular ice templated collagen scaffolds. Scaffolds had either aligned or isotropic porous architecture and were adhered directly to the live epicardial surface with either sutures or cyanoacrylate glue. We demonstrate that the biomechanical characteristics of native myocardial deformation on the epicardial surface can be reproduced by an ex-vivo cardiac model. Furthermore, we identified that scaffolds with unidirectionally aligned pores adhered with suture fixation most accurately recapitulated the deformation of the native epicardium. Our study contributes a translational characterization methodology to assess the physio-mechanical performance of engineered cardiac tissue and adds to the growing body of evidence showing that anisotropic scaffold architecture improves the functional biomimetic capacity of engineered cardiac tissue. STATEMENT OF SIGNIFICANCE: Engineered cardiac tissue offers potential for myocardial repair, but engraftment remains a challenge. In-vivo, engineered scaffolds are subject to complex biomechanical stresses and the mechanical capacity of scaffolds to biomimetically deform is critical. To date, the biomechanical response of engineered scaffolds applied to live myocardium has not been characterized. In this paper, we utilize optical imaging of an ex-vivo cardiac model to characterize the deformation of the native epicardium and scaffolds attached directly to the heart. Comparing scaffold architecture and fixation method, we demonstrate that sutured scaffolds with anisotropic pores aligned with the native alignment of the superficial myocardium best recapitulate native deformation. Our study contributes a physio-mechanical characterization methodology for cardiac tissue engineering scaffolds.

Effect of Physicochemical Surface Properties of Silicon-Substituted Hydroxyapatite on Angiogenesis.

Synthetic hydroxyapatite (HA) is a widely studied bioceramic for bone tissue engineering (BTE) due to its similarity to the mineral component of bone. As bone mineral contains various ionic substitutions that play a crucial role in bone metabolism, the bioactivity of HA can be improved by adding small amounts of physiologically relevant ions into its crystal structure, with silicate-substituted HA (Si-HA) showing particularly promising results. Nevertheless, it remains unclear how distinct material characteristics influence the bioactivity due to the intertwined nature of surface properties. A coculture methodology was optimized and applied for in vitro quantification of the biological response. Initially, HA and Si-HA samples were produced and characterized. To compare the bioactivity of the samples, a method was developed to measure interactions in an increasingly complex environment, first including fibronectin (FN) adsorption and subsequently cell adhesion in mono and coculture using primary human osteoblasts (hOBs) and human dermal microvascular endothelial cells (HDMECs), with and without FN precoating. An experimental set-up was designed to assess to what extent different surface features of the samples contribute to the induced biological response. An 8-nm gold sputter coating was applied to eradicate the electrochemical differences and polishing and abrading was used to reduce the differences in surface topographies. Overall, 1.25 wt% Si-HA exhibited most nanoscale variations in surface potential. In terms of bioactivity, 1.25 wt% Si-HA samples induced the highest osteoblast attachment and vessel formation. Additionally, in vitro vessel formation was established on Si-HA surfaces using a hOB:HDMEC cell ratio of 70:30 and a methodology was established that enabled the assessment of the relative effect of topographical and electrochemical features induced by silicon substitution in the HA lattice on their bioactivity. It was found that the difference in the amount of protein attached to HA and 1.25 wt% Si-HA after 2 h was affected by topographical differences. Conversely, electrochemical differences induced different vessel-like structure formation in coculture with a FN precoating. Without an FN precoating, both topographical and electrochemical differences dictated the differences in angiogenic response. Overall, 1.25 wt% Si-HA surface features appear to induce the most favorable protein adsorption and cell adhesion in mono and coculture with and without FN precoating.

Controlling the Architecture of Freeze-Dried Collagen Scaffolds with Ultrasound-Induced Nucleation.

Collagen is a naturally occurring polymer that can be freeze-dried to create 3D porous scaffold architectures for potential application in tissue engineering. The process comprises the freezing of water in an aqueous slurry followed by sublimation of the ice via a pre-determined temperature-pressure regime and these parameters determine the arrangement, shape and size of the ice crystals. However, ice nucleation is a stochastic process, and this has significant and inherent limitations on the ability to control scaffold structures both within and between the fabrication batches. In this paper, we demonstrate that it is possible to overcome the disadvantages of the stochastic process via the use of low-frequency ultrasound (40 kHz) to trigger nucleation, on-demand, in type I insoluble bovine collagen slurries. The application of ultrasound was found to define the nucleation temperature of collagen slurries, precisely tailoring the pore architecture and providing important new structural and mechanistic insights. The parameter space includes reduction in average pore size and narrowing of pore size distributions while maintaining the percolation diameter. A set of core principles are identified that highlight the huge potential of ultrasound to finely tune the scaffold architecture and revolutionise the reproducibility of the scaffold fabrication protocol.

Extracellular macrostructure anisotropy improves cardiac tissue-like construct function and phenotypic cellular maturation.

Regenerative cardiac tissue is a promising field of study with translational potential as a therapeutic option for myocardial repair after injury, however, poor electrical and contractile function has limited translational utility. Emerging research suggests scaffolds that recapitulate the structure of the native myocardium improve physiological function. Engineered cardiac constructs with anisotropic extracellular architecture demonstrate improved tissue contractility, signaling synchronicity, and cellular organization when compared to constructs with reduced architectural order. The complexity of scaffold fabrication, however, limits isolated variation of individual structural and mechanical characteristics. Thus, the isolated impact of scaffold macroarchitecture on tissue function is poorly understood. Here, we produce isotropic and aligned collagen scaffolds seeded with embryonic stem cell derived cardiomyocytes (hESC-CM) while conserving all confounding physio-mechanical features to independently assess the effects of macroarchitecture on tissue function. We quantified spatiotemporal tissue function through calcium signaling and contractile strain. We further examined intercellular organization and intracellular development. Aligned tissue constructs facilitated improved signaling synchronicity and directional contractility as well as dictated uniform cellular alignment. Cells on aligned constructs also displayed phenotypic and genetic markers of increased maturity. Our results isolate the influence of scaffold macrostructure on tissue function and inform the design of optimized cardiac tissue for regenerative and model medical systems.

Mimicking Transmural Helical Cardiomyofibre Orientation Using Bouligand-like Pore Structures in Ice-Templated Collagen Scaffolds.

The helical arrangement of cardiac muscle fibres underpins the contractile properties of the heart chamber. Across the heart wall, the helical angle of the aligned fibres changes gradually across the range of 90-180°. It is essential to recreate this structural hierarchy in vitro for developing functional artificial tissue. Ice templating can achieve single-oriented pore alignment via unidirectional ice solidification with a flat base mould design. We hypothesise that the orientation of aligned pores can be controlled simply via base topography, and we propose a scalable base design to recapitulate the transmural fibre orientation. We have utilised finite element simulations for rapid testing of base designs, followed by experimental confirmation of the Bouligand-like orientation. X-ray microtomography of experimental samples showed a gradual shift of 106 ± 10°, with the flexibility to tailor pore size and spatial helical angle distribution for personalised medicine.

In vitro angiogenesis in response to biomaterial properties for bone tissue engineering: a review of the state of the art.

Bone tissue engineering (BTE) aims to improve the healing of bone fractures using scaffolds that mimic the native extracellular matrix. For successful bone regeneration, scaffolds should promote simultaneous bone tissue formation and blood vessel growth for nutrient and waste exchange. However, a significant challenge in regenerative medicine remains the development of grafts that can be vascularized successfully. Amongst other things, optimization of physicochemical conditions of scaffolds is key to achieving appropriate angiogenesis in the period immediately following implantation. Calcium phosphates and collagen scaffolds are two of the most widely studied biomaterials for BTE, due to their close resemblance to inorganic and organic components of bone, respectively, and their bioactivity, tunable biodegradability and the ability to produce tailored architectures. While various strategies exist to enhance vascularization of these scaffolds in vivo, further in vitro assessment is crucial to understand the relation between physicochemical properties of a biomaterial and its ability to induce angiogenesis. While mono-culture studies can provide evidence regarding cell-material interaction of a single cell type, a co-culture procedure is crucial for assessing the complex mechanisms involved in angiogenesis. A co-culture more closely resembles the natural tissue both physically and biologically by stimulating natural intercellular interactions and mimicking the organization of the in vivo environment. Nevertheless, a co-culture is a complex system requiring optimization of various parameters including cell types, cell ratio, culture medium and seeding logistics. Gaining fundamental knowledge of the mechanism behind the bioactivity of biomaterials and understanding the contribution of surface and architectural features to the vascularization of scaffolds, and the biological response in general, can provide an invaluable basis for future optimization studies. This review gives an overview of the available literature on scaffolds for BTE, and trends are extracted on the relationship between architectural features, biochemical properties, co-culture parameters and angiogenesis.

Adjusting the physico-chemical properties of collagen scaffolds to accommodate primary osteoblasts and endothelial cells.

Collagen-based biomaterials are used widely as tissue engineering scaffolds because of their excellent bioactivity and their similarity to the natural ECM. The regeneration of healthy bone tissue requires simultaneous support for both osteoblasts and, where angiogenesis is intended, endothelial cells. Hence it is important to tailor carefully the biochemical and structural characteristics of the scaffold to suit the needs of each cell type. This work describes for the first time a systematic study to gain insight into the cell type-specific response of primary human osteoblast (hOBs) and human dermal microvascular endothelial cells (HDMECs) to insoluble collagen-based biomaterials. The behaviour was evaluated on both 2D films and 3D scaffolds, produced using freeze-drying. The collagen was cross-linked at various EDC/NHS concentrations and mono-cultured with hOBs and HDMECs to assess the effect of architectural features and scaffold stabilization on cell behaviour. It was observed that 3D scaffolds cross-linked at 30% of the standard conditions in literature offered an optimal combination of mechanical stiffness and cellular response for both cell types, although endothelial cells were more sensitive to the degree of cross-linking than hOBs. Architectural features have a time-dependent impact on the cell migration profile, with alignment being the most influential parameter overall.

Complex architectural control of ice-templated collagen scaffolds using a predictive model.

The architectural and physiomechanical properties of regenerative scaffolds have been shown to improve engineered tissue function at both a cellular and tissue level. The fabrication of regenerative three-dimensional scaffolds that precisely replicate the complex hierarchical structure of native tissue, however, remains a challenge. The aim of this work is therefore two-fold: i) demonstrate an innovative multidirectional freeze-casting system to afford precise architectural control of ice-templated collagen scaffolds; and ii) present a predictive simulation as an experimental design tool for bespoke scaffold architecture. We used embedded heat sources within the freeze-casting mold to manipulate the local thermal environment during solidification of ice-templated collagen scaffolds. The resultant scaffolds comprised complex and spatially varied lamellar orientations that correlated with the imposed thermal environment and could be readily controlled by varying the geometry and power of the heat sources. The complex macro-architecture did not interrupt the hierarchical features characteristic of ice-templated scaffolds, but pore orientation had a significant impact on the stiffness of resultant structures under compression. Furthermore, our finite element model (FEM) accurately predicted the thermal environment and illustrated the freezing front topography within the mold during solidification. The lamellar orientation of freeze-cast scaffolds was also predicted using thermal gradient vector direction immediately prior to phase change. In combination our FEM and bespoke freeze-casting system present an exciting opportunity for tailored architectural design of ice-templated regenerative scaffolds that mimic the complex hierarchical environment of the native extracellular matrix. STATEMENT OF SIGNIFICANCE: Biomimetic scaffold structure improves engineered tissue function, but the fabrication of three-dimensional scaffolds that precisely replicate the complex hierarchical structure of native tissue remains a challenge. Here, we leverage the robust relationship between thermal gradients and lamellar orientation of ice-templated collagen scaffolds to develop a multidirectional freeze-casting system with precise control of the thermal environment and consequently the complex lamellar structure of resultant scaffolds. Demonstrating the diversity of our approach, we identify heat source geometry and power as control parameters for complex lamellar orientations. We simultaneously present a finite element model (FEM) that describes the three-dimensional thermal environment during solidification and accurately predicts lamellar structure of resultant scaffolds. The model serves as a design tool for bespoke regenerative scaffolds.

Avoiding artefacts in MicroCT imaging of collagen scaffolds: Effect of phosphotungstic acid (PTA)-staining and crosslink density.

X-ray micro-computed tomography (µ-CT) can be used to provide both qualitative and quantitative information on the structure of three-dimensional (3D) bioactive scaffolds. When performed in a dry state, µ-CT accurately reflects the structure of collagen-based scaffolds, but imaging in a wet state offers challenges with radiolucency. Here we have used phosphotungstic acid (PTA) as a contrast agent to visualise fully hydrated collagen scaffolds in a physiologically relevant environment. A systematic investigation was performed to understand the effects of PTA on the results of µ-CT imaging by varying sample processing variables such as crosslinking density, hydration medium and staining duration. Immersing samples in 0.3% PTA solution overnight completely stained the samples and the treatment provided a successful route for µ-CT analysis of crosslinked samples. However, significant structural artefacts were observed for samples which were either non-crosslinked or had low levels of crosslinking, which had a heterogeneous interior architecture with collapsed pores at the scaffold periphery. This work highlights the importance of optimising the choice of processing and staining conditions to ensure accurate visualisation for hydrated 3D collagen scaffolds in an aqueous medium.

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.

A technique for improving dispersion within polymer-glass composites using polymer precipitation.

Particulate reinforcement of polymeric matrices is a powerful technique for tailoring the mechanical and degradation properties of bioresorbable implant materials. Dispersion of inorganic particles is critical to achieving optimal properties, however established techniques such as twin-screw extrusion or solvent casting can have significant drawbacks including excessive thermal degradation or particle agglomeration. We present a facile method for production of polymer-inorganic composites that reduces the time at elevated temperature and the time available for particle agglomeration. Glass slurry was added to a dissolved PLLA solution, and ethanol was added to precipitate polymer onto the glass particles. Characterisation of parts formed by subsequent micro-injection moulding of composite precipitate revealed a significant reduction in agglomeration, with d0.9 reduced from 170 to 43 µm. This drastically improved the ductility (ɛB) from 7% to 120%, without loss of strength or stiffness. The method is versatile and applicable to a wide range of polymer and filler materials.

Collagen Film Activation with Nanoscale IKVAV-Capped Dendrimers for Selective Neural Cell Response.

Biocompatible neural guidance conduits are alternatives to less abundant autologous tissue grafts for small nerve gap injuries. To address larger peripheral nerve injuries, it is necessary to design cell selective biomaterials that attract neuronal and/or glial cells to an injury site while preventing the intrusion of fibroblasts that cause inhibitory scarring. Here, we investigate a potential method for obtaining this selective cellular response by analysing the responses of rat Schwann cells and human dermal fibroblasts to isoleucine-lysine-valine-alanine-valine (IKVAV)-capped dendrimer-activated collagen films. A high quantity of nanoscale IKVAV-capped dendrimers incorporated onto pre-crosslinked collagen films promoted rat Schwann cell attachment and proliferation, and inhibited human dermal fibroblast proliferation. In addition, while pre-crosslinked dendrimer-activated films inhibited fibroblast proliferation, non-crosslinked dendrimer-activated films and films that were crosslinked after dendrimer-activation (post-crosslinked films) did not. The different cellular responses to pre-crosslinked and post-crosslinked films highlight the importance of having fully exposed, non-covalently bound biochemical motifs (pre-crosslinked films) directing certain cellular responses. These results also suggest that high concentrations of nanoscale IKVAV motifs can inhibit fibroblast attachment to biological substrates, such as collagen, which inherently attract fibroblasts. Therefore, this work points toward the potential of IKVAV-capped dendrimer-activated collagen biomaterials in limiting neuropathy caused by fibrotic scarring at peripheral nerve injury sites.

Tunable bioactivity and mechanics of collagen-based tissue engineering constructs: A comparison of EDC-NHS, genipin and TG2 crosslinkers.

Due to its ubiquity and versatility in the human body, collagen is an ideal base material for tissue-engineering constructs. Chemical crosslinking treatments allow precise control of the biochemical and mechanical properties through macromolecular modifications to the structure of collagen. In this work, three key facets regarding the collagen crosslinking process are explored. Firstly, a comparison is drawn between the carbodiimide-succinimide (EDC-NHS) system and two emerging crosslinkers utilising alternate chemistries: genipin and tissue transglutaminase (TG2). By characterising the chemical changes upon treatment, the effect of EDC-NHS, genipin and TG2 crosslinking mechanisms on the chemical structure of collagen, and thus the mechanical properties conferred to the substrate is explored. Secondly, the relative importance of mechanical and biochemical cues on cellular phenomena are investigated, including cell viability, integrin-specific attachment, spreading and proliferation. Here, we observe that for human dermal fibroblasts, long-term, stable proliferation is preconditioned by the availability of suitable binding sites, irrespective of the substrate modulus post-crosslinking. Finally, as seen in the graphical abstract we show that by choosing the appropriate crosslinker chemistries, a materials selection map can be drawn for collagen films, encompassing both a range of tensile modulus and fibroblast proliferation which can be modified independently. Thus, in addition to a range of parameters that can be modified in collagen constructs, we demonstrate a route to obtaining tunable bioactivity and mechanics in collagen constructs is uncovered, that is exclusively driven by the crosslinking process.

MicroCT analysis of connectivity in porous structures: optimizing data acquisition and analytical methods in the context of tissue engineering.

Micro-computed X-ray tomography (MicroCT) is one of the most powerful techniques available for the three-dimensional characterization of complex multi-phase or porous microarchitectures. The imaging and analysis of porous networks are of particular interest in tissue engineering due to the ability to predict various large-scale cellular phenomena through the micro-scale characterization of the structure. However, optimizing the parameters for MicroCT data capture and analyses requires a careful balance of feature resolution and computational constraints while ensuring that a structurally representative section is imaged and analysed. In this work, artificial datasets were used to evaluate the validity of current analytical methods by considering the effect of noise and pixel size arising from the data capture, and intrinsic structural anisotropy and heterogeneity. A novel 'segmented percolation method' was developed to exclude the effect of anomalous, non-representative features within the datasets, allowing for scale-invariant structural parameters to be obtained consistently and without manual intervention for the first time. Finally, an in-depth assessment of the imaging and analytical procedures are presented by considering percolation events such as micro-particle filtration and cell sieving within the context of tissue engineering. Along with the novel guidelines established for general pixel size selection for MicroCT, we also report our determination of 3 µm as the definitive pixel size for use in analysing connectivity for tissue engineering applications.

Poly-l-Lactic Acid Nanotubes as Soft Piezoelectric Interfaces for Biology: Controlling Cell Attachment via Polymer Crystallinity.

It has become increasingly evident that the mechanical and electrical environment of a cell is crucial in determining its function and the subsequent behavior of multicellular systems. Platforms through which cells can directly interface with mechanical and electrical stimuli are therefore of great interest. Piezoelectric materials are attractive in this context because of their ability to interconvert mechanical and electrical energy, and piezoelectric nanomaterials, in particular, are ideal candidates for tools within mechanobiology, given their ability to both detect and apply small forces on a length scale that is compatible with cellular dimensions. The choice of piezoelectric material is crucial to ensure compatibility with cells under investigation, both in terms of stiffness and biocompatibility. Here, we show that poly-l-lactic acid nanotubes, grown using a melt-press template wetting technique, can provide a "soft" piezoelectric interface onto which human dermal fibroblasts readily attach. Interestingly, by controlling the crystallinity of the nanotubes, the level of attachment can be regulated. In this work, we provide detailed nanoscale characterization of these nanotubes to show how differences in stiffness, surface potential, and piezoelectric activity of these nanotubes result in differences in cellular behavior.

Targeted protein delivery: carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds.

Tissue engineering response may be tailored via controlled, sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional (3D) ice-templated collagen scaffolds. However, the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored. Here, we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide) microparticles. We probe the effects of subsequent N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride crosslinking on protein release, using microparticles with different internal protein distributions. Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug. The scaffolds display a homogeneous microparticle distribution, and a reduction in pore size and percolation diameter with increased microparticle addition, although these values did not fall below those reported as necessary for cell invasion. The protein distribution within the microparticles, near the surface or more deeply located within the microparticles, was important in determining the release profile and effect of crosslinking, as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold. Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release. Protein located within the bulk of the microparticles, was protected from the crosslinking reaction and no delay in the overall release profile was seen.

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.

Self-assembly of collagen bundles and enhanced piezoelectricity induced by chemical crosslinking.

The piezoelectricity of collagen is purported to be linked to many biological processes including bone formation and wound healing. Although the piezoelectricity of tissue-derived collagen has been documented across the length scales, little work has been undertaken to characterise the local electromechanical properties of processed collagen, which is used as a base for tissue-engineering implants. In this work, three chemically distinct treatments used to form structurally and mechanically stable scaffolds-EDC-NHS, genipin and tissue transglutaminase-are investigated for their effect on collagen piezolectricity. Crosslinking with EDC-NHS is noted to produce a distinct self-assembly of the fibres into bundles roughly 300 nm in width regardless of the collagen origin. These fibre bundles also show a localised piezoelectric response, with enhanced vertical piezoelectricity of collagen. Such topographical features are not observed with the other two chemical treatments, although the shear piezoelectric response is significantly enhanced upon crosslinking. These observations are reconciled by a proposed effect of the crosslinking mechanisms on the molecular and nanostructure of collagen. These results highlight the ability to modify the electromechanical properties of collagen using chemical crosslinking methods.

Fabrication of free standing collagen membranes by pulsed-electrophoretic deposition.

This work reports an important new development in the production of collagen membranes, based on pulsed electrophoretic deposition (P-EPD), suitable for a wide range of biomedical applications. Collagen membranes are of great interest as a biomaterial and in a range of other industries, though current production techniques suffer from limitations with scaling up, homogeneity, and complex shapes. P-EPD can be used to rapidly create detachable, large-area, homogeneous products with controlled thickness in a wide variety of shapes. We provide a new understanding of the influence of a range of parameters (pulse width, voltage, duty cycle, solvent additions) and their effects on membrane structure. Characterisation by AFM, SEM, and cryoSEM revealed the ability to produce dense, structurally defect-free membranes, and significantly, we show and discuss the ability to produce thicker membranes by sequential deposition without seeing a corresponding increase in cell electrical resistance. We anticipate this novel, rapid, and controllable method for the production of collagen membranes to be of interest for a wide range of fields.