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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.

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.

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 α2β1, 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.

Funder: Department of Materials Science and Metallurgy, University of Cambridge; Id: http://dx.doi.org/10.13039/501100006299, Summary: Micro���CT is often used to assess the characteristics of porous structures such as tissue engineering scaffolds and trabecular bone. Prior to analysis, micro���CT images can be thresholded and filtered to remove noise. Scaffold pore size affects mechanical properties and biological cell behaviour and is a frequently assessed parameter. This paper identifies and characterizes an artefact affecting a commonly used filter which erroneously increases mean pore size. The 3D sweep despeckling filter removes all but the largest object within a volume of interest, and therefore deletes any disconnected objects located at the periphery, increasing measured mean pore size. This artefact is characterized, and effective methods to mitigate its effects are devised, involving despeckling a sufficiently large volume of interest, then reducing the volume of interest in size to remove the error prior to analysis. Techniques to effectively apply this method to other data sets are described. This method eliminates the artefact but is time���consuming and computationally expensive. Alternative, more economical filters which remove objects below a specified size are also assessed but are shown to be affected by the same artefact. These results will help to guide the implementation of future studies investigating the effects of pore size.

Micro-CT is often used to assess the characteristics of porous structures such as tissue engineering scaffolds and trabecular bone. Prior to analysis, micro-CT images can be thresholded and filtered to remove noise. Scaffold pore size affects mechanical properties and biological cell behaviour and is a frequently assessed parameter. This paper identifies and characterizes an artefact affecting a commonly used filter which erroneously increases mean pore size. The 3D sweep despeckling filter removes all but the largest object within a volume of interest, and therefore deletes any disconnected objects located at the periphery, increasing measured mean pore size. This artefact is characterized, and effective methods to mitigate its effects are devised, involving despeckling a sufficiently large volume of interest, then reducing the volume of interest in size to remove the error prior to analysis. Techniques to effectively apply this method to other data sets are described. This method eliminates the artefact but is time-consuming and computationally expensive. Alternative, more economical filters which remove objects below a specified size are also assessed but are shown to be affected by the same artefact. These results will help to guide the implementation of future studies investigating the effects of pore size.

Fused filament fabrication (FFF) is an inexpensive way of producing objects through a programmed layer-by-layer deposition. For multi-layer, macro-scaled prints, acceptable printing is achieved provided, amongst other factors, first layer adhesion is sufficient to fix a part to the surface during printing. However, in the deposition of structures with a single or few layers, first layer consistency is significantly more important and is an issue that has been previously overlooked. As layer-to-bed adhesion is prioritised in first layer printing, thin layer structures are difficult to remove without damage. The deposition of controllable thin structures has potential in tissue engineering through the use of bioactive filaments and incorporation of microfeatures into complex, patient-specific scaffolds. This paper presents techniques to progress the deposition of thin, reproducible structures. The linear thickness variation of 3D-printed single PVA and PLLA layers is presented as a function of extrusion factor and the programmed vertical distance moved by the nozzle between layers (the layer separation). A sacrificial PVA layer is shown to significantly improve first layer consistency, reducing the onus on fine printer calibration in the deposition of single layers. In this way, the linear variation in printed single PLLA layers with bed deviation is drastically reduced. Further, this technique is used to demonstrate the printing of freestanding thin layers of ~25 µm in thickness.

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 d 0 . 9 reduced from 170 to 43 μ m. This drastically improved the ductility ( ɛ B ) 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.

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., Graphical abstract Image 1

Cardiovascular diseases (CVD) constitute a major fraction of the current major global diseases and lead to about 30% of the deaths, i.e., 17.9 million deaths per year. CVD include coronary artery disease (CAD), myocardial infarction (MI), arrhythmias, heart failure, heart valve diseases, congenital heart disease, and cardiomyopathy. Cardiac Tissue Engineering (CTE) aims to address these conditions, the overall goal being the efficient regeneration of diseased cardiac tissue using an ideal combination of biomaterials and cells. Various cells have thus far been utilized in pre-clinical studies for CTE. These include adult stem cell populations (mesenchymal stem cells) and pluripotent stem cells (including autologous human induced pluripotent stem cells or allogenic human embryonic stem cells) with the latter undergoing differentiation to form functional cardiac cells. The ideal biomaterial for cardiac tissue engineering needs to have suitable material properties with the ability to support efficient attachment, growth, and differentiation of the cardiac cells, leading to the formation of functional cardiac tissue. In this review, we have focused on the use of biomaterials of natural origin for CTE. Natural biomaterials are generally known to be highly biocompatible and in addition are sustainable in nature. We have focused on those that have been widely explored in CTE and describe the original work and the current state of art. These include fibrinogen (in the context of Engineered Heart Tissue, EHT), collagen, alginate, silk, and Polyhydroxyalkanoates (PHAs). Amongst these, fibrinogen, collagen, alginate, and silk are isolated from natural sources whereas PHAs are produced via bacterial fermentation. Overall, these biomaterials have proven to be highly promising, displaying robust biocompatibility and, when combined with cells, an ability to enhance post-MI cardiac function in pre-clinical models. As such, CTE has great potential for future clinical solutions and hence can lead to a considerable reduction in mortality rates due to CVD.

Collagen-based constructs have emerged in recent years as ideal candidates for tissue engineering implants. For many biomedical applications, collagen is crosslinked in order to improve the strength, stiffness and stability of the construct. However, the crosslinking process may also result in unintended changes to cell viability, adhesion or proliferation on the treated structures. This review provides a brief overview of some of both the most commonly used and novel crosslinkers used with collagen, and suggests a framework by which crosslinking methods can be compared and selected for a given tissue engineering application.

Phosphate glasses for bioresorbable implants display dissolution rates that vary significantly with composition, however currently their mechanisms of dissolution are not well understood. Based on this systematic study we present new insights into these mechanisms. Two-stage dissolution was observed, with time dependence initially parabolic and later linear, and a two-stage model was developed to describe this behaviour. Dissolution was accelerated by lower Ca concentration in the glass, and lower pH in the dissolution medium. A new dissolution mechanism is proposed, involving an initial stage where diffusion-controlled formation of a conversion layer occurs. Once the conversion layer is stabilised, layer dissolution reactions become rate-limiting. Under this mechanism the transition time is sensitive to the nature of the conversion layer and solution conditions. These results reveal the dependence of P$_{2}$O$_{5}$-CaO-Na$_{2}$O glass dissolution on solution pH, and provide new insight into the dissolution mechanisms, particularly regarding the transition between the two dissolution stages., 21 pages, 17 figures

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., EPSRC, Geistlich Pharma AG, Bill and Melinda Gates Foundation

Porous biomaterials which provide a structural and biological support for cells have immense potential in tissue engineering and cell-based therapies for tissue repair. Collagen biomaterials that can host endothelial cells represent promising tools for the vascularization of engineered tissues. Three-dimensional collagen scaffolds possessing controlled architecture and mechanical stiffness are obtained through freeze–drying of collagen suspensions, followed by chemical cross-linking which maintains their stability. However, cross-linking scaffolds renders their biological activity suboptimal for many cell types, including human umbilical vein endothelial cells (HUVECs), by inhibiting cell–collagen interactions. Here, we have improved crucial HUVEC interactions with such cross-linked collagen biomaterials by covalently coupling combinations of triple-helical peptides (THPs). These are ligands for collagen-binding cell-surface receptors (integrins or discoidin domain receptors) or secreted proteins (SPARC and von Willebrand factor). THPs enhanced HUVEC adhesion, spreading and proliferation on 2D collagen films. THPs grafted to 3D-cross-linked collagen scaffolds promoted cell survival over seven days. This study demonstrates that THP-functionalized collagen scaffolds are promising candidates for hosting endothelial cells with potential for the production of vascularized engineered tissues in regenerative medicine applications.

Diffusion of nutrients to cells cultured within three-dimensional scaffolds is fundamental for cell survival during development of the tissue construct, when no vasculature is present to aid transport. Significant efforts have been made to characterize the effect of structure on solute diffusivity in nanoporous hydrogels, yet a similar thorough characterization has not been attempted for microporous scaffolds. Here, we make use of freeze-dried collagen scaffolds, possessing pore sizes in the range 150–250 μm and isotropic or aligned morphology, to study the diffusivity of fluorescent dextran molecules. Fluorescence recovery after photobleaching is used to measure the self diffusivity of the solutes within single pores, while Fickian diffusion over scales larger than the pore size is studied by assessing the solute concentration profile within the materials over time. We show that, not only do the morphological parameters of the scaffolds significantly affect the diffusivity of the solutes, but also that the assessment of such diffusivity depends on the length scale of diffusion of the molecules under investigation, with the resulting diffusion coefficients being differently affected by the scaffold structure. The results provided can guide the design of scaffolds with tailored diffusivity and nutrient concentration profiles.

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.

Micro-computed X-Ray Tomography (MicroCT) is one of the most powerful techniques available for the three dimensional characterisation 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 characterisation of the structure. However, optimising the parameters for MicroCT data capture and analyses requires a careful balance of feature resolution and computational constraints whilst 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.

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.

Bioresorbable composites consist of micro- and nano-sized α-tricalcium phosphate (α-TCP) particles in a poly(D,L-lactic-co-glycolic acid) (PLGA) matrix were compared for their ability to form bone-like apatite in simulated body fluid (SBF). Due to the uniform distribution of α-TCP nanoparticles over the nanocomposite surface, enhanced apatite formation was observed. Compared with microcomposite with the same α-TCP load, this enhanced apatite formation was achieved through faster and more uniform apatite nucleation. In the first stage of apatite formation, the presence of abundant high energy boundaries between α-TCP nanoparticles and PLGA matrix in nanocomposite provided a large number of suitable sites for calcium phosphate (CaP) nucleation. A homogenous distribution fo CaP nuclei formed after days. The resulting apatite crystals grew to form a flake-like apatite layer. In contrast, CaP nucleation was only observed on the micrometre-size α-TCP particles in the microcomposite over the same period. After 14 days, a dense, flake-like apatite was visible covering the surface of nanocomposite, whilst this suface layer was formed only on α-TCP particles of the microcomposite.

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.

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.

The aim of this study is to explore the importance of the potentially competing effects of buffering effects of the calcium phosphate filler and particle-mediated water sorption on the degradation products of poly(d,l lactide-co-glycolide (50:50))(PLGA)/hydroxyapatite(HA) composites. Further the influence of type of HA on the mechanical properties of the composites was investigated. Phase pure HA was synthesised via a reaction between aqueous solutions of calcium hydroxide and orthophosphoric acid. The powder produced was either used as produced (uncalcined) or calcined in air or calcined in a humidified argon atmosphere. An in-vitro degradation study was carried out in phosphate buffered saline (PBS). The results obtained indicated that the degradation rate of the composite might be better understood if both the buffering effects and the rate of water sorption by the composites are considered.

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.

Adipocytes are one of the major stromal cell components of the human breast. These cells play a key role in the development of the gland and are implicated in breast tumorigenesis. Frequently, directional stromal collagen I fibers are found surrounding aggressive breast tumors. These fibers enhance breast cancer cell migration and are associated with poor patient prognosis. We sought to recapitulate these stromal components in vitro to provide a three-dimensional (3D) model comprising human adipose tissue and anisotropic collagen fibers. We developed a human mesenchymal stem cell (hMSC) cell line capable of undergoing differentiation into mature adipocytes by immortalizing hMSCs, isolated from breast reduction mammoplasties, through retroviral transduction. These immortalized hMSCs were seeded in engineered collagen I scaffolds with directional internal architecture, and adipogenesis was chemically induced, resulting in human adipose tissue being synthesized in vitro in an architectural structure associated with breast tumorigenesis. Subsequently, fluorescently labeled cells from an established breast cancer cell line were seeded into this model, cocultured for 7 days and imaged using multiphoton microscopy. Enhanced breast cancer cell migration was observed in the adipose-containing model over empty scaffold controls, demonstrating an adipocyte-mediated influence on breast cancer cell migration. Thus, this 3D in vitro model recapitulates the migratory effects of adipocytes observed on breast cancer cells and suggests that it could have utility with fresh breast tumor biopsies as an assay for cancer therapeutic efficacy in personalized medicine strategies.

The degradation characteristics of αTCP-PLGA(50:50) nanocomposites containing varying ceramic weight loadings in an aqueous medium have been assessed using X-ray microtomography (XμT). Also measured were bulk density changes, pharmaceutic drug release and medium acidification for the degrading materials. Calcium phosphate addition to the polymer leads to increasing delays in the onset of degradation medium acidification and tetracycline release. Bulk density changes with time for all composite materials measured using a buoyancy method were well described during the initial degradation regime by a t1/2function. PLGA density evolution follows a linear function of time which indicates a differing water absorption process occurring in the pure polymer compared with the nanocomposites. Nanocomposite microtomographic analysis over the same period elucidated a core-periphery structure caused by water imbibition. Peripheral regions closest to the specimen surface exhibit reduced attenuation coefficients compared with the core which may be characteristic of a frontal system caused by a polymer phase transition. The front position and specimen swelling are adequately described by a t1/2and complementary error function respectively which if assessed under the assumption of a diffusion controlled process yields a diffusion coefficient of water in all nanocomposites at 37 °C of 4.8 × 10-14cm2s-1. Nevertheless, a t dependence is a necessary but not sufficient condition of a Fickian diffusion process. For all nanocomposite types both XμT data and bulk density measurements exhibited no variations with ceramic filler content.

Collagen scaffolds are widely used in a range of tissue engineering applications, both in vitro and in vivo, where their permeability to fluid flow greatly affects their mechanical and biological functionality. This paper reports new insights into the interrelationships between permeability, scaffold structure, fluid pressure and deformation in collagen scaffolds, focussing in particular on the degree of closure and the alignment of the pores. Isotropic and aligned scaffolds of different occlusivity were produced by ice templating, and were characterised in terms of their structure and mechanical properties. Permeability studies were conducted using two experimental set-ups to cover a wide range of applied fluid pressures. The permeability was found to be constant at low pressures for a given scaffold with more open structures and aligned structures being more permeable. The deformation of scaffolds under high pressure led to a decrease in permeability. The aligned structures were more responsive to deformation than their isotropic equivalents with their permeability falling more quickly at low strain. For isotropic samples, a broad (1 − ɛ)2 dependence for permeability was observed with the constant of proportionality varying with collagen fraction as the starting structures became more occluded. Aligned scaffolds did not follow the same behaviour, with the pores apparently closing more quickly in response to early deformation. These results highlight the importance of scaffold structure in determining permeability to interstitial fluid, and provide an understanding of scaffold behaviour within the complex mechanical environment of the body. Statement of significance Collagen scaffolds are widely used in tissue engineering applications, for instance to contribute with wound healing. Their permeability to fluid flow, such as water and blood, is important to ensure they perform efficiently when inside the body. The present study reports new insights into the relationships between permeability, scaffold structure, fluid pressure and deformation in collagen scaffolds. It presents in particular the experimental setups used to measure these properties and the result of comparisons between collagen scaffolds with different structures: aligned and isotropic (non-aligned). It indicates quantitative differences in terms of permeability, and the effects of compression on such permeability. The results contribute to the development and understanding of collagen scaffolds and their applications.

Collagen, as the basic building block of native extracellular matrix, possesses an inherent biocompatibility which makes it ideal for regenerative medicine., The basic building block of the extra-cellular matrix in native tissue is collagen. As a structural protein, collagen has an inherent biocompatibility making it an ideal material for regenerative medicine. Cellular response, mediated by integrins, is dictated by the structure and chemistry of the collagen fibers. Fiber formation, via fibrillogenesis, can be controlled in vitro by several factors: pH, ionic strength, and collagen structure. After formation, fibers are stabilized via cross-linking. The final bioactivity of collagen scaffolds is a result of both processes. By considering each step of fabrication, scaffolds can be tailored for the specific needs of each tissue, improving their therapeutic potential.

Dietary silicon is essential in the maintenance of bone and cartilage. However, the mechanism by which silicon, in the form of silicates, triggers a biological response has never been uncovered. Here we demonstrate the incorporation of orthosilicic acid (Si(OH)4), the form of silicon in the body, within collagen scaffolds for use as an in vitro platform to identify key genes affected by silicates. Ice-templated collagen–silicate scaffolds, containing 0.21 wt% silicon, were validated by examining the mRNA levels for an array of genes in human osteoblasts and mesenchymal stromal cells (MSC) after 48 h in culture. Several novel genes, such as tumor necrosis factor alpha (TNF), were identified as having potential links to orthosilicic acid, verifying that collagen–silicate scaffolds are a versatile platform for identifying novel mechanisms in which silicates regulate musculoskeletal tissue.

UNLABELLED: We provide evidence to show that the standard reactant concentrations used in tissue engineering to cross-link collagen-based scaffolds are up to 100 times higher than required for mechanical integrity in service, and stability against degradation in an aqueous environment. We demonstrate this with a detailed and systematic study by comparing scaffolds made from (a) collagen from two different suppliers, (b) gelatin (a partially denatured collagen) and (c) 50% collagen-50% gelatin mixtures. The materials were processed, using lyophilisation, to produce homogeneous, highly porous scaffolds with isotropic architectures and pore diameters ranging from 130 to 260 μm. Scaffolds were cross-linked using a carbodiimide treatment, to establish the effect of the variations in crosslinking conditions (down to very low concentrations) on the morphology, swelling, degradation and mechanical properties of the scaffolds. Carbodiimide concentration of 11.5mg/ml was defined as the standard (100%) and was progressively diluted down to 0.1%. It was found that 10-fold reduction in the carbodiimide content led to the significant increase (almost 4-fold) in the amount of free amine groups (primarily on collagen lysine residues) without compromising mechanics and stability in water of all resultant scaffolds. The importance of this finding is that, by reducing cross-linking, the corresponding cell-reactive carboxylate anions (collagen glutamate or aspartate residues) that are essential for integrin-mediated binding remain intact. Indeed, a 10-fold reduction in carbodiimide crosslinking resulted in near native-like cell attachment to collagen scaffolds. We have demonstrated that controlling the degree of cross-linking, and hence retaining native scaffold chemistry, offers a major step forward in the biological performance of collagen- and gelatin-based tissue engineering scaffolds. STATEMENT OF SIGNIFICANCE: This work developed collagen and gelatine-based scaffolds with structural, material and biological properties suitable for use in myocardial tissue regeneration. The novelty and significance of this research consist in elucidating the effect of the composition, origin of collagen and crosslinking concentration on the scaffold physical and cell-binding characteristics. We demonstrate that the standard carbodiimide concentrations used to crosslink collagenous scaffolds are up to 100 times higher than required for mechanical integrity in service, and stability against dissolution. The importance of this finding is that, by reducing crosslinking, the corresponding cell-reactive carboxylate anions (essential for integrin-mediated binding) remain intact and the native scaffold chemistry is retained. This offers a major step forward in the biological performance of tissue engineered scaffolds.

The potential applications of ice-templating porous materials are constantly expanding, especially as scaffolds for tissue engineering. Ice-templating, a process utilizing ice nucleation and growth within an aqueous solution, consists of a cooling stage (before ice nucleation) and a freezing stage (during ice formation). While heat release during cooling can change scaffold isotropy, the freezing stage, where ice crystals grow and anneal, determines the final size of scaffold features. To investigate the path of heat flow within collagen slurries during solidification, a series of ice-templating molds were designed with varying the contact area with the heat sink, in the form of the freeze drier shelf. Contact with the heat sink was found to be critical in determining the efficiency of the release of latent heat within the perspex molds. Isotropic collagen scaffolds were produced with pores which ranged from 90 μm up to 180 μm as the contact area decreased. In addition, low-temperature ice annealing was observed within the structures. After 20 h at -30 °C, conditions which mimic storage prior to lyophilization, scaffold architecture was observed to coarsen significantly. In future, ice-templating molds should consider not only heat conduction during the cooling phase of solidification, but the effects of heat flow during ice growth and annealing.

This study investigates the influence of silanisation on the mechanical and degradation behaviour of PLGA/HA composites. Three different silanes (mercaptopropyl trimethoxy silane (MPTMS), aminopropyl trimethoxy silane (APTMS) and aminopropyltriethoxy silane (APTES)) were applied to HA substrates in order to study the effect of head group (which binds to the polymer) and tail group (which binds to the surface hydroxyl groups in HA). A composite of hydroxyapatite (HA) and poly( d , l lactide-co-glycolide (50:50)) (PLGA) was investigated. The influence of concentration, the reaction time, drying temperature and substrate surface on silanisation was examined. TGA was used to detect the degree of silanisation. HA with MPTMS (1 wt.% MPTMS with reaction time of 1 h) was used as filler in PLGA-30 wt.% HA composites for an in-vitro degradation study carried out in PBS. In addition, the mechanical properties of the composites were studied. Silanisation affects the properties of the composite by improving the bonding at the interface and hence it was found to influence the plastic mechanical properties rather than the elastic mechanical properties or the degradation profile of the composite.

Coordinating the behaviour of multiple cell types provides a challenge for the tissue engineer, since response to structure is highly cell type dependent. Here, we compare human primary fibroblast invasion with that of the MC3T3 and HT1080 cell lines to demonstrate proof-of-concept that controlled changes in 3D collagen scaffold architecture can modify the invasion response of one cell type, while keeping that of other cells constant. While complete invasion was seen for all three cell types where pore size and percolation diameter were sufficiently high, both primary fibroblasts and MC3T3 required a percolation diameter above roughly 40 µm to permit efficient invasion by day 3. Conversely, substantial HT1080 invasion was observed in all scaffold conditions. By day 7, MC3T3 and fibroblast invasion responses to structure were also distinct, with only MC3T3 inhibited by low percolation diameters. Collagen scaffolds can therefore act as cellular sieves, using structural control to promote the invasion of some cell types, while restricting the movement of others. As well as holding promise for the design of tissue engineering scaffolds for complex applications, this result is highly relevant for the development of in vitro systems for studying in vivo phenomena such as cancer cell invasion and metastasis.

Graphical abstract, 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 α2β1, and rat glioma Rugli cells, with only rat α1β1. 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.

A significant body of research has considered collagen as a scaffold material for soft tissue regeneration. The main structural component of extra-cellular matrix (ECM), collagen’s advantages over synthetic polymers are numerous. However, for applications where higher stiffness and stability are required, significant cross-linking may affect bioactivity. A carbodiimide (EDC) cross-linking route consumes carboxylate groups that are key to collagen’s essential cell recognition motifs (GxOGER). Fibrinogen was considered as a promising additive as it plays a key role in the process of wound repair and contains RGD integrin binding sites which bind to a variety of cells, growth factors and cytokines. Fibrinogen’s binding sites however, also contain the same carboxylate groups as collagen. We have successfully produced highly interconnected, porous collagen-fibrinogen scaffolds using a lyophilisation technique and micro-computed tomography demonstrated minimal influence of either fibrinogen content or cross-linking concentration on the scaffold structure. The specific biological effect of fibrinogen additions into cross-linked collagen are considered by using films as a model for the struts of bulk scaffolds. By considering various additions of fibrinogen to the collagen film with increasing degrees of cross-linking, this study demonstrates a significant biological advantage with fibrinogen addition across the cross-linking concentrations typically applied to collagen-based scaffolds.

Research on the development of collagen constructs is extremely important in the field of tissue engineering. Collagen scaffolds for numerous tissue engineering applications are frequently crosslinked with 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride (EDC) in the presence of N-hydroxy-succinimide (NHS). Despite producing scaffolds with good biocompatibility and low cellular toxicity the influence of EDC/NHS crosslinking on the cell interactive properties of collagen has been overlooked. Here we have extensively studied the interaction of model cell lines with collagen I-based materials after crosslinking with different ratios of EDC in relation to the number of carboxylic acid residues on collagen. Divalent cation-dependent cell adhesion, via integrins α$_{1}$β$_{1}$, α$_{2}$β$_{1}$, α$_{10}$β$_{1}$ and α$_{11}$β$_{1}$, were sensitive to EDC crosslinking. With increasing EDC concentration, this was replaced with cation-independent adhesion. These results were replicated using purified recombinant I domains derived from integrin α$_{1}$ and α$_{2}$ subunits. Integrin α$_{2}$β$_{1}$-mediated cell spreading, apoptosis and proliferation were all heavily influenced by EDC crosslinking of collagen. Data from this rigorous study provides an exciting new insight that EDC/NHS crosslinking is utilising the same carboxylic side chain chemistry that is vital for native-like integrin-mediated cell interactions. Due to the ubiquitous usage of EDC/NHS crosslinked collagen for biomaterials fabrication this data is essential to have a full understanding in order to ensure optimized collagen-based material performance.

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.

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 α2β1, and Rugli expressing α1β1) and a parent cell line C2C12 with gelatin-binding receptors (αvβ3 and α5β1), 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.

Interconnecting pathways through porous tissue engineering scaffolds play a vital role in determining nutrient supply, cell invasion, and tissue ingrowth. However, the global use of the term "interconnectivity" often fails to describe the transport characteristics of these pathways, giving no clear indication of their potential to support tissue synthesis. This article uses new experimental data to provide a critical analysis of reported methods for the description of scaffold transport pathways, ranging from qualitative image analysis to thorough structural parameterization using X-ray Micro-Computed Tomography. In the collagen scaffolds tested in this study, it was found that the proportion of pore space perceived to be accessible dramatically changed depending on the chosen method of analysis. Measurements of % interconnectivity as defined in this manner varied as a function of direction and connection size, and also showed a dependence on measurement length scale. As an alternative, a method for transport pathway parameterization was investigated, using percolation theory to calculate the diameter of the largest sphere that can travel to infinite distance through a scaffold in a specified direction. As proof of principle, this approach was used to investigate the invasion behavior of primary fibroblasts in response to independent changes in pore wall alignment and pore space accessibility, parameterized using the percolation diameter. The result was that both properties played a distinct role in determining fibroblast invasion efficiency. This example therefore demonstrates the potential of the percolation diameter as a method of transport pathway parameterization, to provide key structural criteria for application-based scaffold design.

Chitosan/collagen (Chit/Col) blends have demonstrated great potential for use in tissue engineering (TE) applications. However, there exists a lack of detailed study on the influence of important design parameters (i.e, component ratio or crosslinking methods) on the essential properties of the scaffolds (morphology, mechanical stiffness, swelling, degradation and cytotoxicity). This work entailed a systematic study of these essential properties of three Chit/Col compositions, covering a wide range of component ratios and using different crosslinking methods. Our results showed the possibility of tailoring these properties by changing component ratios, since different interactions occurred between Chit/Col: samples with Chit-enriched compositions showed a hydrogen-bonding type complex (HC), whereas a self-crosslinking phenomenon was induced in Col-enriched scaffolds. Additionally, material and biological properties of the resultant matrices were further adjusted and tuned by changing crosslinking conditions. In such way, we obtained a wide range of scaffolds whose properties were tailored to meet specific needs of TE applications.

The relationship between biological scaffold interconnectivity and cell migration is an important but poorly understood factor in tissue regeneration. Here a scale-independent technique for characterization of collagen scaffold interconnectivity is presented, using a combination of X-ray microcomputed tomography and percolation theory. Confocal microscopy of connective tissue cells reveals this technique as highly relevant for determining the extent of cell invasion.

Ptosis describes a low position of the upper eyelid. When this condition is due to poor function of the levator palpebrae superioris muscle, responsible for raising the lid, "brow-suspension" ptosis correction is usually performed, which involves internally attaching the malpositioned eyelid to the forehead musculature using brow-suspension materials. In service, such materials are exposed to both rapid tensile loading and unloading sequences during blinking, and a more sustained tensile strain during extended periods of closure. In this study, various mechanical tests were conducted to characterise and compare some of commonly-used synthetic brow-suspension materials (Prolene(®), Supramid Extra(®) II, Silicone rods (Visitec(®) Seiff frontalis suspension set) and Mersilene(®) mesh) for their time-dependent response. At a given constant tensile strain or load, all of the brow-suspension materials exhibited stress-relaxation or creep, with Prolene(®) having a statistically different relaxation or creep ratio as compared with those of others. Uniaxial tensile cyclic tests through preconditioning and fatigue tests demonstrated drastically different time-dependent response amongst the various materials. Although the tests generated hysteresis force-strain loops for all materials, the mechanical properties such as the number of cycles required to reach the steady-state, the reduction in the peak force, and the cyclic energy dissipation varied considerably. To reach the steady-state, Prolene(®) and the silicone rod required the greatest and the least number of cycles, respectively. Furthermore, the fatigue tests at physiologically relevant conditions (15% strain controlled at 6.5 Hz) demonstrated that the reduction in the peak force during 100,000 cycles ranged from 15% to 58%, with Prolene(®) and the silicone rod exhibiting the greatest and the least value, respectively. Many factors need to be considered to select the most suitable brow-suspension material for ptosis correction. These novel data on the mechanical time-dependent performance could therefore help to guide clinicians in their decision-making process for optimal surgical outcome.