Mechanical forces play a critical role in cellular behaviour, yet devising systems capable of applying multiaxial strain to three-dimensional (3D) tissue engineering substrates remains challenging. This study introduces an innovative approach using 3D-printed auxetic re-entrant honeycomb meshes to convert uniaxial tension into biaxial straining of cell-laden collagen hydrogel substrates by utilising the unconventional negative Poisson's ratio. Key findings demonstrate that polypropylene (PP) meshes exhibited enhanced compliance and ductility compared to conventional poly-l-lactide (PLA) alternatives, making them particularly suitable for this application. Across all auxetic designs, the tensile moduli of the PP meshes were approximately 6 times lower, and their ultimate tensile strains remained at 0.66-approximately 17 times higher than that of PLA meshes. Critically, when human dermal fibroblasts were cultured on the strained collagen gels, their aspect ratio increased by 59 % while maintaining random alignment, providing proof of concept for true biaxial mechanical stimulation. This approach offers a versatile and accessible tool for advancing research in mechanobiology and tissue engineering by enabling the exploration of cellular responses to physiologically relevant mechanical environments.

Collagen-based soft tissues are often anisotropic in structure, and in regenerative medicine, it is important that scaffolds are designed to closely mimic their architecture and mechanical behaviour. Existing testing standards are not directly applicable to anisotropic structures in physiologically relevant conditions. The challenge is therefore to systematically quantify mechanical anisotropy, nonlinear tensile behaviour, and both in vitro degradation and fatigue in a way appropriate for soft, porous, natural macromolecular structures. In this study, we fabricated collagen scaffolds with elongated porosity via directional freeze-drying and then chemically crosslinked them using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Compressive mechanical behaviour was monitored along two characteristic directions, and distinct stress-strain curves were observed. The elastic modulus was identified as 6.42 kPa and 1.02 kPa along the two directions. Nonlinear tensile behaviour was characterised using the tangent modulus, which varied from 25 kPa to 270 kPa before failure. Standardised methods for monitoring degradation at 37 °C were developed. In vitro degradation was investigated by immersing scaffolds in deionised water for 60 days, during which the critical stress decreased by 30% in the first 30 days. Fatigue was studied by monitoring the stress-strain curves under 20% tensile strain for 1000 cycles. Scaffolds exhibited weaker mechanical integrity when loaded at 1.43 Hz. This study addresses the lack of standardised testing methods for anisotropic ice-templated collagen scaffolds by establishing a set of protocols to characterise the mechanical anisotropy, quantify tensile behaviour, and monitor scaffold degradation. This flexible characterisation toolkit can be adapted to specific sample treatments during tissue culture.

The extracellular matrix changes dramatically during the progression of diseases like cancer. These complex, tissue-specific changes are not adequately replicated by most current biomaterial disease models. This work demonstrates, for the first time, a biomaterial system allowing combined, independent control over stiffness, extracellular matrix composition and 3D collagen architecture. Defined hydrogel formulations are successfully perfused into ice-templated collagen scaffolds, controlling the composition of these hybrid scaffolds at constant stiffness. The Young's moduli of these hybrid scaffolds can also be tuned independently of composition via chemical cross-linking. Encapsulation of human dermal fibroblasts reveals that fibroblast morphology depends on hybrid scaffold composition and on viscoelasticity, highlighting the importance of a system that decouples biophysical from biochemical properties. Finally, these hybrid scaffolds are successfully applied to exert combined control over biochemical and biophysical drivers of cell growth and invasion, focusing on breast cancer as proof-of-concept. The results reveal that collagen fiber patterning enhances breast cancer cell proliferation, also directing the invasion of patient-derived breast cancer cells. These hybrid scaffolds are therefore promising new tools for dissecting the diverse but complementary roles played by the extracellular matrix in regulating cell phenotype, in a range of healthcare applications.

The safety, quality and supply of donor-derived platelet units intended for transfusion have improved over the past decades but significant problems still remain. In vitro-derived platelets offer a possible alternative but up-scaling production is hindered by our limited understanding of thrombopoiesis (the release of platelets by their mother cell, the megakaryocyte [MK]). Here, we have developed an integrated strategy aiming to mimic ex vivo the bone marrow physiological niche that promotes thrombopoiesis by mature MK. The screening of a panel of 259 recombinant transmembrane proteins derived from cells known to promote platelet production through direct contact with MK enabled us to show that ACVR1B, CRTAM, MUCEN and BTN1A1 improve platelet production from either cord blood- (ACVR1B) or pluripotent stem cells-derived (CRTAM, MUCEN and BTN1A1) MK. Using two different methodologies, we functionalize either collagen- or silk-based 3-dimensional scaffolds and confirm increased functional platelet production by up to 2-fold. This unbiased approach has allowed us to identify novel proteins whose role in platelet formation was previously unknown and highlights the potential gain of recreating the MK niche to allow in vitro platelets to become a viable alternative for transfusion.

Auxetic structures studied in the literature are often based on relatively stiff, metallic materials and theories regarding their response to mechanical loading cannot be translated directly to polymeric materials. As "soft" auxetics increase in popularity for applications in tissue engineering further investigation into the joint behaviour and effect on their Poisson's ratio is required. 3D printed polypropylene auxetic mesh structures were produced to compare to the requirements for biological cell-stretching devices while investigating the deformation mechanics. The behaviour of the meshes was characterised with tensile force-strain curves and high-definition imaging and the effect of joint behaviour on the Poisson's ratio was evaluated. Isolated unit cell samples of the re-entrant mesh were produced to characterise the in- and out-of-plane behaviour for geometries comprising re-entrant strut angles of 30, 45, and 60° to the tensile straining direction. Force-strain curves with three distinct phases were observed, with linear, plateau, and terminal regions characteristic of re-entrant honeycomb structures. A constant negative Poisson's ratio was measured up to a critical transition strain, at which point it is theorised that the onset of buckling triggers bending-dominated deformation to occur, out-of-plane. The production of full-scale mesh samples with the same 30, 45, and 60° geometry resulted in consistent values for critical transition strain and Poisson's ratios. An auxetic region of strain was defined, where the force is linear and a homogeneous negative Poisson's ratio can be maintained. This region represents the limit within which a biological cell-stretching device could operate successfully for the current mesh design.

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.

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.

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 μm 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.

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.

Replicating the intricate architecture of native tissues remains a significant challenge in tissue engineering. Icetemplated biomimetic scaffolds possess controlled porosity that conveniently resembles the native parenchyma of many tissues. In this study, we investigate the relationship between the porous architecture of lyophilised collagen scaffolds and key processing parameters during production. We establish a predictive model that correlates specific lyophilisation conditions with the resulting pore sizes. Systematic variations in the freeze-drying conditions resulted in scaffolds with average pore sizes ranging from 46 mu m to 251 mu m, effectively matching the length scale of extracellular matrix features found in native tissues. We introduce the concept of heat flux density (HFD) at equilibrium as a metric for quantifying latent heat extraction efficiency during the freezing process. Our findings reveal a power law relationship between HFD at equilibrium and pore size, with an exponent of-0.44. This approach provides a non-destructive and non-intrusive method for precisely controlling pore architecture, advancing the potential for creating scaffolds that closely emulate the complex structures of native tissues.

A major challenge in regenerative medicine is the development of grafts that can be vascularised successfully. Capillary networks facilitate processes that are essential for tissue survival including the supply of nutrients to cells and waste removal. To prevent core degradation of tissue-engineered grafts, it is important that scaffolds can successfully accommodate vessel ingrowth. Vessel formation is often assessed in vitro using a complex co-culture of osteoblasts and endothelial cells to improve the angiogenic potential of grafts. Despite extensive research, no consensus exist regarding optimal co-culture conditions due in part to the strong contextual dependency of multiple parameters, including the scaffold material and cell types. The aim of his work is to define optimal co-culture conditions on both 2-dimensional (2D) collagen films and 3-dimensional (3D) collagen scaffolds cross-linked using EDC/NHS. The influence of seeding density and cell ratio on cell-metabolic activity and proliferation of primary human osteoblast (hOBs) and human dermal microvascular endothelial cells (HDMECs) was assessed. Systematically studying the hOB:HDMEC cell ratios showed that 70:30 and 50:50 provided the optimal seeding ratio for microvessel formation on 2D films and 3D scaffolds, respectively. Nevertheless, vascularisation of these collagen substrates remained limited. Further assessment in 2D revealed that osteoblast detachment was the main factor restricting vessel formation. Pre-coating of the substrates with a 5 μg/mL fibronectin solution provided appropriate conditions for the formation of a stable osteoblast mono layer which supported abundant vessel formation on collagen substrates. Overall, fibronectin-coated collagen scaffolds seeded with a ratio of 50:50 hOB:HDMEC resulted in abundant microvascular network formation with multiple branching points and lumen formation that did not require exogeneous VEGF application.

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

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.

This paper investigates drug release from a novel series of mPEG-functionalised PLLA polymers whose individual components (PEG and PLLA) have regulatory FDA approval. Two processing methods were explored to understand their effect on the morphology and drug release profiles of the polymers, with and without mPEG functionalisation. In the first method the polymer and Propranolol.HCl drug powders were mixed together before injection moulding. In the second method, supercritical CO 2 was used to mix the polymer and drug before injection moulding. When non-functionalised PLLA was processed through injection moulding alone, there were no signs of polymer-drug interaction, and the drug was confined to crystals on the surface. This resulted in up to 85 wt% burst release of propranolol.HCl after one day of incubation. By contrast, injection moulding of mPEG-functionalised polymers resulted in the partial dissolution of drug in the polymer matrix and a smaller burst (50 wt% drug) followed by sustained release. This initial burst release was completely eliminated from the profile of mPEG-functionalised polymers processed via supercritical CO 2 . The addition of mPEG facilitated the distribution of the drug into the bulk matrix of the polymer. Paired with supercritical CO 2 processing, the drug release profile showed a slow, sustained release throughout the 4 months of the study.

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.

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.

Abstract 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.

This paper presents a novel method to spatially vary the intra‐layer birefringence of Fused Filament Fabricated (FFF) parts by controlling chain alignment during extrusion along individual rasters. The role of print speed, extrusion factor and layer separation on the birefringence of single PLLA layers is explored, at thicknesses ranging from 50–125 µm and print speeds 1000–6000 mm min −1 . The cumulative and subtractive effect of multiple PLLA layers are explored to elicit colours corresponding to a range of retardations, achieve complete extinction, and printing a physical Michel‐Levy chart. By increasing print speed and reducing layer separation and extrusion factor, a birefringence up to Δ n = 9 × 10 −4 could be achieved in single layers. In multi‐layer structures, retardations of 0–800 nm are demonstrated. These results suggest that spatially varied birefringence can be used to store data, text or images, which can be resolved when parts are illuminated between polarizers. This effect is utilized to present a steganographic technique embedding information within bulk printed parts. These techniques might find application in a range of printed optics and devices, where spatial control over molecular alignment and associated influence on the propagation of light is desirable, including the ability to encode information within a print.

Fully bioresorbable polymer matrix composites have long been considered as potential orthopaedic implant materials, however their combination of mechanical strength, stiffness, ductility and bioresorbability is also attractive for cardiac stent applications. This work investigated reinforcement of polylactide-based polymers with phosphate glasses, addressing key drawbacks of current polymer stents, and examined the often-neglected evolution of structure and mechanical properties during degradation. Incorporation of 15-30 wt% phosphate glass led to modulus increases of up to 80% under simulated body conditions, and 15 wt% glass composites retained comparable ductility to pure polymers, crucial for stent applications where ductility and stiffness are required. Two-stage degradation was observed, dominated by interfacial water absorption and glass dissolution. Polymer embrittlement mechanisms (crystallisation, enthalpy relaxation) were suppressed by glass addition, allowing composites to achieve a more controlled loss of mechanical properties during degradation, which could allow gradual transfer of loading to newly healed tissue. These results provide a valuable new system for understanding the structural and mechanical changes occurring during degradation of fully bioresorbable polymer matrix composites, providing important new data to underpin the design of effective cardiac stent materials.