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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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sanjay Sinha reports financial support was provided by British Heart Foundation Oxbridge Centre for Regenerative Medicine. Sanjay Sinha reports financial support was provided by British Heart Foundation Senior Fellowship. Sanjay Sinha reports financial support was provided by British Heart Foundation Centre for Cardiovascular Research Excellence. Maria Colzani reports financial support was provided by British Heart Foundation Oxbridge Centre for Regenerative Medicine. Maria Colzani reports financial support was provided by British Heart Foundation. Jamie A. Cyr reports financial support was provided by the Gates Cambridge Fellowship. Ruth E. Cameron reports financial support was provided by the Engineering and Physical Sciences Research Council Established Career Fellowship. Serena M. Best reports financial support was provided by the Engineering and Physical Sciences Research Council Established Career Fellowship. Vera Graup reports financial support was provided by the British Heart Foundation Cambridge Centre of Excellence. Sanjay Sinha reports financial support was provided by the Wellcome Trust Medical Research Council Cambridge Stem Cell Institute., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2021 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

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., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

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

One of the greatest obstacles to clinical translation of bone tissue engineering is the inability to effectively and efficiently vascularize scaffolds. The goal of this work was to explore systematically whether architecture, at a scale of hundreds of microns, can be used to direct the growth of microcapillary-like structures into the core of scaffolds. Biphasic bioceramic patterned architectures were produced using silicone molds of 3D printed parts. Grooves and ridges were designed to have widths of 330 μm and 660 μm, with periodicities respectively of 1240 μm and 630 μm. Groove depth was varied between 150 μm and 585 μm. Co-cultures of human dermal microvascular endothelial cells (HDMECs) and human osteoblasts (hOBs) were used to grow microcapillary-like structures on substrates. Bioceramic architecture was found to significantly affect microcapillary-like structure location and orientation. Microcapillary-like structures were found to form predominantly in grooves or between convexities. For all patterned samples, the CD31 (endothelial cell marker) signal was at least 2.5 times higher along grooves versus perpendicular to grooves. In addition, the average signal was at least two times higher within grooves than outside grooves for all samples. Grooves with a width of 330 μm and a depth of 300 μm resulted in the formation of individual, highly aligned microcapillary-like structures with lengths around 5 mm. Extensive literature has focused on the role of nano- and micro-topography (on the scale below tens of microns) on cellular response. However, the idea that architecture at a scale much larger than a cell could be used to modulate angiogenesis has not been systematically investigated. This work shows the crucial influence of architecture on microcapillary-like structure self-assembly at the scale of hundreds of microns. Elucidating the precise correspondence between architecture and microcapillary-like structure organization will ultimately allow the engineering of microvasculature by tuning local scaffold design to achieve desirable microvessel properties., Competing Interests: The authors have declared that no competing interests exist.

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.

Breast cancers are highly heterogeneous and their metastatic potential and response to therapeutic drugs is difficult to predict. A tool that could accurately gauge tumour invasiveness and drug response would provide a valuable addition to the oncologist's arsenal. We have developed a 3-dimensional (3D) culture model that recapitulates the stromal environment of breast cancers by generating anisotropic (directional) collagen scaffolds seeded with adipocytes and culturing tumour fragments therein. Analysis of tumour cell invasion in the presence of various therapeutic drugs, by immunofluorescence microscopy coupled with an optical clearing technique, demonstrated the utility of this approach in determining both the rate and capacity of tumour cells to migrate through the stroma while shedding light also on the mode of migration. Furthermore, the response of different murine mammary tumour types to chemotherapeutic drugs could be readily quantified.

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.

Tissue engineering has grown in the past two decades as a promising solution to unresolved clinical problems such as osteoarthritis. The mechanical response of tissue engineering scaffolds is one of the factors determining their use in applications such as cartilage and bone repair. The relationship between the structural and intrinsic mechanical properties of the scaffolds was the object of this study, with the ultimate aim of understanding the stiffness of the substrate that adhered cells experience, and its link to the bulk mechanical properties. Freeze-dried type I collagen porous scaffolds made with varying slurry concentrations and pore sizes were tested in a viscoelastic framework by macroindentation. Membranes made up of stacks of pore walls were indented using colloidal probe atomic force microscopy. It was found that the bulk scaffold mechanical response varied with collagen concentration in the slurry consistent with previous studies on these materials. Hydration of the scaffolds resulted in a more compliant response, yet lesser viscoelastic relaxation. Indentation of the membranes suggested that the material making up the pore walls remains unchanged between conditions, so that the stiffness of the scaffolds at the scale of seeded cells is unchanged; rather, it is suggested that thicker pore walls or more of these result in the increased moduli for the greater slurry concentration conditions., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Involution is a process whereby the mammary gland undergoes extensive tissue remodelling involving exquisitely coordinated cell death, extracellular matrix degradation and adipose tissue regeneration following the weaning of offspring. These processes are mediated in part through Jak/Stat signalling pathways, which can be deregulated in breast cancer. Synthetic in vitro analogues of the breast could become important tools for studying tumorigenic processes, or as personalized drug discovery platforms and predictors of therapeutic response. Ideally, such models should support 3D neo-tissue formation, so as to recapitulate physiological organ function, and be compatible with high-throughput screening methodologies. We have combined cell lines of epithelial, stromal and immunological origin within engineered porous collagen/hyaluronic acid matrices, demonstrating 3D-specific molecular signatures. Furthermore seeded cells form mammary-like branched tissues, with lobuloalveolar structures that undergo inducible involution phenotypes reminiscent of the native gland under hormonal/cytokine regulation. We confirm that autophagy is mediated within differentiated mammary epithelial cells in a Stat-dependent manner at early time points following the removal of a prolactin stimulus (H/WD). In addition, epithelial cells express markers of an M2 macrophage lineage under H/WD, a process that is attenuated with the introduction of the monocyte/macrophage cell line RAW 264.7. Thus, such 3D models are suitable platforms for studying cell-cell interactions and cell death mechanisms in relation to cancer.

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

Collagen-based scaffolds can be used to mimic the extracellular matrix (ECM) of soft tissues and provide support during tissue regeneration. To better match the native ECM composition and mechanical properties as well as tailor the degradation resistance and available cell binding motifs, other proteins or different collagen types may be added. The present study has explored the use of components such as gelatin or elastin and investigated their effect on the bulk physical properties of the resulting scaffolds compared to those made from pure collagen type I. The effect of altering the composition and crosslinking was evaluated in terms of the scaffold structure, mechanical properties, swelling, degradation and cell attachment. Results demonstrate that scaffolds based on gelatin had reduced tensile stiffness and degradation time compared with collagen. The addition of elastin reduced the overall strength and stiffness of the scaffolds, with electron microscopy results suggesting that insoluble elastin interacts best with collagen and soluble elastin interacts best with gelatin. Carbodiimide crosslinking was essential for structural stability, strength and degradation resistance for scaffolds of all compositions. In addition, preliminary cell adhesion studies showed these highly porous structures (pore size 130-160 μm) to be able to support HT1080 cell infiltration and growth. Therefore, this study suggests that the use of gelatin in place of collagen, with additions of elastin, can tailor the physical properties of scaffolds and could be a design strategy for reducing the overall material costs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 α 2 β 1 , 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 α 1. Conversely, binding of the I-domain from integrin α 2 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. 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-α 2 but not -α 1. 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. Image, graphical abstract [ABSTRACT FROM AUTHOR]

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

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. Statement of Significance Carbodiimide stabilised collagen is employed extensively for the fabrication of biologically active materials. Despite this common usage, the effect of carbodiimide crosslinking on cell-collagen interactions is unclear. Here we have found that carbodiimide crosslinking of collagen inhibits native-like, whilst increasing non-native like, cellular interactions. We propose a mechanistic model in which carbodiimide modifies the carboxylic acid groups on collagen that are essential for cell binding. As such we feel that this research provides a crucial, long awaited, insight into the bioactivity of carbodiimide crosslinked collagen. Through the ubiquitous use of collagen as a cellular substrate we feel that this is fundamental to a wide range of research activity with high impact across a broad range of disciplines. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this work was to develop nanocrystalline apatite (Ap) dispersed in a chitosan (CHI) matrix as a material for applications in bone tissue engineering. CHI/Ap composites of different weight ratios (20/80, 50/50 and 80/20) and with CHI of different molecular weights were prepared by a biomimetic stepwise route. Firstly, CaHPO4·2H2O (DCPD) crystals were precipitated from Ca(CH3COO)2 and NaHPO4 in the bulk CHI solution, followed by the formation of CHI/DCPD beads by coacervation. The beads were treated with Na3PO4/Na5P3O10 solution (pH 12–13) to crosslink the CHI and to hydrolyse the DCPD to nanocrystalline Ap. This new experimental procedure ensured that complete conversion of DCPD into sodium-substituted apatite was achieved without appreciable increases in its crystallinity and particle size. In addition, composites with silicon-doped Ap were prepared by substituting Na3PO4 by Na2SiO3 in the crosslinking/hydrolysis step. Characterization of the resultant composites by scanning electron microscopy, X-ray powder diffraction (XRD), thermal analysis and Fourier transform infrared spectroscopy confirmed the formation, within the CHI matrix, of nanoparticles of sodium- and carbonate-substituted hydroxyapatite [Ca10–xNax(PO4)6–x(CO3)x(OH)2] with diameters less than 20nm. Relatively good correspondence was shown between the experimentally determined inorganic content and that expected theoretically. Structural data obtained from its XRD patterns revealed a decrease in both crystal domain size and cell parameters of Ap formed in situ with increasing CHI content. It was found that the molecular weight of CHI and silicate doping both affected the nucleation and growth of apatite nanocrystallites. These effects are discussed in detail. [Copyright &y& Elsevier]