The effects of despeckling filters on pore size measurements in collagen scaffold micro-CT data.

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 an imaging technique commonly used to assess the characteristics of porous structures, such as medical tissue engineering scaffolds and bone. Prior to analysis micro-CT images are often processed by thresholding and filtering to improve the image quality. Scaffold pore size affects biological cell behaviour and mechanical properties, and is a frequently assessed parameter when evaluating medical scaffolds. This paper identifies and characterises an artefact affecting a commonly used filter which erroneously increases measured peripheral mean pore size. The artefact affects the periphery of volumes of interest which have been filtered by a technique called 3D sweep despeckling. This filter removes all but the largest object in the volume of interest, and therefore also deletes small disconnected objects located at the volume of interest periphery. This paper characterises the artefact, 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 ascertain the parameters required to effectively apply this artefact reduction method to other datasets are described. This method eliminates the artefact, but is time consuming and computationally expensive. Alternative, more economical despeckling filters are assessed for their ability to remove the error. Of these, a filter which deletes objects below a prescribed area was found to be most effective when performing 2D pore analysis on scaffolds, and the same filter applied to objects below a set volume was best when 3D pore analysis was used. This filter was found to be afflicted by the same artefact as sweep despeckling. These results will help guide the implementation of future studies investigating the effects of pore size.

Optimising collagen scaffold architecture for enhanced periodontal ligament fibroblast migration.

Design of cell-free scaffolds for endogenous cell recruitment requires an intimate knowledge of precise relationships between structure and biological function. Here, we use morphological analysis by Micro-CT to identify the key structural features necessary for periodontal ligament fibroblast recruitment into collagen scaffolds. By the combined use of time-lapse imaging and end-point invasion analysis, we distinguish the influences of pore size, pore wall alignment, and pore transport pathways (percolation diameter) on the individual cell migration and bulk invasion characteristics of these fibroblasts. Whereas maximising percolation diameter increased individual cell speed, elongation and directionality, and produced the most rapid bulk cell invasion, a pore size of 100 µm was found to be necessary to ensure an even distribution of cells across the scaffold cross-section. These results demonstrate that control of percolation diameter and pore size may be used respectively to tune the efficiency and uniformity of invasion through macroporous scaffolds. Crucially, however, these observations were subject to the condition of pore wall alignment, with low alignment in the direction of travel producing relatively low cell speeds and limited invasion in all cases. Pore wall alignment should therefore be carefully optimised in the design of scaffolds for cell recruitment, such as that required for periodontal ligament regeneration, as a key determining factor for cell movement.

3D imaging of cells in scaffolds: direct labelling for micro CT.

The development of in-vitro techniques to characterise the behaviour of cells in biomedical scaffolds is a rapidly developing field. However, until now it has not been possible to visualise, directly in 3D, the extent of cell migration using a desktop X-ray microCT. This paper describes a new technique based on cell labelling with a radio opacifier (barium sulphate), which permits cell tracking without the need for destructive sample preparation. The ability to track cells is highlighted via a comparison of cell migration through demonstrator lyophilised collagen scaffolds with contrasting pore size and interconnectivity. The results demonstrate the ease with which the technique can be used to characterise the effects of scaffold architecture on cell infiltration.

Correction to: Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry.

The article "Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry", written by Natalia Davidenko, Carlos F. Schuster, Daniel V. Bax, Richard W. Farndale, Samir Hamaia, Serena M. Best and Ruth E. Cameron, was originally published Online First without open access. After publication in volume 27, issue 10, page 148 it was noticed that the copyright was wrong in the PDF version of the article. The copyright of the article should read as "© The Author(s) 2016". The Open Access license terms were also missing.

Correction to: The effect of cationically-modified phosphorylcholine polymers on human osteoblasts in vitro and their effect on bone formation in vivo.

The article "The effect of cationically modified phosphorylcholine polymers on human osteoblasts in vitro and their effect on bone formation in vivo", written by Jonathan M. Lawton, Mariam Habib, Bingkui Ma, Roger A. Brooks, Serena M. Best, Andrew L. Lewis, Neil Rushton and William Bonfield, was originally published Online First without open access. After publication in volume 28, issue 9, page 144 it was noticed that the copyright was wrong in the PDF version of the article. The copyright of the article should read as "

The effect of cationically-modified phosphorylcholine polymers on human osteoblasts in vitro and their effect on bone formation in vivo.

The effect of introducing cationic charge into phosphorylcholine (PC)-based polymers has been investigated in this study with a view to using these materials as coatings to improve bone formation and osseointegration at the bone-implant interface. PC-based polymers, which have been used in a variety of medical devices to improve biocompatibility, are associated with low protein adsorption resulting in reduced complement activation, inflammatory response and cell adhesion. However, in some applications, such as orthopaedics, good integration between the implant and bone is needed to allow the distribution of loading stresses and a bioactive response is required. It has previously been shown that the incorporation of cationic charge into PC-based polymers may increase protein adsorption that stimulates subsequent cell adhesion. In this paper, the effect of cationic charge in PC-based polymers on human osteoblasts (HObs) in vitro and the effect of these polymers on bone formation in the rat tibia was assessed. Increasing PC positive surface charge increased HOb cell adhesion and stimulated increased cell differentiation and the production of calcium phosphate deposits. However, when implanted in bone these materials were at best biotolerant, stimulating the production of fibrous tissue and areas of loosely associated matrix (LAM) around the implant. Their development, as formulated in this study, as bone interfacing implant coatings is therefore not warranted.

The Mechanics of Brow-Suspension Ptosis Repair: A Comparative Study of Fox Pentagon and Crawford Triangle Techniques.

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.

Optimisation of UV irradiation as a binding site conserving method for crosslinking collagen-based scaffolds.

Short wavelength (λ = 254 nm) UV irradiation was evaluated over a range of intensities (0.06 to 0.96 J/cm(2)) as a means of cross-linking collagen- and gelatin-based scaffolds, to tailor their material characteristics whilst retaining biological functionality. Zero-link carbodiimide treatments are commonly applied to collagen-based materials, forming cross-links from carboxylate anions (for example the acidic E of GFOGER) that are an essential part of integrin binding sites on collagen. Cross-linking these amino acids therefore disrupts the bioactivity of collagen. In contrast, UV irradiation forms bonds from less important aromatic tyrosine and phenylalanine residues. We therefore hypothesised that UV cross-linking would not compromise collagen cell reactivity. Here, highly porous (~99 %) isotropic, collagen-based scaffolds were produced via ice-templating. A series of scaffolds (pore diameters ranging from 130-260 µm) with ascending stability in water was made from gelatin, two different sources of collagen I, or blends of these materials. Glucose, known to aid UV crosslinking of collagen, was added to some lower-stability formulations. These scaffolds were exposed to different doses of UV irradiation, and the scaffold morphology, dissolution stability in water, resistance to compression and cell reactivity was assessed. Stabilisation in aqueous media varied with both the nature of the collagen-based material employed and the UV intensity. Scaffolds made from the most stable materials showed the greatest stability after irradiation, although the levels of cross-linking in all cases were relatively low. Scaffolds made from pure collagen from the two different sources showed different optimum levels of irradiation, suggesting altered balance between stabilisation from cross-linking and destabilisation from denaturation. The introduction of glucose into the scaffold enhanced the efficacy of UV cross-linking. Finally, as hypothesized, cell attachment, spreading and proliferation on collagen materials were unaffected by UV cross-linking. UV irradiation may therefore be used to provide relatively low level cross-linking of collagen without loss of biological functionality.

Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry.

Studies of cell attachment to collagen-based materials often ignore details of the binding mechanisms-be they integrin-mediated or non-specific. In this work, we have used collagen and gelatin-based substrates with different dimensional characteristics (monolayers, thin films and porous scaffolds) in order to establish the influence of composition, crosslinking (using carbodiimide) treatment and 2D or 3D architecture on integrin-mediated cell adhesion. By varying receptor expression, using cells with collagen-binding integrins (HT1080 and C2C12 L3 cell lines, expressing α2ß1, and Rugli expressing α1ß1) and a parent cell line C2C12 with gelatin-binding receptors (αvß3 and α5ß1), the nature of integrin binding sites was studied in order to explain the bioactivity of different protein formulations. We have shown that alteration of the chemical identity, conformation and availability of free binding motifs (GxOGER and RGD), resulting from addition of gelatin to collagen and crosslinking, have a profound effect on the ability of cells to adhere to these formulations. Carbodiimide crosslinking ablates integrin-dependent cell activity on both two-dimensional and three-dimensional architectures while the three-dimensional scaffold structure also leads to a high level of non-specific interactions remaining on three-dimensional samples even after a rigorous washing regime. This phenomenon, promoted by crosslinking, and attributed to cell entrapment, should be considered in any assessment of the biological activity of three-dimensional substrates. Spreading data confirm the importance of integrin-mediated cell engagement for further cell activity on collagen-based compositions. In this work, we provide a simple, but effective, means of deconvoluting the effects of chemistry and dimensional characteristics of a substrate, on the cell activity of protein-derived materials, which should assist in tailoring their biological properties for specific tissue engineering applications.

Electrospinning of Bioinspired Polymer Scaffolds.

Electrospinning is a technique used in the production of polymer nanofibre meshes. The use of biodegradable and biocompatible polymers to produce nanofibres that closely mimic the extracellular matrix (ECM) of different tissues has opened a wide range of possibilities for the application of electrospinning in Tissue Engineering. It is believed that nano-features (such as voids and surface cues) present in nanofibre mesh scaffolds, combined with the chemical composition of the fibres, can stimulate cell attachment, growth and differentiation. Despite the widespread use of electrospun nanofibres in tissue engineering, the present chapter will focus on the advances made in the utilisation of these materials in bone, cartilage and tooth related applications. Several aspects will be taken into consideration, namely the choice of polymers, the surface modification of the nanofibres in order to achieve mineralisation, and also the biological application of such materials.

Bioactive IGF-1 release from collagen-GAG scaffold to enhance cartilage repair in vitro.

Tissue engineering is a promising technique for cartilage repair. Toward this goal, a porous collagen-glycosaminoglycan (CG) scaffold was loaded with different concentrations of insulin-like growth factor-1 (IGF-1) and evaluated as a growth factor delivery device. The biological response was assessed by monitoring the amount of type II collagen and proteoglycan synthesised by the chondrocytes seeded within the scaffolds. IGF-1 release was dependent on the IGF-1 loading concentration used to adsorb IGF-1 onto the CG scaffolds and the amount of IGF-1 released into the media was highest at day 4. This initial IGF-1 release could be modelled using linear regression analysis. Osteoarthritic (OA) chondrocytes seeded within scaffolds containing adsorbed IGF-1 deposited decorin and type II collagen in a dose dependent manner and the highest type II collagen deposition was achieved via loading the scaffold with 50 µg/ml IGF-1. Cells seeded within the IGF-1 loaded scaffolds also deposited more extracellular matrix than the no growth factor control group thus the IGF-1 released from the scaffold remained bioactive and exerted an anabolic effect on OA chondrocytes. The effectiveness of adsorbing IGF-1 onto the scaffold may be due to protection of the molecule from proteolytic digestion allowing a more sustained release of IGF-1 over time compared to adding multiple doses of exogenous growth factor. Incorporating IGF-1 into the CG scaffold provided an initial therapeutic burst release of IGF-1 which is beneficial in initiating ECM deposition and repair in this in vitro model and shows potential for developing this delivery device in vivo.

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

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

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

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

The diazirine-mediated photo-crosslinking of collagen improves biomaterial mechanical properties and cellular interactions.

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.

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

Applied to the epicardium in-vivo, regenerative cardiac patches support the ventricular wall, reduce wall stresses, encourage ventricular wall thickening, and improve ventricular function. Scaffold engraftment, however, remains a challenge. After implantation, scaffolds are subject to the complex, time-varying, biomechanical environment of the myocardium. The mechanical capacity of engineered tissue to biomimetically deform and simultaneously support the damaged native tissue is crucial for its efficacy. To date, however, the biomechanical response of engineered tissue applied directly to live myocardium has not been characterized. In this paper, we utilize optical imaging of a Langendorff ex-vivo cardiac model to characterize the native deformation of the epicardium as well as that of attached engineered scaffolds. We utilize digital image correlation, linear strain, and 2D principal strain analysis to assess the mechanical compliance of acellular ice templated collagen scaffolds. Scaffolds had either aligned or isotropic porous architecture and were adhered directly to the live epicardial surface with either sutures or cyanoacrylate glue. We demonstrate that the biomechanical characteristics of native myocardial deformation on the epicardial surface can be accurately 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.

Collagen fibre implant for tendon and ligament biological augmentation. In vivo study in an ovine model.

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.

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

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

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

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

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

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