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.

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.

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.

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.

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.

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.

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.