Fabrication of a corneal-limbal tissue substitute using silk fibroin

Fibroin extracted from silkworm cocoon silk provides an intriguing and potentially important biomaterial for corneal reconstruction. In the present chapter we outline our methods for producing a composite of two fibroin-based materials that supports the co-cultivation of human limbal epithelial (HLE) cells and human limbal stromal (HLS) cells. The resulting tissue substitute consists of a stratified epithelium overlying a three-dimensional arrangement of extracellular matrix components (principally ‘degummed’ fibroin fibers) and mesenchymal stromal cells. This tissue substitute is currently being evaluated as a tool for reconstructing the corneal limbus and corneal epithelium.

Of tails and Tempests : Feminine sexuality and Shakespearean children's texts

This paper reads a range of nineteenth-century texts for children that retell either Shakespeare's The Tempest or mermaid narratives, considering the models of feminine subjectivity and sexuality that they construct. It then moves on to two key contemporary texts — Disney's film adaptation of The Little Mermaid (Clements and Musker 1989) and Penni Russon's Undine (2004) — that combine the Shakespearean heroine with the mermaid, and reads them against the nineteenth-century models. Ultimately, the essay determines that, while these texts seem to perform a progressive appropriation of the two traditions, they actually combine the most conservative aspects of both The Tempest and mermaid stories to produce authoritative (and dangerously persuasive) ideals of passive feminine sexuality that confine girls within patriarchally-dictated familial positions. The new figure for adolescent female subjectivity, the mermaid-Miranda, becomes in turn a model of identification and aspiration for the implied juvenile consumer.

The city is connections : Seoul as an urban network

With the rise of ubiquitous computing in recent years, concepts of spatiality have become a significant topic of discussion in design and development of multimedia systems. This article investigates spatial practices at the intersection of youth, technology, and urban space in Seoul, and examines what the author calls ‘transyouth’: in the South Korean context, these people are between the ages of 18 and 24, situated on the delicate border between digital natives and immigrants in Prensky’s (2001) terms. In the first section, the article sets out the technosocial environment of contemporary Seoul. This is followed by a discussion of social networking processes derived from semi-structured interviews conducted in 2007-8 with Seoul transyouth about their ‘lived experiences of the city.’ Interviewees reported how they interact to play, work, and live with and within the city’s unique environment. The article develops a theme of how technosocial convergence (re)creates urban environments and argues for a need to consider such user-driven spatial recreation in designing cities as (ubiquitous) urban networks in recognition of its changing technosocial contours of connections. This is explored in three spaces of different scales: Cyworld as an online social networking space; cocoon housing – a form of individual residential space which is growing rapidly in many Korean cities – as a private living space; and u-City (ubiquitous City) as the future macro-space of Seoul.

Evaluation of silk sericin as a biomaterial: In vitro growth of human corneal limbal epithelial cells on Bombyx mori sericin membranes

Sericin and fibroin are the two major proteins in the silk fibre produced by the domesticated silkworm, Bombyx mori. Fibroin has been extensively investigated as a biomaterial. We have previously shown that fibroin can function successfully as a substratum for growing cells of the eye. Sericin has been so far neglected as a biomaterial because of suspected allergenic activity. However, this misconception has now been dispelled, and sericin’s biocompatibility is currently indisputable. Aiming at promoting sericin as a possible substratum for the growth of corneal cells in order to make tissue-engineered constructs for the restoration of the ocular surface, in this study we investigated the attachment and growth in vitro of human corneal limbal epithelial cells (HLECs) on sericin-based membranes. Sericin was isolated and regenerated from the silkworm cocoons by an aqueous procedure, manufactured into membranes, and characterized (mechanical properties, structural analysis, contact angles). Primary cell cultures from two donors were established in serum-supplemented media in the presence of murine feeder cells. Membranes made of sericin and fibroin-sericin blends were assessed in vitro as substrata for HLECs in a serum-free medium, in a cell attachment assay and in a 3-day cell growth experiment. While the mechanical characteristics of sericin were found to be inferior to those of fibroin, its ability to enhance the attachment of HLECs was significantly superior to fibroin, as revealed by the PicoGreen® assay. Evidence was also obtained that cells can grow and differentiate on these substrata.

Designing and evaluating mobile multimedia user experiences in public urban places : Making sense of the field

The majority of the world’s population now lives in cities (United Nations, 2008) resulting in an urban densification requiring people to live in closer proximity and share urban infrastructure such as streets, public transport, and parks within cities. However, “physical closeness does not mean social closeness” (Wellman, 2001, p. 234). Whereas it is a common practice to greet and chat with people you cross paths with in smaller villages, urban life is mainly anonymous and does not automatically come with a sense of community per se. Wellman (2001, p. 228) defines community “as networks of interpersonal ties that provide sociability, support, information, a sense of belonging and social identity.” While on the move or during leisure time, urban dwellers use their interactive information communication technology (ICT) devices to connect to their spatially distributed community while in an anonymous space. Putnam (1995) argues that available technology privatises and individualises the leisure time of urban dwellers. Furthermore, ICT is sometimes used to build a “cocoon” while in public to avoid direct contact with collocated people (Mainwaring et al., 2005; Bassoli et al., 2007; Crawford, 2008). Instead of using ICT devices to seclude oneself from the surrounding urban environment and the collocated people within, such devices could also be utilised to engage urban dwellers more with the urban environment and the urban dwellers within. Urban sociologists found that “what attracts people most, it would appear, is other people” (Whyte, 1980, p. 19) and “people and human activity are the greatest object of attention and interest” (Gehl, 1987, p. 31). On the other hand, sociologist Erving Goffman describes the concept of civil inattention, acknowledging strangers’ presence while in public but not interacting with them (Goffman, 1966). With this in mind, it appears that there is a contradiction between how people are using ICT in urban public places and for what reasons and how people use public urban places and how they behave and react to other collocated people. On the other hand there is an opportunity to employ ICT to create and influence experiences of people collocated in public urban places. The widespread use of location aware mobile devices equipped with Internet access is creating networked localities, a digital layer of geo-coded information on top of the physical world (Gordon & de Souza e Silva, 2011). Foursquare.com is an example of a location based 118 Mobile Multimedia – User and Technology Perspectives social network (LBSN) that enables urban dwellers to virtually check-in into places at which they are physically present in an urban space. Users compete over ‘mayorships’ of places with Foursquare friends as well as strangers and can share recommendations about the space. The research field of Urban Informatics is interested in these kinds of digital urban multimedia augmentations and how such augmentations, mediated through technology, can create or influence the UX of public urban places. “Urban informatics is the study, design, and practice of urban experiences across different urban contexts that are created by new opportunities of real-time, ubiquitous technology and the augmentation that mediates the physical and digital layers of people networks and urban infrastructures” (Foth et al., 2011, p. 4). One possibility to augment the urban space is to enable citizens to digitally interact with spaces and urban dwellers collocated in the past, present, and future. “Adding digital layer to the existing physical and social layers could facilitate new forms of interaction that reshape urban life” (Kjeldskov & Paay, 2006, p. 60). This methodological chapter investigates how the design of UX through such digital placebased mobile multimedia augmentations can be guided and evaluated. First, we describe three different applications that aim to create and influence the urban UX through mobile mediated interactions. Based on a review of literature, we describe how our integrated framework for designing and evaluating urban informatics experiences has been constructed. We conclude the chapter with a reflective discussion on the proposed framework.

Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin membranes

Membranes prepared from a protein, fibroin, isolated from domesticated silkworm (Bombyx mori) silk, support the cultivation of human limbal epithelial (HLE) cells and thus display significant potential as biomaterials for ocular surface reconstruction. We presently extend this promising avenue of research by directly comparing the attachment, morphology and phenotype of primary HLE cell cultures grown on fibroin to that observed on donor amniotic membrane (AM), the current clinical standard substrate for HLE transplantation. Fibroin membranes measuring 6.3 ± 0.5 μm (mean ± sd) in thickness and permeable to FITC dextran of a molecular weight up to 70 kDa, were used. Attachment of HLE cells to fibroin was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. Nevertheless, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (K3/K12 expression) and displayed a comparable number and distribution of ΔNp63+ progenitor cells to that seen in cultures grown on AM. These results support the suitability of membranes constructed from Bombyx mori silk fibroin as substrata for HLE cultivation and encourage progression to studies of efficacy in preclinical models.

Incorporation of exogenous RGD peptide and inter-species blending as strategies for enhancing human corneal limbal epithelial cell growth on Bombyx mori silk fibroin membranes

While fibroin isolated from the cocoons of domesticated silkworm Bombyx mori supports growth of human corneal limbal epithelial (HLE) cells, the mechanism of cell attachment remains unclear. In the present study we sought to enhance the attachment of HLE cells to membranes of Bombyx mori silk fibroin (BMSF) through surface functionalization with an arginine-glycine-aspartic acid (RGD)-containing peptide. Moreover, we have examined the response of HLE cells to BMSF when blended with the fibroin produced by a wild silkworm, Antheraea pernyi, which is known to contain RGD sequences within its primary structure. A procedure to isolate A. pernyi silk fibroin (APSF) from the cocoons was established, and blends of the two fibroins were prepared at five different BMSF/APSF ratios. In another experiment, BMSF surface was modified by binding chemically the GRGDSPC peptide using a water-soluble carbodiimide. Primary HLE were grown in the absence of serum on membranes made of BMSF, APSF, and their blends, as well as on RGD-modified BMSF. There was no statistically significant enhancing effect on the cell attachment due to the RGD presence. This suggests that the adhesion through RGD ligands may have a complex mechanism, and the investigated strategies are of limited value unless the factors contributing to this mechanism become better known.

Assessment of freestanding membranes prepared from Antheraea pernyi silk fibroin as a potential vehicle for corneal epithelial cell transplantation

Freestanding membranes created from Bombyx mori silk fibroin (BMSF) offer a potential vehicle for corneal cell transplantation since they are transparent and support the growth of human corneal epithelial cells (HCE). Fibroin derived from the wild silkworm Antheraea pernyi (APSF) might provide a superior material by virtue of containing putative cell- attachment sites that are absent from BMSF. Thus we have investigated the feasibility of producing transparent, freestanding membranes from APSF and have analysed the behaviour of HCE cells on this material. No significant differences in cell numbers or phenotype were observed in short term HCE cell cultures established on either fibroin. Production of transparent freestanding APSF membranes, however, proved to be problematic as cast solutions of APSF were more prone to becoming opaque, displayed significantly lower permeability and were more brittle than BMSF-membranes. Cultures of HCE cells established on either membrane developed a normal stratified morphology with cytokeratin pair 3/12 being immuno-localized to the superficial layers. We conclude that while it is feasible to produce transparent freestanding membranes from APSF, the technical difficulties associated with this biomaterial, along with an absence of enhanced cell growth, currently favours the continued development of BMSF as a preferred vehicle for corneal cell transplantation. Nevertheless, it remains possible that refinement of techniques for processing APSF might yet lead to improvements in the handling properties and performance of this material.

Treatment of silk fibroin with poly(ethylene glycol) for the enhancement of corneal epithelial cell growth

A silk protein, fibroin, was isolated from the cocoons of the domesticated silkworm (Bombyx mori) and cast into membranes to serve as freestanding templates for tissue-engineered corneal cell constructs to be used in ocular surface reconstruction. In this study, we sought to enhance the attachment and proliferation of corneal epithelial cells by increasing the permeability of the fibroin membranes and the topographic roughness of their surface. By mixing the fibroin solution with poly(ethylene glycol) (PEG) of molecular weight 300 Da, membranes were produced with increased permeability and with topographic patterns generated on their surface. In order to enhance their mechanical stability, some PEG-treated membranes were also crosslinked with genipin. The resulting membranes were thoroughly characterized and compared to the non-treated membranes. The PEG-treated membranes were similar in tensile strength to the non-treated ones, but their elastic modulus was higher and elongation lower, indicating enhanced rigidity. The crosslinking with genipin did not induce a significant improvement in mechanical properties. In cultures of a human-derived corneal epithelial cell line (HCE-T), the PEG treatment of the substratum did not improve the attachment of cells and it enhanced only slightly the cell proliferation in the longer term. Likewise, primary cultures of human limbal epithelial cells grew equally well on both non-treated and PEG-treated membranes, and the stratification of cultures was consistently improved in the presence of an underlying culture of irradiated 3T3 feeder cells, irrespectively of PEG-treatment. Nevertheless, the cultures grown on the PEG-treated membranes in the presence of feeder cells did display a higher nuclear-to-cytoplasmic ratio suggesting a more proliferative phenotype. We concluded that while the treatment with PEG had a significant effect on some structural properties of the B. mori silk fibroin (BMSF) membranes, there were minimal gains in the performance of these materials as a substratum for corneal epithelial cell growth. The reduced mechanical stability of freestanding PEG-treated membranes makes them a less viable choice than the non-treated membranes.