Bone tissue engineering : reconstruction of critical sized segmental bone defects in the ovine tibia

Well-established therapies for bone defects are restricted to bone grafts which face significant disadvantages (limited availability, donor site morbidity, insufficient integration). Therefore, the objective was to develop an alternative approach investigating the regenerative potential of medical grade polycaprolactone-tricalcium phosphate (mPCL-TCP) and silk-hydroxyapatite (silk-HA) scaffolds. Critical sized ovine tibial defects were created and stabilized. Defects were left untreated, reconstructed with autologous bone grafts (ABG) and mPCL-TCP or silk-HA scaffolds. Animals were observed for 12 weeks. X-ray analysis, torsion testing and quantitative computed tomography (CT) analyses were performed. Radiological analysis confirmed the critical nature of the defects. Full defect bridging occurred in the autograft and partial bridging in the mPCL-TCP group. Only little bone formation was observed with silk-HA scaffolds. Biomechanical testing revealed a higher torsional moment/stiffness (p < 0.05) and CT analysis a significantly higher amount of bone formation for the ABG group when compared to the silk-HA group. No significant difference was determined between the ABG and mPCL-TCP groups. The results of this study suggest that mPCL-TCP scaffolds combined can serve as an alternative to autologous bone grafting in long bone defect regeneration. The combination of mPCL-TCP with osteogenic cells or growth factors represents an attractive means to further enhance bone formation.

An in-vitro 3-D cell culture model for studying pathomechanisms in AMD

Purpose To develop a novel 3-D cell culture model with the view to studying the pathomechanisms underlying the development of age-related macular degeneration (AMD). Our central hypothesis is that the silk structural protein fibroin used in conjunction with cultured human cells can be used to mimic the structural relationships between the RPE and choriocapillaris in health and disease. Methods Co-cultures of human RPE cells (ARPE-19 cells grown in Miller’s medium) and microvascular endothelial cells (HMEC-1 cells grown in endothelial culture medium) were established on opposing sides of a synthetic Bruch’s membrane (3 microns thick) constructed from B mori silk fibroin. Cell attachment was facilitated by pre-coating the fibroin membrane with vitronectin (for ARPE-19 cells) and gelatin (for HMEC-1 cells) respectively. The effects of tropoelastin on attachment of ARPE-19 cells was also examined. Barrier function was examined by measurement of trans-epithelial resistance (TER) using a voltohmmeter (EVOM-2). The phagocytic activity of the synthetic RPE was tested using vitronectin-coated microspheres (2 micron diameter FluoSpheres). In some cultures, membrane defects were created by puncturing within a 24 G needle. The architecture of the synthetic tissue before and after wounding was examined by confocal microscopy after staining for ZO-1 and F-actin. Results The RPE layer of the 3D model developed a cobblestoned morphology (validated by staining for ZO-1 and F-actin), displayed barrier function (validated by measurement of TER) and demonstrated cytoplasmic uptake of vitronectin-coated microspheres. Attachment of ARPE-19 cells to fibroin was unaffected by tropoelastin. Microvascular endothelial cells attached well to the gelatin-coated surface of the fibroin membrane and remained physically separated from the overlaying RPE layer. The fibroin membranes were amenable to puncturing without collapse thus providing the opportunity to study transmembrane migration of the endothelial cells. Conclusions Synthetic Bruch’s membranes constructed from silk fibroin, vitronectin and gelatin, support the co-cultivation of RPE cells and microvascular endothelial cells. The resulting RPE layer displays functions similar to that of native RPE and the entire tri-layered structure displays potential to be used as an in vitro model of choroidal neovascularization.

Development of an ultra-thin fibroin membrane for RPE cell transplantation

Purpose: One of the challenges associated with cell-based therapies for repairing the retina is the development of suitable materials on which to grow and transplant retinal cells. Using the ARPE-19 cell line, we have previously demonstrated the feasibility of growing RPE-derived cells on membranes prepared from the silk protein fibroin. The present study was aimed at developing a porous, ultra-thin fibroin membrane that might better support development of apical-basal polarity in culture, and to extend this work to primary cultures of human RPE cells. Methods: Ultra-thin fibroin membranes were prepared using a highly polished casting table coated with Topas® (a cyclic olefin copolymer) and a 1:0.03 aqueous solution of fibroin and PEO (Mv 900 000 g/mol). Following drying, the membranes were water annealed to make them water-stable, washed in water to remove PEO, sterilised by treatment with 95% ethanol, and washed extensively in saline. Primary cultures containing human RPE cells were established from donor posterior eye cups and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotics. First passage cultures were seeded onto fibroin membranes pre-coated with vitronectin and grown for 6 weeks in medium supplemented with 1% serum. Comparative cultures were established on porous 1.0 µm pore PET membrane (Millipore) and using ARPE-19 cells. Results: The fibroin membranes displayed an average thickness of 3 µm and contained numerous dimples/pore-like structures of up to 3-5 µm in diameter. The primary cultures predominantly contained pigmented epithelial cells, but mesenchymal cells (presumed fibroblasts) were also often present. Passaged cultures appeared to attach equally well to either fibroin or PET membranes. Over time cells on either material adopted a more cobblestoned morphology. Conclusions: Progress has been made towards developing a porous ultra-thin fibroin membrane that supports cultivation of RPE cells. Further studies are required to determine the degree of membrane permeability and RPE polarity.

Fibroin-based materials support cultivation of limbal stromal cells

Purpose: The silk protein fibroin provides a potential substrate for use in ocular tissue reconstruction. We have previously demonstrated that transparent membranes produced from fibroin support cultivation of human limbal epithelial cells (Tissue Eng A. 14(2008)1203-11). We presently extend this body of work to studies of human limbal stromal cell (HLS) growth on fibroin in the presence and absence of serum. Methods: Primary cultures of HLS cells were established in DMEM/F12 medium supplemented with either 10% fetal bovine serum (FBS) or 2% B27 supplement. Defined keratinocyte serum-free medium (DK-SFM, Invitrogen) was also tested. The resulting cultures were analysed by flow cytometry for expression of CD34, CD90, CD45, and CD141. Cultures grown under each condition were subsequently passaged either onto transparent fibroin membranes prepared from purified fibroin or within 3D scaffolds prepared from partially-solubilised fibroin. Results: HLS cultures were successfully established under each condition, but grew more slowly and passaged poorly in the absence of serum. Cultures grown in 10% FBS were <0.5% CD34+ (keratocytes) and >97% CD90+ (fibroblasts). Cultures established in 2% B27 formed floating spheres and contained >8% CD34+ cells and reduced CD90 expression. Cultures established in DK-SFM displayed traces of epithelial cell growth (CD141), but mostly consisted of CD90+ cells with <1% CD34+ cells. Cells of bone marrow lineage (CD45) were rarely observed under any conditions. Cultures grown in 10% FBS were able to adhere to and proliferate on silk fibroin 3-D scaffolds and transparent films while those grown serum-free could not. Adhesion of HLS cells to fibroin was initially poorer than that displayed on tissue culture plastic. Conclusions: HLS cultures containing cells of predominantly fibroblast lineage can be grown on fibroin-based materials, but this process is dependent upon additional ECM factors such as those provided by serum.

Learner strategy instruction and student uptake of strategies in a Japanese as a foreign language context

The Japanese language is recognised as being more difficult than European languages, needing three times more tuition time to reach comparable levels of proficiency. Encouraging Japanese as a Foreign Language (JFL) students to become aware of, and effectively use, learner strategies is one way to assist them become more controlled, effective learners leading to enhanced language learning. This thesis investigates the development and implementation of a JFL curriculum implemented in a university course for students learning JFL. The curriculum was developed specifically to assist beginner university students with the development of learner strategies appropriate for a JFL reading context. The theoretical underpinning of the study was informed by Educational Criticism (Eisner, 1998), which aims to describe, interpret and evaluate the processes of interaction between the teacher, the learner and the curriculum and the students' learning processes in a tertiary JFL classroom. The study investigated the effect on student learning processes of a JFL reading program that incorporated explicit learner strategy instruction and identified factors that enhanced or impeded the development of learner strategy knowledge. The participants in the study were 29 students enrolled in the course, 10 of whom volunteered to undertake additional tasks, and the two teachers who implemented the curriculum. Data collection involved a number of different strategies to observe the students' participation in the classroom and learning experiences. Learning processes were investigated through TOL protocols, classroom observations, course evaluations, interviews, and learner strategy use measurement instruments (SILL, SILK and SORS) to document student uptake of learner strategies. The design of the study and its applied focus recognised my expertise as a JFL teacher, curriculum writer and researcher, an approach that aligns with the purpose of a Professional Doctorate. Four general thematics, or principles, were identified in this study: „h Explicit learner strategy instruction provides the context for students to develop awareness of learner strategies and take control of their learning; „h Collaborative learning and interaction with teachers offers students the opportunity for shared knowledge construction; „h Reflection offers teachers and students the opportunity to reflect on their own learning style and strategy knowledge, and raises awareness of other available strategies; and „h Diverse cultural and linguistic backgrounds have an impact on curriculum implementation and student uptake of learner strategies. The study¡¦s methodological contribution is that it is one of the first in Australia to use Educational Criticism (Eisner, 1998) as a research methodology. The findings contribute to theoretical knowledge in the fields of Applied Linguistics, Second Language Teaching and Learning, Second Language Acquisition and JFL Teaching and Learning by offering new knowledge on the importance of learner strategies in the beginner JFL classroom, the potential of explicit strategy instruction, the value of reflection for both teachers and students, and the important role of the teacher in the process of curriculum implementation. The general principles identified and the findings of this in-depth study of a JFL classroom can be drawn upon to inform other teaching practice situations, and invite practitioners from not just Japanese, but from other language areas and other disciplines, to examine and improve their own practices, and suggest further research questions to pursue this line of enquiry.

Association between postmenopausal osteoporosis and experimental periodontitis

To investigate the correlation between postmenopausal osteoporosis (PMO) and the pathogenesis of periodontitis, ovariectomized rats were generated and the experimental periodontitis was induced using a silk ligature. The inflammatory factors and bone metabolic markers were measured in the serum and periodontal tissues of ovariectomized rats using an automatic chemistry analyzer, enzyme-linked immunosorbent assays, and immunohistochemistry. The bone mineral density of whole body, pelvis, and spine was analyzed using dual-energy X-ray absorptiometry and image analysis. All data were analyzed using SPSS 13.0 statistical software. It was found that ovariectomy could upregulate the expression of interleukin- (IL-)6, the receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG) and downregulate IL-10 expression in periodontal tissues, which resulted in progressive alveolar bone loss in experimental periodontitis. This study indicates that changes of cytokines and bone turnover markers in the periodontal tissues of ovariectomized rats contribute to the damage of periodontal tissues.

Space within the surface : the patterned surfaces of Florence Broadhurst and Jim Thompson

The Woods Bagot 2007 refurbishment of the Qantas and British Airways Bangkok Business lounge in the Survarnabhumi Airport features wall finishes designed by wallpaper designer, Florence Broadhurst (1899-1977) and Thai Silk trader, Jim Thompson (1906-1967). This distinctive selection, which is proclaimed on the airport’s website, of patterned wall surfaces side by side draws attention to their striking similarities and their defining differences . Thompson and Broadhurst would appear to be worlds apart, but here in the airport their work brings them together. Thompson, the son of a wealthy cotton family in America, worked as an architect before joining the army. He moved to Bangkok to start The Thai Silk Company in 1948. Broadhurst was born on a farm in Mt. Perry, Queensland. She began her career as a performance artist, as part of an Australian troupe in Shanghai, moving onto pursue a career in fashion design, catering to the middle and upper classes in London. Upon her return to Australia, Broadhurst started a print design company in 1959. Both Broadhurst and Thompson pursued multiple careers, lived many lives, and died under mysterious circumstances. Broadhurst was murdered in 1977 at her Sydney print warehouse, which remains an unsolved crime. Thompson disappeared in Malaysia in 1967 and his body has never been found. This chapter investigates the parallels between Thompson and Broadhurst and what lead them to design such popular patterns for wall surfaces towards the end of their careers. While neither designer was a household name, their work is familiar to most, seen in the costume and set design of films, on the walls of restaurants and cafes and even in family homes. The reason for the popularity of their patterns has not previously been analysed. However, this chapter suggests that the patterns are intriguing because they contain something of their designers’ identities. It suggests that the coloured surface provides a way of camouflaging and hiding its subjects’ histories, such that Broadhurst and Thompson, consciously or unconsciously, used the patterned surface as a plane in which their past lives could be buried. The revealing nature of the stark white wall, compared with the forgiveness provided by the pattern in which to hide, is elaborated by painter and advocate for polychromatic architecture, Fernand Léger in his essay, “The Wall, The Architect, The Painter (1965).” Léger writes that, “the modern architect has gone too far in his magnificent attempts to cleanse through emptiness,” and that the resultant white walls of modernity create ‘an impalpability of air, of slick, brilliant new surfaces where nothing can be hidden any longer …even shadows don’t dare to enter’. To counter the exposure produced by the white wall, Thompson and Broadhurst designed patterned surfaces that could harbour their personal histories. Broadhurst and Thompson’s works share a number of commonalities in their design production, even though their work in print design commenced a decade apart. Both designers opted to work more with traditional methods of pattern making. Broadhurst used hand-operated screens, and Thompson outsourced work to local weavers and refrained from operating out of a factory. Despite humble beginnings, Broadhurst and Thompson enjoyed international success with their wall patterns being featured in a number of renowned international hotels in Bahrain, Singapore, Sydney, and London in the 1970s and 1980s. Their patterns were also transferred to fabric for soft furnishings and clothing. Thompson’s patterns were used for costumes in films including the King and I and Ben Hur. Broadhurst’s patterns were also widely used by fashion designers and artists, such as Akira Isogowa‘s costume design for Salome, a 1998 production by the Sydney Dance Company. Most recently her print designs have been used by skin illustrator Emma Hack, in a series of works painting female bodies into Broadhurst’s patterns. Hack’s works camouflage the models’ bodies into the patterned surface, assimilating subject and surface, hinting at there being something living within the patterned wall. More than four decades after Broadhurst’s murder and five decades since Thompson’s disappearance, their print designs persist as more than just a legacy. They are applied as surface finishes with the same fervour as when the designs were first released. This chapter argues that the reason for the ongoing celebration of their work is that there is the impalpable presence of the creator in the patterns. It suggests that the patterns blur the boundary between subject and surface.

Laugh Lines : Exploring Humour in Children's Literature

Humour takes many shapes and forms. It can be as sharp as a surgeon's knife or as gentle as a touch of silk. It can convey uncomfortable truths, point up life's absurdities, challenge the imagination, take us by surprise, release us from fears and anxieties. And while it's true that we don't all agree about what's funny, it's also true that everyone of us finds pleasure in some form of humorous literature.

Tissue-engineered fibroin scaffolds for ocular tissue reconstruction

The silk protein fibroin (Bombyx mori) provides a potential substrate for use in ocular tissue reconstruction. We have previously demonstrated that transparent membranes produced from fibroin support cultivation of human limbal epithelial (HLE) cells (Tissue Eng A. 14(2008)1203-11). We extend this body of work to studies of limbal mesenchymal stromal cell (L-MSC) growth on fibroin. Also, we investigate the ability to produce a fibroin dual-layer scaffold with an upper HLE layer and lower L-MSC layer...

Fibroin-based materials support co-cultivation of limbal epithelial and stromal cells

The silk protein fibroin (Bombyx mori) provides a potential substrate for use in ocular tissue reconstruction. We have previously demonstrated that transparent membranes produced from fibroin support cultivation of human limbal epithelial (HLE) cells (Tissue Eng A. 14(2008)1203-11). We extend this body of work to studies of human limbal stromal cell (HLS) growth on fibroin in the presence and absence of serum. Also, we investigate the ability to produce a bi-layered composite scaffold of fibroin with an upper HLE layer and lower HLS layer.

Mauve: How one man invented the colour that changed the world

In an essay, "The Books of Last Things", Delia Falconer discusses the emergence of a new genre in publishing - microhistories. She cites a number of recent titles in non-fiction and fiction - Longitude, Cod, Tulips, Pushkin's Button, Nathaniel's Nutmeg, Zarafa, The Surgeon of Crowthorne, The Potato, The Perfect Storm. Delia Falconer observes of this tradition: "One has the sense, reading these books, of a surprising weight, of pleasant shock. In part, it is because we are looking at things which are generally present around us, but modestly out of sight and mind - historical nitty gritty like cod, potatoes, longitudinal clocks - which the authors have thrust suddenly, like a Biblical visitation of frogs or locusts, in our face. Things like spice and buttons and clocks are generally seen to enable history on the large scale, but are not often viewed as its worthy subjects. And by the same grand logic of history, more unusual phenomena like cabinets of curiosities or glass-making or farm lore or sailors' knots are simply odd blips on its radar screen, interesting footnotes. These new books, microhistories, reverse the usual order of history, which argues from the general to the particular, in order to prove its inevitable progress. They start from the footnotes. But by reversing the process, and walking through the back door of history, you don't necessarily end up at the front of the same house." Delia Falconer speculates about the reasons for the popularity of microhistories. She concludes: "I would like to think that reading them is not simply an exercise in nostalgia, but a challenge to the present". In Mauve, Simon Garfield provides a new way of thinking and writing about the history of intellectual property. Instead of providing a grand historical narrative of intellectual property, he tells the story of a particular invention, and its exploitation. Simon Garfield relates how English chemist William Perkin accidentally discovered a way to mass-produce colour mauve in a factory. Working on a treatment for malaria in his London home laboratory, Perkin failed to produce artificial quinine. Instead he created a dark oily sludge that turned silk a beautiful light purple. The colour was unique and became the most desirable shade in the fashion houses of Paris and London. ... The book Mauve will have a number of contemporary resonances for intellectual property lawyers and academics. Simon Garfield emphasizes the difficulties inherent in commercialising an invention and managing intellectual property. He investigates the uneasy collaboration between industry and science. Simon Garfield suggests that complaints about the efficacy of patent offices are perennial. He also highlights the problems faced by courts and law-makers in accommodating new technologies within the logic of patent law. In his elegant microhistory of the colour mauve, Simon Garfield confirms the conclusion of Brad Sherman and Lionel Bently that many aspects of modern intellectual property law can only be understood through an understanding of the past: "The image of intellectual property law that developed during the 19th century and the narrative of identity which this engendered played and continue to play an important role in the way we think about and understand intellectual property law".

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order +35 > +1 > -3 > -8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60-72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in 'glowing silkworms'. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.

Development of a cultured tissue substitute to repair the ageing retina

This project provides a foundation for the use of silk membranes in a tissue engineered therapy for the treatment of devastating retinal diseases such as age-related macular degeneration. The three-dimensional tissue model described in this thesis has great potential for use in basic research of retinal pathologies, and the potential to be implemented into clinical approaches after appropriate refinement.

New Perspectives on the Roman Coinage on the Eastern Limes in the Late Republican and Roman Imperial Period

The Ph.D. thesis discusses the monetary development in Roman Syria and Judaea in the Late Republican and the Early Imperial Period, from a numismatic, archaeological and historical point of view. In effect, the work focuses on the 1st century B.C. to the 1st century A.D., that is, the assumed time of introduction of Roman denarii to the region. The work benefits from the silver coin hoards of Khirbet Qumran recently published by the author. Though discovered as early as 1955 at Qumran, where the famous Dead Sea Scrolls had been found prior to that in 1947, most hoards remained unpublished until 2007. A second important source utilized is the so-called Tax Law from Palmyra in Syria. Its significance lies in the fact that Palmyra used to be one of the most important cities on the Silk Road, along which luxury goods were transported into the Roman Empire and Rome itself. During the research conducted, studies of the provincial coinage of Judaea (A.D. 6-66) shed new light on the authority of the Roman governors in economic and monetary matters in eastern Mediterranean regions. Furthermore, a new suggestion as to the length of the mandate period of Pontius Pilate is made. The extent of Emperor Augustus monetary reforms as well as the military history of Judaea are discussed in the light of new analytical studies, which show that the production of Roman base metal coins appears to have been a highly controlled process, contrary to popular opinion. Statistical calculations related to the coin alloy revealed striking similarities with Roman and other local metalwork found in Israel; a fact previously unknown. Results indicate that both Roman and local metalwork consisted of outstandingly systematized practises and may have exploited the same metal sources. Information: Kenneth Lönnqvist (*25.7.1962) has studied at the University of Helsinki since 1981. Furthermore, Lönnqvist has lived in the Mediterranean countries and the Near East, and made research there at various scientific institutions and universities for ca. 7 years. Contact and sales of thesis: kenneth.lonnqvist@helsinki.fi

The story of … the top hat

Throughout the 19th century, the top hat was a mainstay of Victorian life: a man in a topper was well-to-do, respectable, a man of industry. But now the top hat is only a caricature of the upper class privilege it once represented. Its history traces a line through dandies, beavers, silk, and madness...