Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

Measuring reef complexity and rugosity from monocular video bathymetric reconstruction

Topographic structural complexity of a reef is highly correlated to coral growth rates, coral cover and overall levels of biodiversity, and is therefore integral in determining ecological processes. Modeling these processes commonly includes measures of rugosity obtained from a wide range of different survey techniques that often fail to capture rugosity at different spatial scales. Here we show that accurate estimates of rugosity can be obtained from video footage captured using underwater video cameras (i.e., monocular video). To demonstrate the accuracy of our method, we compared the results to in situ measurements of a 2m x 20m area of forereef from Glovers Reef atoll in Belize. Sequential pairs of images were used to compute fine scale bathymetric reconstructions of the reef substrate from which precise measurements of rugosity and reef topographic structural complexity can be derived across multiple spatial scales. To achieve accurate bathymetric reconstructions from uncalibrated monocular video, the position of the camera for each image in the video sequence and the intrinsic parameters (e.g., focal length) must be computed simultaneously. We show that these parameters can be often determined when the data exhibits parallax-type motion, and that rugosity and reef complexity can be accurately computed from existing video sequences taken from any type of underwater camera from any reef habitat or location. This technique provides an infinite array of possibilities for future coral reef research by providing a cost-effective and automated method of determining structural complexity and rugosity in both new and historical video surveys of coral reefs.

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.

Silk fibroin in ocular surface reconstruction : what is its potential as a biomaterial in ophthalmics?

Hardly a month goes by within the scientific literature without some new material “X” being reported as a suitable material on which to grow cell type “Y”, for the potential purpose of treating disease “Z”. Thus when fibroin, a protein found in silk, was first proposed as a biomaterial for cell growth [1] it joined a long list of other materials of both natural as well as synthetic origin. Nevertheless, in the second decade of the Asian Century it is perhaps befitting that a material of so much importance to the continent’s cultural and economic history, should become the focus of cutting-edge biomedical research. Sentiments aside, however, silk fibroin possesses quite a unique combination of properties which make it a promising candidate for repairing the eye and especially for treating damage to the cornea, the transparent window at the front of the eye.

General trajectory prior for non-rigid reconstruction

Trajectory basis Non-Rigid Structure From Motion (NRSFM) currently faces two problems: the limit of reconstructability and the need to tune the basis size for different sequences. This paper provides a novel theoretical bound on 3D reconstruction error, arguing that the existing definition of reconstructability is fundamentally flawed in that it fails to consider system condition. This insight motivates a novel strategy whereby the trajectory's response to a set of high-pass filters is minimised. The new approach eliminates the need to tune the basis size and is more efficient for long sequences. Additionally, the truncated DCT basis is shown to have a dual interpretation as a high-pass filter. The success of trajectory filter reconstruction is demonstrated quantitatively on synthetic projections of real motion capture sequences and qualitatively on real image sequences.

Efficient articulated trajectory reconstruction using dynamic programming and filters

This paper considers the problem of reconstructing the motion of a 3D articulated tree from 2D point correspondences subject to some temporal prior. Hitherto, smooth motion has been encouraged using a trajectory basis, yielding a hard combinatorial problem with time complexity growing exponentially in the number of frames. Branch and bound strategies have previously attempted to curb this complexity whilst maintaining global optimality. However, they provide no guarantee of being more efficient than exhaustive search. Inspired by recent work which reconstructs general trajectories using compact high-pass filters, we develop a dynamic programming approach which scales linearly in the number of frames, leveraging the intrinsically local nature of filter interactions. Extension to affine projection enables reconstruction without estimating cameras.

Prostate gland and extra-capsular tissue 3D reconstruction and measurement

Currently there are little objective parameters that can quantify the success of one form of prostate surgical removal over another. Accordingly, at Old Dominion University (ODU) we have been developing a process resulting in the use of software algorithms to assess the coverage and depth of extra-capsular soft tissue removed with the prostate by the various surgical approaches. Parameters such as the percent of capsule that is bare of soft tissue and where present the depth and extent of coverage have been assessed. First, visualization methods and tools are developed for images of prostate slices that are provided to ODU by the Pathology Department at Eastern Virginia Medical School (EVMS). The visualization tools interpolate and present 3D models of the prostates. Measurement algorithms are then applied to determine statistics about extra-capsular tissue coverage. This paper addresses the modeling, visualization, and analysis of prostate gland tissue to aid in quantifying prostate surgery success. Particular attention is directed towards the accuracy of these measurements and is addressed in the analysis discussions.

Critical Factors for Sustainable Post Natural Disaster Reconstruction : Chinese Data

While scientists continue to explore the level of climate change impact to new weather patterns and our environment in general, there have been some devastating natural disasters worldwide in the last two decades. Indeed natural disasters are becoming a major concern in our society. Yet in many previous examples, our reconstruction efforts only focused on providing short-term necessities. How to develop resilience in the long run is now a highlight for research and industry practice. This paper introduces a research project aimed at exploring the relationship between resilience building and sustainability in order to identify key factors during reconstruction efforts. From extensive literature study, the authors considered the inherent linkage between the two issues as evidenced from past research. They found that sustainability considerations can improve the level of resilience but are not currently given due attention. Reconstruction efforts need to focus on resilience factors but as part of urban development, they must also respond to the sustainability challenge. Sustainability issues in reconstruction projects need to be amplified, identified, processed, and managed properly. On-going research through empirical study aims to establish critical factors (CFs) for stakeholders in disaster prone areas to plan for and develop new building infrastructure through holistic considerations and balanced approaches to sustainability. A questionnaire survey examined a range of potential factors and the subsequent data analysis revealed six critical factors for sustainable Post Natural Disaster Reconstruction that include: considerable building materials and construction methods, good governance, multilateral coordination, appropriate land-use planning and policies, consideration of different social needs, and balanced combination of long-term and short-term needs. Findings from this study should have an influence on policy development towards Post Natural Disaster Reconstruction and help with the achievement of sustainable objectives.

Autologous vs. allogenic mesenchymal progenitor cells for the reconstruction of critical-sized segmental tibial bone defects in aged sheep

Mesenchymal progenitor cells (MPCs) represent an attractive cell population for bone tissue engineering. Their special immunological characteristics suggest that MPCs may be used in an allogenic application. The objective of this study was to compare the regenerative potential of autologous vs. allogenic MPCs in an ovine critical-sized segmental defect model. Ovine MPCs were isolated from bone marrow aspirates, expanded and cultured with osteogenic media for two weeks before implantation. Autologous and allogenic transplantation was performed by using the cell-seeded scaffolds, unloaded scaffolds and the application of autologous bone grafts served as control groups (n=6). Bone healing was assessed twelve weeks after surgery by radiology, micro computed tomography, biomechanical testing and histology. Radiology, biomechanical testing and histology revealed no significant difference in bone formation between the autologous and allogenic group. Both cell groups showed more bone formation than the scaffold alone, whereas the biomechanical data showed no significant differences between the cell-groups and the unloaded scaffolds. The results of the study suggest that scaffold based bone tissue engineering using allogenic cells offers the potential for an off the shelf product. Therefore, the results of this study serve as an important baseline for the translation of the assessed concepts into clinical application.

Critical factors for successful housing reconstruction projects following a major disaster

Post–disaster reconstruction projects are often considered ineffectual or unproductive because on many occasions in the past they have performed extremely poorly during post-contract occupation, or have failed altogether to deliver acceptable outcomes. In some cases, these projects have already failed even before their completion, leading many sponsor aid organisations to hold these projects up as examples of how not to deliver housing reconstruction. Research into some previous unsuccessful projects has revealed that often the lack of adequate knowledge regarding the context and complexity involved in the implementation of these projects is generally responsible for their failure. Post-disaster reconstruction projects are certainly very complex in nature, often very context-specific and they can vary widely in magnitude. Despite such complexity, reconstruction projects can still have a high likelihood of success if adequate consideration is given to the importance of factors which are known to positively influence reconstruction efforts. Good outcomes can be achieved when planners and practitioners ensure best practices are embedded in the design of reconstruction projects at the time reconstruction projects they are first instigated. This paper outlines and discusses factors that significantly contribute to the successful delivery of post-disaster housing reconstruction projects.

Quantitative and qualitative assessment of the regenerative potential of osteoblasts versus bone marrow derived mesenchymal stem cells in the reconstruction of critical sized segmental tibial bone defects in a large animal model

This thesis is about the use of different cells for bone tissue engineering. The cells were used in combination with a novel biomaterial in a large tibial bone defects in a sheep model. Furthermore this study developed a novel cell delivery procedure for bone tissue engineering. This novel procedure of cell delivery could overcome the current problems of cell-based tissue engineering and serve as a baseline for the translation of novel concepts into clinical application.

Post-disaster reconstruction: a statistical overview of the barriers for community participation in Afghanistan

When a community already torn by an event such as a prolonged war, is then hit by a natural disaster, the negative impact of this subsequent disaster in the longer term can be extremely devastating. Natural disasters further damage already destabilised and demoralised communities, making it much harder for them to be resilient and recover. Communities often face enormous challenges during the immediate recovery and the subsequent long term reconstruction periods, mainly due to the lack of a viable community involvement process. In post-war settings, affected communities, including those internally displaced, are often conceived as being completely disabled and are hardly ever consulted when reconstruction projects are being instigated. This lack of community involvement often leads to poor project planning, decreased community support, and an unsustainable completed project. The impact of war, coupled with the tensions created by the uninhabitable and poor housing provision, often hinders the affected residents from integrating permanently into their home communities. This paper outlines a number of fundamental factors that act as barriers to community participation related to natural disasters in post-war settings. The paper is based on a statistical analysis of, and findings from, a questionnaire survey administered in early 2012 in Afghanistan.