Genetically engineered silk and genipin-enhanced fibrin hydrogel for annulus fibrosus repair

INTRODUCTION: Around 80% of people are affected by low back pain at least once in their life, often caused by trauma provoking intervertebral disc (IVD) herniation and/or IVD degeneration. Apart from some promising approaches for nucleus pulposus repair, so far no treatment or repair is available for the outer fibrous tissue, annulus fibrosus (AF). We aimed for sealing and repairing an AF injury in a bovine IVD organ culture model in vitro over 14 days under different loading conditions. For this purpose, a silk fleece composite from Bombyx mori silk was combined with genipin-enhanced fibrin hydrogel [1]. METHODS: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue, followed by cutting out the IVDs [2]. Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described. On the next day, injury was induced using a 2mm biopsy punch (Polymed, Switzerland). The formed cavity was filled with (0.4%) genipin-enhanced human based fibrin hydrogel (35- 55mg/mL human fibrinogen, Baxter, Austria) and sealed with a silk fleece-membrane composite (Spintec Engineering, Germany). Different culture conditions were applied: free swelling, static diurnal load of 0.2MPa for 8h/d and complex loading at 0.2MPa compression combined with ± 2° torsion at 0.2Hz for 8h/d. Complex loading was applied by a custom built 2 degree of freedom bioreactor [3]. After 14 days of culture cell activity was determined with resazurin assay. Additionally, glycosaminoglycan (dimethyl-methylene blue), DNA (Hoechst) and collagen content (hydroxy-proline) were determined. Finally, real-time qPCR of major IVD marker genes was performed. RESULTS: The silk seal closing the injury site could successfully withstand the forces of all three loading conditions with no misplacement over the two weeks’ culture. Nevertheless, disc height of the repaired discs did not significantly differ from the injured group. The disc phenotype could be maintained as demonstrated by biochemical analysis of gene expression, cell activity, DNA-, collagen- and GAG content. The silk itself was evaluated to be highly biocompatible for hMSC, as revealed by cytotoxicity assays. DISCUSSION & CONCLUSIONS: The silk can be considered a highly-elastic and biocompatible material for AF closure and the genipin-enhanced fibrin hydrogel has also good biomechanical properties. However, the cyto-compatibility of genipin seems rather poor and other hydrogels and/or cross-linkers should be looked into. REFERENCES: 1 C.C. Guterl et al. (2014) Characterization of Mechanics and Cytocompatibility of Fibrin Genipin Annulus Fibrosus Sealant with the Addition of Cell Adhesion Molecules, Tissue Eng Part A 2 S.C. Chan, B. Gantenbein-Ritter (2012) Preparation of intact bovine tail intervertebral discs for organ culture, J Vis Exp 3 B Gantenbein et al. (2015) Organ Culture Bioreactors - Platforms to Study Human Intervertebral Disc Degeneration and Regenerative Therapy, Curr Stem Cell Res Ther [epub ahead of print] ACKNOWLEDGEMENTS: This project is supported by the Gebert Rüf Stiftung project # GRS-028/13.

Non-Viral Gene Therapy to Cells of the bovine Intervertebral Disc

Background. Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and degeneration. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM).1 Further studies showed that growth factors from transforming growth factor β (TGFβ) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC).2 Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods. Bovine nucleus pulposus (bNPC) and annulus fibrosus cells (bAFC) were harvested from bovine coccygeal IVD. Primary cells were then electroporized with plasmid GDF6 (Origene, vector RG211366) by optimizing parameters using the Neon Transfection system (Life Technologies, Basel). After transfection, cells were cultured in 2D monolayer or 3D alginate beads for 7, 14 or 21 days. Transfection efficiency of pGDF6 was analyzed by immunohistochemistry and fluorescent microscopy. Cell phenotype was quantified by real-time RT-PCR. To test a non-viral gene therapy applied directly to 3D whole organ culture, coccygeal bovine IVDs were harvested as previously described. Bovine IVDs were transfected by injection of plasmid GDF6 into the center. Electroporation was performed with ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) using 2-needle array electrode or tweezertrodes. 72 h after tranfection discs were fixed and cryosectioned and analyzed by immunofluorescence against GDF6. Results. RT-PCR and immunohistochemistry confirmed up-regulation of GFP and GDF6 in the primary bNPC/bAFC culture. The GFP-tagged GDF6 protein, however, was not visible, possibly due to failure of dimer formation as a result of fusion structure. Organ IVD culture transfection revealed GDF6 positive staining in the center of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GDF6 positive cells. Conclusion. Non-viral transfection is an appealing approach for gene therapy as it fulfills the translational safety aspects of transiency and lacks the toxic effects of viral transduction. We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgements. This project was funded by the Lindenhof Foundation (Funds “Research & Teaching”) Project no. 13-02-F. The imaging part of this study was performed with the facility of the Microscopy Imaging Center (MIC), University of Bern. References. Roughly PJ (2004): Spine (Phila), 29:2691-2699 Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014), Arthritis Research & Therapy, 16:R67

The friction of an oscillating bearing,

"The work described has been carried out in the Engineering Department of the National Physical Laboratory."--Acknowledgements.

Health education; a program for girls in secondary schools.

"Acknowledgements" signed: Esther Sherman.

Skilled nursing facilities : available data show average nursing staff time changed little after Medicare payment increase : report to congressional committees.

"GAO-03-176."

Growing older in Illinois : needs, challenges, opportunities.

"This report was written by the Heartland Center on Aging, Disability and Long Term Care, School of Public and Environmental Affairs, Indiana University and the National Center for Senior Living, South Bend, Indiana. May, 1991." Acknowledgements.

Think small! / curated by Robert Sill.

"The catalog was made possible in part by financial support from Chase Bank and the Illinois State Museum."

The Pilgrim hymnal with responsive readings.

Preface -- Contents -- Order of Subjects -- The hymns -- Sentences and canticles -- Canticles -- Responsive readings and other aids to worship -- Editorial notes -- Acknowledgements -- Indexes of tunes -- Indexes of hymns -- Index of sentences and canticles.

A history of education in the United States,

"With acknowledgements to J. E. Miller, A.M., and to other members of my educational seminar for valuable assistance in the preparation of material."

Federal employees' health benefits : effects of using pharmacy benefit managers on health plans, enrollees, and pharmacies : report to the Honorable Byron L. Dorgan, U.S. Senate.

"GAO-03-196."

Utilising survey data to inform public policy: Comparison of the cost-effectiveness of treatment of ten mental disorders

Background Mental health survey data are now being used proactively to decide how the burden of disease might best be reduced. Aims To study the cost-effectiveness of current and optimal treatments for mental disorders and the proportion of burden avertable by each. Method Data for three affective, four anxiety and two alcohol use disorders and for schizophrenia were compared in terms of cost, burden averted and efficiency of current and optimal treatment. We then calculated the burden unavertable given current knowledge. The unit of health gain was a reduction in the years lived with disability (YLDs). Results Summing across all disorders, current treatment averted 13% of the burden, at an average cost of AUS$30 000 per YLD gained. Optimal treatment at current coverage could avert 20% of the burden, at an average cost of AUS$18 000 per YLD gained. Optimal treatment at optimal coverage could avert 28% of the burden, at AUS$16 000 per YLD gained. Sixty per cent of the burden of mental disorders was deemed to be unavertable. Conclusions The efficiency of treatment varied more than tenfold across disorders. Although coverage of some of the more efficient treatments should be extended, other factors justify continued use of less-efficient treatments for some disorders. Declaration of interest None. Funding detailed in Acknowledgements.

Psychotic symptom and cannabis relapse in recent-onset psychosis - Prospective study

Background: Cannabis use appears to exacerbate psychotic symptoms and increase risk of psychotic relapse. However, the relative contribution of cannabis use compared with other risk factors is unclear. The influence of psychotic symptoms on cannabis use has received little attention. Aims: To examine the influence of cannabis use on psychotic symptom relapse and the influence of psychotic symptom severity on relapse in cannabis use in the 6 months following hospital admission. Method: At baseline, 84 participants with recent-onset psychosis were assessed and 81 were followed up weekly for 6 months, using telephone and face-to-face interviews. Results: A higher frequency of cannabis use was predictive of psychotic relapse, after controlling for medication adherence, other substance use and duration of untreated psychosis. An increase in psychotic symptoms was predictive of relapse to cannabis use, and medication adherence reduced cannabis relapse risk. Conclusions: The relationship between cannabis use and psychosis may be bidirectional, highlighting the need for early intervention programmes to target cannabis use and psychotic symptom severity in this population. Declaration of interest: None. Funding detailed in Acknowledgements.

The Politics of Permaculture: Towards an Ecologically Situated Approach to Resource Policy and Governance

The contributions to environmental politics of Torgerson, Oelschlager, Dryzek, Harrè and others, converge in their respective acknowledgements that the shift towards an ‘ecologically situated’ approach to environmental policy, including resource governance, will require the emergence and consolidation of a new lingua franca of environmental discourse. In this paper, I suggest that an extrapolation of permaculture ethics may provide a gambit through which such a discourse may be assembled and organised. I examine six key signifying elements derived from Orr and Capra’s approach to ecological literacy – network, nested system, flow, cycle, development and dynamic balance – and explore the implications that these might have for resource governance and policy, including the (re)framing of assessment indicators, energy auditing, resource management and integrated planning and development.

The programming language python in earth system simulations

-scale vary from a planetary scale and million years for convection problems to 100km and 10 years for fault systems simulations. Various techniques are in use to deal with the time dependency (e.g. Crank-Nicholson), with the non-linearity (e.g. Newton-Raphson) and weakly coupled equations (e.g. non-linear Gauss-Seidel). Besides these high-level solution algorithms discretization methods (e.g. finite element method (FEM), boundary element method (BEM)) are used to deal with spatial derivatives. Typically, large-scale, three dimensional meshes are required to resolve geometrical complexity (e.g. in the case of fault systems) or features in the solution (e.g. in mantel convection simulations). The modelling environment escript allows the rapid implementation of new physics as required for the development of simulation codes in earth sciences. Its main object is to provide a programming language, where the user can define new models and rapidly develop high-level solution algorithms. The current implementation is linked with the finite element package finley as a PDE solver. However, the design is open and other discretization technologies such as finite differences and boundary element methods could be included. escript is implemented as an extension of the interactive programming environment python (see www.python.org). Key concepts introduced are Data objects, which are holding values on nodes or elements of the finite element mesh, and linearPDE objects, which are defining linear partial differential equations to be solved by the underlying discretization technology. In this paper we will show the basic concepts of escript and will show how escript is used to implement a simulation code for interacting fault systems. We will show some results of large-scale, parallel simulations on an SGI Altix system. Acknowledgements: Project work is supported by Australian Commonwealth Government through the Australian Computational Earth Systems Simulator Major National Research Facility, Queensland State Government Smart State Research Facility Fund, The University of Queensland and SGI.

Catholic literature and secularisation in France and England, 1880-1914

This book is the first comparative study of its kind to explore at length the French and English Catholic literary revivals of the late nineteenth and early twentieth centuries. It compares individual and societal secularisation in France and England and examines how French and English Catholic writers understood and contested secular mores, ideologies and praxis, in the individual, societal and religious domains. It also addresses the extent to which some Catholic writers succumbed to the seduction of secular instincts, even paradoxically in themes which are considered to be emblematic of Catholic literature. The breadth of this book will make it a useful guide for students wishing to become familiar with a wide range of such writings in France and England during this period. It will also appeal to researchers interested in Catholic literary and intellectual history in France and England, theologians, philosophers and students of the sociology of religion. CONTENTS: Preface and acknowledgements Introduction 1. Individual and societal secularisation in France and England 2. Recovering the porous individual 3. Thinking and believing 4. The fragments of secular society 5. Mending secular fragmentation 6. Ultimate societal values 7. Catholic religiosity and the hierarchical Church 8. Catholic religiosity and the charismatic Church Conclusion Bibliography Index