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.

Geochemical data for drilled powder samples from two fossil Porites corals from Integrated Ocean Drilling Program Expedition 310

Fossil corals are unique archives of past seasonal climate variability, providing vital information about seasonal climate phenomena such as ENSO and monsoons. However, submarine diagenetic processes can potentially obscure the original climate signals and lead to false interpretations. Here we demonstrate the potential of laser ablation ICP-MS to rapidly detect secondary aragonite precipitates in fossil Porites colonies recovered by Integrated Ocean Drilling Program (IODP) Expedition 310 from submerged deglacial reefs off Tahiti. High resolution (100 µm) measurements of coralline B/Ca, Mg/Ca, S/Ca, and U/Ca ratios are used to distinguish areas of pristine skeleton from those afflicted with secondary aragonite. Measurements of coralline Sr/Ca, U/Ca and oxygen isotope ratios, from areas identified as pristine, reveal that the seasonal range of sea surface temperature in the tropical south Pacific during the last deglaciation (14.7 and 11 ka) was similar to that of today.

Trends in selected water-quality characteristics, Powder River and tributaries, Montana and Wyoming, water years 1968-88 and 1975-88 /

Mode of access: Internet.

Hearing: in the matter of the minimum wage recommendations of Special Industry Committee no. 17-A for the silk, rayon, and nylon underwear division and the general division of the needlework and fabricated textile products industry in Puerto Rico, August 29 and 30, 1955.

Mode of access: Internet.

The new American gardener; containing practical directions on the culture of fruits and vegetables; including landscape and ornamental gardening, grape-vines, silk, strawberries, &c. &c.

Mode of access: Internet.

Condensed milk and milk powder; prepared for factory, school and laboratory.

Advertising matter: p. 663-696.

The powder metallurgy of uranium /

Uranium is a ductile metal and cannot be comminuted to a fine powder by any mechanical means such as crushing, milling or grinding. Uranium, however, reacts readily with hydrogen and forms UH3, which is a fine powder of less than 400 mesh screen size. The factors controlling the rats of the hydride formation are: (a) The surface area of the metal; (b) the temperature at which the reaction takes place; (c) the pressure of hydrogen. In order to increase the reaction area, one has to hydride small metal pieces rather than a single mass. The hydrogen reacts with uranium metal at temperatures as low as 100 deg to 1500 deg, and the reaction rate becomes quite rapid at approximately 225 deg C. The hydrogen for this purpose has to be of high purity and any small amount of oxygen in hydrogen delays the start of the reaction.

Condensed milk and milk powder; prepared for factory, school and laboratory.

Pages 505-560, advertising matter.

Powder coating for highway safety appurtenances. Final report.

Federal Highway Administration, Washington, D.C.

The family dyer and scourer : being a complete treatise on the arts of dying and cleaning every article of dress, bed and window furniture, silks, bonnets, feathers, &c., whether made of flax, silk, cotton, wool, or hair : also, carpets, counterpanes, and hearth-rugs, ensuring a saving of eighty per cent /

Includes index.

The development of uniform distributions of uranium in beryllium by means of powder metallurgy /

"Contract AT-30-1 GEN 366."

Fire protection precautions for handling uranium turnings, chips, borings, sawdust, and powder /

"4-10-50--825-A11578."

Powder metallurgy in Europe : a trip report /

"Contract AT-30-1-GEN-366."