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.

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.

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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.

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.

Silk culture. A hand-book for silk-growers.

Mode of access: Internet.

Leather stocking and silk; or, Hunter John Myers and his times.

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Journal of the American Silk Society, and rural economist.

Gideon B. Smith, editor.

Observations on the ruinous tendency of the Spitalfields act to the silk manufacture of London: to which is added a reply to Mr. Hale's appeal to the public in defence of the act.

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The silk question settled : the testimony of one hundred and fifty witnesses. Report of the proceedings of the National Convention of Silk Growers and Manufacturers, held in New York, Oct. 13th and 14th, 1843.

"Published under the direction of the American Institute."

A letter to His Grace the Duke of Wellington, on the silk trade, in the year 1829.

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Strictures on the wisdom and policy of the present state measures relative to the allowed importation of silk.

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In silk attire.

Mode of access: Internet.

In silk attire. A novel.

The copy described by Sadleir.

Dyeing artificial silk fabrics /

Mode of access: Internet.