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

Frauchiger, Daniela Angelika; Heeb, Silvan Rolf; Roth, Eva; Wöltje, WM; Benneker, Lorin Michael; Gantenbein, Benjamin (12 February 2016). Genetically engineered silk and genipin-enhanced fibrin hydrogel for annulus fibrosus repair (Unpublished). In: YSBM.ch Symposium 2016. Zürich. 12.02.2016.

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.

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