Repair of Annulus fibrosus with Genipin-enhanced Fibrin Hydrogel and silk membrane- fleece

Frauchiger, Daniela Angelika; Chan, Samantha CW; Tekari, Adel; Benneker, Lorin Michael; Gantenbein, Benjamin (2015). Repair of Annulus fibrosus with Genipin-enhanced Fibrin Hydrogel and silk membrane- fleece (Unpublished). In: Biospine.org, Proceedings of the 5th International Congress on Biotechnologies for Spinal Surgery.

Introduction
Low back pain is often caused by a trauma causing disc herniation and /or disc degeneration. Although there are some promising approaches for nucleus pulposus repair, the inner tissue of the intervertebral disc (IVD) so far no treatment or repair is available for annulus fibrosus (AF) injuries. Here we aimed to develop a new method to seal and repair AF injuries by using a silk fleece composite and a genipin enhanced hydrogel.
Methods
Bovine (b) IVDs were harvested under aseptic conditions and kept in free swelling conditions for 24h in high-glucose DMEM containing 5% bovine serum for equilibration (1). A circular 2mm biopsy punch (Polymed Medical Center, Switzerland) was used to form a reproducible defect in the AF. For filling the defect and keeping the silk composite in place a human-derived fibrin gel (Baxter Tisseel, Switzerland) enhanced with 4.2mg/ml of the cross linker genipin (Wako Chemicals GmbH, Germany) was used. The silk composite consists of a mesh- and a membrane side (Spintec Engineering GmbH, Germany); the membrane is facing outwards to form a seal. bIVDs were cultured in vitro for 14 days either under dynamic load in a custom-built bioreactor under physiological conditions (0.2MPa load and ±2° torsion at 0.2Hz for 8h/day) or static diurnal load of 0.2MPa (2). At the end of culture discs were checked for seal failure, disc height, metabolic activity, cell death by necrosis (LDH assay), DNA content and glycosaminoglycan content.
Results
Silk composite maintained its position throughout the 14 days of culture under loaded conditions. Although repaired discs performed slightly lower in cell activity, DNA and GAG content were in the range of the control. Also LDH resulted in similar values compared to control discs (Fig 1).
Height loss in repaired discs was in the same range as for static diurnal loaded control samples. For dynamically loaded samples the decrease was comparable to the injured, unrepaired discs.
Fig 1 LDH of repaired discs compared to control disc after 24h in free swelling conditions for equilibration and first three loading cycles.
Conclusions
Silk-genipin-fibrin reinforced hydrogel is a promising approach to close AF defects as tested by two degree of freedom loading. In further experiments cytocompatibility of genipin has to be investigated.
References
1. Chan SC, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp 2012, Feb 2;60(60):e3490.
2. Walser J, Ferguson SJ, Gantenbein-Ritter B. Design of a mechanical loading device to culture intact bovine caudal motional segments of the spine under twisting motion. In: Davies J, editors. Replacing animal models: a practical guide to creating and using biomimetic alternatives. Chichester, UK: John Wiley & Sons, Ltd.; 2012. p. 89-105.
Acknowledgements
This project is funded by the Gerbert Rüf Stiftung project # GRS-028/13 and the Swiss National Science Project SNF #310030_153411.

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