Annulus Fibrosus Repair using Genetically Engineered Silk and Genipin-Enhanced Fibringel
Authors: Frauchiger Daniela A. , Benneker Lorin M. , Roth Eva , Gantenbein Benjamin 
Published: April 2016

Introduction Almost 80% of the population suffers from low back pain at least once in their life causing a high socio economic burden. Often these pains originate from a trauma resulting in disc herniation and /or disc degeneration. Despite this high prevalence and associated costs, a satisfying treatment method is not yet on the market. Here we aimed to develop a new method to seal and repair annulus fibrosus (AF) injuries by using genetically engineered silk carrying GDF-6 a growth factor known to lead stem cells toward an intervertebral disc (IVD)-like phenotype and a genipin-enhanced fibrin hydrogel.1 Materials and Methods Bovine IVDs of 14–17 month old animals were harvested under aseptic conditions.2 After inducing an injury by a circular 2mm biopsy punch (Polymed, Switzerland) the defect was filled up with a commercial human based fibrin hydrogel (Baxter Tisseel, Switzerland) enhanced with 4.2mg/ml of the cross linker genipin (Wako Chemicals GmbH, Germany). For closure and acceleration of repair a silk membrane-fleece composite (Spintec Engineering GmbH, Germany) was put on the defect and hold in place by the hydrogel. After 15 minutes allowing for cross linking in vitro organ culture of bIVDs started for 14 days under three different loading schemes 1) dynamic load 0.2MPa load and ±2° torsion at 0.2Hz for 8h/day 2) static diurnal load of 0.2MPa or 3) free swelling. For dynamic loading a custom built two-degree of freedom bioreactor was used.3 At the end of culture discs were checked for seal failure by the eye, disc height, metabolic activity (alamar blue), cell death by necrosis (LDH assay) and apoptosis (Caspase 3/7), DNA, GAG and collagen content (via hydroxyl proline = HYP) and RT-qPCR was performed. Results Throughout the 14 days of culture the silk composite maintained its position under all three loading schemes. Although repaired discs performed slightly lower in alamar blue, DNA and GAG content were in the range of the control. Also LDH resulted in similar values compared with control discs. Height loss in repaired discs was in the same range of static diurnal loaded control samples. Dynamically loaded discs decrease to the level of injured, unrepaired discs. In general discs suspended to dynamic loading showed slightly higher expression of inflammation marker genes measured relative to control discs when compared with free swelling samples. Conclusion Silk-genipin-fibrin reinforced hydrogel is a promising approach to close AF defects as instant loading was possible and composite remained on its position. Further experiments focus on cytocompatibility of genipin-enhanced fibrin hydrogel and organ culture of silk containing covalently linked growth factors. Acknowledgments This project is funded by the Gebert Rüf Stiftung project # GRS-028/13. References Clarke LE, McConnell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA. Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation, composition, and micromechanical properties of nucleus pulposus constructs. Arthritis Res Ther 2014;16(2):R67 Chan SC, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp 2012;60(60):e3490 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, ed. Replacing Animal Models: A Practical Guide to Creating and Using Biomimetic Alternatives. Chichester, UK: John Wiley & Sons Ltd.; 2012:89–105