Structural and Chemical Modification to Improve Adhesive and Material Properties of Fibrin-Genipin for Repair of Annulus Fibrosus Defects in Intervertebral Disks

Abstract

Annulus fibrosus (AF) defects from intervertebral disk (IVD) herniation and degeneration are commonly associated with back pain. Genipin-crosslinked fibrin hydrogel (FibGen) is an injectable, space-filling AF sealant that was optimized to match AF shear properties and partially restored IVD biomechanics. This study aimed to enhance mechanical behaviors of FibGen to more closely match AF compressive, tensile, and shear properties by adjusting genipin crosslink density and by creating a composite formulation by adding Poly(D,L-lactide-co-glycolide) (PDLGA). This study also evaluated effects of thrombin concentration and injection technique on gelation kinetics and adhesive strength. Increasing FibGen genipin concentration from 1 to 36 mg/mL significantly increased adhesive strength (∼5 to 35 kPa), shear moduli (∼10 to 110 kPa), and compressive moduli (∼25 to 150 kPa) with concentration-dependent effects, and spanning native AF properties. Adding PDLGA to FibGen altered the material microstructure on electron microscopy and nearly tripled adhesive strength, but did not increase tensile moduli, which remained nearly 5× below native AF, and had a small increase in shear moduli and significantly decreased compressive moduli. Increased thrombin concentration decreased gelation rate to < 5 min and injection methods providing a structural FibGen cap increased pushout strength by ∼40%. We conclude that FibGen is highly modifiable with tunable mechanical properties that can be formulated to be compatible with human AF compressive and shear properties and gelation kinetics and injection techniques compatible with clinical discectomy procedures. However, further innovations, perhaps with more efficient fiber reinforcement, will be required to enable FibGen to match AF tensile properties.

Fig. 1

Genipin crosslinking increased adhesive strength, compressive and shear moduli, but with a threshold effect. (a) Greater genipin concentration increased crosslinking as measured by significantly reduced free amines. Increased crosslinking increased (b) complex shear modulus (|G*|), (c) Young's compressive modulus (E_C), and (d) adhesive strength. (e) SEM and TEM of selected formulations show that increased crosslinking resulted in structures with larger fibers that were more densely connected. Data displayed as mean±standard deviation. Values of x in FGx correspond to the genipin concentration in mg/mL. * = p < 0.05 compared to FG6, † = p < 0.05 compared to Fibrin, bar = p < 0.05.

Fig. 2

FibGen with PDLGA partially increased adhesive strength but did not increase all moduli. Small (S) and large (L) PDLGA was mixed in with FG6 at 2 mg/mL and 20 mg/mL. FibGen-PDLGA materials were characterized for (a) adhesive strength; (b) |G*| at 0.5 Hz frequency; (c) E_C; and (d) Young's tensile modulus (E_T). Data displayed as mean±standard deviation, “*” indicates significant difference from FG6, p < 0.05.

Fig. 3.

PDLGA FibGen formulations modify hydrogel microstructure. The addition of PDLGA altered FibGen microstructure to increase the fibril density and to create larger structural features. PDLGA had randomly oriented, elongated, and denser whisker-type structures compared to FG6. FG6-L2 and FG-L20 had a roughened surface area with larger bundles than the original FG6 group as visualized on SEM. FG6-S2 and FG6-S20 groups had finer structures compared to the other PDLGA groups. TEM imaging revealed large bundles and thick or long fibrillar whiskers for FG6-L2 and FG6-L20 groups, while FG6-S2 and FG6-S20 groups appeared to have a higher density of fine and short highly branched fibrils.

Fig. 4

Optimization of formulation and injection technique to enhance clinical compatibility. (a) Schematic illustrating pushout testing samples that had a structural cap to increase hydrogel contact area. The structural cap increased (b) adhesive strength. (c) Gelation time was decreased with formulations containing higher concentrations of thrombin. Data displayed as mean±standard deviation, bar indicates significant difference between groups, p < 0.05.