Genipin-Enhanced Fibrin Hydrogel and Novel Silk for Intervertebral Disc Repair in a Loaded Bovine Organ Culture Model

Abstract

(1) Background: Intervertebral disc (IVD) repair represents a major challenge. Using functionalised biomaterials such as silk combined with enforced hydrogels might be a promising approach for disc repair. We aimed to test an IVD repair approach by combining a genipin-enhanced fibrin hydrogel with an engineered silk scaffold under complex load, after inducing an injury in a bovine whole organ IVD culture; (2) Methods: Bovine coccygeal IVDs were isolated from ~1-year-old animals within four hours post-mortem. Then, an injury in the annulus fibrosus was induced by a 2 mm biopsy punch. The repair approach consisted of genipin-enhanced fibrin hydrogel that was used to fill up the cavity. To seal the injury, a Good Manufacturing Practise (GMP)-compliant engineered silk fleece-membrane composite was applied and secured by the cross-linked hydrogel. Then, IVDs were exposed to one of three loading conditions: no load, static load and complex load in a two-degree-of-freedom bioreactor for 14 days. Followed by assessing DNA and matrix content, qPCR and histology, the injured discs were compared to an uninjured control IVD that underwent the same loading profiles. In addition, the genipin-enhanced fibrin hydrogel was further investigated with respect to cytotoxicity on human stem cells, annulus fibrosus, and nucleus pulposus cells; (3) Results: The repair was successful as no herniation could be detected for any of the three loading conditions. Disc height was not recovered by the repair DNA and matrix contents were comparable to a healthy, untreated control disc. Genipin resulted being cytotoxic in the in vitro test but did not show adverse effects when used for the organ culture model; (4) Conclusions: The current study indicated that the combination of the two biomaterials, i.e., genipin-enhanced fibrin hydrogel and an engineered silk scaffold, was a promising approach for IVD repair. Furthermore, genipin-enhanced fibrin hydrogel was not suitable for cell cultures; however, it was highly applicable as a filler material.

Keywords: bioreactor; cell activity; fibre-reinforced hydrogel; fibrin; genipin; histology; intervertebral disc; mechanical loading; organ culture; qPCR; repair; silk.

Figure 1

Stress-Strain curves for two genipin-enhanced fibrin hydrogels (5.7 and 11 mg/mL fibrin) and bovine nucleus pulposus tissue. Nominal compressive stress was calculated from force measurements of 6 mm wide and 4 mm high circular discs under unconfined conditions in pre-warmed PBS, N = 5, mean ± SEM, two-way ANOVA: p-value a < 0.05, b < 0.001.

Figure 2

Mitochondrial activity of genipin cytotoxicity assay. (A) human mesenchymal stem cells and (B) human annulus fibrosus and nucleus pulposus cells were mixed with increasing dimethyl sulfoxide (DMSO) and genipin concentration and the mitochondrial activity was determined after 1, 4, 7 and 14 days of culture.

Figure 3

(A) Disc height; (B) mitochondrial activity; (C) GAG content normalized to dry weight relative to control discs; (D) DNA content normalized to dry weight and relative to control disc. Mean ± SEM, N = 5, t-test: p-value * < 0.05; two-way ANOVA: p-value b < 0.01, c < 0.001.

Figure 4

Relative expression of major anabolic genes. N = 6, mean ± SEM, p-value * < 0.05.

Figure 5

Relative expression of major catabolic genes. N = 6, mean ± SEM, p-value * < 0.05.

Figure 6

Relative expression of major inflammatory genes. N = 6, mean ± SEM, p-value * < 0.05.

Figure 7

Histology of sagittal PMMA and transversal cryosections of IVDs after 14 days of culture under free-swelling conditions. (Left–Right) Hematoxylin & Eosin (H & E), Safranin-O/Fast Green and Picrosirius Red. (Top–Down) Healthy control, injury and repair discs.

Figure 8

(A) Experimental design from healthy to injured to repaired disc. An injury was induced by a 2 mm biopsy punch. The cavity was filled with a genipin-enhanced fibrin hydrogel and closed with an engineered silk-fleece composite with the fleece side facing to the IVD. (B) Loading profiles used for 14 days of organ culture either no load, static load (0.2 MPa for 8 h/d) or complex load (0.2 MPa and 0 ± 2° of torsion at 0.2 Hz both for 8 h/d).