Angle-ply biomaterial scaffold for annulus fibrosus repair replicates native tissue mechanical properties, restores spinal kinematics, and supports cell viability

Abstract

Annulus fibrosus (AF) damage commonly occurs due to intervertebral disc (IVD) degeneration/herniation. The dynamic mechanical role of the AF is essential for proper IVD function and thus it is imperative that biomaterials developed to repair the AF withstand the mechanical rigors of the native tissue. Furthermore, these biomaterials must resist accelerated degradation within the proteolytic environment of degenerate IVDs while supporting integration with host tissue. We have previously reported a novel approach for developing collagen-based, multi-laminate AF repair patches (AFRPs) that mimic the angle-ply architecture and basic tensile properties of the human AF. Herein, we further evaluate AFRPs for their: tensile fatigue and impact burst strength, IVD attachment strength, and contribution to functional spinal unit (FSU) kinematics following IVD repair. Additionally, AFRP resistance to collagenase degradation and cytocompatibility were assessed following chemical crosslinking. In summary, AFRPs demonstrated enhanced durability at high applied stress amplitudes compared to human AF and withstood radially-directed biaxial stresses commonly borne by the native tissue prior to failure/detachment from IVDs. Moreover, FSUs repaired with AFRPs and nucleus pulposus (NP) surrogates had their axial kinematic parameters restored to intact levels. Finally, carbodiimide crosslinked AFRPs resisted accelerated collagenase digestion without detrimentally effecting AFRP tensile properties or cytocompatibility. Taken together, AFRPs demonstrate the mechanical robustness and enzymatic stability required for implantation into the damaged/degenerate IVD while supporting AF cell infiltration and viability.

Statement of significance: The quality of life for millions of individuals globally is detrimentally impacted by IVD degeneration and herniation. These pathologies often result in the structural demise of IVD tissue, particularly the annulus fibrosus (AF). Biomaterials developed for AF repair have yet to demonstrate the mechanical strength and durability required for utilization in the spine. Herein, we demonstrate the development of an angle-ply AF repair patch (AFRP) that can resist the application of physiologically relevant stresses without failure and which contributes to the restoration of functional spinal unit axial kinematics following repair. Furthermore, we show that this biomaterial can resist accelerated degradation in a simulated degenerate environment and supports AF cell viability.

Keywords: Angle-ply laminate; Annulus fibrosus; Collagen scaffold; Intervertebral disc; Tissue engineering.

Fig. 1

Study design for in vitro kinematic testing using AFRP and ABNP repair biomaterials. A) Representative images of assigned testing groups and schematic of preparation for kinematic testing of bovine caudal functional spinal units (FSUs). B) Loading scheme for FSU testing depicting creep, axial cyclic tension-compression, and slow constant-rate ramp tests. C) Representative axial force-displacement curve from axial tension-compression loading and its post-test analysis. D) Representative creep displacement curves from a single FSU. (Methods used to generate data within this figure are described in section 2.4).

Fig. 2

Biomechanical evaluations of non-crosslinked multi-laminate AFRPs subjected to biaxial impact burst and tensile fatigue. A) Representative graph of the average maximum calculated impact burst strength withstood by 2-, 3-, and 6-ply AFRPs. Solid lines connecting different groups on graph indicate a significant difference (p < 0.05). B) S-N curve illustrating the fatigue strength of 3-ply AFRPs, in comparison of native human AF (Dotted horizontal line indicates 70% UTS [46] of measured human AF. Open diamonds and triangles indicate specimens with no mechanical failure observed (i.e. run-out to 10,000 cycles). (Methods used to generate data within this figure are described in sections 2.3.1 and 2.3.2).

Fig. 3

Strength of non-crosslinked AFRP attachment to IVDs. A–C) Representative images of one test specimen illustrating the eventual burst of a 6 mm stainless steel ball through an AFRP (Arrow represents direction of force applied by the ball and rod against the AFRP). D-F) Evaluation of failure mechanism prior to removal of the AFRP (Scale bar = 10 mm). G-I) AFRPs illustrating representative failure modes including; G) AFRP rupture, H) failure by suture break, and I) combined failure modes of suture break and AFRP rupture. (Methods used to generate data within this figure are described in section 2.3.3).

Fig. 4

Kinematic testing results normalized to intact controls. Comparison between groups for A) compressive stiffness and tensile stiffness, B) slow constant-rate slow ramp stiffness C) axial range of motion, D) neutral zone length, and creep parameters E) elastic damping coefficients and F) viscous damping coefficients (i.e. normalized to the intact IVD (dotted black line at y = 1) for each test). Axial biomechanical parameters were partially or completely restored to Intact levels for the AFRP + ABNP repair groups. # and & on graph indicates a significant difference (p < 0.05) compared to the intact or discectomy groups, respectively. (Methods used to generate data within this figure are described in section 2.4).

Fig. 5

Tensile testing of crosslinked AFRPs compared to native human AF lamellae. A-C) Representative graphs illustrating mean elastic modulus (EM), ultimate tensile strength (UTS), and tensile strain at failure (TSF) for crosslinked AFRPs. (Dotted horizontal line indicates human AF tensile properties reported in literature) [34,46,50]. (Methods used to generate data within this figure are described in section 2.5.4).

Fig. 6

Cytotoxicity of crosslinked AFRPS. A) Representative image of bovine AF cells (bAFCs) (P2) (100×) harvested and isolated from skeletally mature bovine caudal IVDs (Insert: Representative image of AF cell fibroblast-like phenotype (400×)). B) Graph illustrating percent lactate dehydrogenase (LDH) produced by bAFC seeded 3-ply AFRPs following seeding and culture of 3, 6, and 12 days relative to a positive cell death control (i.e. bAFC seeded 3-ply AFRPs subjected to snap freezing with liquid nitrogen to induce 100% cell death). Solid lines with (#) connecting different study groups on graphs indicate a significant difference (p < 0.05) from non-crosslinked controls. C) Representative H&E images (200×) of 0.2% and 0.6% GLUT crosslinked AFRPs illustrating minimal to no cell presence at respective time points. (Black arrow head indicates cell nuclei). (Methods used to generate data within this figure are described in section 2.6.3).

Fig. 7

Cell infiltration in crosslinked AFRPS. Representative H&E images (200×) of non-crosslinked, 6 mM EDC, and 30 mM EDC AFRPs following cell seeding. Crosslinked samples illustrate minimal cell infiltration into AFRPs in contrast to non-crosslinked samples at respective time points (Insert: Magnified image highlighting representative region of interest outlined by dotted-line). (Black arrow head indicates cell nuclei). (Methods used to generate data within this figure are described in section 2.6.4).

Fig. 8

Cell infiltration of bAFCs into crosslinked and sonicated AFRPS. A) Representative H&E images (200×) of 6 mM EDC crosslinked AFRPs following 1-, 5-, and 10-min of sonication at the respective time points. (Insert: Magnified image highlighting representative region of interest outlined by dotted-line). (Black arrow head indicates cell nuclei). B–C) Representative graphs of the average cell infiltration depth. Solid connecting lines indicate significant difference (p < 0.05). (Methods used to generate data within this figure are described in section 2.6.7).