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.

Intervertebral disc degeneration (IVDD) is a progressive condition marked by tissue destruction and inflammation. The therapeutic effector functions of mesenchymal stem cells (MSCs) makes them an attractive therapy for patients with IVDD. While several sources of MSCs exist, the optimal choice for use in the inflamed IVD remains a significant question. Adipose (AD)‐ and amnion (AM)‐derived MSCs have several advantages compared with other sources, however, no study has directly compared the impact of IVDD inflammation on their effector functions. Human MSCs were cultured in media with or without supplementation of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α at concentrations reportedly produced by IVDD cells. MSC proliferation and production of pro‐ and anti‐inflammatory cytokines were quantified following 24 and 48 h of culture. Additionally, the osteogenic and chondrogenic potential of AD‐ and AM‐MSCs was characterized via histology and biochemical analysis following 28 days of culture. In inflammatory culture, AM‐MSCs produced significantly more anti‐inflammatory IL‐10 (14.47 ± 2.39 pg/ml; p = 0.004) and larger chondrogenic pellets (5.67 ± 0.26 mm2; p = 0.04) with greater percent area staining positively for glycosaminoglycan (82.03 ± 3.26%; p < 0.001) compared with AD‐MSCs (0.00 ± 0.00 pg/ml; 2.76 ± 0.18 mm2; 34.75 ± 2.49%; respectively). Conversely, AD‐MSCs proliferated more resulting in higher cell numbers (221,000 ± 8,021 cells; p = 0.048) and produced higher concentrations of pro‐inflammatory cytokines prostaglandin E2 (1,118.30 ± 115.56 pg/ml; p = 0.030) and IL‐1β (185.40 ± 7.63 pg/ml; p = 0.010) compared with AM‐MSCs (109,667 ± 5,696 cells; 1,291.40 ± 78.47 pg/ml; 144.10 ± 4.57 pg/ml; respectively). AD‐MSCs produced more mineralized extracellular matrix (3.34 ± 0.05 relative absorbance units [RAU]; p < 0.001) compared with AM‐MSCs (1.08 ± 0.06 RAU). Under identical inflammatory conditions, a different effector response was observed with AM‐MSCs producing more anti‐inflammatories and demonstrating enhanced chondrogenesis compared with AD‐MSCs, which produced more pro‐inflammatory cytokines and demonstrated enhanced osteogenesis. These findings may begin to help inform researchers which MSC source may be optimal for IVD regeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2445–2456, 2019

One major limitation of intervertebral disc (IVD) repair is that no ideal biomaterial has been developed that effectively mimics the angle-ply collagen architecture and mechanical properties of the native annulus fibrosus (AF). Furthermore, it would be beneficial to devise a simple, scalable process by which to manufacture a biomimetic biomaterial that could function as a mechanical repair patch to be secured over a large defect in the outer AF that will support AF tissue regeneration. Such a biomaterial would: (1) enable the employment of early-stage interventional strategies to treat IVD degeneration (i.e. nucleus pulposus arthroplasty); (2) prevent IVD re-herniation in patients with large AF defects; and (3) serve as a platform to develop full-thickness AF and whole IVD tissue engineering strategies. Due to the innate collagen fibre alignment and mechanical strength of pericardium, a procedure was developed to assemble multi-laminate angle-ply AF patches derived from decellularized pericardial tissue. Patches were subsequently assessed histologically to confirm angle-ply microarchitecture, and mechanically assessed for biaxial burst strength and tensile properties. Additionally, patch cytocompatibility was evaluated following seeding with bovine AF cells. This study demonstrated the effective removal of porcine cell remnants from the pericardium, and the ability to reliably produce multi-laminate patches with angle-ply architecture using a simple assembly technique. Resultant patches demonstrated their inherent ability to resist biaxial burst pressures reminiscent of intradiscal pressures commonly borne by the AF, and exhibited tensile strength and modulus values reported for native human AF. Furthermore, the biomaterial supported AF cell viability, infiltration and proliferation.

1Intervertebral disc degeneration (IVDD) is a progressive condition marked by 2 inflammation and tissue destruction. The effector functions of mesenchymal stem cells (MSCs) 3 make them an attractive therapy for patients with IVDD. While several sources of MSCs exist, the 4 optimal choice for use in the inflamed IVD remains a significant question. Adipose (AD)-and 5 amnion (AM)-derived MSCs have several advantages compared to other sources, however, no 6 study has directly compared the impact of IVDD inflammation on their effector functions. Human 7 MSCs were cultured in media with or without supplementation of interleukin-1β and tumor 8 necrosis factor-α at concentrations produced by IVDD cells. MSC proliferation and production of 9 pro-and anti-inflammatory cytokines were quantified following 24-and 48-hours of culture. 10Additionally, the osteogenic and chondrogenic potential of AD-and AM-MSCs was characterized 11 via histology and biochemical analysis following 28 days of culture. In inflammatory culture, AM- 12MSCs produced significantly more anti-inflammatory IL-10 (p=0.004) and larger chondrogenic 13 pellets (p=0.04) with greater percent area staining positively for glycosaminoglycan (p<0.001) 14 compared to AD-MSCs. Conversely, AD-MSCs proliferated more resulting in higher cell numbers 15 (p=0.048) and produced higher concentrations of pro-inflammatory cytokines PGE2 (p=0.030) and 16 IL-1β (p=0.010) compared to AM-MSCs. Additionally, AD-MSCs produced more mineralized 17 matrix (p<0.001) compared to AM-MSCs. These findings begin to inform researchers and 18 clinicians as to which MSC source may be optimal for different IVD therapies including those that 19 may promote regeneration or fusion. Further study is warranted evaluating these cells in systems 20 which recapitulate the nutrient-and oxygen-deprived environment of the degenerate IVD. 21

Focal defects in the annulus fibrosus (AF) of the intervertebral disc (IVD) arising from herniation have detrimental impacts on the IVD's mechanical function. Thus, biomimetic-based repair strategies must restore the mechanical integrity of the AF to help support and restore native spinal loading and motion. Accordingly, an annulus fibrosus repair patch (AFRP); a collagen-based multi-laminate scaffold with an angle-ply architecture has been previously developed, which demonstrates similar mechanical properties to native outer AF (oAF). To further enhance the mimetic nature of the AFRP, interlamellar (ILM) glycosaminoglycan (GAG) was incorporated into the scaffolds. The ability of the scaffolds to withstand simulated impact loading and resist herniation of native IVD tissue while contributing to the restoration of spinal kinematics were assessed separately. The results demonstrate that incorporation of a GAG-based ILM significantly increased (p<0.001) the impact strength of the AFRP (2.57 ± 0.04 MPa) compared to scaffolds without (1.51 ± 0.13 MPa). Additionally, repair of injured functional spinal units (FSUs) with an AFRP in combination with sequestering native NP tissue and a full-thickness AF tissue plug enabled the restoration of creep displacement (p=0.134), short-term viscous damping coefficient (p=0.538), the long-term viscous (p=0.058) and elastic (p=0.751) damping coefficients, axial neutral zone (p=0.908), and axial range of motion (p=0.476) to an intact state. Lastly, the AFRP scaffolds were able to prevent native IVD tissue herniation upon application of supraphysiologic loads (5.28 ± 1.24 MPa). Together, these results suggest that the AFRP has the strength to sequester native NP and AF tissue and/or implants, and thus, can be used in a composite repair strategy for IVDs with focal annular defects thereby assisting in the restoration of spinal kinematics.