Development of an in vitro intervertebral disc innervation model to screen neuroinhibitory biomaterials

Abstract

Pain originating from an intervertebral disc (discogenic pain) is a major source of chronic low back pain. Pathological innervation of the disc by pain-sensing nerve fibers is thought to be a key component of discogenic pain, so treatment with biomaterials that have the ability to inhibit neurite growth will greatly benefit novel disc therapeutics. Currently, disc therapeutic biomaterials are rarely screened for their ability to modulate nerve growth, mainly due to a lack of models to screen neuromodulation. To address this deficit, our lab has engineered a three dimensional in vitro disc innervation model that mimics the interface between primary sensory nerves and the intervertebral disc. Further, herein we have demonstrated the utility of this model to screen the efficacy of chondroitin sulfate biomaterials to inhibit nerve fiber invasion into the model disc. Biomaterials containing chondroitin-4-sulfate (CS-A) decrease neurite growth in a uniform gel and at an interface between a growth-permissive and a growth-inhibitory gel, while chondroitin-6-sulfate (CS-C) is less neuroinhibitory. This in vitro model holds great potential for screening inhibitors of nerve fiber growth to further improve intervertebral disc replacements and therapeutics.

Whole-body Vibration Attenuates Pyroptosis-Mediated Inflammation but Accelerates Progression of Intervertebral Disc Degeneration

“…Pathological innervation of the disc by pain-sensing nerve fibers is thought to be a key component of discogenic pain [35,36]. In this study, we found that IVDD mice exhibited a significant increase in the number of Cgrp + sensory nerves both in outer AF and ectopic bone formation zone, and WBV treatment further increases the density of Cgrp + sensory nerves in outer AF.…”

Injectable decellularized nucleus pulposus tissue exhibits neuroinhibitory properties

“…Inner gels consisted of either regelled NP or type I collagen. Collagen inner gels were used as a positive control due to its neuro‐permissive properties 45,56 . To prepare the model, regelled NP and collagen gels were prepared as previously described herein with a final collagen concentration of 3.6 mg/ml, to match the final regelled NP collagen concentration which equates to the summation of the added type I collagen (2.5 mg/ml), and the collagen from the digested tissue (1.1 mg/ml).…”

“…To accurately model the growth of DRG neurons into a degenerative disc in vitro, we utilized our previously established gel‐within‐gel 3D culture method to model the in vivo environment of the DRG and NP. 45 Cut DRGs were embedded and cultured in type I collagen outer hydrogel that surrounded an inner “NP‐like” gel. Inner gels consisted of either regelled NP or type I collagen.…”

“…ImageJ software, Simple Neurite Tracer tool, and a modified Sholl analysis were used to quantify the: (1) maximum radial distance, (2) total distance traveled in the inner gel, (3) number of neurites extending within the inner gel at specific distances from the gel boundary, and (4) the number of neurites extending away from the inner gel at specific distances from the DRG body. 45 The maximum radial distance was determined by drawing a straight line from the tip of the longest neurite to where it initially crosses the gel boundary. The maximum neurite length was determined by tracing the longest nerves using the simple neurite tracer tool up until the gel boundary.…”

“…The cytotoxicity and neuroinhibitory properties of the regelled NP were tested using primary sensory neurons in a previously developed culture model from our lab. 45 The regelled NP created a suitable environment for NP cells to grow and remodel the matrix while inhibiting nerve growth. These results suggest that a decellularized NP gel could be used as supplemental treatment to prevent nerve growth in a degenerated disc and prevent pain progression.…”