Development of an In Vitro Intervertebral Disc Innervation Model to Screen Neuroinhibitory Biomaterials

S.M.R. performed the experiments described and wrote the manuscript with guidance and feedback from R.A.W. and proofreading by C.A.J. and A.L.R. Data analysis was performed by S.M.R., C.A.J., A.L.R., and R.A.W. Statistical analysis was performed by S.M.R. Funding for the study was obtained by R.A.W. All authors have read and approved the submitted manuscript.

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.

Chronic low back pain (LBP) is a major cause of disability, healthcare costs, and decreased quality of life.1,2 All age groups are affected by chronic LBP, and it is a global epidemic with a lifetime prevalence of 38.9% and increased severity in older populations.3,4 An estimated 26–42% of chronic LBP cases are due to pain originating within the intervertebral disc, which is termed disc-associated or discogenic LBP.5–7 Current treatments for discogenic pain include long‐term pain medication, physical therapy, semi‐invasive treatments such as anti‐inflammatory injections, and surgeries such as spinal fusions; however, these options have limited efficacy for most patients.8–11 Therefore, alternative treatments for chronic discogenic LBP are needed.

Developing new treatments for discogenic LBP requires a mechanistic understanding of its causes, which are closely tied to changes in the disc that occur during disc degeneration. The disc is composed of an outer lamellar ring, the annulus fibrosus (AF), and an inner core called the nucleus pulposus (NP). The NP contains high amounts of chondroitin sulfate proteoglycans (CSPGs) such as aggrecan that maintain the water content of the disc and inhibit nerve growth and vascularization.12,13 The healthy disc is predominantly aneural and avascular, with nerve fibers (i.e., neurites) and blood vessels limited to the outer edges of the AF. During disc degeneration, the matrix of the disc degrades and the concentration of CSPGs decreases, the disc loses water content and height, and other disruptions such as annular fissures occur.8,14 The reduction in CSPG‐rich neuroinhibitory matrix combines with degenerate disc cell secretion of neurotrophic factors (e.g., NGF and BDNF) and proangiogenic factors (e.g., VEGF) to promote sensory neurite and blood vessel growth into the disc.8,15,16 Once the disc has become innervated with pain‐sensing (nociceptive) neurites, the nerve endings may be directly stimulated by inflammatory factors, irritants, or growth factors secreted by disc cells.8,15 Sensory neurites stimulated by the harsh microenvironment within the degenerate disc are the proposed source of discogenic pain.8,15

Clinical evidence from patients with disc degeneration and LBP further supports the role of disc innervation in discogenic pain. For example, in one study 68% of 61 human degenerate discs were innervated.17 Many researchers have found evidence of nociceptive neurite markers deep within discs (sometimes extending into the NP) of patients with LBP.18–22 Specifically, markers of pain sensation such as substance P and calcitonin gene‐related peptide (CGRP) are found in the majority of the neurites identified in degenerate, painful discs.22 Additionally, recent evidence of innervation in discogenic neck pain supports a similar mechanism for discogenic LBP.23,24 Induced and age‐related disc degeneration in mouse and rat models have also exhibited increased disc innervation, including into the NP.25–28 These data demonstrate a strong link between disc degeneration, increased disc innervation, and discogenic pain.

Despite the strong association between disc innervation and discogenic pain, current biomaterials for disc regeneration are often characterized with the disc environment in mind but without direct examination of the effects on sensory neurite growth.29,30 This is due to a lack of in vitro models that mimic the conditions of pathological disc innervation where sensory nerves sprout through the AF into the NP. Current models of neuroinhibition often utilize two‐dimensional (2D) cultures of sensory neurons on a neuroinhibitory substrate12,31,32 or single component hydrogels33 which do not adequately mimic the complex disc environment. Previous in vitro research of the behavior of neurites at a 3D interface was limited to a glial scar model utilizing embryonic chick dorsal root ganglia (DRG), which is not representative of pathologic disc innervation because the gel contents are not similar to the disc.34 Here, we present a novel model that mimics pathologic innervation in the disc by creating an interface between a nerve growth‐permissive hydrogel and a disc‐like gel to screen neuroinhibitory properties of specific materials ( ).

The initial candidate materials chosen for assessment with our in vitro disc innervation model were chondroitin sulfate (CS) biomaterials as there is a robust connection between increased disc innervation and the reduction of neuroinhibitory CSPGs that occurs with age and disc degeneration.12,13,35 The neuroinhibitory properties of CSPGs are determined by the different sulfation patterns of the CS the CSPGs contain because the sulfation pattern determines the neuronal receptors to which the CS can bind.36–38 In bovine aggrecan, the main sulfation patterns are 62.3% chondroitin‐4‐sulfate (CS‐A; ), 25% chondroitin‐6‐sulfate (CS‐C; ), and 12.7% unsulfated chondroitin.33 Human aggrecan from articular sources also consists mainly of CS‐A and CS‐C, with the ratio between the two varying with age and location in the tissue.39 Interestingly, the effects of CS‐A and CS‐C can range from strongly neuroinhibitory to not neuroinhibitory at all depending on the species and neuronal cell type investigated (mouse, rat, or chicken; cerebellar granule neuron, cortical neuron, or DRG neuron).31,33,34,40,41 To maximize relevance to disc physiology, CS‐A and CS‐C were chosen as potential neuroinhibitory biomaterials for screening in our model.

The goals of this work were to (i) engineer a culture model to mimic sensory neurite growth into an intervertebral disc and (ii) screen CS biomaterials for rat DRG neuroinhibition. The hypothesis of this study was that CS‐A and CS‐C would inhibit neurite growth of neonatal rat DRGs in hydrogel culture. The neuroinhibitory properties of CS‐A and CS‐C biomaterials were first assessed in 3D uniform composition hydrogels, demonstrating that CS‐A was more strongly neuroinhibitory than CS‐C. The in vitro disc innervation model assessing neuroinhibition at the interface of two different gels then established that CS‐A is neuroinhibitory at an interface, and enzymatic digestion of the CS‐A significantly decreased the neuroinhibition thereby verifying that CS is necessary for neuroinhibition. Together, these results demonstrate we have established an in vitro innervation model capable of screening neuroinhibitory properties of biomaterials and that CS‐A is an effective neuroinhibitory biomaterial with potential uses in disc biomaterials.