Injectable Hydrogel for Mesenchymal Stem Cell and Protein Delivery to the Degenerate Intervertebral Disk

Abstract

Introduction Instability of the motion segment as a result of intervertebral disk degeneration is well known as a major cause of lower back pain. In recent years, a number of tissue engineering and stem cell approaches have been investigated in an attempt to regenerate the degenerate IVD. However, many of the proposed therapies would entail lengthy/risky operations to “implant” the tissue regeneration scaffold. Our vision is to develop a novel, injectable biomaterial delivery system which will deliver mesenchymal stem cells (MSCs) to the degenerate nucleus pulposus (NP) to stimulate regeneration, it will also deliver inhibitors of degeneration, while providing mechanical support to the motion segment during this process. Here, we investigate a novel hydrogel system, which can be maintained as a liquid ex vivo and can potentially be injected into the IVD where it will gel in situ. Materials and Methods Human MSCs were labelled with a green fluorescent membrane dye to enable visualization before culture within or on hydrogel systems. Effects on cell viability, migration characteristics, matrix production, and differentiation capacity of MSCs were investigated. Additionally, the feasibility of injection through narrow bore needles, into a model degenerate IVDs using collagenase digested bovine caudal disks, was investigated. In addition, effects of the degenerate tissue niche were investigated on MSC behavior to determine whether inhibitors of degeneration would need to be codelivered. MSC were treated with IL-1beta and induction of matrix degrading enzymes and expression of IL-1 family genes investigated. Results Viability of MSCs were unaffected by coculture with hydrogel systems, or following growth on or in such systems over time periods of up to 6 weeks. MSCs adhered to the hydrogel systems and were shown to migrate through the system when applied to the upper surface of a preset hydrogel system. Additionally, MSCs mixed with liquid hydrogel before setting and maintained at 37°C for up to 4 weeks, survived, proliferated, synthesized collagen type II and aggrecan matrix (Fig. 1) similar to that seen within the NP including aggrecan, gene expression analysis demonstrated expression of key genes. Liquid hydrogel was successfully injected into degenerated bovine disks where it fill both the large voids and the smaller branches induced by collagenase digestion. MSC treated with IL-1 induced expression of IL-1, IL-1Ra, MMP3 and MMP13 were also upregulated. IL-Ra was successfully incorporated into the hydrogel system. Conclusion We have developed a hydrogel system with the potential to deliver MSCs via minimally invasive injection via narrow bore needles, decreasing the risk of damage to the annulus fibrosus. The system is nontoxic, supports MSC growth, differentiation and shows potential to provide mechanical support to the motion segment while regeneration takes place. Such a therapy combined with inhibition of degeneration shows immense potential for early and midstages of spinal degeneration. I confirm h...