Elsevier

Acta Biomaterialia

Volume 60, 15 September 2017, Pages 201-209
Acta Biomaterialia

Full length article
Translation of an injectable triple-interpenetrating-network hydrogel for intervertebral disc regeneration in a goat model

https://doi.org/10.1016/j.actbio.2017.07.025Get rights and content

Abstract

Degeneration of the intervertebral discs is a progressive cascade of cellular, compositional and structural changes that is frequently associated with low back pain. As the first signs of disc degeneration typically arise in the disc’s central nucleus pulposus (NP), augmentation of the NP via hydrogel injection represents a promising strategy to treat early to mid-stage degeneration. The purpose of this study was to establish the translational feasibility of a triple interpenetrating network hydrogel composed of dextran, chitosan, and teleostean (DCT) for augmentation of the degenerative NP in a preclinical goat model. Ex vivo injection of the DCT hydrogel into degenerated goat lumbar motion segments restored range of motion and neutral zone modulus towards physiologic values. To facilitate non-invasive assessment of hydrogel delivery and distribution, zirconia nanoparticles were added to make the hydrogel radiopaque. Importantly, the addition of zirconia did not negatively impact viability or matrix producing capacity of goat mesenchymal stem cells or NP cells seeded within the hydrogel in vitro. In vivo studies demonstrated that the radiopaque DCT hydrogel was successfully delivered to degenerated goat lumbar intervertebral discs, where it was distributed throughout both the NP and annulus fibrosus, and that the hydrogel remained contained within the disc space for two weeks without evidence of extrusion. These results demonstrate the translational potential of this hydrogel for functional regeneration of degenerate intervertebral discs.

Statement of Significance

The results of this work demonstrate that a radiopaque hydrogel is capable of normalizing the mechanical function of the degenerative disc, is supportive of disc cell and mesenchymal stem cell viability and matrix production, and can be maintained in the disc space without extrusion following intradiscal delivery in a preclinical large animal model. These results support evaluation of this hydrogel as a minimally invasive disc therapeutic in long-term preclinical studies as a precursor to future clinical application in patients with disc degeneration and low back pain.

Introduction

The intervertebral discs are composite structures composed of a central nucleus pulposus (NP), an outer annulus fibrosus (AF), and two hyaline cartilage end plates that interface with the vertebral bodies. The NP is composed primarily of type II collagen and proteoglycans and in its healthy state is highly hydrated [1], [2]. The AF is composed primarily of type I and II collagen fibers oriented at alternating oblique angles to the long axis of the spine, arranged in concentric lamellae [3]. The function of the disc is primarily mechanical – when the spine is subjected to compressive loading, hydrostatic pressure develops within the NP and places the AF in tension, allowing the discs to bear load, while permitting complex spinal motion [4], [5].

Degeneration of the intervertebral discs is a progressive cascade of cellular, compositional and structural changes that is closely linked with aging [2], [6]. The earliest degenerative changes typically occur in the NP, and are characterized by a loss of proteoglycans, which compromises the ability of the NP to swell and the disc to bear load [7], [8]. This results in a loss of disc height, and progressive structural and mechanical failure of the intervertebral joint [9]. Disc degeneration is frequently associated with low back pain, [10] which is the second most common cause of adult disability in the United States, with a prevalence greater than heart conditions, stroke and cancer combined [10], [11].

For patients with low back pain secondary to disc degeneration, initial clinical treatment is generally conservative [8]. If conservative treatment fails, patients can be treated surgically to immobilize the degenerated motion segment via a fusion procedure or to replace the degenerated disc with a mechanical arthroplasty device [12], [13]. These current surgical treatment options are limited in that they do not address the underlying cause of degeneration, do not restore native disc structure or function and are typically indicated for end-stage degeneration only. In the United States, there are nearly 4 million patients per year with moderate disc degeneration that are unresponsive to conservative treatment, but are not suitable candidates for fusion surgery [14]. For these reasons, there is a pressing need to develop treatment strategies for disc degeneration, particularly those targeting patients with early to mid-stage degeneration and who are unresponsive to conservative therapies.

As progressive degeneration first manifests in the NP, there has been considerable interest in developing hydrogel-base strategies that can synergize with stem cells and bioactive molecules to augment and potentially restore native NP tissue [15]. A candidate hydrogel for NP regeneration would ideally be injectable (to allow for minimally invasive delivery), would rapidly solidify (to prevent extrusion from the disc space with loading), and be able to augment the mechanical function of the degenerative motion segment. The ideal hydrogel would also be cytocompatible with native disc cells, and support delivery of stem cells to potentiate long-term native tissue regeneration.

Towards this goal, several biomaterials have been evaluated in vitro for NP regeneration, including thermoreversible hyaluronan grafted poly(N-isopropylacrylamide) hydrogels [16], [17], hyaluronic acid and collagen II-based hydrogels [18], [19], [20], [21], [22], [23], photocrosslinkable carboxymethylcellulose hydrogels [24], [25], and in situ gelling native extracellular matrices [26], among others. Our group recently developed and characterized a triple-interpenetrating network hydrogel for NP regeneration comprised of N-carboxyethyl chitosan, oxidized dextran and teleostean (DCT), which solidifies in situ via Schiff base formation between the –CHO on the oxidized dextran and the –NH2 on the teleostean and chitosan [27], [28]. This triple component hydrogel possesses improved mechanical properties and degradation profiles as compared to various dual component hydrogel combinations of chitosan, dextran and teleostean [28]. Our previous work showed that the mechanical properties of this DCT hydrogel were similar to that of native NP tissue, and that it supported the survival and differentiation of bovine mesenchymal stem cells (MSCs) towards an NP cell-like phenotype in vitro [29]. We also previously demonstrated that an injectable oxidized hyaluronan and teleostean hydrogel with similar properties was capable of restoring range of motion (ROM) following ex vivo nucleotomy in an ovine disc, and is not expelled from the disc space following cyclic loading of up to 2X body weight [29], [30]. As a platform for the in vivo evaluation of combined stem cell and hydrogel therapeutics for disc regeneration, our group has previously developed a large animal model of intervertebral disc degeneration where a spectrum of degeneration from mild to severe can be reproducibly achieved in goat lumbar discs 12 weeks following intradiscal injection of chondroitinase ABC (ChABC) [31]. The goat lumbar spine is a promising pre-clinical model due to the large size of the lumbar discs (∼5 mm disc height), and similarities in mechanical properties and biochemical composition to human discs [32].

In this study we describe the modification of the DCT hydrogel to impart radiopacity using zirconium dioxide nanoparticles (previously utilized in FDA approved bone cements) [33], thereby facilitating non-invasive assessment of delivery and distribution in vivo. With this modification, we show the feasibility of this material for NP regeneration in our preclinical goat model of disc degeneration by (1) demonstrating the ability of the DCT hydrogel to restore mechanical function to a degenerative disc ex vivo, (2) demonstrating that the radiopaque DCT hydrogel is capable of supporting the survival and matrix producing capacity of both NP cells and MSCs in vitro, and (3) showing delivery, retention and safety of the DCT hydrogel in vivo in degenerate goat lumbar discs.

Section snippets

Hydrogel fabrication and modification to impart radiopacity

To fabricate the DCT hydrogel, oxidized dextran and N-carboxyethyl chitosan (Endomedix Inc, Montclaire, NJ) were synthesized as described previously [27]. Aqueous solutions of 20% teleostean (Sigma Aldrich, St. Louis, MO), 3% N-carboxyethyl chitosan, and 7.5% oxidized dextran were mixed at a ratio of 1:1:2. To render the hydrogel radiopaque and facilitate non-invasive assessment using X-ray fluoroscopy and microcomputed tomography (µCT), we adapted a technique previously developed for

Effects of ex vivo DCT hydrogel injection on degenerate disc mechanics

Ex vivo injection volumes ranged from 0.2 to 0.6 mL of hydrogel. The effect of DCT hydrogel delivery on the mechanical properties of goat lumbar motion segments previously degenerated in vivo via intradiscal ChABC injection was determined via tension-compression mechanical testing. Total ROM (Fig. 1B) was significantly (p = 0.01) reduced in DCT hydrogel injected discs compared to degenerate discs prior to injection, and was not different (p = 0.15) from healthy control discs. There was also a trend

Discussion

This study demonstrates the translational feasibility of a triple interpenetrating network DCT hydrogel for the treatment of mild to moderate disc degeneration. The DCT hydrogel is composed of naturally derived materials and solidifies in vivo without the addition of external crosslinking agents via the formation of Schiff bases, polymerizes rapidly upon mixing of constituents, and achieves peak mechanical properties after approximately 10 h crosslinking time [27], [28], [29]. This hydrogel is

Conclusions

In conclusion, we demonstrated that a radiopaque, injectable, triple interpenetrating network DCT hydrogel significantly improved the mechanical properties of degenerate discs ex vivo, supported the viability and preserved the matrix-producing capacity of MSCs and NP cells in vitro, and could be successfully delivered in vivo to degenerate discs in a clinically-relevant large animal model. Moreover, the hydrogel remained contained within the disc space for 2 weeks of normal activity and resulted

Acknowledgements

This work was supported by the Department of Veterans Affairs (RR&D I01RX00132). The authors would also like to acknowledge the Sharpe Foundation and the Departments of Neurosurgery and Orthopaedic Surgery at the University of Pennsylvania for financial support. Core equipment used in this study was provided by the Penn Center for Musculoskeletal Disorders (National Institutes of Health P30 AR 069619). The authors would also like to acknowledge the veterinary staff at the Penn Vet Center for

Disclosures

The authors declare no potential conflicts of interest with respect to the research, authorship and/or publication of this manuscript.

Role of the funding source

The content is solely the responsibility of the authors and does not necessarily represent the official views of the Department of Veteran’s Affairs. No funding source had a role in the study design, collection, analysis and interpretation of data, writing of the manuscript, or the decision to submit the manuscript for publication.

References (43)

  • J.T. Martin et al.

    A radiopaque electrospun scaffold for engineering fibrous musculoskeletal tissues: Scaffold characterization and in vivo applications

    Acta Biomater.

    (2015)
  • S. Pattnaik et al.

    Chitosan scaffolds containing silicon dioxide and zirconia nano particles for bone tissue engineering

    Int. J. Biol. Macromol.

    (2011)
  • M. Cannella et al.

    The role of the nucleus pulposus in neutral zone human lumbar intervertebral disc mechanics

    J. Biomech.

    (2008)
  • J. Cassidy et al.

    Hierarchical structure of the intervertebral disc

    Connect. Tissue Res.

    (1989)
  • M.H. Walker et al.

    Molecular basis of intervertebral disc degeneration

    Spine J.

    (2004)
  • H. Inoue

    Three-dimensional architecture of lumbar intervertebral discs

    Spine (Phila. Pa. 1976)

    (1976)
  • M.D. Humzah et al.

    Human intervertebral disc: structure and function

    Anat. Rec.

    (1988)
  • D.W. Hukins

    A simple model for the function of proteoglycans and collagen in the response to compression of the intervertebral disc

    Proc. Biol. Sci.

    (1992)
  • M. Haefeli et al.

    The course of macroscopic degeneration in the human lumbar intervertebral disc

    Spine (Phila. Pa. 1976)

    (2006)
  • J.P.G. Urban et al.

    Degeneration of the intervertebral disc

    Arthritis Res. Ther.

    (2003)
  • P.P. Raj, Intervertebral disc : pathophysiology-treatment, 8 (2008)...
  • Cited by (0)

    View full text