Injectable Radiopaque Hyaluronic Acid Granular Hydrogels for Intervertebral Disc Repair

Abstract

Injectable hydrogels offer minimally-invasive treatment options for degenerative disc disease, a prevalent condition affecting millions annually. Many hydrogels explored for intervertebral disc (IVD) repair suffer from weak mechanical integrity, migration issues, and expulsion. To overcome these limitations, we developed an injectable and radiopaque hyaluronic acid granular hydrogel. The granular structure provides easy injectability and low extrusion forces, while the radiopacity enables direct visualization during injection into the disc and non-invasive monitoring after injection. The radiopaque granular hydrogel was injected into rabbit disc explants to investigate restoration of healthy disc mechanics following needle puncture injury ex vivo and then delivered in a minimally-invasive manner into the intradiscal space in a clinically-relevant in vivo large animal goat model of IVD degeneration initiated through degradation by chondroitinase. The radiopaque granular hydrogel successfully halted loss of disc height due to degeneration. Further, the hydrogel not only enhanced proteoglycan content and reduced collagen content in the nucleus pulposus (NP) region compared to degenerative discs, but also helped to maintain the structural integrity of the disc and promote healthy segregation of the NP and annulus fibrosus regions. Overall, this study demonstrates the great potential of an injectable radiopaque granular hydrogel for treatment of degenerative disc disease.

Graphical Abstract

Herein, a radiopaque granular hydrogel is developed from fragmented hyaluronic acid microgels with encapsulated radiopaque nano-powder. The granular structure ensures easy injection and cell invasion, while radiopacity enables non-invasive real-time monitoring. In a large animal goat model, the hydrogel halts disc height loss due to degeneration, showcasing its potential for treating disc degeneration.

1. Introduction

The intervertebral disc (IVD) is a complex fibrocartilaginous connective tissue situated between boney vertebrae in the spine. It consists of three primary elements: the nucleus pulposus (NP), a gel-like tissue located at the core of the IVD that plays a crucial role in maintaining hydrostatic loading and absorbing shocks; the annulus fibrosus (AF), which consists of concentric layers of fibrocartilage that provide mechanical stability under compression and tension; and the cartilaginous endplates (CEP), which separate the disc from the vertebrae and play a role in disc mechanics and the regulation and transportation of nutrients and metabolites.^[1] A healthy IVD is responsible for proper biomechanical response and load distribution in the vertebral column, which is essential for almost all musculoskeletal movements.

Degenerative disc disease (DDD) occurs when the NP loses water and proteoglycan (PG) content, resulting in a reduction of overall disc height. This often leads to compression of nerves and results in chronic back pain. In the United States alone, over 3 million Americans suffer from back pain each year, with DDD being the leading cause.^[2] Globally, lower back pain is the leading cause of disability, and back pain is the second most common reason for a doctor’s visit after the common cold.^[3] Current approaches to treat disc degeneration include either conservative treatments (e.g., physical therapy to improve muscle function/stability) or surgical operations (e.g., spinal disc fusion).^[4] However, there is an unmet clinical need for minimally-invasive therapies for patients who do not respond to physical therapy but are not yet in need of full disc fusion, which limits spine mobility and inhibits future possibilities of tissue repair and recovery of native disc function. Further, DDD is often caused by age-related degeneration in the IVD. As the elderly population continues to expand worldwide, it is predicted that the prevalence of DDD will rise in the coming decades, placing a strain on healthcare providers and insurance company resources.^[3] The development of minimally-invasive therapies, which result in decreased hospital stays and improved recovery times, allows for improvements in healthcare costs while meeting the increased demand for DDD treatment.^[3] Thus, there is an unmet clinical need to develop innovative minimally-invasive therapies to treat DDD.

To this end, NP replacement is a promising treatment option, to alleviate pain and promote healthy NP function.^[5] As the NP degenerates over time or with injury, decreased aggrecan/collagen II levels result in decreased hydrostatic pressure within the disc, attenuating its biomechanical function.^[6] With NP replacement, biomaterials are implanted or injected into the disc with the goal of alleviating pain and restoring disc height. Multiple biomaterial NP replacement strategies have been explored in both preclinical and clinical studies, such as injectable and implantable non-degradable synthetic polymer systems (e.g., PerQdisc, GelStix, Prosthetic Disc Nucleus)^[7–9] and injectable protein-polymer hybrid hydrogels (e.g., NuCore),^[10] as well as ongoing clinical trials exploring injectable NP allografts (e.g., VIA Disc Matrix).^[11] As an example, the Prosthetic Disc Nucleus (PDN) is a dehydrated, porous polyethylene mesh that swells upon insertion into the NP cavity.^[9] However, while the PDN showed promise in the clinic, some complications were observed, including significant over-stiffening of the implant, resulting in “excruciating” back pain and the need for implant removal.^[9] GelStix is another NP replacement strategy in which a synthetic nondegradable hydrogel is injected into the NP cavity. Like the PDN, GelStix injection resulted in some positive clinical outcomes; however, some complications were observed including device migration and fracturing, resulting in the need for device removal.^[7] Overall, current NP replacement technologies are limited by complications such as device migration, back-flow at the injection site, loss of disc height after implantation, over-stiffening of the implant, and immune responses detrimental to tissue healing,^[5,7,9] which are all related to inferior delivery methods and materials.

Building from approaches for NP replacement, researchers have also been evaluating hydrogel biomaterials for NP repair. In this approach, hydrogels are designed to not only alleviate pain and restore disc height, but to also introduce biological function, such as restoring native disc mechanics, increasing tissue adhesion, improving native disc tissue hydration, increasing PG content in the native NP, and/or reducing inflammatory and catabolic responses in the degenerating disc environment.^[12] A variety of promising injectable biopolymer hydrogels for NP repair have been explored. For example, Chen et al. recently developed an injectable hydrogel for siRNA delivery to modulate the inflammatory response in DDD.^[13] In another recent example, Peng et al. developed a hydrogel for delivering extracellular vesicles to the NP in order to alleviate the senescent phenotype of NP stem cells.^[14] While recent studies show the great potential of injectable hydrogels for NP repair, many of these approaches have also faced challenges of expulsion from the injection site, loss of disc height after injection, and hydrogel migration, largely due to inferior materials and lack of thorough characterization methods, particularly of biomechanical outcomes.^[5]

Of the ~300 published studies on biomaterials for use in the NP, only ~30% of studies have assessed biomaterials in vivo, and less than 20% have evaluated IVD biomechanics ex vivo.^[15] Further, there is a need to assess material parameters most relevant to successful clinical translation of the biomaterial, such as injectability, degradation, and retention in the disc upon biomaterial injection, which are all significantly understudied. In addition, since hydrogels consist of mainly water, they often cannot be visualized in vivo using clinically-relevant imaging technologies such as X-ray and CT scans due to a lack of contrast with surrounding tissue.^[16] This makes confirmation of successful delivery into the intradiscal space, as well as long-term monitoring of the hydrogel, a challenging task. Thus, there is an unmet need to engineer injectable biomaterials for NP repair with thorough materials characterization and direct in vivo assessment in animal models using clinically-relevant imaging technologies.

Recently, granular hydrogels have emerged as a promising class of biomaterials for injectable tissue repair and regeneration.^[17,18] Granular hydrogels consist of hydrogel microparticles (i.e., “microgels”) that are assembled into a jammed state.^[17] There are many key properties of granular hydrogels that make them a promising biomaterial for tissue repair. Granular hydrogels have microscale porosity, which can allow for improved cell infiltration when compared to a traditional nanoporous bulk hydrogel.^[19] Further, due to physical interactions and frictional contacts between individual microgels, granular hydrogels have high injectability due to their shear-thinning and self-healing behavior.^[20] In addition, biomedical substances, such as imaging and therapeutic agents, can be directly encapsulated within microgels and incorporated into the granular hydrogel structure.

Herein, we developed an injectable hyaluronic acid (HA) granular hydrogel with encapsulated radiopaque zirconium oxide nano-powder for IVD repair. HA was selected due to it being a native component of the disc ECM, as well as its use in multiple products in the clinic.^[21] Further, HA is the most explored hydrogel biomaterial for NP repair.^[15] Our granular hydrogel design imparts important features for application in the disc, such as microscale porosity, injectability, and radiopacity to allow for direction visualization of hydrogel injection and monitoring of the hydrogel within the disc over time in vivo. In this study, we characterized the material properties of the radiopaque granular hydrogel, determined its capacity to restore disc mechanical properties ex vivo, and demonstrated minimally-invasive delivery in vivo and monitored clinically-relevant outcomes in a large-animal goat model of DDD. Overall, this study highlights the exciting potential of injectable radiopaque granular hydrogels as minimally-invasive treatments for repair of degenerated IVDs.

2. Results and Discussion

2.1. Fragmented radiopaque microgel fabrication and characterization

As a first step towards injectable hydrogel fabrication, HA was modified with norbornene functional groups (NorHA) via anhydrous esterification (Figure 1a, S1).^[22] HA was chosen since it is a native component of the ECM in NP tissue. Further, HA hydrogels are the most commonly explored biomaterial for injectable NP repair.^[15] To form bulk HA hydrogels, NorHA was combined with dithiothreitol (DTT) crosslinker to theoretically consume all norbornene functional groups. To introduce radiopacity, zirconium oxide (ZrO₂) nanoparticles were incorporated into hydrogel precursor solutions at a concentration of 30 wt.%. ZrO₂ nanoparticles have been widely used as a bioinert radiopaque contrast agent for biomedical investigations.^[16] Previous work demonstrated that encapsulating ZrO₂ at a concentration of 30 wt.% in an injectable hydrogel enabled hydrogel visualization post-intradiscal injection in a goat model via radiographic imaging.^[23] In general, visualizing injectable hydrogels using clinically-relevant imaging technology (e.g., radiographs, computed tomography scans) both during- and post-clinical intervention is essential for improving treatment outcomes.^[16] This visualization is essential, as hydrogels delivered to the NP are at risk of migration or being expelled due to the high mechanical demands on the disc; thus, tracking location over time can help improve treatment outcomes.^[5,7,9]

Figure 1. Radiopaque microgel fabrication by extrusion fragmentation.

a) Overview of hydrogel formation, via the crosslinking of norbornene-modified HA (NorHA) with dithiothreitol (DTT) in the presence of ammonium persulfate (APS), tetramethylethylenediamine (TEMED), and zirconia (ZrO₂) nanoparticles. b) Overview of extrusion fragmentation process, depicting the crosslinking of bulk NorHA hydrogels with ZrO₂ inside of a syringe and subsequent extrusion through smaller and smaller gauge needles. c) Representative microscopic image of radiopaque NorHA microgels with ZrO₂ nanoparticles (bright field microscopy). Scale bar = 500 μm. d) Quantified equivalent circular diameter (left) and aspect ratio (right) for radiopaque fragmented NorHA microgels (n ≥ 100).

In previous investigations, NorHA hydrogels have been fabricated using photocrosslinking upon exposure to UV or visible light.^[24] However, in this study, the incorporation of ZrO₂ rendered the hydrogel precursor solution opaque, so photocrosslinking could not be used. Oxidative-reductive (redox) reactions are frequently used to generate free radicals and initiate crosslinking in hydrogels.^[24] One of the most commonly used redox initiator systems in biomedical applications is the combination of ammonium persulfate (APS) and tetramethylethylenediamine (TEMED).^[24] To fabricate bulk hydrogels, NorHA, DTT, APS, and ZrO₂ were combined in phosphate-buffered saline (PBS). TEMED was added while vortexing the hydrogel precursor solution and the mixture was allowed to crosslink to form a bulk hydrogel with encapsulated ZrO₂ nanoparticles (Figure 1b). To investigate the influence of ZrO₂ on biomaterial properties, bulk hydrogels were also fabricated without ZrO₂ as a control (Figure S2).

NorHA hydrogels with and without ZrO₂ both reached full gelation in under 10 minutes, as determined by oscillatory shear rheology time sweeps (Figure S2). Hydrogels with ZrO₂ fully gelled in 3 min, whereas hydrogels without ZrO₂ gelled in about 6 min. For all subsequent experiments, bulk hydrogels were characterized or further processed 30 min after the addition of TEMED to ensure complete gelation. The addition of ZrO₂ into NorHA hydrogels resulted in a slight increase in storage modulus (G’, ~8000 Pa) and compressive modulus (~35 kPa), when compared to a G’ of ~5000 Pa and compressive modulus of ~ 20 kPa for hydrogels without ZrO₂ (Figure S2). This is likely due to the highly dense and ceramic structure of ZrO₂ nanoparticles, which increases the overall mechanical moduli.

Microgels were fabricated from bulk hydrogels with and without encapsulated ZrO₂ using extrusion fragmentation (Figure 1b and Figure S3a,b). Bulk hydrogels were formed within a syringe barrel and subsequently extruded through sequentially smaller gauge needles (18 to 25G). The resulting microgels ranged in diameter from ~10¹ to 10³ μm with aspect ratios ranging from ~1 to 4 (Figure 1c,d). In addition, there was no significant difference in microgel size and shape for samples with and without ZrO₂ (Figure S3c). In previous studies, fragmented microgels were fabricated by extruding bulk hydrogels through needles as small as 30G.^[25] However, due to the presence of ZrO₂ and potential aggregates, bulk hydrogels could not be extruded through needles smaller than 25G without excessive force. Thus, there were some larger microgels present (~300–1000 μm) after the extrusion fragmentation process in this study. If desired, alternate fragmentation methods could be explored to fabricate smaller microgels from bulk hydrogels containing ZrO₂, such as fragmentation by passing through meshes or with a blender.

2.2. Assembling and characterizing radiopaque granular hydrogels

To fabricate granular hydrogels, fragmented NorHA microgels were jammed by vacuum-driven filtration using a water-permeable 0.22 μm membrane (Figure 2a). The resulting granular hydrogel was microporous due to void spaces between microgels.^[18,26] The flow behavior of injectable biomaterials is important to fully characterize for clinical use. Thus, the bulk flow behavior of granular hydrogels was characterized using oscillatory shear rheology (Figure 2a, S3). Radiopaque granular hydrogels were strain yielding with a storage modulus (G’) of ~2200 Pa and a yield strain of ~12%. Interestingly, this storage modulus was significantly higher than most injectable hydrogels, likely due to the frictional interactions between fragmented microgels particles that enhances mechanical integrity under low shear.^[27] Granular hydrogels without ZrO₂ were also strain yielding with a slightly lower G’ of ~1900 Pa and yield strain of ~6% (Figure S3d,e). The slight increase in G’ upon the addition of ZrO₂ nanoparticles is likely a result of the radiopaque bulk hydrogels having a higher compressive modulus (~35 kPa) than those without ZrO₂ (~20 kPa). In our prior work, we demonstrated that the storage modulus of granular hydrogels made from fragmented microgels increases as the initial compressive modulus of the bulk NorHA hydrogel increases, which we previously modulated via crosslinker concentration; interestingly, microgel shape and size did not vary with crosslinker concentration, but rather depended solely on the needles used for fragmentation.^[27,28] In future work, it would be interesting to further investigate the interplay of varied polymer, crosslinker, and ZrO₂ nanoparticle concentrations, as well as microgel shapes and sizes, on granular hydrogel material properties for injectable NP repair.

Figure 2. Granular hydrogel assembly and characterization of rheological behavior and extrusion force.

a) Overview of jamming microgels into granular hydrogels using vacuum-driven filtration and loading onto a rheometer (left), and a representative oscillatory strain sweep (1–250%, 1 Hz) of granular hydrogels (right). b) Representative plots of viscosity versus shear rate (left), frequency sweeps (1–100 Hz, 1% strain, center), and shear recovery (alternating low strain [1%, white] and high [250%, purple] strains, right) for radiopaque granular hydrogels. c) Representative images (left) of the extrusion of radiopaque granular hydrogels through an 18G spinal needle (3.5”), including a bright field microscopy image of extruded filament (scale bar = 500 μm, right), and quantified maximum extrusion force values (right) through 18G, 21G, and 23G needles (1”) and an 18G spinal needle (3.5”, “sp.”). Statistical analysis was conducted using one-way ANOVA and a Tukey’s post hoc comparison. n = 4, ns = no significance, **p < 0.01.

The radiopaque granular hydrogels explored in this work exhibited shear-thinning, demonstrated by a decrease in viscosity with increasing shear rate, as well as frequency-dependent stiffening behaviors (Figure 2b). These are important and beneficial properties towards the application of IVD repair, including for injectability and since the native health NP tissue possesses shock-absorbing properties such as frequency-dependent stiffening.^[29] For example, Pérez-San Vicente et al. demonstrated that injectable hydrogels that exhibit frequency-dependent stiffening (i.e., increases in G’ with increased frequencies) over a sampling range of 0.01–50 Hz improved biomechanical outcomes upon injection into an IVD explant ex vivo when compared to injectable hydrogels that did not exhibit this behavior.^[29] Further, granular hydrogels in this study exhibited complete self-healing behavior, as demonstrated by complete recovery of G’ after extended periods of high strain (Figure 2b).

In clinical practice, injectable hydrogels undergo contraction flow through a syringe and needle, which can lead to unique behaviors that are not observed when characterized with bulk rheology. This is especially important for injectable granular hydrogels, as the size scale of individual microgels (~10–1000 μm in this study) overlaps with the size scale of common needle diameters (e.g., 838 μm for an 18 G needle, 337 μm for a 23G needle). Thus, it is essential to characterize the behavior of granular hydrogels flowing through a clinically relevant syringe and needle. In this study, a custom-built Arduino-based force sensor setup was used, as previously described,^[30] to assess the extrusion forces of granular hydrogels through 18, 21, and 23G needles at clinically-relevant injection speeds (Figure 2c). Needles 1” in length were subsequently used in all ex vivo studies, whereas an 18G spinal needle (3.5”) was used for subsequent percutaneous delivery of the radiopaque hydrogel in vivo. Radiopaque granular hydrogels had extrusion forces well within clinically acceptable limits for extrusion through all 18, 21, and 23G needles (Figure 2c).^[31–33] The 18G spinal needle (3.5”) did require significantly more extrusion force (~7N) than any of the 1” needles, though still was well within clinically-acceptable limits.^[31–33] For subsequent ex vivo studies, a 21G needle (1”) was used to deliver granular hydrogel to the NP cavity, whereas an 18G spinal needle (3.5”) was used in subsequent in vivo studies.

To characterize cytotoxicity in vitro, goat NP cells were plated in 24-well plates and exposed to granular hydrogels with and without radiopaque nanoparticles in a transwell insert for 7 days. There was no significant difference in cell metabolic activity as measured by alamar blue staining between controls (no gel) and cells exposed to granular hydrogels with or without ZrO₂ on Days 1, 5, and 7 (Figure S4a). Further, on Day 7, there was no significant difference between live-dead fluorescence signal intensity ratio between controls (no gel) and cells exposed to granular hydrogels with or without ZrO₂ (Figure S4b,c). Taken together, these results show that radiopaque granular hydrogels are cytocompatible and do not induce significant cytotoxicity in goat NP cells in vitro.

2.3. Characterizing mechanical properties and radiopacity of granular hydrogels after intradiscal delivery to motion segments ex vivo

Ideally, an injectable hydrogel for NP repair should restore disc biomechanical properties, such as range of motion and mechanical moduli, to healthy levels.^[15] Thus, a previously described^[34,35] ex vivo rabbit degenerated disc model was used to assess the ability of radiopaque granular hydrogels to restore healthy disc mechanics. Rabbit motion segments (bone-IVD-bone, L2-L7) explants were isolated and subject to 20 cycles of physiologically-relevant compressive and tensile loads (−42N to +21N) using an Instron setup. The compressive stress applied was 0.48 MPa, which is equivalent to the body weight load on an average human lumbar disc.^[36] To mimic degenerative conditions, discs were radially punctured with a 16G needle, and NP contents were removed (Figure 3a). The radiopaque granular hydrogel was then injected into punctured discs using a 21G needle (Figure 3a). The total range of motion (ROM) as well as total compressive modulus were determined for healthy and punctured discs, as well as discs receiving intradiscal radiopaque granular hydrogel injection (Figure 3a). In addition, the neutral zone (NZ) modulus and ROM were evaluated. The neutral zone is a critically important parameter in disc biomechanics, defined as the mechanical range over which the motion segment moves with minimal resistance.^[37] It has previously been shown that NP tissue is the dominant contributor to healthy NZ mechanics,^[38] and thus it is crucial to investigate the impact of NP repair with injectable hydrogels on NZ parameters.

Figure 3. Ex vivo mechanical testing and fluoroscopic imaging.

a) Overview of the ex vivo mechanical testing process, including, from left to right, removing NP tissue from rabbit motion segment explants with a 16G needle, injecting radiopaque granular hydrogel with a 21G needle, representative force-displacement curves for each testing group, and observing radiopaque granular hydrogels within the intradiscal space post-mechanical testing. b) Quantified neutral zone (NZ) range of motion (ROM), NZ modulus, total ROM, and total compressive modulus for each group. Statistical analysis was conducted using one-way ANOVA and a Tukey’s post hoc comparison. n = 4–5, ns = no significance, #p<0.1 (trending), *p<0.05, **p<0.01, ***p<0.001; each p value explicitly indicated between comparisons when trending. c) Overview of ex vivo intradiscal injection into goat motion segments, including injection of the radiopaque granular hydrogel using an 18G needle (left), fluoroscopic imaging of a goat motion segment containing an intradiscal injection (pink arrow) of the radiopaque granular hydrogel (30 wt.% ZrO₂) and the syringe containing the hydrogel (center), and μCT imaging of a goat motion segment explant containing intradiscal radiopaque granular hydrogel (white, right). Scale bars 1 cm.

Needle puncture injury resulted in a significant increase in both NZ and total ROM (Figure 3b), which are representative of degenerative disc mechanical properties in vivo.^[35,39] Intradiscal injection of radiopaque granular hydrogel resulted in restoration of both NZ and total ROM to levels of healthy control tissues. Restoration of healthy ROM is essential for stabilizing the disc and reestablishing proper load distribution after disc degeneration.^[1,15] Thus, the ability of the radiopaque granular hydrogel to restore healthy ROM levels ex vivo is highly promising for NP repair applications. In addition, the injection of the radiopaque granular hydrogel resulted in a significant increase in NZ modulus compared to punctured and healthy discs (Figure 3b). While NZ modulus was slightly higher than healthy levels, the increase after puncture injury is overall favorable in the restoration of stable and robust disc mechanics. Further, the total compressive modulus was consistent (i.e., no statistical significance) across all three testing groups, though trending towards higher levels upon gel injection when compared to punctured discs (p = 0.06) (Figure 3b). Taken together, the ex vivo evaluation of biomechanical outcomes shows that injection of the radiopaque granular hydrogel into the intradiscal space has the potential to restore healthy disc mechanics after injury.

Introducing radiopacity into injectable hydrogels is highly beneficial for visualizing the hydrogel during surgery and monitoring its location post-operation. Thus, it is important for the radiopacity to be maintained over longer periods of time. To characterize this in vitro, a small volume (200 μL) of radiopaque granular hydrogel was incubated in phosphate buffered saline (PBS, 1 mL) for up to 4 weeks. Upon optical visualization, the PBS appeared clear throughout the incubation period (Figure S5). In addition, upon imaging with a fluoroscope, the ZrO₂ signal remained concentrated in the granular hydrogels (Figure S5). This suggests that ZrO₂ nanoparticles are retained in radiopaque granular hydrogels in vitro over a period of at least 4 weeks. Given that the hydrogel mesh size is on the order of ~10 nm,^[40] and the diameter of the ZrO₂ nanoparticles is on the order of ~100 nm, it is expected that the ZrO₂ will remain entrapped within the microgel particles with limited diffusion while the covalent NorHA network remains intact. Further, our previous work demonstrated that similar covalently crosslinked NorHA hydrogels exhibit very low degradation over periods of weeks to months, highlighting the ability of hydrogel systems like the one used herein to remain stable for longer time periods.^[28,41]

The long-term retention of ZrO₂ nanoparticles demonstrates a key advantage of the granular hydrogel system. In most injectable hydrogels, contrast agents like ZrO₂ are directly encapsulated into a bulk hydrogel that typically consists of polymers held together by weak, reversible crosslinks (i.e., physical associations, dynamic covalent bonds) that allow injectability.^[42] These networks often result in rapid diffusion of encapsulated contract agents, leading to reduced signals within days to weeks.^[43] However, injectable granular hydrogels like the ones used in this study consist of highly stable, covalently crosslinked microgels that can retain encapsulated agents, such as ZrO_2, within the microgels for weeks to months, while the adaptable and flowable granular structure between microgels allows for injectability.

For radiographic imaging of hydrogels injected into the intradiscal space in vivo, it is essential for the hydrogel to possess sufficient signal to be differentiated from surrounding tissue (i.e., AF, cartilage endplates, vertebrae). To evaluate this ex vivo, the radiopaque granular hydrogel was injected into a goat disc explant and visualized using a fluoroscope (Figure 3c). NP tissue was removed from the disc via radial puncture with a 16G needle prior to injection of the radiopaque granular hydrogel. For reference, a healthy goat disc as well as granular hydrogels with varied ZrO₂ concentrations (0–30 wt.%) were visualized in the same fluoroscope scan (Figure S6). A concentration of at least 20 wt.% ZrO₂ was needed to ensure adequate contrast within the goat motion segment tissues, which further validates the use of 30 wt.% ZrO₂ in the radiopaque granular hydrogels used throughout this study, particularly for in vivo imaging where maximal contrast is desired. Further, the radiopaque granular hydrogel was clearly visible upon intradiscal injection in the goat motion segment explant using fluoroscopic imaging (Figure 3c). For further visualization, μCT scanning was used to visualize radiopaque granular hydrogel 3D location in the intradiscal space (Figure 3c). As evident from the μCT imaging, the radiopaque granular hydrogel was present throughout the intradiscal space, confirming its proper placement upon ex vivo injection. These investigations demonstrate the many benefits of granular hydrogel radiopacity in IVD repair.

2.4. In vivo assessment of the injectable radiopaque granular hydrogel in a large animal goat model of disc degeneration

Towards clinical translation of minimally-invasive NP repair strategies, we sought to evaluate our injectable radiopaque granular hydrogel in vivo using a clinically-relevant large animal goat model of cervical disc degeneration (Figure 4a). Goats are often used to model injuries and diseases of the spine, as goat IVDs are similar in size and structure to human spinal discs, and the goat cervical spine has a semi-upright structure (compared to the purely horizontal lumbar spines of quadrupeds) similar to the upright loading of the human spine.^[36,44] To induce degeneration of the goat cervical discs, chondroitinase ABC (ChABC) was percutaneously delivered by intradiscal injection, as in our prior studies.^[23] Four weeks after ChABC injection, radiopaque granular hydrogel was percutaneously injected into the intradiscal space under fluoroscopic guidance using a syringe and 18G needle (Figure 4b). Placement of the radiopaque hydrogel within the intradiscal space in the sagittal, axial, and coronal planes was confirmed using fluoroscopic imaging immediately post-delivery (Figure 4c).

Figure 4. In vivo study design and assessment of disc height index (DHI) in a goat model of disc degeneration.

a) Overview of in vivo study design and outcome measures. b) Representative intra-operative images of percutaneously delivering the injectable radiopaque granular hydrogel into goat cervical spinal discs using a syringe and 18G needle. c) Representative intra-operative fluoroscopic images in the sagittal, coronal, and axial planes depicting hydrogel positioning within the intradiscal space. d) Representative radiograph image of a goat cervical disc pre-injection of the hydrogel and subsequent images of the same disc 2, 4, 6, and 8 weeks after receiving the hydrogel injection. e) Quantification of the average disc height index (DHI, %) over the 12-week duration of the study, where the blue region indicates the 8-week period after hydrogel delivery in a subset of discs. Statistical significance compared to healthy controls at each time point is indicated (degenerative in gray; hydrogel-injected in blue). f) Quantification of the percent change in DHI between week 4 (pre-hydrogel injection) and week 12 (conclusion of the study) in healthy controls, degenerative (“Degen”), and hydrogel-injected (“Degen + Gel”) discs. Statistical analysis was conducted using one-way ANOVA and a Tukey’s post hoc comparison. n ≥ 3, ns = no significance, #p<0.1 (trending), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Lateral plain radiographs of the cervical spine were obtained pre-operatively and every 2 weeks following ChABC injection until completion of the study. Radiograph images confirmed the maintenance of hydrogel radiopacity in vivo as well as the retention of the hydrogel within the intradiscal space over a period of 8 weeks post-injection (Figure 4d). The retention of the hydrogel within the intradiscal space over 8 weeks highlights the promise of our injectable granular hydrogel for disc repair, as many injectable hydrogels suffer from migration and expulsion from the intradiscal space shortly after injection.^[5]

The radiographs were used to calculate the disc height index (DHI, %), which is a measurement of disc height change over time, normalized to adjacent vertebral body length (Figure 4e). DHI serves as an important marker for noninvasively monitoring disc health and to assess potential treatments.^[45,46] In healthy discs (i.e., those that did not receive ChABC), DHI did not change from ~100% over the 12-week duration of the study. After ChABC injection, the DHI decreased to ~80% after a period of 4 weeks and continued to decrease until the completion of the study at 12 weeks. A period of 4 weeks after ChABC injection, the radiopaque granular hydrogel was delivered to a subset of discs. In discs that received the hydrogel treatment, the DHI did not significantly decrease further over the subsequent 8-week period as it did in the degenerative discs. Instead, on average, the DHI increased about 10% between the hydrogel injection at week 4 and the conclusion of the study at week 12 (Figure 4f). This suggests that while degeneration and loss of disc height persists in untreated discs, receiving an injection of the granular hydrogel results in a stabilizing effect that limits continued loss of disc height.

MRI T2 signal is an important marker of overall disc health.^[46] In particular, a high NP T2 relaxation time correlates with high water content and hydration in a healthy disc, whereas a decrease in NP T2 relaxation time correlates with loss of water content and degenerative conditions.^[35,46] To assess the impact of hydrogel injection on NP T2 signal, T2-weighted MR images and quantitative T2 maps were obtained at 12 weeks (Figure S7) to assess disc structure and quantify T2 relaxation times in the NP. Mean NP T2 relaxation times for degenerative discs as well as discs that had received hydrogel injection were obtained and normalized to mean NP T2 relaxation times in healthy controls. NP T2 was not significantly different between degenerative discs and discs that had received hydrogel injection at 12 weeks, suggesting that granular hydrogel injection did not substantially affect NP tissue hydration at this timepoint.

After MRI imaging, isolated goat discs were subject to cyclic compressive loading from 0.5N to −100N, which is within the range of in vivo compressive loading in the goat and human cervical spine.^[47,48] Optical tracking during compressive loading was used to determine strain, and a custom MATLAB program was used to generate a bilinear fit of the 20^th cycle of compression to quantify biomechanical parameters including toe modulus (initial non-linear phase of loading), linear modulus (linear elastic phase), transition strain (between linear and non-linear phase), and maximal compressive strain, per our established methods.^[49] Creep strain was also determined. A representative image of a disc explant subject to mechanical loading is shown in Figure 5a.

Figure 5. Mechanical testing of goat motion segments from in vivo study after 12 weeks (8 weeks post-gel injection.

a) Representative image of goat disc explant subject to mechanical testing via compressive loading and optical tracking. Scale bar = 1 cm. b) Quantified toe modulus (left) and linear modulus in healthy controls (“Ctrl”), degenerative (“Degen”), and hydrogel-injected (“Degen + Gel”) discs after 12 weeks (right). c) Quantified transition strain (%, left), maximum strain (%, center), and creep strain (%, right) for these same groups after 12 weeks. Statistical analysis was conducted using one-way ANOVA and a Tukey’s post hoc comparison. n ≥ 4, ns = no significance, #p<0.1 (trending). p values of ≤ 0.2 are indicated.

There were no significant differences in mean values of biomechanical parameters across controls, degenerative discs, and hydrogel-injected discs at 12 weeks (Figure 5b–c). For degenerative discs, transition strain trended towards increasing relative to controls (p = 0.06), while hydrogel injection trended towards recovering transition strain towards near-healthy levels (i.e., no significant difference between control and hydrogel-injected discs at 12 weeks).

In both the control and the degenerative discs, there was significant variation across mean values for biomechanical parameters, which may be due to inter-animal variability and small differences in the delivery of the ChABC enzyme during surgical procedures.^[46] For example, for the toe modulus, the control and degenerative discs had a coefficient of variation (CV) of 92% and 106%, respectively. For the linear modulus, the CV values were 95% and 113%, respectively. It is interesting to note that, for discs that received hydrogel injections, the CV values for the toe modulus and linear modulus 8-weeks post-gel injection were lower at 58% and 12%, respectively. The CV values being lower for hydrogel-injected discs compared to controls and degenerative discs are in line with the hypothesis that hydrogel injection may have a stabilizing effect on the disc. Stabilizing properties such as disc height and mechanical moduli post-gel injection would be a favorable clinical outcome for hydrogel-based NP repair strategies. Stabilizing the disc and halting or slowing the disc degeneration process could lead to reduced pain, improved spine health, and better quality of life for patients.^[50,51] Future studies could further investigate the ability of injectable granular hydrogels to stabilize disc health by varying properties such as material moduli, injection volume, and stage of degeneration upon hydrogel injection.

To verify retention of the radiopaque granular hydrogel within the intradiscal space 8 weeks post-injection in vivo, micro-CT scans of explanted goat motion segments were performed (Figure 6a). The micro-CT reconstructions clearly demonstrated the presence of the hydrogel within the intradiscal space. This highlights the ability of the hydrogel to occupy and maintain its location within the disc over the 8-week period in vivo.

Figure 6. Micro-CT imaging and histology of goat motion segments after 12 weeks (8 weeks post-gel injection).

a) Representative micro-CT scans in the sagittal and coronal planes for healthy control (left) and hydrogel injection (right) depicting hydrogel positioning within the intradiscal space. Scale bar = 10 mm. b) Representative mid-sagittal histology sections of goat motion segments stained with alcian blue (proteoglycan) and picrosirius red (collagen) for healthy controls (top), degenerative (middle), and hydrogel injection (bottom, “Degen + Gel”), depicting best (left, least degenerative), median (center), and worst (right, most degenerative) outcomes for each group. Scale bar = 5 mm. c) Scoring of mid-sagittal histology sections of goat motion segments, showing total score (left), nucleus pulposus (NP) score (center), annulus fibrosus (AF) score (center), and bone/endplate (EP) score (right) in healthy controls (“Ctrl”), degenerative (“Degen”), and hydrogel-injected (“Degen + Gel”) discs after 12 weeks. Statistical analysis conducted using a Kruskal-Wallis test (due to discrete scoring values). n ≥ 4, ns = no significance, *p<0.05. p values of ≤ 0.2 are indicated.

Histological analysis using alcian blue (proteoglycan), picrosirius red (collagen), and hematoxylin and eosin (H&E) staining revealed crucial insights into degenerative changes in the disc after 12 weeks as well as promising impacts of hydrogel intervention 8 weeks post-injection (Figure 6, Figure S8). Further, semi-quantitative histology scoring on each experimental group was conducted using the ORS Spine Section/JOR Spine histopathology scoring system for large animals, where a higher score indicates more severe degeneration.^[52] Degenerative discs had a significantly higher total histology score, as well as NP and AF scores, indicating more severe degenerative markers compared to healthy controls. In the degenerative discs, a significant loss of PG content within the NP space was observed, as evident by a decrease in alcian blue staining. In addition, increased collagen deposition in the NP space was observed, as evident by increased picrosirius red staining in the NP region. Additionally, the structural integrity of the AF was compromised in the degenerative condition, as supported by the increased AF score for degenerative discs. In healthy controls, the AF layers were convex around the NP region, which is indicative of healthy levels of disc osmotic pressurization and overall disc structural integrity. In the degenerative discs, the normal organization of the AF layers was lost, particularly in the representative “median” and “worst” examples shown in Figure 6b. AF layers appear disoriented and even concave around the NP region, indicative of disc collapse and loss of healthy structure. Histological scoring of the bone and endplates was not significantly different across healthy controls and degenerative discs.

Interestingly, the introduction of granular hydrogel to the degenerative disc appeared to mitigate many of the degenerative changes observed in histological staining. There was no significant difference between total, NP, and AF histology scores of healthy controls and degenerative discs that received granular hydrogel injections (Figure 6c). The increased alcian blue staining within the NP region at 8-weeks post-injection suggests maintenance of PG content, which is important for overall NP health. This is further supported by the maintenance of well-organized AF layers with convex orientation surrounding the NP region, and quantified by a lower AF score, resembling healthy controls. Moreover, picrosirius red staining is reduced within the NP space compared to the degenerative discs, indicating potential suppression of further disc degeneration. Further, hematoxylin and eosin (H&E) staining of the hydrogel-tissue interface suggests that there was no increase in cell density around the interface, which suggests that there was no marked inflammatory response to the hydrogel injection (Figure S8). It is important to note that, across all histological staining, the hydrogel can be distinguished from tissue due to its dark grey coloring due to the encapsulated ZrO₂ nanoparticles (Figure S8). These findings highlight the hydrogel’s capacity to stabilize degenerative discs and present further deterioration.

Overall, our findings strongly suggest that the granular hydrogel intervention played a pivotal role in stabilizing degenerative discs and limiting further loss of structural and mechanical integrity. It is worth emphasizing that these promising outcomes were obtained solely through delivery of the hydrogel itself, without the inclusion of biologic agents or cells. This highlights the potential for injectable granular hydrogel delivery as a simple and streamlined treatment option. Ultimately, this “material-only” approach may offer a straightforward and widely applicable solution to address DDD.

3. Conclusions

Herein, an injectable radiopaque granular hydrogel was explored for degenerative disc repair. Microgels were fabricated by extrusion fragmentation and subsequently jammed by vacuum-driven filtration to form injectable granular hydrogels. ZrO₂ nanoparticles were encapsulated into microgels to introduce radiopacity, enabling direct visualization of the hydrogel using clinically-relevant imaging technologies (i.e., x-ray and CT scan). Material properties, including microgel characteristics, rheological behavior, and injectability were fully characterized. Long-term (i.e., 4-week) retention of radiopaque signal within the granular hydrogel was determined using radiographic imaging in vitro, as well as direct visualization of the radiopaque granular hydrogel within the intradiscal space after injection into a goat motion segment ex vivo. Further, radiopaque granular hydrogels were able to restore healthy disc mechanics in an ex vivo degenerative disc rabbit model. Lastly, radiopaque granular hydrogels were delivered in a minimally-invasive manner into the intradiscal space in a clinically relevant in vivo large animal goat model of IVD degeneration initiated through degradation by chondroitinase. The radiopaque granular hydrogel successfully halted further loss of disc height due to degeneration. Further, the hydrogel not only enhanced PG content and reduced collagen content in the NP region compared to untreated degenerative discs, but also maintained the structural integrity of the disc and promoted healthy segregation of the NP and AF regions. Overall, this study showcases the significant promise of injectable radiopaque granular hydrogels for treating DDD.

4. Methods

Materials:

Sodium hyaluronic acid (HA, MW = 60 kDa) was purchased from Lifecore Biomedical. Chondroitinase ABC was purchased from Amsbio. Unless otherwise indicated, all other reagents were obtained from Sigma-Aldrich.

Chemical modification of HA.

Norbornene-modified HA (NorHA) was prepared as previously described.^[22] Briefly, HA modified with tetrabutylammonium salt (HA-TBA) was dissolved in anhydrous dimethylsulfoxide (DMSO). Dimethyl aminopyridine (DMAP), norbornene carboxylic acid, and ditertbutyl dicarbonate (Boc₂O) were added to the DMSO and allowed to react overnight. NorHA product was then dialyzed for 14 days in DI water, frozen at −80 °C, and subsequently lyophilized for 6 days. Lyophilized polymers were dissolved in deuterium oxide (D₂O) at a concentration of 8 mg/mL and analyzed using ¹H-NMR (Bruker NEO400) to determine the degree of modification. Norbornene degree of modification was determined to be (25 ± 2)%.

Hydrogel precursor solutions and bulk hydrogel fabrication.

Hydrogel precursor solutions consisting of NorHA (2 wt.%), dithiothreitol (DTT, 6 mM), and ammonium persulfate (APS, 10 mM) were dissolved in phosphate buffered saline (PBS). For hydrogels containing zirconium oxide nanoparticles (ZrO₂), 30 wt.% ZrO₂ powder was added to the hydrogel precursor solution. For hydrogels without ZrO₂, high molecular weight (2 MDa) FITC-dextran (0.1 wt.%) was added to the precursor solution for visualization. To initiate gelation by redox radical formation, tetramethylethylenediamine (TEMED, 10 mM) was added by pipette to the hydrogel precursor solution while vigorously vortexing. The hydrogel precursor solution was vortexed for 1 min, then incubated at room temperature for 30 min to allow full gelation.

To prepare a sterilized hydrogel for in vivo studies, dry NorHA polymer was sterilized via UV radiation inside of a biosafety cabinet for 60 min. All aqueous solutions of DTT, APS, and TEMED in PBS were sterile filtered (0.22μm). Subsequently, hydrogel components were mixed and incubated as described above in a sterile environment inside of a biosafety cabinet to form a sterile bulk hydrogel.

Mechanical characterization of bulk hydrogels.

Rheological properties of bulk hydrogels were characterized using an oscillatory shear rheometer (AR2000, TA Instruments) fitted with a 20 mm diameter cone and plate geometry and 27 μm gap. Time sweeps (1% strain, 1 Hz) were performed at 25 °C for 30 min to characterize bulk gelation of hydrogel precursor solutions. Gelation time was determined as the time at which the storage modulus (G’) was equal to 90% of the plateau G’ value.

To determine bulk compressive properties, 50 μL of hydrogel precursor solution was placed into a cylindrical mold (4.6 mm diameter) and then incubated at room temperature for 30 min to allow full gelation. Mechanical testing was performed (TA Instruments, DMA Q800) to determine the compressive moduli of the bulk hydrogel samples. Samples were subject to 0.01 N pre-load force and subsequently compressed until 40% strain at a rate of 0.5 N/min. The compressive moduli were calculated as the slope of the stress-strain curve from 10–20% strain.

Microgel fabrication.

To fabricate microgels, an extrusion fragmentation method was used, as previously described.^[53] A volume of 1 mL of hydrogel precursor solution was added to a 3 mL syringe (BD). The hydrogel precursor solution was then incubated at room temperature inside the syringe for 30 min to allow full gelation. The bulk hydrogel was then extruded by hand through blunt-tip 18G, 20G, 22G, 23G, and 25G needles, sequentially. Excess PBS (1 mL) was added after extruding through the 18G needle to reduce the extrusion forces needed. Microgels were then suspended in pure PBS and centrifuged at 10,000 rpm for 3 min, and the supernatant was removed. This washing step was repeated 5 times to remove any unreacted reagents and to isolate microgels. To prepare sterilized granular hydrogels for in vivo studies, all fragmentation microgel fabrication and washing steps were performed in a sterile biosafety cabinet.

Microgel characterization.

For microgels with ZrO₂, bright field microscopy (Olympus BX51) was used to image the microgels after fabrication. For microgels without ZrO₂ (with FITC-dextran), fluorescence microscopy (Olympus BX51) was used to image the microgels after fabrication. ImageJ was used to quantify microgel size and aspect ratio. Microgel diameters were calculated by treating the area of the microgel as a circle and determining the equivalent circular diameter.

Granular hydrogel formation and rheological characterization.

Microgels suspended in PBS were jammed by vacuum-driven filtration (Steriflip, 0.22μm-pores, Millipore) to form granular hydrogels, as previously described.^[25,27] Rheological properties of granular hydrogels were assessed using an oscillatory shear rheometer (AR2000, TA Instruments) with a 20 mm parallel steel plate geometry set at a 1 mm gap. Strain sweeps (1 – 250 % strain, 1 Hz) were used to assess shear yielding properties. The yield strain was determined as the strain at which G’ < 0.9G’_initial. Frequency sweeps (1 – 100 Hz, 1 % strain) were used to assess frequency-dependent rheological properties. For shear recovery experiments, low (1%) and high (250%) strains were periodically applied at 1 Hz. For flow characterization, the viscosity was measured during a continuous shear rate ramp from 0 – 100 s⁻¹.

Extrusion force measurements.

Granular hydrogels were loaded into a 3 mL syringe (BD) with a needle as specified. The syringe was loaded onto a syringe pump, and a round force-sensitive resistor (Interlink 402) was placed between the syringe plunger and the pump. Data was acquired using a setup built with an Arduino Uno Rev3 as previously described.^[30] The syringe pump was extruded at a rate of 300 μL/min for 20 s to reach a plateau in extrusion force. Voltage output was recorded using the Arduino IDE serial monitor and converted to Newtons using a force calibration curve.

Cytocompatibility.

Goat NP cells were seeded at 4,000 cells per well in a 24-well plate and allowed to equilibrate for 24h while incubating at 37°C. Granular hydrogel with and without ZrO₂ were prepared as described above. A volume of 150 μL of granular hydrogel either with or without ZrO₂ was added to the 24-well plate in transwell inserts (n=6 per group). Goat NP cells exposed to granular hydrogels were incubated in basal media for 7 days. Alamar blue staining was performed on Days 1, 5, and 7. For alamar blue staining, each well was incubated in 500 μL of alamar blue working solution for 3h under mechanical agitation. On Day 7, live-dead staining was conducted according to manufacturer’s instructions (Life Technologies, L3224).

Ex vivo mechanical testing with rabbit motion segment explants.

New Zealand white rabbit lumbar spines (10–12 weeks old) were obtained from Sierra for Medical Science. Spines were dissected into individual bone-IVD-bone motion segments, as previously described.^[35] For healthy controls, motion segments were used as-is. For punctured discs, a 16G needle was used to puncture the AF in a radial direction and scoop out NP contents. For hydrogel injections, a 21G needle was used to deliver the radiopaque granular hydrogel (~50–100 μL) into the punctured discs. The variation in hydrogel volume injected was due to natural variation in NP cavity volumes within tissue explants. Hydrogel was injected until the NP cavity was full, as determined manually by pressure-based feedback from the syringe plunger during injection.

Motion segments (healthy, punctured, and hydrogel injections) were subject to mechanical testing and analysis as previously described.^[34,35,39] Motion segments were potted in an indium casting alloy (McMaster Carr) to enable securing onto the mechanical testing fixtures. The motion segments were subject to 20 cycles of tension and compression (+21 N to −42 N) at 0.5 Hz on an Instron (Instron 5948). To determine displacement of the disc, optical tracking was performed using a high-resolution digital camera (A3800; Basler) and a custom MATLAB tracking program.^[34] All testing was conducted while samples were submerged in a bath of PBS at room temperature.

The force-displacement curve for the 20^th tension-compression cycle was fit to a sigmoid function used to determine the neutral zone (NZ) and total ranges of motion (ROM) and moduli using the custom MATLAB program,^[34,39] according to our established methods. Measurements were normalized to the specific dimensions of the motion segment samples (i.e., disc height and area).

Ex vivo visualization of radiopaque granular hydrogel injections into goat motion segment explants.

Healthy goat lumbar spine motion segments were obtained from animals being utilized for alternate disc-related studies in the cervical spine. A 16G needle was used to puncture the AF in a radial direction and remove NP contents. An 18G needle was used to deliver the radiopaque granular hydrogel (~300–500 μL) into the punctured disc. The variation in hydrogel volume injected was due to natural variation in NP cavity volumes within tissue explants. Hydrogel was injected until the NP cavity was full, as determined manually by pressure-based feedback from the syringe plunger during injection. Motion segments were imaged using a fluoroscope (OrthoScan HD). To visualize the 3D distribution of the radiopaque granular hydrogel within the disc, μCT imaging with 20 μm isotropic scan resolution was used as previously described.^[23] Motion segments with radiopaque granular hydrogel were imaged using a μCT (μCT 50, Scanco).

Animal study and surgical procedures.

The study was approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. Large frame castrated male goats, approximately 2–5 years of age (Thomas D. Morris, Inc.) were used in this study and underwent two surgical procedures, 4 weeks apart. The first surgery was performed to induce degeneration of the goat cervical discs by percutaneous chondroitinase ABC (ChABC, 200 μL, 2U) intradiscal injection, as in our prior studies.^[23] Prior to surgery, animals were fasted between 24–36 hours with small feedings of goat feed up to 12 hours before anesthesia with free access to water. Animals were sedated with diazepam (0.5–1.5 mg/kg, IV/IM) or midazolam (0.3–05 mg/kg IV/IM) followed by induction for general anesthesia with Ketamine (2.2–4.0 mg/kg, IV) and maintained on inhalation anesthesia using 1–5% Isoflurane in oxygen delivered through a circle breathing circuit. Animals were positioned in dorsal recumbency, followed by a prep for aseptic orthopedic surgery. Orthogonal fluoroscopy was utilized to identify the C2-C3, C3-C4 and C4-C5 disc spaces. A 6 inch 22G spinal needle was inserted percutaneously into either the C2-C3 or C4-C5 disc space using fluoroscopic guidance (3D C-model Arcadis Orbis, Siemens). The location of the tip of the spinal needle in the center of the NP was confirmed intraoperatively via a 3D spin of 198°. Following intradiscal injections of ChABC, animals were hand-recovered from anesthesia by veterinary staff until ambulatory, then returned to group housing with unrestricted exercise in a barn for the duration of the study. A combination of opioids (fentanyl transdermal patches, 2.5 μg/kg/hr placed perioperatively and left in place for 72 hrs) and NSAIDs (Flunixin meglumine 1.1 mg/kg, IV/IM, on the day of surgery and for 3 days post-operatively) were used for peri-operative analgesia. Animals were assessed at least daily for clinical signs of pain or distress by a veterinarian.

In one subset of animals (n = 7 goats, 2U ChABC delivered in a randomized fashion to the C2-C3 or C4-C5 disc), animals were euthanized 12 weeks following intradiscal delivery to serve as degenerative (no hydrogel treatment) controls (n = 7 discs). In a second subset of animals (n = 2 goats), both the C2-C3 and C4-C5 discs were injected with 2U ChABC as per above; 4 weeks post-ChABC injection, percutaneous intradiscal granular hydrogel delivery was performed (n = 4 discs). Radiopaque granular hydrogel (300–500 μL) was injected under 3D fluoroscope guidance, as described above, using a syringe and an 18G spinal needle. Hydrogel distribution throughout the disc in the sagittal, axial and coronal planes was confirmed intra-operatively on fluoroscopy via a 3D spin. In all animals, lateral plain radiographs of the cervical spine were obtained pre-operatively, and every two weeks following ChABC injection until completion of the study at 12 weeks. The radiographs were utilized to calculate disc height index (DHI) at the C2-C3, C3-C4 and C4-C5 levels at each time point, utilizing a MATLAB code according to our established methods.^[46] Animals were euthanized 12 weeks following ChABC injection (8 weeks after hydrogel injection) according to the guidelines set forth by the current AVMA Panel on Euthanasia via an overdose of commercially available euthanasia solution (Pentobarbital, 1 ml/5 kg), and lumbar spines harvested for ex vivo analyses. The C3-C4 level was utilized as a healthy control in all animals (n = 5–8 levels depending on outcome assay, taken from both cohorts of animals)

MRI scanning and analysis.

MRI scans were performed on intact whole cervical spines from the in vivo study described above. MRI scans (5 mm slice thickness, 0.5 mm in plane resolution, TR/TE = 4,540/123 ms) of T2-weighted mid-sagittal images were acquired using a 3T scanner (Siemens Magnetom TrioTim). A series of images of the lumbar spine were obtained for T2 mapping, using 6 echoes with an initial TE of 13 ms, a slice thickness of 5 mm, and an in-plane resolution of 0.5 mm. To generate average T2 maps for each experimental group, a custom MATLAB code was used as previously described.^[54] T2 relaxation times in the NP were measured in a manually-contoured region of interest for each experimental disc (degenerative control or hydrogel-injected), and normalized to the T2 relaxation times of the control disc within the same animal.

Mechanical testing and analysis.

Following MRI, lumbar disc motion segments were isolated and the posterior and lateral bony elements were carefully removed using a hand saw to obtain vertebral body – IVD – vertebral body segments. Goat motion segments were potted for mechanical testing as described above for the rabbit motion segments and subjected to 20 cycles of compressive loading from 0.5 to −100 N (~0.24 MPa) in a PBS bath at room temperature. The applied compressive loads are within the range of in vivo loading within the goat and human cervical spine.^[47,48] Force and displacement data were normalized to stress and strain using measures of disc area and height obtained from the cervical spine MRIs. A bilinear fit of the 20^th cycle of compression was utilized to quantify toe modulus, linear modulus, transition strain, and maximal compressive strain, per our established methods.^[49]

Microcomputed tomography (μCT) and histological evaluation.

Following mechanical testing, motion segments were fixed in 10% neutral buffered formalin at 4ºC for 1 week. Samples underwent μCT scanning (Scanco μCT50) at isotropic 24.2 μm resolution to generate 3D reconstructions of the vertebral bone and radiopaque granular hydrogel retained in the disc space 8 weeks following injection (Dragonfly, Comet Technologies, Montreal, Canada). Following μCT scanning, motion segments were decalcified (Formical-2000, Decal Chemical Corporation, Tallman, NY). Subsequently, the segments were processed through paraffin, and 10 μm histologic sections (mid-sagittal) were obtained. Sections were stained with Alcian blue (glycosaminoglycans) and picrosirius red (collagens), or Hematoxylin and Eosin. Stained slides were imaged at 20X magnification using an Aperio slide scanner.

Semi-quantitative histology scoring was completed on each experimental group using the ORS Spine Section/JOR Spine histopathology scoring system for large animals.^[52] This scoring system consists of eight categories scored between 0 and 3 points (a higher score indicates more severe degeneration): NP matrix staining, AF morphology, NP/AF clefts, distinction between the NP and AF, NP cell clusters, NP cell loss and necrosis, bone formation and cartilage endplate morphology. The scoring of each Alcian blue and picrosirius red or H&E-stained histology section was performed by three independent graders. If there was disagreement in the scores between graders, a consensus score was reached. When grading the slides, only native disc tissue was scored, and the granular gel (if present in the section) was disregarded. Total histology score was determined by summing all eight categories. Total NP score was determined by summing three categories relevant to NP properties (NP matrix staining, NP cell clusters, and NP cell loss and necrosis). Total AF score was determined by summing three categories relevant to AF properties (AF morphology, NP/AF clefts, and distinction between the NP and AF).

Statistical analysis.

Data is presented as mean ± standard deviation, unless otherwise indicated. Statistical analysis was conducted in GraphPad Prism 8 using one-way ANOVA and a Tukey’s post hoc comparison. For all samples, n ≥ 3 (specific sample sizes indicated in figure captions), #p < 0.1 (trending), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.