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. 2023 Dec 1;6(4):e1293.
doi: 10.1002/jsp2.1293. eCollection 2023 Dec.

Combining adhesive and nonadhesive injectable hydrogels for intervertebral disc repair in an ovine discectomy model

Christopher J Panebianco  1   2 , Caroline Constant  3 , Andrea J Vernengo  3   4 , Dirk Nehrbass  3 , Dominic Gehweiler  3 , Tyler J DiStefano  1 , Jesse Martin  5 , David J Alpert  5 , Saad B Chaudhary  1 , Andrew C Hecht  1 , Alan C Seifert  6 , Steven B Nicoll  5 , Sibylle Grad  3 , Stephan Zeiter  3 , James C Iatridis  1
Affiliations

Combining adhesive and nonadhesive injectable hydrogels for intervertebral disc repair in an ovine discectomy model

Christopher J Panebianco et al. JOR Spine. .

Abstract

Background: Intervertebral disc (IVD) disorders (e.g., herniation) directly contribute to back pain, which is a leading cause of global disability. Next-generation treatments for IVD herniation need advanced preclinical testing to evaluate their ability to repair large defects, prevent reherniation, and limit progressive degeneration. This study tested whether experimental, injectable, and nonbioactive biomaterials could slow IVD degeneration in an ovine discectomy model.

Methods: Ten skeletally mature sheep (4-5.5 years) experienced partial discectomy injury with cruciate-style annulus fibrosus (AF) defects and 0.1 g nucleus pulposus (NP) removal in the L1-L2, L2-L3, and L3-L4 lumbar IVDs. L4-L5 IVDs were Intact controls. IVD injury levels received: (1) no treatment (Injury), (2) poly (ethylene glycol) diacrylate (PEGDA), (3) genipin-crosslinked fibrin (FibGen), (4) carboxymethylcellulose-methylcellulose (C-MC), or (5) C-MC and FibGen (FibGen + C-MC). Animals healed for 12 weeks, then IVDs were assessed using computed tomography (CT), magnetic resonance (MR) imaging, and histopathology.

Results: All repaired IVDs retained ~90% of their preoperative disc height and showed minor degenerative changes by Pfirrmann grading. All repairs had similar disc height loss and Pfirrmann grade as Injury IVDs. Adhesive AF sealants (i.e., PEGDA and FibGen) did not herniate, although repair caused local endplate (EP) changes and inflammation. NP repair biomaterials (i.e., C-MC) and combination repair (i.e., FibGen + C-MC) exhibited lower levels of degeneration, less EP damage, and less severe inflammation; however, C-MC showed signs of herniation via biomaterial expulsion.

Conclusions: All repair IVDs were noninferior to Injury IVDs by IVD height loss and Pfirrmann grade. C-MC and FibGen + C-MC IVDs had the best outcomes, and may be appropriate for enhancement with bioactive factors (e.g., cells, growth factors, and miRNAs). Such bioactive factors appear to be necessary to prevent injury-induced IVD degeneration. Application of AF sealants alone (i.e., PEGDA and FibGen) resulted in EP damage and inflammation, particularly for PEGDA IVDs, suggesting further material refinements are needed.

Keywords: annulus fibrosus repair; carboxymethylcellulose–methylcellulose; fibrin; hydrogels; injectable biomaterials; intervertebral disc; nucleus pulposus repair; ovine discectomy model; poly(ethylene glycol) diacrylate.

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Figures

FIGURE 1
FIGURE 1
Schematic representation of sheep injury and repair model. Intervertebral discs (IVDs) were injured using a cruciate defect with 0.1 g of nucleus pulposus (NP) removal. L1/L2, L2/L3, and L3/L4 IVDs were injured and systematically assigned to one of the following groups: (1) Injury, (2) repair with poly(ethylene glycol) diacrylate (PEGDA), (3) repair with genipin‐crosslinked fibrin (FibGen), (4) repair with carboxymethylcellulose–methylcellulose (C‐MC), and (5) combination repair with C‐MC as an NP replacement and FibGen as an annulus fibrosus (AF) sealant (FibGen + C‐MC). The L4/L5 IVD was kept as an intact control for all animals.
FIGURE 2
FIGURE 2
Repaired IVDs maintained at least 90% of their preoperative disc height by W12. (A) Representative computed tomography (CT) images at 12 weeks post‐injury (W12). Scale bar = 10 mm. (B) Disc height normalized to preoperative values. *p < 0.05, **p < 0.01, and ***p < 0.001 versus Intact IVDs.
FIGURE 3
FIGURE 3
Repaired and Injured IVDs are similar by Pfirrmann grade, but PEGDA and FibGen IVDs show increased local degeneration by detailed MR scoring. (A) Representative axial and mid‐sagittal T2‐weight magnetic resonance (MR) images. Red arrows indicate radiographic signs of endplate (EP) damage. Scale bar = 1 cm. (B) Pfirrmann grading. (C) Total IVD score using criteria from Table 1. (D) Individual IVD region scores using criteria from Table 1. Individual scores were summed together for the Total IVD Score in (C). *p < 0.05, **p < 0.01, and ***p < 0.001 versus Intact IVDs. † p < 0.05; †† p < 0.01 versus Injury IVDs.
FIGURE 4
FIGURE 4
C‐MC and FibGen + C‐MC IVDs show the least histopathological signs of degeneration. (A) Representative Safranin‐O/Fast Green staining at W12 in the dorsal plane (nondecalcified, resin‐embedded; left is ventrolateral and bottom is caudal). AF lamellae are highly aligned in Intact IVDs, compared to Injury, PEGDA, FibGen, C‐MC, and FibGen + C‐MC IVDs. Scale bar = 2 mm. (B) Semiquantitative analysis of Total Histopathological Score using Shu scoring system. (C) Semiquantitative analysis of individual categories for histopathological findings using the Shu scoring system. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus Intact IVDs.
FIGURE 5
FIGURE 5
Histopathological evaluation showed predominantly lymphocytic inflammation in PEGDA and FibGen IVDs, compared to histiocytic inflammation in C‐MC and FibGen + C‐MC IVDs. Representative inflammatory cell influx observed during histopathological staining at W12. Injury, PEGDA, and FibGen, IVDs showed lymphocytic cells, with round nuclei, aggregated to small follicles near blood vessels. C‐MC and FibGen + C‐MC IVDs showed histiocytic cells, with irregular shapes and peripherally located nuclei. Histiocytic cells (arrowhead) were aggregated around or near the foreign C‐MC material (asterisk), which formed fluid‐filled cysts. Representative images are shown at lower magnification (top, scale bar = 500 μm) with a section (rectangle) shown at higher magnification (bottom, scale bar = 100 μm).
FIGURE 6
FIGURE 6
Histopathological evaluation of EP and vertebral body showed greater damage in PEGDA and FibGen IVDs compared to C‐MC and FibGen + C‐MC IVDs. (A) Representative Safranin‐O/Fast Green images of Intact and Injury IVDs at W12, which had no osteolysis. (B) Representative Safranin‐O/Fast Green images of PEGDA, FibGen, C‐MC, and FibGen + C‐MC IVDs at W12 with varying severity of osteolysis. For each repair, top row images are the lowest grade and bottom row images are the highest grade observed in each group. All images were captured in the dorsal plane (non‐decalcified, resin‐embedded; left is ventrolateral and bottom is caudal). Scale bar = 2 mm. Asterisks mark left‐sided destruction of the trabecular bone tissue and arrowheads mark focal formations in the vertebral bone. (C) Semiquantitative analysis of EP and vertebral bone parameters. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus Intact IVDs. † p < 0.05 and †† p < 0.01 versus Injury IVDs.
FIGURE 7
FIGURE 7
PEGDA and FibGen AF sealants prevent NP herniation and did not experience biomaterial expulsion. Representative Safranin‐O/Fast Green and polarized light images of Intact, Injury, PEGDA, FibGen, C‐MC, and FibGen + C‐MC IVDs at W12 with varying severity of NP herniation or biomaterial expulsion. For each repair, left column images are the least severely herniated and right column images are the most severely herniated IVDs observed in each group. All images were captured in the dorsal plane (non‐decalcified, resin‐embedded; left is ventrolateral and bottom is caudal). Black arrows indicate herniated NP material and pink arrows indicate C‐MC expulsion. Scale bar = 2 mm.
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