An Injectable Engineered Cartilage Gel Improves Intervertebral Disc Repair in a Rat Nucleotomy Model

Abstract

Lower back pain is a major problem caused by intervertebral disc degeneration. A common surgical procedure is lumbar partial discectomy (excision of the herniated disc causing nerve root compression), which results in further disc degeneration, severe lower back pain, and disability after discectomy. Thus, the development of disc regenerative therapies for patients who require lumbar partial discectomy is crucial. Here, we investigated the effectiveness of an engineered cartilage gel utilizing human fetal cartilage-derived progenitor cells (hFCPCs) on intervertebral disc repair in a rat tail nucleotomy model. Eight-week-old female Sprague-Dawley rats were randomized into three groups to undergo intradiscal injection of (1) cartilage gel, (2) hFCPCs, or (3) decellularized extracellular matrix (ECM) (n = 10/each group). The treatment materials were injected immediately after nucleotomy of the coccygeal discs. The coccygeal discs were removed six weeks after implantation for radiologic and histological analysis. Implantation of the cartilage gel promoted degenerative disc repair compared to hFCPCs or hFCPC-derived ECM by increasing the cellularity and matrix integrity, promoting reconstruction of nucleus pulposus, restoring disc hydration, and downregulating inflammatory cytokines and pain. Our results demonstrate that cartilage gel has higher therapeutic potential than its cellular or ECM component alone, and support further translation to large animal models and human subjects.

Keywords: cartilage gel; extracellular matrix; human fetal cartilage-derived progenitor cells; intervertebral disc degeneration; regeneration.

Figure 1

Schematic diagram of the surgical procedure and alleviation of the injury-induced pain phenotype by the cartilage gel. (A) Surgical procedures in a rat tail nucleotomy model; nucleus pulposus aspiration with a 22-gauge catheter through an annulus fibrosus incision. Injection of cartilage gel, hFCPCs, or ECM (15 µL using a 25-gauge catheter). (B) Alleviation of the injury-induced pain phenotype by the cartilage gel. In the von Frey test, the 50% withdrawal thresholds were significantly higher in the cartilage gel-treated group than in the hFCPCs or ECM-treated group. * p < 0.05; *** p < 0.001, significant difference between groups, by one-way ANOVA, n = 10 rats per group. hFCPCs, human fetal cartilage-derived progenitor cells; ECM, extracellular matrix.

Figure 2

Cartilage gel injection promoted the repair of degenerated discs by restoring disc hydration and proteoglycan content in a rat tail nucleotomy model. The Co3-4 discs were healthy controls, the Co5-6 discs were the nucleotomy-only group, and the Co4-5 discs were the groups injected with the cartilage gel, hFCPCs, or ECM after nucleotomy. (A) T2-weighted magnetic resonance imaging (MRI) of rat tail discs of experimental groups taken at 6 weeks after nucleotomy. Black arrows indicate healthy discs (control), orange arrows indicate injected discs (Co4-5), and blue arrows indicate nucleotomy-only discs (Co5-6). (B) Changes in the T2-weighted MRI index. (C) Changes in the Pfirrmann grade as assessed by T2-weighted MRI. (D) (i) Safranin-O (S-O) staining of rat tail discs at 6 weeks after nucleotomy in low-power fields (top). White rectangles indicate the disc nucleus pulposus (NP) area shown in higher-power fields (bottom). (ii) Changes in hematoxylin and eosin (H&E) staining of disc NP spaces (top). White rectangles indicate the disc NP area shown in higher-power fields (bottom). (E) Changes in the histological score by S-O staining. (F) Changes in the positive area of H&E staining in disc NP spaces. (G) Changes in the number of NP cells in the disc NP spaces measured using H&E images. In (B,F,G), data are presented with box plots (n = 7, n = 6 and n = 6 respectively). In (A,E), data are presented with mean ± SEM (n = 7 and n = 6, respectively, * p < 0.05; ** p < 0.01; *** p < 0.001). significant difference by one-way ANOVA. hFCPCs, human fetal cartilage-derived progenitor cells; ECM, extracellular matrix.

Figure 3

The cartilage gel preserved the matrix proteins in disc nucleus pulposus (NP) of a rat nucleotomy model. Immunohistochemical images of the indicated genes are presented for the healthy discs, injury-only discs, and the cartilage gel-injected, hFCPCs-injected, or ECM-injected discs at 6 weeks after surgery. (A) Immunohistochemistry of aggrecan (i) and type II collagen (ii). (B) Changes in the percentage of aggrecan-positive area in the disc NP spaces. The whole IVD images with merged signals are also presented at the bottom. (C) Changes in the percentage of type II collagen-positive area in the disc NP spaces. In (B,C), data are presented with box plots (n = 6, * p < 0.05; ** p < 0.01, *** p < 0.001, significant difference by one-way ANOVA). hFCPCs, human fetal cartilage-derived progenitor cells; ECM, extracellular matrix.

Figure 4

The cartilage gel preserved cells with endogenous phenotype in disc nucleus pulposus (NP) of a rat nucleotomy model. Immunofluorescence images of the indicated genes are presented for the healthy discs, injury-only discs, and the cartilage gel-injected, hFCPCs-injected, or ECM-injected discs at 6 weeks after surgery. (A) Immunofluorescence of brachyury (green) and Tie2 (red), DAPI (blue), and merged signals. (B) Changes in the percentage of Tie2 or brachyury-positive cells in the disc NP spaces. Immunopositivity was counted in disc NP of low-power fields and calculated as relative to the total number of DAPI-positive cells. In (A,B), data are presented with box plots (n = 4, * p < 0.05; ** p < 0.01, *** p < 0.001, significant difference by one-way ANOVA). hFCPCs, human fetal cartilage-derived progenitor cells; ECM, extracellular matrix.

Figure 5

Preservation of implanted cells in the disc nucleus pulposus (NP) of a rat tail nucleotomy model. (A) Immunofluorescence of rat tails in the cartilage gel-injected, hFCPCs-injected, ECM-injected, healthy, and injury-only discs at six weeks after nucleotomy for human nuclei antigen (green), DAPI (blue), and merged signals. (B) Changes in the percentage of human nuclear antigen-positive cells. Immunopositivity was counted in the disc NP with low-power fields and calculated as relative to the total number of DAPI-positive cells. Data are presented with box plots (n = 4, *** p < 0.001, ns, not significant, significant difference by one-way ANOVA). hFCPCs, human fetal cartilage-derived progenitor cells; ECM, extracellular matrix.

Figure 6

Cartilage gel downregulated catabolic and proinflammatory cytokines in the disc nucleus pulposus (NP) of a rat tail nucleotomy model. Immunofluorescence images of the indicated genes are presented for the healthy discs, injury-only discs, and the cartilage gel-injected, hFCPCs-injected, or ECM-injected discs at six weeks after surgery. (A) Immunofluorescence of the rat tail discs for matrix metalloproteinase-13 (MMP-13) (green), DAPI (blue), and merged signals. (B) Immunofluorescence of rat tail discs for tumor necrosis factor-alpha (TNF-α) (red) and interleukin (IL)-1β (purple), DAPI (blue) and merged signals. (C) Changes in the percentage of MMP-13-positive cells in the disc NP spaces. (D) Changes in the percentage of TNF-α- or IL-1 β-positive cells in the disc NP spaces. In (C,D) immunopositivity was counted in the disc NP with low-power fields and calculated as relative to the total number of DAPI-positive cells. Data are presented with box plots (n = 4, ** p < 0.01, *** p < 0.001, significant difference by one-way ANOVA).

Figure 7

Cartilage gel inhibited injury-induced pain in the disc nucleus pulposus (NP) of a rat tail nucleotomy model. (A) Immunofluorescence of rat tails of cartilage gel-injected, hFCPCs-injected, ECM-injected, healthy, and injury-only discs at six weeks after nucleotomy for CGRP (green), DAPI (blue), and merged signals. (B) Changes in the percentage of CGRP-positive cells in the disc NP spaces. Immunopositivity was counted in the disc NP with low-power fields and calculated as relative to the total number of DAPI-positive cells. Data are presented with box plots (n = 4, ** p < 0.01, *** p < 0.001, significant difference by one-way ANOVA). hFCPCs, human fetal cartilage-derived progenitor cells; ECM, extracellular matrix; CCRP, calcitonin gene receptor protein.

Figure 8

Cartilage gel downregulated mRNA levels of proinflammatory cytokines in the disc nucleus pulposus (NP) of a rat tail nucleotomy model. (A) mRNA levels of TNF-α in the disc NP spaces. (B) mRNA levels of iNOS in the disc NP spaces. In (A,B), data are presented in box plots (n = 3, * p < 0.05; ** p < 0.01; *** p < 0.001, significant difference by one-way ANOVA).