Intradiscal transplantation of synovial mesenchymal stem cells prevents intervertebral disc degeneration through suppression of matrix metalloproteinase-related genes in nucleus pulposus cells in rabbits

Takashi Miyamoto, Takeshi Muneta, Takashi Tabuchi, Kenji Matsumoto, Hirohisa Saito, Kunikazu Tsuji, Ichiro Sekiya, Takashi Miyamoto, Takeshi Muneta, Takashi Tabuchi, Kenji Matsumoto, Hirohisa Saito, Kunikazu Tsuji, Ichiro Sekiya

Abstract

Introduction: Synovial mesenchymal stem cells (MSCs) have high proliferative and chondrogenic potentials, and MSCs transplanted into the articular cartilage defect produce abundant extracellular matrix. Because of similarities between the articular cartilage and the intervertebral disc cartilage, synovial MSCs are a potential cell source for disc regeneration. Here, we examined the effect of intradiscal transplantation of synovial MSCs after aspiration of nucleus pulposus in rabbits.

Methods: The nucleus pulposus tissues of rabbit's intervertebral discs were aspirated to induce disc degeneration, and allogenic synovial MSCs were transplanted. At 2, 4, 6, 8, 16, 24 weeks postoperatively, we evaluated with imaging analyses such as X-ray and magnetic resonance imaging (MRI), and histological analysis. To investigate interaction between synovial MSCs and nucleus pulposus cells, human synovial MSCs and rat nucleus pulposus cells were co-cultured, and species specific microarray were performed.

Results: The existence of transplanted cells labeled with DiI or derived from green fluorescent protein (GFP)-expressing transgenic rabbits was confirmed up until 24 weeks. X-ray analyses demonstrated that intervertebral disc height in the MSC group remained higher than that in the degeneration group. T2 weighted MR imaging showed higher signal intensity of nucleus pulposus in the MSC group. Immunohistological analyses revealed higher expression of type II collagen around nucleus pulposus cells in the MSC group compared with even that of the normal group. In co-culture of rat nucleus pulposus cells and human synovial MSCs, species specific microarray revealed that gene profiles of nucleus pulposus were altered markedly with suppression of genes relating matrix degradative enzymes and inflammatory cytokines.

Conclusions: Synovial MSCs injected into the nucleus pulposus space promoted synthesis of the remaining nucleus pulposus cells to type II collagen and inhibition of expressions of degradative enzymes and inflammatory cytokines, resulting in maintaining the structure of the intervertebral disc being maintained.

Figures

Figure 1
Figure 1
Cells from rabbit synovium have characteristics of MSCs. (a) Right hindlimb of GFP transgenic rabbit. (b) Colony forming cells derived from GFP transgenic rabbit synovium. (c) Differentiation potentials.
Figure 2
Figure 2
Intradiscally injected MSCs remain in the nucleus pulposus at 24 weeks. (a) Disc in normal condition and one day after intradiscal injection of DiI-labeld syonovial MSCs into the normal disc. Macroscopic views of interbertebral discs and fluorescent microscopic views of nucleus pulposus are shown. (b) Fluorescent microscopic views for GFP and DiI synovial MSCs. Nucleus pulposus was aspirated in both groups, and synovial MSCs were intradiscally injected into the MSC group.
Figure 3
Figure 3
Intradiscally injected MSCs maintain disc height. (a) X-ray image of normal rabbit spine for measurement of disc height index. (b) Sequential changes of disc height index after transplantation of synovial MSCs. Average percentages of the value are shown with standard deviations. **P < 0.01 between the degeneration group and the normal group or the MSC group (n = 10 at each time point) by two-factor ANOVA and Turkey-Kramer post-hoc test. (c) Disc height index at two weeks after transplantation of bone marrow or synovial MSCs. Average percentages of values with standard deviations. **P < 0.01 between the bone marrow or synovial MSC group and the degeneration group (n = 6 for each group). (d) Representative T2-weighted MR images of intervertebral discs at 2 to 24 weeks after operation.
Figure 4
Figure 4
Intradiscally injected MSCs maintain microstructure of nucleus pulpous. (a) Macroscopic views of the sagittal section of intervertebral discs at two weeks after operation. (b) Sagittal sections with Hematoxylin-Eosin (HE) staining after operation. (c) Higher magnification of the framed area with type II collagen immunostaining.
Figure 5
Figure 5
Synovial MSCs affect gene profile of nucleus pulposus cells in co-culture system. (a) Morphology of mono-culture of rat nucleus pulposus cells and human synovial MSCs at seven days, and co-culture of rat nucleus pulposus cells with human synovial MSCs at one and seven days. (b) Human gene profile of human synovial MSCs in mono-culture and in co-culture with rat nucleus pulposus cells. (c) Rat gene profile of rat nucleus pulposus cells in mono-culture and in co-culture with human MSCs. (d) Number of altered rat genes seven days after co-culture of rat nucleus pulposus cells with human synovial cells by duplicate of microarray analyses.
Figure 6
Figure 6
Possible mechanism of prevention for intervertebral disc degeneration by intradiscal transplantation of synovial MSCs. After aspiration of the nucleus pulposus, intervertebral disc space rapidly decreases. Synovial MSCs injected into the nucleus pulposus space promote synthesis of type II collagen for the remaining nucleus pulposus cells. Also, synovial MSCs affected the remaining nucleus pulposus cells by inhibiting expressions of degradative enzymes and inflammatory cytokines, resulting in maintaining the structure of the vertebral disc.

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