Elsevier

The Spine Journal

Volume 8, Issue 5, September–October 2008, Pages 804-809
The Spine Journal

Clinical Study
In vitro interaction between muscle-derived stem cells and nucleus pulposus cells

https://doi.org/10.1016/j.spinee.2007.07.394Get rights and content

Abstract

Background context

Current therapies for intervertebral disc degeneration (IDD) are aimed at treating the clinical symptoms arising from IDD rather than directly treating the underlying problem. Pathophysiology of IDD is characterized by a progressive decrease in proteoglycan content and cell density in the nucleus pulposus (NP). A cell-based therapy is a promising concept that uses various cell types to repopulate the disc in an attempt to slow, stop, or reverse the progressive loss of proteoglycans. Stem cells appear to be an excellent candidate for this purpose, based on their ability to differentiate into various connective tissue lineages. The muscle tissue could serve as a good source of adult stem cells because of its vast abundance through out the human body.

Purpose

To examine the interaction between the nucleus pulposus cells (NPCs) and the muscle-derived stem cells (MDSCs) in vitro.

Study design

NPCs and MDSCs were cocultured and proteoglycan production and cell proliferation were evaluated.

Methods

Various ratios of human NPCs were cocultured for 2 weeks with murine MDSCs (transduced with retro/LacZ) in a monolayer culture. Each well contained an admixture of cells with NPC-to-MDSC ratios of 0:100, 25:75, 50:50, 75:25, 100:0. Glycosaminoglycan (GAG) content (1,9 dimethylmethylene blue [DMMB]), newly synthesized proteoglycan (35S incorporation), and DNA content were measured, and cultures were stained with 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-Gal) for cell counting.

Results

The NPC-to-MDSC ratio of 75:25 resulted in a significant increase in GAG content compared with NPCs alone. All coculture ratios showed increase in GAG content in comparison with MDSC culture alone. In addition, cocultures showed a significant increase in 35S incorporation normalized to DNA content in comparison with MDSC alone.

Conclusions

The data from this study shows a synergistic effect between MDSCs and NPCs resulting in an upregulated proteoglycan synthesis and NPCs proliferation in vitro.

Introduction

Medical conditions associated with intervertebral disc degeneration (IDD), such as low back pain, disc herniation, and spinal stenosis, are the leading source of disability in people under 45 years of age and result in national economic losses of over 90 billion dollars per year in United States [1]. Despite its clinical importance, treatment options for IDD remain limited, with unpredictable outcomes. In fact, current therapies are aimed at treating the clinical symptoms arising from IDD rather then targeting the pathophysiology involved in the degenerative process.

Although the etiology and pathophysiology of IDD are largely unknown, current evidence indicates that disc degeneration can begin in the nucleus pulposus (NP) with progressive decrease in proteoglycan content leading to dehydration [2], [3]. Furthermore, there is a progressive decrease in the intervertebral disc (IVD) cell density, which starts with the loss of the notochordal cells at a young age in humans [4], [5]. Therefore, the loss of disc cell number is thought to be an important contributor to the pathogenesis of IDD. The ability to replace or augment the cellular constituents of the IVD may have a positive effect to inhibit, halt, or reverse the degenerative process.

Tissue engineering based on cell therapy is one of the most promising new approaches to repair various tissues, including the IVD. This process involves the use of various cell types that have the potential to repair IVD by acting as progenitor cells. Variety of cells available for the use in IVD tissue engineering ranges from undifferentiated pluripotent stem cells [6], [7], [8] to well-differentiated nucleus pulposus cells (NPCs) [9], [10]. Autologous NPCs are a natural and logical choice for IVD repair applications. However, a limited donor-site capacity to provide a large quantity of cells is a major impediment to autologous NPCs transplantation. Furthermore, cells isolated from a degenerated IVD will most likely lack the ability to regenerate the tissue. Therefore, there are ongoing efforts to identify other cell populations that contain chondroprogenitor cells that can be easily isolated and expanded.

In light of its availability and the relative ease of cell isolation, skeletal muscle is an attractive source of progenitor cells for use in IVD tissue engineering applications [11]. Adachi et al. recently reported comparable healing of cartilage defects treated with collagen gel containing either muscle-derived cells or chondrocytes [12], which suggests that skeletal muscle may contain cells that can aid in cartilage repair. Recently, a specific population of highly purified muscle-derived cells using the preplating technique [13] has been characterized to be pluripotent, having the ability to differentiate into various lineages (myogenic, chondrogenic, hematopoietic, osteogenic, endothelial, and neuronal) [14], [15], [16], [17], [18]. Moreover, muscle-derived stem cells (MDSCs) have been shown to be highly chondrogenic when stimulated with bone morphogenetic protein-4 [16].

Based on these studies, we designed this study to determine whether MDSCs could serve as a source of progenitor cells for biological treatment of IDD. The objective of the study was to investigate the change in extracellular matrix (ECM) production by NPCs and MDSCs in an in vitro coculture system.

Section snippets

Human NPCs isolation

Human IVD cells were isolated from three patients during elective surgical procedures performed in the lumbar spine for disc degeneration by one surgeon. Tissue specimens were placed in a sterile saline solution and transferred to the laboratory for isolation. Disc tissues were washed before isolation of NPCs [19]. Any dense annulus tissues or cartilaginous end plate tissues were carefully removed. The specimens were minced with scissors and digested for 90minutes at 37°C under gentle agitation

Proteoglycans synthesis

Coculturing of NPC-to-MDSC at the ratio of 75:25 resulted in a significant increase in total GAG as compared with NPCs alone. In comparison with MDSC alone, all cocultures showed a significant increase in GAG production (Fig. 1). The mean and standard deviations of GAG content were 17±3.71 mg/ml at the NPC-to-MDSC ratios of 75:25, 15.01±5.06 mg/ml at the 50:50 ratio, and 13.24±4.08 mg/ml at the 25:75 ratio, whereas the GAG content of the NPCs and MDSCs alone were 9.57±5.52 mg/ml, 3.7±3.3 mg/ml,

Discussion

Several studies have shown that MDSCs can differentiate into various lineages, including that of muscle, cartilage, blood, bone, blood vessels, and nerve [14], [15], [16], [17], [18]. Muscle tissue could serve as a good source of adult stem cells because of its abundance and the low morbidity of the harvesting site. In the current study, we have described the interaction between MDSCs and disc cells, highlighting the possibility of using MDSCs as a source for cell-mediated treatment of IDD. The

Conclusions

Our data suggests that there was a synergistic effect between MDSCs and NPCs resulting in an upregulation of ECM production and NPCs proliferation in vitro in a coculture system.

References (25)

  • M.F. Pittenger et al.

    Multilineage potential of adult human mesenchymal stem cells

    Science

    (1999)
  • H.E. Gruber et al.

    Autologous intervertebral disc cell implantation: a model using Psammomys obesus, the sand rat

    Spine

    (2002)
  • Cited by (0)

    FDA device/drug status: not applicable.

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