Abstract
Intervertebral disc (IVD) disorder and age-related degeneration are believed to contribute to low back pain. Cell-based therapies represent a promising strategy to treat disc degeneration; however, the cellular and molecular characteristics of disc cells during IVD maturation and aging still remain poorly defined. This study investigated novel molecular markers and their age-related changes in the rat IVD. Affymetrix cDNA microarray analysis was conducted to identify a new set of genes characterizing immature nucleus pulposus (NP) cells. Among these markers, select neuronal-related proteins (Basp1, Ncdn and Nrp-1), transcriptional factor (Brachyury T), and cell surface receptors (CD24, CD90, CD155 and CD221) were confirmed by real-time PCR and immunohistochemical (IHC) staining for differential expression between IVD tissue regions and among various ages (1, 12 and 21 months). NP cells generally possessed higher levels of mRNA or protein expression for all aforementioned markers, with the exception of CD90 in anulus fibrosus (AF) cells. In addition, CD protein (CD24 and CD90) and Brachyury (T) expression in immature disc cells were also confirmed via flow cytometry. Similar to IHC staining, results revealed a higher percentage of immature NP cells expressing CD24 and Brachyury, while higher percentage of immature AF cells was stained positively for CD90. Altogether, this study identifies that tissue-specific gene expression and age-related differential expression of the above markers do exist in immature and aged disc cells. These age-related phenotype changes provide a new insight for a molecular profile that may be used to characterize NP cells for developing cell-based regenerative therapy for IVD regeneration.
Highlights
The human intervertebral disc (IVD), a heterogeneous soft tissue that lies in the space between adjacent vertebral bodies, provides flexibility and load support in the spine [1]
Our current study discovered and confirmed a new set of nucleus pulposus (NP)-markers (Basp1, Ncdn, Nrp-1, CD24, CD155, CD221 and Brachyury T) and one non-NP marker (CD90) through a combination of tools including cDNA microarray, realtime RT-PCR, immuno-histochemical staining and flow cytometry analysis
A novel finding is that the neuronal-associated proteins, Basp-1, Ncdn and Nrp-1 more highly express in immature rat NP, suggesting their relationship to the notochordal-like characteristics of immature NP cells
Summary
The human intervertebral disc (IVD), a heterogeneous soft tissue that lies in the space between adjacent vertebral bodies, provides flexibility and load support in the spine [1]. Significant cell-mediated tissue remodeling occurs in the IVD as a consequence of aging, marked by an increasingly fibrotic nucleus pulposus (NP), disoriented lamellae in the annulus fibrosus (AF), and calcified vertebral endplates [2]. These age-related changes may lead to IVD degenerative disorders, such as internal disc disruption, AF tears, and ‘‘herniated’’ or ‘‘extruded’’ NP [3]. These anatomic features can be associated with symptoms of low back pain, neurological deficits, and disability that affect 30% of the US population annually [4,5]. Several therapeutic advances have been demonstrated in animal models [6,7], a more thorough understanding of molecular phenotype changes in the NP cell population during aging will surely catalyze the development of cell-based therapies for IVD regeneration
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