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Differential Phenotype of Intervertebral Disc Cells: Microarray and Immunohistochemical Analysis of Canine Nucleus Pulposus and Anulus Fibrosus

Sakai, Daisuke, MD, PhD*; Nakai, Tomoko, BSc*; Mochida, Joji, MD, PhD*; Alini, Mauro, PhD; Grad, Sibylle, PhD

doi: 10.1097/BRS.0b013e3181a55705
Basic Science
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Study Design. Microarray gene expression profiling, quantitative gene expression analysis, and immunohistochemistry was used to investigate molecular variations between nucleus pulposus (NP) and anulus fibrosus (AF) of the dog intervertebral disc (IVD).

Objective. To identify specific molecules with differing expression patterns in NP and AF and compare their profile with articular cartilage (AC).

Summary of Background Data. Although experimental and animal studies have demonstrated the potential of cell based approaches for NP regeneration, there is still a deficiency of basic knowledge about the phenotype of IVD cells.

Methods. Comparative microarray analysis of beagle lumbar NP and AF was performed. Molecules of interest were evaluated by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, comparing lumbar and coccygeal NP and AF and AC. To assess interspecies variations, genes that had been found differentially expressed in rat tissues were also investigated.

Results. Forty-five genes with NP/AF signal log ratio ≥1 were identified. α-2-Macroglobulin, cytokeratin-18, and neural cell adhesion molecule (CD56) mRNA were higher in NP compared to AF and AC, and desmocollin-2 mRNA was higher in NP than AF. The expression profiles were similar in lumbar and coccygeal discs, although certain variations were noticed. Interspecies differences between rat and dog were evident in the expression of several genes. Immunohistochemistry confirmed differences in gene expression at the protein level.

Conclusion. This study reports on the expression of molecules that have not been described previously in IVD, in non-notochordal discs comparable with human. Interspecies differences were noted between rat and dog tissues, whereas variations between caudal and lumbar discs were less prominent. The NP of the beagle as a chondrodystrophoid dog breed is potentially more similar to the human than the NP of species whose discs do not naturally degenerate. Therefore, studies on appropriate species may contribute to a better understanding of the cell types residing in the IVD.

Microarray gene expression profiling, quantitative gene expression analysis, and immunohistochemistry was used to investigate differences between dog nucleus pulposus and anulus fibrosus. Comparisons were performed between lumbar and caudal discs and articular cartilage and between rat and dog. The expression of distinct molecules in non-notochordal chondrodystrophoid discs is documented.

From the *Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan; and †Biomaterials and Tissue Engineering Program, AO Research Institute, Davos, Switzerland.

Acknowledgment date: August 5, 2008. First revision date: October 8, 2008. Acceptance date: January 19, 2009.

The manuscript submitted does not contain information about medical device(s)/drug(s).

No funds were received in support of this work. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.

Address correspondence and reprint requests to Sibylle Grad, PhD, AO Research Institute, Clavadelerstrasse 8, CH 7270 Davos, Switzerland; E-mail: sibylle.grad@aofoundation.org

© 2009 Lippincott Williams & Wilkins, Inc.