Download variable expression of neural adhesion molecule (cd56)

VARIABLE EXPRESSION OF NEURAL ADHESION MOLECULE (CD56) IN INTERVERTEBRAL DISCS OF
CHONDRODYSTROPHOID CANINES: ASSOCIATION WITH CLUSTER FORMING NP CELLS
*Tamura,F; +* Sakai, D; * Serigano,K; *Mochida, J
*Dept. of Orthopaedic Surgical Science and Center for Regenerative Medicine, Tokai University School of Medicine, Bohseidai, Kanagwa, Japan,
259-1193, Tel 81463931121, Fax 81463964404, [email protected]
INTRODUCTION:
Despite the significant impairment associated with degenerative disc
disease, a clear understanding of its pathogenesis is still lacking. The
Figure 1
nucleus pulposus (NP) of the intervertebral discs (IVDs) contain a mixed
(a) Caudal NP
(b) Lumbar NP
population of cell types at various stages of differentiation. The NP is
formed either by or with the help of cells from the embryonic notochord,
which appear to be replaced during development by a population of
chondrocyte-like cells. Studies that explore this cellular heterogeneity
are important in determining treatment options and cell types for disc
repair. Recently, neural adhesion molecule (CD56) gene, a marker for
neuron has been reported as one candidate for NP cell marker [1]. CD56
has also been reported as a marker for superior selectivity of
mesenchymal stem cells [2]. In order to understand the relevance of
CD56 in the IVDs, the present study explores the variability of CD56
(c) Caudal (Safranin-O)
(d) Lumbar (Safranin-O)
expression in the IVDs of chondrodystrophoid canines.
METHODS:
Lumbar and caudal IVDs were collected from six mature beagles (age
about 1.5 year). Histological analysis was performed using
Hematoxylin-Eosin(H-E), and Safranin-O stain. Immunohistochemistry
was performed for expression of filamentous actin (F-actin) by
fluorescently tagged phalloidin, for gap junction by connexin-43 and
CD56. In order to assess the difference in matrix production ability
between lumbar and caudal NP cells, NP cells were isolated by
enzymatic digestion and expanded in monolayer culture in standard
(e)Lumbar
(f)Caudal
(g)
Dulbecco’s modified Eagle’s medium (DMEM)/F-12 supplemented with
10% foetal bovine serum, penicillin, and streptomycin. At passage 1,
IVD cells were seeded into alginate beads at a cell density of 106 cells
per milliliter. Proteoglycan (PG) and DNA content were assessed using
DMMB assay and DAPI after 14 days. Influence on cell cycle was
25μm
25μm
50μm
assessed in CD56 (+) NP cells by flow-cytometric BrdU/7AAD assay.
Statistical differences were analyzed by ANOVA using the Fisher’s
PLSD as a post hoc test.
Figure 2 Caudal (CD56)
Figure 3
RESULTS:
Histological sections indicated clusters of large cells in caudal NP,
containing from 3 to over 30 cells. Some of these caudal NP cells
contained vacuole-like inclusions and these clusters stained intensely
with Safranin-O. In contrast, lumbar NP contained relatively few cells,
usually alone or in small clusters (3-8 cells in two-dimensional section).
These cells generally resembled chondrocytes, with smaller diameters
(~15μm) and no inclusions (Fig.1 a-d). F-actin was largely absent in
lumbar IVD but was more prevalent in NP cell clusters of caudal IVD
(Fig.1 e-f). Connexin-43 was scattered over the part of caudal NP cell
Figure 4 (CD56 positivity)
clusters, with a concentration in the vicinity of cell-cell junction (Fig.1
g). CD56 (+) cell was scarce in chondrocyte-like cells but was more
prevalent in caudal NP cell clusters (Fig. 2). In vitro cell culture study
showed significant increase of PG production in caudal NP cells
containing CD 56 (+) cells (lumbar; 0.668±0.212, caudal; 1.30±0.769)
(Fig. 3). Percentage of CD 56(+) NP cells was significantly higher in
cells in S phase compared to other phases in cell cycle analysis (Fig. 4).
DISCUSSION:
Chondrodystrophoid canines are among the few species that possess
chondrocyte-like cells in the lumbar IVDs. Hunter et al. has reported that
NP cell clusters interconnected with F-actin and expressing connexin-43
REFERENCES:
may be a feature of notochordal cell population [3]. We identified these
[1] Sakai D, et al. Differential phenotype of intervertebral disc cells:
NP cell clusters in caudal IVDs of chondrodystrophoid canines but
microarray and immunohistochemical analysis of canine nucleus
significantly less in lumbar IVDs. CD56 a marker identified as highly
pulposus and anulus fibrosus. Spine 2009;34:1448-56.
expressed in caudal IVDs in chondrodystrophoid canines in microarray
[2] Battula VL, et al. Isolation of functionally distinct mesenchymal
analysis was also located to be positive in caudal NP cell clusters. The
stem cell subsets using antibodies against CD56, CD271, and
caudal disc cells which contain more CD56(+) cells and NP cell clusters
mesenchymal stem cell antigen-1. Haematologica 2009;94:173–184.
showed higher PG production ability with high proliferative activity
[3] Hunter CJ, et al. Cytomorphology of notochordal and chondrocytic
shown by the cell cycle analysis. Regarding the fact that there have been
cells from the nucleus pulposus: a species comparison. J Anat.
reports suggesting that notochordal cell population activates other NP
2004;205:357-62.
cells [4], our findings support the fact that presence of NP cell clusters
[4] Erwin MW et al. Erwin, et al: Notochord cells regulate intervertebral
may be important for biological properties of NP cells. Furthermore,
disc chondrocyte proteoglycan production and cell proliferation. Spine
with regard to CD56, a marker for cells with high potential [2], CD56
2006;31:1094-9.
may serve as a useful marker for determining active NP cells that is
associated with proliferating cell clusters.
Poster No. 1434 • 56th Annual Meeting of the Orthopaedic Research Society