J Korean Soc Spine Surg.  2007 Mar;14(1):1-7. 10.4184/jkss.2007.14.1.1.

Notochordal Cells Induce Chemotaxis of Cartilage-Endplate Chondrocytes in In Vitro Motility Assays

• Kim KW
• Lee JS
• Ha KY
• Rhyu KW
• Nam SW
• Woo YK

• KMID: 1527137
• DOI: http://doi.org/10.4184/jkss.2007.14.1.1

Abstract

STUDY DESIGN: In vitro motility assays were carried out using rat intervertebral discs (IVDs).
OBJECTIVES
To demonstrate the motile properties of the cartilage endplate (CE) chondrocytes and the effect of notochordal cells on this property. LITERATURE REVIEW: Although previous in vivo studies have provided evidence for the migration of CE chondrocyte from hyaline CEs into the notochordal nucleus pulposus (NP), it is unclear if CE chondrocytes of the IVD actually have motile properties. In addition, the effect of notochordal cells on these properties has not been reported.
MATERIALS AND METHODS
Notochordal cells and CE chondrocytes were harvested from three-month-old male Wistar rats and cultured separately. The motility was assayed in quadruplicate using a 48-well microchemotaxis chamber and a gelatin-coated 8-micrometer polycarbonate membrane filter. The control medium (serum-free culture medium), notochordal cells (4x, 2x, 1x and 0.5x10(6)) and concentrated conditioned medium (10-, 50-fold) where notochordal cells were cultured were loaded into the wells of the lower chamber, and CE chondrocytes were added to the wells of the upper chamber. At the end of the assays, the CE chondrocytes that migrated to the bottom side of the membrane filter were stained, counted, and compared.
RESULTS
Compared with the control medium, the notochordal cells (N = 4x, 2x, 1x and 0.5x10(6)) and concentrated conditioned medium (10- and 50-fold) significantly increased the chemotactic motility of the CE chondrocytes in a number- and concentration-dependent manner (p<0.05).
CONCLUSION
The CE chondrocytes of the intervertebral disc are motile, and soluble factors produced by notochordal cells induce the chemotaxis of CE chondrocytes.

Keyword

Chemotaxis; Notochordal cell; Chondrocyte; Cartilage endplate; Intervertebral disc

MeSH Terms

Animals
Cartilage
Chemotaxis*
Chondrocytes*
Culture Media, Conditioned
Humans
Hyalin
Intervertebral Disc
Male
Membranes
Notochord*
Rats
Rats, Wistar
Culture Media, Conditioned

Figure

• 

  Fig. 1. Schematic drawings for chemotaxis assays. Test materials (notochordal cells and concentrated conditioned medium) or control medium (α -MEM supplemented with 0.1% bovine serum albumin) is loaded in the lower chamber and cartilage endplate chondrocytes, in the upper chamber. The membrane filter separates the upper and lower chambers.

• 

  Fig. 2. The effect of notochordal cells on the motility of cartilage endplate (CE) chondrocytes. (A), After chemotaxis assays, the membrane filter was stained with Diff-Quik reagents. On visual inspection, darker staining indicates more cell migration. (B-E), The numbers of notochordal cells loaded in the lower chamber were: (B) 4×106, (C) 2×106, (D) 1×106, and (E) 0.5×106. (F), For comparison, control medium (CM, α -MEM supplemented with 0.1% bovine serum albumin) was loaded in the lower chamber. The small round dots are the membrane pores. CE chondrocytes that migrated through the pores and attached to the bottom of the membrane are stained purple; deep purple indicates the nucleus of the CE chondrocyte (×400). The results of this experiment are summarized in Table 1.

• 

  Fig. 3. The effect of soluble factors produced by notochordal cells on the chemotactic motility of cartilage endplate chondrocytes. (A) visual inspection and (B-D) microscopic examinations (×400). Materials loaded in the lower chamber are (B) control medium (CM, α -MEM supplemented with 0.1% bovine serum albumin), (C) 50-fold concentrated conditioned medium and (D) 10-fold concentrated conditioned medium. The results of this experiment are summarized in Table 2. CCM indicates concentrated conditioned medium.

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