Beta irradiation decreases collagen type II synthesis and increases nitric oxide production and cell death in articular chondrocytes

Abstract

Background: When synovitis is proved, intra-articularly injected beta emitting radionuclides like yttrium-90 ((90)Y) are used to treat the inflamed synovium.

Objective: To study the viability, matrix production, and NO production during or after (90)Y treatment of chondrocytes.

Methods: Monolayer, alginate, and explant cultures of primary bovine articular chondrocytes as well as synoviocytes were incubated with 0-3 MBq (90)Y/ml medium for four days from culture day 3 onwards. Cell viability was demonstrated by light and electron microscopy or by trypan blue or ethidium bromide/fluorescein diacetate staining, membrane integrity by measurement of lactate dehydrogenase (LDH) activity in the culture supernatants. Biosynthetic activity was demonstrated by incorporation of [(3)H]proline and immunocytochemical staining of collagen type II. NO production was measured with the Griess reagent.

Results: In chondrocyte and synoviocyte monolayer cultures radiation caused a dose dependent increase in cell death and membrane destruction within four days. In alginate and explant cultures, where proliferation is low, no significantly increased LDH activity was seen, and cell viability was approximately 100% for up to 14 days after irradiation. Collagen type II expression (alginate) and biosynthetic activity (alginate and explants) were decreased dose dependently while there was an increase in NO production. Light and electron microscopy data showed that five weeks after irradiation all cells in alginate and most cells in explants subjected to 3 MBq (90)Y/ml were dead, whereas after lower amounts of irradiation several morphologically intact cells were found.

Conclusions: beta Irradiation may influence the long term maintenance of cartilage tissue or the aetiology of degenerative joint diseases.

Figure 1

Semi-thin sections (A–D) and electron microscopy (E, F) of cartilage explants (A, B) and primary chondrocytes in alginate (C–F) five weeks after a four day period of ß irradiation indicating an induction of cell destruction. (A, C and E) Control cultures. Arrows demonstrate intact cells in A and C or intact organelles like the nucleus (large arrow) and the golgi apparatus and mitochondria (small arrows) in E. (B, D, and F) 3 MBq ⁹⁰Y/ml. Arrows demonstrate damaged cells in B and D. In F remnants of organelles of a dead cell are visible. (A–D) bar=27 µm, (E, F) bar=0.2 µm.

Figure 2

[³H]Proline incorporation in chondrocyte alginate cultures and cartilage explants two weeks after a four day period of ⁹⁰Y irradiation. The bars show the dose dependent decrease of [³H]proline incorporation (p<0.004 by one way ANOVA) related to non-irradiated control cultures. Mean values (SD); n=6 (explants n=3); *p<0.05 v control by two tailed Student's t test.

Figure 3

Immunocytochemical staining of collagen type II on cultures of chondrocytes in alginate beads two weeks after a four day period of ⁹⁰Y irradiation. The figure shows the dose dependent decrease of collagen type II expression (brown staining; arrows demonstrate cells) compared with (A) the non-irradiated control; (B) 0.375 MBq; (C) 1 MBq ⁹⁰Y. Bar=15.38 µm

Figure 4

NO accumulation in supernatants of seven day old chondrocyte alginate and explant cultures, human synovial fluid (synovia) samples, and cell-free culture medium during four days of ⁹⁰Y irradiation. The bars show the dose dependent increase of NO accumulation in the different systems. Mean values (SD); n=6 (explants n=3); *p<0.001 v control by two tailed Student's t test; p<0.001 by one way ANOVA.