Identification of chondrocyte proliferation following laser irradiation, thermal injury, and mechanical trauma

Abstract

Background and objective: Cartilage has a limited regenerative capacity, and there are a lack of reliable techniques and methods to stimulate growth of new tissue to treat degenerative diseases and trauma. This study focused on identifying chondrocyte cell proliferation in ex vivo cartilage tissue following heating Nd:YAG laser using whole-mount analysis and flow cytometry, and compared findings with results produced by contact, and water bath heating methods, mechanical injury, and the addition of transforming growth factor-beta (TGF-beta).

Study design/materials and methods: Ex vivo rabbit nasal septal cartilages were either irradiated with an Nd:YAG laser (lambda = 1.32 microm, 2-16 seconds, 6 W/cm(2)), heated by immersion in a warm saline bath, heated by direct contact with a metal rod, or mechanically damaged by scoring with a scalpel or crushing. After treatment, specimens were incubated for 7 or 14 days in growth media containing 10 microM bromodeoxyuridine (BrdU). Additional specimens were cultured with both BrdU and TGF-beta. Both whole-mount BrdU-double-antibody detection techniques and flow cytometry were used to determine the presence of DNA replication as a marker of proliferation.

Result: An annular region of regenerating chondrocytes was identified surrounding the laser irradiation zone in whole-mount tissue specimens, and the diameter of this region increased with irradiation time. Using whole-mount analysis, no evidence of chondrocyte DNA replication was observed in tissues heated using non-laser methods, grown in TGF-beta, or mechanically traumatized. In contrast, flow cytometry identified the presence of BrdU-positive cells in the S-phase of the cell cycle (synthesis of DNA) for all protocols, indicating chondrocyte proliferation. The percentage of cells that are in S-phase increased with irradiation time.

Conclusion: These data provide evidence that laser irradiation, along with other thermal and mechanical treatments, causes a proliferative response in chondrocytes, and this is observed ex vivo in the absence of cellular and humoral repair mechanisms. The advantage of using optical methods to generate heat in cartilage is that microspot injuries could be created in tissue and scanned across surfaces in clinical applications.