Effects of Estrogen on Chondrocyte Proliferation and Collagen Synthesis in Skeletally Mature Articular Cartilage

Purpose

Estrogen has been shown to have a modulating effect on cartilage thickness. This investigation was performed to determine the effects of estrogen supplementation on cartilage thickness, cellular proliferation, and type II and X collagen production in skeletally mature rat cartilage, both in an organ culture and cell culture system.

Materials and Methods

Mandibular condyles were harvested from 8-week-old female Sprague Dawley rats and placed into tissue culture plates containing culture media with or without 17β-estradiol supplementation. Organ cultures were labeled with 5-bromo-2′-deoxyuridine on culture day 2 or 4 to determine the effects of estrogen supplementation on the cellular mitotic index. Histomorphometric analysis of the organ culture sections was used to determine the thickness (μm) of the various cartilage zones, as well as the total cartilage thickness following estrogen exposure. Type X collagen was immunohistochemically identified in the ECM of hypertrophic chondrocytes using a rabbit anti-rat collagen type X antibody raised against the NCl domain. The reaction was visualized with an avidin-biotin peroxidase detection system (Vector Laboratories, Burlingame, CA). In a separate experiment, articulating cartilage chondrocytes were harvested by collagenase digestion and cultured at 5 × 10⁵ cells per 35 mm tissue culture plate. Second subculture chondrocytes were divided into 2 groups: controls and [10⁻⁸ M] 17β-estradiol (E₂−10⁻⁸ M) and grown to confluence. The cell cultures were used to establish growth curves for each group using cell counts at 2-day intervals.

Results

In the organ culture experiment, 17β-estradiol–treated condyles had a significant decrease in total cartilage thickness after 4 days in culture (P < .05). Estrogen supplementation resulted in a significant reduction in the mitotic index as early as culture day 2 (P < .05). Type X collagen deposition into the extracellular matrix was visibly increased in the hypertrophic chondrocyte zone for the estrogen-supplemented group on experimental days 2 and 4 compared with the control group. In the cell culture system, 17β-estradiol [10⁻⁸ M] decreased chondrocyte proliferation during logarithmic growth (P < .05) and at confluence (P < .05).

Conclusion

These data show that estrogen decreased cartilage thickness by inhibition of chondrocyte proliferation and increased chondrocyte maturation. These observed effects showed the potential role of estrogen in the modulation of skeletally mature cartilage.