Biology of Bone, Cartilage and Connective Tissue Disease [1–2]

Background: Corticosteroid injections are commonly used in the treatment of both acute and chronic tendonopathies. Hydrocortisone is one such corticosteroid that is injected into tendon and other soft tissues to reduce inflammation and pain. Currently little is know about the effect of steroid intervention on the cellular characteristics of the treated tissue. This study aimed to observe the effects of different doses of hydrocortisone on primary cultures of avian tenocyte. Methods: Cell cultures were produced from tendon dissected from the lower limb of white Leghorn chicken embryos at 12–13 days post fertilization. Isolated tendon was prepared in cell culture before being incubated under normal conditions. 0.25 µg, 0.5 µg, 1.0 µg, 2.0 µg and 4.0 µg hydrocortisone acetate were added to separate tenocyte culture dishes. Cell morphology and growth was observed at 3, 5 and 7 days of incubation. Additional experiments looked at high-density micromass cell culture to test the effect of increased cell confluence when 1.0 µg of hydrocortisone was added. Alcian blue / neutral red was used indicate for any evidence of chondrogenesis and immunohistological analysis was performed to identify cellular phenotype. Results: Hydrocortisone treated tenocyte cultures showed obvious morphological differences when compared with untreated controls. In all cultures (including control) there was no difference in morphology noted at 3 days regardless of dose. Following 7 days of incubation, low doses of hydrocortisone (0.25 µg, 0.5 µg and 1.0 µg) showed large cellular aggregation and organization forming multiple nodules. Cultures treated with higher levels of hydrocortisone (2.0 µg and 4.0 µg) demonstrated different morphology when compared with the control and samples treated with lower doses of steroid. Micromass cultures showed numerous organized nodules in comparison with the control which showed no evidence of such morphology. Staining with alcian blue / neutral red showed no evidence of chondrogenesis in the nodules seen. Conclusions: Results suggest that hydrocortisone has an increasing effect on the cells as the culture becomes more established and may be due to rising cell confluence. Organized nodules were seen in culture treated with low doses of steroid (in particular 1.0 µg). This type of morphology was significantly more evident in the micromass cell culture suggesting that high cell density is important for this pattern of development. Alcian blue staining sugges