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Ascorbic Acid Alters Collagen Integrins in Bone Culture¹

Department of Orthopaedics, University of Connecticut Health Center, Farmington, Connecticut 06032

Address all correspondence and requests for reprints to: Gloria Gronowicz, Department of Orthopaedics MC 1110, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: gronowicz{at}NSO1.uchc.edu

The effects of ascorbic acid on collagen synthesis, mineralization, and integrins were investigated in a mineralizing organ culture system derived from 20-day fetal rat parietal bones. A significant dose-dependent decrease in calcification at 96 h was demonstrated with decreasing concentrations of ascorbic acid (100–0 µg/ml). No effect on DNA content, [³H]thymidine incorporation, or dry weight was found in control (100 µg/ml ascorbic acid) bones compared with bones treated with decreased ascorbic acid concentrations (10, 1, and 0 µg/ml). Collagen synthesis, measured by [³H]proline incorporation, and 1(I) procollagen messenger RNA levels were also unaffected. However, ascorbic acid produced a dose-dependent decrease in the hydroxyproline content, with a maximal 76.8% decrease in bones without ascorbic acid compared with the control bones with 100 µg/ml ascorbic acid. Light microscopy of the ascorbic acid-deficient bones revealed a disruption of the osteoblast layer with misshapen osteoblasts and a decrease in the osteoid seam. The loss of osteoblast organization was also confirmed by analyzing the integrins for collagen by Northern and Western blot and immunofluorescence microscopy. A dose-dependent decrease in ₂ and ß₁ integrin messenger RNA levels and in ₁, ₂, and ß₁ protein were found in 96-h bone cultures deficient in ascorbic acid. These integrin subunits mediate the binding of osteoblasts to collagen. Immunofluorescence microscopy also demonstrated a dose-dependent decrease in ₂ and ß₁ staining of the osteoblast layer. However, the protein levels of ₃ and ₅ subunits were not affected. No ß₅ was detected, whereas only bones cultured without ascorbic acid demonstrated a small decrease in _v and ß₃ protein levels. The ₃, ₅, _v, and ß₃ subunits are involved in cell binding to extracellular matrix proteins other than collagen. Thus, the integrins for collagen are down-regulated, probably in response to the underhydroxylated collagen fibrils, which causes a disruption of osteoblast organization leading to a decrease in mineralization of bone. Integrin assays for specific extracellular proteins may be useful tools in detecting matrix defects in various metabolic bone diseases.