Methods: First, RAGE expression was assayed by immunohistochemistry with goat anti-RAGE and non-immune antibodies in 6 µm cryosections made from calvarial bone defects of non-diabetic and diabetic animals harvested after 10 days of healing. Second, N-e-(carboxymethyl)lysine-modified (CML-MSA) or unmodified mouse serum albumin (MSA) were applied to standardized 1.0 mm defects in non-diabetic animals via surgically implanted 100 ml Alzet osmotic micropumps to directly assess for a role of glycated proteins in inhibiting bone formation in the absence of systemic diabetes. Two doses (0.85 and 0.085 mg) of both substances were applied continuously throughout the 14 day healing period (n=8). Animals were then sacrificed and healing defects were prepared for quantitative histomorphometric evaluations.
Results: Quantitative analyses of RAGE immunohistochemistry studies revealed that the number of specific positive-stained cells was up-regulated by three-fold in the periosteum, dura mater, and in the granulation tissue filling the calvarial bone defects of diabetic animals compared to non-diabetic animals (p<0.01).
Analyses of healing defects in non-diabetic animals revealed that bone defects receiving MSA healed almost completely after 14 days of healing. By contrast, 0.85 and 0.085 mg CML-MSA application inhibited the area of healed bone healing 61% and 43%, respectively (p<0.05).
Conclusions: RAGE is up-regulated in calvaria defects in diabetic animals at locations known to participate in bone formation. Glycated proteins inhibit calvarial bone healing in non-diabetic animals. Data suggest that AGE-RAGE interactions may contribute to inhibited bone healing in diabetes.