Advanced glycation of type I collagen and fibronectin modifies periodontal cell behavior.

BACKGROUND: Advanced glycation end products (AGEs) have been linked to pathogenic mechanisms of diabetes mellitus. However, little is known about the contribution of protein glycation to periodontal disease in patients with diabetes. Therefore, this study investigated whether glycation of type I collagen (COLI) and fibronectin (FN) modified the behavior of human gingival fibroblasts (hGFs) and periodontal ligament fibroblasts (hPDLs). METHODS: Procedures for rapid in vitro glycation of COLI and FN used methylglyoxal (MG). Formation of AGEs was analyzed by changes in protein migration using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with antibodies specific for MG-glycated proteins. Experiments then characterized the effects of glycated FN and COLI on the behavior of hGFs and hPDLs. RESULTS: MG glycated COLI and FN in <6 hours. Confirming the specificity of the reactions, antibodies specific for MG-induced AGEs reacted with glycated FN and COLI but not with control proteins. In cell culture experiments, glycated FN was significantly less efficient in supporting the attachment of hGFs and hPDLs (P <0.05). Moreover, the morphologic parameters, including length, area, perimeter, and shape factor, were altered (P <0.001) for cells on both glycated proteins. Finally, cell migration was reduced on glycated FN and COLI (P <0.001). CONCLUSIONS: MG treatment efficiently glycated COLI and FN, providing a new tool to study the effects of diabetes on periodontal disease. The substantial effects of glycated COLI and FN on hGF and hPDL behavior indicated that protein glycation contributed to the pathogenesis and altered periodontal wound healing observed in patients with diabetes.

Fibronectin fragmentation is a feature of periodontal disease sites and diabetic foot and leg wounds and modifies cell behavior.

BACKGROUND: Fibronectin (FN) undergoes fragmentation in periodontal disease sites and in poorly healing diabetic wounds. The biologic effects of FN fragments on wound healing remain unresolved. This study characterized the pattern of FN fragmentation and its effects on cellular behavior compared to intact FN. METHODS: Polyclonal antibodies were raised against FN and three defined recombinant segments of FN and used to analyze gingival crevicular fluid from periodontal disease sites in systemically healthy subjects and in subjects with diabetes, as well as chronic leg and foot wound exudates from subjects with diabetes. Subsequently, the behavior of human gingival fibroblasts (hGFs) and HT1080 reference cells were analyzed by measuring cell attachment, migration, and chemotaxis in the presence of intact FN or recombinant FN fragments. RESULTS: FN fragmentation was evident in fluids from periodontal disease sites and diabetic leg and foot wounds. However, no fragmentation pattern distinguished systemically healthy subjects from subjects with diabetes. hGFs and HT1080 cells required significantly higher concentrations of FN fragments to achieve attachment comparable to intact FN. Cells cultured on FN fragments also were morphologically different from cells cultured on full-length FN. Migration was reduced for hGFs cultured on FN fragments relative to full-length FN. In contrast, FN fragments increased HT1080 fibrosarcoma cell migration over intact FN. CONCLUSIONS: FN fragmentation is a prominent feature of periodontal and chronic leg and foot wounds in diabetes. Furthermore, cell culture assays confirmed the hypothesis that exposure to defined FN fragments significantly alters cell behavior.