Characteristics of newly formed bone during guided bone regeneration: observations by immunohistochemistry and confocal laser scanning microscopy.

The purpose of this study was to investigate the characteristics of new bone formation during guided bone regeneration (GBR) using immunohistochemistry and confocal laser scanning microscopy. e-PTFE membranes were applied to defects created in the tibiae of rats, and some animals were sacrificed 6, 8, or 10 days later. Serial paraffin sections were cut, stained with H-E, and examined to analyze the ratio of new bone formation. Immunohistochemical staining with a monoclonal antibody specific for PCNA was used to evaluate the proliferating activity. In other experimental rats, calcein was injected at 6, 8, and 10 days after the surgery, and the animals were sacrificed 48 hr after injection. Their tibiae were removed, and Villanueva bone staining was performed before observation using confocal laser scanning microscopy to investigate the mineralization of new bones. The bone occupation ratio increased day by day, but the experimental groups had significantly higher ratios than control groups (without membrane) at each of the time periods. However, PCNA positive cells decreased over time in all groups, and there were no significant differences among the groups. Mineralization occurred more rapidly in the experimental groups than in the control groups. These results suggest that GBR accelerates the migration of osteogenic cells, the formation of new bone, and mineralization in the defect created by the e-PTFE membrane.