Longitudinal observation of cementum regeneration through multiple fluorescent labeling

The assessment of new attachment after periodontal treatment has been the focus of continuous research. An approach to longitudinally examine the deposition of cementum was devised by using fluorescence microscopy (FL), contact microradiography (CMR), and toluidine blue staining (TBS) after the injection of three labeling agents known to be incorporated within newly mineralized tissues with different tones: tetracycline, calcein, and alizarin complexion. Three adult Japanese monkeys (male, 6.0 to 8.3 Kg weight) were used for this experiment. Bone defects were surgically created in 24 mandibular sites and a copper plate was inserted for a period of 4 weeks to promote microbial colonization to form periodontal pockets. Scaling and root planing (baseline) were then performed, and the fluorescent agents were administered twice weekly leaving a 1 week interval between the different agents. The mandibular specimens were fixed in neutralized formalin and embedded in polyester resin. Undecalcified sections were prepared 3, 6, and 9 weeks after baseline. Cementum regeneration was confirmed in 18 out of 24 sites; in 6 samples only epithelial proliferation was observed. Regeneration could be seen as early as 2 weeks after debridement. Cementum was identified by observation under FL of a labeled structure, discrimination in the degree of mineralization of dentin by CMR, and by the presence of functional collagen fibers and location of the epithelial border by TBS. In this study the use of three different labeling agents using the three observation techniques was shown to be effective for the longitudinal assessment of cementum regeneration.