At 4 weeks, osseous defects in all groups were filled with granulation tissue and newly formed woven bone that was labelled diffusely by tetracycline and calcein (Fig. 2). Calcium sulphate was not recognized at 4 weeks. Collapsing of the membranes into the osseous defects was not observed. In addition, no sign of tissue reaction to the membrane was observed in any membrane group. However, the internal portion of PLGA membrane was degraded, and cell infiltration linked to the resorption of the collagen membrane was observed (Fig. 3). No signs of root resorption or cementum-like structure on the resected root surface were found.
At 8 weeks in all groups, the new cancellous bone that had formed in the osseous defects was mature. In some specimens, the defects were closed by regenerated cortical bone that was clearly labelled by tetracycline and calcein (Fig. 4a). A meagre amount of bone was formed and a small number of inflammatory cells infiltrated adjacent to the root-end filling. Cementum-like structure was deposited on the resected root surface in some specimens except on the filling material. The PLGA membrane was more degraded than in the 4-week group. Sparse residues of the collagen membrane were observed.
Figure 2. Representative sections of group A at 4 weeks after the surgery.
(a) The osseous defect was being filled with newly formed woven bone. Large arrows, resected surfaces of original cortical bone; Small arrows, resected root surface; Arrowheads, e-PTFE membrane. ( 2.5, Toluidine blue stain) (b, c) High magnifications of newly formed woven bone under a fluorescence microscope. Woven bone was labelled by tetracycline and calcein. Asterisks in (b), labelling lines by tetracycline; arrowheads in (c), labelling lines by calcein. (x10)
Figure 3. Representative section of group C at 4 weeks after the surgery. Arrowheads, sparse residues of collagen membrane; arrows, cell infiltration into the collagen membrane (x20, Toluidine blue stain).
Figure 4. Newly formed cortical bone under a fluorescence microscope. (a) Eight weeks specimen of group A. Arrowheads, e-PTFE membrane. (x5) (b) Sixteen weeks specimen of group D. Calcium sulphate had been resorbed. (x5)
At 16 weeks, newly formed cortical bone had closed the defect in the cortical plate in all groups (Fig. 5). Fluorescent lines, which labelled new cortical bone, were reduced compared with those at 8 weeks (Fig. 4b). Deposition of the cementum-like structure on the resected root surface was observed in almost all samples, and in some cases, fibre bundles were inserted into the newly formed cementum-like structure and bone. However, no such deposition was observed on the root-end filling.
Calcium sulphate had disappeared completely at 16 weeks. However, remnants of the collagen membrane were scattered. Traces of PLGA membrane were still visible, and collapsing into the bone defect was observed. The regenerated bone was observed in contact with the e-PTFE membrane and remnants of the collagen membrane. However, fibrous tissue between the PLGA membrane and the new cortical bone was thicker compared with the e-PTFE membrane and collagen membrane (Fig. 6).
Although root resorption on the resected surface was observed in some samples of all groups at 8 and 16 weeks, the cementum-like structure was formed in root resorption lacunae in almost all samples. There was no evidence of ankylosis on the resected surfaces during any of the periods in this experiment.
The results of concavity of the new cortical bone and BV/TV are shown in Tables 2 and 3, respectively. The degree of concavity of the new cortical bone was slight in groups A and D, but marked in group B (Fig. 5). There were significant differences between groups A and B ( P < 0.01), and between groups B and D ( P < 0.01) using the one-way anova test (Table 2). BV/TV in group A was significantly higher than in groups B ( P < 0.01), C ( P < 0.05) and E ( P < 0.05) when using the two-way anova test. Also, BV/TV in group D was significantly higher than in groups B ( P < 0.01) and E ( P < 0.05) using the two-way anova test (Table 3).
Table 2. Concavity of new cortical bone at 16 weeks (mm).
Table 3. BV/TV in each group at three observation periods (%).