*3.3. In Vivo Test (Animal Model)*

There were no severe inflammations and swellings at the flaps in the defect. The periodontal tissues were healthy on the day of sacrifice. The GTR animal model for cementum and bone height were H&E stained and observed under a light microscope (Figure 4).

**Figure 4.** The GTR animal model for cementum and bone height evaluation of (**A**) empty control group and (**B**) representative experimental group. NB: New bone, T: Tooth, AB: Alveolar bone, CE: Cementum, CEJ: Cementoenamel junction. White scale bar noted as 1 mm. Time of implantation: 8 weeks.

The histological results of various 8-week ES PLA95/β−TCP GTR membranes were shown in Figure 5. The histometric evaluation were carried out and listed in Table 2 and Figure 6. The cementum height of the test and control membranes was significantly different between the empty defect after 8 and 16 weeks (*p* < 0.05). The results of bone height showed difference only at 16 weeks (*p* > 0.05) with respective test and control values of 2.67 (±0.33) mm and 2.58 (±0.15) mm; these values were significantly different from the corresponding value of the empty group at 16 weeks (*p* < 0.05). The ES PLA95/β-TCP membrane was effective to block the migration of fast-growing connective tissue into the defect area and in creating some space for the regeneration of new tissues.

In a typical GTR animal study, the usage of bone grafts usually interferes with the efficacy of the GTR membrane [34,35]. Therefore, we intentionally adopted the similar procedure but without bone grafts to confirm the blocking function of the proposed GTR membrane in this study. The results, however, still showed significant differences between the experimental and empty control groups. In addition, few previous studies have shown more bone formation after GTR as compared to that in the empty control without membrane [36,37]. In this study, the cementum height of the experimental group was larger than that of the empty control group without bone grafts at eight weeks. From these results, we conjectured that the addition of bone graft would affect the growth of new bone tissues. The ES PLA95/β-TCP membrane and Epi-Guide® groups were all with similar PLA materials and mechanical properties, with a limited amount of β-TCP content for buffering properties. Therefore, the osteoconductive effect on tissue growth was not significantly different from ES PLA95/β-TCP membrane and Epi-Guide® groups.

**Figure 5.** The 8-week H&E stain of various ES PLA95/β-TCP GTR barriers (H&E Stain, 40X) T: Tooth, B: Cementum, N: New bone, PDL: Periodontal ligament.



\*,#: Statistical significant differences when *p* < 0.05. Values are reported as mean (SD) (*N* = 3).

**Figure 6.** The cementum and bone height of ES PLA95/β-TCP and commercial membranes were measured in LanYu pigs after 8 and 16 weeks. \*Differences were considered statistically significant when *p* < 0.05.
