Figure 5. Hydroxymethylene diphosphonate (HMDP)-deformable nanoscale vesicles (DNV) topical application normalized tooth extraction wound healing of zoledronate (ZOL)-pretreated mice.

(A) Histological evaluation depicted BRONJ lesion at the tooth extraction site of the untreated ZOL-injected mice (No Tx) exhibiting that the abnormal epithelial hyperplasia (E. Hyp) extending to the necrotic alveolar bone (Nec) appeared to facilitate the sustained open wound, the uneven bone regeneration in the tooth extraction sockets (Soc) and localized infiltration of inflammatory cells (Inf) on the alveolar bone surface. The abnormal wound healing pattern was also observed at 4 weeks after tooth extraction with fistula formation by epithelial hyperplasia reaching to the necrotic bone and a localized pustule lesion (Pus). The unwounded side of ZOL-injected mice did not show any abnormality of remaining tooth (Tooth), alveolar bone (Bone) and overlining gingival tissue (Gingiva). After the HMDP-DNV topical treatment, gingival wound was found closed with more diffused inflammation (Inf) and a sign of bone resorption (white arrowheads) was depicted on the surface of alveola bone. The tooth extraction socket (Soc) showed bone regeneration at week-2, which was further remodeled and matured at week-4. (B) High magnification histology of 2 weeks after tooth extraction demonstrated the dense inflammatory cell infiltration (Inf) in the gingival connective tissue and osteonecrosis area (Nec) in untreated ZOL-injected mice (No Tx). The HMDP-DNV treatment attenuated the inflammatory cell infiltration and increased signs of osteoclastic bone resorption (white arrowheads). At 4 weeks after tooth extraction, the untreated mice continued to show dense inflammatory cell infiltration and osteonecrosis. However, mice with the HMDP-DNV treatment showed subsided inflammation and the minimized osteonecrosis area likely due to bone resorption, which appeared to result in alveolar bone loss (dotted white line and arrow) as compared to the regenerated bone in the tooth extraction socket (Soc). (C) Cathepsin K (Ctsk) immune-stained osteoclasts on the alveolar bone surface of the untreated ZOL-injected mice were observed not only at the proximal area (Zone A) of the tooth extraction socket (Soc) but also under the palatine neuro-vascular complex (Zone B) with inflammation (Inf). Characteristically, these Ctsk-positive osteoclasts were small and flattened (black arrowheads). In some specimens, Ctsk-positive cells were observed away from the bone surface (small arrowheads). The HMDP-DNV-treated mice showed large Ctsk-positive osteoclasts in deep bone lacunae adjacent to the tooth extraction socket (Zone A). (D) Total number of osteoclasts defined as Ctsk-positive multi-nuclear cells on the alveolar bone surface. (Figure 5—source data 1) (E) Osteoclasts in Zone A and Zone B were separately counted. (Figure 5—source data 2) In (D) and (E), the graphs show the mean and SD (n=5 per group), and the Turkey test was used to analyze multiple samples. The statistical significance was determined to be at p<0.05. Different letters (e.g., a, b) are used to show statistically significant differences between multiple groups.