Inhibiting PHD2 in human periodontal ligament cells via lentiviral vector-mediated RNA interference facilitates cell osteogenic differentiation and periodontal repair.

Periodontal defect regeneration in severe periodontitis remains a challenging task in clinic owing to poor survival of seed cells caused by the remaining oxidative stress microenvironment. Recently, the reduction of prolyl hydroxylase domain-containing protein 2 (PHD2), a primary cellular oxygen sensor, has shown an incredible extensive effect on skeletal muscle tissue regeneration by improving cell resistance to reactive oxygen species, whereas its role in periodontal defect repair is unclear. Here, through lentivirus vector-mediated RNA interference, the PHD2 gene in human periodontal ligament cells (hPDLCs) is silenced, leading to hypoxia-inducible factor-1α stabilization in normoxia. In vitro, PHD2 silencing not only exhibited a satisfactory effect on cell proliferation, but also induced distinguished osteogenic differentiation of hPDLCs. Real-time polymerase chain reaction and Western blotting revealed significant up-regulation of osteocalcin, alkaline phosphatase (ALP), runt-related transcription factor 2, and collagen type I (COL I). Under oxidative stress conditions, COL I and ALP expression levels, suppressed by 100 µM H2 O2 , were elevated by PHD2-gene-silencing in hPDLCs. In vivo, periodontal fenestration defects were established in 18 female Sprague-Dawley rats aged 6 wk old, followed by implantation of PHD2 silencing hPDLCs in situ for 21 d. Persistent and stable silencing of PHD2 in hPDLCs promoted better new bone formation according to microcomputed tomography 3D reconstruction and related bone parameter analysis. This work demonstrates the therapeutic efficiency of PHD2 gene interference in osteogenic differentiation and periodontal defect repair for highly efficient periodontal regeneration.

Non-surgical Periodontal Treatment Restored the Gut Microbiota and Intestinal Barrier in Apolipoprotein E-/- Mice With Periodontitis.

Periodontitis has been associated with a variety of systematic diseases via affecting gut microbiota. However, the influence of periodontal treatment on intestinal microbiota is not known. Hyperlipidemia can significantly alter gut microbiota structure. It is proposed that the presence of hyperlipidemia can influence the impact of periodontitis on microbiota. This study was conducted to explore the influence of periodontitis and periodontal treatment on the gut microbiota on the basis of hyperlipidemia. Apolipoprotein E-/-(ApoE-/-) mice were ligatured to induced periodontitis and non-surgical periodontal treatment was performed for half of them after 4 weeks of ligation. Microbiota communities in the feces collected at 4, 5, 8 weeks after ligation were investigated using next-generation sequencing of 16S rDNA. Bone loss at periodontitis sites were analyzed using micro-computed tomography (Micro-CT). Morphology and mucosal architecture injury of ileum tissue were observed with hematoxylin-eosin staining. The serum lipid levels were assayed. The results showed that ß-diversity index in experimental periodontitis group was differed significantly from that of the control group. Significant differences were found in ß-diversity between the non-surgical periodontal treatment group and the ligation group. The samples of the non-surgical periodontal treatment group and the control group were clustered together 4 weeks after periodontal treatment. Intestinal villus height and ratio of villus height to crypt depth was found decreased after ligation and restored after non-surgical periodontal treatment. Non-surgical periodontal treatment induced the colonization and prosper of butyrate-producing bacteria Eubacterium, which was absent/not present in the ligation group. We confirmed that periodontitis led to gut microbiota dysbiosis in mice with hyperlipidemia. Non-surgical periodontal treatment had the trend to normalize the gut microbiota and improved the intestinal mucosal barrier impaired by periodontitis in apoE-/- mice.

Chloroquine and 3-Methyladenine Attenuates Periodontal Inflammation and Bone Loss in Experimental Periodontitis.

Periodontitis is an inflammation characterized by alveolar bone resorption caused by imbalance in bone homeostasis. It is known that autophagy is related to inflammation and bone metabolism. However, whether autophagy inhibitors could be used for periodontitis in animal models remains unknown. We investigated the role of two classical autophagy inhibitors, 3-methyladenine (3-MA) and chloroquine (CQ), on the development of rat experimental periodontitis in terms of the bone loss (micro-CT), the number of inflammatory cells (hematoxylin and eosin staining), and the osteoclastic activity (tartrate-resistant acid phosphatase staining). Expression of autophagy-related genes and nuclear factor kappa B p65 (NF-κB p65) were assessed by immunohistochemistry. Expression of Beclin-1 and microtubule-associated proteins 1A/1B light chain 3 (LC3) were analyzed by Western blot. To further observe the effect of autophagy inhibitors on osteoclasts (OCs) in vitro, bone marrow-derived mononuclear macrophages were used. Together, these findings indicated that topical administration of 3-MA or CQ reduced the infiltration of inflammatory cells and alveolar bone resorption in experimental periodontitis. Furthermore, 3-MA and CQ may attenuate activation of OCs by autophagy. Therefore, 3MA and CQ may have prophylactic and therapeutic potential for inflammation and alveolar bone resorption in periodontitis in the future.