Mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ) revealed by targeted removal of legacy bisphosphonate from jawbone using equilibrium competing inert hydroxymethylene diphosphonate

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) presents as a morbid jawbone lesion in patients exposed to a nitrogen-containing bisphosphonate (N-BP). Although it is rare, BRONJ has caused apprehension among patients and healthcare providers and decreased acceptance of this anti-resorptive drug class to treat osteoporosis and metastatic osteolysis. We report here a novel method to elucidate the pathological mechanism of BRONJ by the selective removal of legacy N-BP from the jawbone using an intra-oral application of hydroxymethylene diphosphonate (HMDP) formulated in deformable nanoscale vesicles (DNV). After maxillary tooth extraction, zoledronate-treated mice developed delayed gingival wound closure, delayed tooth extraction socket healing and increased jawbone osteonecrosis consistent with human BRONJ lesion. Single cell RNA sequencing of mouse gingival cells revealed oral barrier immune dysregulation and unresolved pro-inflammatory reaction. HMDP-DNV topical applications to nascent mouse BRONJ lesions resulted in accelerated gingival wound closure and bone socket healing as well as attenuation of osteonecrosis development. The gingival single cell RNA sequencing demonstrated resolution of chronic inflammation by increased anti-inflammatory signature gene expression of lymphocytes and myeloid-derived suppressor cells. This study suggests that BRONJ pathology was predominantly induced by the oral N-BP and demonstrates the potential of HMDP-DNV as an effective BRONJ therapy.

Influence of Anodized Titanium Surfaces on the Behavior of Gingival Cells in Contact with: A Systematic Review of In Vitro Studies

Electrochemically anodized (EA) surfaces promise enhanced biological properties and may be a solution to ensure a seal between peri-implant soft tissues and dental transmucosal components. However, the interaction between the modified nano-structured surface and the gingival cells needs further investigation. The aim of this systematic review is to analyze the biological response of gingival cells to EA titanium surfaces in in vitro studies with a score-based reliability assessment. A protocol aimed at answering the following focused question was developed: “How does the surface integrity (e.g., topography and chemistry) of EA titanium influence gingival cell response in in vitro studies?”. A search in three computer databases was performed using keywords. A quality assessment of the studies selected was performed using the SciRAP method. A total of 14 articles were selected from the 216 eligible papers. The mean reporting and the mean methodologic quality SciRAP scores were 87.7 ± 7.7/100 and 77.8 ± 7.8/100, respectively. Within the limitation of this review based on in vitro studies, it can be safely speculated that EA surfaces with optimal chemical and morphological characteristics enhance gingival fibroblast response compared to conventional titanium surfaces. When EA is combined with functionalization, it also positively influences gingival epithelial cell behavior.

Effect of nano zinc oxide on proliferation and toxicity of human gingival cells

Background Periodontal dressing is used to cover the gum surface and protect the wound after periodontal surgery. Nanomaterials have been widely applied in dentistry in recent years. Zinc oxide (ZnO) is one of the main components of periodontal dressing. Aim This study aims to explore the toxicity ZnO nanoparticles (ZnO NPs) causes to human gingival fibroblast cells (HGF-1) and its effect on cell proliferation. Methods First, we identified and analyzed HGF-1, including cell morphology, growth curve, and immunohistochemistry staining. Then, we treated HGF-1 with ZnO NP. Cell viability, the integrity of the cell membrane, oxidative damage, and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release assay, fluorescent probe, and flow cytometry. Furthermore, the expression of murine double minute 2 (MDM2) and p53 was determined by quantitative real-time polymerase chain reaction (qPCR) and Western blotting. We finally overexpressed MDM2 in HGF-1 to verify the relationship between MDM2 and cell proliferation. Results Our research indicated ZnO NPs did not affect cell proliferation at low concentrations. However, high-concentration ZnO NP inhibited cell proliferation, destroyed the integrity of cell membranes, and induced oxidative stress and apoptosis. In addition, high concentration of ZnO NPs inhibited the proliferation of HGF-1 by regulating the expression of MDM2 and p53. Conclusion High concentration of ZnO NP caused toxicity to HGF-1 cells and inhibited cell proliferation by regulating MDM2 and p53 expression.

The Biological Effects of Ozone Gas on Soft and Hard Dental Tissues and the Impact on Human Gingival Fibroblasts and Gingival Keratinocytes

Ozone is an allotropic form of oxygen, so in the medical field ozone therapy has special effects. Starting from the premise that bio-oxidative ozone therapy reduces the number of bacteria, in the present study two approaches were proposed: to evaluate the biological effects of ozone gas on the tooth enamel remineralization process and to demonstrate its impact on the morphology and confluence of human primary gingival cells, namely keratinocytes (PGK) and fibroblasts (HGF). The ozone produced by HealOzone was applied in vivo to 68 M1s (first permanent molars), both maxillary and mandibular, on the occlusal surfaces at pit and fissure. The molars included in the study recorded values between 13 and 24 according to the DIAGNOdent Pen 2190 scale, this being the main inclusion/exclusion criterion for the investigated molars. Because the gas can make contact with primary gingival cells during the ozonation process, both human gingival fibroblasts and keratinocytes were exposed to different doses of ozone (20 s, 40 s, 60 s), and its effects were observed with the Olympus IX73 inverted microscope. The contact of ozone with the human primary gingival cells demonstrates cell sensitivity to the action of ozone, this being higher in fibroblasts compared to keratinocytes, but it is not considered toxic because all the changes are reversible at 48 h after exposure.

Oral Mucosa Could Be an Infectious Target of SARS-CoV-2

The World Health Organization reported that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission is caused by respiratory droplets and aerosols from the oral cavity of infected patients. The angiotensin-converting enzyme 2 (ACE2) is considered the host functional protein for SARS-CoV-2 infection. In this article, we first revealed that the positive proportion of ACE2 expression in gingival cells collected from the gingival sulcus was increased to the same level as the tongue. Our data demonstrate that cells in the gingival sulcus may be a new entry point for the SARS-CoV-2 virus via a high expression of ACE2. In addition, we first evaluated the expression of ACE2 in various sites of the oral cavity with noninvasive, convenient liquid-based cytology. The liquid-based cytology evaluation of oral tissue may provide a novel preventive medical avenue against COVID-19.

Regulation of hBD-2, hBD-3, hCAP18/LL37, and Proinflammatory Cytokine Secretion by Human Milk Oligosaccharides in an Organotypic Oral Mucosal Model

Human milk oligosaccharides (HMOs), the third largest solid fraction in human milk, can modulate inflammation through Toll-like receptor signaling, but little is known about their immunomodulatory potential in the oral cavity. In this study, we determined whether the HMOs 2’-fucosyllactose (2’-FL) and 3-fucosyllactose (3-FL) regulate human-beta defensin (hBD)-2 and -3, cathelicidin (hCAP18/LL-37), and cytokine responses in human gingival cells using a three-dimensional oral mucosal culture model. The model was incubated with 0.1% or 1% 2’-FL and 3-FL, alone and in combination, for 5 or 24 h, and hBD-2, hBD-3, and hCAP18/LL-37 were analyzed by immunohistochemistry. The expression profiles of interleukin (IL)-1, IL-1RA, IL-8, and monocyte chemoattractant protein (MCP)-1 were determined by LUMINEX immunoassay. The combination of 1% 2’-FL and 1% 3-FL, and 1% 3-FL alone, for 24 h upregulated hBD-2 protein expression significantly (p < 0.001 and p = 0.016, respectively). No changes in the other antimicrobial peptides or proinflammatory cytokines were observed. Thus, 3-FL, alone and in combination with 2´-FL, stimulates oral mucosal secretion of hBD-2, without effecting a proinflammatory response when studied in an oral mucosal culture model.

Entamoeba gingivalis Exerts Severe Pathogenic Effects on the Oral Mucosa

The protozoan Entamoeba gingivalis colonizes the healthy oral mucosa with a prevalence of 15%. Colonization can be asymptomatic, and it is considered not pathogenic. However, it is able to invade lacerated oral mucosa, where it ingests fragments of live cells, suggesting pathogenous potential. Here, we characterized the transcriptomes of gingival cells after infection with E. gingivalis using RNA sequencing and observed pathogen interaction with the epithelial monolayer barrier by scanning electron microscopy. In epithelial and fibroblast cells, strongest differential expression showed gene set “chemokines and inflammatory molecules in myeloid cells” (area under the curve [AUC] = 0.9, effect size 5.15, adjusted P = 3.1 × 10−19) and “cell cycle and growth arrest” (AUC = 0.91, effect size = 4.56, adjusted P = 4.8 × 10−9), respectively. The most upregulated genes were TNF (fold change 430) and IL8 (fold change 359) in epithelial cells and ZN331 (fold change 18) in fibroblasts. We showed that E. gingivalis killed live epithelial cells by trogocytosis, demonstrating strong pathogenic potential.