3D-printed TCP-HA scaffolds delivering MicroRNA-302a-3p improve bone regeneration in a mouse calvarial model.

OBJECTIVE: To demonstrate hydroxyapatite nanoparticles modified with cationic functional molecules. 3-aminopropyltriethoxysilane (HA-NPs-APTES) carrying microRNA-302a-3p (miR) in the 3D-printed tricalcium phosphate/Hydroxyapatite (TCP/HA) scaffold can increase healing of the critical-sized bone defect. MATERIALS AND METHODS: 3D-printed TCP/HA were modified with HA-NPs-APTES by two methods (M1, M2). The dispersion of particles was visualized by fluorescent microscopy. Biocompatibility of the scaffolds was tested by alizarin assay. Delivery of miR to the cells and osteogenic gene expression were evaluated by qPCR. After selecting best method (M2), scaffolds, scaffolds+HA-NPs-APTES with or without miR were implanted in 4 mm mouse calvarium defect (n = 4 per group). After 2,4 and 6 weeks, bone regeneration were evaluated by microCT and histology sections. RESULTS: Both M1 and M2 scaffolds were biocompatible with cell adhesion on its surface. M2 scaffold showed significant increase of miR, suggesting successful delivery, resulted in downregulation of its target mRNA COUP-TFII, and upregulation of RUNX2 mRNA. Calvarium defect with M2 scaffold also showed significantly higher BV/TV and higher number of filled spaces at all time points. Histomorphometry demonstrated new bone formed at the center of the HA-NPs-APTES-miR scaffold earlier than controls. CONCLUSION: TCP/HA scaffold modified with HA-NPs-APTES facilitated delivery of miR and enhanced bone regeneration.

Digital fabrication of customized intraoral appliances for head and neck radiotherapy.

Introduction: The radiotherapy received by head and neck cancer patients commonly has adverse effects on oral tissue and the muscles of mastication. This short communication describes the digital fabrication of intraoral appliances for radiotherapy and muscle exercises. Methods: Three patients diagnosed with tongue squamous carcinoma were treatment-planned for radiotherapy using different radiation techniques. The patients were referred for oral scanning and digital bite records, and the appliance was collaboratively designed by a radiation oncologist, dentist, and laboratory technician. The appliance covered the occlusal surface of the remaining teeth with a 1-mm engagement. The lingual plate was 2-mm below the occlusal plane, and extended 4-mm distally, and the jaws were opened by 20-mm. The appliances were printed overnight using a rigid and biocompatible 3D printing material. Results: Requiring minimal chair-time, the appliance was easily inserted and adjusted to comfortably fit in the mouth. The patients were trained to insert it themselves. The tongue was at a pre-determined position during daily radiotherapy, and the healthy tissues were separated from the radiation field. The patients had mild adverse effects on their oral mucosa. Additionally, the appliances were used for muscle exercises after the radiation courses to prevent trismus. Conclusions: The interprofessional collaboration to fabricate customized intraoral appliances using digital workflow to maximize patients' benefits is feasible. Clinical significance: The use of intraoral appliances is potentially increased when the fabrication process is facilitated. Using an intraoral appliance precisely targets the tumor are for better treatment outcomes, and the healthy adjacent tissues will be preserved to maintain the patient's quality of life.

Trehalose versus carboxymethylcellulose oral spray for relieving radiation-induced xerostomia in head and neck cancer patients: a randomized controlled trial.

BACKGROUND: The aim of this study was to investigate the effect of trehalose oral spray to relieve radiation-induced xerostomia on a randomized controlled trial (RCT). METHODS: Prior to RCT, the effect of trehalose (5-20%) on the epithelial growth of fetal mouse salivary gland (SG) explants was evaluated to confirm if 10% trehalose exerted the best epithelial outcomes. Participants who completed radiotherapy for head and neck cancer (HNC) treatment were enrolled in a double-blind RCT, according to inclusion and exclusion criteria as per the CONSORT statement. The experimental group (n = 35) received 10% trehalose spray, while the control group (n = 35) received carboxymethylcellulose (CMC) spray to apply intra-orally 4 times/day for 14 days. Salivary pH and unstimulated salivary flow rate were recorded pre- and post-interventions. The Xerostomia-related Quality of Life scale (XeQoLs) was filled, and scores assessed post-interventions. RESULTS: In the SG explant model, pro-acinar epithelial growth and mitosis was supported by 10% topical trehalose. As for RCT outcomes, salivary pH and unstimulated salivary flow rate were significantly improved after use of 10% trehalose spray when compared to CMC (p < 0.05). Participants reported an improvement of XeQoLs dimension scores after using trehalose or CMC oral sprays in terms of physical, pain/discomfort, and psychological dimensions (p < 0.05), but not social (p > 0.05). When comparing between CMC and trehalose sprays, XeQoLs total scores were not statistically different (p > 0.05). CONCLUSIONS: The 10% trehalose spray improved salivary pH, unstimulated salivary flow rate, and the quality-of-life dimensions linked with physical, pain/discomfort, and psychological signs. The clinical efficacy of 10% trehalose spray was equivalent with CMC-based saliva substitutes for relieving radiation-induced xerostomia; therefore, trehalose may be suggested in alternative to CMC-based oral spray.(Thai Clinical Trials Registry; https://www.thaiclinicaltrials.org/ TCTR20190817004).

Delivery of microRNA-302a-3p by APTES modified hydroxyapatite nanoparticles to promote osteogenic differentiation in vitro.

OBJECTIVE: To demonstrate the miRNA delivery by hydroxyapatite nanoparticles modified with APTES (HA-NPs-APTES) and promote osteogenic gene expression. MATERIALS AND METHODS: Osteosarcoma cells (HOS, MG-63) and primary human mandibular osteoblasts (HmOBs) were co-cultured with HA-NPs-APTES conjugated with miRNA-302a-3p. Resazurin reduction assay was performed to evaluate HA-NPs-APTES biocompatibility. Intracellular uptake was demonstrated by confocal fluorescent and scanning electron microscopy. The miRNA-302a-3p and its mRNA targets expression levels including COUP-TFII and other osteogenic genes were assessed by qPCR on day1 or day5 post-delivery. Calcium deposition induced by the osteogenic gene upregulation was shown by alizarin red staining on day7 and 14 post-delivery. RESULTS: Proliferation of HOS cells treated with HA-NPs-APTES was similar to that of untreated cells. HA-NPs-APTES was visualized in cell cytoplasm within 24 hours. MiRNA-302a-3p level was upregulated in HOS, MG-63 and HmOBs as compared to untreated cells. As a result, COUP-TFII mRNA expression was reduced, followed by an increase of RUNX2 and other osteogenic genes mRNA expression. Calcium deposition induced by HA-NPs-APTES-miR-302a-3p in HmOBs was significantly higher than in untreated cells. CONCLUSION: HA-NPs-APTES may support the delivery of miRNA-302a-3p into bone cells, as assessed by osteogenic gene expression and differentiation improvement once this combination is used on osteoblast cultures.

Host Responses in Peri-implant Tissue in Comparison to Periodontal Tissue: A Retrospective Study.

PURPOSE: To investigate inflammatory responses in peri-implant crevicular fluid (PICF) in comparison to periodontal tissue. MATERIALS AND METHODS: Nineteen participants with healthy implants restored with titanium or gold-casting abutment were included. PICF and gingival crevicular fluid (GCF) were collected for inflammatory cytokine detection by ELISA. Cytokine levels in PICF or GCF of the same individual were compared using the paired t-test, and those from titanium or gold-casting (UCLA) abutment were compared using the independent t-test. Human gingival fibroblast responses to PICF and GCF were then evaluated with one-way ANOVA. RESULTS: The results demonstrated that IL-6, IL-8, TNFα, and IFNγ expressed in PICF are similar to GCF in the same individual. However, IL-1ß (p = 0.032) and IL-1α (p = 0.030) was statistically significantly higher in PICF than in GCF. IL-8 level was statistically significantly higher with gold-casting than with titanium abutments (p = 0.003). PICF statistically significantly stimulated higher expression of RANKL, IL-1ß, IL-6, and IL-8 mRNA in human gingival fibroblasts (HGF), while focal adhesion kinase (FAK) suppressed mRNA. CONCLUSION: The inflammatory cytokines, including IL-1α and IL-1ß, are higher in healthy peri-implant tissues. Abutment materials may also influence the level of inflammatory cytokines in PICF. Inflammatory mediators in crevicular fluid may affect HGF inflammatory responses and peri-implant tissue integration.

Epigallocatechin-3-Gallate Protects Pro-Acinar Epithelia Against Salivary Gland Radiation Injury.

Antioxidant agents are promising pharmaceuticals to prevent salivary gland (SG) epithelial injury from radiotherapy and their associated irreversible dry mouth symptoms. Epigallocatechin-3-gallate (EGCG) is a well-known antioxidant that can exert growth or inhibitory biological effects in normal or pathological tissues leading to disease prevention. The effects of EGCG in the various SG epithelial compartments are poorly understood during homeostasis and upon radiation (IR) injury. This study aims to: (1) determine whether EGCG can support epithelial proliferation during homeostasis; and (2) investigate what epithelial cells are protected by EGCG from IR injury. Ex vivo mouse SG were treated with EGCG from 7.5-30 µg/mL for up to 72 h. Next, SG epithelial branching morphogenesis was evaluated by bright-field microscopy, immunofluorescence, and gene expression arrays. To establish IR injury models, linear accelerator (LINAC) technologies were utilized, and radiation doses optimized. EGCG epithelial effects in these injury models were assessed using light, confocal and electron microscopy, the Griess assay, immunohistochemistry, and gene arrays. SG pretreated with EGCG 7.5 µg/mL promoted epithelial proliferation and the development of pro-acinar buds and ducts in regular homeostasis. Furthermore, EGCG increased the populations of epithelial progenitors in buds and ducts and pro-acinar cells, most probably due to its observed antioxidant activity after IR injury, which prevented epithelial apoptosis. Future studies will assess the potential for nanocarriers to increase the oral bioavailability of EGCG.

miR-302a-3p regulates RANKL expression in human mandibular osteoblast-like cells.

Receptor activator of nuclear factor kappa-B ligand (RANKL) is important substance during osteoclastogenesis that resulted in alveolar bone loss of periodontitis. MicroRNAs (miRNAs) regulate gene expression in several biological processes including osteoclastogenesis. We investigated the function of microRNA-302a-3p (miR-302a-3p) to regulate receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human mandibular osteoblast-like cells (HMOBs). HMOBs were incubated with prostaglandin E2 (PGE2 ) to mimic inflammation, or with PGE2 and interferon gamma (IFNγ) to mimic homeostasis. MicroRNA (miRNA) profiles related to RANKL expression were demonstrated by PCR array, and miR-302a-3p was identified. Using TargetScanHuman 7.0, a target of miR-302a-3p was predicted. To confirm its function, miR-302a-3p was overexpressed, or silenced, by transfection with miR-302a-3p mimic, or inhibitor, respectively. Level of miR-302a-3p and RANKL mRNA was assessed by qRT-PCR. Soluble RANKL (sRANKL), and membrane-bound RANKL (mRANKL) were measured by ELISA and by Western blot, respectively. When PGE2 stimulated RANKL in HMOBs, miR-302a-3p was lower than baseline level. However, upregulation of miR-302a-3p is observed when IFNγ suppressed RANKL expression in PGE2 -stimulated HMOBs. miR-302a-3p was predicted to target PRKACB mRNA encoding the catalytic subunit in cAMP/PKA pathway. Overexpression of miR-302a-3p could decrease RANKL expression during PGE2 stimulation. In contrast, silencing of miR-302a-3p by its inhibitor increased RANKL expression in PGE2 -IFNγ conditioned HMOBs. miR-302a-3p regulates RANKL expression in HMOBs within PGE2 -IFNγ regulatory network.

Epithelial Cells Secrete Interferon-γ Which Suppresses Expression of Receptor Activator of Nuclear Factor Kappa-B Ligand in Human Mandibular Osteoblast-Like Cells.

BACKGROUND: Prostaglandin (PG)E2 accumulates in inflamed periodontal tissue and induces receptor activator of nuclear factor kappa-B ligand (RANKL)-RANK-osteoprotegerin (OPG) signaling associated with bone resorption. Although oral epithelial cells maintain tissue homeostasis, the role of these cells in RANKL regulation remains unknown. METHODS: To mimic an inflamed condition, RANKL upregulation in human mandibular osteoblast-like cells (HMOBs) were stimulated with PGE2. Effect of recombinant human interferon (IFN)-γ or epithelial-derived IFN-γ in constitutively released or Porphyromonas gingivalis lipopolysaccharide (PgLPS)-stimulated epithelial supernatant was investigated in HMOBs. Some HMOBs were pretreated with an anti-IFN-γ antibody before PGE2 stimulation. THP-1 human monocytes and HMOBs were cocultured in a transwell system to investigate RANKL-driven THP-1 osteoclastic activity. RESULTS: PGE2 significantly increased RANKL messenger RNA (mRNA) and protein in HMOBs in a dose-dependent manner, while OPG protein remained similar to baseline. Epithelial cells constitutively released IFN-γ, which was substantially increased by PgLPS. HMOBs treated with epithelial supernatant or recombinant IFN-γ, concurrently with PGE2 stimulation, reduced RANKL, but not OPG, expression. In contrast, anti-IFN-γ antibody reversed the effect of epithelial mediators on RANKL expression. When cocultured with THP-1, RANKL released by PGE2-stimulated HMOBs is adequate to drive THP-1 differentiation as osteoclastogenic gene expression and bone resorption pit are increased. However, recombinant IFN-γ, or IFN-γ derived from oral epithelial cells, suppressed RANKL expression at both the mRNA and protein level, resulting in decreased THP-1-derived osteoclastic activity. CONCLUSION: Oral epithelial cells interact with HMOBs by releasing IFN-γ to regulate RANKL expression and contribute to osteoclastogenesis.

The role of microRNA in periodontal tissue: A review of the literature.

MicroRNAs (miRNAs) bind at the 3'UTR of their target mRNA to induce gene silencing. Through this mechanism, number of biological pathways implicated in developmental, physiological, and pathological processes, have been frequently found to involve miRNA functions. MiRNA functions in bone metabolism have also been reported, especially in association with receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis. Expression of RANKL has been related to several inflammatory mediators, and thus some miRNAs may be implicated in the regulatory mechanism of inflammatory-induced RANKL expression as shown in periodontal resident cells such as gingival fibroblasts or periodontal ligament cells. This review aims to review the current miRNA research relating periodontal tissue and its relevance in periodontal inflammation. In miRNA profiling studies of tissues isolated from individuals with periodontal disease, miR-223 has been consistently identified as a potential candidate miRNA to be further investigated in periodontitis-related processes. Although these studies point to an important role of miRNA-mediated epigenetic changes in tissue inflammation and alveolar bone loss, further investigation is still required to determine the function of miRNAs in the complex processes of periodontal tissue homeostasis and pathogenesis. Knowledge gained from future studies will be beneficial in developing alternative therapeutic approaches, especially ones that use miRNA delivery systems to treat periodontal disease.

Periimplant bacteria associated with different transmucosal designs or smoking habits.

PURPOSE: This study is to compare periimplant microbiota associated with implant transmucosal designs or smoking habits. METHODS: Submucosal samples from healthy 52 implants were collected for analysis of bacteria associated with bone-level (n = 37) or tissue-level (n = 15) implants or smoking habits, using quantitative polymerase chain reaction. Profiles of periimplant bacteria of smokers (n = 5) were investigated using PhyloChip Array version G3 and compared with nonsmokers (n = 5). RESULTS: The number of bone-level implants positive for at least 1 pathogen was higher than that of tissue level; however, differences in each bacterium were insignificant. The prevalence and abundance of Treponema denticola in smokers were significantly higher than that in nonsmokers (P < 0.05). Smokers and nonsmokers exhibited similar periimplant microbiota based on the PhyloChip Array, but they could be distinguished by limiting observations to only 18 operational taxonomic units. Streptococcus macedonicus within Firmicutes and Prevotella within Bacteroidetes were more abundant in smokers compared with nonsmokers. CONCLUSION: Prevalence of putative pathogens with bone-level implants was higher than tissue-level implants in nonsmokers. Firmicutes and Bacteroidetes were significantly higher in smokers. Smoking therefore strongly influenced peri-implant bacterial composition of bone-level implant.

PCR detection of Scardovia wiggsiae in combination with Streptococcus mutans for early childhood caries-risk prediction.

The microbial factor is an important determinant in caries risk assessment. This study aimed to use detection, by PCR, of Scardovia wiggsiae, in combination with Streptococcus mutans, for the accurate prediction of caries risk in children. Detection of Lactobacillus, as a caries-specific species, was also performed. Dental plaque, as well as infected dentine when available, was collected from children who were caries-free (n = 30) or diagnosed with early childhood caries (n = 30), and the prevalence and abundance of S. wiggsiae and S. mutans were estimated using quantitative PCR. Lactobacillus was amplified by Lactobacillus genus-specific primers and then sequenced. Both S. wiggsiae and S. mutans were concurrently detected in 19 children diagnosed with early childhood caries, but in none of the caries-free children. The positive predictive value was 1 in children with S. wiggsiae- and S. mutans-positive test results, compared with 0.58 when only S. mutans was detected and 0.9 when only S. wiggsiae was detected. The abundance of S. wiggsiae and S. mutans in infected dentine was higher than that in dental plaque from children. Diverse Lactobacillus species were observed in dental plaque but none appeared to be caries-specific. In conclusion, the detection of S. wiggsiae in combination with S. mutans improves the positive predictive value and the specificity of the test.

Altering host resistance to infections through microbial transplantation.

Host resistance to bacterial infections is thought to be dictated by host genetic factors. Infections by the natural murine enteric pathogen Citrobacter rodentium (used as a model of human enteropathogenic and enterohaemorrhagic E. coli infections) vary between mice strains, from mild self-resolving colonization in NIH Swiss mice to lethality in C3H/HeJ mice. However, no clear genetic component had been shown to be responsible for the differences observed with C. rodentium infections. Because the intestinal microbiota is important in regulating resistance to infection, and microbial composition is dependent on host genotype, it was tested whether variations in microbial composition between mouse strains contributed to differences in "host" susceptibility by transferring the microbiota of resistant mice to lethally susceptible mice prior to infection. Successful transfer of the microbiota from resistant to susceptible mice resulted in delayed pathogen colonization and mortality. Delayed mortality was associated with increased IL-22 mediated innate defense including antimicrobial peptides Reg3γ and Reg3ß, and immunono-neutralization of IL-22 abrogated the beneficial effect of microbiota transfer. Conversely, depletion of the native microbiota in resistant mice by antibiotics and transfer of the susceptible mouse microbiota resulted in reduced innate defenses and greater pathology upon infection. This work demonstrates the importance of the microbiota and how it regulates mucosal immunity, providing an important factor in susceptibility to enteric infection. Transfer of resistance through microbial transplantation (bacteriotherapy) provides additional mechanisms to alter "host" resistance, and a novel means to alter enteric infection and to study host-pathogen interactions.

Oral keratinocytes support non-replicative infection and transfer of harbored HIV-1 to permissive cells.

BACKGROUND: Oral keratinocytes on the mucosal surface are frequently exposed to HIV-1 through contact with infected sexual partners or nursing mothers. To determine the plausibility that oral keratinocytes are primary targets of HIV-1, we tested the hypothesis that HIV-1 infects oral keratinocytes in a restricted manner. RESULTS: To study the fate of HIV-1, immortalized oral keratinocytes (OKF6/TERT-2; TERT-2 cells) were characterized for the fate of HIV-specific RNA and DNA. At 6 h post inoculation with X4 or R5-tropic HIV-1, HIV-1gag RNA was detected maximally within TERT-2 cells. Reverse transcriptase activity in TERT-2 cells was confirmed by VSV-G-mediated infection with HIV-NL4-3Deltaenv-EGFP. AZT inhibited EGFP expression in a dose-dependent manner, suggesting that viral replication can be supported if receptors are bypassed. Within 3 h post inoculation, integrated HIV-1 DNA was detected in TERT-2 cell nuclei and persisted after subculture. Multiply spliced and unspliced HIV-1 mRNAs were not detectable up to 72 h post inoculation, suggesting that HIV replication may abort and that infection is non-productive. Within 48 h post inoculation, however, virus harbored by CD4 negative TERT-2 cells trans infected co-cultured peripheral blood mononuclear cells (PBMCs) or MOLT4 cells (CD4+ CCR5+) by direct cell-to-cell transfer or by releasing low levels of infectious virions. Primary tonsil epithelial cells also trans infected HIV-1 to permissive cells in a donor-specific manner. CONCLUSION: Oral keratinocytes appear, therefore, to support stable non-replicative integration, while harboring and transmitting infectious X4- or R5-tropic HIV-1 to permissive cells for up to 48 h.

Porphyromonas gingivalis induces CCR5-dependent transfer of infectious HIV-1 from oral keratinocytes to permissive cells.

BACKGROUND: Systemic infection with HIV occurs infrequently through the oral route. The frequency of occurrence may be increased by concomitant bacterial infection of the oral tissues, since co-infection and inflammation of some cell types increases HIV-1 replication. A putative periodontal pathogen, Porphyromonas gingivalis selectively up-regulates expression of the HIV-1 coreceptor CCR5 on oral keratinocytes. We, therefore, hypothesized that P. gingivalis modulates the outcome of HIV infection in oral epithelial cells. RESULTS: Oral and tonsil epithelial cells were pre-incubated with P. gingivalis, and inoculated with either an X4- or R5-type HIV-1. Between 6 and 48 hours post-inoculation, P. gingivalis selectively increased the infectivity of R5-tropic HIV-1 from oral and tonsil keratinocytes; infectivity of X4-tropic HIV-1 remained unchanged. Oral keratinocytes appeared to harbor infectious HIV-1, with no evidence of productive infection. HIV-1 was harbored at highest levels during the first 6 hours after HIV exposure and decreased to barely detectable levels at 48 hours. HIV did not appear to co-localize with P. gingivalis, which increased selective R5-tropic HIV-1 trans infection from keratinocytes to permissive cells. When CCR5 was selectively blocked, HIV-1 trans infection was reduced. CONCLUSION: P. gingivalis up-regulation of CCR5 increases trans infection of harbored R5-tropic HIV-1 from oral keratinocytes to permissive cells. Oral infections such as periodontitis may, therefore, increase risk for oral infection and dissemination of R5-tropic HIV-1.

An internal polarity landmark is important for externally induced hyphal behaviors in Candida albicans.

Directional growth is a function of polarized cells such as neurites, pollen tubes, and fungal hyphae. Correct orientation of the extending cell tip depends on signaling pathways and effectors that mediate asymmetric responses to specific environmental cues. In the hyphal form of the eukaryotic fungal pathogen Candida albicans, these responses include thigmotropism and galvanotropism (hyphal turning in response to changes in substrate topography and imposed electrical fields, respectively) and penetration into semisolid substrates. During vegetative growth in C. albicans, as in the model yeast Saccharomyces cerevisiae, the Ras-like GTPase Rsr1 mediates internal cellular cues to position new buds in a prespecified pattern on the mother cell cortex. Here, we demonstrate that Rsr1 is also important for hyphal tip orientation in response to the external environmental cues that induce thigmotropic and galvanotropic growth. In addition, Rsr1 is involved in hyphal interactions with epithelial cells in vitro and its deletion diminishes the hyphal invasion of kidney tissue during systemic infection. Thus, Rsr1, an internal polarity landmark in yeast, is also involved in polarized growth responses to asymmetric environmental signals, a paradigm that is different from that described for the homologous protein in S. cerevisiae. Rsr1 may thereby contribute to the pathogenesis of C. albicans infections by influencing hyphal tip responses triggered by interaction with host tissues.