Effect of 15 days -6° head-down bed rest on microbial communities of supragingival plaque in young men.

Introduction: The microgravity environment astronauts experience during spaceflight can lead to an increased risk of oral diseases and possible changes in oral microecology. In this study, we aimed to assess changes in the microbial community of supragingival plaques to explore the effects of spaceflight microgravity environment on oral microecology. Methods: Sixteen healthy male volunteers were recruited, and supragingival plaque samples were collected under -6° head-down bed rest (HDBR) at five-time points: day 1 before HDBR; days 5, 10, and 15 of HDBR; and day 6 of recovery. Bacterial genomic DNA was sequenced using gene sequencing technology with 16S ribosomal ribonucleic acid V3-V4 hypervariable region amplification and the obtained data were analyzed bioinformatically. Results: Alpha diversity analysis showed a significant increase in species richness in supragingival plaque samples on day 15 of HDBR compared with that at pre-HDBR. Beta diversity analysis revealed that the community composition differed among the groups. Species distribution showed that, compared with those at pre-HDBR, the relative abundances of Corynebacterium and Aggregatibacter increased significantly during HDBR, while those of Veillonella, Streptococcus, and Lautropia decreased significantly. Moreover, compared with those at pre-HDBR, the relative abundance of Leptotrichia increased significantly on day 6 of recovery, whereas the relative abundances of Porphyromonas and Streptococcus decreased significantly. Network analysis showed that the interaction relationship between the dominant genera became simpler during HDBR, and the positive and negative correlations between them showed dynamic changes. Phylogenetic investigation of communities by reconstruction of unobserved states analysis showed that the amino acid metabolism function of plaque microorganisms was more enriched during HDBR. Discussion: In summary, in a 15-day simulated microgravity environment, the diversity, species distribution, interaction relationship, and metabolic function of the supragingival plaque microbial community changed, which suggests that microgravity may affect the oral microecosystem by changing the balance of supragingival plaque microbial communities and further leading to the occurrence and development of oral diseases.

A preliminary study of the salivary microbiota of young male subjects before, during, and after acute high-altitude exposure.

Background: The microbial community structure in saliva differs at different altitudes. However, the impact of acute high-altitude exposure on the oral microbiota is unclear. This study explored the impact of acute high-altitude exposure on the salivary microbiome to establish a foundation for the future prevention of oral diseases. Methods. Unstimulated whole saliva samples were collected from 12 male subjects at the following three time points: one day before entering high altitude (an altitude of 350 m, pre-altitude group), seven days after arrival at high altitude (an altitude of 4,500 m, altitude group) and seven days after returning to low altitude (an altitude of 350 m, post-altitude group). Thus, a total of 36 saliva samples were obtained. 16S rRNA V3-V4 region amplicon sequencing was used to analyze the diversity and structure of the salivary microbial communities, and a network analysis was employed to investigate the relationships among salivary microorganisms. The function of these microorganisms was predicted with a Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. Results: In total, there were 756 operational taxonomic units (OTUs) identified, with 541, 613, and 615 OTUs identified in the pre-altitude, altitude, and post-altitude groups, respectively. Acute high-altitude exposure decreased the diversity of the salivary microbiome. Prior to acute high-altitude exposure, the microbiome mainly consisted of Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria, and Actinobacteria. After altitude exposure, the relative abundance of Streptococcus and Veillonella increased, and the relative abundance of Prevotella, Porphyromonas, and Alloprevotella decreased. The relationship among the salivary microorganisms was also affected by acute high-altitude exposure. The relative abundance of carbohydrate metabolism gene functions was upregulated, while the relative abundance of coenzyme and vitamin metabolism gene functions was downregulated. Conclusion: Rapid high-altitude exposure decreased the biodiversity of the salivary microbiome, changing the community structure, symbiotic relationships among species, and abundance of functional genes. This suggests that the stress of acute high-altitude exposure influenced the stability of the salivary microbiome.

pH-Responsive Selenium Nanoplatform for Highly Efficient Cancer Starvation Therapy by Atorvastatin Delivery.

Recently, starvation-inducing nutrient deprivation has been regarded as a promising strategy for tumor suppression. As a first-line lipid-lowering drug, atorvastatin (ATV) significantly reduces caloric intake, suggesting its potential in starvation therapy for suppressing tumors. Accordingly, we developed a novel starvation therapy agent (HA-Se-ATV) in this study to suppress tumor growth by using hyaluronic acid (HA)-conjugated chitosan polymer-coated nano-selenium (Se) for loading ATV. HA-Se-ATV targets cancer cells, following which it effectively accumulates in the tumor tissue. The HA-Se-ATV nanoplatform was then activated by inducing a weakly acidic tumor microenvironment and subsequently releasing ATV. ATV and Se synergistically downregulate the levels of cellular adenosine triphosphate while inhibiting the expression of thioredoxin reductase 1. Consequently, the starvation-stress reaction of cancer cells is significantly elevated, leading to cancer cell death. Furthermore, the in vivo results indicate that HA-Se-ATV effectively suppresses tumor growth with a low level of toxicity, demonstrating its great potential for clinical translation.

Short-term head-down bed rest microgravity simulation alters salivary microbiome in young healthy men.

Microgravity influences are prevalent during orbital flight and can adversely affect astronaut physiology. Notably, it may affect the physicochemical properties of saliva and the salivary microbial community. Therefore, this study simulates microgravity in space using a ground-based -6° head-down bed rest (HDBR) test to observe the effects of microgravity on oral salivary secretion function and the salivary microbiome. Sixteen healthy young male volunteers were recruited for the 15-day -6° HDBR test. Non-stimulated whole saliva was collected on day 1 (pre-HDBR), on days 5, 10, and 15 of HDBR, and day 6 of recovery. Salivary pH and salivary flow rate were measured, and the V3-V4 region of the 16S rRNA gene was sequenced and analyzed in 80 saliva samples. The results showed that there were no significant differences in salivary pH, salivary flow rate, and alpha diversity between any two time points. However, beta diversity analysis revealed significant differences between pre-HDBR and the other four time points. After HDBR, the relative abundances of Actinomyces, Parvimonas, Peptostreptococcus, Porphyromonas, Oribacterium, and Capnocytophaga increased significantly, whereas the relative abundances of Neisseria and Haemophilus decreased significantly. However, the relative abundances of Oribacterium and Capnocytophaga did not recover to the pre-HDBR level on day 6 of recovery. Network analysis revealed that the number of relationships between genera decreased, and the positive and negative correlations between genera changed in a complex manner after HDBR and did not reach their original levels on day 6 of recovery. PICRUSt analysis demonstrated that some gene functions of the salivary microbiome also changed after HDBR and remained significantly different from those before HDBR on day 6 of recovery. Collectively, 15 days of -6° HDBR had minimal effect on salivary secretion function but resulted in significant changes in the salivary microbiome, mainly manifested as an increase in oral disease-related bacteria and a decrease in oral health-related commensal bacteria. Further research is required to confirm these oral microbial changes and explore the underlying pathological mechanisms to determine the long-term effects on astronauts embarking on long-duration voyages to outer space.

In vitro biocompatibility of biohybrid polymers membrane evaluated in human gingival fibroblasts.

The biohybrid polymer membrane (BHM) is a new biomaterial designed for the treatment of soft periodontal tissue defects. We aimed to evaluate the in vitro biocompatibility of the membrane in human gingival fibroblasts and the capability to induce cell adhesion, migration, differentiation and improving the production of the extracellular matrix. BHM and Mucograft® collagen matrix (MCM) membranes were punched into 6 mm diameter round discs and placed in 96-well plates. Human primary gingival fibroblasts were seeded on the membranes or tissue culture plastic (TCP) serving as the control. Cell proliferation/viability and morphology were evaluated after 3, 7, and 14 days of culture by cell counting kit (CCK)-8 assay and scanning electron microscopy, respectively. Additionally, the gene expression of transforming growth factor (TGF)-ß1, focal adhesion kinase (FAK), collagen type 1 (Col1), alpha-smooth muscle actin (α-SMA), and fibroblasts growth factor (FGF)-2 was analyzed at 3, 7, and 14 days of culture by qPCR. Cell proliferation on BHM was significantly higher than on MCM and similar to TCP. Gene expression of TGF-ß1, FAK, Col1, and α-SMA were significantly increased on BHM compared to TCP at most investigated time points. However, the gene expression of FGF-2 was significantly decreased on BHM at Day 7 and recovered at Day 14 to the levels similar to TCP. The finding of this study showed that BHM is superior for gingival fibroblasts in terms of adhesion, proliferation, and gene expression, suggesting that this membrane may promote the healing of soft periodontal tissue.

Injectable Adhesive Hydrogel through a Microcapsule Cross-Link for Periodontitis Treatment.

In this work, chitosan (CS) decorated metronidazole (MTZ) microcapsules (CS@MTZ) were synthesized and used as a cross-linker for the preparation of a poly(vinyl alcohol) (PVA) injectable hydrogel by dynamic covalent bonding and ionic interaction through a 4-carboxyphenylboronic acid bridge. The use of MTZ microcapsules efficiently slowed down the release rate of the hydrophilic antibiotic from the hydrogel matrix. Besides, the hydrophobicity of the microcapsules endows the PVA@CS@MTZ hydrogel to be sticky to a substrate in wet conditions, under a suggested mechanism of evicting the water boundary layer on the substrate. The sustained release behavior endowed a prolonged bacteriostasis ability of the hydrogel formulation for up to 14 days in vitro, and the bioadhesive property as well as the injectability of the hydrogel benefited the topical delivery of MTZ in periodontal pockets and exhibited desirable antibacterial capacity in 1 week on the rat periodontitis model.

Effects of low-intensity pulsed electromagnetic fields on bone microarchitecture, mechanical strength and bone turnover in type 2 diabetic db/db mice.

Type 2 diabetic patients have impaired bone quality, leading to increased fracture risk. Substantial evidence demonstrates that pulsed electromagnetic fields (PEMF) could resist osteopenia/osteoporosis induced by estrogen deficiency and disuse. However, the effects of PEMF on osteopenia/osteoporosis associated with diabetes, especially for more prevalent type 2 diabetes, remain poorly understood. We herein investigated the skeletal effects and mechanisms of PEMF (15 Hz, 20 Gs) on leptin receptor-deficient db/db mice with typical type 2 diabetic symptoms. Our µCT results showed that 12-week PEMF exposure significantly improved both cancellous and cortical bone microarchitecture in db/db mice. Three-point bending and biomechanical indentation testing demonstrated that PEMF improved whole-bone structural properties and tissue-level material properties in db/db mice. PEMF significantly promoted bone formation in db/db mice evidenced by increased serum osteocalcin and bone mineral apposition rate, whereas PEMF exerted no observable alteration in bone resorption. Real-time PCR showed that PEMF upregulated tibial gene expression of osteoblastogenesis-related of canonical Wnt/ß-catenin signaling but not osteoclastogenesis-related RANKL-RANK signaling in db/db mice. Our findings demonstrate that PEMF improved bone quantity and quality with obvious anabolic activities in db/db mice, and imply that PEMF might become a clinically applicable treatment modality for improving bone quality in type 2 diabetic patients.

[Effects of hypergravity exposure after 30-days simulated weightlessness on chemokine CCL20 and its receptor CCR6 in lingual mucosa of rhesus macaque].

OBJECTIVE: To study the influence of hypergravity exposure after 30-days simulated weightlessness on the expression of chemokine CCL20 and its receptor CCR6 in lingual mucosa of rhesus macaque. METHODS: Twenty-three male rhesus monkeys were divided according to the random number method into one control group (A, n = 3) and three experimental groups, including the weightlessness group (B, n = 3), the hypergravity group (C, n = 3) and hypergravity exposure after 30-days simulated weightlessness group (D, n = 14), which was further divided into four subgroups according to the values of overload as: +11 Gx/270s group (D1, n = 3), +13 Gx/230s group (D2, n = 4) and +15 Gx/200s group (D3, n = 4) and +13 Gx/230s with 9-days recovery group (D4, n = 3). Histopathological changes of lingual mucosa were observed by hematoxylin-eosin staining and the expressions of CCL20 and CCR6 were detected by immunohistochemistry (IHC) and quantitative real-time PCR (Q-PCR). RESULTS: Histological observation showed that no significant histopathological changes were found in the lingual mucosa in all experimental groups of the animals, however, there were more infiltrated lymphocyte and neutrophils in the experimental groups. The immunohistochemical scores of CCL20 in group C, group D2 and group D3 were 1.30 ± 0.11, 1.68 ± 0.62 and 2.26 ± 1.00, respectively, significantly higher than that in A group (0.47 ± 0.12, P < 0.05). The immunohistochemical scores of CCR6 expression in those three groups were 4.40 ± 1.48, 6.67 ± 2.04 and 7.02 ± 2.11, respectively, also remarkably higher than that in A group (1.33 ± 0.66, P < 0.05). As far as the mRNA expression was concerned, the expressions of CCL20 and CCR6 had similar change trend with their protein expressions. CONCLUSION: Hypergravity exposure after 30-days simulated weightlessness will not lead to significant pathological changes in lingual mucosa, but can induce the expression of chemokine CCL20 and its receptor CCR6.

[Effects of hypergravity exposure after 30 days of simulated weightlessness on chemokine CCL20 and its receptor CCR6 in gingival tissue of rhesus macaque].

PURPOSE: To study the effect of hypergravity exposure after 30 days of simulated weightlessness on the expression of chemokine CCL20 and its receptor CCR6 in gingival tissue of rhesus macaque. METHODS: Twenty-three male rhesus monkeys were randomly divided into 4 groups, namely control group (A,n=3), weightlessness group (B,n=3), hypergravity group (C,n=3) and hypergravity exposure after 30 days of simulated weightlessness group (D, n=14). Group D was further divided into 4 subgroups according to the values of overload as: +11 Gx /270 s group (D1, n=3), +13 Gx /230 s group (D2,n=4), +15 Gx/200 s group (D3,n=4) and +13 Gx /230 s with 9 days of recovery group (D4, n=3). Histopathological changes of gingival tissues were observed by hematoxylin-eosin staining and the expressions of CCL20 and CCR6 were detected by immunohistochemistry (IHC) and quantitative real-time PCR (Q-PCR). SPSS 17.0 software package was used for statistical analysis. RESULTS: Histological observation showed that no significant histopathological change was found in the gingival tissues in all experimental groups. However, there were more infiltrated lymphocytes and neutrophils in the experimental groups. Normal gingival epithelial cells were hardly stained by anti-CCL20 but weakly stained by anti-CCR6. In the experimental groups, CCL20 could be detected in the basal layer of the gingival epithelial tissue, and CCR6 could be detected in the spinous layer and the basal layer of the gingival epithelium. The CCL20 and CCR6 expression in the gingival tissues of each experimental group were significantly higher than those of the control group, not only at the protein level but at the mRNA level (P<0.05) except the CCL20 expression in the weightlessness group. CONCLUSIONS: Hypergravity exposure after 30 days of simulated weightlessness will not lead to significant pathological changes in gingival tissues, but can induce the expression of chemokine CCL20 and its receptor CCR6 in gingival tissue.

Prospects application of polypyrrole-based immunosensor to Porphyromonas gingivalis quantification in subgingival plaque samples.

BACKGROUND: Periodontitis still poses a serious threat to oral and systemic health condition of humans. The proportion of the main pathogenic bacteria change in localized sites was associated with the initiation of the disease process. However, the limitations of microbiological diagnostic aids rendered the diagnosis of active periodontitis status point-of-care or chair-side based on microbiological data difficult. METHODS: Porphyromonas gingivalis, a major putative etiological agent in the initiation and progression of chronic periodontitis, was used as the experimental subject. An immunosensor based on polypyrrole-coated interdigitated array microelectrodes was developed to quantify Porphyromonas gingivalis in pure culture, gingival crevicular fluid and saliva samples. The regression equation for the normalized impedance change (NIC) versus Porphyromonas gingivalis concentration (C) was measured. The correlation between results of the immunosensor and quantitative real-time PCR method in quantifying Porphyromonas gingivalis in subgingival plaque samples was evaluated. RESULTS: Results of the study revealed that the lowest detection limits of the immunosensor was 1.9 x 10(4), 2.7 x 10(5), and 2.7 x 10(6) cells/mL in pure culture, gingival crevicular fluid, and saliva samples respectively. The values determined using the immunosensor strongly correlated with those obtained using quantitative real-time PCR method (R2 = 0.91, p < 0.05). The immunosensor did not require any labels and amplification steps, and the total detection time from sampling to measurement was less than one hour. CONCLUSIONS: The immunosensor developed in the present study offered some insight into monitoring the change in the number of periodontal bacteria chair-side during routine clinical practice.

Phenotypic changes in nonfimbriated smooth strains of Aggregatibacter actinomycetemcomitans grown in low-humidity solid medium.

Aggregatibacter actinomycetemcomitans is the primary etiologic agent of localized aggressive periodontitis. In vitro, it can undergo fimbriated rough to nonfimbriated smooth phenotypic transition, accompanied by an increase in invasive ability and a decrease in adhesive ability. No opposite direction phenotypic transition was reported. To better understand its pathogenicity, the authors studied the morphological changes of nonfimbriated smooth strains induced by growth environmental humidity. Transmission electron microscopy was used to identify fimbriae expression change. It was found that the lower medium humidity, the more fimbriae reexpressed. In conclusion, the smooth strain of A. actinomycetemcomitans can reexpress the fimbriae in lower humidity environment.

[The effect of insulin-like growth factor-I on the proliferation and alkaline phosphatase activity of human periodontal ligament cells under three-dimensional culture system].

OBJECTIVE: To investigate the effect of insulin-like growth factor- I (IGF- I) on the proliferation and alkaline phosphatase (ALP) activity of human periodontal ligament cells (hPDLCs) under three-dimensional (3D) culture system. METHODS: The hPDLCs were cultured from periodontium of human teeth by the outgrowth method. Rotary cell culture system (RCCS) was enrolled to set 3D culture system. Samples were set to four groups: Negative control group, positive control group (3D group, IGF-I group), and experimental group (3D with IGF- I group). Proliferation was tested with methylthiazolyl tetrazolium (MTT), and ALP activity was assayed by spectrophotometer at 1, 3, 5, 7 d respectively. RESULTS: Compared with that of negative control group, cell proliferation increased significantly in 3D with IGF-I group since 3 d (P < 0.05). Besides, the cell proliferation of 3D with IGF-I group was significantly higher than that of 3D group (P < 0.05). ALP activity of 3D with IGF- I group was significantly higher than that of negative control group, and 3D group at 3, 5, 7 d (P < 0.05). CONCLUSION: IGF-I significantly promotes the proliferation and ALP activity of hPDLCs under 3D culture system.

[Effect of insulin-like growth factor on the proliferation and early stage osteogenesis of human periodontal ligament stem cells under three-dimensional culture system].

OBJECTIVE: To investigate the effect of insulin-like growth factor-I (IGF-I) on the proliferation and osteogenesis of human periodontal ligament stem cells (hPDLC) under three-dimensional (3D) culture system. METHODS: Human periodontal cells were isolated from the ligament of surgically extracted human teeth, and through the limiting dilution assay, got mono-clone of the cell, hPDLCs were isolated from MesenPRO RS medium. Rotary cell culture system (RCCS) was enrolled to set 3D environment. Control group and experiment groups were assigned according to the concentration of IGF-I. There were 5 level of experiment groups (0.1, 1, 10, 50, 100 µg/L). Proliferation was tested with methyl thiazolyl tetrazolium (MTT), and alkine phosphatase (ALP) level was assayed by spectrophotometer to analyze the osteogenesis of hPDLCs. Gene expression of ostetocalcin (OCN) and type I collagen (Col I) were assayed by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: In 3D culture system, the effect of IGF-I on cell proliferation was significantly different between control group and experiment groups (P < 0.05), and there showed significant differences between the group of 0.1 µg/L (0.219 ± 0.021) IGF-I and the groups of 50, 100 µg/L (0.287 ± 0.011, 0.293 ± 0.012). However, there showed no significant differences among other groups. Significant differences of ALP activity were observed between the control group and experiment groups, and between the groups of 1, 10 µg/L (0.304 ± 0.020, 0.310 ± 0.013) and that of 50, 100 µg/L (0.347 ± 0.011, 0.344 ± 0.010) (P < 0.05). While no significant differences were detected between the group of 1 µg/L and that of 10 µg/L, nor between the group of 50 µg/L and that of 100 µg/L. Expressions of Col I and OCN in mRNA and protein level both showed dose-dependent increase. CONCLUSIONS: In 3D culture system, in the scale of 0.1 - 100 µg/L, the effect of IGF-I on the proliferation of hPDLCs increased dose-dependently. 100 µg/L IGF-I promotes osteogenesis of the cells significantly.

AFM and fluorescence imaging of nanomechanical response in periodontal ligament cells.

Most biologists think that AFM has only a limited use in biological research due to its inability to study other than surface structures. Therefore, a BIO-AFM system has been developed to combine both AFM imaging and fluorescence detection, which acts as a powerful tool for a better understanding of dynamic cell processes. In this study, based on a custom-made BIO-AFM system, the elasticity and ultrastructure of living periodontal ligament cells (PDLCs) were investigated. The cantilever probe with a micron-sized bead was used to exert nano-loading force onto the PDLCs. The related signal of NO was then recorded simultaneously. The results show that PDLCs hold strong networks of stress fibers as well as high elastic modulus value, exhibiting the ability for better counteracting the external forces. In the mechano-transduction studies, an initial increase and subsequent drop in intracellular NO response was found. Furthermore, NO may diffuse from a stimulated cell to adjacent cells. In conclusion, our single-cell nano-mechanical study provides a significant advancement in elucidating the magnitude, location, time scale, and biomolecular mechanisms underlying cell mechano-transduction.

[Primary culture of human periodontal ligament fibroblasts by explants with enzymatic digestion].

OBJECTIVE: To increase the success rate of primary culture of human periodontal ligament fibroblasts (HPLF), and to establish an experimental model for studying HPLF in vitro. METHODS: The primary cells were isolated from human periodontal ligament by explants with enzymatic digestion method. Morphological analysis and immunocytochemical staining were used to characterize the cell lineage, and growth curve assay to evaluate the biological features of HPLF. RESULTS: The success rate of primary culture of HPLF was 77.8%. Cultured cells were spindle-shaped, and had a positive reaction to antibodies against vimentin, and a negative reaction to antibodies against keratin. Their morphological and biological characteristics were similar to those of typical HPLF. Growth of HPLF obtained by this method was satisfactory. CONCLUSION: The success rate of primary culture of HPLF is significantly increased by explants combined with enzymatic digestion. The method is simple and feasible.