Detection of SARS-CoV-2 IgG antibodies and inflammatory cytokines in saliva-a pilot study.

Objective: The pandemic caused by SARS-CoV-2 virus continues to have a profound effect worldwide. However, COVID-19 induced oral facial manifestations have not been fully described. We conducted a prospective study to demonstrate feasibility of anti-SARS-CoV-2 IgG and inflammatory cytokine detection in saliva. Our primary objective was to determine whether COVID-19 PCR positive patients with xerostomia or loss of taste had altered serum or saliva cytokine levels compared to COVID-19 PCR positive patients without those oral symptoms. Our secondary objective was to determine the correlation between serum and saliva COVID-19 antibody levels. Materials and methods: For cytokine analysis, saliva and serum were obtained from 17 participants with PCR-confirmed COVID-19 infection at three sequential time points, yielding 48 saliva samples and 19 paired saliva-serum samples from 14 of the 17 patients. For COVID-19 antibody analyses, an additional 27 paired saliva-serum samples from 22 patients were purchased. Results: The saliva antibody assay had 88.64% sensitivity [95% Confidence Interval (CI) 75.44%, 96.21%] to detect SARS-CoV-2 IgG antibodies compared to serum antibody. Among the inflammatory cytokines assessed - IL-6, TNF-α, IFN-γ, IL-10, IL-12p70, IL-1ß, IL-8, IL-13, IL-2, IL-5, IL-7 and IL-17A, xerostomia correlated with lower levels of saliva IL-2 and TNF-α, and elevated levels of serum IL-12p70 and IL-10 (p < 0.05). Loss of taste was observed in patients with elevated serum IL-8 (p < 0.05). Conclusions: Further studies are needed to construct a robust saliva-based COVID-19 assay to assess antibody and inflammatory cytokine response, which has potential utility as a non-invasive monitoring modality during COVID-19 convalescence.

The Role of Oral Health in the Acquisition and Severity of SARS-CoV-2: A Retrospective Chart Review.

Objective: Studies have shown that gingival crevices may be a significant route for SARS-CoV-2 entry. However, the role of oral health in the acquisition and severity of COVID-19 is not known. Design: A retrospective analysis was performed using electronic health record data from a large urban academic medical center between 12/1/2019 and 8/24/2020. A total of 387 COVID-19 positive cases were identified and matched 1:1 by age, sex, and race to 387 controls without COVID-19 diagnoses. Demographics, number of missing teeth and alveolar crestal height were determined from radiographs and medical/dental charts. In a subgroup of 107 cases and controls, we also examined the rate of change in alveolar crestal height. A conditional logistic regression model was utilized to assess association between alveolar crestal height and missing teeth with COVID-19 status and with hospitalization status among COVID-19 cases. Results: Increased alveolar bone loss, OR = 4.302 (2.510 - 7.376), fewer missing teeth, OR = 0.897 (0.835-0.965) and lack of smoking history distinguished COVID-19 cases from controls. After adjusting for time between examinations, cases with COVID-19 had greater alveolar bone loss compared to controls (0.641 ± 0.613 mm vs 0.260 ± 0.631 mm, p < 0.01.) Among cases with COVID-19, increased number of missing teeth OR = 2.1871 (1.146- 4.174) was significantly associated with hospitalization. Conclusions: Alveolar bone loss and missing teeth are positively associated with the acquisition and severity of COVID-19 disease, respectively.

Estrogen receptor alpha mediates mandibular condylar cartilage growth in male mice.

OBJECTIVES: In the appendicular skeleton, estrogen via ERα signalling has been shown to mediate endochondral growth plate fusion in both males and females. However, the role of ERα in mediating growth of the mandibular condylar cartilage is unknown. Thus, this study focuses on the characterization of the mandibular condylar cartilage phenotype in young and adult male ERαKO mice. SETTING: Columbia University Medical Center. MATERIAL AND METHODS: WT and ERαKO C57BL/6 male mice were sacrificed at 49 days or 9 months for phenotypic analysis. Changes to MCC thickness, cell number and cell density were measured using histomorphometric methods. Cartilage-specific gene expression and OARSI scores were investigated for 49-day and 9-month-old male ERαKO and WT mice. RESULTS: In young mice, a significant increase in the number of mandibular condylar cartilage cells and a significant decrease in the expression of Col10, Runx2 and DMP1 were observed in the male ERαKO mice compared to WT. In 9-month-old mice, we found a similar increase in the number of cells but no change in osteoarthritic histological scoring in ERαKO mice compared to WT mice. CONCLUSION: In summary, estrogen plays a role in mediating mandibular condylar maturation in young male mice. However, according to this study, it does not play a role in mediating long-term growth or age-related mandibular condylar cartilage degeneration in males.

Sex differences in the estrogen-dependent regulation of temporomandibular joint remodeling in altered loading.

OBJECTIVE: Temporomandibular joint (TMJ) diseases predominantly afflict women, suggesting a role of estrogen in the disease etiology. Previously, we determined that decreased occlusal loading (DOL) inhibited collagen type II (Col2) expression in the mandibular condylar cartilage (MCC) of female wild-type (WT) mice whereas no change was observed in males. This decrease in chondrogenesis was abolished by estrogen receptor beta (ERß) deficiency in females. Therefore, the goal of this study was to examine the role of estradiol - ERß signaling in mediating DOL effects in male mice to further decipher sex differences. METHODS: Male 21 day-old WT and ERßKO male mice were treated with either placebo or estradiol and exposed to normal or DOL for 4 weeks. Cartilage thickness and cell proliferation, gene expression and immunohistochemistry of chondrogenic markers and estrogen receptor alpha (ERα), and analysis of bone histomorphometry via microCT were completed to ascertain the effect of estradiol on DOL effects to the TMJ. RESULTS: ERßKO male mice lack a MCC phenotype. In both genotypes, estradiol treatment increased Col2 gene expression and trabecular thickness. DOL in combination with estradiol treatment caused a significant increase in Col2 gene expression in both genotypes. CONCLUSIONS: The sex differences in DOL-induced inhibition of Col2 expression do not appear to be mediated by differences in estradiol levels between male and female mice. Greater understanding on the role of estrogen and altered loading are critical in order to decipher the sex dimorphism of TMJ disorders.

Phospholipid vesicles encapsulated bacteriophage: A novel approach to enhance phage biodistribution.

Phage therapy has been at the centre of attraction for combating multi-drug resistant strains. However, less stability and rapid clearance of phage by mononuclear phagocytic system (MPS) restricts its use in humans. In the present study, aim was to develop a liposomal delivery system for bacteriophage that can assure efficient phage delivery and retention at the site of infection. Different ratios of cholesterol, lipids and surfactant along with different charge inducers were employed to prepare liposomes. Phage was then entrapped in the liposomes and characterized on the basis of morphology, size, entrapment efficiency and stability. Further, in vivo biodistribution of free phage and liposome entrapped phage was compared in different organs of mice. A cationic liposomal formulation showed maximum encapsulation efficiency of 92%. Transmission electron microscopy (TEM) confirmed the entrapment of phages in liposomes. Liposome preparation was found to be most stable at 4°C during storage. Liposome entrapped bacteriophage was retained for longer duration in different organs i.e. upto day 4 in blood, day 6 in liver, lungs and kidney, 14days in spleen of mice as compared to free phage that became undetectable by 36th h in blood as well as lungs and by 48th h in all other organs.

Estrogen via estrogen receptor beta partially inhibits mandibular condylar cartilage growth.

OBJECTIVE: Temporomandibular joint (TMJ) diseases predominantly afflict women, suggesting a role for female hormones in the disease process. However, little is known about the role of estrogen receptor (ER) signaling in regulating mandibular condylar cartilage growth. Therefore, the goal of this study was to examine the effects of altered estrogen levels on the mandibular condylar cartilage in wild type (WT) and ER beta Knockout (KO) mice. MATERIALS AND METHODS: 21-day-old female WT (n = 37) and ER beta KO mice (n = 36) were either sham operated or ovariectomized, and treated with either placebo or estradiol. The mandibular condylar cartilage was evaluated by histomorphometry, proliferation was analyzed by double ethynyl-2'-deoxyuridine/bromodeoxyuridine (EdU/BrdU) labeling, and assays on gene and protein expression of chondrocyte maturation markers were performed. RESULTS: In WT mice, ovariectomy caused a significant increase in mandibular condylar cartilage cell numbers, a significant increase in Sox9 expression and a significant increase in proliferation compared with sham operated WT mice. In contrast, ovariectomy did not cause any of these effects in the ER beta KO mice. Estrogen replacement treatment in ovariectomized WT mice caused a significant decrease in ER alpha expression and a significant increase in Sost expression compared with ovariectomized mice treated with placebo. Estrogen replacement treatment in ovariectomized ER beta KO mice caused a significant increase in Col2 expression, no change in ER alpha expression, and a significant increase in Sost expression. CONCLUSION: Estrogen via ER beta inhibits proliferation and ER alpha expression while estrogen independent of ER beta induces Col2 and Sost expression.

Evaluation of BSP expression and apoptosis in the periodontal ligament during orthodontic relapse: a preliminary study.

OBJECTIVE: To examine the expression of bone sialoprotein (BSP) and apoptosis in an in vivo orthodontic relapse model. MATERIALS AND METHODS: Male mice (10-12 weeks old), either transgenic [green fluorescent protein (GFP) driven by the BSP promoter] or wild type, were used in this study. To achieve orthodontic tooth movement (OTM), maxillary right first molars were moved mesially using closed-coil springs. Animals were divided into an OTM group (14 days continuous orthodontic force - 11 animals) or Relapse group (10 days of force application followed by 4 days of relapse - 8 animals). The control group was comprised of the contralateral maxillary molars. The periodontal ligament (PDL) was analyzed in areas of compression and tension for transgenic expression, osteoclast localization, and the presence of apoptotic cells. RESULTS: There was a significant decrease in GFP-labeled cells on the compression and tension sides of the PDL in the OTM group compared with control. In the relapse group, GFP-labeled cells were significantly decreased only on the old compression side. Osteoclasts were localized on the compression side of the OTM group, whereas in the Relapse group, they were present on both sides. PDL apoptosis significantly increased on the compression side in OTM and Relapse groups. CONCLUSION: Both OTM and Relapse groups exhibited a decreased number of GFP-labeled cells in areas of compression and tension. There was significant PDL apoptosis in regions under compressive forces following OTM and to a lesser extent following relapse.

Differential effects of prenatal chronic high-decibel noise and music exposure on the excitatory and inhibitory synaptic components of the auditory cortex analog in developing chicks (Gallus gallus domesticus).

Proper development of the auditory cortex depends on early acoustic experience that modulates the balance between excitatory and inhibitory (E/I) circuits. In the present social and occupational environment exposure to chronic loud sound in the form of occupational or recreational noise, is becoming inevitable. This could especially disrupt the functional auditory cortex development leading to altered processing of complex sound and hearing impairment. Here we report the effects of prenatal chronic loud sound (110-dB sound pressure level (SPL)) exposure (rhythmic [music] and arrhythmic [noise] forms) on the molecular components involved in regulation of the E/I balance in the developing auditory cortex analog/Field L (AuL) in domestic chicks. Noise exposure at 110-dB SPL significantly enhanced the E/I ratio (increased expression of AMPA receptor GluR2 subunit and glutamate with decreased expression of GABA(A) receptor gamma 2 subunit and GABA), whereas loud music exposure maintained the E/I ratio. Expressions of markers of synaptogenesis, synaptic stability and plasticity i.e., synaptophysin, PSD-95 and gephyrin were reduced with noise but increased with music exposure. Thus our results showed differential effects of prenatal chronic loud noise and music exposures on the E/I balance and synaptic function and stability in the developing auditory cortex. Loud music exposure showed an overall enrichment effect whereas loud noise-induced significant alterations in E/I balance could later impact the auditory function and associated cognitive behavior.

Orthodontic tooth movement causes decreased promoter expression of collagen type 1, bone sialoprotein and alpha-smooth muscle actin in the periodontal ligament.

OBJECTIVE: To evaluate the effects of orthodontic tooth movement on the promoter expression of collagen type 1 (3.6Col1), bone sialoprotein (BSP) and alpha-smooth muscle actin (αSMA) in the periodontal ligament (PDL) using transgenic mice containing transgenes of these promoters fused to green fluorescent proteins (GFP). MATERIALS AND METHODS: The maxillary first molars of 10-12 week-old transgenic mice were loaded with 10-12 g of force for 12, 48 h, or 7 days. Mice were transgenic for one of the following GFP-tagged bone markers of osteoblast lineage cells: 3.6-kb fragment of the rat collagen type 1 promoter (3.6Col1), BSP or α-smooth muscle actin (αSMA). Loaded molars under compression and tension were compared with contra-lateral unloaded controls. RESULTS: On the compression side of the PDL, orthodontic tooth movement caused a significant decrease in GFP expression of all the promoters at each time point. On the tension side, there was a significant increase in BSP-GFP expression, 12 h following loading compared to the contralateral unloaded controls. CONCLUSIONS: An in vivo tooth movement model using transgenic mice with promoter-GFP constructs provides an efficient and effective way of investigating the cellular events underlying orthodontic tooth movement. PDL cells may undergo decreased differentiation in response to the compressive force.

Isolation and characterization of murine mandibular condylar cartilage cell populations.

OBJECTIVES: The mandibular condylar cartilage is a heterogeneous tissue containing cells at various stages of chondrocyte maturation organized into 4 zones: superficial, polymorphic, flattened, and hypertrophic. The goal of this study was to use transgenic mice containing chondrocyte maturation markers fused to fluorescent protein transgenes to isolate and characterize homogenous cell populations of the mandibular condylar cartilage. METHODS: Fluorescent reporter expression in the mandibular condylar cartilage of transgenic mice containing the 3.6-kb fragment of the rat collagen type 1 promoter fused to a topaz-fluorescent protein (Col3.6-tpz), collagen type 2 promoter fused to a cyan-fluorescent protein (Col2-cyan), and/or collagen type 10 promoter fused to cherry-fluorescent protein (Col10-cherry) was examined. Mandibular condylar cartilage cells were analyzed by fluorescence-activated cell sorting (FACS) and either used for gene expression analysis or plated in cell cultures and exposed to adipogenic, osteogenic, or chondrogenic conditions. To determine cell fate, transgenic mice containing the Col3.6-cre recombinase were bred with cre reporter mice. RESULTS: Localization and analysis of gene expression revealed that Col3.6-tpz-positive cells corresponded to the polymorphic/flattened zones and Col2-cyan-positive cells corresponded to the flattened/hypertrophic zones of the mandibular condylar cartilage. Mandibular condylar cartilage FACS-sorted Col3.6-tpz-positive cells have the potential to differentiate into bone, cartilage, and fat. Cell fate mapping revealed that Col3.6 cells are precursors of some of the hypertrophic chondrocytes in the mandibular condylar cartilage. CONCLUSION: Col3.6-tpz cells represent an earlier stage of the mandibular condylar cartilage maturation pathway.

Multivariate optimization of cloud point extraction procedure for zinc determination in aqueous extracts of medicinal plants by flame atomic absorption spectrometry.

Cloud point extraction method has been developed for preconcentration of trace quantities of zinc (Zn) in aqueous extract of medicinal plants and blood samples of liver cancer patients using flame atomic absorption spectrometry. The Zn in aqueous extracts of medicinal plants (MPs) was complexed with 2-methyl-8-hydroxyquinoline (quinaldine) and 1-(2-pyridylazo)-2-naphthol (PAN) separately and entrapped in a non-ionic surfactant Triton X-114. After centrifugation, the surfactant-rich phase was diluted with 0.25mL acidic ethyl alcohol. The multivariate strategy was applied to estimate the optimum values of experimental variables (pH, time temperature, ligands and surfactant concentrations). Interactions between analytical factors and their optimal levels were investigated by two level factorial designs. Student's t-test on the results of factorial design with 16 runs for Zn extraction, demonstrated that the factors, ligands concentrations, pH and temperature were statistically significant. The accuracy was assessed by analysis of certified reference materials, namely, BCR 101 (spruce needles), Clincheck control-lyophilized human whole blood. Enhancement factor of 30 and 26 were achieved for the preconcentration of Zn by 2-methyl-8-hydroxyquinoline (L1) and 1-(2-pyridylazo)-2-naphthol (L2), respectively. The relative standard deviation for six replicate determinations of Zn at 10µg/L level using 2-methyl-8-hydroxyquinoline (L1) and 1-(2-pyridylazo)-2-naphthol (L2) were <4% and >5%, respectively.

Sex differences in chondrocyte maturation in the mandibular condyle from a decreased occlusal loading model.

Temporomandibular joint disorders (TMDs) predominantly afflict women of childbearing age. Defects in mechanical loading-induced temporomandibular joint (TMJ) remodeling are believed to be a major etiological factor in the development of TMD. The goal of this study was to determine if there are sex differences in CD-1 and C57BL/6 mice exposed to a decreased occlusal loading TMJ remodeling model. Male and female CD-1 and C57BL/6 mice, 21 days old, were each divided into two groups. They were fed either a normal pellet diet (normal loading) or a soft diet and had their incisors trimmed out of occlusion (decreased occlusal loading) for 4 weeks. The mandibular condylar cartilage was evaluated by histology, and the subchondral bone was evaluated by micro-CT analysis. Gene expression from both was evaluated by real-time PCR analysis. In both strains and sexes of mice, decreased occlusal loading caused similar effects in the subchondral bone, decreases in bone volume and total volume compared with their normal loading controls. However, in both strains, decreased occlusal loading caused a significant decrease in the expression of collagen type II (Col2) and Sox9 only in female mice, but not in male mice, compared with their normal loading controls. Decreased occlusal loading causes decreased bone volume in both sexes and a decrease in early chondrocyte maturation exclusively in female mice.

Comparative in vitro cytotoxicity study of carbon nanotubes and titania nanostructures on human lung epithelial cells.

The aim of this study is to assess in vitro cytotoxic effects of titania nanostructures and carbon nanotubes (CNTs) by exposing A549 lung epithelial cell line to these materials. Titania nanotubes (TiNTs) were grown by hydrothermal treatment of TiO(2) nanoparticles, followed by annealing them at 400°C. The titania nanostructures obtained on annealing (mixture of nanotubes and nanorods) were hollow and open ended, containing 3-5 layers of titania sheets, with an internal diameter ∼3-5 nm and external diameter ∼8-10 nm, and a specific surface area of 265 m(2)/g. As-supplied single walled (SWCNTs) and microwave plasma enhanced chemical vapour deposition (MPCVD) grown multi walled carbon nanotubes (MWCNTs) were used in this study. The lengths and diameters of the SWCNTs were 5-10nm and 0.5-3 nm respectively. The lengths and diameters of the MWCNTs were 25-30 µm and 10-30 nm respectively. The cell viability was evaluated using the MTT (3-(4,-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium) assay. No significant cytotoxic effects of titania nanostructures were observed over a period of a week of testing time, while the presence of CNTs in some cases demonstrated significant cytotoxic effects. Finally, possible reason of cytotoxicity is discussed in the light of microstructures of materials.

Murine TMJ loading causes increased proliferation and chondrocyte maturation.

The purpose of this study was to examine the effects of forced mouth opening on murine mandibular condylar head remodeling. We hypothesized that forced mouth opening would cause an anabolic response in the mandibular condylar cartilage. Six-week-old female C57BL/6 mice were divided into 3 groups: (1) control, (2) 0.25 N, and (3) 0.50 N of forced mouth opening. Gene expression, micro-CT, and proliferation were analyzed. 0.5 N of forced mouth opening caused a significant increase in mRNA expression of Pthrp, Sox9, and Collagen2a1, a significant increase in proliferation, and a significant increase in trabecular spacing in the subchondral bone, whereas 0.25 N of forced mouth opening did not cause any significant changes in any of the parameters examined. Forced mouth opening causes an increase in the expression of chondrocyte maturation markers and an increase in subchondral trabecular spacing.