Morphologic and gene expression analysis of periodontal ligament fibroblasts subjected to pressure.

INTRODUCTION: Force application (FA) during orthodontic tooth movement is mediated through periodontal ligament (PDL) fibroblasts. FA on deciduous teeth has an inherent risk of root resorption, which is less in permanent teeth. Currently, the root resorption mechanism is poorly understood. We hypothesized that FA alters the morphology and gene expression of PDL fibroblasts. This study was designed to achieve homogenous PDL fibroblast cultures, establish an in-vitro FA model, analyze fibroblast morphology after FA, and compare the gene expressions of PDL fibroblasts of deciduous and permanent teeth after FA. METHODS: Fibroblasts were sorted from primary cultures of deciduous and permanent tooth PDLs. Cell viability was evaluated in the Opticell (Thermo Scientific, Waltham, Mass) FA model. Cellular morphology was analyzed using immunofluorescence staining for actin and focal adhesion complexes. Gene expressions of untreated or pressure-treated PDL fibroblasts of deciduous and permanent teeth were compared by gene array and confirmed by real-time polymerase chain reaction. RESULTS: Cell sorting resulted in cultures containing 98% of PDL fibroblasts. The Opticell model showed 94% cell survival after FA. FA increased fibroblasts' adhesion. Gene arrays and real-time polymerase chain reactions indicated greater up-regulation of DKK2 mRNA in untreated PDL fibroblasts of deciduous teeth and greater up-regulation of ADAMTS1 mRNA in pressurized PDL fibroblasts of deciduous and permanent teeth. CONCLUSIONS: Cell sorting is an efficient method to establish homogenous PDL fibroblast cultures. Using the Opticell FA model allows the maintenance of excellent cell viability. FA increased the surface adherence of fibroblasts. Up-regulation of ADAMTS1 after FA may indicate its involvement in the remodeling of the periodontium during orthodontic tooth movement. Understanding root resorption mechanisms under FA will help to prevent it during orthodontic treatment.

Identification of Sjögren's syndrome oral fluid biomarker candidates following high-abundance protein depletion.

OBJECTIVE: SS is an autoimmune exocrinopathy affecting ∼1 million patients in the USA that is diagnosed mostly in middle-aged women. Oral fluids (OFs) serving as the mirror of the body were suggested as an ideal non-invasive diagnostic tool. Previously we developed depletion techniques for OF high-abundance proteins to increase visualization of low-abundance proteins. Therefore the aim of this study was to examine the effect of depletion pretreatments on the identification potential of SS OF biomarker candidates. METHODS: Unstimulated OFs were collected from 18 female SS patients and 18 healthy age- and gender-matched controls. High-abundance proteins were depleted using affinity and immunodepletion methodologies followed by semi-quantitative two-dimensional gel electrophoresis and quantitative dimethylation liquid chromatography tandem mass spectrometry (LC-MS/MS). To initially validate the MS results, western blotting was performed. RESULTS: The use of depletion strategy before proteomics analysis increased identification ability by 3-fold. Overall, 79 biomarker candidates were identified. Proteins with the most pronounced fold changes were related to SS serum or tissue factors. In addition, bioinformatics analysis of proteins with a >3-fold increase in SS patients showed calcium-binding proteins, defence-response proteins, proteins involved in apoptotic regulation, stress-response proteins and cell motion-related proteins. Preliminary validation by western blotting of profilin and CA-I indicated similar expression profile trends to those identified by quantitative MS. CONCLUSION: The significance of OF novel depletion methodologies is clearly demonstrated for increased visibility of biomarker candidates as well as for unveiling possible mechanisms involved in this syndrome. This represents a major contribution to our ability to use OF as a future diagnostic fluid.

Development of saliva-based cardiac troponin I point-of-care test using alpha-amylase depletion: a feasibility study.

INTRODUCTION: Cardiac troponin (cTn) is the biomarker of choice for detection of myocardial injury. There is a great need for simple point-of-care (POC) troponin testing among patients with chest pain, mainly in the prehospital setting. The purpose of this study was to evaluate the presence of cardiac troponin I (cTnI) in saliva of patients with myocardial injury using alpha-amylase depletion technique. METHODS: Saliva samples were collected from 40 patients with myocardial injury who were tested positive for conventional high-sensitivity cardiac troponin T (cTnT) blood tests, and from 66 healthy volunteers. Saliva samples were treated for the removal of salivary alpha-amylase. Treated and untreated samples were tested with blood cTnI Rapid Diagnostic Test. Salivary cTnI levels were compared to blood cTnT levels. RESULTS: Thirty-six of 40 patients with positive blood cTnT had positive salivary samples for cTnI following alpha-amylase depletion treatment (90.00% sensitivity). Moreover, three of the four negative saliva samples were obtained from patients with relatively low blood cTnT levels of 100 ng/L or less (96.88% sensitivity for 100 ng/L and above). The negative predictive value was 93.65% and rose up to 98.33% considering the 100 ng/L cutoff. Positive predictive values were 83.72% and 81.58%, respectively. Among 66 healthy volunteers and 7 samples yielded positive results (89.39% specificity). CONCLUSION: In this preliminary work, the presence of cTnI in saliva was demonstrated for the first time to be feasibly identified by a POC oriented assay. The specific salivary alpha-amylase depletion technique was shown to be crucial for the suggested assay.

Dental care during the coronavirus disease 2019 (COVID-19) outbreak: operatory considerations and clinical aspects.

OBJECTIVES: This paper is aimed at addressing the urgent need to develop a protocol that will address the operatory and clinical aspects of dental care during the Coronavirus disease 2019 (COVID-19) outbreak. DATA SOURCES: The epidemiology, clinical signs and symptoms, and modes of transmission of COVID-19 are presented. This protocol was established as an international collaboration of three dental universities: Hadassah School of Dental Medicine, Israel; University of Rochester Medical Center, USA; and the University of Pennsylvania, USA. This protocol is based on a detailed review of the existing English language literature as well on the logistic and clinical experience of each facility and the opinion of the authors. The protocol is designed for a hospital setting and includes considerations related to dental treatment in both healthy subjects and those suspected or diagnosed with COVID-19. The first part of this review discusses operatory considerations; the second part discusses general dental clinical aspects; the third part discusses endodontic considerations; and the fourth part discusses surgical aspects. This protocol may be applicable to other future similar pandemics. CONCLUSION: Logistic and clinical steps are required to provide dental care during the COVID-19 outbreak while preventing cross-contamination and protecting the dental team during the provision of care.

Proteomic profiling of whole-saliva reveals correlation between Burning Mouth Syndrome and the neurotrophin signaling pathway.

Burning mouth syndrome (BMS) is characterized by a spontaneous and chronic sensation of burning in the oral mucosa, with no apparent signs. The underlying pathophysiological and neuropathic mechanisms remain unclear. Here, we attempt to elucidate some of these mechanisms using proteomic profiling and bioinformatic analyses of whole-saliva (WS) from BMS patients compared to WS from healthy individuals. Qualitative and quantitative proteomic profiling was performed using two dimensional gel electrophoresis (2-DE) and quantitative mass spectrometry (q-MS). In order to improve protein visibility, 21 high abundance proteins were depleted before proteomic profiling. Quantitative proteomic analysis revealed 100 BMS specific proteins and an additional 158 proteins up-regulated by more than threefold in those with BMS. Bioinformatic analyses of the altered protein expression profile of BMS group indicated high correlations to three cellular mechanisms including the neurotrophin signaling pathway. Based on this finding, we suggest that neurotrophin signaling pathway is involved in the pathophysiology of BMS by amplifying P75NTR activity, which in turn increases neural apoptosis thereby reducing sub-papillary nerve fiber density in the oral mucosa.

Identification of salivary protein biomarkers for orthodontically induced inflammatory root resorption.

PURPOSE: Orthodontically induced inflammatory root resorption (OIIRR) is one of the most prevalent and unavoidable consequence of orthodontic tooth movement. The aim of this study was to discover potential diagnostic protein biomarkers for detection of OIIRR in whole saliva (WS). MATERIAL AND METHODS: Unstimulated WS was collected from 72 subjects: 48 OIIRR patients and 24 untreated, generally healthy, age and gender matched controls. Radiographic assessment of periapical x-rays of four upper incisors taken before and 9 months after bonding was done. High-abundance proteins were depleted followed by two-dimensional-gel-electrophoresis and quantitative mass spectrometry (qMS). Finally, to initially validate qMS results, Western blotting was performed. RESULTS: qMS revealed differentially expressed proteins in the moderate-to-severe OIIRR group, which have never been found in WS before. Additionally, in the moderate-to-severe young OIIRR group, the pathogenetic mechanisms related to actin cytoskeleton regulation and Fc gamma R- mediated phagocytosis were detected, while in adults- to focal adhesion. Preliminary validation by Western blotting of fetuin-A and p21-ARC indicated expression profile trends similar to those identified by qMS. CONCLUSION: The significance of WS novel proteomic methodologies is clearly demonstrated for detecting new OIIRR biomarkers as well as for unveiling possible novel pathogenetic mechanisms in both young and adult patients.

Biocompatibility of tungsten disulfide inorganic nanotubes and fullerene-like nanoparticles with salivary gland cells.

Impaired salivary gland (SG) function leading to oral diseases is relatively common with no adequate solution. Previously, tissue engineering of SG had been proposed to overcome this morbidity, however, not yet clinically available. Multiwall inorganic (tungsten disulfide [WS2]) nanotubes (INT-WS2) and fullerene-like nanoparticles (IF-WS2) have many potential medical applications. A yet unexplored venue application is their interaction with SG, and therefore, our aim was to test the biocompatibility of INT/IF-WS2 with the A5 and rat submandibular cells (RSC) SG cells. The cells were cultured and subjected after 1 day to different concentrations of INT-WS2 and were compared to control groups. Growth curves, trypan blue viability test, and carboxyfluorescein succinimidyl ester (CFSE) proliferation assay were obtained. Furthermore, cells morphology and interaction with the nanoparticles were observed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy. The results showed no significant differences in growth curves, proliferation kinetics, and viability between the groups compared. Moreover, no alterations were observed in the cell morphology. Interestingly, TEM images indicated that the nanoparticles are uptaken by the cells and accumulate in cytoplasmic vesicles. These results suggest promising future medical applications for these nanoparticles.

Expression of tropoelastin in human periodontal ligament fibroblasts after simulation of orthodontic force.

The ability of elastic fibers to respond to mechanical stimuli suggests that they play a central role in physiological adaptation to external stimuli including application of orthodontic force. The purpose of this study was to examine the effect of external pressure simulating orthodontic force on tropoelastin gene expression in cultured human periodontal ligament fibroblasts (hPDLF). External pressure simulation was achieved by centrifugation for 10, 30, 60, 90 and 120 min of hPDLF in a horizontal microplate rotor. Semi-quantitative RT-PCR analysis of tropoelastin mRNA was performed and beta-actin was used as an internal invariant control. While centrifugal force on mRNA levels of beta-actin showed almost no change, the mRNA levels of tropoelastin increased significantly to a peak level of more than four-fold after 30 min. Thereafter, at 60 min, the mRNA levels remained at more than three-fold. After 90 min, mRNA levels decreased to control levels. The finding that no changes in mRNA levels of beta-actin occurred during the first 90 min of centrifugation validates its use as an invariant control gene in such an experimental model. This study demonstrated that tropoelastin is expressed in hPDLF and that the pressure caused significant time-dependent upregulation of the tropoelastin gene. The responsiveness of the tropoelastin gene to force shows its possible clinical importance in orthodontic tooth movement. Further studies, however, are essential in order to learn whether the high expression of the gene in vitro will also be followed by corresponding protein synthesis and deposition in vivo in the extracellular matrix (ECM) of the periodontal ligament (PDL).

Comparison of diverse affinity based high-abundance protein depletion strategies for improved bio-marker discovery in oral fluids.

Oral fluids (OF) have been suggested as a source of biomarkers for oral and systemic health, but as with other bio-fluids, the presence of high-abundance proteins interferes with the detection of lower-abundance biomarkers. Here, we compared the performance of four depletion treatments: triple depletion (TD) of amylases, albumins and immunoglobulin G; multiple depletion (MD) of amylases and a panel of 20 proteins, a combination of the two (EMD) and combinatorial peptide ligand library based depletion termed CPLL. TD, MD, EMD and CPLL removed 76%, 83%, 85% and 94% of total proteins, respectively, coupled with increased low abundance protein detection and narrowed dynamic range. 2-DE revealed that all depletion pretreatments successfully clarified areas hampered by high-abundance proteins; however, EMD and CPLL exposed the highest number of proteins. Quantitative MS of EMD samples relative to none treated samples indicated that most of downregulated proteins (>90%) were EMD target proteins. In conclusion, a multiple step EMD and CPLL depletion approaches bring about the highest number of protein detection ability and the best hampered-area clearance. As CPLL requires at least 10 fold more protein starting material, we suggest EMD pretreatment as a new detection tool in instances of low protein starting material.

Effect of irradiation on cell transcriptome and proteome of rat submandibular salivary glands.

Salivary glands (SGs) are irreversibly damaged by irradiation (IR) treatment in head and neck cancer patients. Here, we used an animal irradiation model to investigate and define the molecular mechanisms affecting SGs following IR, focusing on saliva proteome and global transcription profile of submandibular salivary gland (SSG) tissue.We show that saliva secretion was gradually reduced to 50% of its initial level 12 weeks post-IR. Saliva protein composition was further examined by proteomic analysis following mass spectrometry (MS) analysis that revealed proteins with reduced expression originating from SSGs and proteins with increased expression derived from the serum, both indicating salivary tissue damage. To examine alterations in mRNA expression levels microarray analysis was performed. We found significant alterations in 95 genes, including cell-cycle arrest genes, SG functional genes and a DNA repair gene.Tissue damage was seen by confocal immunofluorescence of α-amylase and c-Kit that showed an increase and decrease, respectively, in protein expression. This was coherent with real-time PCR results.This data indicates that IR damages the SSG cells' ability to produce and secrete saliva and proteins, and maintain the physiological barrier between serum and saliva. The damage does not heal due to cell-cycle arrest, which prevents tissue regeneration. Taken together, our results reveal a new insight into IR pathobiology.

An approach to remove alpha amylase for proteomic analysis of low abundance biomarkers in human saliva.

Proteomic characterization of human whole saliva for the identification of disease-specific biomarkers is guaranteed to be an easy-to-use and powerful diagnostic tool for defining the onset, progression and prognosis of human systemic diseases and, in particular, oral diseases. The high abundance of proteins, mainly alpha amylase, hampers the detection of low abundant proteins appearing in the disease state and therefore should be removed. In the present study a 2-DE was used to analyze human whole saliva following the removal of alpha amylase by affinity adsorption to potato starch. After alpha amylase removal whole saliva was analyzed by SDS-PAGE showing at least sixfold removal efficiency and by an alpha amylase activity assay showing 97% reduced activity. MS identification of the captured alpha amylase after elution demonstrated specific removal; 2-DE analysis showed the selective removal of alpha amylase and consequently increased gel resolution. MS identification of protein spots in the 60 kDa area revealed 15 proteins, which were masked before alpha amylase removal. In conclusion, treatment of human whole saliva with an alpha amylase removal device increases gel resolution and enables a higher protein sample for analysis.

Gene transfer mediated by different viral vectors following direct cannulation of mouse submandibular salivary glands.

The salivary gland has been suggested as an accessible organ for gene transfer to express recombinant proteins locally in the saliva, as well as for secretion to the blood circulation. The aim of this study was to evaluate the efficiency of gene transfer to salivary glands using different viral vectors: adenovirus, vaccinia, herpes simplex type 1 (HSV), and two retroviral vectors (murine leukemia virus (MuLV) and lentivirus). We show, by in situ staining and beta-galactosidase reporter activity assay, that the adenoviral and vaccinia vectors were able to deliver the reporter gene efficiently to acinar and duct cells. The HSV vector was less efficient and infected only the acinar cells. The lentiviral vector infected acinar and duct cells, but at a relatively low efficiency. The MuLV vector did not infect the salivary gland unless cell proliferation was induced. Host immune responses to viral infection, inflammation, apoptosis and lymphocyte infiltration, in the transduced glands, were assessed. The DNA viral vectors induced local lymphocyte infiltration and apoptosis. In contrast, the retroviral vectors did not induce an immune response. Our results describe the outcome of salivary gland infection with each of the five different viral vectors and indicate their advantages and limitations for transferring genes to the salivary glands.

Reduced expression of gamma interferon in serum and marked lymphoid depletion induced by Porphyromonas gingivalis increase murine morbidity and mortality due to cytomegalovirus infection.

Porphyromonas gingivalis, a gram-negative anaerobe, is a major etiological agent of severe forms of periodontal disease. Although periodontal disease is considered a localized disease, accumulating evidence indicates that it may lead to a predisposition to a decline in immunocompetence. Human cytomegalovirus (CMV) commonly infects all human populations without producing significant clinical symptoms. Immunocompromised patients usually develop a primary or reactivated CMV infection, which is associated with high rates of morbidity and mortality. The aim of this study was to determine whether P. gingivalis increases animal susceptibility to CMV infection. Mice were inoculated with CMV and infected locally with P. gingivalis 3 days after the virus inoculation. Mortality rates were monitored, and traces of viral DNA and bacterial infection were detected systemically by using real-time PCR. Local and systemic cytokine secretion was measured, and histological sections were used to assess the pathological state of infected organs. P. gingivalis- and CMV-coinfected mice showed dramatically higher mortality rates than mice infected with P. gingivalis or CMV only. Although the organs of coinfected mice exhibited decreased viral titers, distinct necrosis and tissue damage were more evident in the livers and spleens of these mice than in those of mice infected with CMV only. Furthermore, systemic gamma interferon levels were decreased in coinfected mice, and marked lymphoid depletion was observed in their necrotic organs. In parallel control Escherichia coli-CMV coinfection experiments, the mortality and pathological results were the same as those found in mice infected with CMV only. Our results suggest a specific influence of P. gingivalis on the mouse immune response, causing increased susceptibility to CMV infection.