Candida albicans Mannosidases, Dfg5 and Dcw1, Are Required for Cell Wall Integrity and Pathogenesis.

Candida albicans is an oral mucosal commensal fungus that transforms into an opportunistic pathogen under specific conditions, including immunosuppression. It causes oral and systemic candidiasis, which results in a significant health burden. Furthermore, an alarming rise in antifungal drug resistance in Candida species raises the urgent need for novel drugs and drug targets. C. albicans Dfg5 and Dcw1 are homologous cell wall alpha-1,6-mannosidases with critical functions and represent potential new drug targets. Our past studies have shown that Dfg5 and Dcw1 function in cell wall biogenesis through the cross-linking of glycoproteins into the cell wall, thus playing a key role in cell wall integrity. Additionally, Dfg5 and Dcw1 are required for hyphal morphogenesis. However, the exact functions of Dfg5 and Dcw1 in cell wall integrity, hyphal morphogenesis, and pathogenesis are not known. In this study, we determined the relation of Dfg5 and Dcw1 with Hog1 MAPK, which plays a key role in cell wall integrity via the regulation of chitin synthesis in C. albicans. Additionally, we also determined the effects of dfg5 and dcw1 mutations on the gene expression of transcriptional regulators of hyphal morphogenesis. Furthermore, we determined the effects of dfg5 and dcw1 mutations on pathogenesis in a mouse model of oral candidiasis. Our results demonstrate that dfg5 and dcw1 mutations, as well as a hog1 knockout mutation, result in the dysregulation of chitin synthesis, resulting in a cell separation defect. Heterozygous and conditional mutations in dfg5 and dcw1 resulted in decreased transcriptional levels of cst20, a positive regulator of hyphal morphogenesis. However, dfg5 and dcw1 mutations resulted in increased levels of all the five negative regulators of hyphal morphogenesis-Tup1, Nrg1, Mig1, Rbf1, and Rfg1. Additionally, Tup1 levels were significantly higher than other negative regulators, indicating that Dfg5 and Dcw1 function in hyphal morphogenesis by repressing Tup1. Finally, dfg5 and dcw1 mutations affected the ability of C. albicans to cause oral candidiasis in mice. Thus, the cell wall glycosidases Dfg5 and Dcw1 are required for virulence and pathogenesis and represent novel drug targets.

The relationship between periodontal and kidney disease: a critical review.

Periodontal disease has been associated with various systemic diseases including kidney disease. However, a causal relationship is yet to be established. One possible association is that periodontitis may cause an increased inflammatory response in kidney disease patients which in turn destroys endothelial vasculature. This may contribute to development of risk factors of kidney disease such as diabetic neuropathy and cardiovascular events leading the progression and mortality in kidney disease patients. The role of periodontal inflammation driving kidney disease is still under investigation. This review article highlights the role of periodontal inflammation in the development and progression of kidney disease. It is crucial that dental practitioners and nephrologists understand the association between periodontal and kidney disease. Early periodontal screening and educating patients about the importance of good oral hygiene may play an important role in prevention of progression of kidney disease.

Knowledge, attitude and oral care practices for preventing ventilator-associated pneumonia among critical care nurses - A questionnaire study.

BACKGROUND: A cross-sectional study was conducted among intensive care unit (ICU) nurses in private hospitals in India to identify knowledge and practice of ICU nurses in the prevention of ventilator-associated pneumonia (VAP). METHODS: Knowledge of 135 nurses working in ICU was tested using a questionnaire consisting of 18 questions. Fourteen forms were excluded from the statistical analysis due to incomplete data entry by the participants. The data of 121 filled questionnaires were analyzed. The information letters, consent forms, and questionnaires were handed to ICU nurses by Research assistant. Data were coded and entered into SPSS version for descriptive and inferential statistics. RESULTS: A majority of the participants perceived oral care as a necessity in all critically ill patients. Nurses were generally aware of the most likely mechanism of acquiring pneumonia. The type and frequency of oral care varied widely. Many of them stated that they had adequate supplies to provide oral care. Although a majority of nurses had some formal training in oral care, they would appreciate an opportunity to enhance and improve their knowledge and skills. CONCLUSION: The methods of oral care provided vary widely. In summary, randomized controlled trial to date has demonstrated that tooth brushing is associated with a trend toward lower rates of VAP in intubated mechanically ventilated critically ill patients. But it is also to be noted that there was no clear difference between electric and manual tooth brushing. In-house training and workshop can provide required skills needed for the betterment of the treatment provided.

Treponema denticola stimulates Oncostatin M cytokine release and de novo synthesis in neutrophils and macrophages.

Oncostatin M (OSM) is a pleiotropic cytokine elevated in a number of inflammatory conditions including periodontal disease. OSM is produced by a variety of immune cells and has diverse functionality such as regulation of metabolic processes, cell differentiation, and the inflammatory response to bacterial pathogens. The oral cavity is under constant immune surveillance including complementary neutrophil and macrophage populations, due to a persistent symbiotic bacterial presence. Periodontal disease is characterized by a dysbiotic bacterial community, with an abundance of Treponema denticola. Despite strong associations with severe periodontal disease, the source and mechanism of the release of OSM have not been defined in the oral cavity. We show that OSM protein is elevated in the gingival epithelium and immune cell infiltrate during periodontal disease. Furthermore, salivary and oral neutrophil OSM is elevated in correlation with the presence of T. denticola. In an air pouch infection model, T. denticola stimulated higher levels of OSM than the oral pathogen Porphorymonas gingivalis, despite differential recruitment of innate immune cells suggesting T. denticola has distinct properties to elevate OSM levels. OSM release and transcription were increased in isolated human blood, oral neutrophils, or macrophages exposed to T. denticola in vitro as measured by ELISA, qPCR, and microscopy. Using transcription, translation, and actin polymerization inhibition, we found that T. denticola stimulates both OSM release through degranulation and de novo synthesis in neutrophils and also OSM release and synthesis in macrophages. Differential induction of OSM by T. denticola may promote clinical periodontal disease.

Candidal carriage in saliva and subgingival plaque among smokers and non-smokers with chronic periodontitis-a cross-sectional study.

BACKGROUND AND OBJECTIVES: Studies of gum or periodontal disease have focused mainly on bacterial pathogens. However, information related to fungal species in the saliva and subgingival mileu is particularly lacking in smokers with periodontitis. This cross-sectional study compared the prevalence of various Candida species in saliva and subgingival plaque samples of smokers and non-smokers with periodontal disease. METHODOLOGY: Study subjects were recruited into three group-Group 1: Smokers with chronic periodontitis (N = 30), Group 2: Non-smokers with chronic periodontitis (N = 30) and Group 3: Healthy controls (N = 30). Clinical parameters recorded included plaque index (PI), gingival index (GI), periodontal probing depth (PPD) and clinical attachment loss (CAL). Saliva and subgingival plaque samples were collected from subjects from the above groups. The collected samples were processed for isolation and identification of various Candida species using CHROMagar chromogenic media. Additionally, antifungal susceptibility tests were performed for the isolated Candida species in order to assess antifungal drug resistance to fluconazole and voriconazole. RESULTS: Prevalence of Candida species in saliva samples was quantified as 76.6% in Group 1, 73.3% in Group 2 and 36.6% in Group 3 and statistically significant differences were observed between groups 1 & 3. Prevalence of Candida species in subgingival plaque samples was quantified as 73.3% in Group 1, 66.6% in Group 2 and 60% in Group 3 and no statistically significant differences were observed between groups. Candida albicans was the most frequently isolated species followed by Candida krusei and Candida tropicalis. A positive correlation was observed for smoking exposure, pack years and Candida colonization. A marginally significant positive correlation was observed between Candida colonization and increasing pocket depth and attachment loss. Antifungal drug resistance was mainly observed for Candida krusei in both saliva and subgingival plaque samples. CONCLUSION: Based on the results we can conclude that oral candidal carriage is significantly increased in smokers with periodontal disease. Mechanistic studies are needed to understand the importance of Candida species in periodontal disease.

Candida albicans cell wall integrity transcription factors regulate polymicrobial biofilm formation with Streptococcus gordonii.

Polymicrobial biofilms play important roles in oral and systemic infections. The oral plaque bacterium Streptococcus gordonii is known to attach to the hyphal cell wall of the fungus Candida albicans to form corn-cob like structures in biofilms. However, the role of C. albicans in formation of polymicrobial biofilms is not completely understood. The objective of this study was to determine the role of C. albicans transcription factors in regulation of polymicrobial biofilms and antibiotic tolerance of S. gordonii. The proteins secreted by C. albicans and S. gordonii in mixed planktonic cultures were determined using mass spectrometry. Antibiotic tolerance of S. gordonii to ampicillin and erythromycin was determined in mixed cultures and mixed biofilms with C. albicans. Additionally, biofilm formation of S. gordonii with C. albicans knock-out mutants of 45 transcription factors that affect cell wall integrity, filamentous growth and biofilm formation was determined. Furthermore, these mutants were also screened for antibiotic tolerance in mixed biofilms with S. gordonii. Analysis of secreted proteomes resulted in the identification of proteins being secreted exclusively in mixed cultures. Antibiotic testing showed that S. gordonii had significantly increased survival in mixed planktonic cultures with antibiotics as compared to single cultures. C. albicans mutants of transcription factors Sfl2, Brg1, Leu3, Cas5, Cta4, Tec1, Tup1, Rim101 and Efg1 were significantly affected in mixed biofilm formation. Also mixed biofilms of S. gordonii with mutants of C. albicans transcription factors, Tec1 and Sfl2, had significantly reduced antibiotic tolerance as compared to control cultures. Our data indicates that C. albicans may have an important role in mixed biofilm formation as well as antibiotic tolerance of S. gordonii in polymicrobial biofilms. C. albicans may play a facilitating role than being just an innocent bystander in oral biofilms and infections.

The microbiome and lung cancer.

It has become increasingly clear that we live in a symbiotic relationship with microbes within us. We are just beginning to unravel the nature and strength of this relationship and its impact on both physiology and by extension, pathology. While microorganisms have long been known to have carcinogenic potential, their role may have been underestimated. The knowledge of the role of the microbiome in carcinogenesis is rapidly evolving. This evolution has reached a tipping point with current omics technologies used for cataloguing the microbiome. The lung is an organ constantly exposed to the environment. It is now clear that the lung has a distinct microbiome and that this may influence the development of lung cancer. In addition, evidence suggests that this microbiome originates from the oral microbiome. This review summarizes current knowledge about the role of microbiome, especially the oral and lung microbiome in human lung cancer. The goal of the manuscript is to provide a summary of this rapidly evolving field while providing a context of the general role of the microbiome in carcinogenesis. In addition, a primer of the current technology used in evaluating the microbiome is provided to familiarize the practicing clinician with the experimental methods used to generate the information that will likely impact the field of lung cancer.

Functions of Candida albicans cell wall glycosidases Dfg5p and Dcw1p in biofilm formation and HOG MAPK pathway.

BACKGROUND: Candida albicans is a commensal fungus that inhabits the oral mucosal surface and causes oral and systemic candidiasis. Oral candidiasis most commonly occurs in patients with AIDS, denture wearers and newborn children. Systemic candidiasis occurs mainly in immunocompromised patients and patients admitted to hospitals for prolonged periods. C. albicans homologous genes, DFG5 and DCW1, encode for two closely related cell wall proteins with putative glycosyltransferase enzyme activity and C-terminal GPI-anchors. Past studies have shown that individual DFG5 and DCW1 mutations are viable but simultaneous deletion of DFG5 and DCW1 in C. albicans results in lethality. However, the exact functions of these cell wall based enzymes, which represent potential drug targets, are not understood. METHODS: C. albicans DFG5/DCW1 heterologous and conditional double mutant strains were assessed for growth and biofilm formation in comparison to wild type and parental strains. Cell wall and heat stress susceptibility of the mutant and control strains were assessed using agar spotting assays. Growth was assessed under normal and osmotic stress conditions along with light microscopy imaging. Biofilm dry weight and microscopic imaging analysis of biofilms was performed. Hypha formation in response to serum was analyzed using light microscopy imaging. Western blot analysis of mutant strains and control strains was performed to assess Hog1 basal levels and phosphorylation status. RESULTS: Analysis of the heterologous mutants indicated that Dfg5p is more important for growth while Dcw1p appeared to play a role in cell wall integrity response. The conditional double mutant was observed to be less resistant to cell wall stress. However, growth of the mutants was similar under control and osmotic stress conditions. The mutants were also able to grow similar to wild type under heat stress. Biofilm formation was reduced in the mutants where DFG5 was deleted or suppressed. Hyphal morphogenesis was reduced although germ tube formation was observed in the biofilms of the mutant strains. Basal Hog1 protein levels were reduced or absent in the DFG5 and DCW1 mutants. However, osmotic stress was able to induce Hog1 protein levels comparable to wild type. Hog1 phosphorylation appeared to be slightly reduced although not significantly. In addition to biofilm assays, serum dose response imaging analysis indicated that hyphae formation in DFG5 and DCW1 mutants was defective. CONCLUSIONS: These data indicate that DFG5 and DCW1 are required for hyphal morphogenesis and biofilm formation in C. albicans. These functions may be regulated via basal Hog1 MAPK which is required for transcriptional regulation of chitin synthesis.

Calcium Sulfate as Bone Replacement Graft in Management of Localized Aggressive Periodontitis: A One-year Follow-up Report.

We report a case of localized aggressive periodontitis (LAP), which occurred during fixed orthodontic treatment in a 16-year-old African-American female patient. Oral hygiene instruction, removal of orthodontic bands and nonsurgical periodontal therapy were followed by surgical treatment of multiple sites using calcium sulfate as a synthetic bone graft material and collagen membrane as a barrier to achieve guided tissue regeneration. One-year follow-up of the case demonstrated that use of calcium sulfate as a synthetic bone substitute may provide favorable outcome in LAP patients. Furthermore, LAP patients undergoing orthodontic treatment can be managed successfully without tooth morbidity.

Management of Gingival Overgrowth in a Cardiac Transplant Patient Using Laser-Assisted Gingivectomy/Gingivoplasty.

Drug-induced gingival overgrowth (DIGO) is an oral clinical manifestation associated with certain medications such as immunosuppressants that are administered to organ transplant patients to prevent graft rejection. In patients with cardiac transplants, management of DIGO is critical. In such patients, plaque biofilm accumulation at the gingival interface might be detrimental as it may lead to transient bacteremia as well as systemic inflammation resulting in thromboembolic events. This case report describes the management of DIGO in a cardiac transplant recipient by change of immunosuppressant medication, non-surgical periodontal therapy and laser-assisted gingivectomy.

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall.

A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix.

Oral Candida Carriage and Morphotype Differentiation in Chronic Periodontitis Patients with and without Diabetes in the Indian Sub-Continent.

The aim of this study was to assess the oral Candida carriage and morphotype differentiation of Candida species in chronic periodontitis patients, with and without diabetes mellitus. This cross sectional study included 30 subjects in the age range of 40-60 years, who were divided into two groups: 15 chronic periodontitis only (CP) patients, and 15 chronic periodontitis patients with diabetes (CPD). Clinical measurements included plaque index (PI), gingival index (GI), probing depth (PD), clinical attachment level (CAL), and fasting blood sugar level (FBS). The unstimulated whole saliva samples were collected for fungal analysis. Candida carriage was analyzed by measuring colony forming units (CFU) following the culture of samples. Qualitative morphotype differentiation of Candida species from yeast to hyphal form was analyzed using Periodic acid-Schiff (PAS) staining. There was no statistically significant difference between CP and CPD groups for the periodontal parameters. However, a significantly higher Candida species CFU count was found in CPD (0.33 ± 0.23) as compared to CP (0.05 ± 0.04) group. This pilot study suggests that the occurrence of Candida species is higher in the saliva of chronic periodontitis patients with diabetes as compared to patients with chronic periodontitis alone.

Oral biofilms, oral and periodontal infections, and systemic disease.

PURPOSE: Oral biofilms harbor several hundreds of species of bacteria as well as spirochetes, protozoa, fungi and viruses. The composition of the oral biofilm varies from health to disease. It is the source of microorganisms that cause dental and periodontal infections. Oral infections and periodontal disease have been implicated in the etiopathogenesis of several important chronic systemic diseases.

WSC-1 and HAM-7 are MAK-1 MAP kinase pathway sensors required for cell wall integrity and hyphal fusion in Neurospora crassa.

A large number of cell wall proteins are encoded in the Neurospora crassa genome. Strains carrying gene deletions of 65 predicted cell wall proteins were characterized. Deletion mutations in two of these genes (wsc-1 and ham-7) have easily identified morphological and inhibitor-based defects. Their phenotypic characterization indicates that HAM-7 and WSC-1 function during cell-to-cell hyphal fusion and in cell wall integrity maintenance, respectively. wsc-1 encodes a transmembrane protein with extensive homology to the yeast Wsc family of sensor proteins. In N. crassa, WSC-1 (and its homolog WSC-2) activates the cell wall integrity MAK-1 MAP kinase pathway. The GPI-anchored cell wall protein HAM-7 is required for cell-to-cell fusion and the sexual stages of the N. crassa life cycle. Like WSC-1, HAM-7 is required for activating MAK-1. A Δwsc-1;Δham-7 double mutant fully phenocopies mutants lacking components of the MAK-1 MAP kinase cascade. The data identify WSC-1 and HAM-7 as the major cell wall sensors that regulate two distinct MAK-1-dependent cellular activities, cell wall integrity and hyphal anastomosis, respectively.

The Neurospora crassa dfg5 and dcw1 genes encode α-1,6-mannanases that function in the incorporation of glycoproteins into the cell wall.

The covalent cross-linking of cell wall proteins into the cell wall glucan/chitin matrix is an important step in the biogenesis of the fungal cell wall. We demonstrate that the Neurospora crassa DFG5 (NCU03770) and DCW1 (NCU08127) enzymes function in vivo to cross-link glycoproteins into the cell wall. Mutants lacking DFG5 or DCW1 release slightly elevated levels of cell wall proteins into their growth medium. Mutants lacking both DFG5 and DCW1 have substantially reduced levels of cell wall proteins in their cell walls and release large amounts of known cell wall proteins into the medium. DFG5 and DCW1 are members of the GH76 family of glycosyl hydrolases, which have specificity to recognize and cleave α-1,6-mannans. A model for incorporation of glycoproteins into the cell wall through the α-1,6-mannan core of the N-linked galactomannan is presented. In this model, DFG5 and DCW1 recognize the N-linked galactomannan present on glycoproteins and cross-link it into the cell wall glucan/chitin matrix.

α-1,6-Mannosylation of N-linked oligosaccharide present on cell wall proteins is required for their incorporation into the cell wall in the filamentous fungus Neurospora crassa.

The enzyme α-1,6-mannosyltransferase (OCH-1) is required for the synthesis of galactomannans attached to the N-linked oligosaccharides of Neurospora crassa cell wall proteins. The Neurospora crassa och-1 mutant has a tight colonial phenotype and a defective cell wall. A carbohydrate analysis of the och-1 mutant cell wall revealed a 10-fold reduction in the levels of mannose and galactose and a total lack of 1,6-linked mannose residues. Analysis of the integral cell wall protein from wild-type and och-1 mutant cells showed that the mutant cell wall had reduced protein content. The och-1 mutant was found to secrete 18-fold more protein than wild-type cells. Proteomic analysis of the proteins released by the mutant into the growth medium identified seven of the major cell wall proteins. Western blot analysis of ACW-1 and GEL-1 (two glycosylphosphatidylinositol [GPI]-anchored proteins that are covalently integrated into the wild-type cell wall) showed that high levels of these proteins were being released into the medium by the och-1 mutant. High levels of ACW-1 and GEL-1 were also released from the och-1 mutant cell wall by subjecting the wall to boiling in a 1% SDS solution, indicating that these proteins are not being covalently integrated into the mutant cell wall. From these results, we conclude that N-linked mannosylation of cell wall proteins by OCH-1 is required for their efficient covalent incorporation into the cell wall.

Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans.

Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS-PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and six secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and nine secreted proteins. Most of the C. albicans cell wall proteins are found in the cell walls of both yeast and hyphae cells, but some cell type-specific cell wall proteins were observed. The analyses showed that the pattern of cell wall proteins present in N. crassa vegetative hyphae and conidia (asexual spores) are quite different. Almost all of the cell wall proteins identified in N. crassa have close homologs in the sequenced fungal genomes, suggesting that these proteins have important conserved functions within the cell wall.

Role of the cell wall integrity and filamentous growth mitogen-activated protein kinase pathways in cell wall remodeling during filamentous growth.

Many fungal species including pathogens exhibit filamentous growth (FG) as a means of foraging for nutrients. Genetic screens were performed to identify genes required for FG in the budding yeast Saccharomyces cerevisiae. Genes encoding proteins with established functions in transcriptional activation (MCM1, MATalpha2, PHD1, MSN2, SIR4, and HMS2), cell wall integrity (MPT5, WSC2, and MID2), and cell polarity (BUD5) were identified as potential regulators of FG. The transcription factors MCM1 and MATalpha2 induced invasive growth by promoting diploid-specific bipolar budding in haploid cells. Components of the cell wall integrity pathway including the cell surface proteins Slg1p/Wsc1p, Wsc2p, Mid2p, and the mitogen-activated protein kinase (MAPK) Slt2p/Mpk1p contributed to multiple aspects of the FG response including cell elongation, cell-cell adherence, and agar invasion. Mid2p and Wsc2p stimulated the FG MAPK pathway through the signaling mucin Msb2p and components of the MAPK cascade. The FG pathway contributed to cell wall integrity in parallel with the cell wall integrity pathway and in opposition with the high osmolarity glycerol response pathway. Mass spectrometry approaches identified components of the filamentous cell wall including the mucin-like proteins Msb2p, Flo11p, and subtelomeric (silenced) mucin Flo10p. Secretion of Msb2p, which occurs as part of the maturation of the protein, was inhibited by the ss-1,3-glucan layer of the cell wall, which highlights a new regulatory aspect to cell wall remodeling in this organism. Disruption of ss-1,3-glucan linkages induced mucin shedding and resulted in defects in cell-cell adhesion and invasion of cells into the agar matrix.

Long wave ultrasound may enhance bone regeneration by altering OPG/RANKL ratio in human osteoblast-like cells.

Human osteoblast cell line (MG63) cells were treated with long wave (45 kHz, intensity 30 mW/cm(2)) continuous ultrasound (US) for 5 min and incubated for various time periods following the treatment. The reverse transcriptase polymerase chain reaction (RT-PCR) technique was used for observing the genetic expression and real-time PCR for quantitative analysis of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) along with alkaline phosphatase (ALP), an early bone marker, and osteocalcin (OCN) a late marker. ELISA was performed to estimate the amount of the cytokine released into the culture media. The osteoblasts responded to US by significantly upregulating both the OPG mRNA and protein levels. There was no RANKL mRNA expression observed in both the US and control groups and the protein levels were also very low in both groups. There was also no TNF-alpha expression and the TNF-alpha protein levels were insignificant. ALP and OCN mRNA were significantly upregulated in the US group. To our knowledge, this is the first study that shows the effect of US on OPG, RANKL and TNF-alpha expression. US appears to upregulate OPG and may downregulate RANKL production. From these findings, we conclude that therapeutic ultrasound may increase bone regeneration by altering the OPG/RANKL ratio in the bone microenvironment.