Effect of strontium fluoride on mechanical and remineralization properties of enamel: An in-vitro study on a modified orthodontic adhesive.

OBJECTIVES: Evaluate the ability of strontium fluoride on bond strength and enamel integrity after incorporation within orthodontic adhesive system as a delivery vehicle. METHODS: Experimental orthodontic adhesive system Transbond™ XT were modified with 1% Sr2+, 0.5% SrF2, 1% strontium, 0.5% Sr2+, 1% F-, 0.5% F-, and no additions were control. Mixing of formulation was monitored using Fourier transform infrared spectroscopy. Small-molecule drug-discovery suite was used to gain insights into Sr2+, F-, and SrF2 binding. Shear bond testing was performed after 6-months of ageing. Enamel blocks were cut, and STEM pictures were recorded. Specimens were indented to evaluate elastic modulus. Raman microscope was used to collect Raman spectra and inspected using a scanning electron microscope. Crystal structural analysis was performed using X-ray diffraction. Effect of material on cellular proliferation was determined. Confocal was performed to evaluate the effect of formulation on biofilms. RESULTS: FTIR of modified adhesives depicted peak changes within range due to various functional groups existing within samples. TEM represented structurally optimized hexagonal unit-cell of hydroxyapatite. Mean shear bond strength is recorded highest for Transbond XT with 1% SrF2. Dead bacterial percentage appeared higher in 0.5% SrF2 and 1% F- specimens. Crystal lengths showed an increase in 0.5% and 1% SrF2 specimens. Phase contrast within TEM images showed a union of 0.5% SrF2 crystal with enamel crystal with higher elastic modulus and highly mineralized crystalline hydroxyapatite. Intensity of ν1 PO43- and ν1 CO32- along with carbonate - / ν1PO43- ratio displayed good association with strontium fluoride. The formulation showed acceptable cell biocompatibility (p < 0.353). All specimens displayed characteristic diffraction maxima of different apatite angles within XRD. SIGNIFICANCE: Experimental results suggested good biocompatibility, adequate mechanical strength, and far-ranging crystallization ability. This would provide a new strategy to overcome the two major challenges of fixed orthodontics, biofilm growth, and demineralization of enamel.

Cathepsin-K inhibition enhances anti-cancerous activity within oral squamous cell carcinoma cells: Uncloaking the potency of new K21 formulation.

BACKGROUND: The ability of cancer cells to be invasive and metastasize depend on several factors, of which the action of protease activity takes center stage in disease progression. PURPOSE/OBJECTIVE: To analyze function of new K21 molecule in the invasive process of oral squamous cell carcinoma (OSCC) cell line. MATERIALS & METHODS: The Fusobacterium (ATCC 23726) streaks were made, and pellets were resuspended in Cal27 (ATCC CRL-2095) OSCC cell line spheroid cell microplate. Cells were seeded and Lysotracker staining performed for CathepsinK red channel. Cell and morphology were evaluated using Transmission Electron microscopy. Thiobarbituric acid assay was performed. OSCC was analyzed for Mic60. Raman spectra were collected from the cancer cell line. L929 dermal fibroblast cells were used for Scratch Assay. ELISA muti arrays were used for cytokines and matrix molecules. Internalization ability of fibroblast cells were also analyzed. Structure of K21 as a surfactant molecule with best docked poses were presented. RESULTS: Decrease in lysosomal staining was observed after 15 and 30 min of 0.1% treatment. Tumor clusters were associated with cell membrane destruction in K21 primed cells. There was functional silencing of Mic60 via K21, especially with 1% concentration with reduced cell migration and invasiveness. Raman intensity differences were seen at 700 cm-1, 1200 cm-1 and 1600 cm-1 regions. EVs were detected within presence of fibroblast cells amongst K21 groups. Wound area and wound closure showed the progress of wound healing. CONCLUSION: Over expression of CatK can be reduced by a newly developed targeted K21 based drug delivery system leading to reduced migration and adhesion of oral squamous cell carcinoma cells. The K21 drug formulation can have great potential for cancer therapies due to targeting and cytotoxicity effects.

In-vitro evaluation of the effectiveness of polyphenols based strawberry extracts for dental bleaching.

To formulate a dental bleaching agent with strawberry extract that has potent bleaching properties and antimicrobial efficacy. Enamel specimens (3 × 3 × 2 mm3) were prepared. Quaternary Ammonium Silane (CaC2 enriched) was homogenized with fresh strawberries: Group 1: supernatant strawberry (10 g) extract < Group 2: supernatant strawberry (10 g) extract + 15%HA (Hydroxyapatite) < Group 3: supernatant strawberry (10 g) extract + 15% (HA-2%k21) < Group 4: supernatant strawberry (20 g) extract only (20 g strawberries) < Group 5: supernatant strawberry (20 g) extract + 15% HA < Group 6: supernatant strawberry (20 g) extract + 15% (HA-2%K21) < Group 7: In-office Opalescence Boost 35%. Single-colony lactobacillus was examined using confocal microscopy identifying bacterial growth and inhibition in presence of bleaching agents using 300 µL aliquot of each bacterial culture. Images were analysed by illuminating with a 488 nm argon/helium laser beam. Colour difference (∆E00) was calculated using an Excel spreadsheet implementation of the CIEDE2000 colour difference formula and colour change measured between after staining and after bleaching. Scanning electron microscope was used to image specimens. Raman spectra were collected, and enamel slices were used for STEM/TEM analysis. HPLC was used for strawberry extract analysis. Nano-indentation was performed and X-ray photoelectron spectroscopy. Antioxidant activity was determined along with molecular simulation. hDPSCs were expanded for Alamar Blue Analysis and SEM. Mean colour change was significantly reduced in group 1 compared to other groups (p < 0.05). CLSM showed detrimental effects of different strawberry extracts on bioflms, especially with antimicrobial (p < 0.05). Groups 1, 2 and 3 showed flatter/irregular surfaces with condensation of anti-microbial in group 3. In strawberry specimens, bands predominate at 960 cm-1. HPLC determined the strawberry extracts content. Molecular simulation verified interaction between calcium and polyphenol components. XPS peak-fitted high-resolution corresponding results of Ca2p3/2 and Ca2p1/2 for all k21 groups. Combination of 10 g strawberry extract supernatant and 15% (hydroxyapatite 2%k21) improved the whiteness and provided additional antimicrobial potential. The novel strawberry extract and antimicrobial based dental formulation had immediate bleaching effect without promoting significant changes in enamel morphology.

Establishing a technique for isolation and characterization of human periodontal ligament derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are extensively used in tissue regenerative procedures. One source of MSCs is the periodontal ligament (PDL) of teeth. Isolation of MSCs from extracted teeth is reasonably simple, being less invasive and presenting fewer ethical concerns than does the harvesting of MSC's from other sites. The objectives of this study were to isolate and characterize the PDL stem cells (PDLSC) from healthy adults' extracted teeth and then to characterize them by comparing them with bone-marrow derived MSCs (BMMSC). METHODS: The PDL tissue was scraped from the roots of freshly extracted teeth to enzymatically digest using collagenase. The cells were sub-cultured. Flow-cytometric analysis for the MSC surface-markers CD105, CD73, CD166, CD90, CD34, CD45 and HLA-DR was performed. To confirm the phenotype, total RNA was extracted to synthesize cDNA and which was then subjected to RT-PCR. The gene-expression for Oct4A, Sox2, NANOG and GAPDH was determined by gel-electrophoresis. To assess their multilineage potential, cells were cultured with osteogenic, chondrogenic and adipogenic medium and then stained by Alizarin-red, Alcian-blue and Oil-Red-O respectively. MSCs from the bone-marrow were processed similarly to serve as controls. RESULTS: The cells isolated from extracted teeth expanded successfully. On flow-cytometric analysis, the cells were positive for CD73, CD90, CD105, CD166 and negative for CD34, CD45 and HLA-DR. The PDLSCs expressed Oct4A, Sox2, and NANOG mRNA with GAPDH expression. Cells cultured in the osteogenic, chondrogenic and adipogenic media stained positive for Alizarin-red, Alcian-blue and Oil- Red-O respectively. The surface marker expression and the trilineage differentiation characteristics were comparable to those of the BMMSCs. CONCLUSIONS: The periodontal ligament tissue of extracted teeth is a potential source of therapeutically useful MSCs. Harvesting them is not invasive and are a promising source of MSC as the PDLSCs showed characteristics similar to those of the highly regarded MSC's derived from bone-marrow.

3D Clumps/Extracellular Matrix Complexes of Periodontal Ligament Stem Cells Ameliorate the Attenuating Effects of LPS on Proliferation and Osteogenic Potential.

BACKGROUND: The effects of lipopolysaccharide (LPS) on cell proliferation and osteogenic potential (OP) of MSCs have been frequently studied. OBJECTIVE: to compare the effects of LPS on periodontal-ligament-derived mesenchymal stem cells (PDLSCs) in monolayer and 3D culture. METHODS: The PDLSCs were colorimetrically assessed for proliferation and osteogenic potential (OP) after LPS treatment. The 3D cells were manually prepared by scratching and allowing them to clump up. The clumps (C-MSCs) were treated with LPS and assessed for Adenosine triphosphate (ATP) and OP. Raman spectroscopy was used to analyze calcium salts, DNA, and proline/hydroxyproline. Multiplexed ELISA was performed to assess LPS induced local inflammation. RESULTS: The proliferation of PDLSCs decreased with LPS. On Day 28, LPS-treated cells showed a reduction in their OP. C-MSCs with LPS did not show a decrease in ATP production. Principal bands identified in Raman analysis were the P-O bond at 960 cm-1 of the mineral component, 785 cm-1, and 855 cm-1 showing qualitative changes in OP, proliferation, and proline/hydroxyproline content, respectively. ELISA confirmed increased levels of IL-6 and IL-8 but with the absence of TNF-α and IL-1ß secretion. CONCLUSIONS: These observations demonstrate that C-MSCs are more resistant to the effects of LPS than cells in monolayer cell culture. Though LPS stimulation of C-MSCs creates an early pro-inflammatory milieu by secreting IL-6 and IL-8, PDLSCs possess inactivated TNF promoter and an ineffective caspase-1 activating process.

Antimicrobial and self-crosslinking potential of experimentally developed dioctadecyldimethyl ammonium bromide and riboflavin dentin adhesive.

The aim of the study is to investigate a new formulation, based on dioctadecyldimethyl ammonium-bromide (QA) and riboflavin (RF), combining antimicrobial activities and protease inhibitory properties with collagen crosslinking without interference to bonding capabilities in a rabbit model. Quaternary ammonium riboflavin (QARF) experimental adhesives modified with dioctadecyldimethyl ammonium-bromide and riboflavin were bonded (0.5/1.0/2.0%) to rabbit dentin to investigate for pulpal-histology, interfacial-morphology, transmission electron microscopy, mechanical properties, collagen crosslinking, micro-Raman analysis, antimicrobial, and anti-protease activities. Collagen type-I molecules were generated using molecular-docking. Odontoblasts appeared with normal histology, were seen in controls with no inflammatory cells detected in 0.5% specimens at day 7 and mild inflammatory response at day 30. In QARF 2.0%, inflammatory cells were not detected at day 7 and 30 (p < .05). Dentinal tubules are seen with intact collagen surface in 1% specimens. Resin penetrated inside 1% adhesive specimens with few irregularly funnel-shaped tags. Transmission electron microscopy showed thinner collagen in all specimens except 1% QARF specimens. Biofilms were influenced by QARF adhesives. Elastic moduli were significantly higher in 1.0% and 2.0% QARF adhesive specimens with a significant increase in total crosslinks. Stable amide groups with anti-protease activity was observed in QARF groups. Charged residues were seen in the triple helix hCOL3A1, Gly489-Gly510 after stabilisation with formulation. The 1% QARF modified adhesives improved biochemical and biomechanical properties of rabbit dentin.

Immunohistochemical expression of Tazarotene-induced Gene 3 in oral squamous cell carcinoma.

BACKGROUND: The prognosis of hyperproliferative skin lesions, such as psoriasis, basal cell carcinoma, and non-melanoma skin cancers, is significantly benefited from the levels of tazarotene-induced gene-1 (TIG3) expression and subsequent treatment with tazarotene. Such observations suggest that TIG3 could be used as a biomarker for apoptosis, differentiation, and proliferation. The current study aimed to evaluate the expression of TIG3 in normal oral mucosa (NOM) and oral squamous cell carcinoma (OSCC) compared with normal skin (NS) and skin squamous cell carcinoma (SSCC) using immunohistochemistry. METHODS: Seventeen cases each of SSCC, OSCC, NOM, and NS were evaluated. Each section was immunohistochemically stained with a rabbit polyclonal TIG3 antibody. The entire procedure was blinded and evaluated by 5 observers. Statistical analysis was performed using the chi-square test. RESULTS: There was a significant decrease in TIG3 protein expression in OSCC and SSCC compared with that in NOM and NS (P = 0.008). The progressive loss of expression was observed as the grade of both malignancies increased. However, there was no significant difference in the expression among the normal tissue groups and within SCC groups of similar grades. CONCLUSION: The present study suggests that the loss of TIG3 is an important event in carcinogenesis. TIG3 acts as a regulator of keratinocyte proliferation and terminal differentiation. Therefore, TIG3 could be a potential biomarker to differentiate aggressive and non-aggressive neoplasms.

Effect of lipopolysaccharide on cell proliferation and vascular endothelial growth factor secretion of periodontal ligament stem cells.

PURPOSE: Periodontal ligament stem cells (PDLSCs) have considerable potential for use as a means of achieving periodontal regeneration due to their noteworthy proliferative properties and secretory functions. In particular, PDLSCs secrete vascular endothelial growth factor (VEGF) which enhances angiogenesis and osteogenesis. The resulting repair and development of blood vessels and hard tissues which would occur in the presence of these cells could be central to an effective periodontal regeneration procedure.The bacterial biofilm of tooth surface related to the periodontium might provide either an inhibition or a stimulus to different factors involved in a regenerative process. Cell culture experiments have been investigated in vitro by adding lipopolysaccharide (LPS) to the culture medium but the effect of various concentration of LPS in these circumstances has not been investigated. Therefore, this study aimed to investigate the effect of LPS concentrations on proliferation of PDLSCs in vitro and on their secretion of VEGF. MATERIALS AND METHODS: PDLSCs were treated with 0, 5, 10 and 20 µg/mL of Escherichia coli LPS. At 48 and 96 h, total cell numbers of control and LPS treated PDLSCs were counted by haemocytometer under a microscope. The VEGF concentration in the conditioned media of the PDLSCs was measured by ELISA. RESULTS: Rate of cell proliferation of PDLSCs decreased significantly in all LPS treated groups at both 48 h and 96 h except for the group treated with 5 µg/mL of LPS at 48 h. At both 48 and 96 h, VEGF secretion from PDLSCs was reduced significantly at all three LPS concentrations. There was no statistically significant difference in cell proliferation and the amount of VEGF secretion of PDLSCs among the groups treated with different LPS concentrations. No statistically significant change was found in cell proliferation of LPS treated PDLSCs over time, whereas VEGF secretion of PDLSCs was found to have increased significantly with time despite the LPS treatment. CONCLUSIONS: LPS reduced cell proliferation and VEGF secretion of PDLSCs, suggesting that periodontal pathogens might reduce the capability of PDLSCs in periodontal regeneration. Yet, LPS treated PDLSCs remained viable and VEGF secretion increased significantly over time. Further research is needed to study the potential use of PDLSCs in periodontal regeneration and the relationship of biofilm LPS accumulations.

Image Montaging for Creating a Virtual Pathology Slide: An Innovative and Economical Tool to Obtain a Whole Slide Image.

Background. Microscopes are omnipresent throughout the field of biological research. With microscopes one can see in detail what is going on at the cellular level in tissues. Though it is a ubiquitous tool, the limitation is that with high magnification there is a small field of view. It is often advantageous to see an entire sample at high magnification. Over the years technological advancements in optics have helped to provide solutions to this limitation of microscopes by creating the so-called dedicated "slide scanners" which can provide a "whole slide digital image." These scanners can provide seamless, large-field-of-view, high resolution image of entire tissue section. The only disadvantage of such complete slide imaging system is its outrageous cost, thereby hindering their practical use by most laboratories, especially in developing and low resource countries. Methods. In a quest for their substitute, we tried commonly used image editing software Adobe Photoshop along with a basic image capturing device attached to a trinocular microscope to create a digital pathology slide. Results. The seamless image created using Adobe Photoshop maintained its diagnostic quality. Conclusion. With time and effort photomicrographs obtained from a basic camera-microscope set up can be combined and merged in Adobe Photoshop to create a whole slide digital image of practically usable quality at a negligible cost.

Role of serum ß-carotene in the diagnosis and prevention of oral squamous cell carcinoma - a case control study.

CONTEXT: Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck. It accounts for more than 90% of all malignancies Which occur in the head and neck region. The high incidence and mortality rate of oral cancer stimulates continuation of research on finding new diagnostic tools or markers for it. AIM: To evaluate the possible role of serum ß-carotene as a biochemical parameter in the diagnosis of oral squamous cell carcinoma. MATERIALS AND METHODS: The serumß-carotene levels in 40 patients with clinically diagnosed and histopathologically confirmed oral squamous cell carcinoma were estimated and the levels were compared with those of 40 healthy controls. The serum ß-carotene levels were estimated by using a spectrophotometer. The data which was obtained was analyzed statistically by using unpaired t-test. RESULTS: Subjects with oral squamous cell carcinoma showed significantly lower levels of mean serum ß-carotene (149.95 ± 61.29) as compared to those seen in controls (278.19 ± 90.12). CONCLUSION: The results of the present study are encouraging and they suggest that the estimation of the low levels of ß-carotene in the patients with oral squamous cell carcinoma may be a useful diagnostic tool for making the diagnosis of oral squamous Cell carcinoma and thereby improving the prognosis of this dreaded disease.

Diagnostic efficacy of saliva for dengue - a reality in near future? A piloting initiative.

BACKGROUND: Dengue, a mosquito-transmitted viral infection presents variable symptoms, including death. Due to their increasing incidences, early detection and improved diagnoses of severe cases are of prime importance. Currently, viral antigens and antibodies are detected by traditional serological tests. However, the introduction of oral fluid as an alternative, has led to many researches. Hence, this prompted us to carry out a pilot study to evaluate the diagnostic efficacy of saliva in detecting dengue antibody by using Enzyme Linked Immunosorbent Assay (ELISA). AIM AND OBJECTIVES: To evaluate the presence of Dengue antibody in saliva and its sensitivity and specificity through ELISA. METHODOLOGY AND RESULTS: Twenty seropositive patients and twenty seronegative patients of Dengue were considered individually. Saliva samples collected from these patients were subjected to ELISA test for detection of Dengue antibody. A sensitivity of 100% and a specificity of 100% were obtained for making a diagnosis of Dengue infection. CONCLUSION: Many studies have been conducted by utilizing saliva as a diagnostic tool, especially in western population. Its advantages over venipuncture are many, especially as it is less invasive, safe, less expensive and as it allows large numbers of samples to be collected easily for screening and epidemiological purposes. In a developing tropical country like India, such a diagnostic tool has to be encouraged. Further research necessitates the implementation of saliva as a diagnostic tool.