A Novel Coated Suture Displays Antimicrobial Activity Without Compromising Structural Properties.

BACKGROUND: Treated or coated sutures promise to prevent contamination of wounds. PURPOSE: The purpose of the study was to coat surgical sutures with a new quaternary ammonium silane (QAS) antimicrobial compound at two different application temperatures and then to evaluate the resulting structural, physical, mechanical, and biological properties. STUDY DESIGN, SETTING, SAMPLE: In vitro and in vivo studies were conducted using male albino Wistar rats approved by the Joint Ethical Committee of IMU and Postgraduate Medical Institute, Lahore. Only suture samples, coated uniformly with verified presence of the compound and of adequate length were used. Samples which were not coated uniformly and with inadequate length or damaged were excluded. PREDICTOR VARIABLE: Predictor variables were sutures with and without QAS coatings and different temperatures. Sutures were coated with QAS at 0.5 and 1.0% wt/vol using the dip coating technique and sutures with and without QAS coating were tested at 25 and 40 °C temperatures. MAIN OUTCOME VARIABLE(S): Outcome variables of structural and physico-mechanical properties of QAS-coated and non-coated sutures were measured using Fourier transform infrared spectroscopy (for structural changes), confocal laser and scanning electron (for diameter changes), and tensile strength/modulus (for mechanical testing). Biologic outcome variables were tested (bacterial viability); macrophage cultures from Wistar rats were tested (M1/M2 polarization detecting IL-6 and IL-10). Macrophage cells were analyzed with CD80+ (M1) and CD163+ (M2). Chemotaxis index was calculated as a ratio of quantitative fluorescence of cells. COVARIATES: Not applicable. ANALYSES: Ordinal data among groups were compared using the Wilcoxon Mann-Whitney U test along with the comparison of histological analysis using the Wilcoxon Sign-rank test (P < .05). RESULTS: Fourier transform infrared spectroscopy peak at 1490 cm-1 confirmed the presence of QAS on suture's surfaces with a significant increase (P < .05) in diameter (0.99 ± 0.5-mm) and weight (0.77 ± 0.02-mg) observed for 1% QAS groups treated at 40 °C. Non-coated samples heated at 25 °C had significantly (P < .05) less diameters (0.22 ± 0.03-mm) and weights (0.26 ± 0.06-mg). Highest tensile strength/modulus was observed for 0.5% QAS-coated samples which also had significantly higher antibacterial characteristics than other sutures (P < .05). QAS-coated sutures significantly increased M1 and M2 markers. CONCLUSION AND RELEVANCE: QAS coating conferred antibacterial action properties without compromising the physical and mechanical properties of the suture.

Newly discovered clouting interplay between matrix metalloproteinases structures and novel quaternary Ammonium K21: computational and in-vivo testing.

AIMS AND OBJECTIVES: To analyze anti-MMP mode of action of Quaternary Ammonium Silane (QAS, codenamed as k21) by binding onto specific MMP site using computational molecular simulation and Anti-Sortase A (SrtA) mode of action by binding onto specific site using computational molecular simulation. MATERIALS AND METHODS: In silico Molecular Dynamics (MD) was used to determine the interactions of K21 inside the pocket of the targeted protein (crystal structure of fibroblast collagenase-1 complexed to a diphenyl-ether sulphone based hydroxamic acid; PDB ID: 966C; Crystal structure of MMP-2 active site mutant in complex with APP-derived decapeptide inhibitor. MD simulations were accomplished with the Desmond package in Schrödinger Drug Discovery Suite. Blood samples (~ 0.5 mL) collected into K2EDTA were immediately transferred for further processing using the Litron MicroFlow® PLUS micronucleus analysis kit for mouse blood according to the manufacturer's instructions. Bacterial Reverse Mutation Test of K21 Molecule was performed to evaluate K21 and any possible metabolites for their potential to induce point mutations in amino acid-requiring strains of Escherichia coli (E. coli) (WP2 uvrA (tryptophan-deficient)). RESULTS: Molecular Simulation depicted that K21 has a specific pocket binding on various MMPs and SrtA surfaces producing a classical clouting effect. K21 did not induce micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus, in the peripheral blood reticulocytes of male and female CD-1 mice when administered by oral gavage up to the maximum recommended dose of 2000 mg/kg. The test item, K21, was not mutagenic to Salmonella typhimurium (S. typhimurium) strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2 uvrA in the absence and presence of metabolic activation when tested up to the limit of cytotoxicity or solubility under the conditions of the test. CONCLUSION: K21 could serve as a potent protease inhibitor maintaining the physical and biochemical properties of dental structures.

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.

Biochemical changes and macrophage polarization of a silane-based endodontic irrigant in an animal model.

Silane-based/fully hydrolyzed, endodontic irrigant exhibiting antimicrobial properties, is prepared, and is hypothesized to control macrophage polarization for tissue repair. Albino wistar rats were injected with 0.1 ml root canal irrigant, and bone marrow cells procured. Cellular mitochondria were stained with MitoTracker green along with Transmission Electron Microscopy (TEM) performed for macrophage extracellular vesicle. Bone marrow stromal cells (BMSCs) were induced for M1 and M2 polarization and Raman spectroscopy with scratch assay performed. Cell counting was used to measure cytotoxicity, and fluorescence microscopy performed for CD163. Scanning Electron Microscopy (SEM) was used to investigate interaction of irrigants with Enterococcus faecalis. K21 specimens exhibited reduction in epithelium thickness and more mitochondrial mass. EVs showed differences between all groups with decrease and increase in IL-6 and IL-10 respectively. 0.5%k21 enhanced wound healing with more fibroblastic growth inside scratch analysis along with increased inflammation-related genes (ICAM-1, CXCL10, CXCL11, VCAM-1, CCL2, and CXCL8; tissue remodelling-related genes, collagen 1, EGFR and TIMP-2 in q-PCR analysis. Sharp bands at 1643 cm-1 existed in all with variable intensities. 0.5%k21 had a survival rate of BMSCs comparable to control group. Bacteria treated with 0.5%k21/1%k21, displayed damage. Antimicrobial and reparative efficacy of k21 disinfectant is a proof of concept for enhanced killing of bacteria across root dentin acquiring functional type M2 polarization for ethnopharmacological effects.

Highly Segregated Biocomposite Membrane as a Functionally Graded Template for Periodontal Tissue Regeneration.

Guided tissue regeneration (GTR) membranes are used for treating chronic periodontal lesions with the aim of regenerating lost periodontal attachment. Spatially designed functionally graded bioactive membranes with surface core layers have been proposed as the next generation of GTR membranes. Composite formulations of biopolymer and bioceramic have the potential to meet these criteria. Chitosan has emerged as a well-known biopolymer for use in tissue engineering applications due to its properties of degradation, cytotoxicity and antimicrobial nature. Hydroxyapatite is an essential component of the mineral phase of bone. This study developed a GTR membrane with an ideal chitosan to hydroxyapatite ratio with adequate molecular weight. Membranes were fabricated using solvent casting with low and medium molecular weights of chitosan. They were rigorously characterised with scanning electron microscopy, Fourier transform infrared spectroscopy in conjunction with photoacoustic sampling accessory (FTIR-PAS), swelling ratio, degradation profile, mechanical tensile testing and cytotoxicity using human osteosarcoma and mesenchymal progenitor cells. Scanning electron microscopy showed two different features with 70% HA at the bottom surface packed tightly together, with high distinction of CH from HA. FTIR showed distinct chitosan dominance on top and hydroxyapatite on the bottom surface. Membranes with medium molecular weight showed higher swelling and longer degradation profile as compared to low molecular weight. Cytotoxicity results indicated that the low molecular weight membrane with 30% chitosan and 70% hydroxyapatite showed higher viability with time. Results suggest that this highly segregated bilayer membrane shows promising potential to be adapted as a surface layer whilst constructing a functionally graded GTR membrane on its own and for other biomedical applications.

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.

A quaternary ammonium silane antimicrobial triggers bacterial membrane and biofilm destruction.

To study the antimicrobial effects of quaternary ammonium silane (QAS) exposure on Streptococcus mutans and Lactobacillus acidophilus bacterial biofilms at different concentrations. Streptococcus mutans and Lactobacillus acidophilus biofilms were cultured on dentine disks, and incubated for bacterial adhesion for 3-days. Disks were treated with disinfectant (experimental QAS or control) and returned to culture for four days. Small-molecule drug discovery-suite was used to analyze QAS/Sortase-A active site. Cleavage of a synthetic fluorescent peptide substrate, was used to analyze inhibition of Sortase-A. Raman spectroscopy was performed and biofilms stained for confocal laser scanning microscopy (CLSM). Dentine disks that contained treated dual-species biofilms were examined using scanning electron microscopy (SEM). Analysis of DAPI within biofilms was performed using CLSM. Fatty acids in bacterial membranes were assessed with succinic-dehydrogenase assay along with time-kill assay. Sortase-A protein underwent conformational change due to QAS molecule during simulation, showing fluctuating alpha and beta strands. Spectroscopy revealed low carbohydrate intensities in 1% and 2% QAS. SEM images demonstrated absence of bacterial colonies after treatment. DAPI staining decreased with 1% QAS (p < 0.05). Fatty acid compositions of dual specie biofilm increased in both 1% and 2% QAS specimens (p < 0.05). Quaternary ammonium silane demonstrated to be a potent antibacterial cavity disinfectant and a plaque inhibitor and can be of potential significance in eliminating caries-forming bacteria.

Functionally graded biomimetic biomaterials in dentistry: an evidence-based update.

Design and development of novel therapeutic strategies to regenerate lost tissue structure and function is a serious clinical hurdle for researchers. Traditionally, much of the research is dedicated in optimising properties of scaffolds. Current synthetic biomaterials remain rudimentary in comparison to their natural counterparts. The ability to incorporate biologically inspired elements into the design of synthetic materials has advanced with time. Recent reports suggest that functionally graded material mimicking the natural tissue morphology can have a more exaggerated response on the targeted tissue. The aim of this review is to deliver an overview of the functionally graded concept with respect to applications in clinical dentistry. A comprehensive understanding of spatiotemporal arrangement in fields of restorative, prosthodontics, periodontics, orthodontics and oral surgery is presented. Different processing techniques have been adapted to achieve such gradients ranging from additive manufacturing (three dimensional printing/rapid prototyping) to conventional techniques of freeze gelation, freeze drying, electrospinning and particulate leaching. The scope of employing additive manufacturing technique as a reliable and predictable tool for the design and accurate reproduction of biomimetic templates is vast by any measure. Further research in the materials used and refinement of the synthesis techniques will continue to expand the frontiers of functionally graded membrane based biomaterials application in the clinical domain.

Effect of photodynamic therapy and laser alone as adjunct to scaling and root planing on gingival crevicular fluid inflammatory proteins in periodontal disease: A systematic review.

BACKGROUND: To assess the efficacy of antimicrobial photodynamic therapy (aPDT) or laser therapy (LT) alone as an adjunct to scaling and root planing (SRP) on the gingival crevicular fluid (GCF) inflammatory proteins in periodontal disease. METHODS: Databases (MEDLINE via PubMed; EMBASE; Cochrane Central Register of Controlled Trials and Cochrane Oral Health Group Trials Register databases) were searched from 1980 up to and including July 2016. The addressed PICO question was: "What effect does aPDT and/or LT as an adjunct to SRP have on the GCF inflammatory proteins in periodontal disease patients?" RESULTS: Eight studies used aPDT while 10 studies used laser alone. Eight cytokines including tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, IL-6, IL-8, IL-10, interferon gamma (IFN-γ), matrix metalloproteinase (MMP)-8 and granulocyte colony-stimulating factor (GM-CSF) were eligible for qualitative analysis for aPDT and LT studies. Four aPDT studies showed significant reduction in IL-1ß while one study showed significant reduction in TNF-α levels after aPDT application at follow-up. One study showed significant reduction of IFN-γ, IL-8 and GM-CSF levels after aPDT at follow-up. IL-1ß significantly reduced in 4 LT studies, while one study showed significant decrease for IL-6 and TIMP-1 levels. MMP-8 and TNF-α showed significant reduction in three and one study respectively. CONCLUSION: It remains debatable whether adjunctive aPDT or LT is effective in the reduction of GCF inflammatory proteins in periodontal disease due to non-standard laser parameters and short follow up period. These findings should be considered preliminary and further studies with long-term follow up and standardized laser parameters are recommended.

Bactericidal Efficacy of Photodynamic Therapy Against Periodontal Pathogens in Periodontal Disease: A Systematic Review.

OBJECTIVE: The aim of this study was to assess the bactericidal efficacy of antimicrobial photodynamic dynamic therapy (aPDT) as an adjunct to scaling and root planing (SRP) against periodontal pathogens. BACKGROUND DATA: SRP followed by laser therapy has better clinical outcomes than conventional SRP alone. MATERIALS AND METHODS: The question addressed was "Does aPDT as an adjunct to SRP exhibit better bactericidal effect against periodontal pathogens than the use of SRP alone in periodontal disease?" MEDLINE(®)/PubMed, Embase, Scopus, Web of Science, and Google Scholar databases were searched from 1977 to December 2015, using different combinations of key words. Review articles, in vitro and experimental studies, and articles in languages other than English were excluded. RESULTS: Seventeen clinical studies were included. Laser wavelengths and duration of irradiation ranged between 470 and 810 nm and 60 and 300 sec, respectively. All studies showed that aPDT application was effective in reducing the counts of periodontal microbes at follow-up. Four studies showed significantly reduced bacterial counts for aPDT as an adjunct to SRP compared with SRP alone. Thirteen studies showed comparable reduction in the counts of periodontal bacteria when aPDT alone or as an adjunct to SRP was compared with SRP alone. CONCLUSIONS: The bactericidal efficacy of aPDT as an adjunct to SRP against periodontal pathogens in periodontal disease remains debatable.

Surface characterization analysis of failed dental implants using scanning electron microscopy.

OBJECTIVE: To investigate the failure of 15 dental implants (Paragon/Zimmer) in relation to their surface quality. MATERIALS AND METHODS: The study comprised of 15 dental implants (7 mm D Advent Implant, 3.9 mm D apex design implant), which were followed from surgery to completion of prosthetic restorations. The implants were placed during a 6-year period from 2003-2009 in non-smoking patients (male; 7, females; 5). There were eight upper and seven lower implants. Surface characterization after immersion in SBF of these failed implants was investigated using SEM and EDS compared to that of an unused implant of the same brand. RESULTS: Results revealed that, following immersion in SBF, the implant surfaces showed new components like Ca(+), Na(+) and Cl(-), but in trace quantities. CONCLUSIONS: After SEM observation and EDS analysis, it was concluded that the apatite layer formation could not be verified.