Scanning electron microscopic analysis of adherent bacterial biofilms associated with peri-implantitis.

OBJECTIVES: Peri-implantitis (PI) is caused by bacteria in the peri-implant space but the consensus on microbial profile is still lacking. Current microbial sampling of PI lesions has largely focused on analyzing bacterial species that have been shed from the implant surface and captured in the pocket fluid. The purpose of the present study was to investigate the morphotypes of bacteria in biofilm covering the implant threads and explore whether certain morphotypes were associated with PI. METHODS: Fourteen failed implants were removed and instantly processed for scanning electron microscope analysis. The implants were imaged at three equally divided sub-crestal levels of the exposed area. Bacterial morphotypes were identified and quantified by three examiners. Mobility and years in function were correlated to the presence of different morphotypes. RESULTS: The implants demonstrated the presence of variable bacterial morphotypes that did not correlate to disease progression in our study. Some implants were dominated by filaments and others showed the presence of combinations of cocci/rods or spirilles/spirochetes. In general, all implants showed variable morphologic biofilm composition. However, individual implants tended to have similar composition throughout the entire implant. Rods and filaments were dominant morphotypes throughout the surfaces and cocci showed increased presence toward the apical third. There were some differences in the biofilm morphology with mobility and time in function. CONCLUSIONS: The profiles of bacterial biofilm morphotypes in failing implants with similar clinical presentations were highly variable. While there were significant differences between implants, similar morphotypes in individual implants were often found throughout the entire surface.

Gingival epithelial cell-derived microvesicles activate mineralization in gingival fibroblasts.

Soft tissue calcification occurs in many parts of the body, including the gingival tissue. Epithelial cell-derived MVs can control many functions in fibroblasts but their role in regulating mineralization has not been explored. We hypothesized that microvesicles (MVs) derived from gingival epithelial cells could regulate calcification of gingival fibroblast cultures in osteogenic environment. Human gingival fibroblasts (HGFs) were cultured in osteogenic differentiation medium with or without human gingival epithelial cell-derived MV stimulation. Mineralization of the cultures, localization of the MVs and mineral deposits in the HGF cultures were assessed. Gene expression changes associated with MV exposure were analyzed using gene expression profiling and real-time qPCR. Within a week of exposure, epithelial MVs stimulated robust mineralization of HGF cultures that was further enhanced by four weeks. The MVs taken up by the HGF's did not calcify themselves but induced intracellular accumulation of minerals. HGF gene expression profiling after short exposure to MVs demonstrated relative dominance of inflammation-related genes that showed increases in gene expression. In later cultures, OSX, BSP and MMPs were significantly upregulated by the MVs. These results suggest for the first time that epithelial cells maybe associated with the ectopic mineralization process often observed in the soft tissues.

Effects of prolonged use of over-the-counter bleaching agents on enamel: An in vitro study.

This study evaluated the effects of four over-the-counter (OTC) bleaching products on the properties of enamel. Extracted human molars were randomly assigned into four groups (n = 5): PD: Poladay (SDI), WG: White Teeth Global (White Teeth Global), CW: Crest3DWhite (Procter & Gamble), and HS: HiSmile (HiSmile). The hydrogen peroxide (H2 O2 ) content in each product was analyzed via titration. Twenty teeth were sectioned into quarters, embedded in epoxy resin, and polished. Each quarter-tooth surface was treated with one of the four beaching times: T0: control/no-bleaching, T14: 14 days, T28: 28 days, and T56: 56 days. Materials were applied to enamel surfaces as recommended. Enamel surfaces were examined for ultramicrohardness (UMH), elastic modulus (EM), superficial roughness (Sa), and scanning electron microscopy (SEM). Ten additional teeth were used to evaluate color and degree of demineralization (DD) (n = 5). Data were statistically tested by two-way ANOVA and Tukey's tests (α = 5%). Enamel surfaces treated with PD and WG presented UMH values significantly lower than the controls (p < .05). Elastic modulus (E) was significantly reduced at T14 and T28 for PD, and at T14 for HS (p < .05). A significant increase in Sa was observed for CW at T14 (p < .05). Color changes were observed in the PD and WG groups. Additionally, DD analysis showed significant demineralization at T56 for CW. Overall, more evident morphological alterations were observed for bleaching products with higher concentrations of H2 O2 (p < .05), PD, and WG. Over-the-counter bleaching products containing H2 O2 can significantly alter enamel properties, especially when application time is extended.

Decontamination of multispecies oral biofilm from rough implant surface by airflow with glycine.

OBJECTIVES: Decontamination of biofilm-colonized rough implant surfaces remains challenging. We investigated the effect of airflow with glycine powder (AFG) on decontamination of mature oral multispecies biofilm from a sandblasted and acid etched (SLA) titanium surface. MATERIALS AND METHODS: Subgingival dental plaque was cultured on SLA disks anaerobically for 21 days. AFG with various settings and distances was applied directly on the disks with or without previous rinse of 0.9% NaCl. The specimens were then analyzed through scanning electron microscope and remaining bacteria on the implant surface were quantified and statistically compared. RESULTS: Mature oral biofilm with cocci and rods as major morphotypes, as well as spiral- and filamentous-shaped organisms, was formed on the untreated disks. Saline rinsing removed the thick biofilm layer but left numerous of coccoid bacteria in rough surface pits. AFG effectively removed most of the bacteria from the pits. Both 25% and 50% power settings were equally effective at 3-mm distance. With 50% power, AFG successfully removed bacteria at both 3- and 6-mm distance. When AFG was applied on native biofilm without prior rinsing with saline, it effectively removed the biofilm including bacteria in the pits. CONCLUSION: Application of AFG appears effective in removing bacteria from rough implant surfaces.

Novel Lignin-Capped Silver Nanoparticles against Multidrug-Resistant Bacteria.

The emergence of bacteria resistant to antibiotics and the resulting infections are increasingly becoming a public health issue. Multidrug-resistant (MDR) bacteria are responsible for infections leading to increased morbidity and mortality in hospitals, prolonged time of hospitalization, and additional burden to financial costs. Therefore, there is an urgent need for novel antibacterial agents that will both treat MDR infections and outsmart the bacterial evolutionary mechanisms, preventing further resistance development. In this study, a green synthesis employing nontoxic lignin as both reducing and capping agents was adopted to formulate stable and biocompatible silver-lignin nanoparticles (NPs) exhibiting antibacterial activity. The resulting silver-lignin NPs were approximately 20 nm in diameter and did not agglomerate after one year of storage at 4 °C. They were able to inhibit the growth of a panel of MDR clinical isolates, including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii, at concentrations that did not affect the viability of a monocyte-derived THP-1 human cell line. Furthermore, the exposure of silver-lignin NPs to the THP-1 cells led to a significant increase in the secretion of the anti-inflammatory cytokine IL-10, demonstrating the potential of these particles to act as an antimicrobial and anti-inflammatory agent simultaneously. P. aeruginosa genes linked with efflux, heavy metal resistance, capsular biosynthesis, and quorum sensing were investigated for changes in gene expression upon sublethal exposure to the silver-lignin NPs. Genes encoding for membrane proteins with an efflux function were upregulated. However, all other genes were membrane proteins that did not efflux metals and were downregulated.

Nanoscale flow cytometry for immunophenotyping and quantitating extracellular vesicles in blood plasma.

Extracellular vesicles (EVs) are lipid membrane enclosed nano-sized structures released into the extracellular environment by all cell types. EV constituents include proteins, lipids and nucleic acids that reflect the cell from which they originated. The molecular profile of cancer cells is distinct as compared to healthy cells of the same tissue type, and this distinct profile should be reflected by the EVs they release. This makes EVs desirable candidates for blood-based biopsy diagnosis of cancer. EVs can be time consuming to isolate therefore, a technology that can analyze EVs in complex biological samples in a high throughput manner is in demand. Here nanoscale flow cytometry is used to analyze EVs in whole, unpurified, plasma samples from healthy individuals and breast cancer patients. A known breast cancer marker, mammaglobin-a, was evaluated as a potential candidate for expression on EVs and increased levels in breast cancer. Mammaglobin-a particles were abundantly detected in plasma by nanoscale flow cytometry but only a portion of these particles were validated as bona fide EVs. EVs could be distinguish and characterized from small protein clusters and platelets based on size, marker composition, and detergent treatment. Mammaglobin-a positive EVs were characterized and found to be CD42a/CD41-positive platelet EVs, and the number of these EVs present was dependent upon plasma preparation protocol. Different plasma preparation protocols influenced the total number of platelet EVs and mammaglobin-a was found to associate with lipid membranes in plasma. When comparing plasma samples prepared by the same protocol, mammaglobin-a positive EVs were more abundant in estrogen receptor (ER) positive as compared to ER negative breast cancer patient plasma samples. This study demonstrates the capabilities of nanoscale flow cytometry for EV and small particle analysis in whole, unpurified, plasma samples, and highlights important technical challenges that need to be addressed when developing this technology as a liquid biopsy platform.

Decontamination of rough implant surfaces colonized by multispecies oral biofilm by application of leukocyte- and platelet-rich fibrin.

BACKGROUND: Decontamination of biofilm-infected rough implant surfaces is challenging. Platelet rich blood products have been shown to have anti-microbial properties against periodontal pathogens. Our aim was to investigate the effect of a potential biological implant surface disinfectant, leukocyte- and platelet-rich fibrin (L-PRF), on a mature oral multispecies biofilm on a rough titanium surface. METHODS: Sandblasted, large grit, acid-etched (SLA) titanium disks were inoculated with subgingival dental plaque and cultured anaerobically for 21 days. The L-PRF membranes were collected from 12 donors in three trials (four donors in each trial). The disks were rinsed with 0.9% NaCl and exposed to the cell-rich portion of the L-PRF membranes for 48 hours followed by scanning electron microscope (SEM) analysis immediately or after rinsing with 0.9% NaCl prior to fixation. The presence of platelet factor-4 in the rinse samples was analyzed by Western blotting. Remaining bacteria were quantified from SEM images of the implant surfaces and their numbers statistically compared. RESULTS: The L-PRF-treated samples without rinsing displayed numerous cells with multiple pseudopodia in immediate contact with bacteria that appeared perforated and increased in size. The cells were identified as platelets based on morphological criteria and by positive reaction for platelet factor-4 by Western blotting. After post-treatment rinsing, the L-PRF-treated disks displayed a significant reduction in bacterial counts (in average 92% reduction). CONCLUSION: Application of L-PRF significantly reduced bacterial counts on contaminated SLA titanium surface, most likely through anti-microbial action by platelets.

Hydoxyapatite/beta-tricalcium phosphate biphasic ceramics as regenerative material for the repair of complex bone defects.

Bone is a composite material composed of collagen and calcium phosphate (CaP) mineral. The collagen gives bone its flexibility while the inorganic material gives bone its resilience. The CaP in bone is similar in composition and structure to the mineral hydroxyapatite (HA) and is bioactive, osteoinductive and osteoconductive. Therefore synthetic versions of bone apatite (BA) have been developed to address the demand for autologous bone graft substitutes. Synthetic HA (s-HA) are stiff and strong, but brittle. These lack of physical attributes limit the use of synthetic apatites in situations where no physical loading of the apatite occurs. s-HA chemical properties differ from BA and thus change the physical and mechanical properties of the material. Consequently, s-HA is more chemically stable than BA and thus its resorption rate is slower than the rate of bone regeneration. One solution to this problem is to introduce a faster resorbing CaP, such as ß-tricalcium phosphate (ß-TCP), when synthesizing the material creating a biphasic (s-HA and ß-TCP) formulation of calcium phosphate (BCP). The focus of this review is to introduce the major differences between BCP and biological apatites and how material scientists have overcome the inadequacies of the synthetic counterparts. Examples of BCP performance in vitro and in vivo following structural and chemical modifications are provided as well as novel ultrastructural data. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2493-2512, 2018.

Suppression of αvß6 Integrin Expression by Polymicrobial Oral Biofilms in Gingival Epithelial Cells.

Periodontal diseases manifest by the formation of deep pockets between the gingiva and teeth where multispecies bacterial biofilms flourish, causing inflammation and bone loss. Epithelial cell receptor αvß6 integrin that regulates inflammation by activating the anti-inflammatory cytokine transforming growth factor-ß1, is highly expressed in healthy junctional epithelium that connects the gingiva to the tooth enamel. However, its expression is attenuated in human periodontal disease. Moreover, Itgb6 -/- mice display increased periodontal inflammation compared to wild-type mice. We hypothesized that bacterial biofilms present in the periodontal pockets suppress αvß6 integrin levels in periodontal disease and that this change aggravates inflammation. To this end, we generated three-week-old multi-species oral biofilms in vitro and treated cultured gingival epithelial cells (GECs) with their extracts. The biofilm extracts caused suppression of ß6 integrin expression and upregulation of pro-inflammatory cytokines, including interleukin-1ß and -6. Furthermore, GECs with ß6 integrin siRNA knockdown showed increased interleukin-1ß expression, indicating that αvß6 integrin-deficiency is associated with pro-inflammatory cytokine responsiveness. FSL-1, a synthetic bacterial lipopeptide, also suppressed ß6 integrin expression in GECs. Therefore, biofilm components, including lipopeptides, may downregulate αvß6 integrin expression in the pocket epithelium and thus promote epithelial cell-driven pro-inflammatory response in periodontal disease.

Chemotherapeutic decontamination of dental implants colonized by mature multispecies oral biofilm.

AIM: No studies have tested disinfectants on mature multispecies oral biofilms on titanium substrata. The aim of this study was to investigate the efficacy of commonly used antimicrobial agents in decontamination of multispecies mature oral biofilm on sandblasted, large-grit, acid-etched (SLA) titanium implants. METHODS: SLA titanium disks were inoculated with dental plaque and cultured anaerobically for 21 days. The disks were rinsed with 0.9% NaCl, exposed for 2 min. to tetracycline paste, 1% Chlorhexidine gel (CHX), 35% phosphoric acid gel (Etch) or a novel chemical formula (0.3% cetrimide, 0.1% CHX and 0.5% EDTA) and then rinsed again with 0.9% NaCl. Bacteria were quantified from scanning electron micrographs of the implant surfaces. Living bacteria were quantified with confocal laser scanning microscopy (CLSM). RESULTS AND CONCLUSIONS: Rinsing the surfaces with 0.9% NaCl removed the majority of the biofilm. However, bacteria persisted in all specimens and none of the disinfectants was superior to the double saline rinse group. CLSM analysis showed that CHX and Etch groups had a statistically significant reduction of viable bacteria, although small. Overall the results show that many disinfection agents used in the clinic are ineffective in biofilm removal and leave live bacteria on the surface.

Sterilization of Ceramic Sharpening Stones.

Traditionally, periodontal hand instruments are honed or sharpened during patient care as they dull easily during contact with enamel, calculus and cementum. This approach is taught in dental and hygiene schools around the world and remains the standard of care. Recently, some professional organizations have questioned whether this practice should be abandoned because of safety issues. Questions have been raised whether sharpening stones can be properly sterilized and whether the sharpening of contaminated instruments poses a health hazard for the provider. Using bacteria culture techniques and scanning electron microscopy, we tested whether contaminated ceramic sharpening stones can be sterilized. Our results demonstrate that the stones were sterile after being subjected to the manufacturer's sterilization protocol. In addition, over the last year, no incidents related to periodontal instrument sharpening have been reported among nearly 400 students at the faculty of dentistry, University of British Columbia, where chair-side sharpening is taught. Therefore, we conclude that ceramic sharpening stones can be sterilized using normal office protocols and that chair-side sharpening adds little risk beyond routine handling of operatory or periodontal instruments during patient care when proper protocols are followed.

Keratinocyte Microvesicles Regulate the Expression of Multiple Genes in Dermal Fibroblasts.

Extracellular vesicles released from cells regulate many normal and pathological conditions. Little is known about the role of epidermal keratinocyte microvesicles (KC-MVs) in epithelial-stromal interaction that is essential for wound healing. We investigated, therefore, whether MV-like structures are present in human wounds and whether they affect wound healing-associated gene expression in dermal fibroblasts. In human wounds, MV-like vesicles were observed during active epithelial migration and early granulation tissue formation. When KC-MVs derived from keratinocyte-like cells (HaCaT) were added to fibroblast cultures, expression of 21 genes was significantly regulated (P<0.05) out of 80 genes investigated, including matrix metalloproteinase-1 and -3, interleukin-6 and -8, and genes associated with transforming growth factor-ß signaling. Similar changes were observed at the protein level. MVs from normal epidermal keratinocytes showed similar response to HaCaT cells. KC-MVs activated ERK1/2, JNK, Smad, and p38 signaling pathways in fibroblasts with ERK1/2 signaling having the most prominent role in the MV-induced gene expression changes. KC-MVs stimulated fibroblast migration and induced fibroblast-mediated endothelial tube formation but did not affect collagen gel contraction by fibroblasts. The results demonstrate that keratinocyte microvesicles have a strong and a specific regulatory effect on fibroblasts that may modulate several aspects of wound healing.

Formation of cartilage and synovial tissue by human gingival stem cells.

Human gingival stem cells (HGSCs) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone-marrow-derived mesenchymal stem/stromal cells has been well documented. HGSCs derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSCs can be induced to differentiate to osteoblasts and chondrocytes. HGSCs were cultured either in a classical monolayer culture or in three-dimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSCs cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase (ALPL), and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSCs have the capacity to differentiate to osteogenic, chondrogenic, and synoviocyte lineages. Therefore, HGSCs could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis.

Critical role for αvß6 integrin in enamel biomineralization.

Tooth enamel has the highest degree of biomineralization of all vertebrate hard tissues. During the secretory stage of enamel formation, ameloblasts deposit an extracellular matrix that is in direct contact with the ameloblast plasma membrane. Although it is known that integrins mediate cell-matrix adhesion and regulate cell signaling in most cell types, the receptors that regulate ameloblast adhesion and matrix production are not well characterized. We hypothesized that αvß6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both ß6 integrin mRNA and protein. The maxillary incisors of Itgb6(-/-) mice lacked yellow pigment and their mandibular incisors appeared chalky and rounded. Molars of Itgb6(-/-) mice showed signs of reduced mineralization and severe attrition. The mineral-to-protein ratio in the incisors was significantly reduced in Itgb6(-/-) enamel, mimicking hypomineralized amelogenesis imperfecta. Interestingly, amelogenin-rich extracellular matrix abnormally accumulated between the ameloblast layer of Itgb6(-/-) mouse incisors and the forming enamel surface, and also between ameloblasts. This accumulation was related to increased synthesis of amelogenin, rather than to reduced removal of the matrix proteins. This was confirmed in cultured ameloblast-like cells, in which αvß6 integrin was not an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin αvß6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition and/or turnover and subsequent enamel biomineralization.

Melanoma-associated cancer-testis antigen 16 (CT16) regulates the expression of apoptotic and antiapoptotic genes and promotes cell survival.

Cancer-testis (CT) antigens are predominantly expressed in testis or placenta, but absent in most adult tissues. During malignant transformation CT genes are often activated. CT antigen 16 (CT16, PAGE5) is frequently expressed in advanced melanoma but its biological function has been unknown. To examine the role of CT16 in cell survival we knocked it down in A2058 melanoma cells using specific siRNAs and exposed the cells to cancer drug cisplatin known to induce apoptosis. As a result, cell survival was markedly decreased. To study the effects of CT16 on cell survival in more detail, the cellular gene expression profiles were investigated after CT16 silencing in CT16 positive A2058 melanoma cells, as well as after CT16 overexpression in CT16 negative WM-266-4 melanoma cells. Among the 11 genes both upregulated by CT16 silencing and downregulated by CT16 overexpression or vice versa, 4 genes were potentially apoptotic or antiapoptotic genes. CT16 was recognized as a positive regulator of antiapoptotic metallothionein 2A and interleukin 8 genes, whereas it inhibited the expression of apoptosis inducing dickkopf 1 (DKK1) gene. In addition CT16 enhanced the expression of fatty acid binding protein 7, a known promoter of melanoma progression. The effect of CT16 on DKK1 expression was p53 independent. Furthermore, CT16 did not regulate apoptotic genes via DNA methylation. In twenty melanoma metastasis tissue samples average DKK1 mRNA level was shown to be significantly (p<0.05) lower in high CT16 expressing tumors (n = 3) when compared to the tumors with low CT16 expression (n = 17). Thus, our results indicate that CT16 promotes the survival of melanoma cells and is therefore a potential target for future drug development.

Integrin ß6-deficient mice show enhanced keratinocyte proliferation and retarded hair follicle regression after depilation.

Integrin αvß6 is an epithelial-specific receptor that binds and activates latent transforming growth factor-ß1 (TGF-ß1). TGF-ß1 has been implicated as an endogenous inducer of hair follicle (HF) regression during hair cycling. We hypothesized that αvß6 integrin-mediated TGF-ß1 signaling regulates hair regeneration and HF involution. In wild-type (WT) mice, the expression of integrin αvß6 was strongly upregulated in the outer root sheath (ORS) during early hair regeneration, and was specifically enhanced in the HF bulge region. Expression gradually decreased in late anagen and remained restricted to the bulge region in the catagen and telogen stage HFs. The first spontaneous hair cycle was not altered in ß6 integrin knockout (ß6(-/-)) mice. However, after depilation, ß6(-/-) mice exhibited retarded HF regression compared with WT controls. ß6(-/-) follicles contained significantly higher numbers of proliferating Ki67-positive keratinocytes than WT follicles at an identical cycle stage. The ß6(-/-) follicles also demonstrated significantly reduced levels of TGF-ß1 expression and Smad2 phosphorylation during early anagen and anagen-catagen transition. Our study indicates that αvß6 integrin has an important inhibitory role in keratinocyte proliferation in both HFs and interfollicular epidermis. Thus, downregulated TGF-ß1 signaling in ß6(-/-) mice may affect bulge niche stem cell behavior.

An in vitro study of plasticized poly(lactic-co-glycolic acid) films as possible guided tissue regeneration membranes: material properties and drug release kinetics.

Guided tissue regeneration (GTR), in periodontal therapy, involves the placement of a barrier membrane, to ensure the detached root surface becomes repopulated with periodontal ligament cells capable of regenerating this attachment. GTR procedures exhibit large variability in surgical outcome as a consequence of poor membrane performance. The objective of this study was to evaluate the suitability of plasticized poly(lactic-co-glycolic acid) (PLGA) as a material for GTR membranes. The material was also investigated as a localized controlled release system for the antibiotic, anti-inflammatory agent tetracycline. Films made from PLGA (85:15), plasticized with either 10% w/v methoxypoly(ethyleneglycol) (MePEG) or a diblock copolymer [poly(D,L-lactic acid)-block-methoxypoly(ethyleneglycol)] were loaded with tetracycline base (or hydrochloride salt) and cast by solvent evaporation. Drug release was measured using high performance liquid chromatography (HPLC). The time-course of elasticity changes and swelling were determined using a stress-strain apparatus or gravimetric/dimensional determinations, respectively. Cells extracted from periodontal ligament cell explants were used to evaluate the effect of material and drug loading on cell morphology. Tetracycline·HCl released more rapidly than tetracycline from PLGA films. The addition of either MePEG or diblock caused a concentration dependent increase in release rates for both drugs. Release profiles ranged from a small initial burst phase followed by slow sustained release to almost full drug release after 1 day. After incubation in PBS, the films stiffened and swelled within 30 min. Periodontal ligament cell morphology was not affected by the inclusion of tetracycline. Plasticized PLGA films displayed desired features for possible use as GTR membranes.

Exploring the Nature of Desmosomal Cadherin Associations in 3D.

Desmosomes are a complex assembly of protein molecules that mediate adhesion between adjacent cells. Desmosome composition is well established and spatial relationships between components have been identified. Intercellular cell-cell adhesion is created by the interaction of extracellular domains of desmosomal cadherins, namely, desmocollins and desmogleins. High-resolution methods have provided insight into the structural interactions between cadherins. However, there is a lack of understanding about the architecture of the intact desmosomes and the physical principles behind their adhesive strength are unclear. Electron Tomography (ET) studies have offered three-dimensional visual data of desmosomal cadherin associations at molecular resolution. This review discusses the merits of two cadherin association models represented using ET. We discuss the possible role of sample preparation on the structural differences seen between models and the possibility of adaptive changes in the structure as a direct consequence of mechanical stress and stratification.

Bone formation on rough, but not polished, subcutaneously implanted Ti surfaces is preceded by macrophage accumulation.

Implanted rough surfaces have long been associated with the accumulation of macrophages and other cells of the monocytic lineage such as foreign body giant cells and osteoclasts. As cells of the moncytic lineage are part of the immune system, the response of this cell family to biomaterials has attracted wide concern. This study compared events at the interface of implant surface topographies with varied roughness in a rat subcutaneous model. Titanium-coated epoxy replicas of machined, etched, blasted, titanium-plasma-sprayed (TPS), sandblasted-and-etched (SLA), micromachined, and polished surfaces were implanted for up to 11 weeks, and processed for light or electron microscopy or immunohistochemistry for ED1, a marker for recruited macrophages. Initially, healing appeared similar among all surfaces, the frequency of mineralization followed the order of SLA, micromachined, TPS, machined, etched, blasted, and polished surfaces. On the SLA surface macrophages, as identified by both ultrastructural morphology and immunohistochemistry were the predominant cell type at 1 week and persisted until mineralization occurred as early as 2 weeks. On smoother surfaces collagenous matrix predominated at 2 weeks and subsequently increased with time. There, thus, appears to be two routes to bone-like tissue formation on Ti implants in this rat subcutaneous model; macrophage-mediated and macrophage-independent dense collagenous-matrix-associated.

A reproducible technique for specific labeling of antigens using preformed fluorescent molecular IgG-F(ab')2 complexes from primary antibodies of the same species.

Immunolabeling two different antigens using the indirect approach with antibodies from the same species is not possible as secondary antibodies can bind to either primary target antibodies. In this study, we describe how preformed complexes of primary and secondary labeled antibodies can be used in such circumstances. In this situation, the first antigen is labeled using the conventional indirect method followed by incubation with the preformed primary-secondary antibody complex against the second antigen. To prevent unbound secondary antibody from binding the indirectly-labeled antibodies, resulting in a false positive, we quenched excess secondary antibody with nonimmune murine serum from the species of the primary antibody. Before the formation of the preformed complex, the optimum dilution of both primary and secondary antibodies was determined. Once these concentrations were established, the concentration of nonimmune murine serum required to quench excess unbound secondary was determined. This step was accomplished by first incubating the sample with an antibody against an antigen known to be localized away from the antigen of interest, followed by the preformed complex. If specific staining was seen, other than that expected from the preformed complex, then the concentration of the serum was deemed insufficient for quenching, and increased accordingly. We demonstrate that this approach is successful in determining the optimum conditions for the preformation of ascites and purified monoclonal primary IgG with fluorescently conjugated F(ab')(2). Double immunolabelling of two focal adhesion antigens and two cytoskeletal proteins, with two murine primary antibodies, are presented as examples of the methodology.