We describe a novel approach for quantification and colocalization of immunofluorescence (IF) signals of multiple markers on high-resolution panoramic images of serial histological sections utilizing ...standard staining techniques and readily available software for image processing and analysis. Human gingiva samples stained with primary antibodies against the common leukocyte antigen CD45 and factors related to heparan sulfate glycosaminoglycans (HS GAG) were used. Expression domains and spatial gradients of IF signals were quantified by histograms and 2D plot profiles, respectively. The importance of histomorphometric profiling of tissue samples and IF signal thresholding is elaborated. This approach to quantification of IF staining utilizes pixel (px) counts and comparison of px grey value (GV) or luminance. No cell counting is applied either to determine the cellular content of a given histological section nor the number of cells positive to the primary antibody of interest. There is no selection of multiple Regions-Of-Interest (ROIs) since the entire histological section is quantified. Although the standard IF staining protocol is applied, the data output enables colocalization of multiple markers (up to 30) from a given histological sample. This can serve as an alternative for colocalization of IF staining of multiple primary antibodies based on repeating cycles of staining of the same histological section since those techniques require non standard staining protocols and sophisticated equipment that can be out of reach for small laboratories in academic settings. Combined with the data from ontological bases, this approach to quantification of IF enables creation of in silico virtual disease models.
Since chronically inflamed periodontal tissue exhibits extracellular matrix (ECM) degradation, the possible alternative to standard periodontitis treatment is to restore ECM by supplementing its ...components, including heparan sulfate glycosaminoglycan (HS GAG). Supplementation of the degraded ECM with synthetic derivatives of HS GAGs has been shown to be effective for periodontal tissue regeneration in experimental animal models of periodontitis. However, the potential of HS GAG supplementation for the treatment of periodontal disease in humans is still unknown. Here, we used a statistical model to investigate the role of HS GAG on inflammatory infiltrate formation and alveolar bone resorption in humans with severe periodontitis. The model was based on data from immunofluorescence staining (IF) of human gingiva samples, and reconstruction of a subset of HS GAG -related proteins from STRING reactome database. According to predictions, increased expression of native HS GAG might stabilize the accumulation of gingival inflammatory infiltrate (represented by the general inflammatory cell marker CD45) and alveolar bone resorption (represented by Receptor Activator of Nuclear ΚΒ ligand (RANKL) and osteoprotegerin (OPG) ratio) but could not restore them to healthy tissue levels. Therefore, supplementation of native HS GAG may be of limited benefits for the treatment of sever periodontitis in humans.
Syndecans belong to a four-member family of cell surface heparan sulfate proteoglycans (HSPGs) abundantly present in various tissues. They are primarily recognized as extracellular matrix (ECM) ...receptors able to bind various ECM components and form gradients of morphogens and growth factors. Syndecans are composed of core protein with distinctive cytoplasmic, transmembrane, and extracellular domains to which several HS glycosaminoglycan (GAG) chains are covalently attached. In development of composite organs, such as teeth, expression patterns of syndecans display temporo-spatial shifts between epithelial and mesenchymal tissue compartments. Along with diverse functional properties of syndecans and generally large number of their interactors due to HS GAG chain content, this suggests possible involvement of syndecans in modulation of epithelial-to-mesenchymal crosstalk. Functional versatility of syndecans greatly depends upon the biochemical properties of attached HS GAG chains. These are specifically determined during the HS biosynthesis by the combinatorial action of glycosyl-transferases (Exts/EXTs) and bi-functional sulfotransferases (Ndsts/NDSTs), as well as by post-biosynthetic enzymatic cleavage of HS by the only active endoglucuronidase in mammals, heparanase 1 (Hpse1/HPSE1). Matching the essential requirement for HS during organogenesis, null-mutant animals for genes encoding these enzymes display severe developmental anomalies of mineralized tissues (including teeth) with embryonic or perinatal lethality. In this study, we analyzed expression of syndecan HSPGs (syndecans 1, 2, and 4), enzymes involved in HS biosynthesis (EXT1, NDST1, NDST2) and HS cleavage (HPSE1) in human tooth germs during the early stages of odontogenesis. All of the investigated factors displayed temporo-spatial differences in expression patterns, and some of them showed distinctive asymmetries of expression domains. Our findings suggest that these factors might be differentially involved in cellular processes which take place during the early odontogenic sequence in humans.
Introduction: Bioceramic root canal sealers are the newest generation of root canal sealers. Τhere are contradictory results in the literature about their antimicrobial activity. The aim of the study ...was to evaluate the antimicrobial efficacy of four root canal sealers against Enterococcus faecalis. Materials and Methods: Four root canal sealers were used in order to examine antibacterial efficacy: TotalFill, BioRoot Root Canal Sealer, mineral trioxide aggregate (MTA) Fillapex, and AH Plus. The bacterial suspension was placed on a freshly mixed sealer, one or 3 days set sealer in vertically held microtiter plates. After incubation of 2, 5, 20, and 60 min in 100% humidity at 37 C, Trypticase Soy Broth was added to each well and mixed. Then, bacterial suspension from each well was transferred, serially diluted, and placed on Mitis salivarius agar plates. After incubation, colony-forming units were counted. All experiments were performed in triplicate. The outcomes of antimicrobial properties of tested materials were analyzed by one-way analysis of variance. Results: All bioceramic root canal sealers showed significantly better efficacy than the control group and epoxy resin sealer (P < 0.05). TotalFill presented the highest efficacy comparable with BioRoot RCS (P > 0.05), followed with MTA Fillapex (P < 0.05). Also, freshly mixed sealers showed comparable efficacy as sealers set for 3 days (P > 0.05), but better than sealers set for 1 day (P < 0.05). All sealers exhibited the highest efficacy after 20 min of contact time, independently of materials that were freshly mixed or set for 1 or 3 days. Conclusion: Bioceramic sealers have greater antimicrobial activity than commercially used epoxy resin sealer. These sealers exhibited the strongest efficacy at 20 min of contact time, independently of their setting condition.
Periodontitis is a common degenerative disease initiated by the bacteria in subgingival biofilm. The exposure to bacterial biofilm triggers host inflammatory response whose dysregulation is ...ultimately responsible for the destruction of hard and soft periodontal tissues resulting in tooth loss. To date, significant effort has been invested in the research of the involvement of host cells and inflammatory mediators in regulation of inflammatory response in periodontitis. Syndecans (Sdcs) belong to a four-member family of heparan sulfate proteoglycans (HSPGs). Sdcs are compound molecules comprised of the core protein to which several heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. The role of Sdcs in pathogenesis of periodontitis is poorly investigated despite the numerous reports from experimental studies about the critical involvement of these factors in modulation of various aspects of inflammatory response, such as the formation of inflammatory mediators gradients, leukocyte recruitment and extracellular matrix remodeling in resolution of inflammation. Most of these functions of Sdcs are HS-related and, thus, dependent upon the structure of HS. This, in turn, is determined by the combinatorial action of enzymes for biosynthesis and modification of HS such as exostosis (EXTs), sulfotransferases (NDSTs), and heparanase 1 (HPSE1). The data presented in this study clearly indicate that some Sdcs display different expression profiles in healthy and diseased periodontal tissue. Additionally, the differences in expression profiles of HS GAG biosynthesis and modification enzymes (EXTs, NDSTs, and HPSE1) in healthy and diseased periodontal tissue imply that changes in HS GAG content and structure might also take place during periodontitis. Most notably, expression profiles of Sdcs, EXTs, NDSTs, and HPSE1 differentially correlate with the presence of inflammatory infiltrate in healthy and diseased periodontal tissue, which might imply that these factors could also be involved in modulation of inflammatory response in periodontitis.
Low muscle strength, functional score at discharge, and complications during a ten-day rehabilitation hospital stay can affect mortality rates in bedridden geriatric patients. This was a prospective ...observational study in a cohort of 105 bedridden geriatric patients admitted to the Rehabilitation ward after a major illness or surgery. All participants had a severe dependency on another person (Barthel's Index < 60). The one-year mortality rate in this cohort was 15.2%, with further subdivision according to the number of complications: 61.5% in patients with ≥3 complications during hospitalization, 17.6% in patients with two complications, 9.5% with one complication, and 3% in patients with no complications. The Barthel Index at discharge (OR = 0.95;
= 0.003) and ≥3 medical complications (OR = 8.33;
= 0.005) during rehabilitation ward stay were significant predictors for one-year mortality. The odds of one-year mortality after discharge increased eightfold in patients with ≥3 medical complications. Sarcopenia, age, and sex were not significant predictors of mortality in this cohort. The 10-day acute rehabilitation was too short to achieve progress from severe to moderate independence in 60% of patients. The Barthel Index at discharge and a number of complications affect the mortality rate. These findings provide valuable insights into the complex dynamics of mortality and functional outcomes in bedridden geriatric patients.
Insulin-Like Growth Factor 2 (IGF-2) is a peptide hormone essential for prenatal growth and development. IGF-2 exerts its mitogenic effects via Insulin-Like Growth Factor 1 Receptor (IGF-1R), and is ...eliminated by binding to Insulin-Like Growth Receptor 2 (IGF-2R). IGF-2 is also negatively regulated by Phosphatase and Tensin Homolog (PTEN), a phosphatase mutated in various tumors. Not much is known about the interplay between these factors during human odontogenesis. In this study, expression patterns of IGF-2, IGF-1R, IGF-2R and PTEN were analyzed by double immunofluorescence in incisor human tooth germs during the foetal period of development between the 7
th
and 20
th
gestational week. Throughout the investigated period, IGF-2 was mostly expressed in enamel organ, whereas mild to moderate expression of PTEN could be seen in dental papilla and parts of enamel organ. Expression of IGF-1R was ubiquitous and displayed strong intensity throughout the entire enamel organ. In contrast, expression of IGF-2R had rather erratic pattern in enamel organ and dental papilla alike. Expression patterns of IGF-2, IGF-1R, IGF-2R and PTEN in highly proliferative cervical loops, as well as in differentiating pre-ameloblasts and pre-odontoblasts of cusp tip region during the early and late bell stages when enamel organ acquires definitive shape, indicate importance of these factors in crown morphogenesis of human incisor. Taken together, our data suggest the involvement of IGF-2, IGF-1R, IGF-2R and PTEN in temporo-spatial patterning of basic cellular processes (proliferation, differentiation) during normal tooth development. They are also relevant for improving knowledge of molecular basis of human odontogenesis.
Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation ...patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19
INK4d
. p19
INK4d
induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19
INK4d
by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19
INK4d
in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19
INK4d
throughout the investigated period indicates that p19
INK4d
plays active role during human tooth development. Furthermore, comparison of expression domains of p19
INK4d
with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.
The markers of cell proliferation (Ki‐67) and apoptosis caspase‐3, TdT‐mediated biotin–dUTP nick‐end labelling (TUNEL) and the expression of syndecan‐1 and heat shock protein 70 (Hsp70) were analyzed ...immunohistochemically in 11 developing human palates, from developmental weeks 6 to 10. During fusion of the primary palate, the proportion of proliferating cells decreased from 42 to 32% and the proportion of apoptotic cells decreased from 11 to 7% in the medial‐edge epithelium. At later stages, the proportions of both types of cells decreased in the ectomesenchyme, except for proliferating cells in its non‐condensing part. At developmental weeks 9–10, the epithelial seam in the secondary palate comprised 28% proliferative cells and 5% apoptotic cells. While condensing ectomesenchyme contained more apoptotic cells than proliferating cells, the opposite was observed for the non‐condensing ectomesenchyme. Co‐expression of syndecan‐1 and Hsp70 was detected in cells budding from the epithelial seam. Our study indicates similar principles for human primary palate and secondary palate fusion, and parallel persistence of proliferation and apoptotic activity. While proliferation enables growth and fusion of different palatal primordia, apoptosis results in the removal of of large numbers epithelial cells at the fusion point. The disintegration of seam remnants seems to be executed through the processes of change in protein content and cell migration, probably leading to cell death as their final outcome.
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