Effects of glycation end-products on the dental pulp in patients with type 2 diabetes.

AIM: This ex vivo study aimed to compare protein expression of advanced glycation end-products (AGE) and receptor (RAGE), and the levels of selected genes associated with inflammation and collagen within dental pulp tissue from patients with type 2 (T2D) diabetes and non-T2D. METHODOLOGY: Noncarious extracted permanent molar teeth from patients with well-controlled T2D (n = 19) and non-T2D (controls) (n = 19) were collected and compared. The coronal pulp was examined using immunohistochemistry (IHC) (n = 10 per group) for anti-AGE and anti-RAGE. Quantitative PCR (n = 9 per group) was used to analyse the gene expression levels of NFKB, S100A12 and COLIA1. Data analyses were performed between the groups using GraphPad Prism using Pearson correlation, Shapiro-Wilk and Mann-Whitney U-tests, and multiple regression using SPSS. RESULTS: AGEs were distributed diffusely throughout the pulp extracellular matrix associated with collagen fibres and were present on several cell types. RAGE was expressed at the pulp-dentine interface and was observed on odontoblasts, immune cells, endothelial cells and fibroblasts. Semi-quantitative analysis of IHC samples showed significantly increased expression of AGE (p < .0001) and RAGE (p = .02) in T2D samples compared with controls. The expression of NFKB (p < .0001), S100A12 (p < .0001) and COLIA1 (p = .01) genes were significantly higher in the T2D pulp, and multivariate logistic regression analysis showed that these findings were not affected by age. CONCLUSION: T2D may exert a similar glycation response in the dental pulp to other body sites. This could occur through activation of NF-κB pathways with a concomitant increase in genes associated with inflammation and collagen.

Bone remodelling marker expression in grafted and ungrafted sheep tooth extraction sockets: A comparative randomised study.

OBJECTIVE: To compare key markers of bone remodelling in a sheep tooth extraction model for sockets left to heal naturally or grafted with the bovine-derived xenograft Bio-Oss® covered with a collagen Bio-Gide® membrane. DESIGN: Right side premolar teeth were removed from thirty Romney-cross ewes. Standardised sockets in each sheep were randomly allocated treatments, a grafted test and an empty control. At 4-, 8- and 16-weeks sheep were euthanized and tissue collected (N = 10/group). RANK, RANKL and OPG immunohistochemical analysis was performed (n = 3). RANK, RANKL, OPG, COL1A1, TIMP3, SP7 and MSX2 mRNA expression levels were determined using RT2-qPCR assays (n = 3). RESULTS: Histologically, more new woven bone was observed in the test group at all time points. Strong RANK and RANKL expression was found in both groups; at all time points with stronger RANK staining in the test group at 8 and 16 weeks. Strong OPG staining was localized to both osteoblasts and connective tissues. RANK receptor mRNA was expressed at a lower level in the test group (-4.26-fold; p = 0.02) at 4 weeks and SP7 at 16 weeks (-2.89-fold; p = 0.04). COL1A1 and TIMP3 mRNA expression increased significantly over time in the control group (p = 0.045, F = 5.4 and p = 0.003, F = 42.2 respectively). CONCLUSION: Socket healing over time was comparable. The sheep tooth extraction model was found to be suitable for the evaluation of changes in the alveolar bone at the molecular level.

Expression of the pleiotrophin-midkine axis in a sheep tooth socket model of bone healing.

OBJECTIVE AND BACKGROUND: Resorption of alveolar bone after tooth extraction is a common problem often requiring bone grafting. The success of the grafting procedures is dependent on multiple factors including the presence of growth factors. This is the first in vivo study to investigate the role of the pleiotrophin family of cytokines in alveolar bone regeneration. This research investigated the role of the pleiotrophin-midkine (PTN-MDK) axis during osteogenesis, with and without a grafting material, after tooth extraction in a sheep model. METHODS: Thirty Romney-cross ewes were anesthetized, and all premolar teeth on the right side were extracted. The sockets were randomized to controls sites with no treatment and test sites with Bio-Oss® graft material and Bio-Gide® membrane. Samples were harvested after sacrificing animals 4, 8, and 16 weeks post-grafting (n = 10 per time-point). Tissue for qRT2 -PCR gene analysis was recovered from the socket next to the first molar using a trephine (Ø = 2 mm). Each socket was fixed, decalcified, paraffin-embedded, and sectioned. Immunohistochemistry was conducted to localize both PTN and MDK along with their receptors, protein tyrosine phosphatase receptor type Z1 (PTPRZ1), ALK receptor tyrosine kinase (ALK), and notch receptor 2 (NOTCH2). RESULTS: Within the healing sockets, high expression of genes for PTN, MDK, NOTCH2, and ALK was found at all time-points and in both grafted and non-grafted sites, while PTPRZ1 was only expressed at low levels. The relative gene expression of the PTN family of cytokines was not statistically different at the three time-points between test and control groups (p > .05). Immunohistochemistry found PTN and MDK in association with new bone, NOTCH2 in the connective tissue, and PTPRZ1 and ALK in association with cuboidal osteoblasts involved in bone formation. CONCLUSIONS: The PTN-MDK axis was highly expressed in both non-grafted and grafted sockets during osteogenesis in a sheep model of alveolar bone regeneration with no evidence that grafting significantly affected expression. The activation of NOTCH2 and PTPRZ1 receptors may be important during bone regeneration in vivo. The discovery of the PTN-MDK axis as important during alveolar bone regeneration is novel and opens up new avenues of research into these stably expressed highly active cytokines. Growth factor supplementation with PTN and/or MDK during healing may be an approach for enhanced regeneration or to initiate healing where delayed.

Culturing Adipose-Derived Stem Cells Under Serum-Free Conditions.

Growing adipose-derived stem cells (ADSC) in serum-free conditions is important as it represents a way of expanding multipotent cells in a clinical grade medium. Most cultured ADSC are expanded and tested in serum-containing media, which can pose significant health risks if these cells were used in clinical applications. Moreover, cells grown in serum-free conditions behave very different than those cultured in serum-containing media. Here, we present a technique to culture adipose-derived stem cells in serum-free conditions. The methods described in this chapter were optimized for ovine ADSC. The appropriate optimization should be done for other cell lines.

Quantitative Real-Time Gene Profiling of Human Alveolar Osteoblasts Using a One-Step System.

The use of quantitative real-time reverse transcriptase PCR (qRT2-PCR) for the identification of differentially regulated genes is a powerful technology. The protocol presented here uses qRT2-PCR gene arrays to investigate the regulation of 84 angiogenic related genes in human primary alveolar osteoblasts following treatment with the bisphosphonate, zoledronic acid, and geranylgeraniol (GGOH). GGOH has potential as a therapeutic agent for medication-related osteonecrosis of the jaw, a serious side effect resulting from treatment for metastatic cancer (Zafar S, Coates DE, Cullinan MP, Drummond BK, Milne T, Seymour GJ. J Oral Pathol Med 43:711-721, 2014). The isolation of the primary osteoblast cells follows the methods described by Dillon et al. (Method Mol Biol 816:3-18, 2012) with a new RNA extraction technique described fully. The method highlights the importance of obtaining high-quality RNA which is DNA-free. Relative levels of gene expression are normalized against selected reference genes (HKG) and a number of examples of how fold regulation (2-ΔΔCq) and gene expression level (2-ΔCq) data can be presented are given.

Differential behaviour and gene expression in 3D cultures of femoral- and calvarial-derived human osteoblasts under a cyclic compressive mechanical load.

The aim of the study was to compare the response of calvarial and femoral osteoblasts cultured in a 3D hydrogel environment to cyclic compressive mechanical loading. Human foetal femoral and calvarial osteoblasts were encapsulated in a semi-synthetic thiol-modified hyaluronan gelatin polyethylene glycol diacrylate (PEGDA) cross-linked HyStemC hydrogel. Constructs were subjected to a cyclic compressive strain of 33.4 kPa force every second for 5 s every hour for 6 h per day using FlexCell BioPress culture plates and compared to non-compressed constructs. Cell viability, mineralisation, and morphological changes were observed over 21 days. BMP2, ALP, COL1A1, COL2A1, and OCN gene expression levels were quantified. Encapsulated osteoblast numbers increased and formed hydroxyapatite over a 21-day period. Cell viability decreased under a cyclical strain when compared to cells under no strain. Femoral osteoblasts under strain expressed increased levels of BMP2 (53.9-fold) and COL1A1 (5.1-fold) mRNA compared to no strain constructs. Surprisingly, no BMP2 mRNA was detected in calvarial osteoblasts. Osteoblasts derived from endochondral (femoral) and intra-membranous (calvarial) processes behaved differently in 3D-constructs. We therefore recommend that site-specific osteoblasts be used for future bone engineering and bone replacement materials and further research undertaken to elucidate how site-specific osteoblasts respond to cyclic compressive loads.

An in-vitro mechanical strain three-dimensional culture model: periodontal ligament cell viability, apoptosis, and endoplasmic reticulum stress response.

To develop a model to investigate a potential relationship between mechanical strain, cell responses, and endoplasmic reticulum stress in periodontal ligament (PDL) cells, primary PDL cell cultures were obtained from extracted premolars. Cells were cultured in hydrogel and subjected to 24 h of static mechanical strain, resulting in 18% dimensional substrate elongation. Cell viability, caspase-3/7 activity, and mRNA levels for 28 genes, including unfolded protein response (UPR)-related and mechanically responsive genes, serving as positive controls for stress induction, were examined. Compared with unstrained cultures, no difference in caspase activity was observed; however, viability responses differed between cell lines. Multiple UPR-related genes were differentially upregulated, with marginal statistical significance, including cAMP responsive element binding protein 3 like 3 (CREB3L3) (mean fold-regulation = 1.91), an adenosine monophosphate-dependent transcription factor with roles in UPR activation and the acute inflammatory response; and the pro-apoptotic UPR gene, endoplasmic reticulum to nucleus signaling 2 (ERN2) (mean fold-regulation = 4.01). The observed effect on cell viability following strain with no change in caspase activity suggests that reduction in viability may be mediated via caspase-3/7-independent mechanisms. Three-dimensional mechanical strain PDL cell culture models offer a method to study the role of endoplasmic reticulum stress and UPR, and provide a framework and potential UPR targets for future investigations.

A TaqMan™-based quantitative PCR screening assay for the probiotic Streptococcus salivarius K12 based on the specific detection of its megaplasmid-associated salivaricin B locus.

In order to assess the colonization efficacy of the oral probiotic Streptococcus salivarius K12, a rapid method for specific detection and enumeration of the strain was developed. Here, we describe a two-step TaqMan™ quantitative PCR assay using primer-probe combinations targeting genes of the locus encoding the lantibiotic bacteriocin salivaricin B.

Postnatal expression of chondrogenic and osteogenic regulatory factor mRNA in the rat condylar cartilage.

OBJECTIVES: The condylar cartilage is a key site of growth and development of the mandible. The aim of this research was to determine the mRNA expression levels of a number of chondrogenic and osteogenic regulatory factors in the condylar cartilage of the postnatal rat. MATERIALS AND METHODS: Condyles were extracted from 40 rats aged 4, 10, 21 or 90 days with 10 rats assigned to each age group. The condyles from one rat from each age group was fixed and decalcified in 10% EDTA for histology. Using cryogenic grinding combined with QIAzol reagent total RNA was purified from pooled samples collected for each age group. Each pool contained six condyles (N = 3). mRNA expression levels for 28 genes were determined using qPCR. RESULTS: Histological analysis revealed distinct morphological differences in the condyle tissue of the 4, 10, 21 and 90 day old postnatal rats. Expression of all examined genes was detected. High levels of mRNA for Alpl, Bglap, Col1a1, Col2a1, Runx2, Sox9 and Sp7 but not Msx1 were detected. Fgf1 and Fgf2 were expressed at a similar level. No significant difference (defined as ±â€¯fold-regulation > 2 and P < 0.05) in the gene mRNA expression levels was found when days 10, 21 or 90 were compared to day 4. CONCLUSIONS: Apparent morphological changes of the rat condylar cartilage are not reflected in a change in the expression levels of the chondrogenic and osteogenic regulatory factor mRNA investigated in this study.

A Protocol for the Determination of the Methylation Status of Gingival Tissue DNA at Specific CpG Islands.

Tissue biopsies are very precious. A method that allows the isolation of a high quality and quantity of genomic DNA, total RNA, and total protein from a single biopsy that is suitable for downstream applications (e.g., DNA methylation analysis, quantitative PCR, and gel electrophoresis techniques) is very desirable. The method described here combines a tissue stabilization reagent combined with a spin-column method for the simultaneous purification of gingival tissue DNA, RNA, and protein. The genomic DNA is then used for quantitative analysis of DNA methylation using real-time PCR (the qAMP method). Subsequent data analysis is very straightforward using online software. Future analyses may include the RNA transcript analysis as well as protein expression levels of genes identified as differentially methylated.

Quantitative Real-Time Gene Profiling of Human Alveolar Osteoblasts.

The use of quantitative real-time reverse transcriptase PCR (qRT2-PCR) for the identification of differentially regulated genes is a powerful technology. The protocol presented here uses qRT2-PCR gene arrays to investigate the regulation of 84 angiogenic related genes in human primary alveolar osteoblasts following treatment with the bisphosphonate, zoledronic acid (ZA), and geranylgeraniol (GGOH). GGOH has potential as a therapeutic agent for Bisphosphate-Related Osteonecrosis of the Jaw (BRONJ), a serious side-effect resulting from the treatment for metastatic cancer (Zafar et al., J Oral Pathol Med 43:711-721, 2014; Ruggiero, Ann NY Acad Sci 1218:38-46, 2011). The isolation of the primary osteoblast cells follows the methods previously described (Dillon et al., Methods Mol Biol 816:3-18, 2012) with a new RNA extraction technique described fully. The method highlights the importance of obtaining high-quality RNA which is DNA-free. Relative levels of gene expression are normalized against selected housekeeping genes (HKG) and a number of examples of how fold regulation (2-∆∆Cq) and gene expression level (2-∆Cq) data can be presented are given.

A comparison of salivary IgA in children with Down syndrome and their family members.

The aim of this study was to compare total IgA in the whole saliva of children with Down syndrome with levels in sibling and parent groups. IgA measurements were presented as the concentration in saliva (µg/ml) and also adjusted for salivary flow rate (SFR; µg/min). Twenty children with Down syndrome, ten siblings and twenty parents were recruited. Stimulated whole saliva was collected from the participants and SFR calculated. The measurement of salivary IgA (sIgA) was carried out using an indirect competitive Enzyme-Linked Immunosorbent Assay. The difference in the mean SFR between children with Down syndrome, parents and siblings were not statistically significant. The mean salivary concentration of IgA was higher in children with Down syndrome (95.1 µg/ml) compared with siblings (48.3 µg/ml; p=0.004). When adjusted for SFR children with Down syndrome had mean sIgA levels of 98.8 µg/min and the siblings 48.6 µg/min (p=0.008). The children with Down syndrome had sIgA levels similar to those of the parents (92.5 µg/ml; 93.2 µg/min). There was a positive correlation between age and sIgA concentration in the siblings (p=0.008) but not for children with Down syndrome (p=0.363). This suggests that under similar environmental influences, the levels of sIgA in children with Down syndrome are higher than in the siblings, from a very young age.

Real-time PCR focused-gene array profiling of gingival and periodontal ligament fibroblasts.

The techniques for the establishment of primary gingival and periodontal ligament fibroblast cultures have been well established for over 30 years. It is only more recently, with the commercial availability of real-time PCR (RT-PCR) gene arrays that the expression profiles of up to 84 genes can be carried out simultaneously. Each focused panel of genes can identify the up- or down-regulation of genes associated with any one of over 100 biological pathways or specific disease states. Fibroblasts for RNA extraction and subsequent gene expression analysis can be collected under various experimental conditions and stored in RNA-preserving solution (e.g., RNAlater) for processing at a later date or extracted immediately. The "gold standard" method for the extraction of RNA from fibroblasts for RT-PCR purposes is the TRIzol reagent method. With the addition of a spin-column clean-up step, any phenol carried over from the TRIzol step is removed, thus ensuring a high yield of quality RNA. The RNA is then reverse transcribed to cDNA and analyzed using the RT-PCR focused-gene arrays. Data analysis is made easy using on-line array analysis software packages.

Induction of osteopenia during experimental tooth movement in the rat: alveolar bone remodelling and the mechanostat theory.

Increases in bone strains above a certain threshold have a positive effect on bone mass, whereas reductions in strain magnitude lead to bone loss and osteopenia; the term 'mechanostat' has been introduced to describe this tissue-level negative feedback mechanism. The mechanobiology of bone and particularly alveolar bone is poorly understood, and whether the mechanostat theory has any relevance to explaining the osseous changes that occur during orthodontic tooth movement remains unclear. To investigate the relationship further, an expansile force of 0.2 N was applied to the maxillary molars of 36, 6-week-old Wistar rats by helical coil springs. The animals were sacrificed at 1, 2, 4, and 8 days and the tissue response analyzed by histological, biochemical, and finite element (FE) methods. Differences between groups were determined by Student's t-test (two-tailed). The appliance produced an increase in the intermolar width averaging 0.5 mm after 8 days. Tetracycline uptake in the control rats suggested a rapid turnover of bone in both the interradicular domain and the bone-periodontal ligament interface. In the experimental group, however, incorporation of tetracycline into the interradicular domain was reduced and conventional histology revealed evidence of bone loss and osteopenia, in both the experimental and a group of sham-treated positive controls (with inactive, annealed springs). Serum alkaline phosphatase declined significantly in both experimental and sham-treated groups over the 8-day time course, indicating decreased bone formation. Serum acid phosphatase also declined, suggesting a concomitant decrease in bone resorption. Three-dimensional FE analysis of the stresses generated in the bone following occlusal (2 N) and orthodontic loading showed that the orthodontic force created a constant loading condition shielding some areas of bone from mechanical stress. Areas of low mechanical stimulation were coincident with sites of bone loss observed histologically, while bone mass was preserved in areas with higher levels of loading. These findings suggest that (1) the osteopenia resulted from stress shielding of the interradicular bone by the appliance, and a consequent reduction in occlusal loading below the critical threshold required for maintaining normal osseous architecture and (2) the mechanostat model can be employed to explain, at least in part, the response of the bone to orthodontic loading.

Cultured human periodontal ligament cells constitutively express multiple osteotropic cytokines and growth factors, several of which are responsive to mechanical deformation.

BACKGROUND AND OBJECTIVE: A role for cytokines and growth factors in mediating the cellular and molecular events involved in orthodontic tooth movement is well established. The focus to date, however, has been largely on individual mediators, rather than to study cytokines in terms of complex interacting networks. Our objective was to expand our knowledge of the cytokines and growth factors expressed by human periodontal ligament (PDL) cells and to identify new genes that are responsive to mechanical deformation. MATERIAL AND METHODS: Human PDL cells were strained with a cyclic deformation of 12% for 6-24 h, and the differential expression of 79 cytokine and growth factor genes was quantified using real-time RT-PCR arrays. For statistical comparison, t-tests were used with mean critical threshold (CT) values derived from triplicate samples. RESULTS: Forty-one genes were detected at CT values < 35 and, of these, 15 showed a significant change in relative expression. These included seven interleukins (IL): IL1A, IL1F7, IL6 and IL7 (down), IL8, IL11 and IL12A (up). Eight genes representing other cytokine and growth factor families showed comparable mechanical sensitivity, including VEGFD and OPG (down) and PDGFA, INHBA, GDF8 and two transforming growth factor beta genes, TGFB1 and TGFB3 (up). The genes CSF2/GMCSF and IL11 were found to be consistently stimulated across all three time points. Genes that were not expressed included: (1) the immunoregulatory lymphokines (IL2-IL5), IL17 and IL17B; (2) IL10 and other members of the IL-10 family of anti-inflammatory cytokines (IL19, IL20, IL22 and IL24); and (3) TNF and RANKL. CONCLUSION: Human PDL cells constitutively express numerous osteotropic cytokines and growth factors, many of which are mechanoresponsive.

Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily.

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.