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.

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.

Formulation of a Semisolid Emulsion Containing Leptospermum scoparium Essential Oil and Evaluation of In Vitro Antimicrobial and Antibiofilm Efficacy.

Manuka oil, an essential oil derived from the Leptospermum scoparium, has been traditionally used for wound care and as a topical antibacterial, antifungal, and anti-inflammatory. However, the essential oil is not well retained at mucosal sites, such as the oral cavity, where the benefits of the aforementioned properties could be utilized toward the treatment of persistent biofilms. Within this study, L. scoparium essential oil was incorporated into a semisolid emulsion for improved delivery. The safety profile of L. scoparium essential oil on human gingival fibroblasts was determined via cell viability, cytotoxicity, and caspase activation. The minimal bactericidal concentration of L. scoparium essential oil was determined, and the emulsion's antibiofilm effects visualized using confocal laser scanning microscopy. L. scoparium essential oil demonstrated a lower IC50 (0.02% at 48 h) when compared to the clinical control chlorhexidine (0.002% at 48 h) and displayed lower cumulative cytotoxicity. Higher concentrations of L. scoparium essential oil (≥ 0.1%) at 6 h resulted in higher caspase 3/7 activation, suggesting an apoptotic pathway of cell death. A minimal bactericidal concentration of 0.1% w/w was observed for 6 oral bacteria and 0.01% w/v for Porphyromonas gingivalis. Textural and rheometric analysis indicated increased stability of emulsion with a 1 : 3 ratio of L. scoparium essential oil: Oryza sativa carrier oil. The optimized 5% w/w L. scoparium essential oil emulsion showed increased bactericidal penetrative effects on Streptococci gordonii biofilms compared to oil alone and to chlorhexidine controls. This study has demonstrated the safety, formulation, and antimicrobial activity of L. scoparium essential oil emulsion for potential antibacterial applications at mucosal sites.

Oral hygiene behaviours and readiness to change using the TransTheoretical Model (TTM).

OBJECTIVE: The aim of this pilot study was to investigate the Transtheoretical Model (TTM) in relation to measures of readiness to change oral hygiene behaviours. RESEARCH DESIGN: Participants (N = 105) were recruited from a dental hygiene patient waiting list. A self-administered questionnaire was designed; it included four measures related to inter-dental cleaning used for TTM staging, confidence and frequency measures of future interdental cleaning and toothbrushing, together with items seeking demographic details. Data collection occurred before a dental hygiene appointment where oral health advice was offered, and then at three and six months afterwards, in order to measure readiness to change post-intervention. RESULTS: All three questionnaires were returned by 91.4% of participants. The confidence measures for maintaining toothbrushing twice per day and for interdental cleaning were associated with TTM staging at baseline (respective correlation coefficients of 0.200; P = 0.042 and 0.584; P < 0.001). Participants were likely to be in a higher TTM stage at 3 months after attendance at the dental hygiene clinic and then decline to a lower TTM stage by 6 months (baseline to 3 months and 6 months: Wilcoxon signed rank tests of p= 0.024 and p = 0.627). Of the 31 participants (33%) who improved their TTM staging between baseline and 3 months, 11 (35%) fell back to a lower category between 3 months and 6 months, 14 (45%) maintained their improvement, and 6 (19%) improved further. CONCLUSIONS: Understanding a person's readiness to change could improve the way in which oral hygiene interventions and advice are given in the clinical setting. The TTM staging measurement tool used here provides insight into people's readiness to change their oral hygiene behaviours, and its use would aid practitioners in the delivery of oral health messages. The initial improvement in TTM stage and subsequent regression was consistent with the TTM's relapse phenomenon and reinforces the concept that on-going support is crucial to maintaining behaviour change.

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.

Effect of the Rho-Kinase/ROCK Signaling Pathway on Cytoskeleton Components.

The mechanical properties of cells are important in tissue homeostasis and enable cell growth, division, migration and the epithelial-mesenchymal transition. Mechanical properties are determined to a large extent by the cytoskeleton. The cytoskeleton is a complex and dynamic network composed of microfilaments, intermediate filaments and microtubules. These cellular structures confer both cell shape and mechanical properties. The architecture of the networks formed by the cytoskeleton is regulated by several pathways, a key one being the Rho-kinase/ROCK signaling pathway. This review describes the role of ROCK (Rho-associated coiled-coil forming kinase) and how it mediates effects on the key components of the cytoskeleton that are critical for cell behaviour.

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.

Meeting the challenges and clinical requirements for dental regeneration; the New Zealand experience.

Disease and trauma leading to tooth loss and destruction of supporting bone is a significant oral handicap, which may be addressed through surgical therapies that aim to regenerate the lost tissue. Whilst complete regeneration of teeth is still aspirational, regeneration of supporting structures (dental pulp, cementum, periodontal ligament, bone) is becoming commonplace, both for teeth and for titanium dental implants that are used to replace teeth. Most grafting materials are essentially passive, however the next generation of oral regenerative devices will combine non-antibiotic antimicrobials and/or osteogenic or inductive factors and/or appropriate multipotential stem cells. The review gives an overview of the approaches taken, including fabrication of novel scaffolds, incorporation of growth factors and cell-based therapies, and discusses the preclinical animal models we employ in the development pathway.

Effects of galectin-1 inhibitor OTX008 on oral squamous cell carcinoma cells in vitro and the role of AP-1 and the MAPK/ERK pathway.

OBJECTIVE: To investigate the in vitro effects of inhibiting galectin-1 using the small-molecule inhibitor OTX008 on oral squamous cell carcinoma (OSCC) cell lines and the role of the MAPK pathway. METHODS: One normal oral keratinocyte (NOK) and three OSCC cell lines were cultured in vitro and the expression of galectin-1 protein by each quantified using ELISA. Cell lines were treated with galectin-1 (50, 100 and 150 ng/mL) or OTX008 (12.5, 25, 50 and 100 µg/mL) and cell viability assayed (n = 3). OSCC cell lines with and without 25 µg/mL OTX008 (n = 3) treatment for 48 h, were analysed using qRT2-PCR with a custom array, to assess relative gene expression. RESULTS: All cell lines were found to express galectin-1 protein. Exogenous galectin-1 significantly reduced cell viability in one OSCC cell line over time while the others were only minimally affected. OTX008 treatment reduced cell viability in a dose and time-dependent manner in all cell lines and this was associated with significant regulation of FOS gene expression in the OSCC cell lines. CONCLUSION: OTX008 decreased the viability of OSCC and NOK cells in a dose-dependent manner. The significant regulation of FOS suggests OTX008 causes early induction of the MAPK pathway via the immediate response gene FOS as a subunit of the AP-1 complex.

New Zealand's oral health students--education and workforce implications.

OBJECTIVES: To obtain background information on the Oral Health (OH) students at Auckland University of Technology (AUT) and the University of Otago in order to aid in the recruitment of students; to determine the extent of the students' professional knowledge; and to determine their future employment preferences. METHOD: Cross-sectional survey of all OH students at AUT and the University of Otago in 2008. A questionnaire was given to all 165 OH students at both Universities, and the response rate was 100%. RESULTS: Most students came from Cities. Prior to commencing their OH course, they had been engaged in full-time work, in tertiary education or at school. Their main sources of information about the courses were websites, the Universities, friends and dental practitioners. The students' professional knowledge improved significantly as they progressed through the OH courses. Students were likely to want to return to work in the type of community that they had come from. Most (90.3%) would consider working in private practice, while 56.4% would consider working for the School Dental Service (SDS). Overall, 49.7% of students would consider working in both environments. CONCLUSION: This study provides information on recruitment of students into OH courses, and the OH students' preferences for employment after graduation. The findings have implications for OH education and workforce planning in New Zealand.

HGF/MET in osteogenic differentiation of primary human palatal periosteum-derived mesenchymal stem cells.

PURPOSE: This study aimed to determine expressions of hepatocyte growth factor (HGF) and MET proto-oncogene receptor tyrosine kinase (MET) in palatal periosteum (PP) and to examine the effect of HGF/MET on osteogenic differentiation of human palatal periosteum-derived mesenchymal stem cells (PD-MSCs). METHODS: HGF/MET proteins in human palatal periosteum (n = 3) were localized using immunohistochemistry. PD-MSCs (n = 3) were cultured in serum-free Essential 8 (E8) medium or osteogenic medium with and without Capmatinib, a selective ATP-inhibitor of MET. HGF concentration in vitro was measured with ELISA. Relative gene expression was quantified from PD-MSCs by quantitative reverse transcription real-time polymerase chain reaction. RESULTS: Immunohistochemistry detected co-localization of HGF and MET protein in PP. HGF protein levels were significantly higher (P < 0.05) in osteogenic media (day 21: 12.19 ± 8.36 ng/mL) than in E8 medium (day 21: 0.42 ± 0.72 ng/mL). MET inhibitor had a limited feedback effect on the expression profile of the osteogenic genes tested. Gene expression levels for all but three genes were comparable in serum-free and osteogenic media at all time points. CONCLUSION: HGF/MET present in human PP and HGF is upregulated in vitro during osteogenesis; however the targeted pathways controlled by MET may not involve osteoblast maturation.

Growing 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 significantly 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.

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.

Unfolded protein response-related gene regulation in inflamed periodontal tissues with and without Russell bodies.

OBJECTIVE: To examine the expression of unfolded protein response (UPR) genes, a set of genes that are activated to assist in protein trafficking and cellular homeostasis when endoplasmic reticulum (ER) stress occurs, in inflamed and uninflamed periodontal tissues, with or without Russell bodies (RB). RB are a histologically apparent extension of the ER that represents an accumulation of abnormal proteins that cannot be secreted or degraded and may serve as a marker of ER stress. DESIGN: Periodontal tissue specimens were collected and categorised histologically based on the presence of inflammation and the quantity of RB. The differential regulation of 84 UPR-related genes was examined by qRT(2)-PCR. RESULTS: UPR genes related to the inositol-requiring ER-to-nucleus signal kinase (IRE)-1 pathway, molecular chaperones and ER quality control were up-regulated in RB(+) tissues compared with RB(-) tissues, irrespective of inflammation. Inflamed periodontal tissues showed a marked down-regulation of heat shock protein (HSP)-70 family members. CONCLUSION: The presence of RB in inflamed periodontal tissues correlated with the expression of a unique set of ER stress-related genes and therefore may serve as a marker of UPR response in periodontal inflammation. Inflamed periodontal tissues showed a marked down-regulation of UPR genes, in particular HSP70. This may be contributory to disease progression in periodontal disease.

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.

Dental therapists and dental hygienists educated for the New Zealand environment.

New Zealand has a long history of dental care provided by school dental nurses, now known as dental therapists. The nature of their training courses, although delivered in different centers, had remained relatively constant until 1999 when educational responsibility was transferred to the universities. Dental hygienists were not trained in New Zealand until 1994, with the exception of the New Zealand Army hygienists. Since 2001, the education of both dental therapists and dental hygienists has been the responsibility of the universities. Significant and progressive changes in educational delivery have occurred since then, which have culminated in three-year degree qualifications for dual-trained oral health professionals. Factors influencing this change included increased professionalism associated with the new legislative requirements for registration, workforce shortages, and enhanced educational and clinical practice requirements. The Bachelor of Oral Health degree at the University of Otago has an added emphasis on social sciences and incorporates aspects of learning relating to New Zealand's cultural heritage. We explore in this article the rationale for the introduction of a Bachelor of Oral Health in New Zealand and how it is designed to equip graduates as professionals in oral health.