Calcium ions promote osteogenic differentiation and mineralization of human dental pulp cells : implication for pulp capping materials

Calcium (Ca) is the main element of most pulp capping materials and plays an essential role in mineralization. Different pulp capping materials can release various concentrations of Ca ions leading to different clinical outcomes. The purpose of this study was to investigate the effects of various concentrations of Ca ions on the growth and osteogenic differentiation of human dental pulp cells (hDPCs). Different concentrations of Ca ions were added to growth culture medium and osteogenic inductive culture medium. A Cell Counting Kit-8 (CCK-8) was used to determine the proliferation of hDPCs in growth culture medium. Osteogenic differentiation and mineralization were measured by alkaline phosphatase (ALP) assay, Alizarin red S/von kossa staining, calcium content quantitative assay. The selected osteogenic differentiation markers were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Within the range of 1.8–16.2 mM, increased concentrations of Ca ions had no effect on cell proliferation, but led to changes in osteogenic differentiation. It was noted that enhanced mineralized matrix nodule formation was found in higher Ca ions concentrations; however, ALP activity and gene expression were reduced. qRT-PCR results showed a trend towards down-regulated mRNA expression of type I collagen (COL1A2) and Runx2 at elevated concentrations of Ca ions, whereas osteopontin (OPN) and osteocalcin (OCN) mRNA expression was significantly up-regulated. Ca ions content in the culture media can significantly influence the osteogenic properties of hDPCs, indicating the importance of optimizing Ca ions release from dental pulp capping materials in order to achieve desirable clinical outcomes.

Pre-clinical evaluation of amphiphilic silicone oligomers for scar remediation

The formation of hypertrophic scars is a frequent outcome of wound repair and often requires further therapy with treatments such as silicone gel sheets (SGS; Perkins et al., 1983). Although widely used, knowledge regarding SGS and their mechanism of action on hypertrophic scars is limited. Furthermore, SGS require consistent application for at least twelve hours a day for up to twelve consecutive months, beginning as soon as wound reepithelialisation has occurred. Preliminary research at QUT has shown that some species of silicone present in SGS have the ability to permeate into collagen gel skin mimetics upon exposure. An analogue of these species, GP226, was found to decrease both collagen synthesis and the total amount of collagen present following exposure to cultures of cells derived from hypertrophic scars. This silicone of interest was a crude mixture of silicone species, which resolved into five fractions of different molecular weight. These five fractions were found to have differing effects on collagen synthesis and cell viability following exposure to fibroblasts derived from hypertrophic scars (HSF), keloid scars (KF) and normal skin (nHSF and nKF). The research performed herein continues to further assess the potential of GP226 and its fractions for scar remediation by determining in more detail its effects on HSF, KF, nHSF, nKF and human keratinocytes (HK) in terms of cell viability and proliferation at various time points. Through these studies it was revealed that Fraction IV was the most active fraction as it induced a reduction in cell viability and proliferation most similar to that observed with GP226. Cells undergoing apoptosis were also detected in HSF cultures exposed to GP226 and Fraction IV using the Tunel assay (Roche). These investigations were difficult to pursue further as the fractionation process used for GP226 was labour-intensive and time inefficient. Therefore a number of silicones with similar structure to Fraction IV were synthesised and screened for their effect following application to HSF and nHSF. PDMS7-g-PEG7, a silicone-PEG copolymer of low molecular weight and low hydrophilic-lipophilic balance factor, was found to be the most effective at reducing cell proliferation and inducing apoptosis in cultures of HSF, nHSF and HK. Further studies investigated gene expression through microarray and superarray techniques and demonstrated that many genes are differentially expressed in HSF following treatment with GP226, Fraction IV and PDMS7-g-PEG7. In brief, it was demonstrated that genes for TGFβ1 and TNF are not differentially regulated while genes for AIFM2, IL8, NSMAF, SMAD7, TRAF3 and IGF2R show increased expression (>1.8 fold change) following treatment with PDMS7-g-PEG7. In addition, genes for αSMA, TRAF2, COL1A1 and COL3A1 have decreased expression (>-1.8 fold change) following treatment with GP226, Fraction IV and PDMS7-g-PEG7. The data obtained suggest that many different pathways related to apoptosis and collagen synthesis are affected in HSF following exposure to PDMS7-g-PEG7. The significance is that silicone-PEG copolymers, such as GP226, Fraction IV and PDMS7-g-PEG7, could potentially be a non-invasive substitute to apoptosis-inducing chemical agents that are currently used as scar treatments. It is anticipated that these findings will ultimately contribute to the development of a novel scar therapy with faster action and improved outcomes for patients suffering from hypertrophic scars.

Genetic evidence for the vital function of osterix in cementogenesis

To date, attempts to regenerate a complete tooth, including the critical periodontal tissues associated with the tooth root, have not been successful. Controversy still exists regarding the origin of the cell source for cellular cementum (epithelial or mesenchymal). This disagreement may be partially due to a lack of understanding of the events leading to the initiation and development of the tooth roots and supportive tissues, such as the cementum. Osterix (OSX) is a transcriptional factor essential for osteogenesis, but its role in cementogenesis has not been addressed. In the present study, we first documented a close relationship between the temporal- and spatial-expression pattern of OSX and the formation of cellular cementum. We then generated 3.6 Col 1-OSX transgenic mice, which displayed accelerated cementum formation vs. WT controls. Importantly, the conditional deletion of OSX in the mesenchymal cells with two different Cre systems (the 2.3 kb Col 1 and an inducible CAG-CreER) led to a sharp reduction in cellular cementum formation (including the cementum mass and mineral deposition rate) and gene expression of dentin matrix protein 1 (DMP1) by cementocytes. However, the deletion of the OSX gene after cellular cementum formed did not alter the properties of the mature cementum as evaluated by backscattered SEM and resin-cast SEM. Transient transfection of Osx in the cementoblasts in vitro significantly inhibited cell proliferation and increased cell differentiation and mineralization. Taken together, these data support 1) the mesenchymal origin of cellular cementum (from PDL progenitor cells); 2) the vital role of OSX in controlling the formation of cellular cementum; and 3) the limited remodeling of cellular cementum in adult mice.

Proteasome inhibitors as anti-cancer agents

The ubiquitin (Ub)-proteasome pathway is the major nonlysosomal pathway of proteolysis in human cells and accounts for the degradation of most short-lived, misfolded or damaged proteins. This pathway is important in the regulation of a number of key biological regulatory mechanisms. Proteins are usually targeted for proteasome-mediated degradation by polyubiquitinylation, the covalent addition of multiple units of the 76 amino acid protein Ub, which are ligated to 1-amino groups of lysine residues in the substrate. Polyubiquitinylated proteins are degraded by the 26S proteasome, a large, ATP-dependent multicatalytic protease complex, which also regenerates monomeric Ub. The targets of this pathway include key regulators of cell proliferation and cell death. An alternative form of the proteasome, termed the immunoproteasome, also has important functions in the generation of peptides for presentation by MHC class I molecules. In recent years there has been a great deal of interest in the possibility that proteasome inhibitors, through elevation of the levels of proteasome targets, might prove useful as a novel class of anti-cancer drugs. Here we review the progress made to date in this area and highlight the potential advantages and weaknesses of this approach.

The cementogenic differentiation of periodontal ligament cells via the activation of Wnt/β-catenin signalling pathway by Li+ ions released from bioactive scaffolds

Lithium (Li) has been widely used as a long-term mood stabilizer in the treatment of bipolar and depressive disorders. Li+ ions are thought to enhance the remyelination of peripheral nerves and also stimulate the proliferation of neural progenitor cells and retinoblastoma cells via activation of the Wnt/β-catenin signalling pathway. Until now there have been no studies reporting the biological effects of released Li+ in bioactive scaffolds on cemetogenesis in periodontal tissue engineering applications. In this study, we incorporated parts of Li+ ions into the mesoporous bioactive glass (MBG) scaffolds and showed that this approach yielded scaffolds with a favourable composition, microstructure and mesopore properties for cell attachment, proliferation, and cementogenic differentiation of human periodontal ligament-derived cells (hPDLCs). We went on to investigate the biological effects of Li+ ions themselves on cell proliferation and cementogenic differentiation. The results showed that 5% Li+ ions incorporated into MBG scaffolds enhanced the proliferation and cementogenic differentiation of hPDLCs on scaffolds, most likely via activation of Wnt/β-catenin signalling pathway. Further study demonstrated that Li+ ions by themselves significantly enhanced the proliferation, differentiation and cementogenic gene expression of PDLCs. Our results indicate that incorporation of Li+ ions into bioactive scaffolds is a viable means of enhancing the Wnt canonical signalling pathway to stimulate cementogenic differentiation of PDLCs.

The roles of the preproghrelin-derived peptides - ghrelin, desacyl ghrelin and obestatin - in prostate cancer

Prostate cancer is the second most common cause of cancer related deaths in Western men. Despite the significant improvements in current treatment techniques, there is no cure for advanced metastatic, castrate-resistant disease. Early detection and prevention of progression to a castrate-resistant state may provide new strategies to improve survival. A number of growth factors have been shown to act in an autocrine/paracrine manner to modulate prostate cancer tumour growth. Our laboratory has previously shown that ghrelin and its receptors (the functional GHS-R1a and the non-functional GHS-R1b) are expressed in prostate cancer specimens and cell lines. We have shown that ghrelin increases cell proliferation in the PC3 and LNCaP prostate cancer cell lines through activation of ERK1/2, suggesting that ghrelin could regulate prostate cancer cell growth and play a role in the progression of the disease. Ghrelin is a 28 amino-acid peptide hormone, identified to be the natural ligand of the growth hormone secretagogue receptor (GHS-R1a). It is well characterised as a growth hormone releasing and as an orexigenic peptide that stimulates appetite and feeding and regulates energy expenditure and bodyweight. In addition to its orexigenic properties, ghrelin has been shown to play a regulatory role in a number of systems, including the reproductive, immune and cardiovascular systems and may play a role in a number of pathological conditions such as chronic heart failure, anorexia, cachexia, obesity, diabetes and cancer. In cancer, ghrelin and its receptor are expressed in a range of tumours and cancer cell lines and ghrelin has been demonstrated to modulate cell proliferation, apoptosis, migration and invasion in some cell types. The ghrelin gene (GHRL) encodes preproghrelin peptide, which is processed to produce three currently known functional peptides - ghrelin, desacyl ghrelin and obestatin. Prohormone convertases (PCs) have been shown to cleave the preproghrelin peptide into two primary products - the 28 amino acid peptide, ghrelin, and the remaining 117 amino acid C-terminal peptide, C-ghrelin. C-ghrelin can then be further processed to produce the 23 amino acid peptide, obestatin. Ghrelin circulates in two different forms - an octanoylated form (known as ghrelin) and a non-octanoylated form, desacyl ghrelin. The unique post-translational addition of octanoic acid to the serine 3 residue of the propeptide chain to form acylated ghrelin is catalysed by ghrelin O-acyltransferase (GOAT). This modification is necessary for binding of ghrelin to its only known functional receptor, the GHS-R1a. As desacyl ghrelin cannot bind and activate the GHS-R1a, it was initially thought to be an inactive peptide, despite the fact that it circulates at much higher levels than ghrelin. Further research has demonstrated that desacyl ghrelin is biologically active and shares some of the actions of ghrelin, as well as having some opposing and distinct roles. Interestingly, both ghrelin and desacyl ghrelin have been shown to modulate apoptosis, cell differentiation and proliferation in some cell types, and to stimulate cell proliferation through activation of ERK1/2 and PI3K/Akt pathways. The third known peptide product of the ghrelin preprohormone, obestatin, was initially thought to oppose the actions of ghrelin in appetite regulation and food intake and to mediate its effects through the G protein-coupled receptor 39 (GPR39). Subsequent research failed to reproduce the initial findings, however, and the possible anorexigenic effects of obestatin, as well as the identity of its receptor, remain unclear. Obestatin plays some important physiological roles, including roles in improving memory, the inhibition of thirst and anxiety, increased secretion of pancreatic juice, and regulation of cell proliferation, survival, apoptosis and differentiation. Preliminary studies have also shown that obestatin stimulates cell proliferation in some cell types through activation of ERK1/2, Akt and PKC pathways. Overall, however, at the commencement of this PhD project, relatively little was known regarding the functions and mechanisms of action of the preproghrelin-derived functional peptides in modulating prostate cancer cell proliferation. The roles of obestatin, and desacyl ghrelin as potential growth factors had not previously been investigated, and the potential expression and regulation of the preproghrelin processing enzymes, GOAT and prohormone convertases was unknown in prostate cancer cell lines. Therefore, the overall objectives of this study were to: 1. investigate the effects of obestatin on cell proliferation and signaling in prostate cancer cell lines 2. compare the effects of desacyl ghrelin and ghrelin on cell proliferation and signaling in prostate cancer cell lines 3. investigate whether prostate cancer cell lines possess the necessary enzymatic machinery to produce ghrelin and desacyl ghrelin and if these peptides can regulate GOAT expression Our laboratory has previously shown that ghrelin stimulates cell proliferation in the PC3 and LNCaP prostate cancer cell line through activation of the ERK1/2 pathway. In this study it has been demonstrated that treatments with either ghrelin, desacyl ghrelin or obestatin over 72 hours significantly increased cell proliferation in the PC3 prostate cancer cell line but had no significant effect in the RWPE-1 transformed normal prostate cell line. Ghrelin (1000nM) stimulated cell proliferation in the PC3 prostate cancer cell line by 31.66 6.68% (p<0.01) with the WST-1 method, and 13.55 5.68% (p<0.05) with the CyQUANT assay. Desacyl ghrelin (1000nM) increased cell proliferation in PC3 cells by 21.73 2.62% (p<0.01) (WST-1), and 15.46 7.05% (p<0.05) (CyQUANT) above untreated control. Obestatin (1000nM) induced a 28.37 7.47% (p<0.01) (WST-1) and 12.14 7.47% (p<0.05) (CyQUANT) significant increase in cell proliferation in the PC3 prostate cancer cell line. Ghrelin and desacyl ghrelin treatments stimulated Akt and ERK phosphorylation across a range of concentrations (p<0.01). Obestatin treatment significantly stimulated Akt, ERK and PKC phosphorylation (p<0.05). Through the use of specific inhibitors, the MAPK inhibitor U0126 and the Akt1/2 kinase inhibitor, it was demonstrated that ghrelin- and obestatin-induced cell proliferation in the PC3 prostate cancer cell line is mediated through activation of ERK1/2 and Akt pathways. Although desacyl ghrelin significantly stimulated Akt and ERK phosphorylation, U0126 failed to prevent desacyl ghrelin-induced cell proliferation suggesting ghrelin and desacyl ghrelin might act through different mechanisms to increase cell proliferation. Ghrelin and desacyl ghrelin have shown a proliferative effect in osteoblasts, pancreatic -cells and cardiomyocytes through activation of ERK1/2 and PI3K/Akt pathways. Here it has been shown that ghrelin and its non-acylated form exert the same function and stimulate cell proliferation in the PC3 prostate cancer cell line through activation of the Akt pathway. Ghrelin-induced proliferation was also mediated through activation of the ERK1/2 pathway, however, desacyl ghrelin seems to stimulate cell proliferation in an ERK1/2-independent manner. As desacyl ghrelin does not bind and activate GHSR1a, the only known functional ghrelin receptor, the finding that both ghrelin and desacyl ghrelin stimulate cell proliferation in the PC3 cell line suggests that these peptides could be acting through the yet unidentified alternative ghrelin receptor in this cell type. Obestatin treatment also stimulated PKC phosphorylation, however, a direct role for this pathway in stimulating cell proliferation could not be proven using available PKC pathway inhibitors, as they caused significant cell death over the extended timeframe of the cell proliferation assays. Obestatin has been shown to stimulate cell proliferation through activation of PKC isoforms in human retinal epithelial cells and in the human gastric cancer cell line KATO-III. We have demonstrated that all of the prostate-derived cell lines examined (PC3, LNCaP, DU145, 22Rv1, RWPE-1 and RWPE-2) expressed GOAT and at least one of the prohormone convertases, which are known to cleave the proghrelin peptide, PC1/3, PC2 and furin, at the mRNA level. These cells, therefore, are likely to possess the necessary machinery to cleave the preproghrelin protein and to produce the mature ghrelin and desacyl ghrelin peptides. In addition to prohormone convertases, the presence of octanoic acid is essential for acylated ghrelin production. In this study octanoic acid supplementation significantly increased cell proliferation in the PC3 prostate cancer cell line by over 20% compared to untreated controls (p<0.01), but surprisingly, not in the DU145, LNCaP or 22Rv1 prostate cancer cell lines or in the RWPE-1 and RWPE-2 prostate-derived cell lines. In addition, we demonstrated that exogenous ghrelin induced a statistically significant two-fold decrease in GOAT mRNA expression in the PC3 cell line (p<0.05), suggesting that ghrelin could pontentially downregulate its own acylation and, therefore, regulate the balance between ghrelin and desacyl ghrelin. This was not observed, however, in the DU145 and LNCaP prostate cancer cell lines. The GOAT-ghrelin system represents a direct link between ingested nutrients and regulation of ghrelin production and the ghrelin/desacyl ghrelin ratio. Regulation of ghrelin acylation is a potentially attractive and desirable tool for the development of better therapies for a number of pathological conditions where ghrelin has been shown to play a key role. The finding that desacyl ghrelin stimulates cell proliferation in the PC3 prostate cancer cell line, and responds to ghrelin in the same way, suggests that this cell line expresses an alternative ghrelin receptor. Although all the cell lines examined expressed both GHS-R1a and GHS-R1b mRNA, it remains uncertain whether these cell lines express the unidentified alternative ghrelin receptor. It is possible that the varied responses seen could be due to the expression of different ghrelin receptors in different cell lines. In addition to GOAT, prohormone convertases and octanoic acid availability may regulate the production of different peptides from the ghrelin preprohormone. The studies presented in this thesis provide significant new information regarding the roles and mechanisms of action of the preproghrelin-derived peptides, ghrelin, desacyl ghrelin and obestatin, in modulating prostate cancer cell line proliferation. A number of key questions remain to be resolved, however, including the identification of the alternative ghrelin/desacyl ghrelin receptor, the identification of the obestatin receptor, a clarification of the signaling mechanisms which mediate cell proliferation in response to obestatin treatment and a better understanding of the regulation at both the gene and post-translational levels of functional peptide generation. Further studies investigating the role of the ghrelin axis using in vivo prostate cancer models may be warranted. Until these issues are determined, the potential for the ghrelin axis, to be recognised as a novel useful target for therapy for cancer or other pathologies will be uncertain.

Preparation, characterization, and in vitro bioactivity of nagelschmidtite bioceramics

The aim of this study is to prepare Ca, P and Si-containing ternary oxide nagelschmidtite (NAGEL, Ca7Si2P2O16) bioceramics and explore their in vitro bioactivity for potential bone tissue regeneration. We prepared dense NAGEL ceramics through high-temperature sintering of NAGEL ceramic powders. The apatite-mineralization ability, dissolution rate, and human osteoblast response (including cytotoxicity analysis, attachment, morphology, proliferation, and bone-related gene expression) to NAGEL ceramics have been systematically studied by comparing with conventional β-tricalcium phosphate (β-TCP) ceramics. The results showed that NAGEL ceramics possessed more obvious apatite mineralization and dissolution (degradation) and stimulated bone-related gene expression (OCN and OPN) of osteoblasts than β-TCP ceramics. NAGEL ceramics also showed no significant cytotoxicity. NAGEL ceramics supported osteoblast attachment, proliferation, and osteogenic gene expression, with a comparable cell proliferation activity with β-TCP ceramics. These results indicate that novel NAGEL bioceramics with the specific composition of Ca7Si2P2O16, are a promising biomaterial for bone tissue regeneration application.

Fibroblast growth factor receptor inhibition synergizes with paclitaxel and doxorubicin in endometrial cancer cells

OBJECTIVE: The fibroblast growth factor (FGF) family of signaling molecules has been associated with chemoresistance and poor prognosis in a number of cancer types, including lung, breast, ovarian, prostate, and head and neck carcinomas. Given the identification of activating mutations in the FGF receptor 2 (FGFR2) receptor tyrosine kinase in a subset of endometrial tumors, agents with activity against FGFRs are currently being tested in clinical trials for recurrent and progressive endometrial cancer. Here, we evaluated the effect of FGFR inhibition on the in vitro efficacy of chemotherapy in endometrial cancer cell lines. METHODS: Human endometrial cancer cell lines with wild-type or activating FGFR2 mutations were used to determine any synergism with concurrent use of the pan-FGFR inhibitor, PD173074, and the chemotherapeutics, doxorubicin and paclitaxel, on cell proliferation and apoptosis. RESULTS: FGFR2 mutation status did not alter sensitivity to either chemotherapeutic agent alone. The combination of PD173074 with paclitaxel or doxorubicin showed synergistic activity in the 3 FGFR2 mutant cell lines evaluated. In addition, although nonmutant cell lines were resistant to FGFR inhibition alone, the addition of PD173074 potentiated the cytostatic effect of paclitaxel and doxorubicin in a subset of FGFR2 wild-type endometrial cancer cell lines. CONCLUSIONS: Together these data suggest a potential therapeutic benefit to combining an FGFR inhibitor with standard chemotherapeutic agents in endometrial cancer therapy particularly in patients with FGFR2 mutation positive tumors.

GABAergic modification of myopic and hyperopic ocular tissue effects on scleral fibroblast growth

Purpose: Myopia is a common eye disorder affecting up to 90% of children in South East Asia and 30% of the population worldwide. Myopia of high severity is a leading cause of blindness around the world (4th to 5th most common). Changes and remodelling of the sclera i.e. increase cellular proliferation & increase protein synthesis within scleral cells (↑ scleral DNA) and thinning and lose of extracellular matrix of sclera (↓ scleral GAG synthesis) have been linked to myopic eye growth in animal models. Signals acting on the sclera are thought to originate in the retina, and are modulated by the retinal pigment epithelium (RPE) with limited evidence suggesting that the RPE can modify scleral cell growth in culture. However, the mechanism of retinal signal transmission and the role of posterior eye cup tissue, including the RPE, in mediating changes in scleral fibroblast growth during myopia development are unclear. Retinal transmitter systems are critically involved in pathways regulating eye growth, which ultimately lead to alterations in the sclera if eye size is to change. A dopaminergic agonist and muscarinic antagonists decrease the proliferation of scleral chondrocytes when co-cultured with chick’s retinal pigment epithelium (RPE). GABA receptors have recently been localised to chick sclera. We therefore hypothesised that posterior eye cup tissue from myopic eyes would stimulate and from hyperopic eyes would inhibit growth of scleral fibroblasts in vitro and that GABAergic agents could directly interact with scleral cells or indirectly modify the effects of myopic and hyperopic posterior eye cup tissue on scleral fibroblast growth. Method: Fibroblastic cells obtained from 8-day-old chick sclera were used to establish cell banks. Two major experiments were performed. Experiment 1: To determine if posterior eye cup tissues from myopic eye stimulates and hyperopic eye inhibits scleral cell proliferation, when co-cultured with scleral cells in vitro. This study comprised two linked experiments, i) monocular visual treatments of FDM (form-deprivation myopia), LIM (lens-induced myopia) and LIH (lens-induced hyperopia) with assessment of the effect of full punch eye cup tissue on DNA and GAG synthesis by cultured chick scleral fibroblasts, and ii) binocular visual treatments comprising LIM and LIH with assessment of the effect of individual layers of eye cup tissues (neural retina, RPE and choroid) on cultured chick scleral fibroblasts. Visual treatment was applied for 3 days. Experiment 2: To determine the direct interaction of GABA agents on scleral cell growth and to establish whether GABA agents modify the stimulatory/inhibitory effect of myopic and hyperopic posterior eye cup tissues on cultured scleral cell growth in vitro. Two linked experiments were performed. i) GABA agonists (muscimol and baclofen) and GABA antagonists (bicuculine (-), CGP46381 and TPMPA) were added to scleral cell culture medium to determine their direct effect on scleral cells. ii) GABAergic agents (agonists and antagonists) were administered to scleral fibroblasts co-cultured with posterior eye cup tissue (retina, RPE, retina/RPE, RPE/choroid). Ocular tissues were obtained from chick eyes wearing +15D (LIH) or -15D lenses (LIM) for 3 days. In both experiments, tissues were added to hanging cell culture insert (pore size 1.0ìm) placed over each well of 24 well plates while scleral cells were cultured in DMEM/F12, Glutamax (Gibco) plus 10% FBS and penicillin/streptomycin (50U/ml)) and fungizone (1.25ug/ml) (Gibco), at seeding density of 30,000 cells/well at the bottom of the well and allowed to grow for 3 days. Scleral cells proliferation rate throughout the study was evaluated by determining GAG and DNA content of scleral cells using Dimethylmethylene blue (DMMB) dye and Quant-iTTm Pico Green® dsDNA reagent respectively. Results and analysis: Based on DNA and GAG content, there was no significant difference in tissue effect of LIM and LIH eyes on scleral fibroblast growth (DNA: 8.4 ± 1.1μg versus 9.3 ± 2.3 μg, p=0.23; GAG: 10.13 ± 1.4 μg versus 12.67 ± 1.2 μg, F2,23=6.16, p=0.0005) when tissues were obtained from monocularly treated chick eyes (FDM or +15D lens or -15D lens over right eyes with left eyes untreated) and co-cultured as full punch. When chick eyes were treated binocularly with -15D lens (LIM) right eye and +15D lens (LIH) left eyes and tissue layers were separated, the retina from LIM eyes did not stimulate scleral cell proliferation compared to LIH eyes (DNA: 27.2 ± 6.7 μg versus 23.2 ± 1.5 μg, p=0.23; GAG: 28.1 ±3.7 μg versus 28.7 ± 4.2 μg, p=0.21). Similarly, the LIH and LIM choroid did not produce a differential effect based on DNA (LIM 46.9 ± 6.4 μg versus LIH 53.5 ± 4.7 μg, p=0.18), however the choroid from LIH eyes induced higher scleral GAG content than from LIM eyes (32.5 ± 6.7 μg versus 18.9 ± 1.2 μg, p=0.023). In contrast, the RPE from LIM eyes caused a significant increase in fibroblast proliferation whereas the RPE from LIH eyes was relatively inhibitory (72.4 ± 6.3 μg versus 27.9 ± 2.3 μg, F1, 6=69.99, p=0.0005). GAG data were opposite to DNA data e.g. the RPE from LIH eyes increased (33.7 ± 7.9 μg) while the RPE from LIM eyes decreased (28.2 ± 3.0 μg) scleral cell growth (F1, 6=13.99, p=0.010). Based on DNA content, GABA agents had a small direct effect on scleral cell growth; GABA agonists increased (21.4 ± 1.0% and 18.3 ± 1.0% with muscimol and baclofen, p=0.0021), whereas GABA antagonists decreased fibroblast proliferation (-23.7 ± 0.9% with bicuculine & CGP46381 and -28.1 ± 0.5% with TPMPA, p=0.0004). GABA agents also modified the effect of LIM and LIH tissues (p=0.0005).The increase in proliferation rate of scleral fibroblasts co-cultured with tissues (RPE, retina, RPE/retina and RPE/choroid) from LIM treated eyes was enhanced by GABA agonists (muscimol: 27.4 ± 1.2%, 35.8 ± 1.6%, 8.4 ± 0.3% and 11.9 ± 0.6%; baclofen: 27.0 ± 1.0%, 15.8 ± 1.5%, 16.8 ± 1.2% and 15.4 ± 0.4%, p=0.014) whereas GABA antagonists further reduced scleral fibroblasts growth (bicuculine: -52.5 ± 2.5%, -36.9 ± 1.4%, -37.5 ± 0.6% and -53.7 ± 0.9%; TPMPA: 57.3 ± 1.3%, -15.7 ± 1.2%, -33.5 ± 0.4% and -45.9 ± 1.5%; CGP46381: -51.9 ± 1.6%, -28.5 ± 1.5%, -25.4 ± 2.0% and -45.5 ± 1.9% respectively, p=0.0034). GAG data were opposite to DNA data throughout the experiment e.g. GABA agonists further inhibited while antagonists relatively enhanced scleral fibroblasts growth for both LIM and LIH tissue co-culture. The effect of GABA agents was relatively lower (p=0.0004) for tissue from LIH versus LIM eyes but was in a similar direction. There was a significant drug effect on all four tissue types e.g. RPE, retina, RPE/retina and RPE/choroid for both LIM and LIH tissue co-culture (F20,92=3.928, p=0.0005). However, the effect of GABA agents was greatest in co-culture with RPE tissue (F18,36=4.865, p=0.0005). Summary and Conclusion: 1) Retinal defocus signals are transferred to RPE and choroid which then exert their modifying effect on scleral GAG and DNA synthesis either through growth stimulating factors or directly interacting with scleral cells in process of scleral remodeling during LIM and LIH visual conditions. 2) GABAergic agents affect the proliferation of scleral fibroblasts both directly and when co-cultured with ocular tissues in vitro.

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3alpha mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.

Contribution of DEAF1 structural domains to the interaction with the breast cancer oncogene LMO4

The proteins LMO4 and DEAF1 contribute to the proliferation of mammary epithelial cells. During breast cancer LMO4 is upregulated, affecting its interaction with other protein partners. This may set cells on a path to tumour formation. LMO4 and DEAF1 interact, but it is unknown how they cooperate to regulate cell proliferation. In this study, we identify a specific LMO4-binding domain in DEAF1. This domain contains an unstructured region that directly contacts LMO4, and a coiled coil that contains the DEAF1 nuclear export signal (NES). The coiled coil region can form tetramers and has the typical properties of a coiled coil domain. Using a simple cell-based assay, we show that LMO4 modulates the activity of the DEAF NES, causing nuclear accumulation of a construct containing the LMO4-interaction region of DEAF1.

Nagelschmidtite bioceramics with osteostimulation properties : material chemistry activating osteogenic genes and WNT signalling pathway of human bone marrow stromal cells

Bioactive materials with osteostimulation properties are of great importance to promote osteogenic differentiation of human bone marrow stromal cells (hBMSCs) for potential bone regeneration. We have recently synthesized nagelschmidtite (NAGEL, Ca7Si2P2O16) ceramic powders which showed excellent apatite-mineralization ability. The aim of this study was to investigate the interaction of hBMSCs with NAGEL bioceramic bulks and their ionic extracts, and to explore the osteostimulation properties of NAGEL bioceramics and the possible molecular mechanism. The cell attachment, proliferation, bone-related gene expression (ALP, OPN and OCN) and WNT signalling pathways (WNT3a, FZD6, AXIN2 and CTNNB) of hBMSCs cultured on NAGEL bioceramic disks were systematically studied. We further investigated the biological effects of ionic products from NAGEL powders on cell proliferation and osteogenic differentiation of hBMSCs by culturing cells with NAGEL extracts. Furthermore, the effect of NAGEL bioceramics on the osteogenic differentiation in hBMSCs was also investigated with the addition of cardamonin, a WNT inhibitor. The results showed that NAGEL bioceramic disks supported the attachment and proliferation of hBMSCs, and significantly enhanced the bone-related gene expression and WNT signalling pathway of hBMSCs, compared to conventional beta-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic products from NAGEL powders also significantly promoted the proliferation, bone and WNT-related gene expression of hBMSCs. It was also identified that NAGEL bioceramics could bypass the action of the WNT inhibitor (10 μM) to stimulate the selected osteogenic genes in hBMSCs. Our results suggest that NAGEL bioceramics possess excellent in vitro osteostimulation properties. The possible mechanism for the osteostimulation may be directly related to the released Si, Ca and P-containing ionic products from NAGEL bioceramics which activate bone-related gene expression and WNT signalling pathway of hBMSCs. The present study suggests that NAGEL bioceramics are a potential bone regeneration material with significant osteostimulation capacity.

The Ghrelin axis : does it have an appetite for cancer progression?

Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHSR), is a peptide hormone with diverse physiological roles. Ghrelin regulates GH release, appetite and feeding, gut motility, and energy balance and also has roles in the cardiovascular, immune, and reproductive systems. Ghrelin and the GHSR are expressed in a wide range of normal and tumor tissues, and a fluorescein-labeled, truncated form of ghrelin is showing promise as a biomarker for prostate cancer. Plasma ghrelin levels are generally inversely related to body mass index and are unlikely to be useful as a biomarker for cancer, but may be useful as a marker for cancer cachexia. Some single nucleotide polymorphisms in the ghrelin and GHSR genes have shown associations with cancer risk; however, larger studies are required. Ghrelin regulates processes associated with cancer, including cell proliferation, apoptosis, cell migration, cell invasion, inflammation, and angiogenesis; however, the role of ghrelin in cancer is currently unclear. Ghrelin has predominantly antiinflammatory effects and may play a role in protecting against cancer-related inflammation. Ghrelin and its analogs show promise as treatments for cancer-related cachexia. Further studies using in vivo models are required to determine whether ghrelin has a role in cancer progression.

Loss of breast epithelial marker hCLCA2 promotes epithelial to mesenchymal transition and indicates higher risk of metastasis

Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of epithelial-to-mesenchymal transition by cell dilution, TGFβ or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral small hairpin RNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18-year period among breast cancer patients compared with lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells.

Growth of confined cancer spheroids : a combined experimental marker, critical modelling approach

A critical step in the dissemination of ovarian cancer is the formation of multicellular spheroids from cells shed from the primary tumour. The objectives of this study were to apply bioengineered three-dimensional (3D) microenvironments for culturing ovarian cancer spheroids in vitro and simultaneously to build on a mathematical model describing the growth of multicellular spheroids in these biomimetic matrices. Cancer cells derived from human epithelial ovarian carcinoma were embedded within biomimetic hydrogels of varying stiffness and grown for up to 4 weeks. Immunohistochemistry, imaging and growth analyses were used to quantify the dependence of cell proliferation and apoptosis on matrix stiffness, long-term culture and treatment with the anti-cancer drug paclitaxel. The mathematical model was formulated as a free boundary problem in which each spheroid was treated as an incompressible porous medium. The functional forms used to describe the rates of cell proliferation and apoptosis were motivated by the experimental work and predictions of the mathematical model compared with the experimental output. This work aimed to establish whether it is possible to simulate solid tumour growth on the basis of data on spheroid size, cell proliferation and cell death within these spheroids. The mathematical model predictions were in agreement with the experimental data set and simulated how the growth of cancer spheroids was influenced by mechanical and biochemical stimuli including matrix stiffness, culture duration and administration of a chemotherapeutic drug. Our computational model provides new perspectives on experimental results and has informed the design of new 3D studies of chemoresistance of multicellular cancer spheroids.