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Introduction: Recent successes in dental pulp engineering indicate that regenerative treatment strategies in endodontics are feasible. Clinically, revascularization procedures render completion of root formation in immature teeth. The generation of a pulp-like tissue after seeding of dental pulp stem cells into dentin discs or cylinders and transplantation in vivo is possible. In this experimental setup, which mimics the situation in the root canal, the pretreatment of dentin might influence cellular behavior at the cell-dentin interface. Thus, the objective of this study was to investigate whether dentin conditioning can determine cell fate., Methods: Dental pulp stem cells (DPSCs) were seeded into a growth factor-laden peptide hydrogel, transferred into dentin cylinders, and transplanted subcutaneously into immunocompromised mice. Before cell seeding, dentin cylinders were either pretreated with sodium hypochloride (NaOCl) or conditioned with EDTA. The constructs were explanted after 6 weeks and subjected to histological and immunohistochemical analysis., Results: In dentin treated with NaOCl, resorption lacunae were found at the cell-dentin interface created by multinucleated cells with clastic activity. After conditioning with EDTA, DPSCs adjacent to the dentin formed an intimate association with the surface, differentiated into odontoblasts-like cells that expressed dentin sialoprotein, and extended cellular processes into the dentinal tubules. A vascularized soft connective tissue similar to dental pulp was observed inside the dentin cylinder., Conclusions: Dentin conditioning considerably influences DPSC fate when seeded in close proximity to dentin. This information might be critical for optimized strategic planning for future regenerative endodontic treatment., (Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the inductive potential of scaffold material combing with transforming growth factor-β1 (TGF-β1), and to induce odontoblast differentiation and dentin formation from dental pulp cells both in vitro and in vivo., Methods: Primarily cultured dental pulp cells were used for MTT, ALP activity assay and Alizarin red staining in the presence of TGF-β1. Pelleted cells were put on the filters combining with or not with TGF-β1 and cultured in vitro or in vivo. The in vitro and in vivo cell response and tissue formation were analysed with Haematoxylin-Eosin (HE), transmission electron microscopy (TEM) and immunohistochemical staining., Results: TGF-β1 increased the mineralization and ALP activity of dental pulp cells as revealed by Alizarin red staining and ALP activity assay. After in vitro culture for 7 days, cells polarized in the TGF-β1 group and expressed dentin sialoprotein (DSP), osteopontin (OPN) and type I collagen (Col I). After in vivo transplantation for 7 days, columnar odontoblast formed on the surface of filter in experimental group, and tubular dentin expressing DSP formed after 3 months transplantation., Conclusion: It was concluded that TGF-β1 combining with transfilter could induce odontoblast differentiation and dentin formation. Our results implied that suitable substrate for the progenitors of odontoblast to anchor on and inductive signals to initiate the differentiation of odontoblast should be taken into consideration when designing scaffold material for inducing dentin tissue engineering., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Introduction: This study was to investigate the effects of tricalcium silicate (Ca(3)SiO(5)) on proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) in vitro., Methods: The hDPCs were seeded in culture medium with or without Ca(3)SiO(5) extract and calcium hydroxide (Ca(OH)(2)) extract. Proliferation of the hDPCs was measured by methyl-thiazol-tetrazolium (MTT) assay. Odontogenic differentiation of hDPCs was evaluated by real-time polymerase chain reaction by using odontogenic marker genes such as dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP 1), osteocalcin (OC), alkaline phosphatase (ALP), and collagen type I (Col I), which were verified by ALP activity assessment, mineralization assay, and immunocytochemistry staining for dentin sialoprotein (DSP)., Results: The MTT assay showed that hDPCs cultured with Ca(3)SiO(5) extract proliferated more significantly as compared with Ca(OH)(2) extract. Analysis of odontogenic marker genes indicated that Ca(3)SiO(5) enhanced the expression of those genes. Moreover, the extract of Ca(3)SiO(5) stimulated mineralization and increased ALP and DSP production conspicuously., Conclusions: These results reveal that Ca(3)SiO(5) can induce the proliferation and odontogenic differentiation of hDPCs in vitro and might be a potential candidate for preparation of a new type of Ca(3)SiO(5-)based cement as a pulp-capping agent., (Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: Dental pulp stem cells (DPSCs) have been receiving more attentions recently as an important biomaterial for tissue engineering. Notch signalling plays a key role in regulating self-renewal and differentiation of a variety of cells. The objective of this study is to investigate the effects of Notch-Delta1 RNA interference (RNAi) on the proliferation and differentiation of human dental pulp stem cells in vitro., Design: In the present study, we performed gene knockdown of Notch ligand Delta1 in DPSCs using lentivirus-mediated Delta1-RNAi. Changes of proliferation in DPSCs/Delta1-RNAi were examined by cell cycle analysis, Cell viability assay (CCK-8) and Western blot analysis of proliferating cell nuclear antigen (PCNA). Cells were cultured in odontoblast differentiation-inducing medium, and the differentiation of cells was detected with Alkaline phosphatase ALP activity assay, Alizarin red S staining, calcium concentration measurement, and Western blot analysis of Dentine sialophosphoprotein (DSPP)., Results: Lentivirus-mediated Delta1-RNAi stably knocked-down the expression of Delta1 and Notch signalling, and some of DPSCs/Delta1-RNAi displayed changes in morphology or DSPP expression. The growth rate of Delta1-deficient DPSCs was significantly suppressed as compared with wild type DPSCs and control lentivirus vector transfected DPSCs. Furthermore, the differentiating capability of DPSCs/Delta1-RNAi into odontoblasts is much higher than the two control groups., Conclusions: Notch signalling plays a crucial role in regulating self-renewal and differentiation in DPSCs. The deficient Notch signalling inhibits the self-renewal capacity of DPSCs and tends to induce DPSCs differentiation under odontoblast differentiation-inducing conditions. These findings suggested that DPSCs/Delta1-RNAi might be applicable to stem cell therapies and tooth tissue engineering., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Introduction: Signaling pathways responsible for dentin regeneration in a dental pulp are not fully understood. In this study, we determined the effects of the mammalian target of rapamycin (mTor) on the differentiation and mineralization of dental pulp stem cells. We hypothesized that the two known mTor complexes Torc1 and Torc 2 play pivotal roles in the differentiation of odontoblasts and that they modulate deposition of a mineralized extracellular matrix. Therefore, we investigated the effects of Torc1 and Torc 2 signaling on the differentiation and mineralization of stem cells from human exfoliated deciduous teeth (SHED)., Methods: We used Western blot analysis to examine the expression of markers of dental differentiation in SHED (+/-) inhibition of either Torc1 or Torc 2 complex proteins raptor or rictor, respectively. In addition, the deposition of a mineralized matrix was determined under these conditions via alkaline phosphatase and alizarin red staining., Results: Results show that the inhibition of Torc 1, via reduced expression of either raptor or mTor, severely restricts the synthesis of dentin sialoprotein and inhibits deposition of a mineralized matrix. Inhibition of Torc 2, via reduction of rictor, has the opposite effect, enhancing mineralization. This latter effect disappears when both rictor and mTor are inhibited, showing that the Torc 2 effect is Torc 1 dependent., Conclusions: These results strongly suggest an important role for mTor in dental pulp stem cell differentiation and provide evidence that the mechanisms involved in protein synthesis could prove an interesting target for dental pulp tissue engineering., (Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Dogs are commonly used animal models for regenerative endodontics research. Although several studies have used stem cells isolated from dog teeth to investigate the dentin/pulp regeneration in vivo, less attention has been paid for the characterization of these cells. Therefore, this study aimed to characterize the dental pulp stem cells isolated from dog teeth (cDPSCs) in order to further define the dog as an animal model for regenerative endodontics., Methods: Stem cells were isolated from freshly extracted premolars of 10-month-old Beagles. The isolated cells were investigated for their stem cell properties by analysis of their clonogenic and growth characteristics; expression of mesenchymal stem cell markers; and evaluation of their osteo/odontogenic, adipogenic, and neurogenic potential., Results: A colony formation assay showed the existence of a clonogenic cell population in cDPSCs isolated. The growth curves revealed a higher proliferation rate of cDPSCs compared with hBMMSCs. cDPSCs expressed mesenchymal stem cell markers STRO-1, CD146, and Nanog. However, they were negative for CD73, CD105, and CD45. cDPSCs were able to differentiate into odontoblast-like cells as shown by increased alkaline phosphatase activity, dentin sialoprotein expression, and formation of mineralized nodules. cDPSCs also showed the neurogenic and adipogenic differentiation potential at a lower level compared with those of hDPSCs and hBMMSCs., Conclusions: The results of this study confirmed the stem cell properties of cDPSCs at a comparable level to those of hDPSCs and hBMMSCs. Overall, the data presented in this study provide evidence in supportive of using cDPSCs and dogs as an animal model in dental tissue engineering via stem cell-based approaches., (Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Direct application of dentin bonding agents onto the exposed pulp has been advocated, but in vivo studies indicate a lack of reparative dentin formation. Our objective was to investigate the role of triethylene glycol dimethacrylate (TEGDMA), a commonly used compound in dentin bonding agents, as a potential inhibitor of mineralization. Human pulp cells were exposed to different concentrations of TEGDMA, and expression of the mineralization-related genes collagen I, alkaline phosphatase, bone sialoprotein, osteocalcin, Runx2, and dentin sialophosphoprotein was analyzed. Gene expression studies by real-time polymerase chain-reaction revealed a concentration- and time-dependent decrease of mineralization markers. A subtoxic TEGDMA concentration (0.3 mM) reduced expression levels by 5 to 20% after 4 hrs and by 50% after 12 hrs. Furthermore, alkaline phosphatase activity and calcium deposition were significantly lower in dental pulp cells treated with TEGDMA over 14 days. These findings indicate that even low TEGDMA concentrations might inhibit mineralization induced by dental pulp cells, thus impairing reparative dentin formation after pulp capping with dentin bonding agents.

Introduction: Odontogenic ameloblast-associated protein (ODAM) has been shown to be specifically expressed in ameloblasts and odontoblasts and has been suggested to play a role in the mineralization of the enamel, possibly through the regulation of matrix metalloproteinase 20. However, its function in dentin is not clear. The purpose of this study was to evaluate the effect of ODAM on tertiary dentin formation., Methods: MDPC-23 odontoblastic cells were cultured, and the effect of recombinant ODAM (rODAM) on mineralized nodule formation was evaluated. Pinpoint pulp exposures were made in rat teeth and then capped with rODAM mixed with a carrier (rODAM group), carrier only (Carrier group), or white mineral trioxide aggregate (WMTA group). After 1, 2, and 4 weeks, odontoblasts and tertiary dentin were investigated histologically and immunohistochemically., Results: Nodule formation in MDPC-23 cells was enhanced by rODAM treatment. Odontoblasts were polarized and showed a palisade arrangement in the remaining pulp from the rODAM group, but not the Carrier or WMTA groups. In the WMTA group, extensive tertiary dentin along the entire pulp-dentin border obliterated the pulp chamber. In contrast, in the rODAM group, limited reaction of odontoblasts resulted in normal pulp tissue appearance without excessive tertiary dentin formation and obliteration of the pulp cavity. In the Carrier and WMTA groups, bone sialoprotein was immunostained in most of the tertiary dentin, whereas in the rODAM group, dentin sialoprotein expression was immunostained primarily in newly formed reactionary dentin., Conclusions: These results suggest that rODAM accelerates reactionary dentin formation close to the pulp exposure area, thereby preserving normal odontoblasts in the remaining pulp., (Copyright © 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The hypoxia condition was expected to be suitable for the establishment and maintenance of human dental pulp cells (hDPCs), because they reside in a low-oxygen environment in vivo. Therefore, we presently examined the effects of hypoxia on the proliferation and differentiation of hDPCs in vitro. hDPCs grown under 3% O(2) showed a significantly higher proliferation rate than those under 21% O(2). Then, we prepared hypoxic cultures of hDPCs from older patients' teeth having inflammation and succeeded in recovering and expanding a small number of hDPCs. These cells were confirmed to have capability for osteo/odontogenic differentiation. Hypoxia suppressed the osteo/odontogenic differentiation of hDPCs in vitro and increased the number of cells expressing STRO-1, an early mesenchymal stem cell marker. This simple method will increase the possibility to obtain living hDPCs from damaged and/or aged tissues, from which it is ordinarily difficult to isolate living stem cells with differentiation capability., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: In the clinical field of jawbone formation, the use of autogenous bone as the graft material is the gold standard. However, there are some problems with this technique, such as risk of infection on the donor side, the limited amount of available bone mass, and marked resorption of the grafted bone. We investigated the potential for using teeth as a bone graft material for jawbone formation because the dental pulp contains stem cells, including undifferentiated neural crest-derived cells., Methods: Alveolar bone defects were created in Wistar rats, and the defects were filled with either tooth or iliac bone graft material, or left as controls. The potential for using teeth as a bone graft material for jawbone formation was measured using real-time polymerase chain reaction, microcomputed tomography, and histologic analysis., Results: Polymerase chain reaction revealed that the expressions of P75, P0, nestin, and musashi-1 were significantly higher in teeth than in mandibular bone and iliac bone grafts. Hematoxylin and eosin staining and microcomputed tomography showed that at 8 weeks, tooth graft material produced a similar amount of new bone compared to iliac bone graft material. Osteopontin was expressed in both the tooth and iliac bone graft material at 6 and 8 weeks after surgery. Dentin sialoprotein was expressed in the tooth graft material in the new bone at 6 weeks only., Conclusion: These results indicate that teeth may be an alternative material to autogenous bone for treating alveolar bone defects by grafting.

Studies on mechanisms underlying the differentiation of dental pulp stem cells are critical for the understanding of the biology of odontogenesis and for dental tissue engineering. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) differentiate into functional odontoblasts and endothelial cells. SHED were seeded in tooth slice/scaffolds and implanted subcutaneously into immunodeficient mice. SHED differentiated into functional odontoblasts that generated tubular dentin, as determined by tetracycline staining and confocal microscopy. These cells also differentiated into vascular endothelial cells, as determined by beta-galactosidase staining of LacZ-tagged SHED. In vitro, vascular endothelial growth factor (VEGF) induced SHED to express VEGFR2, CD31, and VE-Cadherin (markers of endothelium) and to organize into capillary-like sprouts. VEGF induced ERK and AKT phosphorylation (indicative of differentiation), while inhibiting phosphorylation of STAT3 (indicative of 'stemness'). Collectively, this work demonstrates that SHED can differentiate into angiogenic endothelial cells and odontoblasts capable of generating tubular dentin.

Aim: To examine gene expression levels within the cells of dental pulp tissue in fluorotic rats and normal subjects and to identify fluoride-susceptible genes., Methodology: Female wistar rats were given 0.16 (control) or 100 parts per 10(6) fluoride ion in their drinking water. After 3 months, the teeth in the fluoride-exposed animals showed signs of fluorosis. The animals were killed, and RNA was extracted from incisor pulp tissue and pooled into three fluorosis and three control pools. The females were analysed for gene expression profiles using microarray analysis and semi-quantitative reverse transcriptase polymerase chain reaction (Sq-RT-PCR)., Results: Of the 26 962 probe sets, duplicate hybridisation data indicated that 5443 genes were detected as being present by both targets. Two hundred and forty seven genes, that is 4.53% of detected genes, were 1.5-fold or greater differentially expressed after fluoride treatment. Three interesting genes, dentine matrix protein 1, dentine sialophosphoprotein and Cadherin-11, were confirmed as being down-regulated by Sq-RT-PCR., Conclusions: The gene expression levels in rat pulp tissue changed during fluorosis. The gene expression profiles between the fluorotic model and the normal group will help to understand the multiple pathogenic mechanisms for the altered mineralization patterns observed in fluorotic dentine.

Purpose: This study was designed to investigate the effect of enamel derivative matrix (EMD) on the proliferation, mineralization, and differentiation of human mesenchymal stem cells (hMSCs)., Material and Methods: For the proliferation assay, water-soluble tetrazolium salt-8 tests were carried out after culturing for 24 and 48 h. For the evaluation of mineralization, Alizarin red S (ARS) tests were performed after 21 days of culturing in an osteogenic medium. In order to investigate some of the bone-related proteins, namely type I collagen (Col I A2), bone sialoprotein (BSP), and bone gamma-carboxyglutamate (Gla) protein (BGLAP, osteocalcin), real-time polymerase chain reaction (RT-PCR) tests were carried out after 2, 3, and 4 weeks of culturing, respectively., Results: The activity of proliferation and mineralization increased significantly depending on the concentration of EMD (P<0.05). In the control group, the expression of Col I A2 decreased, but EMD enhanced its expression over time and was correlated to the concentration. The amount of expression of BSP in this group increased over time, but EMD strikingly suppressed its expression in the fourth week. As well, the amount of expression of BGLAP increased as the culture duration lengthened in the control group. However, the expression of BGLAP was suppressed in the experimental group with EMD., Conclusion: Within the limits of this study, EMD enhanced the proliferation of hMSCs. After evaluation with ARS staining, EMD seemed to enhance mineralization, and the RT-PCR test revealed that EMD promoted early-stage osteoblast differentiation by enhancing Col I A2 expression, but exerted an inhibitory effect on the mineralization by lowering the gene expression of BSP and BGLAP. Mineralized nodules formed with EMD may be composed of substances other than normal bone. Because most of the organic matrix of bone is type I collagen, which acts as the mineralization site, bone or bone-like mineralized mass might have been formed in spite of the different components of the non-collagenous proteins.

Objective: To test the ability of Bio-Oss in inducing growth factors and proinflammatory cytokines that may have a role in inflammation after grafting, bone resorption, remodeling and in the homeostasis of osteoblasts., Material and Methods: Normal human osteoblasts were seeded in Petri dishes containing granules of Bio-Oss, cells were harvested after confluency and RNA was extracted. Reverse transcriptase polymerase chain reaction was performed using specific primers for osteonectin, bone sialoprotein (BSP), bone morphogenetic protein (BMP)-2 and BMP-7, platelet-derived growth factor (PDGF), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and integrin beta1. Glycerol-3-phosphate dehydrogenase was used as the housekeeping gene and normal human osteoblasts grown on Petri dishes without Bio-Oss granules were used as negative controls., Results: Osteoblast grown on Bio-Oss showed a normal RNA expression of osteonectin, integrin beta1 and PDGF. However, compared with control osteoblasts it showed a reduced expression of BSP, BMP-2 and BMP-7, IL-6 and TNF-alpha., Conclusions: Our findings further support the evidence that Bio-Oss is an excellent biomaterial that does not enhance the production of proinflammatory cytokines.

N-Glycolylneuraminic acid (Neu5Gc) is the second most populous sialic acid (Sia). The only known biosynthetic pathway of Neu5Gc is the hydroxylation of cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac), catalyzed by CMP-Neu5Ac hydroxylase (CMAH). Neu5Gc is abundantly found in mammals except for human, in which CMAH is inactivated due to mutation in the CMAH gene. Evidence has accumulated to show occurrence of Neu5Gc-containing glycoconjugates in sera of cancer patients, human cancerous tissues and cultured human cell lines. Recently, occurrence of natural antibodies against Neu5Gc was shown in healthy humans and is a serious problem for clinical xenotransplantation and stem cell therapies. Studying human occurrence of Neu5Gc is of importance and interest in a broad area of medical sciences. In this study, using a fluorometric high performance liquid chromatography method, we performed quantitative analyses of Sias both inside and in the external environment of the cell and found that (i) incorporation of Neu5Gc was most prominent in soluble glycoproteins found both in the extracellular space and inside the cell as the major Sia compounds. (ii) Of the total Neu5Gc in the Sia compounds that the cells synthesized, 90% was found in the secreted sialoglycoproteins, whereas for Neu5Ac, 70% was found in the secreted sialoglycoproteins. (iii) The Neu5Gc ratio was higher in the secreted sialoglycoproteins (as high as 40% of total Sias) than in intracellular sialoglycoproteins. (iv) The majority of the secreted sialoglycoproteins was anchored on the culture dishes and solubilized by brief trypsin treatment. Based on these findings, a new idea on the mechanism of accumulation of Neu5Gc in cancer cells was proposed.

Background: The aim of this study is to investigate the potential use of periosteum-derived cells (PCs) for tissue engineering in peri-implant defects., Methods: Bone marrow cells (BMCs) and PCs were harvested from seven adult beagle dogs, cultured in vitro, and phenotypically characterized with regard to their osteogenic properties. The animals were then subjected to teeth extraction, and 3 months later, two implant sites were drilled, bone dehiscences created, and dental implants placed. Dehiscences were randomly assigned to one of two groups: PCs (PCs + carrier) and BMCs (BMCs + carrier). After 3 months, the animals were sacrificed and the implants with adjacent hard tissues were processed for undecalcified sections. Bone-to-implant contact, bone fill within the limits of implant threads, and new bone area in a zone lateral to the implant were histometrically obtained., Results: In vitro, phenotypic characterization demonstrated that both cell populations presented osteogenic potential, as identified by the mineral nodule formation and the expression of bone markers. Histometrically, an intergroup analysis showed that both cell-treated defects had similar bone fill within the limits of implant threads and bone-to-implant contact (P >0.05), and although a trend toward higher new bone area values was found for the PC group, there was no significant difference between the experimental groups (P >0.05)., Conclusions: Periosteal and bone marrow cells presented a similar potential for bone reconstruction. As such, periosteum may be considered as an alternative source of osteogenic cells in implant dentistry.

Introduction: Although human dental pulp stem cells isolated from healthy teeth have been extensively characterized, it is unknown whether stem cells also exist in clinically compromised teeth with irreversible pulpitis. Here we explored whether cells retrieved from clinically compromised dental pulp have stem cell-like properties., Methods: Pulp cells were isolated from healthy teeth (control group) and from teeth with clinically diagnosed irreversible pulpitis (diseased group). Cell proliferation, stem cell marker STRO-1 expression, and cell odonto-osteogenic differentiation competence were compared., Results: Cells from the diseased group demonstrated decreased colony formation capacity and a slightly decreased cell proliferation rate, but they had similar STRO-1 expression and exhibited a similar percentage of positive ex vivo osteogenic induction and dentin sialophosphoprotein expression from STRO-1-enriched pulp cells., Conclusions: Our study provides preliminary evidence that clinically compromised dental pulp might contain putative cells with certain stem cell properties. Further characterization of these cells will provide insight regarding whether they could serve as a source of endogenous multipotent cells in tissue regeneration-based dental pulp therapy., (Copyright (c) 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Bone sialoprotein (BSP) is a major non-collagenous, extracellular matrix glycoprotein associated with mineralized tissues. Fibroblast growth factor 2 (FGF2) is recognized as a potent mitogen for a variety of mesenchymal cells. FGF2 produced by osteoblasts accumulates in the bone matrix and acts as an autocrine/paracrine regulator of osteoblasts. We previously reported that FGF2 regulates BSP gene transcription through the FGF2 response element (FRE) and activator protein 1 (AP1) binding site overlapping with the glucocorticoid response element in the rat BSP gene promoter. In the present study, FGF2 (10 ng/ml) increased BSP and Runx2 mRNA levels at 6 h in MCF7 human breast cancer cells. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of MCF7 cells with FGF2 (10 ng/ml) increased the luciferase activity of the constructs between -84LUC and -927LUC. Gel mobility shift analyses showed that FGF2 increased the binding of AP1 and CRE2. The CRE2- and AP1-protein complexes were disrupted by antibodies against CREB1, c-Fos, c-Jun, Fra2, p300 and Runx2. These studies demonstrate that FGF2 stimulates BSP transcription in MCF7 human breast cancer cells by targeting the AP1 and CRE2 elements in the human BSP gene promoter.

Of the approximately 8,400 children born each year in the US with cytomegalovirus (CMV)-induced birth defects, more than one third exhibit hypoplasia and hypocalcification of tooth enamel. Our prior studies indicated that CMV severely delayed, but did not completely interrupt, early mouse mandibular first molar morphogenesis in vitro. The aim of the present study was to examine the effects of CMV infection on progressive tooth differentiation and amelogenesis. Since initial CMV infection in human fetuses can occur at different developmental times, we varied the stage of initial viral infection (that is, Cap stage, Early Bell stage and Bell stage), as well as the duration of infection. CMV infection of embryonic mouse mandibular first molars in vitro induces tooth dysmorphogenesis and enamel defects in a developmental stage- and duration-dependent manner. Cap stage- and Early Bell stage-infected molars exhibit enamel agenesis and Bell stage-infected molars exhibit enamel hypoplasia. This viral-induced pathology is coincident with stage-dependent changes in Amelx, Enam and Dspp gene expression, distribution of amelogenin, enamelin and DSP proteins, cell proliferation localization and dedifferentiation of secretory ameloblasts. Importantly, our data indicate that specific levels of Amelx and Dspp gene expression define whether mouse CMV induces enamel agenesis or hypoplasia., (Copyright © 2010 S. Karger AG, Basel.)

Background: Bone is one of the most common sites of metastasis in patients with non-small-cell lung cancer (NSCLC). Over-expression of bone sialoprotein (BSP) and osteopontin (OPN) in tumour samples has shown prognostic significance in bone metastasis (BM) of breast and prostate cancer, respectively. However, their importance in BM of NSCLC has not been verified. Therefore, we planned a large cohort retrospective study to investigate the relationship between the expression of these two biomarkers (BSP and OPN) and BM in surgically resected NSCLC patients., Methods: 180 completely resected NSCLC patients were included in this study. 40 patients subsequently developed BM. Paraffin-embedded primary tumour tissues of patients were supplied to produce a tissue microarray, and immunohistochemistry method was used for evaluation of the expression of BSP and OPN. Different expressions of these two biomarkers among BM group and non-BM group were estimated by chi(2) test. BM-free survival was analyzed by Kaplan-Meier method. The prognostic impact of clinicopathologic variables and biomarker expression was evaluated by Cox proportional hazards model., Results: BSP expression was associated with BM (p=0.007), whereas OPN expression did not reach statistical significance (p=0.245). Univariate analysis showed that expression of BSP (p=0.010) and N staging (p<0.005) was associated with BM-free survival. Multivariate analyses showed BSP expression (HR=3.322, p=0.003), N staging (HR=1.879, p=0.001), and T staging (HR=1.618, p=0.024) were independent prognostic factors for BM., Conclusions: BSP protein expression in the primary resected NSCLC is strongly associated with BM and could be used to identify high-risk patients. Correlation of OPN protein expression and BM needs further investigation.

Objectives: Bacterial metabolites demineralize dental hard tissues, and soluble factors lead to tertiary dentinogenesis in the area of the dentin-pulp complex. However, it is unclear whether the oral bacteria are directly involved in the differentiation of dental pulp cells. In this study, we evaluated the effect of oral bacterial extracts on cellular differentiation in human dental pulp-derived cells (hDPC)., Study Design: The hDPC were obtained from third molar teeth, and the cells were subcultured. The sonicated extracts were obtained from Porphyromonas gingivalis (gram-negative) and Streptococcus mutans (gram-positive). The effect of bacterial extracts on cellular growth and differentiation in hDPC were tested., Results: Alkaline phosphatase activity and bone sialoprotein (BSP) gene expression were increased in hDPC exposed to low concentrations of both sonicated extracts, whereas the activity was decreased upon exposure to high concentrations of sonicated extracts from P. gingivalis., Conclusion: This is the first evidence that oral bacteria have a positive effect on cellular differentiation in hPDC., (Copyright 2010 Mosby, Inc. All rights reserved.)

ClC-5 is one of the voltage-dependent chloride channel (ClC) family members. Mutations involving CLCN5 cause an X-linked nephropathy associated with Dent's disease. Some Clcn5 gene knockout (ClC-5 KO) mice have abnormal growth of the teeth; however, the expression and function of ClC-5 during tooth development is still unknown. Herein we report abnormal dentin structure, decreased DSPP and increased TGF-beta1 protein level in ClC-5 KO teeth. In odontoblast-like MDPC-23 cells, the mRNA levels of Tgfb1, Dspp and Dmp-1 were upregulated with Clcn5 RNAi after 48h treatment; whilst there was no change in those of TGF-beta receptor Tgfbr1 and Tgfbr2. We suggest that the dentin changes in ClC-5 KO mice might be a result of increasing TGF-beta1, and the interplay between ClC-5 and TGF-beta1 needs further identified.

Objective: Primary pulp cell cultures are frequently used to study cellular responses, odontogenic potential and stem cell responses. Their isolation and expansion via a range of technical approaches are widely reported. The purpose of this study was to investigate the influence of isolation approach and extended expansion on cell phenotype and behaviour., Design: To determine viable cell isolation, enzymatic dissociation was performed on rodent incisor pulps using collagenase, trypsin, hyaluronidase and ficin. Extended expansion culture of released cells was performed in DMEM and alpha-MEM media. Cultures were subsequently analysed for gene expression, cell proliferation, cell morphology and differentiation capacity up to passage 20., Results: Data indicated that incubation of extirpated and mechanically minced rodent pulpal tissue with 0.25% Trypsin:EDTA and subsequent culture in alpha-MEM medium provided optimal conditions for maximal cell growth and expansion. Under these conditions, extended culture decreased cellular proliferative capacity up to passage 7, whilst higher passages demonstrated recovered growth rates. In general gene expression analysis of osteogenic and dentinogenic associated markers decreased with increasing passage number. Notably expression of TGFbetas-1, -2 and -3 increased up to passage 10 as did the stem cell and pericyte/myofibroblast markers, CD74, Neuroserpin and alpha-SMA. Analysis of molecular phenotypes indicated little difference in lineage differentiation capacity between earlier and later passages., Conclusions: The present study characterizes conditions for primary pulp cell isolation and expansion and indicates that both earlier and later passages maintain differentiation capacity. Continued passage however may result in selection for cells with a pericyte/myofibroblast phenotype.

Background: Sialic acid is a deoxy uronic acid with a skeleton of nine carbons which is mostly found on cell surface in animals. This sialic acid on cell surface performs various biological functions by acting as a receptor for microorganisms, viruses, toxins, and hormones; by masking receptors; and by regulating the immune system. In order to synthesize an artificial sialoglycoprotein, we developed a large-scale production of rat alpha2,6-sialyltransferase (ST6Gal1). The ST6Gal1 was expressed in fifth instar silkworm larval hemolymph using recombinant both cysteine protease- and chitinase-deficient Bombyx mori nucleopolyhedrovirus (BmNPV-CP--Chi-) bacmid. The expressed ST6Gal1 was purified, characterized and used for sialylation of asialoglycopolypeptide. We tested the inhibitory effect of the synthesized alpha2,6-sialoglycopolypeptide on hemagglutination by Sambucus nigra (SNA) lectin., Results: FLAG-tagged recombinant ST6Gal1 was expressed efficiently and purified by precipitation with ammonium sulphate followed by affinity chromatography on an anti-FLAG M2 column, generating 2.2 mg purified fusion protein from only 11 silkworm larvae, with a recovery yield of 64%. The purified ST6Gal1 was characterized and its N-glycan patterns were found to be approximately paucimannosidic type by HPLC mapping method. Fluorescently-labelled N-acetyllactosamine (LacNAc) glycoside containing dansyl group was synthesized chemo-enzymatically as high-sensitivity acceptor substrate for ST6Gal1. The acceptor substrate specificity of the enzyme was similar to that of rat liver ST6Gal1. The fluorescent glycoside is useful as a substrate for a highly sensitive picomole assay of ST6Gal1. Asialoglycopolypeptide was regioselectively and quantitatively sialylated by catalytic reaction at the terminal Gal residue to obtain alpha2,6-sialoglycopolypeptide using ST6Gal1. The alpha2,6-sialoglycopolypeptide selectively inhibited hemagglutination induced by Sambucus nigra (SNA) lectin, showing about 780-fold higher affinity than the control fetuin. Asialoglycopolypeptide and gamma-polyglutamic acid did not affect SNA lectin-mediated hemagglutination., Conclusion: The recombinant ST6Gal1 from a silkworm expression system is useful for the sialylation of asialoglycopeptide. The sialylated glycoprotein is a valuable tool for investigating the molecular mechanisms of biological and physiological events, such as cell-cell recognition and viral entry during infection.

Overexpression of disease-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) on peripheral blood mononuclear cells (PBMC) of visceral leishmaniasis (VL) patients (PBMC(VL)) compared to their levels of expression in healthy individuals has been demonstrated using a lectin, achatinin-H, with specificity toward 9-O-acetylated sialic acid derivatives alpha2-6 linkage with subterminal N-acetylgalactosamine (9-O-AcSAalpha2-6GalNAc). The decreased presence of disease-associated 9-O-AcSGPs on different immune cells of parasitologically cured individuals after successful treatment relative to the levels in patients with active VL prior to treatment was demonstrated. However, their contributory role as immunomodulatory determinants on PBMC(VL) remained unexplored. Accordingly, 9-O-AcSGPs on PBMC(VL) were sensitized with achatinin-H, leading to their enhanced proliferation compared to that observed with different known mitogens or parasite antigen. This lymphoproliferative response was characterized by evaluation of the TH1/TH2 response by intracellular staining and enzyme-linked immunosorbent assay for secreted cytokines, and the results were corroborated by their genetic expression. Sensitized PBMC(VL) evidenced a mixed TH1/TH2 cellular response with a predominance of the TH1 response, indicating the ability of 9-O-AcSGPs to modulate the host cell toward a favorable response. Interestingly, the humoral and cellular responses showed a good correlation. Further, high levels of anti-9-O-AcSGP antibodies with an order of distribution of immunoglobulin M (IgM) > IgG1 = IgG3 > IgG4 > IgG2 > IgE could be explained by a mixed TH1/TH2 response. A good correlation of enhanced 9-O-AcSGPs with both the cell-mediated (r = 0.98) and humoral (r = 0.99) response was observed. In summary, it may be concluded that sensitization of 9-O-AcSGPs on PBMC(VL) may provide a basis for the modulation of the host's immune response by their controlled expression, leading to a beneficial immune response and influencing the disease pathology.

Introduction: The current study investigated the effectiveness of enamel matrix derivative (EMD) and mineral trioxide aggregate (MTA) in inducing the differentiation of human dental pulp cells (HDPCs) into odontoblast-like cells in vitro., Methods: The effects of MTA and EMD on odontoblastic differentiation were indexed by alkaline phosphatase (ALP) activity and the expression of odontoblastic/osteoblastic markers, as determined by reverse-transcription polymerase chain reaction analysis. Mineralization was also evaluated by the staining of calcium deposits with Alizarin red. Cells were treated with a combination of MTA and EMD or with MTA alone., Results: Compared with MTA-treated cells on day 3, MTA/EMD-treated cells exhibited significantly greater increases in ALP activity and in dentin sialophosphoprotein and bone sialoprotein expression. Mineralization was significantly greater on day 7 in MTA/EMD-treated cells than in MTA-treated cells., Conclusions: These results suggest that a combination of MTA and EMD promotes more rapid differentiation in HDPCs than MTA alone and that this combination is thus a potential pulp-capping agent.

The purpose of this study was to investigate the effects of mineral trioxide aggregate (MTA) on survival, mineralization, and expression of mineralization-related genes of cementoblasts. Immortalized cementoblasts (OCCM) were maintained with Dulbecco modified Eagle medium containing 10% fetal bovine serum. Methyl-thiazol-diphenyl-tetrazolium experiments were performed at 24 and 72 hours to evaluate bioactive components released by MTA (0.002-20 mg/mL) on the cell survival of OCCM. Von Kossa staining was used to evaluate biomineralization of OCCM cells. Images of cementoblasts were taken on day 3 by using inverted microscopy. Gene transcripts for bone sialoprotein (BSP), OCN, collagen type I (COL I), and osteopontin (OPN) were evaluated on days 3 and 5 by using semiquantitative reverse transcriptase polymerase chain reaction. The 20 mg/mL concentration of MTA was toxic for OCCM cells, whereas other concentrations of MTA tested exhibited similar cell numbers when compared with control group, and the 0.02 mg/mL concentration of MTA increased OCCM cell survival at 72 hours. Although an apparent decrease in mineralization was observed in the highest 3 concentrations of MTA used, 0.02 and 0.002 mg/mL concentrations of MTA induced greater biomineralization of OCCM cells than seen in the control. Moreover, increased BSP and COL I mRNA expression was observed at 0.02 and 0.002 mg/mL concentrations of MTA. MTA did not have a negative effect on the viability and morphology of cementoblasts and induced biomineralization of cementoblasts at the concentrations of 0.02 and 0.002 mg/mL. Based on these results MTA can be considered as a favorable material regarding cell-material interaction.

Progenitor cells play an important biological role in tooth and bone formation, and previous analyses during bone and dentine induction have indicated that they may be a good alternative for tissue engineering. Thus, to clarify the influence of the microenvironment on protein and gene expression, MDPC-23 cells (mouse dental papilla cell line) and KUSA/A1 cells (bone marrow stromal cell line) were used, both in vitro cell culture and in intra-abdominal diffusion chambers implanted in 4-week-old male immunodefficient mice (SCID mice). Our results indicate that KUSA/A1 cells differentiated into osteoblast-like cells and induced bone tissue inside the chamber, whereas, MDPC-23 showed odontoblast-like characteristics but with a low ability to induce dentin formation. This study shows that MDPC-23 cells are especial cells, which possess morphological and functional characteristics of odontoblast-like cells expressing dentin sialophosphoprotein in vivo. In contrast, dentin sialophosphoprotein gene and protein expression was not detected in both cell lines in vitro. The intra-abdominal diffusion chamber appears as an interesting experimental model for studying phenotypic expression of dental pulp cells in vivo.

Objectives: This study aims to investigate the effects of anatase-structured titania surface on the response of osteoblast-like cells., Methods: Three kinds of titanium (Ti) disks were prepared: the polished Ti (PT), the roughened Ti (RT) formed by sandblasting and acid etching, and the anatase-coated roughened Ti (ART) formed on the RT via H(2)O(2)/HCl solution treatment followed by heating at 400 degrees C. The surface topography of Ti disks was observed under a scanning electron microscope. MC3T3-E1 cells were cultured on the Ti disks. Cell morphology was observed by fluorescence microscopy. Cell adhesion and proliferation were assessed by MTT testing. To evaluate the cell differentiation on RT and ART, the expressions of core-binding factor alpha, osterix, bone sialoprotein, collagen type 1, osteocalcin and alkaline phosphatase-2 were measured by real-time reverse transcriptase polymerase chain reaction., Results: The three kinds of Ti disks had distinct surface topographic characteristics. The cells on the ART more rapidly displayed well-defined stress fibers and numerous processes and pseudopodi than the cells on the PT and RT. At 1 and 3h after culture, there were more cells adhering to the ART than to PT and RT. Cell proliferation did not differ between groups after 1-5 days of culture. In addition, ART generally enhanced the osteogenic gene expression in a time-dependent manner compared with RT., Significance: Anatase-structured titania promotes initial adhesion and spreading of osteoblast-like cells, but does not influence cell proliferation. Anatase could also enhance cell differentiation by enhancing osteogenic gene expression.

Leukocytes and tumor cells use E-selectin binding ligands to attach to activated endothelial cells expressing E-selectin during inflammation or metastasis. The cysteine-rich fibroblast growth factor receptor (CFR) represents the main E-selectin ligand (ESL-1) on granulocytes and its expression is exclusively modified by alpha(1,3)-fucosyltransferases IV or VII (FucT4 and FucT7). Hepatic stellate cells (HSC) are pericytes of liver sinusoidal endothelial cells. The activation of HSC and transdifferentiation into a myofibroblastic phenotype is involved in the repair of liver tissue injury, liver regeneration and angiogenesis of liver metastases. In the present study, we demonstrated that HSC expressed CFR together with FucT7 and exhibited a functional E-selectin binding activity on their cell surface. Since HSC appear to be oxygen-sensing cells, the expression of E-selectin binding activity was analyzed in HSC under a hypoxic atmosphere. While the expression of the glycoprotein CFR was unaffected by hypoxia, the cell-associated E-selectin binding activity decreased. However, under the same conditions, mRNA expression of the modifying enzyme FucT7 increased. The loss of E-selectin binding activity, therefore, appears to be neither the result of a reduced expression of the modifying transferase nor the expression of the backbone glycoprotein. After the transient transfection of HSC with CFR cDNA, the E-selectin binding activity (ESL-1) was efficiently released into the supernatant. Therefore, we hypothesize that under hypoxia, ESL-1 is shed from activated HSC. Our findings provide a novel perspective on the function of HSC in liver metastasis and inflammatory liver diseases.

A new bioactive titanium surface treatment, labeled Ti-ASD, was developed using the electrochemical anodic spark deposition (ASD) technique and results in a thickened titanium oxide layer with higher levels of calcium and phosphorus typical of newly deposited mineral phase. This study was aimed at extending the knowledge on Ti-ASD treatment, by means of evaluation of the attachment, morphology, proliferation, metabolic activity, differentiation, and mineralization of osteoblast-like cells (SaOS-2) after growth on Ti-ASD treated titanium compared with nontreated titanium (Ti) and with chemically etched titanium (Ti-ETC). This novel type of titanium coating supported cell attachment, cell proliferation, and mineralization, revealing no cytotoxicity effects. The expression of differentiation markers on Ti-ASD treated titanium shows that genes related to the proliferation phase (Collagen type I, Coll I; Cbfa-1) were early expressed, whereas genes related to the mineralization phase (alkaline phosphatase, osteopontin, bone sialo protein) increased in a time-related way. Mineralization occurred on all analyzed surfaces, but on Ti-ASD the number of bone-like nodules and the amount of mineralized area was higher. In conclusion, Ti-ASD resulted to be a good surface for osteoblast attachment and proliferation, also promoting the maintenance of cell differentiation and matrix mineralization, a fundamental requirement for sustain the osseointegration and the clinical success of dental implants., ((c) 2008 Wiley Periodicals, Inc.)

Objective: Podocalyxin expression on Ter119(+) erythroblasts is induced following administration of erythropoietin (Epo) or phenylhydrazine treatment, but is notably absent on committed erythroid progenitors during homeostatic red cell turnover. Following high-dose Epo administration in vivo, podocalyxin surface expression is upregulated, in part, via a signal transducers and activators of transcription 5-dependent pathway and this expression has been postulated to play a role in the release of reticulocytes from hematopoietic organs into the periphery under conditions of increased erythropoietic rate. Here we have thoroughly addressed this hypothesis and further examined the expression profile of podocalyxin during Epo-induced erythroblast expansion and stress erythropoiesis., Materials and Methods: Following Epo induction, progenitor cells were sorted to characterize podocalyxin expression during stress. In addition, as podocalyxin-deficient mice die perinatally, we used chimeric mice reconstituted with wild-type or podocalyxin-deficient hematopoietic cells to analyze differences in response to high dose Epo administration and chemically induced anemia., Results: Podocalyxin surface expression is rapidly upregulated in response to stress and marks early erythroid progenitors and erythroblasts. Despite loss of podocalyxin, chimeras exhibit normal basal erythropoiesis and no differences in erythroid progenitor proportions in the spleen and marrow in response to Epo. Further, podocalyxin is dispensable for efficient recovery from models of anemia., Conclusions: We demonstrate that podocalyxin is a highly specific marker of stress-induced blast-forming unit erythroid and colony-forming unit erythroid progenitors in mouse bone marrow and spleen. In addition, our findings suggest that podocalyxin is not necessary for efficient erythroblast expansion, erythroid differentiation, or reticulocyte release in response to Epo stimulation in vivo.

SST-VEDI-1(VEDI-1) is a new synthetic compound that is synthesized from tryptamine, and has structural similarity to the SSH-BM family of compounds. However, the biological effects of VEDI-1 have yet to be well characterized. A recent report has demonstrated that SSH-BM-type compounds can stimulate osteoblast activity in cultured scales of goldfish. In this study, we examined the effects of VEDI-1 on osteoblastic differentiation as well as its effects on apoptosis, which is known to be closely related to osteoblastic differentiation. We found that VEDI-1 enhanced the formation of mineralized nodules in rat osteoblast cell lines, including ROS17/2.8 cells, and in mouse pre-osteoblast cell lines, including MC3T3-E1 cells, in a dose dependent manner, which was accompanied by increased expression of late osteoblast markers, bone sialoprotein (BSP) and osteocalcin (OC). Furthermore, VEDI-I inhibited apoptotic cell death and regulated the expression of proteins in the Bcl-2 family. These results suggest that VEDI-1 may facilitate late differentiation of osteoblasts and may have an inhibitory effect on apoptosis.

Aim: The primary goal of periodontal treatment is regeneration of the periodontium. Current theories suggest that the periodontal ligament (PDL) cells have the capacity to participate in restoring connective and mineralized tissues, when appropriately triggered. We evaluated whether human PDL cell sheets could reconstruct periodontal tissue., Material and Methods: To obtain the cell sheet, human PDL cells were cultured on temperature-responsive culture dishes with or without osteogenic differentiation medium. The cell sheets were transplanted on periodontal fenestration defects of immunodeficient rats. Forty rats were divided in two groups: in one group, cell sheets cultured with control medium were transplanted and in the other, cell sheets cultured with osteogenic differentiation medium were transplanted. The defects were analysed histologically and histomorphologically after healing., Results: Most of the experimental group exhibited a new cementum-like layer and new attachment of collagen fibres to the layer. Histomorphological analyses indicated significant periodontal regeneration. The control group revealed dense extracellular matrix and fibre formation, but an obvious cementum layer was not observed., Conclusions: Transplanted PDL cell sheets cultured with osteogenic differentiation medium induced periodontal regeneration containing an obvious cementum layer and Sharpey's fibres. Thus, the method could be feasible as a new therapeutic approach for periodontal regeneration.

Histopathologic and clinical findings suggest that small cell lung cancer is derived from a multipotent proximal airway epithelial cell. In order to investigate the histogenetic origin of small cell lung cancer, we compared stem cell marker expression in human fetal lung tissue, human adult bronchial tissue, and a cohort of 64 small cell lung cancers. Supporting derivation of a multipotent precursor cell, 87.5% (56/64) of small cell lung cancers showed a dot-like expression of podocalyxin-like protein 1 (PODXL-1), a marker of embryonic and hematopoetic stem cells. Of small cell lung cancers, 98.4% (63/64) ubiquitously expressed Bmi-1, a key player in self-renewal of stem cells. Oct4 and AP2gamma were not expressed. Although podocalyxin-like protein 1 did not correlate with p53 or Wilms tumor suppressor 1, known regulators of podocalyxin-like protein 1, we could demonstrate demethylated CpG islands in the podocalyxin-like protein 1 promoter in small cell lung cancer, indicating epigenetic regulation. During fetal lung development and within adult bronchial mucosa, Bmi-1 was expressed ubiquitously. In contrast, podocalyxin-like protein 1 was detected in few stromal cells during the pseudoglandular phase (n = 7) and, importantly, in clustered epithelial cells within proximal bronchi and the trachea during the canalicular phase (n = 10). Interestingly, podocalyxin-like protein 1 was not expressed in normal or metaplastic adult bronchial epithelium (n = 36) but was expressed in sparse epithelial cells in half of the cases of normal tumor adjacent bronchial mucosa (20/40). Taken together, we show that small cell lung cancers and clustered epithelial cells in developing proximal bronchi share the expression of stem cell markers, suggesting a possible histogenetic link.

Runx2 has been identified as "a master gene" for the differentiation of osteoblasts and Runx2-deficient mice has demonstrated a complete absence of mature osteoblast and ossification. To further characterize the Runx2 responsive elements within the bone sialoprotein (BSP) promoter and further investigate into the role of Runx2 haploinsufficiency in osteoblast differentiation, mBSP9.0Luc mice and mBSP4.8Luc mice were crossed with Runx2-deficient mice respectively. Luciferase assay, micro CT scan, and histological analysis were performed using tissues isolated from mBSP9.0luc/Runx2+/- mice, mBSP4.8luc/Runx2+/- mice and their corresponding Runx2+/+ littermates. Alkaline phosphatase activity, mineralization assays and RT-PCR analysis using calvarial osteoblasts isolated from these transgenic mice were also performed. Luciferase assay demonstrated an early increase in luciferase expression in mBSP9.0luc/Runx2+/- mice before the expression level of luciferase dramatically decreased and turned lower than that in their control littermates in later stages. In contrast, luciferase expression in mBSP4.8luc/Runx2+/- failed to show such an early increase. Micro CT scan and histological analysis showed that BMD and trabecular bone volume were decreased and bone formation was delayed in Runx2+/- mice. Furthermore, mineralization assay and semi-quantitative RT-PCR assay demonstrated a gene-dose-dependent decrease in bone nodule formation and bone marker genes expression levels in cultured calvarial osteoblasts derived from Runx2 knockout mice. Reconstitution of Runx2-null cells with Runx2 vector partially rescued the osteoblast function defects. In conclusion, the 9.0 kb BSP promoter demonstrated a higher tissue-specific regulation of the BSP gene by Runx2 in vivo and full Runx2 gene dose is essential for osteoblast differentiation and normal bone formation., ((c) 2008 Wiley-Liss, Inc.)

Purpose: In previous work from our laboratory, we demonstrated that the three-dimensional (3D) cell cultures developed in simulated microgravity environments enhanced osseous-like aggregate formation and accelerated preosteoblast cell differentiation. Thus, as described here, we hypothesize that aggregate formation and mineralization would occur with fewer than 10 x 10(6) cells as previously described., Materials and Methods: Human preosteoblastic cells were cultured at different concentrations in a rotary wall vessel to simulate microgravity for 7 days. Aggregate size was assessed, and mineralization and collagen expression detected using Von Kossa and Masson Trichrome staining. Scanning electron microscopy was used for structural and elemental analysis. Immunohistochemistry was used to detect expression of the osteogenic markers BSPII and osteopontin (OP)., Results: Size and calcium expression were dependent upon cultured cell number (p < 0.01). Calcium and collagen expression were detected throughout the aggregate, but organization was independent of cell number. Aggregates had similar microscopic structural patterns demonstrating organized development. Presence of BSPII and OP showed that the aggregates share common differentiation proteins with in vivo bone formation., Conclusions: These results may lead to novel bone engineering techniques associated with dental treatment.

Podocalyxin is an anti-adhesive mucin-like transmembrane sialoglycoprotein that has been implicated in the development of aggressive forms of cancer. Podocalyxin is also known as keratan sulfate (KS) proteoglycan. Recently, we revealed that highly sulfated KS or another mucin-like transmembrane sialoglycoprotein podoplanin/aggrus is upregulated in malignant astrocytic tumors. The aim of this study is to examine the relationship between podocalyxin expression and malignant progression of astrocytic tumors. In this study, 51 astrocytic tumors were investigated for podocalyxin expression using immunohistochemistry, Western blot analysis, and quantitative real-time PCR. Immunohistochemistry detected podocalyxin on the surface of tumor cells in six of 14 anaplastic astrocytomas (42.9%) and in 17 of 31 glioblastomas (54.8%), especially around proliferating endothelial cells. In diffuse astrocytoma, podocalyxin expression was observed only in vascular endothelial cells. Podocalyxin might be associated with the malignant progression of astrocytic tumors, and be a useful prognostic marker for astrocytic tumors.

Nicotine is a major component of tobacco smoke, and signals via nicotinic acetylcholine receptors (nAChR). However, little is known about the effects of nicotine on human dental pulp cells (HDPCs). In this study, we assessed the effects of nicotine on mineralization in HDPCs. We confirmed messenger RNA expression of nAChR subunits and examined the effects of nicotine on expression of extracellular matrices (ECMs), alkaline phosphatase (ALP) activity, and mineralized nodule formation by HDPCs. Gene expression of nAChR subunits alpha1, alpha2, alpha 4, alpha 5, alpha 6, alpha 7, beta1, beta2, and beta 4 was detected in HDPCs. Interestingly, the messenger RNA expression of dentin matrix acidic phosphoprotein-1, bone sialoprotein, and ALP activity were significantly reduced in nicotine-treated HDPC. In addition, mineralized nodule formation, which was examined by alizarin red staining, was also inhibited in HDPCs by the same treatment. These results indicate that nicotine suppresses the cytodifferentiation and mineralization of HDPCs, possibly via nAChR.

The homeodomain protein Dlx5 is an activator of Runx2 (a key regulator of osteogenesis) and is thought to be an important regulator of bone formation. At present, however, the perinatal lethality of Dlx5-null mice has hampered the elucidation of its function in osteogenesis. Here we provide the first analysis of the effects of Dlx5 inactivation on bone development. Femurs of Dlx5-null mouse embryos at the end of gestation exhibit a reduction in both total and trabecular bone volume associated with increased trabecular separation and reduced trabecular number. These parameters are often associated with pathological conditions characterized by reduced osteoblast activity and increased bone resorption. Dlx5(-/-) osteoblasts in culture display reduced proliferation and differentiation rate and reduction of Runx2, Osx, Osteocalcin and Bone Sialoprotein expression. In addition to impaired osteoblast function, Dlx5(-/-) femurs exhibit significant increases in osteoclast number. As Dlx5 is not expressed by osteoclasts, we suggest that its osteoblastic expression might control osteoblast/osteoclast coupling. Cultured Dlx5(-/-) osteoblasts displayed a higher RANKL/OPG ratio. Furthermore, Dlx5(-/-) osteoblasts induced a higher number of TRAP-positive multinucleated cells in normal spleen cultures with a globally increased resorption activity. These findings suggest that Dlx5 is a central regulator of bone turnover as it activates bone formation directly and bone resorption indirectly.

gammadelta T cells are distinct with respect to tissue localisation, phenotype and biological functions and similarities between species are not very apparent. To elucidate local and functional heterogeneity of non-stimulated avian gammadelta T cells, the CD8-characterised gammadelta T cell subsets [CD8alpha(+high) (CD8alphaalpha(+) and CD8alphabeta(+)); CD8alpha(+dim); CD8(-)] of blood, spleen and caecum were flow cytometrically quantified and analysed for proliferation state as well as sorted for determination of immune-relevant gene expression by quantitative real-time RT-PCR. The number of avian CD8-characterised gammadelta T cell subsets differed in dependence on tissue and age of bird. Compared to blood and spleen, caecum showed the highest percentage of gammadelta T cells as well as of the CD8alpha(+high) gammadelta T cell subset in 7-week-old birds. Generally, the CD8alphabeta(+) cells significantly outnumbered the CD8alphaalpha(+) lymphocytes within the CD8alpha(+high) gammadelta T cell population of all organs. Additionally, the splenic CD8alphabeta(+) subpopulation revealed the highest proliferation activity. By RT-PCR, mRNA expression of immune-relevant genes was proved in non-stimulated gammadelta T cell subsets, but on different levels. Generally, both CD8alpha(+high) cell subsets (CD8alphaalpha(+) and CD8alphabeta(+)) of blood and spleen showed elevated expression levels for Fas ligand (FasL), XCL1 (lymphotactin) and interferon-gamma (IFNgamma) compared to the CD8alpha(-) gammadelta T cell subset. In contrast, all caecal gammadelta T cell subsets showed similar high levels of these transcripts. Notably, the CD8alphaalpha(+) cells of all locations showed unique expression of TLR4 and interleukin (IL)-2. The results demonstrated that avian gammadelta T cells are not only heterogeneous concerning their CD8 antigen characteristics and tissue localisation, but also with regard to functional features such as proliferation and mRNA expression.

Osterix (Osx) is an osteoblast-specific transcription factor required for the differentiation of pre-osteoblasts into functional osteoblasts. This study sought to examine the changes of Osx expression in periodontal ligament cells (PDLC) subjected to mechanical force, and to investigate whether Osx is involved in the mechanical stress-induced differentiation of PDLC. Human PDLC were exposed to centrifugal force for 1-12 h. Real-time polymerase chain reaction (PCR), western blot, and immunofluorescence assays were used to examine the mRNA and protein expression of Osx and its subcellular localization. Furthermore, PDLC were transfected with the expression vector pcDNA3.1 flag-Osx and subjected to mechanical force for 6 h. The changes in alkaline phosphatase (ALP) activity and in the expression of core-binding factor alpha1 (Cbfa1), ALP, osteopontin, bone sialoprotein, osteocalcin, and collagen I were measured. After the application of mechanical force, Osx was upregulated in a time-dependent manner at both mRNA and protein levels, and Osx protein was translocated from the cytosol into the cell nuclei. Overexpression of Osx did not affect the expression of Cbfa1, but it significantly enhanced the ALP activity and the mRNA expression of all the aforementioned osteogenic marker genes, all of which increased further under mechanical stress. These results suggest that Osx might play an important role in the mechanical stress-induced osteogenic differentiation of PDLC and therefore be involved in alveolar bone remodeling during orthodontic therapy.

Background and Objective: The aim of this study was to investigate the responses of periodontal ligament cells under hypoxia and re-oxygenation conditions in vitro., Material and Methods: Periodontal ligament fibroblasts were isolated from rat incisors. In the hypoxia group, cells were incubated in 2% O(2) for 1-3 d. In the re-oxygenation group, cells were first incubated under the same conditions as the hypoxia group for 24 h and then were returned to normoxic conditions and cultured for 1-2 additional days., Results: Proliferation ratios increased in all groups in a time-dependent manner. Proliferation ratios in both the hypoxia and re-oxygenation groups were significantly higher than in the control group on days 2 and 3. Alkaline phosphatase activity was significantly higher in the hypoxia group than in the control and the re-oxygenation groups. The expression of bone sialoprotein mRNA was significantly higher in the hypoxia group than in the control group on days 1 and 2. The expression of vascular endothelial growth factor mRNA was significantly higher in the hypoxia group than in the control group on days 1 and 2. In the re-oxygenation group, the level of expression of bone sialoprotein mRNA and vascular endothelial growth factor mRNA were similar to those of the control group. The expression of heat shock protein 70 mRNA in the hypoxia group was similar to that in the control group, whereas in the re-oxygenation group it was statistically higher than in the other groups., Conclusion: These results suggest that periodontal ligament cells maintain their osteogenic ability in hypoxia and re-oxygenation conditions in vitro.

Aim: To analyze the process of appositional bone formation using our original rat experimental model., Materials and Methods: Rats were anesthetized and a ring made of polytetrafluorethylene was placed on the parietal bone surface in the surgical procedure. The time course of appositional bone formation was analyzed with histomorphometry and in situ hybridization for type I collagen and bone sialoprotein., Results: The rat experimental model allowed new bone to be formed on the pre-existing bone surface and persist for 12 weeks. We demonstrated that bone is apposed actively for the first 4 weeks and less actively thereafter., Conclusions: The experimental model may contribute to biological analysis for appositional bone formation expected to occur in clinical procedures such as alveolar bone augmentation and sinus lifting.

Muscle satellite cells have long been considered a distinct myogenic lineage responsible for postnatal growth, repair and maintenance of skeletal muscle. Recent studies have demonstrated that they are multi-potential. Osterix (Osx), a novel zinc-finger-containing transcription factor of the sp family, is required for osteoblast differentiation and bone formation. It was hypothesized that Osx overexpression would enhance osteoblast differentiation of muscle satellite cells in vitro. Recombinant adenovirus-mediated Osx gene (Ad-Osx) was constructed and used to transfect muscle satellite cells. Osx overexpression inhibited myogenesis, as demonstrated by suppression of MyoD and myogenin mRNA levels and reduced myotube formation. Muscle satellite cells transduced with Ad-Osx exhibited apparent osteoblast differentiation as determined by the expression of related osteoblastic genes, increased activity of alkaline phosphatase and the formation of mineralized nodules. These results confirmed the ability of Osx to enhance osteoblast differentiation of muscle satellite cells in vitro, and the competence of muscle satellite cells as promising seed cells for bone tissue engineering.

Background and Objective: Bone sialoprotein is a mineralized tissue-specific noncollagenous protein that is glycosylated, phosphorylated and sulfated. The temporo-spatial deposition of bone sialoprotein into the extracellular matrix of bone, and the ability of bone sialoprotein to nucleate hydroxyapatite crystal formation, indicates a potential role for bone sialoprotein in the initial mineralization of bone, dentin and cementum. Bone sialoprotein is also expressed in breast, lung, thyroid and prostate cancers., Material and Methods: We used osteoblast-like cells (rat osteosarcoma cell lines ROS17/2.8 and UMR106, rat stromal bone marrow RBMC-D8 cells and human osteosarcoma Saos2 cells), and breast and prostate cancer cells to investigate the transcriptional regulation of bone sialoprotein. To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene, we conducted northern hybridization, transient transfection analyses with chimeric constructs of the bone sialoprotein gene promoter linked to a luciferase reporter gene and gel mobility shift assays., Results: Bone sialoprotein transcription is regulated by hormones, growth factors and cytokines through tyrosine kinase, mitogen-activated protein kinase and cAMP-dependent pathways. Microcalcifications are often associated with human mammary lesions, particularly with breast carcinomas. Expression of bone sialoprotein by cancer cells could play a major role in the mineral deposition and in preferred bone homing of breast cancer cells., Conclusion: Bone sialoprotein protects cells from complement-mediated cellular lysis, activates matrix metalloproteinase 2 and has an angiogenic capacity. Therefore, regulation of the bone sialoprotein gene is potentially important in the differentiation of osteoblasts, bone matrix mineralization and tumor metastasis. This review highlights the function and transcriptional regulation of bone sialoprotein.

Leucine-rich amelogenin peptide (LRAP), an alternatively spliced amelogenin protein, possesses a signaling property shown to induce osteogenic differentiation. In the current study, we detected LRAP expression during osteogenesis of wild-type (WT) embryonic stem (ES) cells and observed the absence of LRAP expression in amelogenin-null (KO) ES cells. We explored the signaling effect of LRAP on wild-type ES cells, and the ability of LRAP to rescue the impaired osteogenesis phenotype observed in KO ES cells. Our data indicate that LRAP treatment of WT and KO ES cells induces a significant increase in mineral matrix formation, and significant increases in bone sialoprotein and osterix gene expression. In addition, the amelogenin KO phenotype is partially rescued by the addition of exogenous LRAP. These data suggest a unique function of LRAP during ES cell differentiation along osteogenic lineage.

Background: Previous data demonstrated that root cementum may affect periodontal regeneration. As such, this study aimed to explore further possible mechanisms involved in this process by investigating in humans whether root cementum modulates gene expression in the regenerating tissue formed under membrane-protected intrabony defects., Methods: Thirty subjects with deep intrabony defects (> or =5 mm; 2- or 3-wall) were selected and assigned to the control or test group. The control group received scaling and root planing with the removal of granulation tissue and root cementum; the test group underwent removal of granulation tissue and soft microbial deposits by cleaning the root surface with a microbrush and saline solution, aiming at cementum preservation. Guided tissue regeneration (GTR) was applied to both groups. Twenty-one days later, the newly formed tissue under the membrane was assessed for the expression of the following genes: alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), platelet-derived growth factor-alpha (PDGFA), bone sialoprotein (BSP), and basic fibroblast growth factor (bFGF)., Results: Data analysis demonstrated that mRNA levels for PDGFA, BSP, and bFGF were higher in the sites where root cementum was kept in place compared to the sites where root cementum was removed completely as part of the periodontal therapy (P <0.05); in contrast, OCN levels were lower (P <0.05). No difference for ALP or OPN was observed between the control and test groups (P >0.05)., Conclusion: Root cementum may modulate the expression of growth and mineral-associated factors during periodontal regeneration.

Background: Surface topography is important in the creation of a scaffold for tissue engineering. Chemical etching of poly(l-lactide-co-glycolide) with sodium hydroxide has been shown to enhance adhesion and function of numerous cell types. The authors investigated the effects of sodium hydroxide pretreatment of three-dimensional poly(l-lactide-co-glycolide) scaffolds on the adhesion, differentiation, and proliferation of MC3T3-E1 murine preosteoblasts., Methods: MC3T3-E1 cells were seeded onto three-dimensional poly(l-lactide-co-glycolide) scaffolds with and without 1 M sodium hydroxide pretreatment. Cells were then cultured in osteogenic medium and harvested at varying time points for RNA extraction. Quantitative real-time reverse-transcriptase polymerase chain reaction was performed to measure mRNA expression of several osteogenic marker genes. In addition, cell numbers were determined at varying time points during the culture period. All experiments were performed in triplicate., Results: Pretreatment of three-dimensional poly(l-lactide-co-glycolide) scaffolds with sodium hydroxide resulted in statistically significant up-regulation of mRNA expression of alkaline phosphatase, bone sialoprotein, osteocalcin, and vascular endothelial growth factor during the first 10 days of culture. Histologic analysis demonstrated a striking increase in mineralized cell matrix deposition in the sodium hydroxide-treated group. Cell number was statistically higher in the sodium hydroxide-treated group immediately after cell seeding, suggesting improved adhesion. During the first 24 hours of culture, cells grew faster in the control group than in the sodium hydroxide-treated group., Conclusions: Chemical etching of poly(l-lactide-co-glycolide) scaffolds with sodium hydroxide strongly influences the behavior of MC3T3-E1 preosteoblasts in vitro by enhancing adhesion and differentiation and slowing proliferation. Sodium hydroxide treatment may represent a simple and inexpensive way of improving scaffolds for use in bone tissue engineering.

While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation., ((c) 2007 Wiley-Liss, Inc.)