[Show abstract][Hide abstract] ABSTRACT: Dental fluorosis is characterized by subsurface hypomineralization and increased porosity of enamel, associated with a delay in the removal of enamel matrix proteins. To investigate the effects of fluoride on ameloblasts, A/J mice were given 50 ppm sodium fluoride in drinking water for four weeks, resulting serum fluoride levels of 4.5 µM, a four-fold increase over control mice with no fluoride added to drinking water. MicroCT analyses showed delayed and incomplete mineralization of fluorosed incisor enamel as compared to control enamel. A microarray analysis of secretory and maturation stage ameloblasts microdissected from control and fluorosed mouse incisors showed that genes clustered with Mmp20 appeared to be less downregulated in maturation stage ameloblasts of fluorosed incisors as compared to control maturation ameloblasts. One of these Mmp20 co-regulated genes was the global chromatin organizer, special AT-rich sequence-binding protein-1 (SATB1). Immunohistochemical analysis showed increased SATB1 protein present in fluorosed ameloblasts compared to controls. In vitro, exposure of human ameloblast-lineage cells to micromolar levels of both NaF and AlF3 led to a significantly increase in SATB1 protein content, but not levels of Satb1 mRNA, suggesting a fluoride-induced mechanism protecting SABT1 from degradation. Consistent with this possibility, we used immunohistochemistry and Western blot to show that fluoride exposed ameloblasts had increased phosphorylated PKCα both in vivo and in vitro. This kinase is known to phosphorylate SATB1, and phosphorylation is known to protect SATB1 from degradation by caspase-6. In addition, production of cellular diacylglycerol (DAG) was significantly increased in fluorosed ameloblasts, suggesting that the increased phosphorylation of SATB1 may be related to an effect of fluoride to enhance Gαq activity of secretory ameloblasts.
PLoS ONE 08/2014; 9(8):e103994. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Highly mineralized tooth enamel develops from an extracellular matrix chiefly comprised of amelogenins formed by splicing of 7 (human) or 9 (rodent) exons secreted from specialized epithelia cells known as ameloblasts. Here we examined the role of the 59 amino acid alternatively spliced amelogenin known as Leucine Rich Amelogenin Peptide (LRAP) on enamel formation, using transgenic murine models in which LRAP overexpression is driven by an amelogenin promotor (TgLRAP). Beginning in the secretory stage of mouse amelogenesis, we found a reduced thickness of enamel matrix and a loss of Tomes' processes, followed by upregulated amelogenin mRNA expression, inhibited amelogenin secretion and loss of cell polarity. In the prescretory stage (P0) amelogenin m180 mRNA expression was increased 58 fold along with a 203 fold increase in MMP-20 expression and 3.5 and 3.2 fold increased in respectively enamelin and ameloblastin. When LRAP was overexpressed on an amelogenin knockout mouse model, the ameloblasts were not affected. Further, expression of the global chromatin organizer and transcription factor SATB1 was reduced in secretory stage TgLRAP ameloblasts. These findings identify a cellular role for LRAP in enamel formation that is not directly related to directing enamel crystal formation as is reported to be the primary function of full length amelogenins. The effect of LRAP overexpression in upregulating amelogenins, MMP-20 and SATB1, suggests a role in protein regulation critical to ameloblast secretion and matrix processing, to form a mineralized enamel matrix.
Matrix biology: journal of the International Society for Matrix Biology 06/2013; · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: LRAP, leucine rich amelogenin peptide, is a 59 amino acid alternatively spliced amelogenin lacking the large central hydrophobic domain of full-length amelogenin. LRAP has been shown to promote differentiation of dental mesenchyme, but its role in enamel formation is not known. The objective of this study was to determine if LRAP plays a direct role in the differentiation of ameloblasts from pre-ameloblasts to matrix secreting cells.
Method: Molars from transgenic mice with LRAP overexpressed by the amelogenin promotor (TgLRAP) and WT mice were collected at postnatal day 0 (primarily presecretory) to 5 (primarily secretory). The tooth organs were embedded in paraffin, sectioned, and analyzed morphologically. Immunohistochemistry was used to characterize expression of enamel matrix proteins. Amelogenin expression was characterized by in situ hybridization and by PCR analysis of molars and ameloblast lineage cells transfected with LRAP. A Tunel assay was used to detect apoptosis. The morphology of molars from transgenic mice overexpressing full-length amelogenin (M180) was compare to the TgLRAP mouse molars.
Result: Secretory ameloblasts from TgLRAP mice showed layering of cells in the enamel organ and a loss of cell polarization. Immunohistochemistry showed increased amelogenin and cytokeratin 14 protein in presecretory and secretory ameloblasts of TgLRAP mice. PCR and in situ hybridization showed upregulation of amelogenin mRNA in TgLRAP as compared to WT mice. Analysis of alternative splicing by PCR showed a specific upregulation of exon 4 both in vivo and in vitro. Late secretory ameloblasts showed a marked increase in apoptosis in TgLRAP mice. Developing molars from mice overexpressing m180 did not show a histological phenotype similar to TgLRAP mice.
Conclusion: LRAP has a key role in modulating ameloblast differentiation, possibly through regulation of alternative splicing of exon 4 in the amelogenin gene.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: Objective: Low-level light therapy (LLLT) uses low energy laser in the near infrared spectrum of 760-1500 nm to promote tissue repair and wound healing. However, the mechanism remains unknown. In this study, we investigated the effects of 810-nm laser treatment on human adult dental pulp cells in-vitro.
Method: Dental pulp cells were isolated from freshly extracted human permanent teeth for orthodontic treatment. The cells were cultured in 24-well plates at a density of 1x105 cells/well in DMEM media supplemented with 10% FBS. As cells reached 75% confluence, the Nexus 7W laser (810 nm) was used to treat the cells with a 10-sec exposure/day for 4 consecutive days using energy levels at 0 (control), 0.7, 1.0 and 1.4 Watts (W). Treated cells were photographed, followed by cell quantification, and immunoblot analysis to evaluate the protein expression levels of alkaline phosphatase (ALP), dentin sialophosphoprotien (DSP), and type I collagens. Experiments were conducted in triplicate. Statistical analyses were done by one-way ANOVA.
Result: Exposures at 0.7 and 1.0 W resulted in approximately 2 and 2.5 folds increase in cell counts, respectively, as compared to the control group. However, at 1.4-W treatment, cell count was not significantly different from the control. Increased amount and size of mineralization-like nodules were detected in cells exposed with 1.0 and 1.4 W. Western blots showed that ALP, DSP and type-I collagen were significantly up-regulated in an energy-dose dependent manner, while significant increase of DSP was detected only at 1.4-W treatment as compared to the control groups.
Conclusion: Low-level light therapy (LLLT) in the near infrared (810 nm) promotes adult dental pulp cell proliferation and differentiation, suggesting the potential clinical application of LLLT in accelerating pulpal wound healing and dentin formation.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to investigate virulence factors associated with maternal transmission of mutans streptococci (MS).
Saliva samples were collected from 10 mothers with active caries and their 2- to 5-year-old children. Ten MS colonies were isolated from each subject. Transmission of MS was identified by arbitrarily primed polymerase chain reactions. Biofilm formation and mutacin production of the isolates against Streptococcus gordonii 10558, Streptococcus sanguinis 10557, Streptococcus mutans 25175, and Streptococcus sobrinus 6715 were analyzed.
All mothers and children had MS colonization. Only 7 of the 36 maternal genotypes (33 Streptococcus mutans genotypes and 3 Streptococcus sobrinus genotypes) were transmitted. Maternal transmission was found in 4 mother-child pairs, whereas 9 children had nonmaternal genotypes. There was no difference in biofilm formation between transmitted and nontransmitted genotypes (P>.05). Transmitted genotypes, however, produced more mutacin against Streptococcus sobrinus 6715 than nontransmitted genotypes.
This pilot study showed that there may be nonmaternal as well as maternal mutans streptococci transmission.
[Show abstract][Hide abstract] ABSTRACT: The role of the prion protein (PrP) in transmissible spongiform encephalopathies has been the focus of intense investigation. However, less is known about the physiological function of normal cellular PrP (PrP(C)). In adult human teeth, PrP(C) has been identified in odontoblasts, cementoblasts and epithelial remnants of Malassez. In this study, we have localized PrP(C) in developing human and mouse teeth, and investigated the function of PrP using a PrP-knockout (Prnp(0/0) ) mouse model. PrP(C) was detected in developing human and mouse ameloblasts and odontoblasts. In vitro, undifferentiated dental mesenchymal cells from embryonic day 18 (E18) Prnp(0/0) mouse molars proliferated much more rapidly compared to age-matched, wild-type (wt) mouse molar dental mesenchymal cells. Histochemistry and immunohistochemical analyses showed a subtle but measurable phenotype, with the absence of PrP resulting in earlier initiation of both dentin and enamel formation. Consistent with this finding, laser microdissected odontoblasts from newborn Prnp(0/0) mouse incisors had a reduced proliferation rate, as measured by the expression of proliferating cell nuclear antigen (PCNA), and increased type 1 collagen mRNA expression. Dentin microhardness of the fully erupted molars was reduced and incisal enamel mineralization was delayed in Prnp(0/0) compared to age-matched wt mouse teeth. Taken together, these results suggest that PrP(C) affects multiple processes involved in tooth formation, through regulating the differentiation of ameloblasts and odontoblasts.
The International journal of developmental biology 01/2011; 55(10-12):953-60. · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The studies reported here examines stress-related psychobiological processes that might account for the high, disproportionate rates of dental caries, the most common chronic disease of childhood, among children growing up in low socioeconomic status (SES) families. In two 2004-2006 studies of kindergarten children from varying socioeconomic backgrounds in the San Francisco Bay Area of California (Ns = 94 and 38), we performed detailed dental examinations to count decayed, missing or filled dental surfaces and microtomography to assess the thickness and density of microanatomic dental compartments in exfoliated, deciduous teeth (i.e., the shed, primary dentition). Cross-sectional, multivariate associations were examined between these measures and SES-related risk factors, including household education, financial stressors, basal and reactive salivary cortisol secretion, and the number of oral cariogenic bacteria. We hypothesized that family stressors and stress-related changes in oral biology might explain, fully or in part, the known socioeconomic disparities in dental health. We found that nearly half of the five-year-old children studied had dental caries. Low SES, higher basal salivary cortisol secretion, and larger numbers of cariogenic bacteria were each significantly and independently associated with caries, and higher salivary cortisol reactivity was associated with thinner, softer enamel surfaces in exfoliated teeth. The highest rates of dental pathology were found among children with the combination of elevated salivary cortisol expression and high counts of cariogenic bacteria. The socioeconomic partitioning of childhood dental caries may thus involve social and psychobiological pathways through which lower SES is associated with higher numbers of cariogenic bacteria and higher levels of stress-associated salivary cortisol. This convergence of psychosocial, infectious and stress-related biological processes appears to be implicated in the production of greater cariogenic bacterial growth and in the conferral of an increased physical vulnerability of the developing dentition.
Social Science [?] Medicine 11/2010; 71(9):1644-52. · 2.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: Infectious prion protein's (PrP) can cause a transmissible neurodegenerative disease. However the role of normal PrP found to be present in tissues including, odontoblasts, cementoblasts and epithelial remnants of Malassez, are not known. This study investigated the role of PrP in developing dental mesenchyme.
METHODS: Mandibles from newborn PrP knockout (K/O) mice and wild type (WT) mice were isolated. The dentin / pulp complex was compared following H&E, Von Kossa staining, and immunohistochemistry for nPrP protein. Microhardness of dentin from molars of age and gender matched K/O and WT mice, under dry conditions, were compared. Cell proliferation of dental mesenchymal cells isolated from E18 mouse molar tooth germs of K/O and WT mice was compared by BrdU immunoassay. Expression of type I collagen, matrix extracellular phosphoglycoprotein (MEPE), and alkaline phosphatase (ALP) in W/T and K/O cells rescued by the addition of recombinant PrP was compared.
RESULTS: PrP was immunolocalized to the least mature mesenchymal cells at the root apex and dental follicle of the molars, odontoblast, amelobast, and cervical loop area of the incisors. Mesenchymal cells isolated from E18 PrP K/O mice showed significantly increased proliferation when compared to WT cells. Addition of recombinant normal PrP upregulated collagen and down regulated MEPE and ALP expression to levels below that expressed by either the WT or K/O cells. Microhardness of PrP KO (predentin: 0.670.11GPa, dentin: 0.820.10GPa) was significantly lower when compared to WT (predentin: 0.880.16GPa, dentin: 1.070.14GPa) (P<0.01).
CONCLUSIONS: This study shows that normal PrP protein in early dental mesenchyme inhibits mesenchymal cell proliferation and upregulates collagen expression. Absence of PrP protein results reduced dentin microhardness, suggesting that, PrP has a significant role in collagen and mineral regulation during development of dentin.
[Show abstract][Hide abstract] ABSTRACT: Objectives: To determine the efficacy of SpiffiesTM xylitol-containing wipes on transmission of mutans streptococci(MS) and lactobacilli(LB) to infants.
Methods: In a double-blind randomized controlled clinical trial, 44 mothers with active caries and their infants aged 6-24 months were recruited. DMFS/dmfs scores were recorded using WHO criteria. Saliva samples were collected from mothers and infants at baseline. The infants were randomized into xylitol-wipe or placebo-wipe groups(GrA and GrB, n=22/group). All subjects and investigators were blinded for group assignment except the PI(JDBF). Mothers were instructed to use the wipes 3 times daily for their infants. Saliva samples were taken from each infant at 3 and 6 months. MS and LB counts were enumerated using traditional culture.
Results: No statistically significant differences were found in caries status, age, gender, and cariogenic bacteria levels between groups at baseline(P>0.05, Student t test). All but one mother had MS infection with log10 meanSD as 5.41.4(GrA) and 5.30.9(GrB). Six infants(GrA) and 7 infants(GrB) had MS and 1 infant in each group had LB at baseline. At 3 months, 0 in GrA and 3 in GrB had new MS infection, while 1 in GrA cleared MS infection. At 6 months, 3 in GrA and 1 in GrB had new MS infection, while 3 in GrA and 2 in GrB cleared MS infection. Two in GrA had new LB infection at 6 months and 2 in GrB had transient infection at 3 months. No significant differences were found between the groups for MS or LB changes at 3 or 6 months(P>0.05, Student t test). New MS or LB infection in each group was very low ranging from 0-15% of subjects at 6 months.
Conclusions: Tooth wipes may contribute to the low MS and LB infection rate in both groups. Supported by CSPD Research Grant and DR Products.
[Show abstract][Hide abstract] ABSTRACT: This study's purpose was to characterize dental pulp cells from human primary teeth and determine their ability to induce differentiation of oral epithelial cells.
Dental pulp cells were isolated from freshly extracted primary incisors, digested with 4 mg/ml collogenase/dispase, and grown in Dulbecco's modified Eagle's medium with 10 percent fetal bovine serum. Stem cell populations were identified by immunocytochemical staining for STRO-1 and CD146 and fluorescence activated cell sorting. To determine whether primary pulp cells can signal epithelium, the pulp cells were grown in coculture with human fetal oral epithelial cells. After 3 days, the cocultured cells were collected and analyzed for amelogenin expression by polymerAse chain reaction (PCR) and immunocytochemical staining.
Immunofluorescence and fluorescence activated cell sorting of STRO-1+ cells showed this stem cell population to be approximately 2 percent of the total population. Growth-arrested primary dental pulp cells grown in coculture with oral epithelial cells showed expression of Amelogenin by immunocytochemistry and PCR. Oral epithelial cells alone were amelogenin immunonegative.
Primary tooth dental pulp cells contain less than 2 percent stem cells. Cells within the primary tooth pulp can promote epithelial cell differentiation toward an ameloblast phenotype, suggesting the potential use of this heterogeneous population of cells in cell-mediated enamel tissue engineering.
[Show abstract][Hide abstract] ABSTRACT: We conducted a study among pediatric renal (RTRs) and liver transplant recipients (LTRs) to determine: a) the overall burden of oral disease; and b) the frequency with which this population utilizes dental care services in relation to sociodemographic factors and oral disease burden.
In this cross-sectional survey, study procedures included the completion of a standardized questionnaire (by parents/guardians), oral mucosal examination, assessment of caries, gingival enlargement, and plaque index.
The 142 children (82 RTRs and 60 LTRs) enrolled from April 2002 to November 2005 were predominantly Latino (41 percent) and Caucasian (34 percent). Forty-three percent had at least one carious surface (in either a deciduous or permanent tooth), 19 percent had five or more carious surfaces, and 25 percent had gingival enlargement. We found only one case of oral candidiasis. Even though 72 percent of parents/guardians reported their child had a regular source of dental care, only 49 percent had a dental cleaning and 44 percent had dental radiographs in the past year, reflecting a low prevalence of preventive dental care. Among children with no regular source of dental care, there were statistically significantly higher proportions of Latinos, younger children, and families with an annual household income <$35,000.
While the prevalence of oral mucosal disease and gingival enlargement was low, the prevalence of children with caries was high, and there was low use of preventive dental care. Strategies to improve this population's utilization of preventive dental care are needed.
Journal of Public Health Dentistry 07/2008; 69(1):48-55. · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To assess the effects of a single 10% povidone iodine application as an adjunct to extensive surgical procedures in the clinical treatment of children with early childhood caries.
Twenty-two children scheduled for dental treatment under general anesthesia were randomized into either an intervention group (10% povidone iodine), or a control group (phosphate buffered saline). Either povidone iodine or phosphate buffered saline was applied to teeth and soft tissues after prophylaxis and all operative dental procedures, followed by 1.23% acidulated phosphate fluoride gel. Saliva samples taken at baseline, and after 1 hour, 3 weeks and 3 months were assayed for mutans streptococci, lactobacilli and total viable bacteria. Caries lesions were recorded at baseline and at one year.
Mutans streptococci and lactobacilli levels in the povidone iodine group were significantly reduced relative to baseline at 1 hour, 3 weeks and 3 months. At one year at least 60% of subjects had new caries lesions in each group, and there was no significant difference in caries increment between the two groups.
Even prophylaxis, fluoride gel application and complete surgical treatment of caries at baseline were insufficient to prevent new caries in over 60% of the patients in these high caries risk infants. Although the one-time treatment with povidone iodine reduced mutans streptococci and lactobacilli levels for up to 3 months this therapy failed to additionally reduce future caries formation over one year, indicating that repeated antibacterial treatments will be needed to control high levels of cariogenic bacteria.
Journal of Public Health Dentistry 02/2006; 66(3):174-9. · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tacrolimus, in contrast to cyclosporine, has not been found to be associated with gingival enlargement (GE) among adult transplant recipients. The purpose of this study was to explore the prevalence of GE in relation to tacrolimus and cyclosporine-based immunosuppressive regimens among pediatric solid-organ transplant recipients, controlling for the use of calcium channel blockers (CCB) and the presence of supragingival plaque.
A standardized questionnaire was administered and a comprehensive oral examination was performed among pediatric renal and liver transplant recipients who were at least 6 months post-transplant.
The prevalence of GE among 133 participants was 26%, with the highest incidence among subjects receiving cyclosporine and CCB (60%) and the lowest among those receiving tacrolimus without CCB (13%). A multivariate model showed that the odds of having GE were 5 times higher among children receiving cyclosporine than in those not receiving this medication, and 4 times higher among boys than girls. Supragingival plaque and the use of CCB, however, were not found to be associated with GE.
This study revealed that tacrolimus was not associated with gingival enlargement while cyclosporine remains a risk factor for the development of this condition in pediatric renal and liver transplant recipients.