VDR deficiency affects alveolar bone and cementum apposition in mice.

OBJECTIVE: To compare the mineralisation density (MD), morphology and histology of alveolar bone and cementum amongst VDR +/+, VDR -/-, and VDR -/- groups supplemented with a diet TD 96348, containing 20% lactose, 2.0% calcium and 1.25% phosphorous. METHODS: Four groups of mice (6 mice/group) were identified by genotyping: VDR +/+ mice (VDR wild type), VDR -/- mice (VDR deficient), VDR -/- offsprings derived from VDR -/- parents receiving a supplemental diet (early rescued), and VDR -/- mice fed with a supplemental diet beginning at age one month (late rescued). All mice were sacrificed at age 70.5 days. Micro-CT was used to compare MD and morphology of alveolar bone and cementum. H-E and Toluidine blue staining was used to examine the ultrastructure of the alveolar bone and cementum at matched locations. RESULTS: In VDR -/- group, alveolar bone and cementum failed to mineralise normally. Early rescue increased MD of alveolar bone in VDR -/- mice with excessive alveolar bone formation, but which not observed in late rescue group. MD and morphology of cementum-dentine complex in both early and late rescue groups were comparable with VDR +/+ group when feeding with high-calcium rescue diet. CONCLUSIONS: VDR affects alveolar bone mineralisation and formation systemically and locally. However, cementum apposition and mineralisation is mainly regulated by calcium concentrations in serum.

A randomized clinical trial to compare diagnostic casts made using plastic and metal trays.

STATEMENT OF PROBLEM: Virtually every prosthodontic rehabilitation involves making diagnostic casts for analysis and prosthesis fabrication. Frequently, these casts are produced using irreversible hydrocolloid (IH) impression materials in stock metal or plastic trays. However, it is unclear whether one technique produces a more accurate cast. PURPOSE: The purpose of this randomized clinical trial was to compare the linear accuracy of diagnostic casts produced using IH with 1 of 3 different tray types: (1) perforated metal trays, (2) stock plastic trays, and (3) directed-flow stock plastic trays. All groups were compared to casts produced with custom trays and vinyl polysiloxane (VPS) impression materials, which were considered the control. MATERIAL AND METHODS: Seven subjects participated in this trial. IH impressions were made in a random order using 1 of 3 tray types: stock plastic, perforated metal, or plastic directed flow. These were compared to VPS impressions using custom trays (control group). Each impression technique was repeated 3 times per subject, for a total of 84 observations. Impressions were disinfected and poured in a type IV stone. Linear accuracy of casts was measured using computer software analysis of scanned images of the casts at x30 magnification. Three linear measurements were made on each cast: second molar to second molar, right second molar to left first premolar, and left second molar to right first premolar. Measurements were compared among techniques using mixed-model analysis of variance to account for correlation among the multiple measurements made on each subject. Dunnett's adjustment for multiple comparisons with control was used (α=.05). RESULTS: For molar-to-molar and right second molar to the left first premolar measurements, there were no significant differences in linear dimensions between casts made from different trays. However, linear measurements from the left second molar to the right first premolar demonstrated significant differences for casts made with stock metal, directed-flow, and stock plastic trays compared to custom trays. In this group, casts produced by stock metal, directed-flow, and stock plastic trays differed from controls by 102, 68, and 71 um, respectively. Generally, casts made with plastic trays (stock plastic and directed flow) had values closer to those of custom trays than did casts made with metal trays. CONCLUSIONS: Impressions made with irreversible hydrocolloid produced casts that were significantly different in linear dimension than casts produced by custom trays and VPS impressions. These differences were not uniform, but varied by location on the cast.

Regulation of enamel and dentin mineralization by vitamin D receptor.

BACKGROUND: Vitamin D plays an important role in bone mineralization. Enamel and dentin are two mineralized tissues of different origins that are part of the tooth structure, but the mechanism by which vitamin D regulates the mineralization of these tissues remains unclear. We examined the mineral deposition pattern of enamel and dentin in continuously erupting incisors in a vitamin D receptor (VDR) deficient mouse model to determine the effect of vitamin D receptor pathway on enamel and dentin mineralization. METHODS: VDR wild-type mice (VDR+/+) and VDR-deficient (VDR--/--) littermates were sacrificed at 70.5 days of age, and their mandibles were dissected. Immunostaining of biglycan and decorin was used to evaluate the dentin maturation. Micro-computerized tomography (micro-CT) was used to compare the mineral density (MD) of enamel and dentin of the two groups at different regions along the axis of the mandibular incisors. Scanning electronic microscopy (SEM) was employed to examine the ultrastructure of enamel and dentin at the levels corresponding to those examined in the micro-CT studies. Furthermore, an accelerated eruption procedure was performed to exclude the effect of delayed eruption on enamel and dentin mineralization. RESULTS: Different mineral deposition patterns of enamel and dentin were observed at different levels of the incisors in the VDR+/+ and VDR--/-- groups. Early enamel maturation and mineralization, and dentin hypomineralization were observed in the VDR--/-- group. CONCLUSION: Vitamin D affects enamel and dentin mineralization through different mechanisms. It may affect the mineralization of dentin systemically while enamel mineralization may be regulated locally.

Normalisation of calcium status reverses the phenotype in dentin, but not in enamel of VDR-deficient mice.

OBJECTIVE: To determine the effects of vitamin D receptor (VDR) deficiency on mouse dentin and enamel mineralisation, and how normalisation of serum calcium level affects dentin and enamel phenotypes in VDR knockout mice. MATERIALS AND METHODS: Groups of VDR wild-type (VDR+/+), VDR deficient (VDR-/-) and VDR-/- rescued mice were sacrificed at 70.5 days of life. The rescued group was established by a high-calcium diet feeding the VDR-/- mice from postnatal 19 days. Micro-CT was used to compare enamel and dentin mineralisation density (MD) at different levels of mandibular incisors among the groups. The scanning electron microscope (SEM) was used to examine the ultrastructure of the enamel and dentin in the corresponding levels and of surface enamel after acidic treatment. RESULTS: Micro-CT showed that in VDR-/- rescued group, dentin phenotype was reversed and dentin MD was reversed to normal; however, enamel mineralisation was not reversible, and remained as hypermineralisation in molar region and apical region of the incisors. SEM also revealed enamel hypermineralisation in the VDR-/- rescued group. This early enamel hypermineralisation was more susceptible to acidic erosion. CONCLUSION: Vitamin D affects dentin mineralisation systemically, and it regulates enamel mineralisation locally.

Enhancement of bonding to enamel and dentin prepared by Er,Cr:YSGG laser.

BACKGROUND AND OBJECTIVE: Erbium lasers are potential tools to remove caries and dental hard tissue but bond strengths of composites to those preparations are reported to be lower than conventional methods. The purpose of this study was to evaluate the effect of mechanical excavation and/or chemical alteration on bond strength of composites to laser irradiated enamel and dentin. MATERIALS AND METHODS: Seventy-two premolars were ground to obtain flat enamel (E, n = 36) or dentin (D, n = 36) surfaces in both buccal and lingual cusps, divided into: LaserExcavation (LEx), LaserNo-excavation (LNex), and Bur (B) groups. The laser groups were irradiated for 10 seconds by Er,Cr:YSGG laser [4.5 W, 60% air, 80% water (enamel) 3 W, 60% air, 70% water (dentin)]. Irradiated surfaces in the excavation groups (Ex) were then mechanically smoothed with a dental excavator, prepared surfaces were then etched (37% H(3)PO(4)) for 20 or 40 seconds (enamel) and 15 or 30 seconds (dentin), washed (20 seconds), adhesive was applied(Single Bond Plus), and light cured (20 seconds). A composite cylinder (Filtek Supreme Plus) formed, placed and light cured (40 seconds). The specimens were stored (37 degrees C,48 hours), shear bond tested (1 mm/minute), and statistically analyzed (P < 0.05). RESULTS: Mixed-model ANOVA showed significant differences between enamel (P = 0.0091) and between dentin groups (P = 0.0035). Tukey/Kramer showed mean shear bond strength (SBS+/-SE) of EB40 (27.01+/-2.38 MPa) was significantly higher than ELNoExc20 (14.39+/-2.5 MPa) and ELExc40 (14.90+/-2.28 MPa). Also DB30 (17.57+/- 1.67 MPa) and DLExc30 (18.6+/-1.74 MPa) were significantly higher than DLNoExc15 (9.56+/-1.86 MPa). CONCLUSION: Increasing the etching time up to 40 seconds or excavation of the laser prepared surface (but not the combination) may increase the bond strength to the level of conventional methods in enamel but excavation has a greater influence in dentin. Also the combination of both methods [excavation+longer etching time (30 seconds)] exhibit significantly better results in dentin.Mode of failure study showed mechanical excavation in both enamel and dentin can significantly reduce the cohesive failure in tooth structure.

Different enamel and dentin mineralization observed in VDR deficient mouse model.

UNLABELLED: Vitamin D plays an important role in the bone mineralization process. Enamel and dentin are two mineralized tissues of different origins that combine to form teeth, but the mechanism by which vitamin D regulates these tissues remains unclear. We hypothesized that vitamin D affects enamel and dentin mineralization through different mechanisms. OBJECTIVE: To examine enamel and dentin mineralization in a vitamin D receptor (VDR) deficient mouse model by micro-computerized tomography (micro-CT) and scanning electronic microscopy (SEM). METHODS: VDR wild type mice (VDR+/+) and VDR deficient (VDR-/-) littermates were sacrificed at 70.5 days old, and their mandibles were dissected. Micro-CT was used to compare mineral density (MD) of enamel and dentin of the two groups at different levels along the axis of mandibular incisors. SEM was employed to examine the ultrastructure of incisors at the levels corresponding to the levels used for the micro-CT studies. Furthermore, an accelerated eruption procedure was performed to exclude the effect of delayed eruption on enamel and dentin mineralization. RESULTS: Different distribution patterns of enamel and dentin MD were observed between VDR+/+ and VDR-/- groups. Early enamel maturation, mineralization, and hypomineralization in dentin were observed in the VDR deficient mice. CONCLUSION: Vitamin D may affect the mineralization of dentin systemically, and enamel mineralization may be regulated locally.

Efficacy of platelet-rich plasma on wound healing in rabbits.

BACKGROUND: The purpose of this study was to evaluate if the healing of full-thickness skin wounds was accelerated by platelet-rich plasma (PRP). METHODS: Four 2.5 x 2.5-cm full-thickness skin wounds were created on the backs of 15 New Zealand white rabbits. One wound on each animal received 0.3, 0.6, or 0.9 ml PRP, and the fourth wound served as a control. Seven and eight animals were sacrificed after 1 or 2 weeks, respectively, to determine histomorphometrically the epithelialization rate, contraction rate, healing rate, tissue fill, and volume fractions of fibroblasts, neutrophils, macrophages, and blood vessels. RESULTS: Only the 0.6- and 0.9-ml groups had significantly lower contraction rates than the controls after 2 weeks (P <0.05). Although no statistically significant differences were found in other parameters between the PRP-treated wounds and the controls, the PRP treatment led to increases in average epithelialization rates and volume fraction of blood vessels at both time periods. The PRP also seemed to have the most positive effect on healing rate, tissue fill, and volume fraction of fibroblasts during week 1 compared to week 2. CONCLUSIONS: The PRP treatment enhanced healing in full-thickness wounds by reducing the contraction rate with a trend toward acceleration of the epithelial migration and the angiogenic response. Further studies with larger sample sizes should be conducted to improve statistical sensitivity. Longer time intervals and modifications of PRP volume should also be explored to evaluate the long-term efficacy of PRP on wound healing.

Vitamin D receptor deficiency affects dentin maturation in mice.

Mutation of vitamin D receptors (vdr) results in resistance to the vitamin's normal effects which may compromise dentin formation. The objective of this study was to investigate the effect of vdr deficiency on post-natal dentin maturation in mice. The dentin in mandibular incisors of 70.5-day-old vdr wild-type and vdr knockout mice was compared at different levels along the long axis. Expression of biglycan and decorin was detected by immunolocalisation. Scanning electron microscopy was used to observe the ultrastructure of the dentin, and micro-computerised tomography was used to determine the degree of dentin mineralisation density. In the vdr knockout mice, the pulp chamber was larger and the dentin wall was thinner compared with the wild-type mice. In addition, the pre-dentin layer was thickened with an irregular front line and diffuse expression of biglycan and decorin. Fewer tubules, lower mineralisation density and pore-like defects were observed in the dentin at the eruptive region and level with the first molar. In conclusion, vdr deficiency compromises dentin maturation.

Surface analysis and biocorrosion properties of nanostructured surface sol-gel coatings on Ti6Al4V titanium alloy implants.

Surfaces of biocompatible alloys used as implants play a significant role in their osseointegration. Surface sol-gel processing (SSP), a variant of the bulk sol-gel technique, is a relatively new process to prepare bioreactive nanostructured titanium oxide for thin film coatings. The surface topography, roughness, and composition of sol-gel processed Ti6Al4V titanium alloy coatings was investigated by atomic force microscopy (AFM) and X-ray electron spectroscopy (XPS). This was correlated with corrosion properties, adhesive strength, and bioreactivity in simulated body fluids (SBF). Electroimpedance spectroscopy (EIS) and polarization studies indicated similar advantageous corrosion properties between sol-gel coated and uncoated Ti6Al4V, which was attributed to the stable TiO2 composition, topography, and adhesive strength of the sol-gel coating. In addition, inductive coupled plasma (ICP) and scanning electron microscopy with energy dispersive spectrometry (SEM-EDS) analysis of substrates immersed in SBF revealed higher deposition of calcium and phosphate and low release rates of alloying elements from the sol-gel modified alloys. The equivalent corrosion behavior and the definite increase in nucleation of calcium apatite indicate the potential of the sol-gel coating for enhanced bioimplant applications.

Cytotoxicity of a new root canal filling material on human gingival fibroblasts.

This study evaluated the cytotoxicity of the root canal sealing materials Resilon and Epiphany versus gutta-percha, Grossman's sealer, Thermaseal, and Sealapex. Using human gingival fibroblasts the fibroblasts cultures were incubated for either 1 or 24 h to test the cytotoxicity after freshly mixing or after 24 h of setting. Fibroblasts were then stained with trypan blue, to determine number of dead cells. Data were analyzed using ANOVA and t tests. Resilon was similar to gutta-percha and the control. Epiphany was less cytotoxic than Grossman's sealer at both the 1 and 24 h time periods. Epiphany was more cytotoxic than Sealapex at the 1-h time period but less cytotoxic at the 24 h time period. These results indicated that Resilon had a lower cytotoxicity and that Epiphany was more cytotoxic than conventional materials.

Expression and localization of versican during postnatal development of rat temporomandibular joint disc.

To analyze the growth-related changes in extracellular matrix components in temporomandibular joint (TMJ) discs, the expression and localization of the core protein of a large chondroitin sulphate proteoglycan, versican, in rat TMJ discs during postnatal development (2-32 weeks) were examined using Western blot analysis, real-time quantitative PCR and immunohistochemistry. Western blot analysis showed that rat TMJ discs predominantly expressed one isoform (V1) and the core protein sharply increased after birth, reached a peak at 8 weeks, and then gradually decreased up to 32 weeks. Real-time quantitative PCR with TaqMan probes indicated that mRNA expression of versican was highest at 2 weeks and gradually decreased with growth. An immunohistochemical study showed that staining for versican was weak and evenly distributed in TMJ discs at 2 weeks. Regional differences in staining for versican became prominent after 8 weeks; staining was intense in the anterior and posterior peripheral attachments, and weak in the central part of the discs. These results demonstrate that growth-related changes and regional differences exist in the expression of versican in the TMJ discs of growing rats, and these probably reflect the changes in the biomechanical environment caused by the development of orofacial functions.

Osteoblast adhesion and matrix mineralization on sol-gel-derived titanium oxide.

The biological events occurring at the bone-implant interface are influenced by the topography, chemistry and wettability of the implant surface. The surface properties of titanium alloy prepared by either surface sol-gel processing (SSP), or by passivation with nitric acid, were investigated systematically using X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy and contact angle metrology. The bioreactivity of the substrates was assessed by evaluating MC3T3-E1 osteoblastic cell adhesion, as well as by in vitro formation of mineralized matrix. Surface analysis of sol-gel-derived oxide on Ti6Al4V substrates showed a predominantly titanium dioxide (TiO(2)) composition with abundant hydroxyl groups. The surface was highly wettable, rougher and more porous compared to that of the passivated substrate. Significantly more cells adhered to the sol-gel-coated surface, as compared with passivated surfaces, at 1 and 24h following cell seeding, and a markedly greater number of mineralized nodules were observed on sol-gel coatings. Collectively our results show that the surface properties of titanium alloy can be modified by SSP to enhance the bioreactivity of this biomaterial.

A novel approach to xenotransplantation combining surface engineering and genetic modification of isolated adult porcine islets.

BACKGROUND: Effective cytoprotection to xenoislets would circumvent the major tissue limitation for pancreatic islet transplantation (PIT). Cell-surface engineering with poly[ethylene glycol] (PEG) derivatives can successfully prevent antibody binding to the surface antigens. Gene transfer of the antiapoptotic Bcl-2 gene has been shown to decrease cytotoxicity mediated by xenoreactive natural antibodies and complement. In this study, we assessed survival and function of surface-engineered porcine islets genetically modified to overexpress Bcl-2. METHODS: Incorporation of PEG derivatives into the islet surface and adenovirus-mediated gene transfer of Bcl-2 (AdBcl-2) was accomplished within 24 hours post-isolation. Cytotoxicity induced by human xenoreactive natural antibodies was evaluated by islet intracellular lactate dehydrogenase release and microscopic analysis using membrane-integrity staining. Islet functionality was assessed by static incubation and after intraportal infusion (5000 IEQ) into diabetic NOD-SCID mice reconstituted with human lymphocytes (5 x 10 8 /intraperitoneally/15 days before PIT). RESULTS: No significant change in islet viability, morphology, and functionality was demonstrated after the incorporation of PEG-mono-succimidyl-succinate (MSPEG), or PEG-di-succimidyl-succinate "end"-capped with albumin (DSPEG) with or without gene transfer of Bcl-2. Islets treated with MSPEG presented a significant reduction in lactate dehydrogenase release compared with controls (41.2 +/- 3 vs 72.1 +/- 7, respectively, P <.05). Further protection was accomplished by DSPEG or AdBcl-2. The maximal cytoprotection was achieved by DSPEG +AdBcl-2 (15.5 +/- 4.9%, P <.001). Nonfasting glucose >200 mg/dL was found in 100% of the animals given control islets (n = 6) within 48 hours post-transplant. In contrast, euglycemia was achieved in 100% of the animals given islets modified with DSPEG + AdBcl-2 during the observation time. CONCLUSIONS: Surface-engineering with functionalized PEG derivatives in combination with genetic modification with Bcl-2 significantly reduced islet loss after PIT. Application of this novel technology may improve results in xenoislet transplantation.

Enhanced isolated pancreatic islet recovery and functionality in rats by 17beta-estradiol treatment of brain death donors.

BACKGROUND: Current isolation techniques recover only 20% to 50% of the pancreatic islets. Brain death (BD) is characterized by activation of proinflammatory cytokines (PICs) with reduced islet yields and functionality. We previously reported that 17beta-estradiol (E2) induces cytoprotection to human islets exposed to PICs. Furthermore, inhibition of PIC release has been demonstrated after E2 treatment. In the present study, we evaluated if E2 treatment to BD donors would improve pancreatic islet recovery and functionality. METHODS: BD was induced in male, 250- to 350-g Lewis rats by inflation of a Fogarty catheter placed intracranially. Rats were mechanically ventilated for 6 hours. Only rats with mean arterial blood pressure > 75 mm Hg were used. Animals (n = 6) received E2 (1 mg/kg/iv immediately after BD induction), vehicle (V), or the combination of 17beta-estradiol and a selective estrogen receptor antagonist ICI 182,780 (ICI, 3 mg/kg/ip/1 hour before BD induction). Islet viability was determined by ethidium bromide-acridine orange. PICs were assessed by ELISA. Islet functionality was determined by static incubation and glucose disposal rate (Kg) after intraportal transplantation (3000 islet equivalent[IEQ]/syngeneic streptozotocin-induced diabetic rat). RESULTS: A 2- to 3-fold reduction in TNF-alpha, IL-1beta, and IL-6 was demonstrated in BD donors given E2; this effect reversed by ICI 182,780. Pancreatic sections from control BD donors presented 26.5% +/- 4% TUNEL-positive beta-cells compared with 15.1% +/- 3% in 17beta-estradio-treated animals. Islet recovery was enhanced in E2-treated donors (1233.4 +/- 123 IEQ/pancreas) compared with controls (725 +/- 224 IEQ, P < .05). Islet viability was significantly enhanced by E2. Higher islet functionality was demonstrated in vitro and in vivo after transplantation in islets recovered from E2-treated BD donors. CONCLUSIONS: Islet recovery and functionality in vitro and in vivo were significantly improved by 17beta-estradiol treatment to BD donors. These observations may lead to strategies to reduce the effects of BD on isolated islets and improve the results in clinical islet transplantation.

Brain death significantly reduces isolated pancreatic islet yields and functionality in vitro and in vivo after transplantation in rats.

Although approximately 1 million islets exist in the adult human pancreas, current pancreas preservation and islet isolation techniques recover <50%. Presently, cadaveric donors remain the sole source of pancreatic tissue for transplantation. Brain death is characterized by activation of proinflammatory cytokines and organ injury during preservation and reperfusion. In this study, we assessed the effects of brain death on islet isolation yields and functionality. Brain death was induced in male 250- to 350-g Lewis rats by inflation of a Fogarty catheter placed intracranially. The rats were mechanically ventilated for 2, 4, and 6 h before removal of the pancreas (n = 6). In controls, the catheter was not inflated (n = 6). Shortly after brain death induction, a significant increase in serum tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 was demonstrated in a time-dependent manner. Upregulation of TNF-alpha, IL-1beta, and IL-6 mRNA was noted in the pancreas. Brain death donors presented lower insulin release after glucose stimulation assessed by in situ perfusion of the pancreas. Islet recovery was reduced in brain death donors compared with controls (at 6 h 602.3 +/- 233.4 vs. 1,792.5 +/- 325.4 islet equivalents, respectively; P < 0.05). Islet viability assessed in dissociated islet cells and in intact cultured islets was reduced in islets recovered from brain death donors, an effect associated with higher nuclear activities of NF-kappaB p50, c-Jun, and ATF-2. Islet functionality evaluated in vitro by static incubation and in vivo after intraportal transplantation in syngeneic streptozotocin-induced diabetic rats was significantly reduced in preparations obtained from brain death donors. In conclusion, brain death significantly reduced islet yields and functionality. These observations may lead to strategies to reduce the effects of brain death on pancreatic islets and improve the results in clinical transplantation.

Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro.

Epithelial cells lining the oral cavity are exposed to HIV-1 through breast-feeding and oral-genital contact. Genital secretions and breast milk of HIV-1-infected subjects contain both cell-free and cell-associated virus. To determine if oral epithelial cells can be infected with HIV-1 we exposed gingival keratinocytes and adenoid epithelial cells to cell-free virus and HIV-1-infected peripheral blood mononuclear cells and monocytes. Using primary isolates we determined that gingival keratinocytes are susceptible to HIV-1 infection via cell-free CD4-independent infection only. R5 but not X4 viral strains were capable of infecting the keratinocytes. Further, infected cells were able to release infectious virus. In addition, primary epithelial cells isolated from adenoids were also susceptible to infection; both cell-free and cell-associated virus infected these cells. These data have potential implications in the transmission of HIV-1 in the oral cavity.

Immunohistochemical localization of large chondroitin sulfate proteoglycan in odontogenic tumor.

The present study investigated the localization of versican in odontogenic tumors by immunohistochemistry, using paraffin-embedded sections obtained from 27 patients with odontogenic tumors (17 ameloblastomas, 1 adenomatoid odontogenic tumor, 4 odontogenic keratocysts, 1 calcifying odontogenic cyst, 2 ameloblastic fibromas, and 2 malignant ameloblastomas). Deparaffinized sections were immersed in a buffered 1 : 1000 solution of an antibody, 5D5 (raised against a large chondroitin sulfate proteoglycan from bovine sclera), which mainly recognizes versican. All samples showed a positive reaction for versican in connective tissues, whereas positive staining of epithelial nests was observed in only some samples. The positive staining in epithelial nests was in areas showing stellate reticulum-like, cuboidal, columnar cells at the periphery, and tear-drop structures. These results indicated that versican might be involved in, at least in part, the morphogenesis of neoplastic epithelium and mesenchymal tissues in odontogenic tumors.