Overexpression of heat-shock protein 47 impacts survival of patients with oral squamous cell carcinoma.

BACKGROUND: The expression of heat-shock protein 47 (HSP47) has been linked to collagen synthesis control and implicated in fibrotic disorders, but more recent studies have demonstrated its role in solid tumors. In this study, we explored the prognostic impact of HSP47 in oral squamous cell carcinomas (OSCC) and determined the in vitro effects of its loss-of-function on viability, proliferation, migration, invasion, and resistance to cisplatin of OSCC cells. METHODS: The HSP47 expression in tumor samples was assessed by immunohistochemistry in two independent cohorts totaling 339 patients with OSCC, and protein levels were associated with clinicopathological features and survival outcomes. The OSCC cell lines HSC3 and SCC9 were transduced with lentivirus expressing short hairpin RNA to stably silence HSP47 and used in assays to measure cellular viability, proliferation, migration, and invasion. RESULTS: HSP47 was overexpressed in OSCC samples, and its overexpression was significantly and independently associated with poor disease-specific survival and shortened disease-free survival in both OSCC cohorts. The knockdown of HSP47 showed no effects on cell viability or cisplatin sensitivity, but impaired significantly proliferation, migration, and invasion of OSCC cells, with stronger effects on SCC9 cells. CONCLUSION: Our results show a significant prognostic impact of HSP47 overexpression in OSCC and reveal that HSP47 inhibition impairs the proliferation, migration, and invasion of OSCC cells. HSP47 may represent a potential therapeutic target for OSCC.

Stress induced phosphoprotein 1 overexpression controls proliferation, migration and invasion and is associated with poor survival in oral squamous cell carcinoma.

Objective: Although there have been remarkable achievements in the molecular landscape of oral squamous cell carcinoma (OSCC) in recent years, bringing advances in the understanding of its pathogenesis, development and progression, little has been applied in the prognosis and choosing the optimal treatment. In this study, we explored the influence of the stress induced phosphoprotein 1 (STIP1), which is frequently reported to be highly expressed in many cancers, in OSCCs. Methods: STIP1 expression was assessed in the TCGA database and in two independent cohorts by immunohistochemistry. Knockdown strategy was applied in OSCC cell lines to determine the impact of STIP1 on viability, proliferation, migration and invasion. The zebrafish model was applied for studying tumor formation and metastasis in vivo. The association of STIP1 and miR-218-5p was explored by bioinformatics and mimics transfection. Results: STIP1 was highly expressed in OSCCs and significantly associated with shortened survival and higher risk of recurrence. STIP1 down-regulation decreased proliferation, migration and invasion of tumor cells, and reduced the number of metastases in the Zebrafish model. STIP1 and miR-218-5p were inversely expressed, and the transfection of miR-218-5p mimics into OSCC cells decreased STIP1 levels as well as proliferation, migration and invasion. Conclusion: Our findings show that STIP1 overexpression, which is inversely associated with miR-218-5p levels, contributes to OSCC aggressiveness by controlling proliferation, migration and invasion and is a determinant of poor prognosis.

Contribution of Bone Marrow-derived Cells to Reparative Dentinogenesis Using Bone Marrow Transplantation Model.

INTRODUCTION: The aim of this study was to analyze the contribution of bone marrow-derived cells (BMDCs) to reparative dentinogenesis using bone marrow transplantation (BMT) and pulp capping as an in vivo model. METHODS: A chimeric mouse model was created through the injection of BMDCs expressing green fluorescent protein (GFP+ BMDCs) from C57BL/6 GFP+ transgenic donor mice into irradiated C57BL/6 wild-type recipient mice (GFP- mice). These GFP- chimeric mice (containing transplanted GFP+ BMDCs) were subjected to microscopic pulp exposure and capping with white mineral trioxide aggregate (n = 18) or Biodentine (Septodont, St Maur-des-Fossés, France) (n = 18) in the maxillary first molar. Maxillary arches from GFP- chimeric mice (with the capped tooth) were isolated and histologically processed 5 (n = 9) and 7 (n = 9) weeks after BMT. Confocal laser microscopy and immunohistochemical analysis were performed to assess the presence of GFP+ BMDCs and the expression of dentin sialoprotein, an odontoblast marker, for those cells contributing to reparative dentinogenesis in the dental pulp. RESULTS: Confocal laser microscopic analyses evidenced the presence of GFP+ BMDCs in close association with reparative dentin synthesized at the site of pulp exposure in GFP- mice 5 and 7 weeks after BMT. Immunohistochemical analysis revealed that GFP+ BMDCs in close association with reparative dentin expressed DSP, suggesting the contribution of nonresident GFP+ BMDCs to reparative dentinogenesis. CONCLUSIONS: These data suggest the presence of nonresident BMDCs in reparative dentinogenesis and its contribution to dental pulp regeneration in the pulp healing process.

5-Aza-CdR promotes partial MGMT demethylation and modifies expression of different genes in oral squamous cell carcinoma.

OBJECTIVE: Treatment strategies for oral squamous cell carcinoma (OSCC) vary, depending on the stage of diagnosis. Surgery and radiotherapy are options for localized lesions for stage I patients, whereas chemotherapy is the main treatment for metastatic OSCC. However, aggressive tumors can relapse, frequently causing death. In an attempt to address this, novel treatment protocols using drugs that alter the epigenetic profile have emerged as an alternative to control tumor growth and metastasis. Therefore, the objective in this study was to investigate the effect of the demethylating drug 5-aza-CdR in SCC9 OSCC cells. STUDY DESIGN: SCC9 cells were treated with 5-Aza-CdR at concentrations of 0.3µM and 2µM for 24hours and 48hours. DNA methylation of the MGMT, BRCA1, APC, c-MYC, and hTERT genes were investigated by using the methylation-specific high-resolution melting technique. Real time-polymerase chain reaction and quantitative polymerase chain reaction were performed to analyze gene expression. RESULTS: 5-Aza-CdR promoted demethylation of MGMT and modified the transcription of all analyzed genes. Curiously, 5-aza-CdR at the concentration of 0.3µM was more efficient than 2µM in SCC9 cells. CONCLUSIONS: We observed that 5-aza-CdR led to MGMT demethylation, upregulated the transcription of 3 important tumor suppressor genes, and promoted the downregulation of c-Myc.

Spatial and Quantitative Detection of BMP Activity in Mouse Embryonic Limb Buds.

Modulation of bone morphogenetic protein (BMP) activity is essential to the progression of limb development in the mouse embryo. Genetic disruption of BMP signaling at various stages of limb development causes defects ranging from complete limb agenesis to oligodactyly, polydactyly, webbing, and chondrodysplasia. To probe the state of BMP signaling in early limb buds, we designed two sets of primers to measure both spatially and quantitatively the transcription of nine key genes indicative of canonical BMP activity. One set is used to generate digoxigenin (DIG)-labeled antisense RNA probes for whole-mount mRNA in situ hybridization, while the second set is used for SYBR® Green-based quantitative PCR on limb bud cDNA. Here we describe step-by-step protocols for both methods around this specific set of genes.

Biological response to self-etch adhesive after partial caries removal in rats.

OBJECTIVES: The purposes of this study were to evaluate a model of slow caries progression and to investigate the performance of a self-etch adhesive system for partial caries removal. MATERIALS AND METHODS: Rat molars were infected with Streptococcus sobrinus 6715 culture. Different time points were analyzed: days 78, 85, and 95 (± 2). After this, the samples were processed for morphological analysis. Additionally, the first molars were restored with zinc oxide and eugenol (IRM™; Dentsply; Brazil) or adhesive system (Clearfil SE Bond™; Kuraray Medical; Japan) 78 days after caries induction. After, 3 or 15 days post-treatment, the animals were euthanized, and their mandibles were processed for morphological analysis, classified by means of scores, and submitted to statistical analysis. Subsequently, immunohistochemical analysis was performed for osteonectin (OSN) and transforming growth factor-ß1 (TGF-ß1) expression. RESULTS: According to the caries induction model used, on day 95 greater inflammatory infiltration (p < 0.001), and more extensive degradation of secondary/primary dentin were demonstrated than on day 78 (p < 0.05). Furthermore, the restorative materials presented similar performance (p > 0.05) and proved to be fundamental to control the carious lesion. The TGF-ß1 and OSN were shown to be active during the caries process. CONCLUSIONS: The slow caries lesion model was feasible for morphological analysis of the dentin-pulp complex. The self-etch adhesive system triggered no acute inflammatory infiltration or pulp necrosis, instead it seemed to stimulate early pulp repair. CLINICAL RELEVANCE: Clearfil SE Bond™ applied directly on caries-affected dentin did not predispose to pulp inflammation; instead, it appeared to provide early biological benefits.

In situ analysis of gelatinolytic activity in human dentin.

Matrix metalloproteinases (MMPs) such as gelatinases are differentially expressed in human tissues. These enzymes cleave specific substrates involved in cell signaling, tissue development and remodeling and tissue breakdown. Recent evidences show that gelatinases are crucial for normal dentin development and their activity is maintained throughout the entire tooth function in the oral cavity. Due to the lack of information about the exact location and activity of gelatinases in mature human dentin, the present study was designed to examine gelatinolytic levels in sound dentin. In situ zymography using confocal microscopy was performed on both mineralized and demineralized dentin samples. Sites presenting gelatinase activity were identified throughout the entire biological tissue pursuing different gelatinolytic levels for distinct areas: predentin and dentinal tubule regions presented higher gelatinolytic activity compared to intertubular dentin. Dentin regions with higher gelatinolytic activity immunohistochemically were partially correlated with MMP-2 expression. The maintenance of gelatinolytic activity in mature dentin may have biological implications related to biomineralization of predentin and tubular/peritubular dentinal regions, as well as regulation of defensive mechanisms of the dentin-pulp complex.

Required Time for Migration of Bone Marrow-derived Cells to Dental Pulp after Bone Marrow Transplantation.

INTRODUCTION: This study aimed to evaluate the time required for bone marrow-derived cells (BMDCs) from transgenic green fluorescent protein (GFP)+ donor mice (GFP+ mice) to migrate into the dental pulp of wild-type GFP- recipient mice (GFP- mice) by using bone marrow transplantation (BMT) as an in vivo model for tracking BMDCs from GFP+ mice (GFP+ BMDCs). METHODS: GFP+ BMDCs were injected into irradiated GFP- mice. Maxillary arches, tibiae, and femora from GFP- mice were isolated and processed at 24 hours, 48 hours, 4, 7, and 14 days, and 7 weeks after BMT. Confocal laser microscopy analyses were performed to assess the presence of GFP+ BMDCs in the dental pulp, and flow cytometry of BM was performed to confirm the efficiency of engraftment of GFP+ BMDCs. RESULTS: Confocal laser microscopy analyses evidenced the presence of GFP+ BMDCs in the dental pulp of GFP- mice from 14 days to 7 weeks after BMT. There was no presence of GFP+ BMDCs at 24 hours, 48 hours, 4 days, and 7 days. Flow cytometry of the BM of GFP- mice demonstrated a constant increase in the presence of GFP+ BMDCs at 24 hours, 48 hours, and 4 days after BMT, which stabilized from 7 days to 7 weeks. CONCLUSIONS: The study demonstrated the presence of GFP+ BMDCs in the dental pulp from 14 days to 7 weeks after BMT and the feasibility of using GFP+ animals and BMT as an in vivo model for tracking GFP+ BMDCs.

Antioxidant therapy enhances pulpal healing in bleached teeth.

OBJECTIVES: The purpose of this study was to evaluate the histopathological effects of an antioxidant therapy on the pulp tissue of rat teeth exposed to a bleaching gel with 35% hydrogen peroxide. MATERIALS AND METHODS: Forty rats were subjected to oral ingestion by gavage of distilled water (DW) or ascorbic acid (AA) 90 min before the bleaching therapy. For the bleaching treatment, the agent was applied twice for 5 min each to buccal surfaces of the first right mandibular molars. Then, the animals were sacrificed at 6 hr, 24 hr, 3 day, or 7 day post-bleaching, and the teeth were processed for microscopic evaluation of the pulp tissue. RESULTS: At 6 hr, the pulp tissue showed moderate inflammatory reactions in all teeth of both groups. In the DW and AA groups, 100% and 80% of teeth exhibited pulp tissue with significant necrosis and intense tissue disorganization, respectively. At 24 hr, the AA-treated group demonstrated a greater regenerative capability than the DW group, with less intense inflammatory reaction and new odontoblast layer formation in 60% of the teeth. For up to the 7 day period, the areas of pulpal necrosis were replaced by viable connective tissue, and the dentin was underlined by differentiated odontoblast-like cells in most teeth of both groups. CONCLUSIONS: A slight reduction in initial pulpal damage during post-bleaching was promoted by AA therapy. However, the pulp tissue of AA-treated animals featured faster regenerative potential over time.

Intermittent administration of parathyroid hormone improves the repairing process of rat calvaria defects: A histomorphometric and radiodensitometric study.

BACKGROUND: The aim of this study was to evaluate the effects of intermittent treatment of parathyroid hormone (PTH (1-34)) on the bone regeneration of critically-sized rat calvarial bone defects. MATERIAL AND METHODS: Thirty-two male rats were trephined (4mm full-thickness diameter), in the central part of the parietal bones and divided into 2 groups of 16. The PTH group received subcutaneous injections of PTH (1-34) at 40µg/kg, 3 times a week and the control (CTL) group received the vehicle in the same regimen. The rats were sacrificed at 4 weeks post-treatment regimen, the parietal bones were extracted and samples were evaluated through histomorphometry and radiodensitometry. RESULTS: The histological observations showed that the PTH group presented more "island-like" new bone between the defect margins with fibrous tissues than did the CTL group. The PTH group significantly exhibited greater histologic bone formation than did the CTL group (1.5mm ±0.7; 1.9 mm ± 0.6, p<0.05/ for residual bone defect). The radiodensitometry analysis revealed significant differences among the PTH and CTL groups (2.1 Al eq. ±0.04; 1.8Al eq. ±0.06, p<0.05), demonstrating an increase in bone mineral density. The PTH treatment contributed to the bone formation with a higher amount of mineral and/or fibrous tissue when compared with the CTL group. CONCLUSIONS: The results suggest that it was possible to increase the process of bone regeneration by accelerating the healing process in rat calvarial defects through intermittent administration of the PTH treatment.

Effect of dimethyl sulfoxide wet-bonding technique on hybrid layer quality and dentin bond strength.

OBJECTIVES: This study examined the effect of a dimethyl sulfoxide (DMSO) wet bonding technique on the resin infiltration depths at the bonded interface and dentin bond strength of different adhesive systems. METHODS: Flat dentin surfaces of 48 human third molars were treated with 50% DMSO (experimental groups) or with distilled water (controls) before bonding using an etch-and-rinse (SBMP: Scotchbond Multi-Purpose, 3M ESPE) or a self-etch (Clearfil: Clearfil SE Bond, Kuraray) adhesive system. The restored crown segments (n=12/group) were stored in distilled water (24h) and sectioned for interfacial analysis of exposed collagen using Masson's Trichrome staining and for microtensile bond strength testing. The extent of exposed collagen was measured using light microscopy and a histometric analysis software. Failure modes were examined by SEM. Data was analyzed by two-way ANOVA followed by Tukey Test (α=0.05). RESULTS: The interaction of bonding protocol and adhesive system had significant effects on the extension of exposed collagen matrix (p<0.0001) and bond strength (p=0.0091). DMSO-wet bonding significantly reduced the extent of exposed collagen matrix for SBMP and Clearfil (p<0.05). Significant increase in dentin bond strength was observed on DMSO-treated specimens bonded with SBMP (p<0.05), while no differences were observed for Clearfil (p>0.05). SIGNIFICANCE: DMSO-wet bonding was effective to improve the quality of resin-dentin bonds of the tested etch-and-rinse adhesives by reducing the extent of exposed collagen matrix at the base of the resin-dentin biopolymer. The improved penetration of adhesive monomers is reflected as an increase in the immediate bond strength when the DMSO-wet bonding technique is used with a water-based etch-and-rinse adhesive.

G-quadruplex formation enhances splicing efficiency of PAX9 intron 1.

G-quadruplexes are secondary structures present in DNA and RNA molecules, which are formed by stacking of G-quartets (i.e., interaction of four guanines (G-tracts) bounded by Hoogsteen hydrogen bonding). Human PAX9 intron 1 has a putative G-quadruplex-forming region located near exon 1, which is present in all known sequenced placental mammals. Using circular dichroism (CD) analysis and CD melting, we showed that these sequences are able to form highly stable quadruplex structures. Due to the proximity of the quadruplex structure to exon-intron boundary, we used a validated double-reporter splicing assay and qPCR to analyze its role on splicing efficiency. The human quadruplex was shown to have a key role on splicing efficiency of PAX9 intron 1, as a mutation that abolished quadruplex formation decreased dramatically the splicing efficiency of human PAX9 intron 1. The less stable, rat quadruplex had a less efficient splicing when compared to human sequences. Additionally, the treatment with 360A, a strong ligand that stabilizes quadruplex structures, further increased splicing efficiency of human PAX9 intron 1. Altogether, these results provide evidences that G-quadruplex structures are involved in splicing efficiency of PAX9 intron 1.

High-throughput DNA analysis shows the importance of methylation in the control of immune inflammatory gene transcription in chronic periodontitis.

BACKGROUND: Chronic periodontitis represents a complex disease that is hard to control and is not completely understood. Evidence from past studies suggests that there is a key role for DNA methylation in the pathogenesis of periodontitis. However, all reports have applied technologies that investigate genes in a low throughput. In order to advance in the knowledge of the disease, we analyzed DNA methylation variations associated with gene transcription using a high-throughput assay. Infinium® HumanMethylation450 (Illumina) was performed on gingival samples from 12 periodontitis cases and 11 age-matched healthy individuals. Methylation data of 1,284 immune-related genes and 1,038 cell cycle-related genes from Gene Ontology (GO) and 575 genes from a dataset of stably expressed genes (genes with consistent expression in different physiological states and tissues) were extracted from a microarray dataset and analyzed using bioinformatics tools. DNA methylation variations ranging from -2,000 to +2,000 bp from the transcription start site (TSS) were analyzed, and the results were tested against a differential expression microarray dataset between healthy and periodontitis gingival tissues. Differences were evaluated using tests from the R Statistical Project. RESULTS: The comparison of probes between periodontitis and normal gingival tissues showed that the mean methylation scores and the frequency of methylated probes were significantly lower in genes related to the immune process. In the immune group, these parameters were negatively correlated with gene expression (Mann-Whitney test, p < 2.2e - 16). CONCLUSIONS: Our results show that variations in DNA methylation between healthy and periodontitis cases are higher in genes related to the immune-inflammatory process. Thus, DNA methylation must be modulating chromatin regions and, consequently, modulating the mRNA transcription of immune-inflammatory genes related with periodontitis, impacting the prognosis of disease.

Parathyroid hormone (1-34) modulates odontoblast proliferation and apoptosis via PKA and PKC-dependent pathways.

Parathyroid hormone (PTH) plays a key role in the development and homeostasis of mineralized tissues such as bone and dentine. We have reported that PTH (1-34) administration can increase dentine formation in mice and that this hormone modulates in vitro mineralization of odontoblast-like cells. The purpose of the present study was to investigate whether PTH (1-34) participates in the proliferative and apoptotic signaling of odontoblast-like cells (MDPC23). MDPC23 cells were exposed to 50 ng/ml hPTH (1-34) or vehicle for 1 (P1), 24 (P24), or 48 (P48) hours, and the cell proliferation, apoptosis, and cell number were evaluated. To examine whether changes in the proliferative and apoptotic signaling in response to PTH involve protein kinases A (PKA) and/or C (PKC), MDPC23 cells were exposed to PTH with or without PKC or PKA signaling pathway inhibitors. Overall, the results showed that the PKA pathway acts in response to PTH exposure maintaining levels of cell proliferation, while the PKC pathway is mainly involved for longer exposure to PTH (24 or 48 h), leading to the reduction of cell proliferation and increase of apoptosis. The exposure to PTH reduced the cell number in relation to the control group in a time-dependent manner. In conclusion, PTH modulates odontoblast-like cell proliferative and apoptotic response in a time-dependent manner. Both PKC and PKA pathways participate in PTH-induced modulation in an antagonist mode.

Outcome of enamel matrix derivative treatment in the presence of chronic stress: histometric study in rats.

BACKGROUND: Psychologic stress and clinical hypercortisolism have been related to direct effects on bone metabolism. However, there is a lack of information regarding the outcomes of regenerative approaches under the influence of chronic stress (CS). Enamel matrix derivative (EMD) has been used in periodontal regenerative procedures, resulting in improvement of clinical parameters. Thus, the aim of this histomorphometric study is to evaluate the healing of periodontal defects after treatment with EMD under the influence of CS in the rat model. METHODS: Twenty Wistar rats were randomly assigned to two groups; G1: CS (restraint stress for 12 hours/day) (n = 10), and G2: not exposed to CS (n = 10). Fifteen days after initiation of CS, fenestration defects were created at the buccal aspect of the first mandibular molar of all animals from both groups. After the surgeries, the defects of each animal were randomly assigned to two subgroups: non-treated control and treated with EMD. The animals were euthanized 21 days later. RESULTS: G1 showed less bone density (BD) compared to G2. EMD provided an increased defect fill (DF) in G1 and higher BD and new cementum formation (NCF) in both groups. The number of tartrate-resistant acid phosphatase-positive osteoclasts was significantly higher in G1 when compared to G2 and in EMD-treated sites of both groups. CONCLUSIONS: CS may produce a significant detrimental effect on BD. EMD may provide greater DF compared to non-treated control in the presence of CS and increased BD and NCF in the presence or absence of CS.

Effects of Cachaça, a typical Brazilian alcoholic beverage, on alveolar bone loss and density: a study in peripubertal rats.

OBJECTIVE: The aim of the present study was to assess the impact of chronic consumption of Cachaça on alveolar bone loss (BL) induced by ligature and on alveolar bone density (BD) in peripubertal rats. DESIGN: Male Wistar rats were assigned into one of the following groups: CONTROL: non-ingestion of Cachaça (n=15); Cachaça: ingestion of ascending concentrations of Cachaça during 100 days (n=15). 70th day after the beginning of Cachaça ingestion, one first mandibular molar received a ligature while the contralateral tooth was left unligated. After 30 days, the rats were killed. BL, BD, the positive cells for tartrate-resistant acid phosphatase (TRAP), receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) were analyzed in the furcation area of the ligated and unligated mandibular molars. RESULTS: The Cachaça group presented greater BL (0.75±0.1mm(2) for Cachaça and 0.66±0.1mm(2) for control group, respectively) and number of RANKL and OPG+ cells and lower BD (60.3±4.2% for Cachaça and 76.8±3.8% for control group, respectively) and number of TRAP+ cells around ligated teeth (p<0.05), when compared to the control group. The Cachaça group (0.42±0.02mm(2)) also presented a higher BL around unligated teeth when compared to control group (0.31±0.05mm(2)). CONCLUSIONS: Cachaça consumption per se and in the presence of ligature negatively affects alveolar bone by increasing the alveolar BL and reducing BD.

Intermittent parathyroid hormone administration improves periodontal healing in rats.

BACKGROUND: Intermittent administration of parathyroid hormone (PTH) promotes new bone formation in patients with osteoporosis and bone fractures. It was shown previously that PTH also reduces periodontitis-related bone loss. The aim of this study is to evaluate the effect of treatment with PTH on periodontal healing in rats. METHODS: Fenestration defects were created at the buccal surface of the distal root of the mandibular first molars, and both periodontal ligament (PDL) and cementum were removed. Animals were then assigned to two groups (eight animals per group): group 1: control, placebo administration; and group 2: test, human PTH (hPTH) 1-34 administration at a concentration of 40 µg/kg. For both groups, the animals were injected every 2 days, and the animals were sacrificed at 14 and 21 days after surgery. Specimens were harvested and processed for routine decalcified histologic sections. The following parameters were assessed: 1) remaining bone defect extension (RBDE); 2) newly formed bone density (NFBD); 3) total callus area (TCA); 4) osteoclast number (ON) in the callus region; and 5) newly formed dental cementum-like tissue (NFC). Birefringence of root PDL reattachment was also evaluated. RESULTS: Birefringence analysis showed root PDL reattachment for both groups 21 days after treatment. Intermittent hPTH 1-34 administration decreased RBDE (P <0.01) and increased NFBD (P <0.01), TCA (P <0.01), area of NFC (P <0.01), and ON in the callus region (P <0.01). CONCLUSION: Within the limits of the present study, intermittent administration of hPTH 1-34 led to an enhanced periodontal healing process compared with non-treated animals.

Effects of lercanidipine on bone density and bone repair in spontaneously hypertensive rats.

PURPOSE: To evaluate the effects of the lercanidipine on bone healing (BH) and bone density (BD) in the tibiae of spontaneously hypertensive rats (SHR), using histometric and tartrate-resistant acid phosphatase (TRAP) expression analyses. MATERIALS AND METHODS: Wistar and SHR were assigned to one of the following groups: normotensive rats (NTR) (n = 15), untreated SHR (n = 15), and lercanidipine-treated SHR (n = 15). The latter group was treated daily with lercanidipine for 6 weeks. Two weeks after the beginning of drug administration, a critical-sized surgical defect was created in the right tibia of all groups, whereas the contralateral tibia remained without defect. The animals were killed 30 days after the creation of the bone defect. RESULTS: There were no significant differences among the groups for BH, trabecular BD, and the number of TRAP+ cells in the newly formed cortical bone (P > 0.05). SHR presented significantly lower cortical BD and increased cortical levels of TRAP+ cells, when compared with NTR and lercanidipine-treated SHR (P < 0.05). CONCLUSION: SHR presented a lower cortical BD and increased levels of TRAP+ cells. In addition, the treatment of SHR with lercanidipine during 6 weeks was able to revert the deleterious effects of hypertension on cortical BD and on the number of TRAP+ cells in the tibia of SHR.

In situ study of the gelatinase activity in demineralized dentin from rat molar teeth.

Matrix metalloproteinases (MMPs) in dentin are believed to participate in various physiological and pathological events in coronal dentin, but their exact source and location is not clear. The purpose of this study was to evaluate the activity of gelatinases in decalcified rat molars crowns by in situ zymography. Hemi-mandibles of five male Wistar rats were fixed in paraformaldehyde, decalcified in EDTA and glycerol solution and embedded in paraffin. Sections from the region of molar teeth were incubated with or without DQ gelatin in 50mM Tris-CaCl2 at 37°C for 2h and observed by means of confocal microscopy. Gelatinolytic activity was observed throughout the coronal dentin with varying intensities in different locations. High gelatinase activity was observed in the dentinal tubules, dentin-enamel junction (DEJ) and predentin, and it was weaker and less uniform in the intertubular dentin. This study shows that the location of gelatinase and relative activity can be detected by means of in situ zymography and confocal microcopy, and this methodology may provide a useful tool in studies on the role of gelatinases in tooth development, maturation and in pathological conditions.

Effects of estrogen deficiency and/or caffeine intake on alveolar bone loss, density, and healing: a study in rats.

BACKGROUND: The aim of this study is to evaluate the effects of caffeine and/or estrogen deficiency on ligature-induced bone loss (BL), trabecular bone area (TBA), and postextraction bone healing (BH). METHODS: Rats were assigned into one of the following groups (15 each): 1) control = non-ingestion of caffeine/sham surgery; 2) caffeine = ingestion of caffeine/sham surgery); 3) ovariectomized (OVX) = non-ingestion of caffeine/ovariectomy; or 4) caffeine/OVX = ingestion of caffeine/ovariectomy. The rats were under caffeine administration for 65 days and/or estrogen deficiency for 51 days. On day 21 after ovariectomy, one first mandibular molar received a ligature and the contralateral tooth was not ligated. The first maxillary molars were extracted 8 days before sacrifice. BL, TBA, the positive cells for tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG) were analyzed in the furcation area of mandibular molars. Histometric BH and gene expression of bone morphogenetic protein (BMP)-2, BMP-7, osteopontin, and bone sialoprotein were evaluated in alveolar sockets. RESULTS: The caffeine group presented the greatest BL and the OVX group the highest number of TRAP-positive (TRAP(+)) cells around ligated teeth (P <0.05). The control group presented higher TBA and BH than the other groups (P <0.05). All test groups presented higher RANKL/OPG(+) cells than the control group around ligated/unligated teeth. The OVX and caffeine/OVX groups presented a greater number of TRAP(+) cells around unligated teeth than the control group (P <0.05). There were no differences among groups for gene expression (P >0.05). CONCLUSIONS: Caffeine increased BL in ligated teeth. Caffeine and/or estrogen deficiency decreased TBA in the unligated teeth and reduced BH after tooth extraction.