The Effect of TAK-778 on Gene Expression of Osteoblastic Cells Is Mediated Through Estrogen Receptor

This study evaluated the effect of TAK-778 [(2R, 4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide)] on in vitro osteogenic events and on gene expression of osteoblastic cells derived from human alveolar bone and the participation of estrogen receptors (ERs) on such effect. Osteoblastic cells were subcultured, with or without TAK-778 (10(-5) M), to evaluate cell growth and viability, total protein content, and alkaline phosphatase (ALP) activity at 7, 14, and 21 days; bone-like formation at 21 days; and gene expression, using cDNA microarray, at 7 days. Also, osteoblastic cells were exposed to TAK-778 (10-5 M) combined to ICI182,780, a nonspecific ER antagonist (10(-6) M), and gene expression was evaluated by real-time polymerase chain reaction (PCR) at 7 days. TAK-778 induced a reduction in culture growth and an increase in cell synthesis, ALP activity, and bone-like formation. The cDNA microarray showed genes associated with cell adhesion and differentiation, skeletal development, ossification, and transforming growth factor-P receptor signaling pathway, with a tendency to be higher expressed in cells exposed to TAK-778. The gene expression of ALP, osteocalcin, Msh homeobox 2, receptor activator of NF-kappa B ligand, and intercellular adhesion molecule 1 was increased by TAK-778 as demonstrated by real-time PCR, and this effect was antagonized by ICI182,780. The present results demonstrated that TAK-778 acts at a transcriptional level to enhance the in vitro osteogenic process and that its effect on gene expression of osteoblastic cells is mediated, at least partially, through ERs. Based on these findings, TAK-778 could be considered in the treatment of bone metabolic disorders. Exp Biol Med 234:190-199, 2009

High concentration of residual aluminum oxide on titanium surface inhibits extracellular matrix mineralization

In the present study we characterized titanium (Ti) surfaces submitted to different treatments and evaluated the response of osteoblasts derived from human alveolar bone to these surfaces. Five different surfaces were evaluated: ground (G), ground and chemical etched (G1-HF for 60 s), sand blasted (SB-Al2O3 particles 65 pm), sand blasted and chemical etched (SLA1-HF for 60 s and SLA2-HF for 13 s). Surface morphology was evaluated under SEM and roughness parameters by contact scanning instrument. The presence of Al2O3 was detected by EDS and the amount calculated by digital analyses. Osteoblasts, were cultured on these surfaces and it was evaluated: cell adhesion, proliferation, and viability, alkaline phosphatase activity, total protein content, and matrix mineralization formation. Physical and chemical treatments produced very different surface morphologies. Al2O3 residues were detected on SB and SLA2 surfaces. Only matrix mineralization formation was affected by different surface treatments, being increased on rough surface (SLA1) and reduced on surface with high amount of Al2O3 residues (SB). On the basis of these findings, it is possible to conclude that high concentration of residual Al2O3 negatively interfere with the process of matrix mineralization formation in contact with Ti implant surfaces. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 87A: 588-597, 2008

In vitro osteogenesis on fluorcanasite glass-ceramic with three different chemical compositions

This study aimed at investigating in vitro osteogenesis on three fluorcanasite glass-ceramic compositions with different solubilities (K3, K5, and K8). Osteoblastic cells were obtained from human alveolar bone fragments and cultured under standard osteogenic condition until subconfluence. First passage cells were cultured on K3, K5, and K8 and on Bioglass (R) 45S5 (45S5-control). Cell adhesion was evaluated at 24 h. For proliferation and viability, cells were cultured for 1, 4, and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 7, 14, and 21 days. Cultures were stained with Alizarin red at 21 days, for detection of mineralized matrix. Data were compared by ANOVA followed by Duncan`s test. Cell adhesion, cell proliferation, viability, total protein content, and ALP activity were not affected by fluorcanasite glass-ceramic composition and solubility. Bone-like formation was similar on all fluorcanasite-glass ceramics and was reduced compared to 45S5. The changes in the chemical composition and consequently solubility of the fluorcanasite glass-ceramics tested here did not significantly alter the in vitro osteogenesis. Further modifications of the chemical composition of the fluorcanasite glass-ceramic would be required to improve bone response, making this biomaterial a good candidate to be employed as a bone substitute.

In vitro biocompatibility of a novel membrane of the composite poly(vinylidene-trifluoroethylene)/barium titanate

This study was aimed at investigating the in vitro biocompatibility of a novel membrane of the composite poly(vinylidene-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT). Osteoblastic cells were obtained from human alveolar bone fragments and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured on P(VDF-TrFE)/BT and expanded polytetrafluoroethylene (e-PTFE - control) membranes in 24-well plates. Cell adhesion and spreading were evaluated at 30 min, and 4 and 24 h. For proliferation assay, cells were cultured for 1, 7, and 10 days. Cell viability was detected by trypan blue at 7 and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 7, 14, and 21 days. Cultures were stained with Alizarin red at 21 days, for detection of mineralized matrix. Data were compared by ANOVA and Student t test. Cell attachment (p = 0.001), cell number (p = 0.001), and ALP activity (p = 0.0001) were greater on P(VDF-TrFE)/BT. Additionally, doubling time was greater on P(VDF-TrFE)/BT (p = 0.03), indicating a decreased proliferation rate. Bone-like nodule formation took place only on P(VDF-TrFE)/BT. The present results showed that both membranes are biocompatible. However, P(VDF-TrFE)/BT presented a better in vitro biocompatibility and allowed bone-like nodule formation. Therefore, P(VDF-TrFE)/BT could be an alternative membrane to be used in guided tissue regeneration. (c) 2006 Wiley Periodicals, Inc.

The influence of pore size on osteoblast phenotype expression in cultures grown on porous titanium

This study investigated the effect of pore size on osteoblastic phenotype development in cultures grown on porous titanium (Ti). Porous Ti discs with three different pore sizes, 312 mu m (Ti 312), 130 mu m (Ti 130) and 62 mu m (Ti 62) were fabricated using a powder metallurgy process. Osteoblastic cells obtained from human alveolar bone were cultured on porous Ti samples for periods of up to 14 days. Cell proliferation was affected by pore size at day 3 (p = 0.0010), day 7 (p = 0.0005) and day 10 (p = 0.0090) in the following way: Ti 62 < Ti 130 < Ti 312. Gene expression of bone markers evaluated at 14 days was affected, RUNX2 (p = 0.0153), ALP (p = 0.0153), BSP (p = 0.0156), COL (p = 0.0156), and OPN (p = 0.0156) by pore size as follows: Ti 312 < Ti 130 < Ti 62. Based on these results, the authors suggest that porous Ti surfaces with pore sizes near 62 mu m, compared with those of 312 mu m and 130 mu m, yield the highest expression of osteoblast phenotype as indicated by the lower cell proliferation rate and higher gene expression of bone markers.

Evaluation of Maxillary Alveolar Reconstruction Using a Resorbable Collagen Sponge with Recombinant Human Bone Morphogenetic Protein-2 in Cleft Lip and Palate Patients

Introduction: A resorbable collagen matrix with recombinant human bone morphogenetic protein (rhBMP-2) was compared with traditional iliac crest bone graft for the closure of alveolar defects during secondary dental eruption. Methods: Sixteen patients with unilateral cleft lip and palate, aged 8 to 12 years, were selected and randomly assigned to group 1 (rhBMP-2) or group 2 (iliac crest bone graft). Computed tomography was performed to assess both groups preoperatively and at months 6 and 12 postoperatively. Bone height and defect volume were calculated through Osirix Dicom Viewer (Pixmeo, Apple Inc.). Overall morbidity was recorded. Results: Preoperative and follow-up examinations revealed progressive alveolar bone union in all patients. For group 1, final completion of the defect with a 65.0% mean bone height was detected 12 months postoperatively. For group 2, final completion of the defect with an 83.8% mean bone height was detected 6 months postoperatively. Dental eruption routinely occurred in both groups. Clinical complications included significant swelling in three group 1 patients (37.5%) and significant donor-site pain in seven group 2 patients (87.5%). Conclusions: For this select group of patients with immature skeleton, rhBMP-2 therapy resulted in satisfactory bone healing and reduced morbidity compared with traditional iliac crest bone grafting.

Human Bone Morphogenetic Protein-2 Use for Maxillary Reconstruction in Cleft Lip and Palate Patients

Background: The conventional methods of maxillary alveolar reconstruction in patient with cleft are the periosteoplasty and autologous bone grafting. As an important alternative of bone substitution, there is the recombinant human bone morphogenetic protein-2 (rhBMP-2). This study compares the rhBMP-2 with periosteoplasty and autologous bone grafting. Methods: Patients with cleft and alveolar defect were divided into 3 groups of 6 patients who underwent to autologous iliac crest bone grafting, resorbable collagen sponge with rhBMP2, and periosteoplasty, respectively. The analysis was performed through computed tomographic scan preoperatively and at months 3, 6, and 12 postoperatively. The variables analyzed were the alveolar defect volume, formed bone volume, bone formation rate, maxillary height repair rate, and the formed bone density mean. Results: The formed bone volume was similar comparing the bone graft and BMP groups at 1-year postoperative analysis (P = 0.58). Both of them had the formed bone volume significantly larger than the periosteoplasty group at 3 and 6 months postoperatively. In this last group, the 1-year follow-up was canceled because the bone formation was insufficient. The bone formation rate, the maxillary height repair rate, and the mean of density of the formed bone were similar in the bone graft and BMP groups at 1-year follow-up with P values of 0.93, 0.90, and 0.81, respectively. Conclusions: The amount of formed bone in the periosteoplasty group was insufficient. There was no difference among the bone graft and rhBMP-2 therapy considering the parameters analyzed.

Gene array of primary human osteoblasts exposed to enamel matrix derivative in combination with a natural bone mineral

OBJECTIVES The application of an enamel matrix derivative (EMD) for regenerative periodontal surgery has been shown to promote formation of new cementum, periodontal ligament, and alveolar bone. In intrabony defects with a complicated anatomy, the combination of EMD with various bone grafting materials has resulted in additional clinical improvements, but the initial cellular response of osteoblasts coming in contact with these particles have not yet been fully elucidated. The objective of the present study was to evaluate the in vitro effects of EMD combined with a natural bone mineral (NBM) on a wide variety of genes, cytokines, and transcription factors and extracellular matrix proteins on primary human osteoblasts. MATERIAL AND METHODS Primary human osteoblasts were seeded on NBM particles pre-coated with versus without EMD and analyzed for gene differences using a human osteogenesis gene super-array (Applied Biosystems). Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules. RESULTS EMD promoted gene expression of various osteoblast differentiation markers including a number of collagen types and isoforms, SMAD intracellular proteins, osteopontin, cadherin, alkaline phosphatase, and bone sialoprotein. EMD also upregulated a variety of growth factors including bone morphogenetic proteins, vascular endothelial growth factors, insulin-like growth factor, transforming growth factor, and their associated receptor proteins. CONCLUSION The results from the present study demonstrate that EMD is capable of activating a wide variety of genes, growth factors, and cytokines when pre-coated onto NBM particles. CLINICAL RELEVANCE The described in vitro effects of EMD on human primary osteoblasts provide further biologic support for the clinical application of a combination of EMD with NBM particles in periodontal and oral regenerative surgery.

Botulinum toxin in masticatory muscles of the adult rat induces bone loss at the condyle and alveolar regions of the mandible associated with a bone proliferation at a muscle enthesis

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Digital subtraction radiographic analysis of the combination of bioabsorbable membrane and bovine morphogenetic protein pool in human periodontal infrabony defects

OBJECTIVES: This study assessed the bone density gain and its relationship with the periodontal clinical parameters in a case series of a regenerative therapy procedure. MATERIAL AND METHODS: Using a split-mouth study design, 10 pairs of infrabony defects from 15 patients were treated with a pool of bovine bone morphogenetic proteins associated with collagen membrane (test sites) or collagen membrane only (control sites). The periodontal healing was clinically and radiographically monitored for six months. Standardized pre-surgical and 6-month postoperative radiographs were digitized for digital subtraction analysis, which showed relative bone density gain in both groups of 0.034 ± 0.423 and 0.105 ± 0.423 in the test and control group, respectively (p>0.05). RESULTS: As regards the area size of bone density change, the influence of the therapy was detected in 2.5 mm² in the test group and 2 mm² in the control group (p>0.05). Additionally, no correlation was observed between the favorable clinical results and the bone density gain measured by digital subtraction radiography (p>0.05). CONCLUSIONS: The findings of this study suggest that the clinical benefit of the regenerative therapy observed did not come with significant bone density gains. Long-term evaluation may lead to a different conclusions.

Vertical alveolar crest bone maintenance around implants in two-stage surgery: an in situ study in dogs

The aim of this study was to evaluate in situ changes in the alveolar crest bone height around immediate implant-supported crowns in comparison to tooth-supported crowns (control) with the cervical margins located at the bone crest level, without occlusal load. In Group I, after extraction of 12 mandibular premolars from 4 adult dogs, implants from Branemark System (MK III TiU RP 4.0 x 11.5 mm) were placed to retain complete acrylic crowns. In Group II, premolars were prepared to receive complete metal crowns. Sixteen weeks after placement of the crowns (38 weeks after tooth extraction), the height of the alveolar bone crest was measured with a digital caliper. Data were analyzed statistically by the Mann-Whitney test at 5% significance level. The in situ analysis showed no statistically significant difference (p=0.880) between the implant-supported and the tooth-supported groups (1.528 + 0.459 mm and 1.570 + 0.263 mm, respectively). Based on the findings of the present study, it may be concluded that initial peri-implant bone loss may result from the remodeling process necessary to establish the biological space, similar to which occurs with tooth-supported crowns.

Three-Dimensional Stress Distribution in the Human Periodontal Ligament in Masticatory, Parafunctional, and Trauma Loads: Finite Element Analysis

Background: The presence of the periodontal ligament (PDL) makes it possible to absorb and distribute loads produced during masticatory function and other tooth contacts into the alveolar process via the alveolar bone proper. However, several factors affect the integrity of periodontal structures causing the destruction of the connective matrix and cells, the loss of fibrous attachment, and the resorption of alveolar bone. Methods: The purpose of this study was to evaluate the stress distribution by finite element analysis in a PDL in three-dimensional models of the upper central incisor under three different load conditions: 100 N occlusal loading at 45 degrees (model 1: masticatory load); 500 N at the incisal edge at 45 degrees (model 2: parafunctional habit); and 800 N at the buccal surface at 90 degrees (model 3: trauma case). The models were built from computed tomography scans. Results: The stress distribution was quite different among the models. The most significant values (harmful) of tensile and compressive stresses were observed in models 2 and 3, with similarly distinct patterns of stress distributions along the PDL. Tensile stresses were observed along the internal and external aspects of the PDL, mostly at the cervical and middle thirds. Conclusions: The stress generation in these models may affect the integrity of periodontal structures. A better understanding of the biomechanical behavior of the PDL under physiologic and traumatic loading conditions might enhance the understanding of the biologic reaction of the PDL in health and disease. J Periodontol 2009;80:1859-1867.

Evidence of the presence of T helper type 17 cells in chronic lesions of human periodontal disease

Periodontal disease is a chronic inflammation of the attachment structures of the teeth, triggered by potentially hazardous microorganisms and the consequent immune-inflammatory responses. In humans, the T helper type 17 (Th17) lineage, characterized by interleukin-17 (IL-17) production, develops under transforming growth factor-beta (TGF-beta), IL-1 beta, and IL-6 signaling, while its pool is maintained by IL-23. Although this subset of cells has been implicated in various autoimmune, inflammatory, and bone-destructive conditions, the exact role of T lymphocytes in chronic periodontitis is still controversial. Therefore, in this study we investigated the presence of Th17 cells in human periodontal disease. Gingival and alveolar bone samples from healthy patients and patients with chronic periodontitis were collected and used for the subsequent assays. The messenger RNA expression for the cytokines IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 in gingiva or IL-17 and receptor activator for nuclear factor-kappa B ligand in alveolar bone was evaluated by real-time polymerase chain reaction. The production of IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 proteins was evaluated by immunohistochemistry and the presence of Th17 cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and IL-17 colocalization. Our data demonstrated elevated levels of IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 messenger RNA and protein in diseased tissues as well as the presence of Th17 cells in gingiva from patients with periodontitis. Moreover, IL-17 and the bone resorption factor RANKL were abundantly expressed in the alveolar bone of diseased patients, in contrast to low detection in controls. These results provided strong evidence for the presence of Th17 cells in the sites of chronic inflammation in human periodontal disease.

Assessment of linear measurements of bone for implant sites in the presence of metallic artefacts using cone beam computed tomography and multislice computed tomography

The objective was to evaluate the influence of dental metallic artefacts on implant sites using multislice and cone-beam computed tomography techniques. Ten dried human mandibles were scanned twice by each technique, with and without dental metallic artefacts. Metallic restorations were placed at the top of the alveolar ridge adjacent to the mental foramen region for the second scanning. Linear measurements (thickness and height) for each cross-section were performed by a single examiner using computer software. All mandibles were analysed at both the right and the left mental foramen regions. For the multislice technique, dental metallic artefact produced an increase of 5% in bone thickness and a reduction of 6% in bone height; no significant differences (p > 0.05) were detected when comparing measurements performed with and without metallic artefacts. With respect to the cone-beam technique, dental metallic artefact produced an increase of 6% in bone thickness and a reduction of 0.68% in bone height. No significant differences (p > 0.05) were observed when comparing measurements performed with and without metallic artefacts. The presence of dental metallic artefacts did not alter the linear measurements obtained with both techniques, although its presence made the location of the alveolar bone crest more difficult.

Human osteoblastic cell response to a Ca- and P-enriched titanium surface obtained by anodization

The aim of the present study was to evaluate the in vitro osteogenic potential of subcultured human osteoblastic cells derived from alveolar bone on a titanium (Ti) surface produced by an anodized alkali treatment (BSP-AK). Primary osteoblastic cells were subcultured on BSP-AK and machined Ti discs (control) and grown for periods of up to 21 days under osteogenic conditions. Morphologic and biochemical methods were used to assess important parameters of in vitro bone-like tissue formation. Although no major differences were observed between the BSP-AK and the control Ti surface in terms of cell attachment and mineralized matrix formation, a significant increase in cell population, ALP activity, and collagen content was detected in cultures on BSP-AK surface. Our results demonstrate that human osteoblastic cells are sensitive to the BSP-AK-modified Ti surface during the transitional stage between the end of the proliferative phase and the onset of the differentiation /matrix maturation ones. Together with the good mechanical properties exhibited by the Ca- and P- coating, our findings suggest that BSP-AK treatment could be useful for the development of a new surface for dental and orthopedic implants. (c) 2008 Wiley Periodicals, Inc.J Biomed Mater Res 88A: 841-848, 2009