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 µm (Ti 312), 130 µm (Ti 130) and 62 µm (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

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane.

This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron- and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.

In vitro biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate composite using cultures of human periodontal ligament fibroblasts and keratinocytes.

The aim of this work was to evaluate the biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane to be used in guided tissue regeneration (GTR). Fibroblasts from human periodontal ligament (hPDLF) and keratinocytes (SCC9) were plated on P(VDF-TrFE)/BT and polytetrafluorethylene membranes at a cell density of 20,000 cells well(-1) and cultured for up to 21 days. Cell morphology, adhesion and proliferation were evaluated in hPDLF and keratinocytes, while total protein content and alkaline phosphatase (ALP) activity were assayed only for hPDLF. Using a higher cell density, real-time polymerase chain reaction (PCR) was performed to assess the expression of typical genes of hPDLF, such as periostin, PDLs17, S100A4 and fibromodulin, and key phenotypic markers of keratinocytes, including involucrin, keratins 1, 10 and 14. Expression of the apoptotic genes bax, bcl-2 and survivin was evaluated for both cultures. hPDLF adhered and spread more on P(VDF-TrFE)/BT, whereas keratinocytes showed a round shape on both membranes. hPDLF adhesion was greater on P(VDF-TrFE)/BT at 2 and 4h, while keratinocyte adhesion was similar for both membranes. Whereas proliferation was significantly higher for hPDLF on P(VDF-TrFE)/BT at days 1 and 7, no signs of keratinocyte proliferation could be noticed for both membranes. Total protein content was greater on P(VDF-TrFE)/BT at 7, 14 and 21 days, and higher levels of ALP activity were observed on P(VDF-TrFE)/BT at 21 days. Real-time PCR revealed higher expression of phenotypic markers of hPDLF and keratinocytes as well as greater expression of apoptotic genes in cultures grown on P(VDF-TrFE)/BT. These results indicate that, by favoring hPDLF adhesion, spreading, proliferation and typical mRNA expression, P(VDF-TrFE)/BT membrane should be considered an advantageous alternative for GTR.

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

INTRODUCTION: 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-1beta, 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. METHODS: 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-1beta, IL-6, and IL-23 in gingiva or IL-17 and receptor activator for nuclear factor-kappaB ligand in alveolar bone was evaluated by real-time polymerase chain reaction. The production of IL-17, TGF-beta, IL-1beta, 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. RESULTS: Our data demonstrated elevated levels of IL-17, TGF-beta, IL-1beta, 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. CONCLUSION: These results provided strong evidence for the presence of Th17 cells in the sites of chronic inflammation in human periodontal disease.

Mechanisms involved in the pressor response to noradrenaline injection into the cingulate cortex of unanesthetized rats.

The cingulate cortex (CC) is involved in cardiovascular modulation. CC electrical or chemical stimulation may evoke either pressor or depressor responses, depending on the stimulated site and experimental conditions such as anesthesia. Noradrenaline (NA) is involved in cardiovascular regulation and it is present throughout the cortex. However, there is no report on the cardiovascular effects of intracortical injections of NA. We attempted to verify the effect of NA injection into the CC and to identify possible receptor and peripheral mechanisms involved. NA injection caused pressor responses accompanied by bradycardia, in unanesthetized rats. These responses were markedly reduced under urethane anesthesia. The pressor response was blocked by intracortical pretreatment with phenoxybenzamine or the selective alpha(1)-antagonist WB4101, and it was not affected by pretreatment with the selective alpha(2)-antagonist RX821002, suggesting that alpha(1)-adrenoceptors mediate the response. The pressor response was potentiated by pretreatment with the ganglion blocker mecamylamine and it was abolished by pretreatment with the vasopressin antagonist, dTyr(CH(2)) (5)(Me)AVP or by hypophysectomy. Circulating vasopressin levels were increased after NA injection into the CC. The present results indicate that the pressor response to local injection of NA within the CC is independent of sympathetic nerve activation and is mediated by vasopressin release.

The lateral hypothalamus is involved in the pathway mediating the hypotensive response to cingulate cortex-cholinergic stimulation.

1. The injection of acetylcholine (ACh) into the medial prefrontal cortex (MPFC) caused marked hypotensive response in either unanesthetized or anesthetized rats. 2. The present experiment was designed to investigate anatomical connections of the ACh injection site in the MPFC with putative autonomic-related brain nuclei, as well as their possible involvement in the mediation of the hypotensive response to ACh. 3. For the above purpose, the bidirectional neuronal tracer biotinylated dextran amine (BDA) was injected into Cg1 and Cg3 areas, within the MPFC of male Wistar rats. Five days later the animals were sacrificed and brain slices were processed and analyzed to determine neuronal projections efferent from as well afferent to the MPFC. 4. Neuronal staining was more prominent in regions ipsilateral to the BDA injection site. Prominent efferent projections of the MPFC were observed in the contralateral MPFC: ipsi- and contralateral amygdala and hypothalamus; ipsilateral septal area, diagonal band, and zona incerta. 5. Similar but not equal patterns of neuronal labeling were observed when BDA injections were performed within the two adjacent MPFC areas. BDA injections centered in the ACh injection site in the Cg3 area caused strong labeling in the septal area and diagonal band as well as an overall hypothalamic labeling. Within the hypothalamus an intense cortical projection was observed in the lateral hypothalamus (LH). BDA injections into the Cg1 area caused a more evident labeling of the amygdaloid complex. 6. Neuronal cell bodies were evident throughout the MPFC as well as in the sensory-motor cortex when BDA was injected into the LH, thus indicating a massive ipsilateral cortical projection from the Cg3 to the LH. 7. Bilateral NMDA-induced lesions within the LH caused a significant attenuation of the depressor responses to ACh injection in the MPFC, whereas unilateral lesions were marginally effective. These results indicate the involvement of the LH in the mediation of the hypotensive response to ACh injection into the MPFC as well as the bilateral distribution of the hypotensive pathway.

Medial prefrontal cortex acetylcholine injection-induced hypotension: the role of hindlimb vasodilation.

The injection of acetylcholine (ACh) into the cingulate region of the medial prefrontal cortex (MPFC) causes a marked fall in arterial blood pressure which is not accompanied by changes in heart rate. The purpose of the present study was to investigate the hemodynamic basis for this stimulus-induced hypotension in Sprague-Dawley rats. The study was designed to determine whether a change in the vascular resistance of hindlimb, renal or mesenteric vascular beds contributes to the fall in arterial pressure in response to ACh injection into the cingulate cortex. Miniature pulsed-Doppler flow probes were used to measure changes in regional blood flow and vascular resistance. The results indicated that the hypotensive response was largely due to a consistent and marked vasodilation in the hindlimb vascular bed. On this basis, an additional experiment was then undertaken to determine the mechanisms that contribute to hindlimb vasodilation. The effect of interrupting the autonomic innervation of one leg on the hindlimb vasodilator response was tested. Unilateral transection of the lumbar sympathetic chain attenuated the cingulate ACh-induced vasodilation in the ipsilateral, but not in the contralateral hindlimb. These results suggest that the hypotensive response to cingulate cortex-ACh injection is caused by skeletal muscle vasodilation mediated by a sympathetic chain-related vasodilator system.

Cardiovascular response to the injection of acetylcholine into the anterior cingulate region of the medial prefrontal cortex of unanesthetized rats.

Injection of acetylcholine (ACh) (2.5-60 nmol) into the anterior cingulate cortex caused dose-dependent hypotensive responses (Emax = -25.3 mmHg) and no change in the heart rate. The hypotensive response to 30 nmol of ACh was blocked by local pretreatment with atropine (3 nmol) or 4-DAMP (6.7 nmol), a non-tropine muscarinic antagonist. When the same dose of atropine was injected i.v., no changes were observed in the hypotensive response to intracortical ACh. This observation rules out the possible leakage of ACh into the peripheral circulation and favors the idea of a cortical site of action. The injection of the same dose of ACh into the corpus callosum or the occipital cortex did not cause changes in the cardiovascular system. The present results confirm earlier evidence that the cingulate cortex is involved in the control of the autonomic system and indicate that cholinergic muscarinic receptors in the cingulate cortex mediate a hypotensive response without a change in heart rate.