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Inducible nitric oxide synthase (iNOS) is one of three enzymes generating nitric oxide (NO) from the amino acid L-arginine. iNOS-derived NO plays an important role in several physiological and pathophysiological conditions. NO is a free radical which produces many reactive intermediates that account for its bioactivity. In the human lung, the alveolar macrophage is an important producer of cytokines and this production may be modified by NO. Moreover, high concentrations of NO have been shown to increase nuclear factor KB (NF-kB) activation. Recent investigations of NO expression in tumor tissue indicated that, at least for certain tumors, NO may mediate one or more roles during the growth of human cancer. We have studied iNOS in two tissue groups: normal human lung tissue and human lung cancer tissue. We localized iNOS in these tissues by immunohistochemistry and tested the mRNA expression by RT-PCR, the protein level by Western blot, and the protein activity by radiometric analysis. The results demonstrate different expression, localization and activity of iNOS in normal versus tumor tissue. This is suggestive of a role for NO production from iNOS in human lung cancer because high concentrations of this short molecule may transform to highly reactive compounds such as peroxynitrite (ONOO-); moreover, through the upregulator NF-kB, they can induce a chronic inflammatory state representing an elevated risk for cell transformation to cancer.

Objectives: The aim of this study is to evaluate the Fibroblast Growth Factor 1 (FGF1) protein expression role during human dental pulp development. Methods: Human dental papilla and dental pulp samples obtained from tooth germs and impacted third molars were extracted at Chieti University Oral Science Department in patients undergoing orthodontic treatment. All samples showed no clinical or radiographic caries evidence, periodontal disease or restoration. FGF1 expression was investigated performing Microarray, Western Blot and RT-PCR analysis. Microarray analysis was carried out using high density array containing 21.329 transcripts in replicates and data obtained were statistically analyzed using the S.A.M system (Significance Analysis of Microarray) and I.P.A. (Ingenuity Pathways analysis). Results: RT-PCR and Western Blot analysis data showed FGF1 expression was higher in human dental papilla than in dental pulp samples. Moreover, FGF main isoforms were mapped to genetic networks available in the S.A.M system and IPA database and then ranked by score to show how their expression change during dental pulp development. Conclusions: In our research we observed the expression profile activity changes of FGF1 and FGF gene family in different dental pulp maturity stages. The FGF1 gene behavior at different tooth stage growth seem to play an important role as key regulator of genes family and proteins involved in human dental pulp and tooth tissues development

Objectives: The purpose of this study was to investigate, by morphostructural and immunohistochemical analysis, the process of bone remodeling during the integration of two different extraoral autogenous bone grafts: calvaria and iliac crest. Methods:The donor sites were Calvaria and Iliac Crest, used to fill bone defects in edentulous sites, in posterior maxilla, for future implant therapy. Samples were withdrawn from donor sites at the moment of grafts insertion (T0), while samples from regenerated sites were taken four months (T1) after. Bone specimens were processed to achieve Hematoxilin-Eosin staining to perform light microscope analysis, to investigate the expression of Bone Sialoprotein and the number of apoptotic cells. Results: Both samples at T1 showed strong remodeling phenomena with the presence of new blood vessels, even if in sites regenerated with calvaria grafts a more dynamic aspect could be detected. BSP and BAX were significantly more expressed in samples T1 compared to that observed in samples T0. In any samples from sites treated with calvaria grafts, the investigated molecule BSP showed higher levels of expression than in samples with iliac crest bone grafts. Similarly, the number of apoptotic cells, evaluated by TUNEL analysis, in samples T1 it results higher in calvaria, while in all specimens it seems to be significantly elevated compared to t0. Conclusions: The calvaria graft microscopic aspect at T1 seems to be more organized and regular than that observed in sites regenerated with iliac crest graft at the same experimental time, suggesting a better clinical performance. Those phenomena of bone apposition and osteoblast differentiation are indicated by rising levels of BSP in T1, that it's specifically higher in calvaria bone graft. The results of Tunel analysis, strong indicators of bone remodeling, although giving higher value for the calvaria, showed an equal number of apoptotic cells between calvaria and iliac crest.

Objectives: The aim of the study was to investigate angiogenesis occurence, by analyzing MMPs and VEGF expression, after autologous extraoral bone grafts from two different donor sites: calvaria and iliac crest. Methods: Calvaria and iliac crest bone grafts were used to recreate adequate bone volumes in the posterior maxilla before implant therapy. Specimens from the donor sites were withdrawn from regenerated sites four months after grafting (T1) and compared to bone biopsy obtained at the moment of grafts insertion (T0). Samples at different experimental times were processed in order to investigate MMP2, MMP9 and VEGF expression by immunohistochemical analysis. Results: Immunohistochemical analyses showed MMP2, MMP9, VEGF expression at T0 was higher in samples from iliac crest than in calvaria ones, while in T1 the levels of the investigated molecules were significantly higher in samples from calvaria bone graft than in those regenerated with iliac crest bone graft. Moreover, in both samples all the investigated molecules showed higher levels of expression in T1 than in T0. Conclusions: Autologous extraoral bone grafts are considered suitable biomaterials for pre-prosthetic regenerative techniques, according to their good clinical features and to the elevated success rate associated with their use. In this study, the capability of host tissue to initiate graft revascularization was investigated by analyzing changes in expression of molecules specifically involved in this important phase, like the MMPs and VEGF. Their expression increase after four months from the grafting, that is at the moment of implant insertion, for both investigated graft, but they resulted significantly more expressed in calvaria bone grafts, indicating a more rapid and intense angiogenic and regenerative response of this graft

Objectives: The aim of the our study was to evaluate how sheep amniotic fluid could be a potential source for stem cells able to differentiate into osteogenic pathways. Methods: Amniotic fluid samples (about 5ml) were withdrawn from sheep in pregnancy. Each sample contains a cell number ranging from 2x103 to 2x106. The pellets of amniotic fluid were directly resuspended in osteogenic medium without the previous selection of Amniotic Fluid Mesenchymal Stem Cells. At established times, the expression of typical osteogenic markers was investigated by RT-PCR. Moreover, presence of calcium deposition in cell culture was observed at light microscope after Alizarin Red staining. Results: sAFCs isolated and treated according to this protocol are able to produce nodules of calcium mineralization within 18 days from withdrawal. After 30 days from withdrawal, the cluster of cells showed at RT-PCR analyses a full expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2). Moreover at the same time, the sample was investigated at light microscope to highlight the presence of calcium deposition with Alizarin Red staining. Conclusion: Our research showed osteoblast-like cells differentiation obtained from sheep amniotic fluid. The presence of calcium phosphate granules around osteoblast-like cells suggests the potential role of these stem cells population to grow and repair pathological or genetic deseases. Further investigation may be necessary to better understand the mechanisms outstanding tissues engineering and bone repair treatment.

Objectives: The aim of this work was to investigate the effects of the peptide VEGF-A165 on the proliferation and osteogenic differentiation of human Dental Pulp Stem Cells (hDPSCs). Methods: Mesenchymal stem cells were obtained from dental pulp of enclosed thirds molars of young healthy patients. Mesenchymal markers expression was investigated by FACS analysis. Cell differentiation towards osteogenic or adipogenic lineages was evaluated by means of Alizarin Red S or Red Oil O staining, respectively. The expression of receptors (VEGF-1 and -2) and co-receptors (neuropilin-1 and -2) was estimated by RT-PCR. The effects of VEGF-A165 on cell proliferation was evaluated by MTT assay whereas Western Blot analysis was used to measure VEGF-induced changes in ALP and PECAM-1 protein content. Results: Cells, isolated from dental pulp, originated a homogenous population expressing typical mesenchymal markers (CD29, CD90, CD146, CD166 and STRO-1), able to differentiate into osteoblasts and adypocytes. RT-PCR analysis in undifferentiated or differentiating hDPSCs showed an equivalent expression of known VEGF receptors (VEGFR-1, VEGFR-2) and co-receptors (neuropilin-1 and -2), able to bind VEGF-A165. The VEGF-A165 (10-40 ng/ml) stimulation of undifferentiated hDPSCs for different periods (1-8 days) caused a dose- and time-dependent increase of cell proliferation rate. Moreover, the exposure of differentiating hDPSCs to 40 ng/VEGF-A165 increased ALP protein content without inducing the endothelial marker PECAM-1 expression. Additionally, VEGF-A165 stimulation induced CD90 decrease, not associated with variations of other investigated mesenchimal markers. Conclusions: The exposure of hDPSCs to the peptide VEGF-A165 stimulates their proliferation. The increase of ALP cell content coupled to the CD90 decrement suggests that VEGF activity on cell division does not reduce their osteogenic differentiation potential, thus confirming a property previously shown in cells deriving from bone marrow. Moreover, VEGF does not favor cell differentiation into a endothelial phenotype, likely due to a lack of an artificial in vitro support.

OBJECTIVE: The aim of this study was to determine and compare the (NO) Nitric Oxide expression in oral cancer. METHODS: twelve (12) patients (8 male and 4 female), aged 30-65 years were included in this study after informed consent, were afflicted with oral cancer. Five (5) healthy patients to be treated for impacted third molar extraction were also included in the study as control group. Immunohystochemical, RT-PCR and Western Blot analysis were used to localize Nitric Oxide in the examined samples. RESULTS: Our results showed that inducible nitric oxide synthase (iNOS) activity was higher in oral cancer when compared to healthy mucosa. Endotelial nitric oxide synthase (eNOS) was not expressed in healty oral mucosa. It was also found a gradual increase of neuronal nitric oxide synthase (nNOS) activity together with the progression of the lesions. CONCLUSIONS: These results could ascribe to nitric oxide a determinant role in growth and development of oral cancer, so we hypothesize the use of NO as a marker in the precocious diagnosis of precancerous and cancerous oral lesions.

Objectives: In this study was evaluated by immunohistochemical analysis the activity of molecules involved in the process of bone remodeling during the integration period (T1) and at a distance of 10 years (T2) of extraoral autologous bone grafts from two different donor sites: calvaria and iliac crest. Methods: Calvaria and Iliac Crest bone grafts were used to fill bone defects in edentulous sites. Specimens from the donor sites were withdrawn at the moment of grafts insertion (T0), and from regenerated sites four months (T1) and ten years (T2) after grafting. Samples at different experimental times were processed in order to investigate the levels of expression of Bone Sialoprotein (BSP) and pro-apoptotic molecule BAX by immunohystochemical analysis. Moreover was investigated the number of apoptotic cells by the Tunel analysis. Results: In our study, it was possible to evaluate that BSP and BAX were significantly more expressed in samples T1 compared to that observed in samples T0. The higher level of BSP in samples T1 indicates an intense activity of osteoblast differentiation and mineralization of the extracellular matrix; while the highest levels of BAX indicate an increase in apoptosis associated with remodeling. In T2 samples their expression return to levels comparable to that recorded in specimens from the donor sites. Moreover, in any samples from sites treated with calvaria grafts, all the investigated molecules showed higher levels of expression than in samples from sites regenerated with iliac crest bone grafts. Conclusions: These data confirm that calvaria and iliac crest bone graft, because of its biomechanical characteristics, but also biomolecular intrinsic qualities, is the most suitable to realize bone grafts since it shows, several years later insertion, complete integration with host tissue.

Objectives: In this study we evaluated the role of VEGF-A165 and CD90 peptides on proliferation and differentiation of osteoblast-like cells from human Dental Papilla Stem Cells (hDPaSCs). Methods: hDPaSCs were selected after dental bud extraction in 20 young patients (11-15 years) during orthodontic treatment. A stem cells cluster was analyzed with light microscope and SEM before and after confluence and was selected in homogeneous mesenchymal pool with FACS analysis markers (CD29, CD90, CD146, CD166 and STRO-1). Cell samples have been cultured in osteogenic Dulbecco modified medium and the differentiation was evaluated with Alizarin Red and Red Oil. Before and after differentiation, the presence of receivers and co-receivers VEGF-A165 receptors was evaluated in the hDPSCs with RT-PCR, proliferation test, Western Blot for ALP and PECAM-1 and FACS analysis. Results: Cells isolated from dental papilla showed a homogenous cell expressing mesenchymal markers. RT-PCR on undifferentiated hDPSCs highlights the presence of cellular receptors VEGFR-1, VEGFR-2, VEGFR-3, NRP-1 and NRP-2 for the VEGF-A165. The proliferation test showed an increase to 8 days of 70% of the proliferation speed in the differentiated cells dealt with 40ng/ml of VEGF-A165. Western Blot showed an increase of ALP and the absence of PECAM-1. FACS analysis showed a decrease of CD90, not associated with variations of the other investigated mesenchymal markers. Conclusion: The ALP increase and the absence of PECAM-1 confirmed the differentiation in osteogenic sense and not in hematopoietic sense. The CD90 decrease suggests that the VEGF increase the proliferation mechanism and not reduce the differentiation processes. Moreover, VEGF-A165 does not induce shift towards an endothelial phenotype in hDPaSCs, suggesting in stimulating cellular proliferation during hDPaSCs differentiation into osteoblast-like cells, as confirmed from elevated levels of ALP expression. Then, VEGF determine an increase of cell proliferation and differentiation and the results appear in relationship with its concentration

Objectives: The aim of this study was to evaluate changes in VEGF and MMPs expression at different times after grafting in extraoral autologous bone grafts from two different donor sites: calvaria and iliac crest. Methods: Calvaria and Iliac Crest bone grafts were used to fill bone defects in edentulous sites, in prediction of implant therapy. Specimens from the donor sites were withdrawn at the moment of grafts insertion (T0), and from regenerated sites six months (T1) and ten years (T2) after grafting. Samples at different experimental times were processed in order to investigate VEGF, MMP2 and MMP9 expression by immunohystochemical analysis. Results: Immunohistochemical analyses showed VEGF, MMP2 and MMP9 significantly increase their expression at T1, while at T2 their expression return to levels comparable to that recorded in specimens from the donor sites (T0). Moreover, in any samples from sites treated with calvaria grafts, all the investigated molecules showed higher levels of expression than in samples from sites regenerated with iliac crest bone grafts. Conclusions: Autologous bone grafts are considered the best biomaterials for pre-prosthetic regenerative techniques, according to their good clinical features and to the elevated success rate associated with their use. In this study, the capability of host tissue integration with the graft was investigated from a microscopical and biological point of view. VEGF and MMPs are known to be involved in the process of neoangiogenesis and revascularization of the graft. Their expression increase after six months from the grafting, that is at the moment of implant insertion, while after ten years it returns to basal levels indicating the graft has been successfully integrated with the host tissue

Background: Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a nove! single step protocol for the osteoblastic differentiation of human arrmiotic fluid cells. Results: The described protocol is able to previde osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid sarnples. These cells display a full expression of markers of osteogenic differentiation (COLl, OC, OPN, OCN, OPG, BSP, Runx2) within 30 days from withdrawal. In arder to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, narnely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium). Electron microscopy analysis evidenced the best celi gro'Nth on this latter surface. Conclusions: The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in ora! osteointegrated implantology.

Objectives: The aim of this study was to investigate the different behaviour of autologous bone (AB) and heterologous-deproteinized bone (HDB) as grafting in pre-prosthetic bone regenerative procedures. AB was obtained from calvaria (ABc) and mandibular ramus (ABm), while HDB was swine commercial bone substitute. Methods: AB and HDB were used to fill bone defects in edentulous sites, in patients requiring implant insertion. Specimens from the regenerated sites were withdrawn at the moment of implant insertion, six months after grafting. Samples underwent light microscope observation. Then, immunohistochemical analyses were performed to assess expression of VEGF, procollagen I, bone sialoprotein (BSP), iNOS and Bax. Finally, TUNEL analysis was conducted to measure the main number of apoptotic cells in the different samples. Results: At light microscope observation, the investigated bone substitutes showed a good integration with the host tissue. Immunohistochemical analyses showed VEGF expression was higher in ABc than in ABm and HDB. In ABm samples, BSP was over-expressed while iNOS appeared down-expressed compared to ABc and HDB. Procollagen I and Bax resulted highly expressed in HDB compared to AB samples. TUNEL analysis showed high number of apoptotic cells in HDB samples. Conclusions: Clinically, all the grafts appeared integrated with the host tissue, indicating they can be successfully used in pre-prosthetic regenerative procedures. However, after six months, specimens from HDB grafting exhibit remaining resorption phenomena, demonstrated by the over-expression of Bax and by the presence of high quantity of procollagen I. Presence of diffuse apoptotic cells in HDB samples was confirmed by TUNEL analysis. In contrast, higher ossification, forwarding neoangiogenesis and lesser inflammation could be observed in AB grafting