Filiberto Mastrangelo

Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol

BackgroundStem 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 novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells.ResultsThe described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2) within 30 days from withdrawal. In order 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, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium). Electron microscopy analysis evidenced the best cell growth on this latter surface.ConclusionThe 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 oral osteointegrated implantology.

Microarray Evaluation of Age-related Changes in Human Dental Pulp

INTRODUCTION The dental pulp undergoes age-related changes that could be ascribed to physiological, defensive, or pathological irritant-induced changes. These changes are regulated by pulp cell activity and by a variety of extracellular matrix (ECM) macromolecules, playing important roles in growth regulation, tissue differentiation and organization, formation of calcified tissue, and defense mechanisms and reactions to inflammatory stimuli. The aim of this research was to better understand the genetic changes that underlie the histological modification of the dental pulp in aging. METHODS The gene expression profile of the human dental pulp in young and older subjects was compared by RNA microarray analysis that allowed to simultaneously analyze the expression levels of thousands of genes. Data were statistically analyzed by Significance Analysis of Microarrays (SAM) Ingenuity Pathway Analysis (IPA) software. Semiquantitative and real-time reverse-transcriptase polymerase chain reaction analyses were performed to confirm the results. RESULTS Microarray analysis revealed several differentially expressed genes that were categorized in growth factors, transcription regulators, apoptosis regulators, and genes of the ECM. The comparison analysis showed a high expression level of the biological functions of cell and tissue differentiation, development, and proliferation and of the immune, lymphatic, and hematologic system in young dental pulp, whereas the pathway of apoptosis was highly expressed in older dental pulp. CONCLUSIONS Expression profile analyses of human dental pulp represent a sensible and useful tool for the study of mechanisms involved in differentiation, growth and aging of human dental pulp in physiological and pathological conditions.

Bevacizumab-related osteneocrosis of the jaw.

We describe a case of jaw bone necrosis after a lung adenocarcinoma bone metastasis, treated the first time in 2004 by means of pneumonectomy and lymph node ablation. One week after a dental extraction, the patient experienced pain in the mandibular region, in conjunction with alveolar bone exposure. Treatment with amoxicillin and clavulanate every 12 hours for 15 days and 0.2% chlorhexidine rinses was administered and there was a remission of infective complications, but not the closure of the exposed alveolar bone. Only at this time did the patient refer that he was treated with bevacizumab therapy immediately after the extraction. A preventive dental assessment of patients scheduled for bevacizumab therapy should be useful as for the zoledronic acid therapy. Dental surgery procedures for patients during bevacizumab therapy should be carefully evaluated and considered as the last choice, to reduce all possible risks and prevent complications.

A macro- and nanostructure evaluation of a novel dental implant.

Success in implant dentistry also comes from the implant macrodesign and nanostructure of its surface. Titanium implant surface treatments have been shown to enhance osseointegration, maximize bone healing, and bone-to-implant contact for predictable clinical results. The aim of the study, was to evaluate the geometric macrodesign and the surface nanostructure of a novel dental implant full contact covering (FCC) obtained by electrochemical procedures. FCC implants were analyzed by scanning electronic microscope, profilometer, and x-ray photoelectron spectroscopy and compared with commercial sandblasted and sandblasted, large-grit acid-etched dental implants. Sample analysis allowed to distinguish the different implant macrodesigns, the step and the profile of the coils that cover the fixture, and the surface characteristics. FCC implant showed novel macro-characteristic of crestal module, coils, and apical zone compared with sandblasted and sandblasted and acid-etched dental implants. Moreover, the FCC nanostructure surface showed roughness values statistically higher than the 2 other surfaces, with a more homogeneity in a peaks and valleys arrangement. Finally, the x-ray photoelectron spectroscopy analysis detected differences between the examined surfaces, with the presence of several contaminants according to the different treatment procedures. Research on new macrostructures and nano morphology should result in a better qualitative and quantitative osseointegration response, with a predictability of the clinical results and long-term success of the implants.

Vascular endothelial growth factor and e-nitric oxide synthase-mediated regenerative response occurring upon autologous and heterologous bone grafts.

Bone regeneration procedures allow oral rehabilitation with dental implants also in edentulous ridges with severe bone atrophy. The integration of grafted materials with the host tissue can initiate regenerative, inflammatory and apoptotic response. Since molecular mechanisms exist at the basis of such response, the aim of this work is to investigate, by immunohistochemical analyses, the expression of proteins involved in the graft integration process, in parallel to clinical and histological modifications, occurring on sites treated with extraoral autologous bone graft deriving from the parietal region of the calvaria (eAB), intraoral autologous bone graft deriving from mandibular ramus (iAB) and heterologous bone graft from swine (hB) in human patients. In our study, the immunohistochemical expression of BSP, VEGF, eNOS in eAB samples was significantly higher (p< 0.05) compared to values recorded in iAB and hB samples. The inflammatory response, investigated by iNOS expression, was found lower in all autologous samples (eAB and iAB) compared to hB, at statistically significant values. Moreover, the expression of the pro-apoptotic molecule, Bax, resulted significantly lower (p< 0.05) in eAB than in iAB and hB samples. These values, together with the low number of apoptotic cells detected in autologous samples, suggest a good regenerative response when extraoral autologous bone graft is used in comparison to the response from the other grafts, and also suggest the use of calvaria graft as a predictable therapeutic procedure for repairing severe bone defects in oral and maxillofacial surgery, not only by clinical and biomechanical criteria, but also from a biomolecular aspect.

The role of anti-cyclic citrullinated peptide antibody in periodontal disease

The anti-Cyclic Citrullinated Peptide Antibodies (anti-CCP) are produced locally in the inflamed synovium of Rheumatoid arthritis (RA) patients, suggesting that citrullinated proteins are located in the inflamed synovium. In scientific literature were find periodontal bacterial DNA in serum and synovial fluid of RA with PD patients. RA and adult periodontitis share common pathogenetic mechanisms and immunologic and pathological findings RA. One oral pathogen strongly implicated in the pathogenesis of periodontal disease (PD), Porphyromonas. gingivalis, possesses a unique microbial enzyme, peptidylarginine deiminase (PAD), the human equivalent of which has been identified as a susceptibility factor for RA. Under this point of view, we speculate about the presence of anti-CCP antibodies in sera of PD with RA patients. We conducted this study to evaluate and compare the diagnostic and predictive utility of anti-CCP antibodies in patients with PD and patients with PD and RA. Anti-CCP antibody was not found in 21 sera (U/ml<10), included RA controls, while only 1 patient with chronic PD and probing depth of 7,1 mm was identified positive for anti-CCP (22.2 U/ml). Our data do not support a role for anti-CCP in diagnoses of periodontal disease.

Influence of novel nano-titanium implant surface on human osteoblast behavior and growth.

Purpose:The aim of the study is to investigate human osteoblast-like cell behavior and growth in the presence of 3 different titanium implant surfaces. Materials:Human stem cells were first obtained and then sorted by fluorescence-activated cell sorter from mesenchymal stem cell clusters of human dental papilla. The obtained human dental papilla stem cells were induced to differentiate into osteoblast-like cells and were then analyzed by reverse transcriptase polymerase chain reaction and Western blot analyses. The cells proliferated and were cultured onto 3 different titanium discs (sandblasted, sandblasted and large-grit acid-etched, and full contact coverage [FCC]) and analyzed by scanning electron microscope. Results:In all analyses samples, a high cell activity was observed, with typical osteoblast mature morphostructural response on rough surface. The high number of osteoblast-like cells was found on titanium FCC discs. At the same time, scanning electron microscope analysis confirmed the high biocompatibility of this surface. Conclusion:The rapid maturation of the osteoblast-like cells on FCC titanium surface suggests that this structure could play a central role during initial phases of bone healing processes.

Vascular endothelial growth factor (VEGF) in human tooth germ center.

Many oncogenis and tumour suppressor genes found inside normal and pathological cells are fundamental for the processes of development, proliferation and tissue differentiation. The purpose of our study is to show the presence and a possible relationship of the VEGF protein during different phases of the development of human dental germ centers. After cephalometric investigation in 8 orthodontic patients with a mean age of 13 years, (4 females and 4 males), hyperdivergence of the third molars were extracted. The 40 surgical samples were tested with monoclonal human anti-VEGFs antibodies carrying out a semi-quantitative analysis to look for a positive reaction. Reaction for anti-VEGF antibodies was detected in normal embryological tissues and in microvessels near odontogenic cells. During different phases of embryologic development of the dental bud our search showed intracytoplasmatic positive immunoreactions both in the ameloblastic and odontoblastic cells. Additionally, a positive reaction was observed for the VEGF protein in the cells of the stellate reticulum and in those endothelial tissue surrounding the microvessels in all the samples examined.

Localization and activity of iNOS in normal human lung tissue and lung cancer tissue

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 chiB (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.

Morphostructural Analysis of Human Follicular Stem Cells on Highly Porous Bone Hydroxyapatite Scaffold

In this study we investigated the in vitro behaviour, morphostructure and extracellular matrix synthesis of human dental follicular stem cells (hDFSCs) isolated from human dental bud, which resulted to be positive for mesenchymal markers (CD29, CD90, CD146 and CD166) by FACS analysis. Cells were analysed by light and electronic microscopy to evaluate their biological response either at week 1, that is before differentiation, or at weeks 3–6, when they had been cultured in osteogenic medium onto a highly porous natural scaffold material (Bio-Oss®). Microscopy analysis of primary culture cells showed they had a mesenchymal stem cell-like morphostructure, spindle shaped, similar to the culture of mesenchymal stem cells derived from adult bone marrow. Also, after osteogenic differentiation, these analyses indicate typical osteoblast morphostructure and reveale a tri-dimensional organization of the cells and deposition of extracellular matrix (ECM) in close contact with biomaterial. This approach would allow to personalize the scaffold for bone tissue engineering in order to accelerate the process of osteogenesis.