Use of supercharged cover screw as static magnetic field generator for bone healing, 1st part: in vitro enhancement of osteoblast-like cell differentiation.

Since 1979, Pulsed electromagnetic fields (PEMFs) have been approved by the Food and Drug Administration as an effective method in the treatment of non-unions. As well as PEMFs, also static magnetic fields (SMFs) have been widely investigated in orthopaedic studies. Even if the exact mechanism of action is not well understood, a large number of studies showed specific effects both at cellular and tissue levels. As bone fracture healing and osseointegration share the same biological events, the application of magnetic field stimulation in order to facilitate the osseointegration process has been suggested. In this study we investigated the proliferation rate and gene expression profile of MG63 osteoblastic-like cells after a 24, 48 and 72-hour SMF stimulation, generated by a small, customized cover screw-shaped neodymium-iron-bore magnet placed in the inner cavity of a dental implant. As a result, we found that the application of a SMF to osteoblastic-like cells does slightly decrease cell proliferation rate while enhancing the expression of those genes correlated to differentiation and mineralization. Our findings represent, to our knowledge, the first clinical ready technique for dental implants showing the ability of SMF to promote the osteogenesis process in vitro.

Use of supercharged cover screw as static magnetic field generator for bone healing, 2nd part: in vivo enhancement of bone regeneration in rabbits.

In 1979, Pulsed electromagnetic fields (PEMFs) were approved by the Food and Drug Administration as an effective method in the treatment of non-unions. As well as PEMFs, also static magnetic fields (SMFs) have been widely investigated in orthopaedic studies. Even if the exact mechanism of action is not well understood, a large number of studies showed specific effects both at cellular and tissue levels. As bone fracture healing and osseointegration share the same biological events, the application of magnetic field stimulation in order to facilitate the osseointegration process has been suggested. In this study we investigated BIC and newly formed bone volume around dental implants inserted in the tibia of New Zealand rabbits after SMF stimulation, generated by a small-customized cover-screw-shaped neodymium-iron-bore magnet placed in the inner cavity of dental implants. As a result, we found that the SMF field generated around dental implants enhanced bone healing in the animal model. Our findings represent, to our knowledge, the first ready clinical technique for dental implants showing the ability of SMF to promote the osteogenesis process in vivo.

Stem cell-based approaches in dentistry.

Repair of dental pulp and periodontal lesions remains a major clinical challenge. Classical dental treatments require the use of specialised tissue-adapted materials with still questionable efficacy and durability. Stem cell-based therapeutic approaches could offer an attractive alternative in dentistry since they can promise physiologically improved structural and functional outcomes. These therapies necessitate a sufficient number of specific stem cell populations for implantation. Dental mesenchymal stem cells can be easily isolated and are amenable to in vitro expansion while retaining their stemness. In vivo studies realised in small and large animals have evidenced the potential of dental mesenchymal stem cells to promote pulp and periodontal regeneration, but have also underlined new important challenges. The homogeneity of stem cell populations and their quality control, the delivery method, the quality of the regenerated dental tissues and their integration to the host tissue are some of the key challenges. The use of bioactive scaffolds that can elicit effective tissue repair response, through activation and mobilisation of endogenous stem cell populations, constitutes another emerging therapeutic strategy. Finally, the use of stem cells and induced pluripotent cells for the regeneration of entire teeth represents a novel promising alternative to dental implant treatment after tooth loss. In this mini-review, we present the currently applied techniques in restorative dentistry and the various attempts that are made to bridge gaps in knowledge regarding treatment strategies by translating basic stem cell research into the dental practice.

Osteopontin, osteocalcin and OB-cadherin expression in Synthetic nanohydroxyapatite vs bovine hydroxyapatite cultured Osteoblastic-like cells.

Calcium phosphate ceramics have been applied in bone replacement for several decades due to their excellent biocompatibility, bioactivity, osteo-conductivity and mechanical strength. Several studies have demonstrated that porous hydroxyapatite (HA) is an excellent scaffold for osteogenic proliferation and differentiation of the osteoprogenitor cells. However, different methods of synthesis and production of HA ceramic-based materials may have considerable effect on the physical and biological properties. In the present work, two hydroxyapatite-based materials, a natural hydroxyapatite ceramic of bovine origin and a synthetic nano-cristalline hydroxyapatite were tested in vitro with MG63 cell line. The results displayed that both the materials demonstrated a good biocompatibility. The immunocytochemical stain revealed a different positivity of the osteogenic markers between the cultures with the biomaterials, and the control culture. Western blot data confirmed the immunocytochemical stain. Both the materials tested in the present study demonstrated a good biocompatibility with the osteoblastic cells allowing, at the same time, the osteogenic differentiation, and they may be useful in clinical use.

Antiproliferative effects of 5-fluorouracil and oxaliplatin in colon cancer cell lines: comparison of three different cytotoxicity assays.

Adjuvant therapy in colorectal cancer has evolved to become the standard of care, whereas the tumor capability of activating effective mechanisms of defence against both chemical and physical cytotoxic agents represents a serious obstacle to the successful therapy of human tumors. Therefore, the possibility to have an assay useful to measure the drug sensitivity of tumor cells has a great importance. A number of cytotoxicity assays are currently available, each of them using a specific approach to detect different aspects of cell viability, such as cell integrity, proliferation and metabolic functions. The purpose of this study is to compare, under identical experimental conditions, three common cytotoxicity assays (ATP-lite, MTT and CCK-8 assays) in the assessment of the anti-proliferative effects of 5-fluorouracil (5-FU) and oxaliplatin (OHP) on three colon cancer cell lines (WiDr, SW620 and HT-29). Regarding 5-FU, the three assays were found to be significantly correlated with a moderate or high correlation coefficient, whereas in the case of OHP we found different outcomes among the assays. Our study demonstrates that the CCK-8 is the most sensitive assay for detecting changes of cell viability, suggesting that the viability measured in cells after drug exposure depends on several parameters like the drug used, the biological characteristics of the target cell and the specific approach employed by the method to detect distinct cell growth and metabolic functions.

In vitro evaluation of mesenchymal stem cell isolation possibility from different intra-oral tissues.

Mesenchymal stem cells (MSCs) are of great interest for the regeneration of tissues and organs. Bone marrow is the first sources of MSCs, but in the recent years there has been interest in other tissues for the isolation of these pluripotent cells. In this study, we investigated the features of MSCs isolated from different oral regions in order to evaluate their potential application in the regeneration of damaged maxillofacial tissues. Sampling from human periodontal ligament, dental pulp, maxillary periosteum as well as bone marrow were collected in order to obtain different stem cell populations. Cells were morphologically and immunophenotipically characterized. Their proliferation potential and their ability to differentiate in osteoblasts were also assessed. All tested cell population showed a similar fibroblast-like morphology and superimposable immunophenotype. Slight differences were observed in proliferation and differentiation potential. Cells isolated from human periodontal ligament, dental pulp, maxillary periosteum had the characteristics of stem cells. Considering their peculiar feature they may alternatively represent interesting cell sources in stem cell-based bone/periodontal tissue regeneration approaches.

Giant basal cell carcinoma of the skin: literature review and personal experience.

As the most common form of skin cancer, basal cell carcinoma (BCC) is typified by locally infiltrative growth and a very low risk of metastasis. On occasion, however, this otherwise indolent neoplasm may behave aggressively, demonstrating deep tissue invasion and a high rate of postsurgical recurrence. The pathogenesis and determinants of such tenacious growth are not completely understood. Only 1% of all BCC's achieve the status of 'giant', as defined in 1988 by the American Joint Committee on Cancer. In this article, the authors provide a comprehensive review of the scientific literature on giant basal cell carcinoma (GBCC) of the skin and report their experience with this rare tumour subtype.

Evaluation of in vitro cytotoxicity of oxaliplatin and 5-fluorouracil in human colon cancer cell lines: combination versus sequential exposure.

Adjuvant therapy has evolved to become the standard care of colon cancer, but the tumor capability of activating effective mechanisms of defence against both chemical and physical cytotoxic agents represents a serious obstacle to the successful therapy. Furthermore, the possibility to have an assay useful to measure the drug sensitivity of tumor cells could be of a great importance. As primary human colon cancer cultures from fresh tumor are technically difficult to obtain, experiments with human cancer cell lines remain essential to explore new adjuvant chemotherapy drugs, to investigate the individual responsiveness to the known agents, and particularly to clarify how these chemotherapeutic agents could be used in maximizing outcomes. In the present study we evaluate the cytotoxic effects of 5-fluorouracil (5-FU) and oxaliplatin (OHP) and of their pharmacological interaction in three human colon cancer cell lines (WiDr, HT-29 and SW620), by using an ATP luminescence assay (ATPlite; Perkin Elmer), displaying high sensitivity, linearity and reproducibility. Cell cycle, apoptosis and CD44 expression were investigated with flow cytometry. Our results show that the drug combinations inhibited the cell growth more than each drug alone in all colorectal cancer cell lines. Interestingly, the sequential exposure of OHP and 5-FU resulted in the most cytotoxic effect in all colon cancer cell lines, when compared to the simultaneous one. Our results focus on the powerful cytotoxic effect of 5-FU-OHP combination, when used in sequential exposure, suggesting interesting implications for a rational use of 5-FU, OHP combination in colon-rectal cancer therapy.

Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix.

Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule.Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated.

Immunohistochemical localization of dentin matrix protein 1 in human dentin.

Dentin matrix protein 1 (DMP1) is a non-collagenous matrix protein with a recognized role in the formation of mineralized tissues such as dentin. The aim of this study was to analyze the distribution of DMP1 in human dentin by means of immunofluorescence and high-resolution immunogold labeling. Fully developed, sound human dentin specimens were submitted to fluorescence labeling and post-embedding immunolabeling techniques with a rabbit polyclonal antihuman DMP1 antibody followed by corresponding fluorochrome-conjugated or gold-conjugated secondary antibodies. Both immunofluorescence and immunogold labeling showed an intense labeling associated with the peritubular dentin. In addition, at the ultrastructural level, there was also a moderate and diffuse immunoreaction over intertubular dentin, and a weak labeling within predentin which increased in density towards the mineralization front. This study suggests that in adult human teeth, like in rodents, DMP1 is prevalently concentrated at the level of peritubular dentin and this feature is preserved also in fully developed-teeth. These data are consistent with what has been observed in rodents and suggest that DMP1 plays a role in maintenance of the dentin tubular space.

The performance of human periodontal ligament mesenchymal stem cells on xenogenic biomaterials.

Mesenchymal stem cells from periodontal ligament (PDL-MSCs) hold great promise for bone regeneration. Most studies regarding the osteogenic differentiation of stem cells from periodontal tissue suggest that PDL cells may have many osteoblast-like properties, including the ability to form calcified nodules in vitro. This study investigated the morphological and histochemistry aspects of human PDL-MSCs, induced for osteogenic differentiation and seeded on a xenogenic porcine bone substitute in vitro, at different times of incubation. This biomaterial seems physically identical to human bone, and it has been reported to be osteoconductive. Our results indicated that the cells had a high affinity for the three-dimensional biomaterials; in fact, cellular proliferation and colonization was evident, and after 21 days the adherent cells started to detach themselves from the substrate, and at 30 days of incubation in differentiation medium, the cells completely lost the adhesion to the Petri's disk, englobing all bioparticles. In conclusion, the in vitro behaviour of PDL-MSCs and their relationship with three-dimensional scaffold biomaterials encourage in vivo investigations for their use in dental tissue regeneration.

Enamel hypoplasia in coeliac children: a potential clinical marker of early diagnosis.

AIM: To assess at the scansion electron microscope (SEM) the structural aspects of enamel hypoplasia (EH) in coeliac disease (CD) with the aim to investigate our hypothesis of a possible significant difference between structural characteristics of EH in CD affected patients and EH of non-coeliac patients. If the presence of specific features of the EH associated with CD were demonstrated, these findings would represent for the dentist early non-invasive clinical markers of diagnosis of CD in case of suspected disease. METHODS: We analysed at SEM two samples of enamel fragments from hypoplasic teeth, both deciduous and permanent, harvested from 10 coeliac children (18 permanent teeth, 6 deciduous teeth; study group) and 10 non-coeliac children (16 permanent teeth, 4 deciduous teeth; control group) treated for dental caries, dental extractions for extensive caries lesions or deciduous teeth exfoliation. RESULTS: Significant structural differences were noted between EH of non-coeliac patients and same dental ? lesion in the group with CD. In the study group, EH defects were predominantly located on the central and lateral incisors, upper and lower, both deciduous and permanent, and on the first permanent molars, and were always simmetrical. EH of permanent teeth of CD affected patients was characterised by prisms more irregularly distributed with irregular margins and less interprismatic substance than observed in non-coeliac EH. The deciduous teeth of the study group showed shorter enamel prisms with a non-parallel direction up to convergence and less interprismatic substance than observed in the control group. CONCLUSION: This morphological analysis at SEM of the hypoplasic enamel defects of a group of coeliac children, the first published in literature, demonstrates that the EH of deciduous and permanent teeth in CD is highly hypomineralised with shorter prisms, more irregularly distributed and less interprismatic substance than observed in the non-coeliac EH. More data are needed to validate the significance OF our observations with the aim to assess if this simple, non-invasive microscopic analysis can be considered effective for the early identification of silent cases of CD that otherwise would not be diagnosed in the paediatric age.

Adult mesenchymal stem cells in dental research: a new approach for tissue engineering.

Many adult tissues contain a population of stem cells that have the ability to regenerate after trauma, disease or aging. Recently, there has been great interest in mesenchymal stem cells and their roles in maintaining the physiological structure of tissues. The studies on stem cells are thought to be very important and, in fact, it has been shown that this cell population can be expanded ex vivo to regenerate tissues not only of the mesenchymal lineage, such as intervertebral disc cartilage, bone and tooth-associated tissues, but also other types of tissues. Several studies have focused on the identification of odontogenic progenitors from oral tissues, and it has been shown that the mesenchymal stem cells obtained from periodontal ligament and dental pulp could have similar morphological and phenotypical features of the bone marrow mesenchymal cells. In fact a population of homogeneous human mesenchymal stem cells derived from periodontal ligament and dental pulp, and proliferating in culture with a well-spread morphology, can be recovered and characterized. Since these cells are considered as candidates for regenerative medicine, the knowledge of the cell differentiation mechanisms is imperative for the development of predictable techniques in implant dentistry, oral surgery and maxillo-facial reconstruction. Thus, future research efforts might be focused on the potential use of this cell population in tissue engineering. Further studies will be carried out to elucidate the molecular mechanisms involved in their maintenance and differentiation in vitro and in vivo.

[Primary genital herpes infection in pregnancy]. / Infezione erpetica primaria in gravidanza.

A case report of primary genital herpes infection in a 36-years old pregnant woman during her second pregnancy is presented; the patient was treated according to American College of Obstetricians and Gynecologists guidelines. Maternal primary herpes simplex virus (HSV) genital infection arose at the 16th week of gestation and treated orally with acyclovir 400 mg 3 times a day for a period of 10 days. Nested polymerase chain reaction (PCR), positive on vaginal swab, resulted negative on the maternal serum and on the amniotic fluid (at the 21st week). The patient was monitored for the rest of her pregnancy and she received a suppressive oral acyclovir therapy (400 mg 3 times daily) from the 36th week of gestation until the vaginal delivery, occurred spontaneously at the 38th week with an HSV negative vaginal swab. The live male infant, weighing 3,050 g and with Apgar scores of 7-9 at 1 and 5 min, was delivered and in repeated clinical examinations resulted negative to HSV laboratory investigations with no evidence of herpes infection or adverse effects due to acyclovir therapy.

Evaluation of Humoral Response and Protective Efficacy of an Inactivated Vaccine Against Peste des Petits Ruminants Virus in Goats.

Four goats were inoculated with an inactivated peste des petits ruminants virus (PPRV) vaccine. Three unvaccinated goats were kept as controls. After 36 days, the four goats were revaccinated. The immune response was monitored by virus neutralization test showing that two doses of the vaccine were able to stimulate strong immune response in all the vaccinated animals. The vaccinated goat and the controls were challenged with virulent PPRV intranasally. After PPRV challenge, the three control goats showed fever, viremia and virus excretion through mucosal surfaces, whereas the vaccinated goats were fully protected against PPRV infection and replication.

[Stapled hemorrhoidectomy: critical observation on state of art]. / Emorroidopessi con stapler: note critiche sullo stato dell'arte.

The authors review pros and cons of stapled hemorrhoidectomy (SH). Postoperative primary lower than after hemorrhoidectomy, but no data are available on the long term recurrences in large prospective series. Severe postoperative complications have been reported and SH seems less effective in patients with 4th degree files. SH is a useful technique, provided that proper indication are strictly followed and the procedure is carried out by specialists.

Benthic foraminifera for environmental monitoring: a case study in the central Adriatic continental shelf.

A study of benthic foraminifera was carried out in sediment samples collected from the central Adriatic coast of Italy, near the Ancona harbour and the Falconara Marittima oil refinery, in order to validate and support their use as bioindicators of ecosystem quality. On the basis of a principal component analysis (PCA), three biotopes (following the bathymetric gradient) have been documented, showing that the distribution pattern of benthic foraminifera is principally related to riverine inputs, organic matter contents at the seafloor, and sediment grain size. We observed higher abundances of opportunistic, low-oxygen tolerant taxa along the coastline, thus being representative of polluted environmental conditions. Near the Falconara Marittima oil refinery, the microfaunal assemblages is characterized by the absence of living specimens and by a low diversity associated with the dominance of opportunistic species. At this site, aberrant tests were also found. The data point out that Ammonia parkinsoniana and Quinqueloculina seem to be the most sensitive taxa and can be considered as good bioindicators of environmental stress in this area. This study confirms that faunal composition and morphology of benthic foraminifera respond to human-induced environmental perturbations, making their study potentially useful for biomonitoring in coastal-marine areas.

Relationship of the phenotypic expression of the A-IMilano apoprotein with plasma lipid and lipoprotein patterns.

The plasma lipoprotein and apolipoprotein profile of 29 adult A-IMilano (A-IM) carriers and 29 age- and sex-matched non-affected subjects of the same kindred was examined, in order to investigate linkages between the lipid and apoprotein abnormalities and the phenotypic expression of the biochemical disorder. Carriers (A-IM+) showed a higher prevalence of hypertriglyceridemia (12 out of 29); they also had lower plasma total cholesterol, esterified cholesterol and phospholipids, compared to non-carriers. Lipoproteins were characterized by a significant enrichment of triglycerides in low and high density fractions (LDL and HDL), and by the expected striking reduction of HDL mass and cholesterolemia. Conversely, no significant alterations of the major circulating apolipoprotein levels, except for apo A-I and apo A-II, were noted in the A-IM+. The increased free cholesterol/esterified cholesterol ratio in plasma (most marked in HDL), was accompanied by a significant reduction of the lecithin cholesterol acyl transferase molar activity. Several correlations pertaining to lipids, lipoproteins and apoproteins were examined: cholesterol and triglycerides in HDL and, more remarkably, apoprotein A-I and C-III levels in plasma were significantly correlated in the A-IM+. While there was no significant prevalence of specific apo E phenotypes, plasma triglycerides and apo C-II levels were highly correlated in the carriers. The A-IM subjects, while in the presence of severe lipoprotein risk factors, may have alternative mechanisms of cholesterol disposal, potentially responsible for the apparently low prevalence of atherosclerosis.

Synthesis of 3-hydroxy-3-cyclohexylbutyric acid derivatives. 1. Cyclic homologues of 3-hydroxy-3-methylglutaric acid.

Z and E isomers of 3-methyl-3-(carboxymethyl)hexahydro-1 (3H)-isobenzofuranones (I), lactones of 3-hydroxy-3-(2-carboxycyclohexyl) butyric acids (II), were prepared and tested on cholesterol biosynthesis in vitro. Compound I of the Z series was prepared through its ethyl ester by hydrogenation, over Rh/Al2O3 catalyst, of the phenyl ring of 3-methyl-3-[(ethoxycarbonyl)methyl]-1(3H)-isobenzofuranone. Compound I of the E series was prepared, through its ethyl ester, by Reformatsky reaction from ethyl (E)-2-acetylcyclohexanecarboxylate. 3-Methyl-3-(carboxymethyl)-5,6,7,8-tetrahydro-1(3H)-isobenzofuranone, 3-methyl-3-ethyl-5,6,7,8-tetrahydro-1(3H)-isobenzofuranone, and 3-methyl-3-(carboxymethyl)-1(3H)-isobenzofuranone were also prepared and tested. The above compounds inhibited acetate incorporation in cholesterol and fatty acids in rat liver slices at 5 X 10(-3) M but lack specific inhibitory activity on HMG-CoA reductase.