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Suppression of oxidative phosphorylation combined with enhanced aerobic glycolysis and the resulting increased generation of protons are common features of several types of cancer. An efficient mechanism to escape cell death resulting from intracellular acidification is proton pump activation. In Ewing sarcoma (ES), although the tumor-associated chimeric gene EWS-FLI1 is known to induce the accumulation of hypoxia-induced transcription factor HIF-1α, derangements in metabolic pathways have been neglected so far as candidate pathogenetic mechanisms. In this paper, we observed that ES cells simultaneously activate mitochondrial respiration and high levels of glycolysis. Moreover, although the most effective detoxification mechanism of proton intracellular storage is lysosomal compartmentalization, ES cells show a poorly represented lysosomal compartment, but a high sensitivity to the anti-lysosomal agent bafilomycin A1, targeting the V-ATPase proton pump. We therefore investigated the role of V-ATPase in the acidification activity of ES cells. ES cells with the highest GAPDH and V-ATPase expression also showed the highest acidification rate. Moreover, the localization of V-ATPase was both on the vacuolar and the plasma membrane of all ES cell lines. The acidic extracellular pH that we reproduced in vitro promoted high invasion ability and clonogenic efficiency. Finally, targeting V-ATPase with siRNA and omeprazole treatments, we obtained a significant selective reduction of tumor cell number. In summary, glycolytic activity and activation of V-ATPase are crucial mechanisms of survival of ES cells and can be considered as promising selective targets for the treatment of this tumor.

Ethnopharmacological relevance Four Indian plants, traditionally used in Ayurvedic medicine: Asparagus racemosus Willd., Emblica officinalis Gaertn., Hemidesmus indicus R. Br., and Rubia cordifolia L. were selected on the basis of their ethnobotanical use and of scientific evidence that suggests a potential efficacy in the treatment of bone-loss diseases. The antiresorptive properties of the four plants have been investigated. The aim was to provide adequate evidence for the exploitation of natural compounds as alternative therapeutics for the treatment of diseases caused by increased osteoclast activity. Materials and methods Decoctions were prepared from dried plant material according to the traditional procedure and standardization by HPLC was performed using marker compounds for each species. Total polyphenols, flavonoids and radical scavenging activity of the decoctions were also determined. The bioactivity of the plant decoctions was evaluated in subsequent phases. (1) A cytotoxicity screening was performed on the mouse monocytic RAW 264.7 cell line to define the concentrations that could be utilized in the following step. (2) The antiresorptive properties of plant decoctions were compared with that of a “gold standard” drug (alendronate) by measuring osteoclastogenesis inhibition and osteoclast apoptosis. (3) The toxic effect on bone forming cells was excluded by evaluating the impact on the proliferation of osteogenic precursors (mesenchymal stem cells, MSC). Results All the decoctions inhibited osteoclastogenesis similarly to alendronate at the highest doses, but Hemidesmus indicus and Rubia cordifolia were also effective at lower concentrations. Apoptosis increased significantly when cells were exposed to the highest concentration of Emblica officinalis, Hemidesmus indicus, and Rubia cordifolia. All concentrations of Emblica officinalis tested inhibited the proliferation of osteogenic precursors, while only the highest doses of Asparagus racemosus and Rubia cordifolia were toxic. On the contrary, Hemidesmus indicus did not affect osteogenic precursor growth at any concentration tested. Conclusion Among the medicinal plants included in the study, Hemidesmus indicus showed the greatest antiosteoclastic activity without toxic effect on osteogenic precursors. Therefore, Hemidesmus indicus exhibits the properties of an antiresorptive drug and represents the ideal candidate for further clinical investigations.

Osteogenesis is the result of a complex sequence of events that involve the differentiation of mesenchymal stem cells (MSC) into osteoblasts. MSCs are multipotent adult stem cells that can give rise to different cell types of the mesenchymal germ layer. The differentiation fate of MSCs depends on the microenvironmental signals received by these cells and is tightly regulated by multiple pathways that lead to the activation of specific transcription factors. Among the transcription factors involved in osteogenic differentiation Osterix (Sp7) plays a key role and has been shown to be fundamental for bone homeostasis. However, the molecular events governing the expression of this transcription factor are not fully understood. In this study we set out to investigate the changes in the microRNA (miRNA) expression that occur during the osteogenic differentiation of bone marrow-derived MSCs. To this purpose, we analyzed the miRNA expression profile of MSCs deriving from 3 donors during the differentiation and mineralization processes by microarray. 29 miRNAs were significantly and consistently modulated during the osteogenic differentiation and 5 during the mineralization process. Interestingly, most of the differentially expressed miRNAs have been reported to be implicated in stemness maintenance, differentiation and/or oncogenesis. Subsequently, we focused our attention on the regulation of Osterix by miRNAs and demonstrated that one of the miRNAs differentially modulated during osteogenic differentiation, miR-31, controls Osterix expression through association to the 3' untranslated region of this transcription factor. By analyzing miR-31 and Osterix expression levels we found an inverse miRNA-target expression trend during osteogenic differentiation and in osteosarcoma cell lines. Moreover, the inhibition of the microRNA activity led to an increase in the endogenous expression of Osterix. Our results define a miRNA signature characterizing the osteogenic differentiation of MSCs and provide evidence for the involvement of miR-31 in the regulation of the bone-specific transcription factor Osterix.

BACKGROUND AND AIMS: Congenital pseudarthrosis of the tibia (CPT) is a rare orthopedic disease presenting spontaneous fractures that do not heal. The treatment of CPT is characterized by repeated surgical procedures that often fail, with the inevitable outcome of severe disability and amputation. We tested the hypothesis that CPT may benefit from regenerative strategies based on mesenchymal stromal cells (MSC) combined with platelet-rich fibrin (PRF) as a source of growth factors. The aim of the study was to verify whether laboratory testing to assess the osteogenic properties of MSC and the osteo-inductive activity of PRF correlated with the clinical outcome.METHODS: Ten patients affected by refractory CPT were treated by using MSC derived from the iliac crest (IC-MSC), PRF and lyophilized bone. In six patients, CPT was associated with type 1 neurofibromatosis (NF1). Biochemical, functional and molecular assays were performed to assess the intrinsic osteogenic potential of IC-MSC (cells cultured with fetal calf serum) and the osteo-inductive properties of PRF (cells cultured with autologous serum).RESULTS: Bone consolidation was obtained in three patients who had CPT and NF1. In these patients, the IC-MSC exposed to autologous serum were able to form mineral nodules in vitro, while the mineralizing ability was totally abrogated in patients with a poor clinical outcome.CONCLUSIONS: Cell therapy may be a useful tool for the treatment of refractory CPT because it increases the opportunity to achieve effective bone tissue regeneration. Our data suggest that the presence of pro-osteogenic growth factors is an essential requirement for bone healing.

The insulin-like growth factors 2 (IGF2) is a peptide hormone that binds to the insulin-like growth factor 1 receptor (IGF1R) and is abundantly stored in bone. IGF1R is deeply involved in the pathogenesis of many cancers that growth within bone and is also involved in osteoclast biology. Among different cell lines representative of osteolytic tumors, we found a very high expression of IGF2 in SH-SY5Y cells derived from neuroblastoma (NB). We previously showed that NB cells induce an osteolytic process through the Osteoprotegerin/RANKL/RANK and the canonical Wnt pathway system. Here, we hypothesized that NB promotes osteoclastogenesis also via IGF2. First, we demonstrated the presence of IGF1R on the osteoclast basolateral membrane, and we observed a cyclic IGF1R activation along with the differentiation process, also when induced by SH-SY5Y. Moreover, we found that IGF2 mRNA expression in SH-SY5Y cells was further increased when co-cultured with mesenchymal stromal cells, suggesting that IGF2 is important for NB interaction with the bone microenvironment. Finally, the treatment of SH-SY5Y cells with an anti-IGF2 siRNA or the addition of anti-IGF1R molecules impaired NB-induced osteoclastogenesis, even though the chemoattraction of monocytes by NB cells was unaffected. Our findings suggest that in IGF2-producing osteolytic tumors IGF1R is a good candidate for targeted therapies in combination with conventional drugs.

The study was designed to establish the biological basis for the use of autologous bone-marrow stromal cells (MSC) in order to improve the curing opportunities of congenital pseudarthrosis of the tibia (CPT). The investigation was planned by taking into account that the pathophysiology of bone healing mainly depends on the osteogenic potential of the resident cells, although several factors play a crucial role in restoring the normal bone structure. Bone marrow samples were collected from the lesion site (P) and the iliac crest (IC) of 7 patients affected by CPT and type 1 neurofibromatosis (NF1+) and 6 patients affected by CPT without NF1 (NF1-). Four patients without CPT served as control group. Biochemical, functional and molecular assays showed that the ability to generate bone-forming cells was higher in IC-MSC than in P-MSC, but lower in CPT patients than in control group. We evaluated whether host factors, such as autologous serum and the microenvironment surrounding the pseudarthrosis lesion, could impair the osteogenic differentiation of IC-MSC. Autologous serum was less effective than FBS in promoting the IC-MSC differentiation, but the damage was more evident in NF1- than in NF1+ patients. Additionally, the supernatant of osteoblast cultures obtained from bone fragments close to the lesion site favoured the differentiation of IC-MSC in NF1- patients. In summary, our results suggest that MSC transplantation could be a promising strategy for the therapy of CPT. Further studies are warranted to confirm the clinical effectiveness in comparison to standard surgical treatment.

Materials used for total knee arthroplasty (TKA), may elicit an immune response whose role in the outcome of the arthroplasty is still unclear. The aim of this study was to evaluate the frequency of sensitization in patients who had undergone TKA, and the clinical impact of this event on the outcome of the implant. Ninety-four subjects were recruited, including 20 patients who had not yet undergone arthroplasty, 27 individuals who had a well-functioning TKA, and 47 patients with loosening of TKA components. Sensitization was detected by using patch testing including haptens representative of cobalt-based alloys (CoCrMo), titanium-based alloys (TiAlV), and bone cements. The frequency of positive skin reactions to metals increased significantly after TKA, either stable or loosened (No Implant 20%; Stable TKA 48.1%, p = 0.05; Loosened TKA 59.6%, p = 0.001, respectively). We found a higher frequency of positive patch testing to vanadium in patients who had a Stable TKA with at least one TiAlV component (39.1%, p = 0.01). The medical history for metal allergy seems to be a risk factor, because the TKA failure was fourfold more likely in patients who had symptoms of metal hypersensitivity before TKA. The prognostic value was supported by survival analysis, because in these individuals the outcome of the implant was negatively influenced (the logrank test Chi square 5.1, p = 0.02). This study confirms that in patients with a TKA the frequency of positive patch testing is higher than in the normal population, although no predictive value is attributable to the sensitization because patch testing was not able to discriminate between stable and loose implants. On the contrary, the presence of symptoms of metal allergy before implantation should be taken into account as a potential risk factor for TKA failure.

Current methods for the replacement of skeletal tissue involve the use of autografts, allografts and, recently, synthetic substitutes, which provide a proper amount of material to repair large bone defects. Engineered bone seems a promising approach, but a number of variables have to be set prior to any clinical application. In this study, four different poly caprolactone-based polymers (PCL) were prepared and tested in vitro using osteoblast-like Saos-2 cells. Differences among three-dimensional polymers include porosity, addition of hydroxyapatite (HA) particles, and treatment with simulated body fluid. Biochemical parameters to assess cell/material interactions include viability, growth, alkaline phosphatase release, and mineralization of osteoblastic cells seeded onto three-dimensional samples, while their morphology was observed using light microscopy and SEM. Preliminary results show that the polymers, though degrading in the medium, have a positive interaction with cells, as they support cell growth and functions. In the short-term culture (3-7 days) of Saos-2 on polymers, little differences were found among PCL samples, with the presence of HA moderately improving the number of cells onto the surfaces. In the long term (3-4 weeks), it was found that the HA-added polymers obtained the best colonization by cells, and more mineral formation was observed after coating with SBF. It can be concluded that PCL is a promising material for three-dimensional scaffold for bone formation, and the presence of bone-like components improves osteoblast activity.

Nineteen patients who had undergone hip revision surgery for aseptic loosening of joint prostheses were studied. Tissue samples were harvested at the interface between bone and implant, either at the stem or at the cotyle level. Immunohistochemistry was performed on tissue sections to detect nitric oxide synthase (NOS), the enzyme which enables the synthesis of nitric oxide (NO), a molecule which can activate bone resorption. Quantitative analysis of the positive cells and correlation with the presence of particulate wear debris and radiological data were performed. The authors observed a trend towards a moderate increase in positive cells due to inducible NOS in tissues containing particulate wear debris, especially of a plastic material. This increase, however, did not achieve statistical significance. On the contrary, there was a statistical correlation between iNOS (inducible NOS) and the severity of osteolysis around the prosthetic implant. Pharmacological control of the biosynthesis of NO may be considered in the prevention or treatment of loosening.

The expression of thrombomodulin after contact with CMW 1 bone cement extracts was studied in human umbilical vein endothelial cells. Cement extracts after 1 h and 7-day curing induced no significant variations in thrombomodulin antigen levels and in mRNA expression. Significant increase of thrombomodulin was observed when endothelial cells were treated with all-trans retinoic acid (ATRA). ATRA induced the increase of thrombomodulin also in cells incubated with cement extracts. These results suggest that CMW 1 bone cement does not impair the expression of thrombomodulin in endothelial cells.

The aim of the study was to evaluate some aspects of the immunocompatibility of 10 acrylic bone cements. Mononuclear cells harvested from healthy individuals were cultured with cement extracts which were tested to assess their effect on the viability of lymphocytes, unstimulated and phytohaemoagglutinin (PHA)-stimulated, activating resting lymphocytes, and changing the reactivity of PHA-stimulated lymphocytes. After 24 h the extracts did not increase the percentage of dead cells in unstimulated or PHA-stimulated lymphocytes. The early apoptotic events of culture were evaluated after 4 and 24 h in PHA-stimulated lymphocytes: at 4 h three cements, namely Zimmer-dough type, Palacos s R and CMW-1, increased significantly the percentage of apoptotic cells, while at 24 h no differences were found. Cement extracts did not activate the resting lymphocytes, whereas the response of the PHA-stimulated cells was significantly modified. All cements decreased the expression of the interleukin 2 receptor (CD25) and the lymphocyte proliferation, whereas only two materials (Zimmer-dough type, CMW 1) affected the expression of early activation antigen (CD69). These findings show that the products released from bone cement are not able, by themselves, to elicit a specific immune response; on the contrary they hamper the function of lymphocytes activated by an exogenous stimulus. r 2001 Elsevier Science Ltd. All rights reserved.

The purpose of this study was to investigate in vitro the apoptosis- and/or necrosis-inducing potential of polymethylmethacrylate (PMMA)-based bone cements for prosthetic surgery. Four bone cements widely used in orthopedics were tested as extracts onto osteoblast-like MG-63 cells and for comparison, HL-60 cells, which are remarkably sensitive to apoptotic stimuli. Neutral red uptake (NRU) was used to measure cell viability while Hoechst 33258 staining was used to detect DNA content. Apoptosis was characterized using a BrdU-based ELISA assay for DNA fragmentation and examined by fluorescence microscopy using acridine orange and propidium iodide staining of nuclei. The generation of reactive oxygen species (ROS), which could mediate apoptosis, was verified using dichlorofluorescein-diacetate (DCFH-DA) oxidation to DCF. After 24 h of challenge of the cells with the four cement extracts, the viability of either MG-63 or HL-60 cells was found to be unaltered, as recorded by NRU. Apoptotic cell death was induced by three cements in HL-60, whereas MG-63 cells were significantly affected by the four cements tested: the finding of DNA fragments both in the cytoplasm and supernatants of MG-63 after 24 h demonstrated that these cells underwent late-apoptosis secondary necrosis. Fluorescent staining of the nuclei confirmed the results obtained with the ELISA test. Oxygen free radicals were elicited by two cements in HL-60 cells, while MG-63 did not generate ROS in response to cements. This study helps to gain more insight into the mechanism of cell death induced by PMMA-based cements and suggests apoptosis of osteoblasts as a part of the tissue reaction around cemented prostheses.

Trace elements such as zinc (Zn) and selenium (Se) play an important role in maintaining the metabolic homeostasis in elderly people and the risk of deficiency seems to increase in proportion to the age. Zn and Se concentrations, as indices of the micronutrient status in healthy subjects over 90 years, are scarcely analyzed and could represent a model for studying the physiology of successful aging. Our aim was to investigate Zn and Se concentrations in the healthy persons over the age of 90 years. One hundred and fifty two subjects volunteered for the study. They were divided into two groups: 90 non-institutionalized nonagenarians/centenarians (91-110 years) (group A) and 62 elderly subjects (60-90 years) used for comparison (group B). Serum concentrations of Zn and Se were determined, respectively, by flame atomic absorption spectrophotometry (FAAS) and electrothermal atomic absorption spectrophotometry (ETAAS). The effect of age and sex on ion concentrations was investigated. Mean values+/-standard deviation of Zn and Se concentrations in the group A were 11.97+/-2.00 and 0.87+/-0.28 micromol/l, respectively. A significant decrease of Se and Zn values was demonstrated in group A, when compared with group B, in both males and females. However, 84.4% of the 'healthy' nonagenarians/centerians had both Zn and Se concentrations equal to or greater than the lowest values of the elderly group and only 3.3% of cases showed both Zn and Se deficiencies. Consequently, a prospective and follow-up evaluation of Zn and Se could be proposed as a good index for a correct monitoring of the micronutrient deficiencies, that could represent an early sign of disease.

The aim of the study was to evaluate the sensitization to metals in patients with Co-Cr hip prosthesis. Peripheral blood mononuclear cells (PBMC) were collected from 14 healthy donors and three groups of patients: 10 candidates for primary total joint replacements, 11 patients with well-fixed implant and 13 patients with aseptic loosening of the hip prosthesis. PBMCs were cultured with the metal ions employed for implant manufacturing and the expression of CD69 activation antigen on CD3/T lymphocytes was detected by flow cytometry. Chromium extract increased significantly the expression of CD3/CD69 phenotype in patients with loosening of hip prosthesis. The chromium-induced 'activation index' was higher in patients with loosening of hip prosthesis than in healthy donors and in pre-implant patients. The cobalt-stimulated PBMC of patients with either well-fixed or loosened prosthesis had an 'activation index' significantly higher than healthy donors. The activation index values were used to graduate the PBMC-response as 'normal' (or = 0.9 and2), 'low' (0.9) and 'high' (or = 2): an high-activation index was observed only in chromium-exposed PBMC of patients with prosthesis. Our data show that chromium released from orthopedic implants could be responsible for the lymphocyte sensitization and flow cytometry is an easy and reliable method for monitoring the hypersensitivity state in patients with metal prostheses. Activated lymphocytes in the peri-implant tissue are likely to elicit a localized immune response and contribute to maintain the inflammatory process evolving in the implant failure.

The authors evaluated the ability of bone cement to modify the pro"le of pro-in#ammatory cytokines secreted by the immune cells. Peripheral blood mononuclear cells (PBMC) collected from healthy individuals were cultured with cement extracts and tested to assess the release of IL-1b, TNFa, GM-CSF and IL-6 in both unstimulated and PHA-stimulated PBMC. The cytokine release of unstimulated PBMC was very poor, and in particular the IL-1b was undetectable: the addition of cement extract increased both TNFa and GM-CSF release and decreased IL-6, sometimes signi"cantly. The most recurrent observation in PHA-stimulated PBMCs exposed to bone cement extract was the increase in both IL-1b and IL-6 release, while both the mean concentration and the index of release of TNFa and GM-CSF were changeable. In conclusion our results showed that leachable components of some bone cements can induce in vitro the release of pro-in#ammatory cytokines which are known to be involved in the bone resorption associated with aseptic loosening of hip prostheses. These "ndings allowed us to identify materials endowed with the highest in#ammatory power. ( 2000 Elsevier Science Ltd. All rights reserved.

The authors examined 54 biopsies taken from the tissue surrounding loosened hip joint prostheses. In situ apoptotic cell identification was performed by the detection of single- and double-stranded DNA breaks that occurred in the early stages of apoptosis. Both types of breaks can be revealed by labeling the free 3′-OH termini with modified nucleotides (fluoresceine-dUTP) in an enzymatic reaction catalyzed by terminal deoxynucleotidyl transferase (TdT). Results were correlated with the presence of wear debris in the tissue and with the use of bone cement for prosthesis fixation. Apoptotic cells were present in a higher percentage in tissue sections where metal particles were present (24% apoptotic cells) if compared to areas where no wear (6%), or plastic wear (2.8%) or ceramic wear (1.5%) was observed. Apoptosis is neither related to bone cement, nor to the time it takes for the implant to fail. Cell death by apoptosis may be important in implants which release metal ions by corrosion or wear and may have been underestimated up to now, as it is a `clean’ way of cell death, leading to limited damage in the surrounding tissues.

In testing for the cytotoxicity of medical devices, crucial parameters are the type of cells, the duration of the exposure, the physical form of the device and the method of evaluation. Using cell cultures the changes of cell functions induced by artificial materials may be evaluated. In screening tests the effect of biomaterials on functions common to all cell types is investigated through both continuous lines and primary cultures. In supplementary tests the effect of biomaterials on specific functions is studied, employing cells of the same type of those which will be in contact with the device in vivo. Osteoblasts, either from primary culture or an established line, are used to study the interaction with materials for bone implants. We evaluated whether dental cements affect the replication cycle of osteoblast-like cells MG63. The S phase was inhibited by the cements with a phenolic group, even though to a different extent. Mononuclear cell cultures are used to investigate the immune response, which is important in the host reaction to implant. We investigated whether the chromium ions released in a biological fluid may induce PBMC to produce cytokines involved in bone resorption. Chromium extract did not impair TNFα release significantly, but inhibited IL-6 release significantly in stimulated PBMC. Endothelial cell cultures are useful to the evaluation of materials for vascular grafts. We studied the influence of some types of Dacron on endothelial functions. Collagen-coated Dacron decreased significantly the production of 6-keto-prostaglandin F1α. Knitted Dacron caused a significant increase of tissue factor, a significant reduction in PECAM-1 and a significant increase in ELAM-1.

The aim of this study was the evaluation of the release of bone-resorbing cytokines in peripheral blood mononuclear cells (PBMC) of patients with aseptic loosening of Co–Cr hip prostheses. TNF-α, IL-6, and GM-CSF were measured in both unstimulated and PHA-stimulated PBMC, and in PBMC cultured in the presence of chromium and cobalt extracts. Blood samples from healthy donors were used as controls. Serum samples of both patients and healthy donors were tested to determine the concentration of metal ions. The proportion of lymphocyte, monocyte and lympocyte subpopulation in cultured PBMC did not differ in patients and the control group. In unstimulated PBMC the release of TNF was significantly higher in patients than in the control group, while IL-6 was significantly decreased and no change was observed for GM-CSF. When the PBMC were challenged with chromium extract, all the ‘index of cytokine release’ resulted higher in patients than in the control group; cobalt extract increased both the TNF and GM-CSF index, but not the index of IL-6 release. Metal concentrations in serum from patients were significantly increased and correlated with the TNF release in PBMC stimulated with both metal extract. Our results suggest that a CoCr-implant releases a large amount of metal ions which could mediate the priming or the renewal of a cell-mediated hypersensitivity reaction. The prevalence of circulating lymphocytes responsible for the delayed hypersensitivity, namely Th1, would justify both the significant increase of TNF and the significant decrease of IL6 in unstimulated PBMC of patients, as well as the significant increase of the ‘index of cytokine release’ after the challenge with metal ions.

In this research adhesive proteins are studied in order to evaluate the interference of woven Dacron in the endothelialization process and in the ability of endothelial cells to bind circulating leucocytes. Endothelial cells from human umbilical vein (HUVEC) were put in contact with woven Dacron for 24 h. PECAM-1, ELAM-1, ICAM-1 and VCAM-1 expression was then evaluated by flow cytometry, using indirect immunofluorescence reaction with monoclonal antibodies. The study of adhesive proteins was completed with the quantitative determination of surface antigens expressed as the antibody binding capacity (ABC). Antigenic density was calculated by the DAKO QFIT calibration system for indirect immunofluorescence. After contact with woven Dacron no significant change was observed in the percentage of positive cells or in the fluorescence intensity of the adhesins. No significant variation was also noted by calculating the surface antigen density by means of calibration fluorospheres. It can be concluded that the material examined does not significantly affect leucocyte adhesion to the endothelium.

The use of a wound dressing with covering and haemostatic properties significantly improves wound healing. In this study, a lyophilized bovine collagen sponge used for the treatment of wounds and ulcerae has been tested in a cell culture system. Phagocytosis of collagen fragments by human blood monocytes/macrophages has been investigated. For the assessment of collagen ingestion by mononuclear phagocytes, a picrosirius dye specific for collagen molecules has been used. By adapting this histochemical technique to microplate cell culture system, replicate monocyte cultures are assayed. Collagen content is determined by evaluating spectrophotometrically at 540 nm the absorbance of a sirius red/picric acid solution. Using this simple and sensitive method, the phagocytosis of bovine collagen by LPS-stimulated monocytes/macrophages has been ascertained.

The effects of orthopaedic cements on the proliferation and cell cycle of in vitro cultured MG63 osteoblast-like cells were examined. Five different cements were mixed and extracted at different time intervals (15 and 60 min, 6, 24 and 48 h). Cell proliferation inhibition (CPI) was evaluated after 72 h culture as the toxicity parameter. As for the toxicity degree, the extracts were considered to have ‘high toxicity’ (CPI ⩾ 50%), ‘medium toxicity’ (50% > CPI > 25%), ‘slow toxicity’ (CPI ⩽ 25%) and ‘no toxicity’ (CPI = 0). Cell cycle phases of MG63 cells were evaluated at 24, 48 and 72 h by flow cytometry; the DNA content was assessed using the propidium iodide uptake and the percentage of cells in the S phase was determined using 5′-bromodeoxyuridine uptake. According to our results, the toxicity is inversely correlated with the time interval between polymerization and extract preparation, and is different according to the cement type. For some cements the effects are still observed 48 h after polymerization. The damaging effect is not linked to a specific phase of the cell cycle, nor does it hamper the restarting of cell proliferation at 72 h.

The effects of endodontic cements on the cell cycle of MG63 osteoblasts cultured in vitro have been examined. Three groups of compounds were tested. Group I encompassed zinc oxide- and eugenol-based cements (Tubliseal, Argoseal, N2), group II consisted of cements with a phenol group other than eugenol (AH26, Forfenan, Methode R/R), and group III included CaOH-based cements (Biocalex, Endocalex). The cell cycle of MG63 cells was analyzed by flow cytometry; the DNA content was evaluated by means of the propidium iodide uptake method, whereas the proportion of cells in the S phase was defined by the incorporation of bromodeoxyuridine later revealed by a specific antibody. The results showed that some root canal sealers could hamper the periapex healing processes by inhibiting the cell proliferation through a selective action on different phases of the cell cycle.

This research aims at evaluating the expression of some adhesive proteins on endothelial cell surface after contact with polyethylene terephthalate coated with pyrolytic carbon (PET + PC). Twenty-two different cultures of human umbilical vein endothelial cells (HUVECs) were put in contact with PET + PC. Both HUVECs grown without the biomaterial and HUVECs incubated with endotoxin were used as control. After 24 h, platelet endothelial cell adhesion molecule-1 (PECAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were evaluated on the cells by monoclonal antibodies and flow cytometry. In agreement with the literature, after 24 h the culture incubated with endotoxin determined a significant increase in the percentage of positive cells for ELAM-1, a significant increase in fluorescence intensity for ICAM-1, and a significant increase in the percentage of positive cells and fluorescence intensity for VCAM-1. After 24 h of culture with PET + PC, no significant variations in the antigens examined were observed. This demonstrates that such material does not activate in vitro the proteins involved in the adhesion between leucocytes and endothelium or in the adhesion between endothelial cells themselves.