Bioactivation of calcium deficient hydroxyapatite with foamed gelatin gel. A new injectable self-setting bone analogue.

An alternative approach to bone repair for less invasive surgical techniques, involves the development of biomaterials directly injectable into the injury sites and able to replicate a spatially organized platform with features of bone tissue. Here, the preparation and characterization of an innovative injectable bone analogue made of calcium deficient hydroxyapatite and foamed gelatin is presented. The biopolymer features and the cement self-setting reaction were investigated by rheological analysis. The porous architecture, the evolution of surface morphology and the grains dimension were analyzed with electron microscopy (SEM/ESEM/TEM). The physico-chemical properties were characterized by X-ray diffraction and FTIR analysis. Moreover, an injection test was carried out to prove the positive effect of gelatin on the flow ensuing that cement is fully injectable. The cement mechanical properties are adequate to function as temporary substrate for bone tissue regeneration. Furthermore, MG63 cells and bone marrow-derived human mesenchymal stem cells (hMSCs) were able to migrate and proliferate inside the pores, and hMSCs differentiated to the osteoblastic phenotype. The results are paving the way for an injectable bone substitute with properties that mimic natural bone tissue allowing the successful use as bone filler for craniofacial and orthopedic reconstructions in regenerative medicine.

Modifications of the 1H NMR metabolite profile of processed mullet (Mugil cephalus) roes under different storage conditions.

(1)H NMR spectroscopy was employed to study the modifications over time of the water-soluble low molecular weight metabolites extracted from samples of salted and dried mullet (Mugil cephalus) roes (mullet bottarga) stored at different conditions. Samples of grated mullet bottarga were stored for 7 months at -20 °C, at 3 °C, and at room temperature in the presence and in the absence of light and then timely extracted and analyzed by NMR. Principal component multivariate data analysis applied to the spectral data indicated that samples stored at -20 °C maintained similar features over time whereas, along PC1, samples stored at room temperature in the presence and in the absence of light showed, over time, marked metabolite modifications. The comparative analysis of the integrated areas of the selected regions of the (1)H NMR spectra indicated that the major compositional changes due to storage conditions were (i) the increase of the derivatives of the breakdown of phosphatidylcholine (choline, phosphorylcholine, and glycerol), (ii) the breakdown of nucleosides, (iii) the decrease of methionine, tryptophan, and tyrosine, and (iv) the cyclization of creatine. These changes were observed at different storage conditions, with more pronounced trends in the samples stored at room temperature. The role of metabolites in food aging is discussed.

[Exclusive intra-operative radiation therapy (IORT) for early stage breast cancer: pilot study of feasibility]. / Radioterapia intraoperatoria (IORT) esclusiva nel carcinoma iniziale della mammella: studio pilota di fattibilità.

AIM: To evaluate feasibility, tolerability and cosmetic outcome of intra-operative radiation therapy (IORT) as an exclusive post-surgery treatment of early stage breast cancer. PATIENTS AND METHODS: From October 2008 to October 2009 30 patients underwent wide breast cancer excision or quadrantectomy followed by IORT on tumor bed with accelerated electrons at the dose of 21Gy. The characteristics of the patients were: ductal breast cancer or invasive lobular cT1, cT2 ≤ 2,5 cm, cN0, G1-2, age over 35 years, M0. RESULTS: The average age was 51.7 (range 38 - 75) with an average follow up of 11.7 months (range 6 - 18). The pathologic stage of the lesions resulted pT1 in 29 cases (96,6%), in particular: one case pT1a (3,3%), 21 cases pT1b (70,0%) and 7 cases pT1c (23,3%). One case (3,3%) was pT2 with a diameter of 2.5 cm. The grading was G2 in 20 cases (66,6%) and G1 in 10 cases (33,3%). The toxicity, evaluated according to the EORTC-RTOG criteria, was G0 (33.3%) in 10 cases, G1 (63,3%) in 19 cases, G2 in one case (3,4%); there was no G3 toxicity. The time needed for a complete healing of the wound was less than 10 days in 96,7% of the cases, with one case of limphocele (3,3%). There were no infections of the surgical wound nor any mastitis, neither in the treated quadrant nor in the other ones. We observed a light fibrosis in 5 cases (16,6%), moderate in 2 cases (6,6%) but never severe. Cosmetics, evaluated in four levels, according to Danoff et al., was excellent in 3 cases (43,3%), good in 15 cases (50%), sufficient in 2 cases (6,7%), never insufficient. As regards local control, there was no local relapse. The global survival was 100%. CONCLUSIONS: The IORT in early breast cancer, at the doses used in this study, proved itself as a secure technique, repeatable, with limited complications. The advantages of its use are the possibility of a direct control, by the surgeon and the radiotherapist, of the structures to treat and those to protect; the absence of time needed for cellular repopulation between surgery and radiotherapy; a good cosmetic outcome; and logistic advantages. It is necessary to have a long term follow up to evaluate the efficacy in terms of long term cosmetic and local control.

Manual orthodontic vs. oscillating-rotating electric toothbrush in orthodontic patients: a randomised clinical trial.

AIM: To compare the efficacy of manual and oscillating-rotating electric toothbrushes in removing plaque and reducing gingivitis in patients with fixed orthodontic appliances. STUDY DESIGN: Randomised clinical trial. MATERIALS AND METHODS: 20 subjects aged 10 to 14 years (8 males; 12 females) scheduled for fixed orthodontic treatment were enrolled, randomly divided into two groups, A and B, and later assigned electric or manual toothbrushes, respectively. Three months after orthodontic appliance bonding (T1), and again after a further 4 (T2) and 8 (T3) weeks, plaque levels (PI), gingival bleeding (GBI) and hypertrophy were assessed. RESULTS: Between the two toothbrush types, statistically significant differences in PI were detected from T2 to T3 (P value 0.010), but not from T1 to T2. In contrast, the GBI was significantly different from T1 to T2 (P value 0.025), but not from T2 to T3. CONCLUSION: With respect to the manual orthodontic toothbrush, the electric oscillating-rotating toothbrush was found to better improve both PI and GBI.

The usefulness of the prognostic inflammatory and nutritional index (PINI) in a haemodialysis population.

BACKGROUND AND AIM: Protein-Energy Wasting and inflammation are the principal risk factors of haemodialysis complications. We evaluated the reliability of a simple and non expensive test, the Prognostic Inflammatory and Nutritional Index (PINI), for regular screening of maintenance haemodialysis (MHD) patients in order to detect early onset of inflammation and malnutrition. METHODS AND RESULTS: 121 adult patients on maintenance dialysis were followed up for 32 months and screened every 6 months for PINI, calculated as alpha1-Acid Glycoprotein (alpha1-AG)xC-Reactive Protein (CRP)/AlbuminxTransthyretin. PINI score < or =1 was considered normal. Patients were stratified according to their PINI score: 86 patients (71.66%) had a normal score, whereas 35 (28.33%) had PINI > or = 1. The latter also had higher CRP levels, despite no clinical evidence of inflammation at the time of enrolment. Survival in patients with normal PINI was similar to patients with normal CRP, while in patients with abnormal PINI it was significantly lower than in patients with low serum albumin (p<0.05) or elevated CRP (p<0.05). After follow-up, all surviving MHD patients with PINI > or = 1 had at least one cardiovascular event vs 2.5% of patients with PINI > or = 1. CONCLUSION: The assessment of PINI can reliably identify MHD patients at higher risk of mortality and morbidity even in the absence of overt Malnutrition-Inflammation Complex Syndrome (MICS). This simple test appears to be more sensitive and specific of the single components, and not expensive, so that it could be routinely used to identify patients with sub-clinical inflammation and/or malnutrition.

13C NMR, GC and HPLC characterization of lipid components of the salted and dried mullet (Mugil cephalus) roe "bottarga".

13C NMR spectroscopy, in conjunction with HPLC and GC techniques, has been used to study the molecular composition of lipids extracted from commercial products of bottarga. To this goal, both the saponifiable and unsaponifiable fractions of lipid extracts were also examined by 13C NMR. Among the major lipid classes wax esters (WE) showed a concentration of more than 50mol%, triacylglycerols (TAG) and phospholipids (PL) represented a minor fraction. Concentrations up to 29mol% of free fatty acids (FFA) were found. The most represented fatty alcohol was 16:0 that accounted for more than 50%, among fatty acids the most represented were 16:1 n-7, 22:6 n-3, 18:1 n-9, 16:0, and 20:5 n-3, in particular the n-3 polyunsaturated fatty acids (PUFA) averaged 40mg/g of the edible portion. 13C NMR spectroscopy put in evidence that cholesterol was present in its free and esterified forms and its total content was measured as ca. 10mg/g of the edible portion.

Protective effect of the oligomeric acylphloroglucinols from Myrtus communis on cholesterol and human low density lipoprotein oxidation.

Myrtle (Myrtus communis L.), a culinary spice and flavouring agent for alcoholic beverages widespread in the Mediterranean area and especially in Sardinia, contains the structurally unique oligomeric non-prenylated acylphloroglucinols, semimyrtucommulone and myrtucommulone A, whose antioxidant activity was investigated during the oxidative modification of lipid molecules implicated in the onset of cardiovascular diseases. Both acylphloroglucinols showed powerful antioxidant properties during the thermal (140 degrees C), solvent-free degradation of cholesterol. Moreover, the pre-treatment with semimyrtucommulone and myrtucommulone A significantly preserved LDL from oxidative damage induced by Cu(2+) ions at 2h of oxidation, and showed remarkable protective effect on the reduction of polyunsaturated fatty acids and cholesterol, inhibiting the increase of their oxidative products (conjugated dienes fatty acids hydroperoxides, 7beta-hydroxycholesterol, and 7-ketocholesterol). Taking into account the widespread culinary use of myrtle leaves, the results of the present work qualify the natural compounds semimyrtucommulone and myrtucommulone A as interesting dietary antioxidants with potential antiatherogenicity.

Protective effect of vanilloids against tert-butyl hydroperoxide-induced oxidative stress in vero cells culture.

This study investigated the effect of synthetic capsiate, a simplified analogue of capsiate, and vanillyl alcohol on the oxidative stress induced by tert-butyl hydroperoxide (TBH) in a line of fibroblasts derived from monkey kidney (Vero cells). In response to the TBH-mediated oxidative stress, a reduction of the levels of total unsaturated fatty acids and cholesterol was observed, and a rise in the concentrations of conjugated dienes fatty acids hydroperoxides and 7-ketocholesterol. Pretreatment with both synthetic capsiate and vanillyl alcohol preserved Vero cells from oxidative damage and showed a remarkable protective effect on the reduction of the levels of total unsaturated fatty acids and cholesterol, inhibiting the increase of MDA, conjugated dienes fatty acids hydroperoxides, and 7-ketocholesterol. Both compounds were effective against peroxidation of cell membrane lipids induced by TBH, with synthetic capsiate essentially acting as a pro-drug of vanillyl alcohol, its hydrophilic hydrolytic derivative.

Protective effect of hydroxytyrosol and its metabolite homovanillic alcohol on H(2)O(2) induced lipid peroxidation in renal tubular epithelial cells.

We investigated the capacity of hydroxytyrosol (HT), 3,4-dihydroxyphenylethanol, and homovanillic alcohol (HVA), 4-hydroxy-3-methoxy-phenylethanol, to inhibit H(2)O(2) induced oxidative damage in LLC-PK1, a porcine kidney epithelial cell line, studying the effect of H(2)O(2) on specific cell membrane lipid targets, unsaturated fatty acids and cholesterol. Exposure to H(2)O(2) induced a significant increase of the level of MDA together with a disruption of the membrane structure, with the loss of unsaturated fatty acids, cholesterol and alpha-tocopherol, and the formation of fatty acids hydroperoxides and 7-ketocholesterol. Pretreatment with HT protected renal cells from oxidative damage: the level of membrane lipids was preserved and there was no significant detection of oxidation products. HVA exerted a comparable activity, thus both HT and HVA were able to prevent in renal cells the lipid peroxidation process that plays a central role in tubular cell injury.

Extraction and separation of volatile and fixed oils from berries of Laurus nobilis L. by Supercritical CO2.

Isolation of volatile and fixed oils from dried berries of Laurus nobilis L. from Tunisia have been obtained by supercritical fractioned extraction with carbon dioxide. Extraction experiments were carried out at a temperature of 40 degrees C and pressures of 90 and 250 bar. The extraction step performed at 90 bar produced a volatile fraction mainly composed of (E)-beta-ocimene (20.9%), 1,8-cineole (8.8%), alpha-pinene (8.0%), beta-longipinene (7.1%), linalool acetate (4.5%), cadinene (4.7%), beta-pinene (4.2%), alpha-terpinyl acetate (3.8%) and alpha-bulnesene (3.5%). The oil yield in this step of the process was 0.9 % by weight charged. The last extraction step at 250 bar produced an odorless liquid fraction, in which a very small percentage of fragrance compounds was found, whereas triacylglycerols were dominant. The yield of this step was 15.0 % by weight. The most represented fatty acids of the whole berry fixed oil were 12:0 (27.6%), 18:1 n-9 (27.1%), 18:2 n-6 (21.4%), and 16:0 (17,1%), with the 18:1 n-9 and 18:2 n-6 unsaturated fatty acids in particular averaging 329 microg/mg of oil.

Evaluation of the antioxidant and cytotoxic activity of arzanol, a prenylated alpha-pyrone-phloroglucinol etherodimer from Helichrysum italicum subsp.microphyllum.

Various phenolics and (mero)terpenoids from Helichrysum italicum subsp. microphyllum, a plant endemic to Sardinia, were investigated for their capacity to inhibit non-enzymatic lipid peroxidation. These compounds were studied in simple in vitro systems, under conditions of autoxidation and of iron (EDTA)-mediated oxidation of linoleic acid at 37 degrees C. Arzanol, a pyrone-phloroglucinol etherodimer, and helipyrone, a dimeric pyrone, showed antioxidant activity, and could protect linoleic acid against free radical attack in assays of autoxidation and EDTA-mediated oxidation. Methylarzanol, as well as the sesquiterpene alcohol rosifoliol, showed a decreased, but still significant, protective effect against linoleic acid oxidation. Arzanol and helipyrone were also tested in an assay of thermal (140 degrees C) autoxidation of cholesterol, where arzanol showed significant antioxidant activity. The cytotoxicity of arzanol was further evaluated in VERO cells, a line of fibroblasts derived from monkey kidney. Arzanol, at non-cytotoxic concentrations, showed a strong inhibition of TBH-induced oxidative stress in VERO cells. The results of the present work suggest that the natural compound arzanol exerts useful antioxidant properties in different in vitro systems of lipid peroxidation.

Protective effect of olive oil minor polar components against oxidative damage in rats treated with ferric-nitrilotriacetate.

The phenolic fraction of virgin olive oil exerts preventive effects against reactive oxygen species mediated degenerative diseases. To investigate its action as inhibitor of lipid peroxidation in vivo, we treated Wistar rats with olive oil minor polar components (MPC) (25-50 mg/kg bw) prior to the administration of a sublethal dose (15 mg Fe/kg bw) of ferric-nitrilotriacetate (Fe-NTA). Intraperitoneal injection (i.p.) of Fe-NTA lead to increased oxidative stress associated with extensive peroxidation of membrane lipids in plasma, kidney, and liver of treated rats. Fe-NTA treatment induced a significant decrease of the major oxidizable membrane lipids, alpha-tocopherol, fatty acids and cholesterol, together with an increase of fatty acids hydroperoxides (HP) and 7-ketocholesterol (7-keto). I.p. administration of MPC significantly inhibited fatty acids and cholesterol oxidation, and reduced the levels of HP and 7-keto. In summary, MPC administration protects organs against lipid peroxidation and conserves the endogenous alpha-tocopherol.

Phase II study of preoperative irradiation and chemotherapy with capecitabine in patients with locally advanced rectal carcinoma.

Preoperative chemoradiotherapy has demonstrated to improve resectability and local control in locally advanced rectal cancer (LARC). 5-fluorouracil (5FU) has traditionally been the drug of choice in combination with radiation therapy. Early studies of capecitabine (CAP) have shown its potential to replace 5FU. Between March 2002 and April 2005, 31 patients with newly diagnosed LARC (T2 N+ 2 cases, T3 N0-N+ 25 cases, T4 N0-N+ 4 cases) received the combined treatment. Surgery was planned 6-8 weeks after chemoradiation. Adjuvant chemotherapy with 5FU plus leucovorin for 6 courses was given in pN+ patients. All patients completed the planned treatment. Grade 3 acute toxicity was observed in 5 patients (16%). Nineteen patients (61%) had a downstaging. A complete pathological remission was observed in 3 cases (10%). Median follow-up is of 23 months (range; 6-36 months). The results of this experience confirm the data of the literature about the feasibility and efficacy of a neoadjuvant treatment with radiation and CAP in LARC.

[Hyperhomocysteinemia and alternate vitamin supplementation]. / Iperomocisteinemia nei pazienti uremici: effetti della supplementazione alternata di acido folico e vitamina B 12.

BACKGROUND: Treatment with folic acid and vitamin B 12 appears to be effective in lowering total plasma Homocysteine (tHcy) concentration, but whether vitamin B 12 alone decreases tHcy in patients with normal vitamin B 12 status is still unknown. The aims of the present study were to explore the effect of alternate vitamin supplementation with folic acid or vitamin B 12 on tHcy concentrations in haemodialysis (HD) patients, and to compare changes in tHcy concentrations with MTHFR genotype. METHODS: 74 patients, 44 men and 30 women, were recruited and randomized blindly into two groups of 37 subjects each. The first group was initially treated with vitamin B 12 for two months, and with folic acid for the following two months; the second group was supplemented in the reverse order. In both groups the treatment was followed by a 2-month washout period. tHcy levels were measured at the beginning of treatment (T0), after two months (T1), four months (T2), and at the end of the washout period (T3). Vitamin B 12 and folate were taken at T0 and T3. RESULTS: The genotype frequency was: C/C 37%, C/T 34%, T/T 29%. tHcy decreased in both groups following the alternate vitamins therapy. This decrease was greater for the T/T genotype (p<0.05) and was more significant when the treatment start-ed with folic acid (p<0.01). Moreover, after the washout period, tHcy increased remarkably without significant differences between diffusive and convective techniques. Folate levels at the end of study appeared to be reduced in haemodialysis patients. Vitamin B 12 concentration significantly increased in patients on diffusive haemodialysis, confirming the fundamental role of membrane performance. CONCLUSION: The alternate vitamin treatment demonstrated the importance of folate therapy and the secondary contribution of vitamin B 12 in lowering tHcy in HD patients.

KFLC Index utility in multiple sclerosis diagnosis: Further confirmation.

The Multiple Sclerosis (MS) diagnosis is based on dissemination of focal lesions in time and space. The free light chains (FLCs) determination might be a sensitive alternative to oligoclonal bands assay. The study aim was to redefine sensitivity, specificity of the kFLC Index cut-off. We analyzed serum and cerebrospinal fluid of 176 patients, with different neurological disorders. We obtained a cut off of 12,3 for kFLC Index with a sensitivity and specificity of 93% and 100% respectively. Our data confirm that the kFLC Index is a valid tool in the diagnosis of MS.

The fate of olive oil polyphenols in the gastrointestinal tract: implications of gastric and colonic microflora-dependent biotransformation.

We have conducted a detailed investigation into the absorption, metabolism and microflora-dependent transformation of hydroxytyrosol (HT), tyrosol (TYR) and their conjugated forms, such as oleuropein (OL). Conjugated forms underwent rapid hydrolysis under gastric conditions, resulting in significant increases in the amount of free HT and TYR entering the small intestine. Both HT and TYR transferred across human Caco-2 cell monolayers and rat segments of jejunum and ileum and were subject to classic phase I/II biotransformation. The major metabolites identified were an O-methylated derivative of HT, glucuronides of HT and TYR and a novel glutathionylated conjugate of HT. In contrast, there was no absorption of OL in either model. However, OL was rapidly degraded by the colonic microflora resulting in the formation of HT. Our study provides additional information regarding the breakdown of complex olive oil polyphenols in the GI tract, in particular the stomach and the large intestine.

Protective effect of capsinoid on lipid peroxidation in rat tissues induced by Fe-NTA.

The activity of a single IP administration (15 or 30 mg/Kg body weight) of vanillyl nonanoate, a simplified analog of capsiate, on ferric nitrilotriacetate (Fe-NTA)-mediated oxidative damage was investigated. A sub-lethal dose of Fe-NTA (15 mg Fe/Kg body weight) was administered IP to rats; animals were sacrificed, and kidney and plasma were collected 1 h after injection. In response to the Fe-NTA administration, a reduction of the levels of total lipids, total unsaturated fatty acids and cholesterol was observed, accompanied by a rise in the concentrations of malondialdehyde (MDA), conjugated dienes fatty acids hydroperoxides and 7-ketocholesterol in plasma and kidney 1 h after administration. A pre-treatment with synthetic capsiate (SCPT) showed remarkable protective effect on the reduction of the levels of total lipids, total unsaturated fatty acids and cholesterol, and the cellular antioxidant vitamin E, inhibiting the increase of MDA, conjugated dienes fatty acids hydroperoxides and 7-ketocholesterol in the plasma and kidney. The protective effect of SCPT and two analogues (vanillyl alcohol and vanillin) during the linoleic acid and cholesterol oxidation was investigated in in vitro systems, providing evidence of definite structure-activity relationships.

Cholesterol as target of Fe-NTA-induced lipid peroxidation in rat tissues.

Intraperitoneal injection of the iron-chelate, ferric-nitrilotriacetate (Fe-NTA), induces renal proximal tubular damage associated with oxidative damage in vivo. A sub-lethal dose of Fe-NTA (15 mg Fe/kg body weight) was administered IP to rats; animals were sacrificed and liver, kidney and plasma were collected 1-4 h after injection. In response to the Fe-NTA administration, there were significant time-dependent reductions of the levels of total lipids, cholesterol and total unsaturated fatty acids, and a rise in the concentrations of conjugated dienes, 7-ketocholesterol and fatty acids hydroperoxides, showing a pattern inversely correlated in plasma, kidney and liver. Cholesterol level decreased significantly from 1 h after injection in the kidney and 3-4 h in the plasma and liver of treated rats. This is the first report on cholesterol reduction and accumulated 7-ketocholesterol in the tissues of rats treated with Fe-NTA as a consequence of lipid peroxidation.

Conjugated dienes detected in tissue lipid extracts by second derivative spectrophotometry.

By studying lipid peroxidation induced by tetrachloromethane in rat liver microsomal PUFA, it has recently been shown that the primary products formed are conjugated diene hydroperoxides having either cis, trans (c,t) or trans,trans (t,t) stereochemistry. Both c,t and t,t hydroperoxidienes present distinct absorbances at 242 nm and 233 nm, respectively. The reaction is kinetically controlled in relation to the total H-atom donating ability of the cell environment. These results have been confirmed in vivo and in vitro experiments performed under different experimental conditions. The need for a precise and objective method to detect conjugated diene signals, the inherent difficulties with current techniques, and the availability of new spectrophotometric techniques have led us to devise a new method based on the second derivatization of the spectrum.

Inhibition of peroxynitrite dependent DNA base modification and tyrosine nitration by the extra virgin olive oil-derived antioxidant hydroxytyrosol.

Hydroxytyrosol is one of the o-diphenolic compounds in extra virgin olive oil and has been suggested to be a potent antioxidant. The superoxide radical (O2*-) and nitric oxide (NO*) can react very rapidly to form peroxynitrite (ONOO ), a reactive tissue damaging species thought to be involved in the pathology of several chronic diseases. Hydroxytyrosol was highly protective against the peroxynitrite-dependent nitration of tyrosine and DNA damage by peroxynitrite in vitro. Given that extra virgin olive oil is consumed daily by many humans, hydroxytyrosol derived from this diet could conceivably provide a defense against damage by oxidants in vivo. The biological activity of hydroxytyrosol in vivo will depend on its intake, uptake and access to cellular compartments.