Germana Martinasso

TNF‐α TGF‐β2 and IL‐1β levels in gingival and peri‐implant crevicular fluid before and after de novo plaque accumulation

AIMS The aim of this split-mouth study was to investigate levels of tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta2) and interleukin-1 beta (IL-1beta) in gingival crevicular fluid (GCF) and peri-implant crevicular fluid (PICF) after a 21-day-period of de novo plaque accumulation in the same patient. MATERIAL AND METHODS In 25 patients, samples of GCF and PICF were collected in the sulcus of the tooth and of the implant after professional hygiene. After the no-hygiene phase (21 days), second samples of GCF and PICF were taken. Third samples were collected after 69 days of re-establishment oral hygiene techniques. The crevicular fluids were used to determine the volume and the levels of TNF-alpha, TGF-beta2 and IL-1beta. RESULTS The volume of the crevicular fluids increased significantly after 21 days of plaque accumulation around teeth and implants and decreased significantly by 69 days. TNF-alpha and TGF-beta2 did not change significantly among the three different samples. A significant increase of IL-1beta was observed after plaque accumulation around the teeth GCF, whereas in the PICF the increase was not statistically significant. CONCLUSIONS These data suggest that increased volumes of GCF and PICF could be useful markers of early inflammation in gingival and peri-implant tissues. In the presence of de novo plaque, implants showed lower, and nearly significant, levels of IL-1beta compared with teeth.

Superpulsed Laser Irradiation Increases Osteoblast Activity Via Modulation of Bone Morphogenetic Factors

Laser therapy is a new approach applicable in different medical fields when bone loss occurs, including orthopedics and dentistry. It has also been used to induce soft‐tissue healing, for pain relief, bone, and nerve regeneration. With regard to bone synthesis, laser exposure has been shown to increase osteoblast activity and decrease osteoclast number, by inducing alkaline phosphatase (ALP), osteopontin, and bone sialoprotein expression. Studies have investigated the effects of continuous or pulsed laser irradiation, but no data are yet available on the properties of superpulsed laser irradiation. This study thus aimed to investigate the effect of superpulsed laser irradiation on osteogenic activity of human osteoblast‐like cells, paying particular attention to investigating the molecular mechanisms underlying the effects of this type of laser radiation.

Involvement of PPARs in Cell Proliferation and Apoptosis in Human Colon Cancer Specimens and in Normal and Cancer Cell Lines.

PPAR involvement in cell growth was investigated “in vivo” and “in vitro” and was correlated with cell proliferation and apoptotic death. “In vivo” PPARγ and α were evaluated in colon cancer specimens and adjacent nonneoplastic colonic mucosa. PPARγ increased in most cancer specimens versus mucosa, with a decrease in c-Myc and in PCNA proteins, suggesting that colon cancer growth is due to increased cell survival rather than increased proliferation. The prevalence of survival over proliferation was confirmed by Bcl-2 or Bcl-XL increase in cancer versus mucosa, and by decreased PPARα. “In vitro” PPARγ and PPARα were evaluated in human tumor and normal cell lines, treated with natural or synthetic ligands. PPARγ was involved in inhibiting cell proliferation with a decrease in c-Myc protein, whereas PPARα was involved in inducing apoptosis with modulation of Bcl-2 and Bad proteins. This involvement was confirmed using specific antagonists of two PPARs. Moreover, the results obtained on treating cell lines with PPAR ligands confirm observations in colon cancer: there is an inverse correlation between PPARα and Bcl-2 and between PPARγ and c-Myc.

The impact of plasma rich in growth factors on clinical and biological factors involved in healing processes after third molar extraction

Extraction of an impacted mandibular third molar is a common surgical procedure, although it still leads to several postoperative symptoms and complications. The study assessed the efficacy of autologous plasma rich in growth factors (PRGF) in the healing process by checking the difference of tissue cytokines and other healing factors produced by the mucosa after extraction between sites treated with PRGF and control sites and, at the same time, by evaluating the clinical efficacy of PRGF in terms of reduced pain and facial swelling. This study was a split-mouth study, in which the patient becomes his/her own control, to eliminate any individual response differences toward PRGF treatment. The parameters regarding inflammation and subsequent wound healing were all significantly higher at PRGF sites than at control sites. The increase at PRGF sites of the two proinflammatory cytokines evaluated, interleukin (IL)-1β and IL-6, was accompanied by the increase of two anti-inflammatory cytokines, IL-10 and transforming growth factor-β. Furthermore, IL-1β and IL-6 induce fibroblast and keratinocyte proliferation, important events in wound healing. Postoperative pain and the swelling, measured at all experimental times, were reduced in the presence of PRGF.

Effects of di(2-ethylhexyl) phthalate, a widely used peroxisome proliferator and plasticizer, on cell growth in the human keratinocyte cell line NCTC 2544.

Phthalate esters are a widely used class of water-insoluble organic chemicals. The adverse effects of di(2-ethylhexyl) phthalate (DEHP) were chiefly studied in animals, while their potential toxicity to humans has not been properly evaluated. It was hypothesized that the effect of DEHP on human cells depends on the concentration, and this study examined the effects of different concentrations of DEHP on cell growth in cultured human keratinocytes NCTC 2544, together with the possible involvement of peroxisome proliferator-activated receptors (PPARs) in mediating the effects. After exposure to DEHP, the number of NCTC 2544 cells in the monolayer decreased in a concentration-dependent and time-dependent manner, whereas the cells that were detached from the monolayer increased, and died via necrosis. The decrease of cell growth was confirmed by the inhibition of pErk1, pErk2, and changes in the c-myc protein content. With regard to PPARs, the PPARbeta protein content increased, whereas PPARalpha decreased. To demonstrate the involvement of PPARbeta in inhibiting cell growth, the use of an antisense oligonucleotide against this receptor revealed the prevention of DEHP-induced cell growth inhibition. In addition, the treatment of keratinocytes with a specific ligand of PPARbeta (L165041) showed a concentration-dependent inhibition of cell growth, as with DEHP. In conclusion, the effect of DEHP on human keratinocytes is concentration dependent, and this effect is mediated via PPARs.

Synthesis and characterisation of bioactive and antibacterial glass–ceramic Part 1 – Microstructure, properties and biological behaviour

Abstract A glass–ceramic composition has been studied to realise a highly bioactive material, suitable for stimulate the bone regeneration, which has been subjected to a patented ion exchange process with silver ions, to impart antibacterial properties. The obtained material has been characterised by SEM, EDS and XRD analyses, before and after the introduction of Ag+ ions, and has been subjected to mechanical tests. Ag+ release was verified by GF-AAS analysis. The influence of silver on material wettability and bioactivity was evaluated through contact angle measurements and in vitro test on SBF solution. Finally, biocompatibility with osteoblast like cells and antibacterial test on Staphylococcus Aureus, have been realised to demonstrate the effective antimicrobial behaviour and the safety of silver doped glass–ceramic. On the basis of this study, it was evinced that ion exchange technique, optimised on glasses in previous research works, allows the controlled introduction of Ag+ ions and can be transferred on medical devices totally or partially realised with bioactive glass–ceramic.

Oral mucosa produces cytokines and factors influencing osteoclast activity and endothelial cell proliferation, in patients with osteonecrosis of jaw after treatment with zoledronic acid

ObjectivesThe intravenous injection of bisphosphonates, currently used as treatment for osteoporosis, bone Paget’s disease, multiple myeloma, or bone metastases, can cause jaw bone necrosis especially in consequence of trauma. The present research aimed to clarify the mechanisms underlying bone necrosis, exploring involvement of the oral mucosa “in vivo.”Patients and methodsSpecimens of oral mucosa were removed from bisphosphonate-treated patients with or without jaw bone necrosis. In mucosa specimens, expression was evaluated of: cytokines involved in the inflammatory process, factors involved in osteoclast activity, i.e., receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin, a factor involved in cell proliferation, namely hydroxymethylglutaryl coenzyme A reductase, and a factor involved in angiogenesis, namely vascular endothelial growth factor (VEGF).ResultsInterleukin (IL)-6 and the RANK/osteoprotegerin ratio were significantly elevated in mucosa from patients with versus without jaw necrosis, whereas hydroxymethylglutaryl coenzyme A reductase and VEGF were significantly decreased.ConclusionsOur results suggest that mucosa, stimulated by bisphosphonate released from the bone, can contribute to the development of jaw necrosis, reducing VEGF, and producing IL-6 in consequence of hydroxymethylglutaryl coenzyme A reductase reduction. In turn, IL-6 stimulates osteoclast activity, as shown by the increased RANKL/osteoprotegerin ratio.Clinical relevanceThe results of this study suggest the importance of evaluating during bisphosphonate treatment the production of IL-6, RANKL, osteoprotegerin, and VEGF, in order to monitor the jaw osteonecrosis onset. To avoid repeated mucosa excisions, the determination of these factors could be carried out in crevicular fluid.

Hydroxyapatite paste Ostim®, without elevation of full‐thickness flaps, improves alveolar healing stimulating BMP‐ and VEGF‐mediated signal pathways: an experimental study in humans

OBJECTIVE Tooth extraction is considered as the starting point of jaw atrophy via osteoclast activity stimulation. The maintenance of dental alveolar bone depends on surgery procedure and use of materials to maintain prior space favoring bone regeneration. Among substitutes used in dentistry to fill bone defects, Ostim-Pastes (Ostim) is a nanocrystalline paste tested for treatment of severe clinical conditions. This research first investigated the effect of Ostim on alveolar healing, comparing in the same healthy subjects, an Ostim-filled socket with a not-filled one. Moreover, it also proposed a new surgical protocol for the post-extractive socket treatment using the graft materials without elevation of full-thickness flaps. MATERIAL AND METHODS Fourteen patients were enrolled to bilateral maxillary or mandibular extraction that was performed without elevation of full-thickness flaps. In each patient, one socket was filled using Ostim, and the other one was allowed to undergo natural healing. No suture was carried out. Clinical and biologic parameters were screened at 1, 7, and 14 days. RESULTS Obtained results evidenced that nanocrystalline hydroxyapatite supports bone regeneration, increasing the synthesis of pro-osteogenic factors as bone morphogenetics protein (BMP)-4, BMP-7, alkaline phosphatase, and osteocalcin. Moreover, filling post-extractive socket with nanocrystalline hydroxyapatite paste leads to a complete epithelialization already at 7 days after extraction, despite the fact that the teeth were extracted without elevation of full-thickness flaps . The improved epithelialization is mediated by increased vascular endothelial growth factor (VEGF) expression. No significant change was observed in inflammatory parameters, with exception of an early and transient IL-1β induction, that could trigger and improve alveolar healing. CONCLUSIONS Clinical and biomolecular observations of this explorative study evidenced that nanocrystalline hydroxyapatite improves alveolar socket healing, increasing angiogenesis, epithelialization, and osteogenesis, also in absence of elevation of full-thickness flaps.

Increase in class 2 aldehyde dehydrogenase expression by arachidonic acid in rat hepatoma cells

Aldehyde dehydrogenase (ALDH) is a family of several isoenzymes important in cell defence against both exogenous and endogenous aldehydes. Compared with normal hepatocytes, in rat hepatoma cells the following changes in the expression of ALDH occur: cytosolic class 3 ALDH expression appears and mitochondrial class 2 ALDH decreases. In parallel with these changes, a decrease in the polyunsaturated fatty acid content in membrane phospholipids occurs. In the present study we demonstrated that restoring the levels of arachidonic acid in 7777 and JM2 rat hepatoma cell lines to those seen in hepatocytes decreases hepatoma cell growth, and increases class 2 ALDH activity. This latter effect appears to be due to an increased gene transcription of class 2 ALDH. To account for this increase, we examined whether peroxisome-proliferator-activated receptors (PPARs) or lipid peroxidation were involved. We demonstrated a stimulation of PPAR expression, which is different in the two hepatoma cell lines: in the 7777 cell line, there was an increase in PPAR alpha expression, whereas PPAR gamma expression increased in JM2 cells. We also found increased lipid peroxidation, but this increase became evident at a later stage when class 2 ALDH expression had already increased. In conclusion, arachidonic acid added to the culture medium of hepatoma cell lines is able to partially restore the normal phenotype of class 2 ALDH, in addition to a decrease in cell growth.

Involvement of PPARalpha in the growth inhibitory effect of arachidonic acid on breast cancer cells.

Epidemiological studies suggest that dietary PUFA may influence breast cancer progression. n-3 PUFA are generally known to exert antitumour effects, whereas reports relative to n-6 PUFA anti-carcinogen effects are controversial. Arachidonic acid (AA; 20:4n-6) and its metabolites have been shown to inhibit the growth of human breast cancer cell lines, even if the downstream mechanisms by which AA may influence carcinogenesis remain unresolved. We explored the molecular basis for AA influence on proliferation, signal transduction and apoptosis in two human breast cancer cell lines, MCF-7 and MDA-MB-231. In both cell lines AA inhibited cell growth in a dose-dependent manner, even if MDA-MB-231 was somewhat more growth-inhibited than MCF-7. AA decreased extracellular signal-regulated protein kinase 1/2 phosphorylation level, and positively modulated PPARgamma and PPARalpha expression, with only a slight effect against PPARbeta/delta. In addition, AA increased Bak (an apoptosis-regulating protein) expression and reduced procaspase-3 and -9 levels only in MDA-MB-231 cells, thus indicating that the growth inhibitory effect can be correlated with apoptosis induction. In both cell lines the use of a specific antagonist made it possible to establish a relationship between AA growth inhibitory effect and PPARalpha involvement. AA decreases cell proliferation most likely by inducing apoptosis in MDA-MB-231 cells, while in the MCF-7 cell line the growth inhibitory activity can be attributed to the inhibition of the signal transduction pathway involved in cell proliferation. In both cases, the results here presented suggest PPARalpha as a possible contributor to the growth inhibitory effect of AA.