Ramon Boninsegna

Growth factors, CD34 positive cells, and fibrin network analysis in concentrated growth factors fraction.

An interesting clinical option for optimizing healing tissue is the use of platelet concentrate. Platelets contain high quantities of growth factors, among these TGF‐β1 and VEGF, which are known to be implicated in tissue regeneration. CGF is produced by processing blood samples with a special centrifuge device; three layers are formed: top acellular plasma (PPP), middle CGF and bottom red blood cells (RBC) layers. Given that to date there are no data concerning the biological characteristic of CGF, the aim of this study was to evaluate the presence of TGF‐β1 and VEGF in CGF and also in PPP and RBC layers. In addition, since circulating stem cells are recruited from blood to injured tissue for healing we also evaluated the presence of CD34 positive cells. Our data show the presence of TGF‐β1 and VEGF in CGF and RBC layers. In addition, we show CD34 positive cells in CGF. Microsc. Res. Tech., 2011.

Endothelial Nitric Oxide Synthase in Dorsal Root Ganglia during Chronic Inflammatory Nociception

Nitric oxide (NO) is a gaseous molecule implicated both in vascular tone and nociceptive transmission. The capillary blood supply to the dorsal root ganglia (DRG) is unique because it is highly permeable to several low and high molecular-weight compounds. This anatomical situation leads to a potential role of endothelial nitric oxide synthase (eNOS) in inflammatory nociception, which is not well established. Therefore, we examined the role of eNOS in DRG in a murine chronic inflammatory pain model induced by complete Freund’s adjuvant using L-N5-(1-iminoethyl)ornithine (L-NIO), a potent inhibitor of eNOS activity. Pain state was examined using a behavioral test. The expression of eNOS, platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial growth factor (VEGF) was examined by immunofluorescence. In control animals, CD31 was detected in vessels; VEGF was localized both in vessels and neurons while a weak eNOS immunopositivity was detected in both vessels and in neurons. Under inflammatory pain conditions, eNOS, CD31 and VEGF immunopositivity increased. Administration of L-NIO significantly attenuated thermal hyperalgesia by 24 h and decreased eNOS activity and CD31 immunopositivity by 7 days. VEGF was unaffected. Our results show that eNOS plays a nociceptive role in the early phases of inflammation while in the later phases it may be involved in neurotrophic support.

Nitroxidergic system in human trigeminal ganglia neurons: A quantitative evaluation

The trigeminal ganglia are involved in transmission of orofacial sensitivity. The free radical gas nitric oxide (NO) has recently been found to function as a messenger molecule in both central and peripheral trigeminal primary afferent neurons. NO is produced within neurons mainly by two enzymes: a constitutive (neuronal) form of NO synthase (nNOS) or an inducible form of NOS (iNOS). The aim of the study was to evaluate the distribution of trigeminal neurons according to size (small, medium and large neurons) and to correlate the percentage of NOS-immunopositive neurons with regard to neuronal size. The results showed a significant relationship between the percentage of nNOS-immunopositive neurons and the size of neurons. Evaluation of the percentage of nNOS-immunopositive neurons showed that they constitute about 50% of the total number of neurons and that they are represented mainly as large-sized neurons. The iNOS immunolabelling was very faint in all neuronal types. Since the nitroxidergic system is well represented in human trigeminal ganglia, this study indicates that it could play a relevant role in trigeminal neurotransmission.

Sodium-DNA for Bone Tissue Regeneration: An Experimental Study in Rat Calvaria

Surgical techniques in dental and maxillofacial surgery request fast bone tissue regeneration, so there is a significant need to improve therapy for bone regeneration. Several studies have recently underlined the importance of nucleotides and nucleosides to increase cell proliferation and activity; in particular, the ability of polydeoxyribonucleotide (PDRN) to induce growth and activity of human osteoblasts was demonstrated. Sodium-DNA is the deoxyribonucleic acid (DNA) extracted from the gonadic tissue of male sturgeon and then purified, depolymerized, and neutralized with sodium hydroxide. To date, there are no evidences about the use of Sodium-DNA for bone tissue regeneration. Consequently, our question is about the efficacy of Sodium-DNA in bone healing. For testing the role of Sodium-DNA in bone healing we used a rat calvarial defect model. Sodium-DNA at different concentrations used alone or in association with Fibrin and/or Bio-Oss was used for healing treatments and the bone healing process was evaluated by histomorphometric and immunohistochemical analyses. Our results suggested a positive effect of Sodium-DNA in bone regeneration, providing a useful protocol and a model for the future clinical evaluation of its osteogenic properties.

In vitro effects of Concentrated Growth Factors on BMP-2 synthesis by human osteoblasts

Bone morphogenetic proteins, and especially BMP-2, play an important role in bone homeostasis and regeneration, stimulating osteoblasts differentiation. Concentrated Growth Factors (CGF) is an autologous platelet preparation, obtained from the patient’s own blood, with a specific protocol of centrifugation and containing several different growth factors, including BMP-2, with an average release of 5-10 pg (1). So, in this study, we investigated the in vitro effect of CGF on BMP-2 synthesis by human osteoblasts (HOBs). Cells were cultured in presence of CGF for 9 days and then the cell number were determined by using an automated cell counter. To test the effect of CGF on BMP2 release from osteoblasts, the supernatants were collected, centrifuged at 1200 rpm for 10 min at room temperature and used to perform the BMP-2 ELISA assay. In addition, the expression of BMP-2 on fixed HOBs was also evaluated by performing an immunocytochemical analysis. Our results showed that CGF significantly enhances HOBs proliferation and increases BMP2 synthesis by HOBs that could act also in paracrine way, together with CGF derived BMP2, to better promote bone regrowth.This work was supported by grants from Silfradent Srl

In vitro effects of silicon and a platelet rich plasma preparation

Cells, growth factors and scaffolds represent the key elements in tissue engineer - ing strategies. Platelet concentrates are blood derivatives, prepared from patient’s own blood, containing autologous platelets, growth factors and cytokines (1). The use of these autologous preparations, like CGF (Concentrated Growth Factor), repre - sents a promising approach in tissue regeneration and some clinical applications have already been made. Some evidences suggest a pivotal role of trace elements in tis- sue regeneration (2); in particular Silicon plays an important role in bone homeostasis and regeneration (3); so Silicon and CGF could represent an innovative approach for tissue regeneration. In this study, the in vitro effects of Silicon (Sodium Orthosilicate) and CGF, on the proliferation of three different human cell lines (endothelial cells, fibroblasts and osteoblasts) were investigated. Cells were treated with Sodium Ortho - silicate and CGF for 24, 48 and 72 hours respectively. After each incubation period, cell proliferation was evaluated, using the Scepter cell counter; some proliferation markers (Collagen type I, Osteopontin) were evaluated using immunohistochemical methods. In addition we examined the influence of Sodium Orthosilicate and CGF on osteoblasts differentiation. Human osteoblasts were grown until confluence in com - plete growth medium. After 24-72 hours cell confluency, the medium was replaced with osteoblast mineralization medium for 14 days and the osteoblasts differentiation was quantitatively evaluated by immunohistochemistry, using Osteocalcin and ALP, and by Alizarin and Von Kossa stainings. Treatment with Silicon and CGF increases proliferation of all cell lines and promotes osteoblasts differentiation. So, these data could represent a potential and innovative tool for tissue regeneration.

Different preparation of Sodium-DNA for bone tissue regeneration

Current strategies for bone tissue regeneration involve the use of a wide range of biomaterials and synthetic bone substitutes; among them, Sodium-DNA could represent a new chance considering its osteoinductive properties (Nakamura et al., 2000; Bowler et a., 2001; Guizzardi et al., 2003; Guizzardi et al., 2007). The aim of this study was to evaluate the regenerative properties of two different preparation of Sodium- DNA (paste or liquid form) in a rat calvarial defect model. The cranium of each rat was shaved and a skin incision from the naso-frontal area to the external occipital protuberance was performed. The skin and the subcutaneous tissues were reflected to expose the full extent of the calvaria. Full-thickness 5X8 mm bone skull defects were made on each parietal region using piezoelectric surgery. Bone defects were filled with Sodium-DNA (paste or liquid form, Veritas, Brescia, Italy) alone or mixed with Bio-Oss (Geistlich, Wolhusen, Switzerland). Histomorphometric evaluation of bone regeneration was performed at the end of the treatments. The data obtained showed a time-dependent active bone healing process; however, differences in the use of past or liquid form were evident. These results suggest that Sodium-DNA could be considered an active biomaterial in bone regeneration, but an adequate formulation to obtain a better regenerative efficacy is needed.

Pineal gland and neuropathic pain

The pineal gland is a small neuroendocrine organ involved primarily in the circadian rhythm by the secretion of melatonin [1]. In addition, the pain modulatory properties of melatonin are generally recognized but its involvement in neuropathic pain regulation is not fully understood. In fact, it is known that the activation of the endogenous melatonin system in the spinal cord can reduce the generation, development and maintenance of central sensitization [2]. Moreover, melatonin showed an analgesic effect, in fact several works in animals [2] and in humans [3] underline its ability to inhibit hyperalgesia. In particular, intracerebroventricular and intraperitoneal melatonin, with its higher doses, produces a blockade of thermal hyperalgesia in mice with partial tight ligation of the sciatic nerve. The aim of our work is to characterize the morphological changes in peripheral structures, such as plantar skin and dorsal root ganglia (DRG) of rats in a neuropathic pain model (chronic constriction injury) after a single melatonin treatment monitoring the behaviour and the changes in NO-system using immunohistochemical techniques. The behavioural results show an increase of withdrawal latency during plantar test already after 30 min from melatonin administration. The immunohistochemical results suggest that melatonin plays a crucial role in keratinocytes-mediated neuropathic pain transmission through the modulation of nitroxidergic system, which could have also a protective role at this site. In addition, at DRG level the NO-system is maintained at low level. These results suggest that melatonin administration or modulation of pineal gland activity may have clinical utility in neuropathic pain therapy in the future.

Protective role of melatonin against obesity-associated vascular dysfunctions

Obesity is a chronic, multifactorial and complex disorder common in industrialized countries and strictly associated with increased mortality and morbidity of cardiovascular diseases. Moreover, it results from a long-term positive energy balance associated with chronic low-grade inflammation, in which both genetic and environmental factors are involved (Wang and Nakayama, 2010). Obesity is associated with endothelial dysfunction, arterial stiffness and vasoconstriction, these effects on vascular functions are mediated by low-grade inflammation expansion of adipose tissue and secretion from fat depots of adipokines that directly affect heart and blood vessels (Singhal, 2005; Ouwens et al., 2010). New emerging data show that melatonin is an affordable, cheap and non-toxic molecule with exceptional potential in body weight regulation and energy metabolism (Reiter and Korkmaz, 2008). However, the exact mechanism of action is not yet fully understood. In this study, we hypothesized that melatonin administration (kindly provided by Chronolife S.r.l., Roma, Italy) can minimize vascular morphological changes and dysfunctions and ameliorate fat accumulation in a mice model of obesity. In particular, the animals were divided in four groups: (i) control mice without treatment, (ii) control mice treated with melatonin for 8 weeks, (iii) obese mice without treatment and (iiii) obese mice treated with melatonin for 8 weeks. Compared with the obese group, intake of melatonin was associated with a statistically significant decrease in body weight, blood glucose level, inflammation, fat accumulation and hyperplasia and it restored the correct vascular cytoarchitecture, reducing also inflammation. These data suggest that melatonin has no toxic effects in mice with normal body weight, but its administration in obese mice reduce fat accumulation and vascular dysfunctions, suggesting its protective role as a tool for effective management of obesity- induced complications, including cardiovascular diseases.

Epithelial expression of vanilloid and cannabinoid receptors: a potential role in burning mouth syndrome pathogenesis

Burning mouth syndrome is an intraoral burning sensation in which the oral mucosa has a normal appearance and no medical or dental causes can be found. It remains an unknown disease for which long-term treatment is still lacking. The aim of this study is to assess in epithelial human tongue the expression of three receptors involved in pain transmission, such as a transient receptor potential vanilloid receptor type 1 (TRPV1) which mediates the sensation produced by chilli peppers, cannabinoid receptors type 1 (CB1) and type 2 (CB2), which are pathway-related to TRPV1. Epithelial cells express TRPV1, CB1 and CB2 receptors suggesting a role for these cells in sensory transduction. The study was performed on 8 healthy and 9 BMS patients. All patients underwent a 3-mm punch biopsy at the anterolateral aspect of the tongue close to the tip. Specimens were included in paraffin and serially cut to obtain 6um thick sections. The sections were processed for TRPV1, CB1 and CB2 immunohistochemistry. The analysis showed an altered expression of the studied receptors. In particular we observed an increase of TRPV1, a decrease of CB1 and an increase of CB2 expression in epithelial cells of the tongue with a change in morphological localization. So, these receptors seem to be correlated with BMS. These data could be useful for future characterization of BMS markers and specific therapies.