Veronica Bonazza

A review of the effects of dietary silicon intake on bone homeostasis and regeneration

OBJECTIVE Increasing evidences suggest that dietary Silicon (Si) intake, is positively correlated with bone homeostasis and regeneration, representing a potential and valid support for the prevention and improvement of bone diseases, like osteoporosis. This review, aims to provide the state of art of the studies performed until today, in order to investigate and clarify the beneficial properties and effects of silicates, on bone metabolism. METHODS We conducted a systematic literature search up to March 2013, using two medical databases (Pubmed and the Cochrane Library), to review the studies about Si consumption and bone metabolism. RESULTS We found 45 articles, but 38 were specifically focused on Si studies. CONCLUSION RESULTS showed a positive relationship between dietary Si intake and bone regeneration.

Biological Characterization and In Vitro Effects of Human ConcentratedGrowth Factor Preparation: An Innovative Approach to Tissue Regeneration

Scientific background: Platelet concentrates are nowadays widely applied in different clinical fields to improve soft tissue and bone regeneration. “Concentrated Growth Factors” (CGF) is a new generation of platelet concentrate products, which exhibits an interesting clinical and biotechnological application potential. Aim of the study: The aim of this study is to assess the biological rationale for the use of CGF, by evaluating blood cell localization, the in vitro cumulative release of seven growth factors (PDGF-AB, VEGF, TNF-α, TGF-β1, BDNF, BMP- 2 and IGF-1), its in vitro effects on cell proliferation and its mechanical behavior. Methods: CGFs were obtained from volunteer donors. Blood cell localization was evaluated after properly morphological staining and immunohistochemistry. The amount of growth factors release was measured at 5 hours, 1, 3, 6, 7 and 8 days, using ELISA assay. Cells were cultured with and without CGF and their proliferation were evaluated after 72 hours, performing the quantification of Ki-67, using flow cytometry (FACS). The mechanical response of CGF under compression was also attempted. Results: The results showed that platelets and leukocytes were found in a very thin space called “buffy coat”, localized between the white and red part of CGF. Each growth factor evaluated, had a specific kinetic release with a great variability among subjects. The in vitro cell proliferation was stimulated. CGF showed an “apparent plasticity” and its mechanical response was influenced by fibrin network structure. Conclusion: These findings support the CGF’s clinical use and will allow us to better understand and improve the clinical outcomes.

Single administration of melatonin modulates the nitroxidergic system at the peripheral level and reduces thermal nociceptive hypersensitivity in neuropathic rats

Neuropathic pain is a severe condition with unsatisfactory treatments. Melatonin, an indolamine, seems to be a promising molecule suitable for this purpose due to its well-known anti-inflammatory, analgesic, and antioxidant effects, as well as its modulation of the nitroxidergic system. Nevertheless, the data on its mechanism of action and potentialities are currently insufficient in this pathology, especially at the peripheral level. Thus, this work evaluated the effect of a single administration of melatonin in an established mononeuropathy pain model that monitors the behaviour and the changes in the nitroxidergic system in dorsal root ganglia and skin, which are affected by nervous impairment. Experiments were carried out on Sprague Dawley rats subdivided into the sham operated (control) and the chronic constriction injured animals, a model of peripheral neuropathic pain on sciatic nerve. Single administrations of melatonin (5–10 mg/kg) or vehicle were injected intraperitoneally on the 14th day after surgery, when the mononeuropathy was established. The animals were behaviourally tested for thermal hyperalgesia. The dorsal root ganglia and the plantar skin of the hind-paws were removed and processed for the immunohistochemical detection of neuronal and inducible nitric oxide synthases. The behavioural results showed an increase of withdrawal latency during the plantar test as early as 30 min after melatonin administration. The immunohistochemical results indicated a modulation of the nitroxidergic system both at dorsal root ganglia and skin level, permitting speculate on a possible mechanism of action. We showed that melatonin may be a possible therapeutic strategy in neuropathic pain.

How the different material and shape of the blood collection tube influences the Concentrated Growth Factors production

Platelet concentrates, such as Concentrated Growth Factors (CGF), are autologous preparations obtained from the patients own blood and rich in platelets, growth factors and cytokines involved in the key processes of tissue regeneration. These autologous concentrates differ in the way of preparation and also in the content of platelets, growth factors and leucocytes, as well as in the fibrin network architecture. So it is difficult to have a standardized product. The aim of the present study was to evaluate how the use of test tubes of different material, for blood collection, could influence the CGF production. Three different test tubes were used and the obtained CGFs were subjected to histomorphological and immunohistochemical analyses. Results showed that the tube material and shape influenced the CGF composition. In fact, according to the type of tube used, the obtained CGFs showed differences in morphology, in the fibrin network architecture and in blood cell localization and distribution.

In vitro treatment with Concentrated Growth Factors (CGF) and sodium orthosilicate positively affects cell renewal in three different human cell lines

The aim of this study was to investigate the in vitro effect of Silicon, in the soluble form of sodium orthosilicate, combined and not with the concentrated growth factors (CGF), a platelet‐rich preparation, on three different human cell lines of fibroblasts (NHDF), endothelial cells (HUVEC), and osteoblasts (HOBs). Each cell type was treated with sodium orthosilicate at the final concentration of 0.5 mM and 1 mM, CGF, and sodium orthosilicate combined with CGF, for 72 h. At the end of the experimental period, the in vitro effect on cell growth, proliferation, and metabolic activity was evaluated by performing a simple cell count, using an automated cell counter and by evaluating the expression of the intracellular proliferation marker Ki‐67, using Fluorescence‐activated cell sorting (FACS) analysis. Moreover, the expression of other cell markers and active molecules, such as Collagen type I, Osteopontin, Vascular Endothelial Growth Factor, and endothelial Nitric Oxide Synthase, was evaluated, through immunohistochemistry. Results obtained showed that the combined use of CGF and sodium orthosilicate stimulates cell growth, proliferation, and metabolic activity, suggesting that this treatment could be effective in tissue regeneration.

Beneficial Effects of Concentrated Growth Factors and Resveratrol on Human Osteoblasts In Vitro Treated with Bisphosphonates

Bisphosphonates are primary pharmacological agents against osteoclast-mediated bone loss and widely used in the clinical practice for prevention and treatment of a variety of skeletal conditions, such as low bone density and osteogenesis imperfecta, and pathologies, such as osteoporosis, malignancies metastatic to bone, Paget disease of bone, multiple myeloma, and hypercalcemia of malignancy. However, long-term bisphosphonate treatment is associated with pathologic conditions including osteonecrosis of the jaw, named BRONJ, which impaired bone regeneration process. Clinical management of BRONJ is controversy and one recent approach is the use of platelet concentrates, such as Concentrated Growth Factors, alone or together with biomaterials or antioxidants molecules, such as resveratrol. The aim of the present study was to investigate the in vitro effects of Concentrated Growth Factors and/or resveratrol on the proliferation and differentiation of human osteoblasts, treated or not with bisphosphonates. Human osteoblasts were stimulated for 3 days in complete medium and for 21 days in mineralization medium. At the end of the experimental period, the in vitro effect on osteoblast proliferation and differentiation was evaluated using different techniques such as MTT, ELISA for the quantification/detection of osteoprotegerin and bone morphogenetic protein-2, immunohistochemistry for sirtuin 1 and collagen type I, and the Alizarin Red S staining for the rate of mineralization. Results obtained showed that Concentrated Growth Factors and/or resveratrol significantly increased osteoblast proliferation and differentiation and that the cotreatment with Concentrated Growth Factors and resveratrol had a protective role on osteoblasts treated with bisphosphonates. In conclusion, these data suggest that this approach could be promised in the clinical management of BRONJ.

Platelet preparations in neuronal cell differentiation

Concentrated Growth Factors (CGF) is a platelet rich preparation that has the important feature of a tight fibrin network and containing a large number of growth factors possessing great regenerative potentialities [1]. The regeneration of nervous system is one of the mail goal of regenerative medicine. The aim of this study is to test the in vitro CGF effects on both differentiated and undifferentiated SH-SY5Y cells, derived from human neuroblastoma. To induce differentiation, SH-SY5Y cells have been treated with Retinoic Acid (RA) 10µM, in both basal and complete medium and in the presence and absence of CGF. After 72 hours, different parameters have been investigated: the morphological characteristics of the cells, the cell proliferation, the cellular vitality using the MTT test, the CGF and/or RA differentiation property and the immunocytochemical analysis of neuronal specific markers (NeuN, Sinaptophisine, β-III-tubulin, Nestin). Moreover the NGF (Nerve Growth Factor) and BDNF (Brain Derived Growth Factor) release have been assayed by ELISA test. Our results obtained suggest that treatment with CGF, also used alone, positively affects cell differentiation and neuronal phenotype regulating the expression of the neuronal markers and improving the outgrowth of neurites. Taken together these results seems to be promised into new approaches for neuronal regeneration using platelet preparations.

H2O2 stress damage is reversed by melatonin in a spinal cord organotypic model

Spinal cord injury (SCI) is characterized to be a two-step process: the primary lesion consisting of the initial trauma; the secondary damage, characterized by multiple processes including inflammation, oxidative stress and cell death that lead to a significant expansion of the original damage and to an increase of the functional deficit (1). Among the aforementioned processes, the oxidative stress plays a significant role in pathophysiology of SCI. In this study, we evaluated the role of the melatonin, an indoleamine recognized as a potent antioxidant and immunomodulator (2, 3 )Reiter et al., 1995, Favero et al., 2015), on the oxidative stress, the tissue vitality and the neuritic plasticity in an experimental model of organotypic cultures of Sprague Dawley rat spinal cord slice (SPS) treated with hydrogen peroxide (H2O2) and/or melatonin. Five experimental protocols were performed: 1) control; 2) H2O2 exposure (50 μM); 3) melatonin treatment (5-10M for 24 hours); 4) H2O2 exposure and post-treatment with melatonin; 5) H2O2 exposure after pre-treatment with melatonin. Cellular death was investigated by propidium iodide (PI) assay and the vitality by MTT assay. The total thiols (SH) levels, contrasting the oxidative stress, the neuronal specific nuclear protein (NeuN) and the synaptophysin (Syp) immunopositivity were also evaluated. Melatonin significantly decreases the number of dead cells and increases slice vitality, mainly in slices treated before H2O2 exposure. Moreover, melatonin attenuates total thiols decrease and NeuN and Syp immunopositivity reduction. Overall, these findings suggest that melatonin may exert a potential beneficial effect upon the progression of SCI secondary damage, protecting the tissue from a further degeneration.This work was supported by grants from Giorgio Brunelli Foundation for Spinal Cord Injuries Research.

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

Concentrated growth factors (CGF): morphological and biochemical characterization

Concentrated growth factors (CGF) represents the novel generation of solid plate - let concentrate preparations (1-2). These 100% autologous preparations, obtained from a venous blood sample, not only enhance tissue healing but also improve the clinical outcomes of various surgical procedures, reducing complications like pain, inflammation and morbidity (3). Considering the few data on CGF morphology and its biological properties, the aim of this study was to analyse the CGF structure (blood cell localization and fibrin matrix architecture) and the in vitro cumulative release of seven growth factors (PDGF-AB, VEGF, TNF-α, TGF-β1, IGF-I, BDNF and BMP-2). CGF was obtained from volunteer donors using a specific protocol of cen - trifugation. Blood cell localization and fibrin architecture were evaluated after prop - erly staining and immunostaining protocols. The kinetics of the growth factor release were performed by incubation of the CGF in a free growth factors cell medium, at 37°C for 5 hours, 1, 3, 6, 7 and 8 days. After each incubation period, the medium was collected, centrifuged and stored at −80 °C until analysis. The total quantity of growth factors was checked using ELISA kits. After venous blood centrifugation, the CGF obtained consisted in three parts: the upper white part (PPP), the lower red part (RBC) and the middle “buffy coat” part (interface between white and red part). The results showed that platelets and leukocytes were localized in the buffy coat, where - as the erythrocytes were present only in the red part of CGF. Moreover, in the white part, the fibrin network and architecture changed moving far from the buffy coat becoming less compact. The in vitro cumulative release of growth factors revealed that each of them had a specific kinetic. Considering the mean value obtained for each time point from all volunteers, PDGF-AB, TGF-β1 and IGF-1 had a constant kinetic release, reaching the maximum accumulation at day 3 rd and 6 th respectively; VEGF and BMP-2 had a slow kinetic release, reaching the maximum accumulation at day 8 th ; TNF-α and BDNF had a fast kinetic release, reaching the maximum accumu - lation at day 1 st and 3 rd respectively. These findings support the clinical use of CGF and will allow us to better understand and improve the clinical outcomes.