Tissue engineering is widely recognized as a promising approach for bone repair and reconstruction. Several attempts have been made to achieve materials that must be compatible, osteoconductive, and osteointegrative and have mechanical strength to provide a structural support. Composite scaffolds consisting in biodegradable natural polymers are very promising constructs. Hydroxyapatite (HAp) can support alginate as inorganic reinforcement and osteoconductive component of alginate/HAp composite scaffolds. Therefore, HAp-strengthened polymer biocomposites offer a solid system to engineer synthetic bone substitutes. In the present work, HAp was incorporated into an alginate solution and internal gelling was induced by addition of slowly acid-hydrolyzing D-gluconic acid delta-lactone for the direct release of calcium ions from HAp. It has been previously demonstrated that alginate-based composites efficiently support adhesion of cancer bone cell lines. Human dental pulp stem cells (DPSCs) identified in human dental pulp are clonogenic cells capable of differentiating in multiple lineage. Thus, this study is aimed at verifying the mineralization and differentiation potential of human DPSCs seeded onto scaffolds based on alginate and nano-hydroxyapatite. For this purpose, gene expression profile of early and late mineralization-related markers, extracellular matrix components, viability parameters, and oxidative stress occurrence were evaluated and analyzed. In summary, our data show that DPSCs express osteogenic differentiation-related markers and promote calcium deposition and biomineralization when growing onto Alg/HAp scaffolds. These findings confirm the use of Alg/HAp scaffolds as feasible composite materials in tissue engineering, being capable of promoting a specific and successful tissue regeneration as well as mineralized matrix deposition and sustaining natural bone regeneration.

Dental pulp stem cells (DPSCs) represent a population of stem cells which could be useful in oral and maxillofacial reconstruction. They are part of the periendothelial niche, where their crosstalk with endothelial cells is crucial in the cellular response to biomaterials used for dental restorations. DPSCs and the endothelial cell line EA.hy926 were co-cultured in the presence of Chitlac-coated thermosets in culture conditions inducing, in turn, osteogenic or angiogenic differentiation. Cell proliferation was evaluated by 3-[4,5-dimethyl-thiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT) assay. DPSC differentiation was assessed by measuring Alkaline Phosphtase (ALP) activity and Alizarin Red S staining, while the formation of new vessels was monitored by optical microscopy. The IL-6 and PGE2 production was evaluated as well. When cultured together, the proliferation is increased, as is the DPSC osteogenic differentiation and EA.hy926 vessel formation. The presence of thermosets appears either not to disturb the system balance or even to improve the osteogenic and angiogenic differentiation. Chitlac-coated thermosets confirm their biocompatibility in the present co-culture model, being capable of improving the differentiation of both cell types. Furthermore, the assessed co-culture appears to be a useful tool to investigate cell response toward newly synthesized or commercially available biomaterials, as well as to evaluate their engraftment potential in restorative dentistry.

Cell therapy with a variety of stem populations is increasingly being investigated as a promising regenerative strategy for cardiovascular (CV) diseases. Their combination with adequate scaffolds represents an improved therapeutic approach. Recently, several biomaterials were investigated as scaffolds for CV tissue repair, with decellularized extracellular matrices (dECMs) arousing increasing interest for cardiac tissue engineering applications. The aim of this study was to analyze whether dECMs support the cardiac differentiation of CardiopoieticAF stem cells. These perinatal stem cells, which can be easily isolated without ethical or safety limitations, display a high cardiac differentiative potential. Differentiation was previously achieved by culturing them on Matrigel, but this 3D scaffold is not transplantable. The identification of a new transplantable scaffold able to support CardiopoieticAF stem cell cardiac differentiation is pivotal prior to encouraging translation of in vitro studies in animal model preclinical investigations. Our data demonstrated that decellularized extracellular matrices already used in cardiac surgery (the porcine CorTMPATCH and the equine MatrixPatchTM) can efficiently support the proliferation and cardiac differentiation of CardiopoieticAF stem cells and represent a useful cellular scaffold to be transplanted with stem cells in animal hosts.

In the present study, we investigated the water extract of Harpagophytum procumbens DC. ex Meisn. in an experimental model of inflammatory bowel diseases (IBDs). Additionally, a microbiological investigation was carried out to discriminate the efficacy against bacterial and fungal strains involved in IBDs. Finally, an untargeted proteomic analysis was conducted on more than one hundred colon proteins involved in tissue morphology and metabolism. The extract was effective in blunting the production of oxidative stress and inflammation, including serotonin, prostaglandins, cytokines, and transcription factors. Additionally, the extract inhibited the growth of Candida albicans and C. tropicalis. The extract was also able to exert a pro-homeostatic effect on the levels of a wide plethora of colon proteins, thus corroborating a protective effect. Conversely, the supraphysiological downregulation of cytoskeletal-related proteins involved in tissue morphology and antimicrobial barrier function suggests a warning in the use of food supplements containing H. procumbens extracts.

Mentha spicata is one of the most popular species in the genus, and it is of great interest as a gastrointestinal and sedative agent in the folk medicine system. In this study, different M. spicata extracts, obtained by the use of four solvents (hexane, chloroform, acetone and acetone/water) were chemically characterized using HPLC-ESI-MS n, which allowed for identification of 27 phenolic compounds. The extracts' antioxidant and enzyme inhibitory properties were investigated. In addition, neuroprotective effects were evaluated in hypothalamic HypoE22 cells, and the ability of the extracts to prevent the hydrogen peroxide-induced degradation of dopamine and serotonin was observed. The best antioxidant effect was achieved for all the extraction methods using acetone/water as a solvent. These extracts were the richest in acacetin, eriodictyol, hesperidin, sagerinic acid, naringenin, luteolin, chlorogenic acid, chrysoeriol and apigenin. The intrinsic antioxidant and enzyme inhibition properties of the acetone/water extract could also explain, albeit partially, its efficacy in preventing prostaglandin E2 overproduction and dopamine depletion (82.9% turnover reduction) in HypoE22 cells exposed to hydrogen peroxide. Thus, our observations can provide a scientific confirmation of the neuromodulatory and neuroprotective effects of M. spicata.

The aim of the present study was to investigate the role of Cyclic-nucleotide Response Element-Binding (CREB) family members and related nuclear transcription factors in the radiation response of human B lymphoma cell lines (Daudi and Ramos). Unlike the more radiosensitive Daudi cells, Ramos cells demonstrated only a moderate increase in early apoptosis after 3-5 Gy irradiation doses, which was detected with Annexin V/PI staining. Moreover, a significant and dose-dependent G2/M phase accumulation was observed in the same cell line at 24 h after both ionizing radiation (IR) doses. Western blot analysis showed an early increase in CREB protein expression that was still present at 3 h and more evident after 3 Gy IR in Ramos cells, along with the dose-dependent upregulation of p53 and NF-κB. These findings were consistent with real-time RT-PCR analysis that showed an early- and dose-dependent upregulation of NFKB1, IKBKB and XIAP gene expression. Unexpectedly, pre-treatment with SN50 did not increase cell death, but cell viability. Taken together, these findings let us hypothesise that the early induction and activation of NF-κB1 in Ramos cells could mediate necrotic cell death and be linked to other molecules belonging to CREB family and involved in the cell cycle regulation.

Pterygium is a relatively common eye disease that can display an aggressive clinical behaviour. To evaluate the in vitro effects of Curcuma longa on human pterygium-derived keratinocytes, specimens of pterygium from 20 patients undergoing pterygium surgical excision were collected. Pterygium explants were put into culture and derived keratinocytes were treated with an alcoholic extract of 1.3% Curcuma longa in 0.001% Benzalkonium Chloride for 3, 6, and 24 h. Cultured cells were examined for CAM5.2 (anti-cytokeratin antibody) and CD140 (anti-fibroblast transmembrane glycoprotein antibody) expression between 3th and 16th passage to assess cell homogeneity. TUNEL technique and Annexin-V/PI staining in flow cytometry were used to detect keratinocyte apoptosis. We showed that Curcuma longa exerts a proapoptotic effect on pterygium-derived keratinocytes already after 3 h treatment. Moreover, after 24 h treatment, Curcuma longa induces a significant increase in TUNEL as well as Annexin-V/PI positive cells in comparison to untreated samples. Our study confirms previous observations highlighting the expression, in pterygium keratinocytes, of nuclear VEGF and gives evidence for the first time to the expression of nuclear and cytoplasmic VEGF-R1. All in all, these findings suggest that Curcuma longa could have some therapeutic potential in the treatment and prevention of human pterygium.

Industrial hemp is a multiuse crop whose phytocomplex includes terpenophenolics and flavonoids. In the present study, the phenolic and terpenophenolic compounds were assayed in the water extract of the hemp variety Futura 75. Protective effects were also investigated in human fibroblast and keratinocytes and isolate mouse skin specimens, which were exposed to hydrogen peroxide and/or to the extract (1-500 µg/mL). The results of phytochemical analysis suggested the cannabidiol, cannabidiolic acid and rutin as the prominent phytocompounds. In the in vitro system represented by human keratinocytes and fibroblasts, the hemp extract was found to be able to protect cells from cytotoxicity and apoptosis induced by oxidative stress. Moreover, modulatory effects on IL-6, a key mediator in skin proliferation, were found. In isolated rat skin, the extract reduced hydrogen peroxide-induced l-dopa turnover, prostaglandin-E2 production and the ratio kynurenine/tryptpophan, thus corroborating anti-inflammatory/antioxidant effects. The in silico docking studies also highlighted the putative interactions between cannabidiol, cannabidiolic acid and rutin with tyrosinase and indoleamine-2,3-dioxygenase, involved in l-dopa turnover and tryptophan conversion in kynurenine, respectively. In conclusion, the present findings showed the efficacy of hemp water extract as a skin protective agent. This could be partly related to the extract content in cannabidiol, cannabidiolic acid and rutin.

Cholesterol accumulation in macrophages leads to the formation of foam cells and increases the risk of developing atherosclerosis. We have verified whether hydroxytyrosol (HT), a phenolic compound with anti-inflammatory and antioxidant properties, can reduce the cholesterol build up in THP-1 macrophage-derived foam cells. We have also investigated the potential mechanisms. Oil Red O staining and high-performance liquid chromatography (HPLC) assays were utilized to detect cellular lipid accumulation and cholesterol content, respectively, in THP-1 macrophages foam cells treated with HT. The impact of HT on cholesterol metabolism-related molecules (SR-A1, CD36, LOX-1, ABCA1, ABCG1, PPARγ and LRX-α) in foam cells was assessed using real-time PCR (RT-qPCR) and Western blot analyses. Finally, the effect of HT on the adhesion of THP-1 monocytes to human vascular endothelial cells (HUVEC) was analyzed to study endothelial activation. We found that HT activates the PPARγ/LXRα pathway to upregulate ABCA1 expression, reducing cholesterol accumulation in foam cells. Moreover, HT significantly inhibited monocyte adhesion and reduced the levels of adhesion factors (ICAM-1 and VCAM-1) and pro-inflammatory factors (IL-6 and TNF-α) in LPS-induced endothelial cells. Taken together, our findings suggest that HT, with its ability to interfere with the import and export of cholesterol, could represent a new therapeutic strategy for the treatment of atherosclerotic disease.

Vitis vinifera (grape) contains various compounds with acknowledged phytochemical and pharmacological properties. Among the different parts of the plant, pomace is of particular interest as a winemaking industry by-product. A characterization of the water extract from grape pomace from Montepulciano d'Abruzzo variety (Villamagna doc) was conducted, and the bioactive phenolic compounds were quantified through HPLC-DAD-MS analysis. HypoE22, a hypothalamic cell line, was challenged with an oxidative stimulus and exposed to different concentrations (1 µg/mL-1 mg/mL) of the pomace extract for 24, 48, and 72 h. In the same conditions, cells were exposed to the sole catechin, in a concentration range (5-500 ng/mL) consistent with the catechin level in the extract. Cell proliferation was investigated by MTT assay, dopamine release through HPLC-EC method, PGE2 amount by an ELISA kit, and expressions of neurotrophin brain-derived neurotrophic factor (BDNF) and of cyclooxygenase-2 (COX-2) by RT-PCR. The extract reverted the cytotoxicity exerted by the oxidative stimulus at all the experimental times in a dose-dependent manner, whereas the catechin was able to revert the oxidative stress-induced depletion of dopamine 48 h and 72 h after the stimulus. The extract and the catechin were also effective in preventing the downregulation of BDNF and the concomitant upregulation of COX-2 gene expression. In accordance, PGE2 release was augmented by the oxidative stress conditions and reverted by the administration of the water extract from grace pomace and catechin, which were equally effective. These results suggest that the neuroprotection induced by the extract could be ascribed, albeit partially, to its catechin content.

Epidermal Growth Factor Receptor (EGFR), a member of the ErbB family of receptor tyrosine kinase (RTK) proteins, is aberrantly expressed or deregulated in tumors and plays pivotal roles in cancer onset and metastatic progression. ZNF216 gene has been identified as one of Immediate Early Genes (IEGs) induced by RTKs. Overexpression of ZNF216 protein sensitizes 293 cell line to TNF-α induced apoptosis. However, ZNF216 overexpression has been reported in medulloblastomas and metastatic nasopharyngeal carcinomas. Thus, the role of this protein is still not clearly understood. In this study, the inverse correlation between EGFR and ZNF216 expression was confirmed in various human cancer cell lines differently expressing EGFR. EGF treatment of NIH3T3 cells overexpressing both EGFR and ZNF216 (NIH3T3-EGFR/ZNF216), induced a long lasting activation of EGFR in the cytosolic fraction and an accumulation of phosphorylated EGFR (pEGFR) more in the nuclear than in the cytosolic fraction compared to NIH3T3-EGFR cells. Moreover, EGF was able to stimulate an increased expression of ZNF216 in the cytosolic compartment and its nuclear translocation in a time-dependent manner in NIH3T3-EGFR/ZNF216. A similar trend was observed in A431 cells endogenously expressing the EGFR and transfected with Znf216. The increased levels of pEGFR and ZNF216 in the nuclear fraction of NIH3T3-EGFR/ZNF216 cells were paralleled by increased levels of phospho-MAPK and phospho-Akt. Surprisingly, EGF treatment of NIH3T3-EGFR/ZNF216 cells induced a significant increase of apoptosis thus indicating that ZNF216 could sensitize cells to EGF-induced apoptosis and suggesting that it may be involved in the regulation and effects of EGFR signaling.

Premature newborns are frequently exposed to hyperoxic conditions and experimental data indicate modulation of liver metabolism by hyperoxia in the first postnatal period. Conversely, nothing is known about possible modulation of growth factors and signaling molecules involved in other hyperoxic responses and no data are available about the effects of hyperoxia in postnatal liver haematopoiesis. The aim of the study was to analyse the effects of hyperoxia in the liver tissue (hepatocytes and haemopoietic cells) and to investigate possible changes in the expression of Vascular Endothelial Growth Factor (VEGF), Matrix Metalloproteinase 9 (MMP-9), Hypoxia-Inducible Factor-1α (HIF-1α), endothelial Nitric Oxide Synthase (eNOS), and Nuclear Factor-kB (NF-kB). Experimental design of the study involved exposure of newborn rats to room air (controls), 60% O2 (moderate hyperoxia), or 95% O2 (severe hyperoxia) for the first two postnatal weeks. Immunohistochemical and Western blot analyses were performed. Severe hyperoxia increased hepatocyte apoptosis and MMP-9 expression and decreased VEGF expression. Reduced content in reticular fibers was found in moderate and severe hyperoxia. Some other changes were specifically produced in hepatocytes by moderate hyperoxia, i.e., upregulation of HIF-1α and downregulation of eNOS and NF-kB. Postnatal severe hyperoxia exposure increased liver haemopoiesis and upregulated the expression of VEGF (both moderate and severe hyperoxia) and eNOS (severe hyperoxia) in haemopoietic cells. In conclusion, our study showed different effects of hyperoxia on hepatocytes and haemopoietic cells and differential involvement of the above factors. The involvement of VEGF and eNOS in the liver haemopoietic response to hyperoxia may be hypothesized.

Composites and porous scaffolds produced with biodegradable natural polymers are very promising constructs which show high biocompatibility and suitable mechanical properties, with the possibility to be functionalized with growth factors involved in bone formation. For this purpose, alginate/hydroxyapatite (Alg/HAp) composite scaffolds using a novel production design were successfully developed and tested for their biocompatibility and osteoconductive properties in vitro. Redox homeostasis is crucial for dental pulp stem cell (DPSC) differentiation and mineralized matrix deposition, and interleukin-6 (IL-6) was found to be involved not only in immunomodulation but also in cell proliferation and differentiation. In the present study, we evaluated molecular pathways underlying the intracellular balance between redox homeostasis and extracellular matrix mineralization of DPSCs in the presence of composite scaffolds made of alginate and nano-hydroxyapatite (Alg/HAp). Prostaglandin-2 (PGE2) and IL-6 secretion was monitored by ELISA assays, and protein expression levels were quantified by Western blotting. This work aims to demonstrate a relationship between DPSC capacity to secrete a mineralized matrix in the presence of Alg/HAp scaffolds and their immunomodulatory properties. The variation of the molecular axis Nrf2 (nuclear factor erythroid 2-related factor 2)/PGE2/IL-6 suggests a tight intracellular balance between oxidative stress responses and DPSC differentiation in the presence of Alg/HAp scaffolds.

Cannabidiol (CBD) and cannabigerol (CBG) are Cannabis sativa terpenophenols. Although CBD's effectiveness against neurological diseases has already been demonstrated, nothing is known about CBG. Therefore, a comparison of the effects of these compounds was performed in two experimental models mimicking the oxidative stress and neurotoxicity occurring in neurological diseases. Rat astrocytes were exposed to hydrogen peroxide and cell viability, reactive oxygen species production and apoptosis occurrence were investigated. Cortexes were exposed to K+ 60 mM depolarizing stimulus and serotonin (5-HT) turnover, 3-hydroxykinurenine and kynurenic acid levels were measured. A proteomic analysis and bioinformatics and docking studies were performed. Both compounds exerted antioxidant effects in astrocytes and restored the cortex level of 5-HT depleted by neurotoxic stimuli, whereas sole CBD restored the basal levels of 3-hydroxykinurenine and kynurenic acid. CBG was less effective than CBD in restoring the levels of proteins involved in neurotransmitter exocytosis. Docking analyses predicted the inhibitory effects of these compounds towards the neurokinin B receptor. Conclusion: The results in the in vitro system suggest brain non-neuronal cells as a target in the treatment of oxidative conditions, whereas findings in the ex vivo system and docking analyses imply the potential roles of CBD and CBG as neuroprotective agents.

Human second trimester Amniotic Fluid Stem Cells (hAFSCs) harbour the potential to differentiate into cells of each of the three germ layers and to form Embryoid Body (EB)-like aggregates, without inducing teratoma formation and with no ethical concerns. However, in spite of the number of reports on hAFSCs-EBs and their characterization, a thorough evaluation in light and electron microscopy of morphological and morphometric features of hAFSCs-EBs development in vitro has not been reported yet. Apart from a superficial layer of epithelial-like flat cells, displaying rare microvilli on the free surface, hAFSCs-EBs enclose inner material, abundant in vesicles and secretory granules, showing early characteristics of connective extracellular matrix dispersed among different types of inner cells. The observation of a number of microvesicles mainly represented by microparticles and, to a lower extent, by exosomes indicates the presence of a complex cellular communication system within this structure. According to morphological analysis, after 7 days of in vitro culture hAFSCs-EB appears as a well-organized corpuscle, sufficiently young to be a carrier of stemness and at the same time, when appropriately stimulated, able to differentiate. In fact, 7-day hAFSCs-EB represents itself an initial cellular transformation towards a specialized structure both in recording and in providing different stimuli from the surrounding environment, organizing structures and cells towards a differentiation fate.

Cannabidiol (CBD) and cannabigerol (CBG) are non-psychotropic terpenophenols isolated from Cannabis sativa, which, besides their anti-inflammatory/antioxidant effects, are able to inhibit, the first, and to stimulate, the second, the appetite although there are no studies elucidating their role in the hypothalamic appetite-regulating network. Consequently, the aim of the present research is to investigate the role of CBD and CBG in regulating hypothalamic neuromodulators. Comparative evaluations between oxidative stress and food intake-modulating mediators were also performed.

Inducible nitric oxide synthase (iNOS) is a crucial enzyme involved in monocyte cell response towards inflammation, and it is responsible for the production of sustained amounts of nitric oxide. This free radical molecule is involved in the defense against pathogens; nevertheless, its continuous and dysregulated production contributes to the development of several pathological conditions, including inflammatory and autoimmune diseases. In the present study, we investigated the effects of two new iNOS inhibitors, i.e., 4-(ethanimidoylamino)-N-(4-fluorophenyl)benzamide hydrobromide (FAB1020) and N-{3-[(ethanimidoylamino)methyl]benzyl}-l-prolinamidedihydrochloride (CM554), on human LPS-stimulated monocytes, using the 1400 W compound as a comparison. Our results show that CM544 and FAB1020 are selective and decrease cytotoxicity, IL-6 secretion and LPS-stimulated monocyte migration. Furthermore, the modulation of iNOS, nitrotyrosine and Nrf2 were analyzed at the protein level. Based on the collected preliminary results, the promising therapeutic value of the investigated compounds emerges, as they appear able to modulate the pro-inflammatory LPS-stimulated response in the low micromolar range in human monocytes.