Recommendations from the COST action CA17116 (SPRINT) for the standardization of perinatal derivative preparation and in vitro testing.

Many preclinical studies have shown that birth-associated tissues, cells and their secreted factors, otherwise known as perinatal derivatives (PnD), possess various biological properties that make them suitable therapeutic candidates for the treatment of numerous pathological conditions. Nevertheless, in the field of PnD research, there is a lack of critical evaluation of the PnD standardization process: from preparation to in vitro testing, an issue that may ultimately delay clinical translation. In this paper, we present the PnD e-questionnaire developed to assess the current state of the art of methods used in the published literature for the procurement, isolation, culturing preservation and characterization of PnD in vitro. Furthermore, we also propose a consensus for the scientific community on the minimal criteria that should be reported to facilitate standardization, reproducibility and transparency of data in PnD research. Lastly, based on the data from the PnD e-questionnaire, we recommend to provide adequate information on the characterization of the PnD. The PnD e-questionnaire is now freely available to the scientific community in order to guide researchers on the minimal criteria that should be clearly reported in their manuscripts. This review is a collaborative effort from the COST SPRINT action (CA17116), which aims to guide future research to facilitate the translation of basic research findings on PnD into clinical practice.

Role of Epigenetics and Metabolomics in Predicting Endothelial Dysfunction in Type 2 Diabetes.

Type 2 diabetes (T2D) is a worldwide health problem and cardiovascular disease (CVD) is a leading cause of morbidity and mortality in T2D patients, making the prevention of CVD onset a major priority. It is therefore crucial to optimize diagnosis and treatment to reduce this burden. Endothelial dysfunction is one of the most important prognostic factors for CVD progression, thus novel approaches to identify the early phase of endothelial dysfunction may lead to specific preventive measures to reduce the occurrence of CVD. Nowadays, multiomics approaches have provided unprecedented opportunities to stratify T2D patients into endotypes, improve therapeutic treatment and outcome and amend the survival prediction. Among omics strategies, epigenetics and metabolomics are gaining increasing interest. Recently, a dynamic correlation between metabolic pathways and gene expression through chromatin remodeling, such as DNA methylation, has emerged, indicating new perspectives on the regulatory networks impacting cellular processes. Thus, a better understanding of epigenetic-metabolite relationships can provide insight into the physiological processes altered early in the endothelium that ultimately head to disease development. Here, recent studies on epigenetics and metabolomics related to CVD prevention potentially useful to identify disease biomarkers, as well as new therapies hopefully targeting the early phase of endothelial dysfunction are highlighted.

Essential Oils from Mediterranean Plants Inhibit In Vitro Monocyte Adhesion to Endothelial Cells from Umbilical Cords of Females with Gestational Diabetes Mellitus.

Essential oils (EOs) are mixtures of volatile compounds belonging to several chemical classes derived from aromatic plants using different distillation techniques. Recent studies suggest that the consumption of Mediterranean plants, such as anise and laurel, contributes to improving the lipid and glycemic profile of patients with diabetes mellitus (DM). Hence, the aim of the present study was to investigate the potential anti-inflammatory effect of anise and laurel EOs (AEO and LEO) on endothelial cells isolated from the umbilical cord vein of females with gestational diabetes mellitus (GDM-HUVEC), which is a suitable in vitro model to reproduce the pro-inflammatory phenotype of a diabetic endothelium. For this purpose, the Gas Chromatographic/Mass Spectrometric (GC-MS) chemical profiles of AEO and LEO were first analyzed. Thus, GDM-HUVEC and related controls (C-HUVEC) were pre-treated for 24 h with AEO and LEO at 0.025% v/v, a concentration chosen among others (cell viability by MTT assay), and then stimulated with TNF-α (1 ng/mL). From the GC-MS analysis, trans-anethole (88.5%) and 1,8-cineole (53.9%) resulted as the major components of AEO and LEO, respectively. The results in C- and GDM-HUVEC showed that the treatment with both EOs significantly reduced: (i) the adhesion of the U937 monocyte to HUVEC; (ii) vascular adhesion molecule-1 (VCAM-1) protein and gene expression; (iii) Nuclear Factor-kappa B (NF-κB) p65 nuclear translocation. Taken together, these data suggest the anti-inflammatory efficacy of AEO and LEO in our in vitro model and lay the groundwork for further preclinical and clinical studies to study their potential use as supplements to mitigate vascular endothelial dysfunction associated with DM.

Pro­differentiating compounds for human intervertebral disc cells are present in Violina pumpkin leaf extracts.

Intervertebral disc (IVD) degeneration (IDD) is closely associated with inflammation, oxidative stress and loss of the discogenic phenotype, which current therapies are unable to reverse. In the present study, the effects of acetone extract from Violina pumpkin (Cucurbita moschata) leaves on degenerated IVD cells were investigated. IVD cells were isolated from the degenerated disc tissue of patients undergoing spinal surgery and were exposed to acetone extract and three major thin layer chromatography subfractions. The results revealed that, in particular, the cells benefited from exposure to subfraction Fr7, which consisted almost entirely of p­Coumaric acid. Western blot and immunocytochemical analysis showed that Fr7 induced a significant increase in discogenic transcription factors (SOX9 and tricho­rhino­phalangeal syndrome type I protein, zinc finger protein), extracellular matrix components (aggrecan, collagen type II), cellular homeostasis and stress response regulators, such as FOXO3a, nuclear factor erythroid 2­related factor 2, superoxide dismutase 2 and sirtuin 1. Two important markers related to the presence and activity of stem cells, migratory capacity and OCT4 expression, were assessed by scratch assay and western blotting, respectively, and were significantly increased in Fr7­treated cells. Moreover, Fr7 counteracted H2O2­triggered cell damage, preventing increases in the pro­inflammatory and anti­chondrogenic microRNA (miR), miR­221. These findings strengthen the hypothesis that adequate stimuli can support resident cells to repopulate the degenerated IVD and restart the anabolic machinery. Taken together, these data contribute to the discovery of molecules potentially effective in slowing the progression of IDD, a disease for which there is currently no effective treatment. Moreover, the use of part of a plant, the pumpkin leaves, which is usually considered a waste product in the Western world, indicated that it contains substances with potential beneficial effects on human health.

CAR-T-Derived Extracellular Vesicles: A Promising Development of CAR-T Anti-Tumor Therapy.

Extracellular vesicles (EVs) are a heterogenous population of plasma membrane-surrounded particles that are released in the extracellular milieu by almost all types of living cells. EVs are key players in intercellular crosstalk, both locally and systemically, given that they deliver their cargoes (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells, crossing biological barriers. Those mechanisms further trigger a wide range of biological responses. Interestingly, EV phenotypes and cargoes and, therefore, their functions, stem from their specific parental cells. For these reasons, EVs have been proposed as promising candidates for EV-based, cell-free therapies. One of the new frontiers of cell-based immunotherapy for the fight against refractory neoplastic diseases is represented by genetically engineered chimeric antigen receptor T (CAR-T) lymphocytes, which in recent years have demonstrated their effectiveness by reaching commercialization and clinical application for some neoplastic diseases. CAR-T-derived EVs represent a recent promising development of CAR-T immunotherapy approaches. This crosscutting innovative strategy is designed to exploit the advantages of genetically engineered cell-based immunotherapy together with those of cell-free EVs, which in principle might be safer and more efficient in crossing biological and tumor-associated barriers. In this review, we underlined the potential of CAR-T-derived EVs as therapeutic agents in tumors.

Innovation in the Development of Synthetic and Natural Ocular Drug Delivery Systems for Eye Diseases Treatment: Focusing on Drug-Loaded Ocular Inserts, Contacts, and Intraocular Lenses.

Nowadays, ocular drug delivery still remains a challenge, since the conventional dosage forms used for anterior and posterior ocular disease treatments, such as topical, systemic, and intraocular administration methods, present important limitations mainly related to the anatomical complexity of the eye. In particular, the blood-ocular barrier along with the corneal barrier, ocular surface, and lacrimal fluid secretion reduce the availability of the administered active compounds and their efficacy. These limitations have increased the need to develop safe and effective ocular delivery systems able to sustain the drug release in the interested ocular segment over time. In the last few years, thanks to the innovations in the materials and technologies employed, different ocular drug delivery systems have been developed. Therefore, this review aims to summarize the synthetic and natural drug-loaded ocular inserts, contacts, and intraocular lenses that have been recently developed, emphasizing the characteristics that make them promising for future ocular clinical applications.

Diabetes and Its Cardiovascular Complications: Potential Role of the Acetyltransferase p300.

Diabetes has been shown to accelerate vascular senescence, which is associated with chronic inflammation and oxidative stress, both implicated in the development of endothelial dysfunction. This condition represents the initial alteration linking diabetes to related cardiovascular (CV) complications. Recently, it has been hypothesised that the acetyltransferase, p300, may contribute to establishing an early vascular senescent phenotype, playing a relevant role in diabetes-associated inflammation and oxidative stress, which drive endothelial dysfunction. Specifically, p300 can modulate vascular inflammation through epigenetic mechanisms and transcription factors acetylation. Indeed, it regulates the inflammatory pathway by interacting with nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit (NF-κB p65) or by inducing its acetylation, suggesting a crucial role of p300 as a bridge between NF-κB p65 and the transcriptional machinery. Additionally, p300-mediated epigenetic modifications could be upstream of the activation of inflammatory cytokines, and they may induce oxidative stress by affecting the production of reactive oxygen species (ROS). Because several in vitro and in vivo studies shed light on the potential use of acetyltransferase inhibitors, a better understanding of the mechanisms underlying the role of p300 in diabetic vascular dysfunction could help in finding new strategies for the clinical management of CV diseases related to diabetes.

Oleanolic acid rescues critical features of umbilical vein endothelial cells permanently affected by hyperglycemia.

Skin wound healing is a physiological process that involves several cell types. Among them, endothelial cells are required for inflammation resolution and neo-angiogenesis, both necessary for tissue restoration after injury. Primary human umbilical vein endothelial cells (C-HUVECs) are derived from the umbilical cord. When women develop gestational diabetes, chronic exposure to hyperglycemia induces epigenetic modifications in these cells (GD-HUVECs), leading to a permanent pro-inflammatory phenotype and impaired angiogenesis in contrast to control cells. Oleanolic acid (OA) is a bioactive triterpenoid known for its epithelial cell migration promotion stimulation and higher tensile strength of wounds. However, the potentially anti-inflammatory and pro-angiogenic properties of OA are still under investigation. We tested OA on C- and GD-HUVECs under inflammatory conditions induced by low levels of the inflammatory cytokine TNF-α. Reduced expression of adhesion molecules VCAM1, ICAM1, and SELE was obtained in OA-pre-treated C- and GD-HUVECs. Additionally, protein VCAM1 levels were also decreased by OA. Coherently, monocyte adhesion assays showed that a lower number of monocytes adhered to GD-HUVEC endothelium under OA pre-treatment when compared to untreated ones. It is noteworthy that OA improved angiogenesis parameters in both phenotypes, being especially remarkable in the case of GD-HUVECs, since OA strongly rescued their poor tube formation behavior. Moreover, endothelial cell migration was improved in C- and GD-HUVECs in scratch assays, an effect that was further confirmed by focal adhesion (FA) remodeling, revealed by paxillin staining on immunocytochemistry assays. Altogether, these results suggest that OA could be an emergent wound healing agent due to its capacity to rescue endothelial malfunction caused by hyperglycemia.

Effect of a 5-aminolevulinic acid gel and 660 nm red LED light on human oral osteoblasts: a preliminary in vitro study.

This study aimed to evaluate the effects of a new photodynamic protocol (ALAD-PDT) on primary human osteoblasts (hOBs). The ALAD-PDT protocol consists of a heat-sensitive gel with 5% 5-delta aminolevulinic acid commercialized as Aladent (ALAD), combined with 630 nm LED. For this purpose, the hOBs, explanted from human mandible bone fragments, were used and treated with different ALAD concentrations (10%, 50%, 100% v/v) incubated for 45 min and immediately afterwards irradiated with a 630 nm LED device for 7 min. The untreated and unirradiated cells were considered control (CTRL). The cellular accumulation of the photosensitizer protoporphyrin IX (PpIX), the proliferation, the alkaline phosphatase (ALP) activity, and the calcium deposition were assessed. All concentrations (10, 50, 100%) determined a significant increment of PpIX immediately after 45 min of incubation (0 h) with the highest peak by ALAD (100%). The consequent 7 min of light irradiation caused a slight decrease in PpIX. At 48 h and 72 h, any increment of PpIX was observed. The concentration 100% associated with LED significantly increased hOB proliferation at 48 h (+ 46.83%) and 72 h (+ 127.75%). The 50% and 100% concentrations in combination to the red light also stimulated the ALP activity, + 12.910% and + 14.014% respectively. The concentration 100% with and without LED was selected for the assessment of calcium deposition. After LED irradiation, a significant increase in calcium deposition was observed and quantified (+ 72.33%). In conclusion, the ALAD-PDT enhanced proliferation, the ALP activity, and mineralized deposition of human oral osteoblasts, highlighting a promising potential for bone tissue regeneration.

Perinatal derivatives: How to best characterize their multimodal functions in vitro. Part C: Inflammation, angiogenesis, and wound healing.

Perinatal derivatives (PnD) are birth-associated tissues, such as placenta, umbilical cord, amniotic and chorionic membrane, and thereof-derived cells as well as secretomes. PnD play an increasing therapeutic role with beneficial effects on the treatment of various diseases. The aim of this review is to elucidate the modes of action of non-hematopoietic PnD on inflammation, angiogenesis and wound healing. We describe the source and type of PnD with a special focus on their effects on inflammation and immune response, on vascular function as well as on cutaneous and oral wound healing, which is a complex process that comprises hemostasis, inflammation, proliferation (including epithelialization, angiogenesis), and remodeling. We further evaluate the different in vitro assays currently used for assessing selected functional and therapeutic PnD properties. This review is a joint effort from the COST SPRINT Action (CA17116) with the intention to promote PnD into the clinics. It is part of a quadrinomial series on functional assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer activities, anti-inflammation, wound healing, angiogenesis, and regeneration.

The Effects of 5% 5-Aminolevulinic Acid Gel and Red Light (ALAD-PDT) on Human Fibroblasts and Osteoblasts.

This study aimed to evaluate the effects of a new photodynamic protocol (ALAD-PDT), consisting of 5% 5-aminolevulinic acid-gel and 630 nm-LED, already used for antibacterial effects in the treatment of periodontitis, on human gingival fibroblasts (HGF) and primary human osteoblasts (HOB). HGF and HOB were incubated with different ALAD concentrations for 45 min, and subsequently irradiated with 630 nm-LED for 7 min. Firstly, the cytotoxicity at 24 h and proliferation at 48 and 72 h were assessed. Then the intracellular content of the protoporphyrin IX (PpIX) of the ROS and the superoxide dismutase (SOD) activity were investigated at different times. Each result was compared with untreated and unirradiated cells as the control. Viable and metabolic active cells were revealed at any concentrations of ALAD-PDT, but only 100-ALAD-PDT significantly enhanced the proliferation rate. The PpIX fluorescence significantly increased after the addition of 100-ALAD, and decreased after the irradiation. Higher ROS generation was detected at 10 min in HGF, and at 30 min in HOB. The activity of the SOD enzyme augmented at 30 min in both cell types. In conclusion, ALAD-PDT not only showed no cytotoxic effects, but had pro-proliferative effects on HGF and HOB, probably via ROS generation.

Estrogen Receptor 1 (ESR1) and the Wnt/ß-Catenin Pathway Mediate the Effect of the Coumarin Derivative Umbelliferon on Bone Mineralization.

Bone physiology is regulated by osteoblast and osteoclast activities, both involved in the bone remodeling process, through deposition and resorption mechanisms, respectively. The imbalance between these two phenomena contributes to the onset of bone diseases. Among these, osteoporosis is the most common metabolic bone disorder. The therapies currently used for its treatment include antiresorptive and anabolic agents associated with side effects. Therefore, alternative therapeutic approaches, including natural molecules such as coumarin and their derivatives, have recently shown positive results. Thus, our proposal was to investigate the effect of the coumarin derivative umbelliferon (UF) using an interesting model of human osteoblasts (hOBs) isolated from osteoporotic patients. UF significantly improved the activity of osteoporotic-patient-derived hOBs via estrogen receptor 1 (ESR1) and the downstream activation of ß-catenin pathway. Additionally, hOBs were co-cultured in microgravity with human osteoclasts (hOCs) using a 3D system bioreactor, able to reproduce the bone remodeling unit in bone loss conditions in vitro. Notably, UF exerted its anabolic role by reducing the multinucleated cells. Overall, our study confirms the potential efficacy of UF in bone health, and identified, for the first time, a prospective alternative natural compound useful to prevent/treat bone loss diseases such as osteoporosis.

Bioengineered Human Stromal Lenticule for Recombinant Human Nerve Growth Factor Release: A Potential Biocompatible Ocular Drug Delivery System.

Small incision lenticule extraction (SMILE), is a surgical procedure for the myopia correction, during which a corneal stromal lenticule is extracted. Given that we have previously demonstrated how this discarded tissue could be repurposed as a bio-scaffold for stromal engineering, this study aimed to explore its use as an ocular drug delivery system of active molecules, using neurotrophic factor Nerve Growth Factor (NGF). We employed human stromal lenticules directly collected from healthy donors undergoing SMILE. Following a sodium dodecylsulfate (SDS) treatment, decellularized lenticules were incubated with a suspension of polylactic-co-glycolic-acid (PLGA) microparticles (MPs) loaded with recombinant human NGF (rhNGF-MPs). Fluorescent MPs (Fluo-MPs) were used as control. Data demonstrated the feasibility to engineer decellularized lenticules with PLGA-MPs which remain incorporated both on the lenticules surface and in its stromal. Following their production, the in vitro release kinetic showed a sustained release for up to 1 month of rhNGF from MPs loaded to the lenticule. Interestingly, rhNGF was rapidly released in the first 24 h, but it was sustained up to the end of the experiment (1 month), with preservation of rhNGF activity (around 80%). Our results indicated that decellularized human stromal lenticules could represent a biocompatible, non-immunogenic natural scaffold potential useful for ocular drug delivery. Therefore, combining the advantages of tissue engineering and pharmaceutical approaches, this in vitro proof-of-concept study suggests the feasibility to use this scaffold to allow target release of rhNGF in vivo or other pharmaceutically active molecules that have potential to treat ocular diseases.

Effect of the Human Amniotic Membrane on the Umbilical Vein Endothelial Cells of Gestational Diabetic Mothers: New Insight on Inflammation and Angiogenesis.

One of the most relevant diabetes complications is impaired wound healing, mainly characterized by reduced peripheral blood flow and diminished neovascularization together with increased inflammation and oxidative stress. Unfortunately, effective therapies are currently lacking. Recently, the amniotic membrane (AM) has shown promising results in wound management. Here, the potential role of AM on endothelial cells isolated from the umbilical cord vein of gestational diabetes-affected women (GD-HUVECs), has been investigated. Indeed, GD-HUVECs in vivo exposed to chronic hyperglycemia during pregnancy compared to control cells (C-HUVECs) have shown molecular modifications of cellular homeostasis ultimately impacting oxidative and nitro-oxidative stress, inflammatory phenotype, nitric oxide (NO) synthesis, and bioavailability, thus representing a useful model for studying the mechanisms potentially supporting the role of AM in chronic non-healing wounds. In this study, the anti-inflammatory properties of AM have been assessed using a monocyte-endothelium interaction assay in cells pre-stimulated with tumor necrosis factor-α (TNF-α) and through vascular adhesion molecule expression and membrane exposure, together with the AM impact on the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB) pathway and NO bioavailability. Moreover, GD-HUVEC migration and tube formation ability were evaluated in the presence of AM. The results showed that AM significantly reduced TNF-α-stimulated monocyte-endothelium interaction and the membrane exposure of the endothelial vascular and intracellular adhesion molecules (VCAM-1 and ICAM-1, respectively) in both C- and GD-HUVECs. Strikingly, AM treatment significantly improved vessel formation in GD-HUVECs and cell migration in both C- and GD-HUVECs. These collective results suggest that AM positively affects various critical pathways in inflammation and angiogenesis, thus providing further validation for ongoing clinical trials in diabetic foot ulcers.

Dietary Bioactives: Their Role in the Prevention and Treatment of Cardiovascular and Metabolic Bone Diseases.

Cardiovascular and metabolic bone diseases are demanding health problems with high morbidity and mortality [...].

Circulating Extracellular Vesicles Are Increased in Newly Diagnosed Celiac Disease Patients.

Extracellular vesicles (EVs) are a class of circulating entities that are involved in intercellular crosstalk mechanisms, participating in homeostasis maintenance, and diseases. Celiac disease is a gluten-triggered immune-mediated disorder, characterized by the inflammatory insult of the enteric mucosa following local lymphocytic infiltration, resulting in villous atrophy. The goal of this research was the assessment and characterization of circulating EVs in celiac disease patients, as well as in patients already on an adequate gluten-free regimen (GFD). For this purpose, a novel and validated technique based on polychromatic flow cytometry that allowed the identification and enumeration of different EV sub-phenotypes was applied. The analysis evidenced that the total, annexin V+, leukocyte (CD45+), and platelet (CD41a+) EV counts were significantly higher in both newly diagnosed celiac disease patients and patients under GFD compared with the healthy controls. Endothelial-derived (CD31+) and epithelial-derived (EpCAM+) EV counts were significantly lower in subjects under gluten exclusion than in celiac disease patients, although EpCAM+ EVs maintained higher counts than healthy subjects. The numbers of EpCAM+ EVs were a statistically significant predictor of intraepithelial leukocytes (IEL). These data demonstrate that EVs could represent novel and potentially powerful disease-specific biomarkers in the context of celiac disease.

Cytotoxic and Genotoxic Effects of Composite Resins on Cultured Human Gingival Fibroblasts.

The aim of the study was to evaluate the cytotoxic and genotoxic potential of five commercially available dental composite resins (CRs), investigating the effect of their quantifiable bisphenol-A-glycidyl-methacrylate (Bis-GMA) and/or triethylene glycol dimethacrylate (TEGDMA) release. Experiments were performed using the method of soaking extracts, which were derived from the immersion of the following CRs in the culture medium: Clearfil-Majesty-ES-2, GrandioSO, and Enamel-plus-HRi (Bis-GMA-based); Enamel-BioFunction and VenusDiamond (Bis-GMA-free). Human Gingival Fibroblasts (hGDFs) were employed as the cellular model to mimic in vitro the oral cavity milieu, where CRs simultaneously release various components. Cell metabolic activity, oxidative stress, and genotoxicity were used as cellular outcomes. Results showed that only VenusDiamond and Enamel-plus-HRi significantly affected the hGDF cell metabolic activity. In accordance with this, although no CR-derived extract induced a significantly detectable oxidative stress, only VenusDiamond and Enamel-plus-HRi induced significant genotoxicity. Our findings showed, for the CRs employed, a cytotoxic and genotoxic potential that did not seem to depend only on the actual Bis-GMA or TEGDMA content. Enamel-BioFunction appeared optimal in terms of cytotoxicity, and similar findings were observed for Clearfil-Majesty-ES-2 despite their different Bis-GMA/TEGDMA release patterns. This suggested that simply excluding one specific monomer from the CR formulation might not steadily turn out as a successful approach for improving their biocompatibility.

Influence of Nano, Micro, and Macro Topography of Dental Implant Surfaces on Human Gingival Fibroblasts.

Current research on dental implants has mainly focused on the influence of surface roughness on the rate of osseointegration, while studies on the development of surfaces to also improve the interaction of peri-implant soft tissues are lacking. To this end, the first purpose of this study was to evaluate the response of human gingival fibroblasts (hGDFs) to titanium implant discs (Implacil De Bortoli, Brazil) having different micro and nano-topography: machined (Ti-M) versus sandblasted/double-etched (Ti-S). The secondary aim was to investigate the effect of the macrogeometry of the discs on cells: linear-like (Ti-L) versus wave-like (Ti-W) surfaces. The atomic force microscopy (AFM) and scanning electron microscopy (SEM) analysis showed that the Ti-S surfaces were characterized by a significantly higher micro and nano roughness and showed the 3D macrotopography of Ti-L and Ti-W surfaces. For in vitro analyses, the hGDFs were seeded into titanium discs and analyzed at 1, 3, and 5 days for adhesion and morphology (SEM) viability and proliferation (Cck-8 and MTT assays). The results showed that all tested surfaces were not cytotoxic for the hGDFs, rather the nano-micro and macro topography favored their proliferation in a time-dependent manner. Especially, at 3 and 5 days, the number of cells on Ti-L was higher than on other surfaces, including Ti-W surfaces. In conclusion, although further studies are needed, our in vitro data proved that the use of implant discs with Ti-S surfaces promotes the adhesion and proliferation of gingival fibroblasts, suggesting their use for in vivo applications.

Pro-Osteogenic Properties of Violina pumpkin (Cucurbita moschata) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures.

Traditional medicines rely mainly on use of plant extracts to mitigate or treat a wide range of disorders, including those that affect skeletal homeostasis. In this study, we investigated for the first time the potential pro-osteogenic effects of hexane, acetone and methanol extracts of the leaves of Cucurbita moschata, a very popular pumpkin cultivar in Western countries. We found that in Cucurbita moschata leaves, there are acetone-extractable substances-in particular, fatty acids such as 13-OH-9Z,11E,15E-octadecatrienoic acid (PU-13OH-FA), which is capable of both stimulating the function of human primary osteoblasts, which are responsible for bone formation, and inhibiting the differentiation of human osteoclasts, which are responsible for bone resorption. This dual effect was monitored by analyzing Runx2 expression, deposition of mineralized matrix, ALP activity, TRAP and actin ring staining respectively. This study suggests that bioactive chemicals from Cucurbita moschata leaves are potentially suitable as therapeutics for managing metabolic bone disorders such as osteoporosis and rheumatoid arthritis, and promoting tissue healing and functional recovery after bone fractures. The data we obtained increase knowledge on the biological activities of Cucurbita moschata, and in particular underline the potential benefits of consuming leaves which are a part of the plant currently little considered in the Western world.

Three-Dimensional Co-Culture System of Human Osteoblasts and Osteoclast Precursors from Osteoporotic Patients as an Innovative Model to Study the Role of Nutrients: Focus on Vitamin K2.

Several natural compounds, such as vitamin K2, have been highlighted for their positive effects on bone metabolism. It has been proposed that skeletal disorders, such as osteoporosis, may benefit from vitamin K2-based therapies or its regular intake. However, further studies are needed to better clarify the effects of vitamin K2 in bone disorders. To this aim, we developed in vitro a three-dimensional (3D) cell culture system one step closer to the bone microenvironment based on co-culturing osteoblasts and osteoclasts precursors obtained from bone specimens and peripheral blood of the same osteoporotic patient, respectively. Such a 3-D co-culture system was more informative than the traditional 2-D cell cultures when responsiveness to vitamin K2 was analyzed, paving the way for data interpretation on single patients. Following this approach, the anabolic effects of vitamin K2 on the osteoblast counterpart were found to be correlated with bone turnover markers measured in osteoporotic patients' sera. Overall, our data suggest that co-cultured osteoblasts and osteoclast precursors from the same osteoporotic patient may be suitable to generate an in vitro 3-D experimental model that potentially reflects the individual's bone metabolism and may be useful to predict personal responsiveness to nutraceutical or drug molecules designed to positively affect bone health.