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PURPOSE: The aim of this ex vivo study was to assess the ability to remove oral biofilm by different combinations of mechanical and chemical treatments on smooth and rough titanium surfaces, as well as their impact on osteoconduction. MATERIALS AND METHODS: Forty-eight sandblasted acid-etched (SLA) and 48 machined titanium disks were contaminated with oral bacterial biofilm and exposed to the following treatments: (1) titanium brush (TB), (2) TB + 40% citric acid (CA), (3) TB + 5.25% sodium hypochlorite (NaOCl), (4) air polishing with glycine powder (AP), (5) AP + 40% CA, and (6) AP + 5.25% NaOCl. Residual bacteria and chemical contamination were assessed using viable bacterial count assay, scanning electron microscopy (SEM), and x-ray spectroscopy (XPS). Human primary osteoblast (hOB) adhesion and osteocalcin (OC) release were also evaluated. RESULTS: The microbiologic, SEM, and XPS analysis indicate a higher biofilm removal efficiency of combined mechanical-chemical treatments compared with exclusively mechanical approaches, especially on SLA surfaces. SEM analysis revealed significant alterations of surface microtopography on the disks treated with TB, while no changes were observed after AP treatment. OC release by hOBs was mainly decreased on disks treated with CA and NaOCl. CONCLUSION: The combination of mechanical and chemical treatments provides effective oral biofilm removal on both SLA and machined implant surfaces. NaOCl and CA may have a negative effect on osteoblasts cultured on SLA samples.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy burdened by poor prognosis. While huge progress of immunotherapy has recently improved the outcome of B-cell malignancies, the lack of tumor-restricted T-cell antigens still hampers its progress in T-ALL. Therefore, innovative immunotherapeutic agents are eagerly awaited. To this end, we generated a novel asymmetric (2 + 1) bispecific T-cell engager (BTCE) targeting CD1a and CD3ε (CD1a x CD3ε) starting from the development of a novel mAb named UMG2. UMG2 mAb reacts against CD1a, a glycoprotein highly expressed by cortical T-ALL cells. Importantly, no UMG2 binding was found on normal T-cells. CD1a x CD3ε induced high T-cell mediated cytotoxicity against CD1a+ T-ALL cells in vitro, as demonstrated by the concentration-dependent increase of T-cell proliferation, degranulation, induction of cell surface activation markers, and secretion of pro-inflammatory cytokines. Most importantly, in a PBMC-reconstituted NGS mouse model bearing human T-ALL, CD1a x CD3ε significantly inhibited the growth of human T-ALL xenografts, translating into a significant survival advantage of treated animals. In conclusion, CD1a x CD3ε is a novel BTCE highly active against CD1a-expressing cortical-derived T-ALL cells suitable for clinical development as an effective therapeutic option for this rare and aggressive disease.

The ultraviolet (UV) component of solar radiation is the driving force of life on earth, but it can cause photoaging and skin cancer. In this study, we investigated the effects of the glucosamine-derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA) on human primary fibroblasts (FBs) stimulated in vitro with environmental levels of UVB radiation. FBs were irradiated with 0.04 J cm-2 UVB dose, which resulted a mild dosage as shown by the cell viability and ROS production measurement. This environmental UVB dose induced activation of MAP kinase ERK 1/2, the stimulation of c-fos and at lower extent of c-jun, and in turn AP-1-dependent up-regulation of pro-inflammatory factors IL-6 and IL-8 and suppression of collagen type I expression. On the contrary, 0.04 J cm-2 UVB dose was not able to stimulate metalloprotease production. NAPA treatment was able to suppress the up-regulation of IL-6 and IL-8 via the inhibition of MAP kinase ERK phosphorylation and the following AP-1 activation, and was able to attenuate the collagen type I down-regulation induced by the UVBs. Taken together, our results show that NAPA, considering its dual action on suppression of inflammation and stimulation of collagen type I production, represents an interesting candidate as a new photoprotective and photorepairing agents.

Mammary serine protease inhibitor or Maspin has been characterized as a class II tumor suppressor gene in several cancer types, among them prostate cancer (CaP). Androgen ablation is an effective therapy for CaP, but with short-term effectiveness, thus new therapeutic strategies are actively sought. The present study is aimed to explore the effects of a glucosamine derivative, 2-(N-Carbobenzyloxy) l -phenylalanylamido-2-deoxy-β- d -glucose (NCPA), on two CaP cell lines, PC3 and LNCaP. In particular we analyzed the impact of NCPA on Maspin production, cell viability and cell cycle progression and apoptosis/necrosis pathway activation in PC3 and LNCaP cell lines. NCPA is able to stimulate Maspin production in PC3 and not in LNCaP cell lines. NCPA blocks the PC3 cell cycle in G1 phase, by inhibiting Cyclin D1 production and induces the apoptosis, therefore interfering with aggressiveness of this androgen-insensitive cell line. Moreover, NCPA is able to induce the expression of Maspin in LNCaP cell line treated with androgen receptor inhibitor, Bicalutamide, and in turn to stimulate the apoptosis of these cells. These findings suggest that NCPA, stimulating the endogenous production of a tumor suppressor protein, could be useful in the design of new therapeutic strategies for treatment of CaP.

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA), was shown to inhibit the kinase activity of IKKα, one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKKα by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKKα and IKKtide, a 20 amino acid peptide substrate derived from IkBα kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKKα and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKKα kinase activity with an IC50 of 0.5 mM, to decrease the Km value from 0.337 mM to 0.402 mM and the Vmax from 0.0257 mM·min−1 to 0.0076 mM·min−1. The computational analyses indicate the region between the KD, ULD and SDD domains of IKKα as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between Km and Ki whereas there is a statistically significant difference between the two Vmax values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide.

Liquid fibrinogen is an injectable platelet concentrate rich in platelets, leukocytes, and fibrinogen obtained by blood centrifugation. The aim of this study was to analyze the release of different growth factors in the liquid fibrinogen at different times and to assess possible correlations between growth factors and cell counts. The concentration of transforming growth factor beta 1 (TGF-&beta, 1), platelet-derived growth factor-AB (PDGF-AB), platelet-derived growth factor-BB (PDGF-BB), bone morphogenetic protein 2 (BMP-2), fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF) released by liquid fibrinogen were examined with ELISA at three time points (T0, time of collection, T7, 7 days, T14, 14 days). The cellular content of the liquid fibrinogen and whole blood was also calculated for each volunteer. A mean accumulation of platelets of almost 1.5-fold in liquid fibrinogen compared to whole blood samples was found. An increase of TGF-&beta, 1, PDGF-AB, FGF-2, and VEGF levels was detected at T7. At T14, the level of TGF-&beta, 1 returned to T0 level, PDGF-AB amount remained high, the levels of FGF-2 and VEGF decreased with respect to T7, but remained higher than the T0 levels, PDGF-BB was high at all time points, BMP-2 level was low and remained constant at all time points. TGF-&beta, 1, PDGF-AB, and PDGF-BB showed a correlation with platelet amount, whereas BMP-2, FGF-2, and VEGF showed a mild correlation with platelet amount. Due to the high concentration of platelets, liquid fibrinogen does contain important growth factors for the regeneration of both soft and hard tissue. The centrifugation protocol tested in this study provides a valid solution to stimulate wound healing in oral and periodontal surgery.

Pegbovigrastim is a commercial long-acting analog of bovine granulocyte colony-stimulating factor (rbG-CSF) that promotes the increased count and functionality of polymorphonuclear cells in dairy cows around the time of parturition. We hypothesized that pegbovigrastim administered to periparturient cows at approximately seven days before parturition and within 24 hours after calving could affect the profiles of gene networks involved in leukocyte function. Blood was collected on Day 3 after calving from treated groups (pegbovigrastim (PEG), 13 Simmental (seven multiparous and six primiparous) and 13 Holstein (seven multiparous and six primiparous) cows) that received pegbovigrastim (Imrestor, Elanco Animal Health) and controls (CTR, 13 Simmental (seven multiparous and six primiparous) and 13 Holstein (six multiparous and seven primiparous) cows) that received saline solution. Blood from all cows was sampled from the jugular vein in a PAXgene Blood RNA System tube (Preanalytix, Hombrechtikon, Switzerland) for RNA extraction. The RT-qPCR analysis was performed to investigate a panel of 34 genes of interest, representing recognition, immune mediation, migration, cell adhesion, antimicrobial strategies, inflammatory cascade, oxidative pattern, and leukotrienes in whole blood leukocytes. Normalized data were subjected to the MIXED model of SAS (ver. 9.4) with treatment, breed, parity, and their interaction as fixed effects. Compared with CTR, whole blood leukocytes of PEG cows had higher expression of genes involved in recognition and immune modulation (CD14, CD16, MYD88, TLR2, and TLR4), cell adhesion (ITGB2, ITGAL, TLN1, SELL, SELPLG, and CD44), antimicrobial activity (MMP9, LTF, and LCN2), and inflammatory cascade (CASP1, TNFRSF1A, IL1B, IL1R, IL18, IRAK1, NLRP3, and S100A8). This suggested an improvement of migration, adhesion, and antimicrobial ability and an enhanced inflammatory response, which in turn could trigger immune cell activation and enhance function. Expression of SOD2 and ALOX5 was also greater in the PEG group. In contrast, compared with CTR cows, PEG led to lower expression of RPL13A, ALOX15, IL8, and TNF. Overall, leukocytes from Simmental compared with Holstein cows had greater expression of IDO1, RPL13A, ALOX5, CD44, CX3CR1, ITGB2, and TNFA, whereas expression of CD16 and TLR2 was lower. Overall, compared with multiparous cows, primiparous cows had higher expression of IL1B, IL18, MYD88, SELL, and TLR2 and lower expression of MMP9. Simmental cows seemed more sensitive to induction of the immune system after calving, as revealed by the greater abundance of genes involved in immune system adaptation, regardless of pegbovigrastim treatment. Primiparous cows undergoing a new stress condition with respect to older cows were characterized by leukocytes with a higher inflammatory response. In conclusion, pegbovigrastim led to higher expression levels of most genes involved in the processes investigated, suggesting a thorough activation of the immune machinery during the critical post-partum period.

Wound healing is a dynamic process that can be seriously delayed by many factors including infectious complications. The development of dressings with intrinsic wound healing activity and/or releasing bioactive compounds may help with addressing such an issue. In this study, hyaluronic acid (HA) at different percentages (1&ndash, 35%) was used to modify chitosan (CS) biological and physico-chemical properties in order to obtain 2D-matrices able to promote healing and protect from infection. HA incorporation in the CS matrix decreased film transparency and homogeneity, but improved film water uptake and surface wettability. The water vapor transmission rate (WVTR) increased up to a 5% HA content, where it reached the highest value (672 g/m2 day), and decreased for higher HA contents. At all of the tested HA concentrations, HA affected mechanical properties providing matrices more flexible than pure CS with benefit for wound care. Pure CS films permitted S. epidermidis adhesion and biofilm formation. That was not true for CS/HA matrices, where HA at concentrations equal to or greater than 5% was able to avoid S. epidermidis adhesion. Fibroblasts adhesion also took benefit from the HA presence in the film, especially at 5% content, where the best adhesion and proliferation was found.

Tissue engineering is a highly interdisciplinary field of medicine aiming at regenerating damaged tissues by combining cells with porous scaffolds materials. Scaffolds are templates for tissue regeneration and should ensure suitable cell adhesion and mechanical stability throughout the application period. Chitosan (CS) is a biocompatible polymer highly investigated for scaffold preparation but suffers from poor mechanical strength. In this study, graphene oxide (GO) was conjugated to chitosan at two weight ratios 0.3% and 1%, and the resulting conjugates were used to prepare composite scaffolds with improved mechanical strength. To study the effect of GO oxidation degree on scaffold mechanical and biological properties, GO samples at two different oxygen contents were employed. The obtained GO/CS scaffolds were highly porous and showed good swelling in water, though to a lesser extent than pure CS scaffold. In contrast, GO increased scaffold thermal stability and mechanical strength with respect to pure CS, especially when the GO at low oxygen content was used. The scaffold in vitro cytocompatibility using human primary dermal fibroblasts was also affected by the type of used GO. Specifically, the GO with less content of oxygen provided the scaffold with the best biocompatibility.

During the last decade the interest for the mesenchymal cells is growing due to their possible uses in therapies to treat certain degenerative pathologies. Mesenchymal stem cells have been found in the bone marrow and they have been shown to be responsible for bone repair and fat cells production. Mesenchymal stromal cells can be obtained from a wide variety of tissues in addition to bone marrow and can differentiate into many other cell types. The study of cell differentiation and programming provides new models for drug discovery and cell therapy that now overcomes gene therapy. Senescence, cancer development and degenerative diseases depend on mesenchymal cells contribution to tissue homeostasis. On the other hand, diet and life style are included among risk factors, which can contribute to the success of pharmacological treatments. This review focuses on nutrients from Mediterranean diet and supplements, which have been shown to influence mesenchymal stem cells and cells derived from them. Dietary intake of nutrients impairs both in vitro and in vivo observations, this review aims to gather the results about the effects of food compounds on mesenchymal cells from which adipocytes and osteoblasts derive. Amino acids and proteins, carbohydrates, lipids, fatty acids and vegetable secondary metabolites, differently act on mesenchymal cells bearing on modulation of gene expression and controlling the fate of cell lineages. Remarkable, the analysis of literature shows that the main effect of nutrients on mesenchymal cells is the stimulation of transcription factors which address the cells toward proliferation or differentiation. For instance, carbohydrates, simple or complex, and lipids appear to stimulate the PPAR receptors, whereas proteins and amino acids result to act on the mTOR system and they can also stimulate the MyoD-1 transcription factor and cooperating proteins. In conclusion, nutrients can promote cell growth and differentiation of mesenchymal cells.

The aim of the present study was to investigate how different extravirgin olive oils (EVOOs), obtained by blending Olea europea cultivars, could influence the cell growth, the response to inflammatory stimuli, and oxidative stress in a culture of the osteosarcoma cell line Saos-2.Three different extravirgin olive oils were physicochemically characterized, determining the free acidity, the oxidation status, the polyphenols content, and the antioxidative activity. Moreover, the effects on Saos-2 cell culture were determined, studying the mRNA expression level by real-time polymerase chain reaction (PCR) assays and the antioxidative activity using fluorescent probes.The cultivars used in the south of Italy, yield extravirgin oils with different amount of fatty acids and polyphenols, which counteract induction of proinflammatory cytokines and regulate free radical production in hydrogen peroxide-stimulated cells. In vitro analysis using the human osteoblast cell line Saos-2 showed that the addition of oils to cell culture simulated a hypoxic stress followed by a reoxygenation period, during which the antioxidant activity of extravirgin olive oils protected cells from oxidative damages. On the other hand, the mRNA expression levels of factors involved in inflammatory processes, cell growth recovery, and antioxidant response, as heme oxygenase-1, were differently stimulated by EVOOs. Moreover, peroxisome proliferator activated receptor γ (PPARγ) was differently modulated by EVOOs.These findings show that the blending of different extravirgin olive oil can impact an osteoblast cell line, in particular regarding cell growth recovery and oxidative stress.

Biocompatible and degradable poly(α-hydroxy acids) are one of the most widely used materials in scaffolds for tissue engineering. Nevertheless, they often need surface modification to improve interaction with cells. Aminolysis is a common method to increase the polymer hydrophilicity and to introduce surface functional groups, able to covalently link or absorb, through electrostatic interaction, bioactive molecules or macromolecules. For this purpose, multi-functional amines, such as diethylenediamine or hexamethylenediamine are used. However, common drawbacks are their toxicity and the introduction of positive charges on the surface. Thus, these kind of modified surfaces are unable to link directly proteins, such as collagens, a promising substrate for many cell types, in particular chondrocytes and osteoblasts. In this work, poly(L-lactide) (PLLA) film surface was labelled with negatively charged sulfonate groups by grafting taurine (TAU) through an aminolysis reaction. The novel modified PLLA film (PLLA-TAU) was able to interact directly with collagen. The reaction was carried out in mild conditions by using a solution of tetrabutylammonium salt of TAU in methanol. ATR-FTIR, XPS and contact angle measurements were used to verify the outcome of the reaction. After the exchange of tetrabutylamonium cation with Na+, collagen was absorbed on the TAU grafted PLLA film (PLLA-TAU-COLL). In vitro biological tests with human primary chondrocytes showed that PLLA-TAU and PLLA-TAU-COLL improved cell viability and adhesion, compared to the unmodified polymer, suggesting that these modifications make PLLA substrate suitable for cartilage repair.