PLA Nanofibers for Microenvironmental-Responsive Quercetin Release in Local Periodontal Treatment.

The management of periodontitis remains a vital clinical challenge due to the interplay between the microorganisms of the dental biofilm and the host inflammatory response leading to a degenerative process in the surrounding tissues. Quercetin (QUE), a natural flavonol found in many foods, including apples, onions and tea, has exhibited prolonged and strong antibiofilm and anti-inflammatory effects both in vitro and in vivo. However, its clinical application is limited by its poor stability and water solubility, as well as its low bioavailability. Thus, in the present study, electrospun polylactic acid (PLA) nanofibers loaded with different amounts (5−10% w/w) of QUE were produced to rapidly respond to the acidic microenvironment typical of periodontal pockets during periodontal disease. This strategy demonstrated that PLA-QUE membranes can act as a drug reservoir releasing high QUE concentrations in the presence of oral bacterial infection (pH < 5.5), and thus limiting Pseudomonas aeruginosa PAO1 and Streptococcus mutans biofilm maturation. In addition, released QUE exerts antioxidant and anti-inflammatory effects on P. gingivalis Lipopolysaccharide (LPS)-stimulated human gingival fibroblast (HGFs). The reported results confirmed that PLA-QUE membranes could inhibit subgingival biofilm maturation while reducing interleukin release, thereby limiting host inflammatory response. Overall, this study provided an effective pH-sensitive drug delivery system as a promising strategy for treating periodontitis.

Antimicrobial and Antibiofilm Activity of Curcumin-Loaded Electrospun Nanofibers for the Prevention of the Biofilm-Associated Infections.

Curcumin extracted from the rhizome of Curcuma Longa has been used in therapeutic preparations for centuries in different parts of the world. However, its bioactivity is limited by chemical instability, water insolubility, low bioavailability, and extensive metabolism. In this study, the coaxial electrospinning technique was used to produce both poly (ε-caprolactone) (PCL)-curcumin and core-shell nanofibers composed of PCL and curcumin in the core and poly (lactic acid) (PLA) in the shell. Morphology and physical properties, as well as the release of curcumin were studied and compared with neat PCL, showing the formation of randomly oriented, defect-free cylindrical fibers with a narrow distribution of the dimensions. The antibacterial and antibiofilm potential, including the capacity to interfere with the quorum-sensing mechanism, was evaluated on Pseudomonas aeruginosa PAO1, and Streptococcus mutans, two opportunistic pathogenic bacteria frequently associated with infections. The reported results demonstrated the ability of the Curcumin-loading membranes to inhibit both PAO1 and S. mutans biofilm growth and activity, thus representing a promising solution for the prevention of biofilm-associated infections. Moreover, the high biocompatibility and the ability to control the oxidative stress of damaged tissue, make the synthesized membranes useful as scaffolds in tissue engineering regeneration, helping to accelerate the healing process.

Regeneration of dentin-pulp complex: Effect of calcium-based materials on hDPSCs differentiation and gene expression.

OBJECTIVE: Dentin-pulp complex is object of interest in the regenerative endodontic field as well as the natural function of human dental pulp stem cells (hDPSCs) that may differentiate into specific cells able to repair and/or regenerate both hard and soft dental structures. The aim of the present study was to evaluate the capacity of hDPSCs to differentiate in odontoblastic-like cells by evaluating the expression of specific odontogenic-related genes and to prove the ability of treatment with calcium-based materials such as calcium carbonate (CaCO3), calcium hydroxide (Ca(OH)2), and mineral trioxide aggregate (MTA). METHODS: hDPSCs were obtained and isolated from a third molar of a young patient. Odontogenic-related gene expression was assessed unti1 28 days of culture as well as alkaline phosphatase activity (ALP). hDPSCs were cultured in odontoblastic-induction medium used as control, and in presence of different concentrations of CaCO3, Ca(OH)2, and MTA. RESULTS: The results demonstrated an upregulation in odontoblastic cell-related genes, in particular of the early differentiation marker known as matrix extracellular phosphoglycoprotein (MEPE), as well as increased ALP activity and the presence of calcium deposits, mainly by stimulation with calcium derivatives. In this regard, treatment of pulp tissue with CaCO3, Ca(OH)2 and even better with MTA seemed to be effective for dentinogenesis. SIGNIFICANCE: The ease of isolation of hDPSCs from discarded or extracted teeth offers a promising source of autologous cells that may be applied for regenerative purpose in combination with selected bioactive materials. However, further investigations should be conducted to confirm the obtained results.

An in-vitro study investigating the effect of air-abrasion bioactive glasses on dental adhesion, cytotoxicity and odontogenic gene expression.

OBJECTIVE: To assess the microtensile bond strength (MTBS) and interfacial characteristics of universal adhesives applied on dentine air-abraded using different powders. The analysis includes the cytotoxicity of the powders and their effect on odontogenic gene expression. METHODS: Sound human dentine specimens were air-abraded using bioglass 45S5 (BAG), polycarboxylated zinc-doped bioglass (SEL), alumina (AL) and submitted to SEM analysis. Resin composite was bonded to air-abraded or smear layer-covered dentine (SML) using an experimental (EXP) or a commercial adhesive (ABU) in etch&rinse (ER) or self-etch (SE) modes. Specimens were stored in artificial saliva (AS) and subjected to MTBS testing after 24 h and 10 months. Interfacial nanoleakage assessment was accomplished using confocal microscopy. The cytotoxicity of the powders was assessed, also the total RNA was extracted and the expression of odontogenic genes was evaluated through RT-PCR. RESULTS: After prolonged AS storage, specimens in the control (SML) and AL groups showed a significant drop in MTBS (p > 0.05), with degradation evident within the bonding interface. Specimens in BAG or SEL air-abraded dentine groups showed no significant difference, with resin-dentine interfaces devoid of important degradation. The metabolic activity of pulp stem cells was not affected by the tested powders. SEL and BAG had no effect on the expression of odontoblast differentiation markers. However, AL particles interfered with the expression of the odontogenic markers. SIGNIFICANCE: The use of bioactive glass air-abrasion may prevent severe degradation at the resin-dentine interface. Unlike alumina, bioactive glasses do not interfere with the normal metabolic activity of pulp stem cells and their differentiation to odontoblasts.

Thermoresponsive Copolymer Nanovectors Improve the Bioavailability of Retrograde Inhibitors in the Treatment of Leishmania Infections.

Clinical manifestations of leishmaniasis range from self-healing, cutaneous lesions to fatal infections of the viscera. With no preventative Leishmania vaccine available, the frontline option against leishmaniasis is chemotherapy. Unfortunately, currently available anti-Leishmania drugs face several obstacles, including toxicity that limits dosing and emergent drug resistant strains in endemic regions. It is, therefore, imperative that more effective drug formulations with decreased toxicity profiles are developed. Previous studies had shown that 2-(((5-Methyl-2-thienyl)methylene)amino)-N-phenylbenzamide (also called Retro-2) has efficacy against Leishmania infections. Structure-activity relationship (SAR) analogs of Retro-2, using the dihydroquinazolinone (DHQZ) base structure, were subsequently described that are more efficacious than Retro-2. However, considering the hydrophobic nature of these compounds that limits their solubility and uptake, the current studies were initiated to determine whether the solubility of Retro-2 and its SAR analogs could be enhanced through encapsulation in amphiphilic polymer nanoparticles. We evaluated encapsulation of these compounds in the amphiphilic, thermoresponsive oligo(ethylene glycol) methacrylate-co-pentafluorostyrene (PFG30) copolymer that forms nanoparticle aggregates upon heating past temperatures of 30°C. The hydrophobic tracer, coumarin 6, was used to evaluate uptake of a hydrophobic molecule into PFG30 aggregates. Mass spectrometry analysis showed considerably greater delivery of encapsulated DHQZ analogs into infected cells and more rapid shrinkage of L. amazonensis communal vacuoles. Moreover, encapsulation in PFG30 augmented the efficacy of Retro-2 and its SAR analogs to clear both L. amazonensis and L. donovani infections. These studies demonstrate that encapsulation of compounds in PFG30 is a viable approach to dramatically increase bioavailability and efficacy of anti-Leishmania compounds.

Controlled delivery of the heparan sulfate/FGF-2 complex by a polyelectrolyte scaffold promotes maximal hMSC proliferation and differentiation.

Growth factors and other regulatory molecules are required to direct differentiation of bone marrow-derived human mesenchymal stem cells (hMSC) along specific lineages. However, the therapeutic use of growth factors is limited by their susceptibility to degradation, and the need to maintain prolonged local release of growth factor at levels sufficient to stimulate hMSC. The aim of this study was to investigate whether a device containing heparan sulfate (HS), which is a co-factor in growth factor-mediated cell proliferation and differentiation, could potentiate and prolong the delivery of fibroblast growth factor-2 (FGF-2) and thus enhance hMSC stimulation. To this aim, we synthesized cationic polyelectrolyte polymers covalently and non-covalently anchored to HS and evaluated their effect on hMSC proliferation. Polymers non-covalently bound to HS resulted in the release of an HS/FGF-2 complex rather than FGF-2 alone. The release of this complex significantly restored hMSC proliferation, which was abolished in serum-free medium and only partially restored by the release of FGF-2 alone as occurred with polymer covalently bound to HS. We also demonstrate that exposure to HS/FGF-2 during early growth but not during post-confluence is essential for hMSC differentiation down the fibroblast lineage, which suggests that both factors are required to establish the correct stem cell commitment that is necessary to support subsequent differentiation. In conclusion, the delivery platform described here is a step towards the development of a new class of biomaterial that enables the prolonged, non-covalent binding and controlled delivery of growth factors and cofactors without altering their potency.

Multifunctional Bioactive Resin for Dental Restorative Materials.

Resin-based composites are widely used as dental restorative materials due to their excellent properties. They must have high modulus, high hardness, and be chemically inert while minimizing moisture uptake. To fulfill these higher standard prerequisites and properties, continuous improvements in each of their components are required. This study develops novel composites with multiple biofunctions. Light-cured Bis-GMA/TEGDMA dental resin (RK)/layered double hydroxide intercalated with fluoride ions (LDH-F)/calcium bentonite (Bt) hybrid composites were prepared. The loading ratio of LDH-F to Bt was varied, ranging from 2.5/2.5 to 10/10 parts per hundred RK and structural, mechanical, and biological properties were studied. The incorporation of even small mass fractions (e.g., 2.5 wt % of LDH-F and 2.5 wt % of Bt) in RK dental resin significantly improved the mechanical properties of the pristine resin. The synthetized materials showed antibacterial and antibiofilm effects against three bacterial strains isolated from healthy volunteers' saliva (Streptococcus spp., Bacteroides fragilis, and Staphylococcus epidermidis) without affecting its ability to induce dental pulp stem cells differentiation into odontoblast-like cells. The capability to balance between the antibiofilm activity and dental pulp stem cells differentiation in addition with improved mechanical properties make these materials a promising strategy in preventive and restorative dentistry.

Compatibilized polymer blends based on PDLLA and PCL for application in bioartificial liver.

Porous scaffolds for tissue engineering applications based on poly(D,L-lactide)/poly(epsilon-caprolactone) compatibilized blends are described. The addition of a third polymer, namely poly( D, L-lactide-co-caprolactone) copolymer, has a profound effect on morphological properties of the blends scaffolds. In fact, the copolymer acts as compatibilizing agent and reduces the dimension of the dispersed phase of an order of magnitude. Such effect is function of the polymer composition. The efficiency of scaffolds obtained with poly( D, L-lactide) based blends containing 30% by weight of poly(epsilon-caprolactone) as dispersed phase toward hepatocytes has been tested by several biological assays and we found that they are able to promote a perfect adhesion, proliferation and growth of cells. Moreover, the addition of the copolymer significantly improves the biomedical performance of the scaffold.

Positively charged polymers modulate the fate of human mesenchymal stromal cells via ephrinB2/EphB4 signaling.

Understanding the mechanisms by which mesenchymal stromal cells (MSCs) interact with the physical properties (e.g. topography, charge, ζ-potential, and contact angle) of polymeric surfaces is essential to design new biomaterials capable of regulating stem cell behavior. The present study investigated the ability of two polymers (pHM1 and pHM3) with different positive surface charge densities to modulate the differentiation of MSCs into osteoblast-like phenotype via cell-cell ephrinB2/EphB4 signaling. Although pHM1 promoted the phosphorylation of EphB4, leading to cell differentiation, pHM3, characterized by a high positive surface charge density, had no significant effect on EphB4 activation or MSCs differentiation. When the MSCs were cultured on pHM1 in the presence of a forward signaling blocking peptide, the osteoblast differentiation was compromised. Our results demonstrated that the ephrinB2/EphB4 interaction was required for MSCs differentiation into an osteoblast-like phenotype and that the presence of a high positive surface charge density altered this interaction.

Effect of layered double hydroxide intercalated with fluoride ions on the physical, biological and release properties of a dental composite resin.

OBJECTIVES: The aim of this work was the preparation of a new fluoride-releasing dental material characterized by a release of fluoride relatively constant over time without any initial toxic burst effect. This type of delivery is obtained by a matrix controlled elution and elicits the beneficial effect of a low amount of fluoride on human dental pulp stem cells (hDPSCs) towards mature phenotype. METHODS: The modified hydrotalcite intercalated with fluoride ions (LDH-F), used as filler, was prepared via ion exchange procedure and characterized by X-ray diffraction and FT-IR spectroscopy. The LDH-F inorganic particles (0.7, 5, 10, 20wt.%) were mixed with a photo-activated Bis-GMA/TEGDMA (45/55wt/wt) matrix and novel visible-light cured composites were prepared. The dynamic thermo-mechanical properties were determined by dynamic mechanical analyzer. The release of fluoride ions in physiological solution was determined using a ionometer. Total DNA content was measured by a PicoGreen dsDNA quantification kit to assess the proliferation rate of hDPSCs. Alkaline phosphatase activity (ALP) was measured in presence of fluoride resins. RESULTS: Incorporation of even small mass fractions (e.g. 0.7 and 5wt.%) of the fluoride LDH in Bis-GMA/TEGDMA dental resin significantly improved the mechanical properties of the pristine resin, in particular at 37°C. The observed reinforcement increases on increasing the filler concentration. The release of fluoride ions resulted very slow, lasting months. ALP activity gradually increased for 28 days in hDPSCs cell grown, demonstrating that low concentrations of fluoride contributed to the cell differentiation. CONCLUSIONS: The prepared composites containing different amount of hydrotalcite filler showed improved mechanical properties, slow fluoride release and promoted hDPSCs cell proliferation and cell differentiation.

The genotoxicity of PEI-based nanoparticles is reduced by acetylation of polyethylenimine amines in human primary cells.

The ultrasmall size and unique properties of polymeric nanoparticles (NPs) have led to raising concerns about their potential cyto- and genotoxicity on biological systems. Polyethylenimine (PEI) is a highly positive charged polymer and is known to have varying degree of toxic effect to cells based on its chemical structure (i.e., amount of primary and secondary amine). Herein, drug delivery carriers such as PEI-PLGA nanoparticles (PEI-NPs) and acetylated PEI-PLGA nanoparticles (AcPEI-NPs) were utilized to examine the effect of acetylation on NPs biocompatibility and genotoxicity, using human primary cells as in vitro model. Cell uptake of NPs was characterized along with their effects on cellular viability. The results indicate that both NPs showed an equivalent behavior in terms of uptake and biocompatibility. In depth analysis of NP uptake on cell biology evidenced that these nanoparticles induced dose dependant genotoxic effects. This phenomenon was significantly reduced by PEI acetylation. Endocytosed PEI-NPs trigger an oxidative stress on cells by inducing the production of reactive oxygen species (ROS), which cause DNA damage without apparently affecting cell viability. Thus, the genotoxicity of nanoparticles, that could be used as non-viral drug carriers, should be evaluated based on the intracellular level of ROS generation and DNA damage even in absence of a significant cell death.

Enhanced in vitro antitumor activity of a titanocene complex encapsulated into polycaprolactone (PCL) electrospun fibers.

PURPOSE: The purpose of this work was to achieve detailed biomaterials characterization of a drug delivery system for local cancer treatment based on electrospun titanocene trichloride-loaded resorbable polycaprolactone (PCL) fibers. METHODS: The PCL fibers were characterized for their structural, morphologic and physical properties. The drug release kinetics of the titanocene complex was investigated at different concentrations, to obtain a set of correlations between structure and tuneable release. After exposing cancer cells directly onto the surface of PCL fibers, the anti-proliferative effects of titanocene-loaded PCL were assessed by: (i) counting viable cells via live/dead staining methods, and (ii) analyzing cell apoptosis. RESULTS AND CONCLUSIONS: Titanocene concentration influenced fiber diameters reduced for PCL filled with titanocene. X-ray analysis suggested that the titanocene, encapsulated into the PCL fibers, is not allowed to crystallize and exists as amorphous aggregates into the fibers. The titanocene release curves presented two stages unrelated to PCL degradation: an initial burst release followed by a release linear with time, extending for a very long time. All of the titanocene-loaded fibers revealed sustained drug release properties suggesting their potential clinical applicability for the treatment of local cancer diseases.

The effect of the surface charge of hydrogel supports on thermophilic biohydrogen production.

The effect of the surface charge of different immobilizing hydrogels on biohydrogen production in batch cultures was investigated using a novel isolate associated to the genus Thermoanaerobacterium. Two crosslinked polysaccharide-based hydrogels and two acrylic hydrogels were tested as polymeric carriers for cell adsorption. Immobilization improved both substrate conversion and hydrogen cumulative production compared to the suspended culture, and a yield of 1.9 mol H(2)/mol glucose was observed after 24h for alginate-supported cultures. Cationic carriers dramatically increased cell immobilization, leading to markedly faster kinetics of substrate degradation and hydrogen production in batch operation, with a peak of 3.6 mol H(2)/mol glucose for the acrylic hydrogel HM92. Accumulation of gaseous and acidic metabolites inhibited further H(2) production, shifting the carbon flow to reduced end-products and biomass synthesis. Preliminary tests showed that all the tested hydrogels had good durability and allowed hydrogen production on repeated batch runs.

Cationic copolymers nanoparticles for nonviral gene vectors: synthesis, characterization, and application in gene delivery.

The major aim of nonviral delivery systems for gene therapy is to mediate high levels of gene expression with low toxicity. Nowadays, one of the most successful synthetic polycations used in gene delivery research is poly(ethylenimine) (PEI) in its high-molecular weight (HMW) branched form. However, PEI is not the ideal transfection agent in vivo because of its overwhelming cytotoxicity. To overcome its toxic effects with a minimal impact on transfection efficiency, PEI has been conjugated with several nonionic biocompatible polymers. Here, we describe the synthesis of nanosized particles consisting of HMW PEI (25 kDa) crosslinked with poly(epsilon-caprolactone) (PCL, 50-60 kDa), a biodegradable aliphatic polyester. PCL was modified by the insertion of glycidyl groups able to condense with the amines of PEI to chemically bind PEI onto PCL. The nanoparticles obtained have been characterized in relation to their physicochemical and biological properties, and the results are extremely promising in terms of low cell toxicity and high transfection efficiency. These biological effects might be related to the peculiar DNA binding to covalently connected polymeric nanoparticles, without the formation of entangled DNA/polymer-soluble aggregates.