Hydrothermal processing of Sarcopeltis skottsbergii and study of the potential of its carrageenan for tissue engineering.

The red macroalga Sarcopeltis skottsbergii was subjected to hydrothermal processing to maximize the solubilization and recovery of carrageenan. Once isolated by ethanol precipitation, the carrageenan was further chemically (oligosaccharides composition), and structurally (TGA/DTG, DSC, HPSEC, FTIR-ATR, 1H NMR, SEM, etc.) characterized, as well as employed as source for the synthesis of hydrogels. The rheological properties of the carrageenan showed promising results as biopolymer for food applications due to the high molecular weight (500 kDa) presenting higher cell viability than 70 %. The evaluation of immune activation using ELISA test reflected a lower inflammatory response for concentrations of 0.025 % of carrageenan. Conversely, the cell viability of the synthesized hydrogels did not surpass 50 %. This work represents a considerable step forward to obtain a biopolymer from natural sources and a thorough study of their chemical, structural and biological properties.

Hyaluronic acid association with bacterial, fungal and viral infections: Can hyaluronic acid be used as an antimicrobial polymer for biomedical and pharmaceutical applications?

The relationships between hyaluronic acid (HA) and pathological microorganisms incite new understandings on microbial infection, tissue penetration, disease progression and lastly, potential treatments. These understandings are important for the advancement of next generation antimicrobial therapeutical strategies for the control of healthcare-associated infections. Herein, this review will focus on the interplay between HA, bacteria, fungi, and viruses. This review will also comprehensively detail and discuss the antimicrobial activity displayed by various HA molecular weights for a variety of biomedical and pharmaceutical applications, including microbiology, pharmaceutics, and tissue engineering.

Curcumin encapsulated polylactic acid nanoparticles embedded in alginate/gelatin bioinks for in situ immunoregulation: Characterization and biological assessment.

Curcumin is a known naturally occurring anti-inflammatory agent derived from turmeric, and it is commonly used as a herbal food supplement. Here, in order to overcome the inherent hydrophobicity of curcumin (Cur), polylactic acid (PLA) nanoparticles (NPs) were synthesised using a solvent evaporation, and an oil-in-water emulsion method used to encapsulate curcumin. Polymeric NPs also offer the ability to control rate of drug release. The newly synthesised NPs were analysed using a scanning electron microscope (SEM), where results show the NPs range from 50 to 250 nm. NPs containing graded amounts of curcumin (0 %, 0.5 %, and 2 %) were added to cultures of NIH3T3 fibroblast cells for cytotoxicity evaluation using the Alamar Blue assay. Then, the curcumin NPs were incorporated into an alginate/gelatin solution, prior to crosslinking using a calcium chloride solution (200 nM). These hydrogels were then characterised with respect to their chemical, mechanical and rheological properties. Following hydrogel optimization, hydrogels loaded with NP containing 2 % curcumin were selected as a candidate as a bioink for three-dimensional (3D) printing. The biological assessment for these bioinks/hydrogels were conducted using THP-1 cells, a human monocytic cell line. Cell viability and immunomodulation were evaluated using lactate dehydrogenase (LHD) and a tumour necrosis factor alpha (TNF-α) enzyme-linked immunosorbent (ELISA) assay, respectively. Results show that the hydrogels were cytocompatible and supressed the production of TNF-α. These bioactive hydrogels are printable, supress immune cell activation and inflammation showing immense potential for the fabrication of tissue engineering constructs.

Towards the Development of a Female Animal Model of T1DM Using Hyaluronic Acid Nanocoated Cell Transplantation: Refinements and Considerations for Future Protocols.

Female mice (Black 6 strain) (C57BL/6) aged 6 weeks were subject to low dose streptozotocin (STZ) treatment for five consecutive days to mimic type 1 diabetes mellitus (T1DM) with insulitis. At two weeks after STZ injections, evaluation of the elevated glucose levels was used to confirm diabetes. The diabetic mice were then subject to the transplantation of pancreatic ß-cells (MIN-6 line). Four groups of mice were studied. The first group was injected with saline-only acting as the placebo surgery control, also known as SHAM group, the second and third groups were injected with MIN-6 single cells and polyethylene glycol-modified dipalmitoyl-glycerol-phosphatidyl ethanolamine (PEG-DPPE) modified MIN-6 single cells (500 µg per 1.106 cells), respectively, while the fourth group was injected with hyaluronic acid (HA)-coated MIN-6 single cells (5 bilayers). At seven- and fourteen-days following transplantation, the mice were euthanised. The renal and pancreatic tissues were then collected and histologically analysed. The induction of diabetes in female mice, through five-consecutive daily STZ injections resulted in inconsistent glycaemic levels. Interestingly, this shows an incomplete diabetes induction in female mice, of which we attribute to sex dimorphism and hormonal interferences. Transplantation failure of free-floating encapsulated cells was unable to decrease blood glucose hyperglycaemia to physiological ranges. The result is attributed to deprived cell-cell interactions, leading to decreased ß-cells functionality. Overall, we highlight the necessity of refining T1DM disease models in female subjects when using multiple low-dose STZ injections together with transplantation protocols. Considerations need to be made regarding the different developmental stages of female mice and oestrogen load interfering with pancreatic ß-cells susceptibility to STZ. The use of pseudo islets, cell aggregates and spheroids are sought to improve transplantation outcome in comparison to free-floating single cells.

On the bacteriostatic activity of hyaluronic acid composite films.

Biofilm-related infections and contamination of biomaterials are major problems in the clinic. These contaminations are frequently caused by Staphylococcus aureus and are a pressing issue for implantable devices, catheters, contact lenses, prostheses, and wound dressings. Strategies to decrease contamination and biofilm related infections are vital for the success of implantable biomaterials. In this context, hyaluronic acid (HA), a naturally derived carbohydrate polymer, known to be biocompatible, degradable, and immunomodulatory, has shown some antimicrobial activity effects. Due to its poor structural stability, crosslinking strategies, and the incorporation of reinforcing fibres in HA gels is required to produce tailored gels for varying applications. Whilst carbon-based reinforcing materials, such as carbon nanofibers (CNF), present some intrinsic antimicrobial activity related to their high surface area, herein, a crosslinking strategy to enhance the mechanical properties and regulate the rate of degradation of HA is presented. We utilise bis-(ß-isocyanatoethyl) disulphide (BIED) as the crosslinker with the gel reinforced using 0.25 wt% CNF. The effects of CNF and BIED on the structural, mechanical, thermal, and swelling behaviour are examined. These new HA derivatives exhibit excellent mechanical properties and are capable of withstanding physiological stresses in vivo. Antimicrobial activity of the HA derivatives were tested against Staphylococcus aureus and the results reveal antibacterial effect. These carbohydrate based materials have potential application on surfaces within clinical settings where staphylococcal contamination is currently an issue.

Labile crosslinked hyaluronic acid via urethane formation using bis(ß-isocyanatoethyl) disulphide with tuneable physicochemical and immunomodulatory properties.

Here, we synthesise a new isocyanate-based crosslinker with the aim of obtaining novel tailored gels. The newly crosslinked hyaluronic acid (HA) gels were analysed in terms of rheological properties, chemical structure, swelling, biocompatibility and immunological response. Results show that the synthesised di-isocyanate outperforms other more conventional di-isocyanates due to its alkyl-based which has been shown to be slower to degrade by hydrolysis than aryl isocyanates. The alkyl-based isocyanate reacts readily with hydroxyl groups on the HA to form stable carbamate crosslinkages. Importantly and uniquely, a centrally located disulphide bond allows versatility with respect to reversible crosslinking and this allows potentially controlled layer-by-layer deposition of the HA for tailored pharmaceutical applications.

Radiological Advances in Pancreatic Islet Transplantation.

Type 1 diabetes mellitus (T1DM) is characterized by hyperglycemia, owing to the loss of pancreatic ß cells in response to an autoimmune reaction leading to a state of absolute insulin deficiency. T1DM treatment is shifting from exogenous insulin replacement therapy toward pancreatic ß-cell replacement, to restore physiologically responsive insulin secretion to variations in blood glucose levels. ß-cell replacement strategies include human whole pancreas transplantation, islet transplantation with cell encapsulation and bioengineered pancreas. Interventional radiology and imaging modalities including positron emission tomography, single-photon emission computed tomography, magnetic resonance imaging, ultrasonography, and molecular imaging are imperative to enable successful ß-cell replacement. Herein, the role of radiological modalities in the treatment of T1DM and its prospective use for noninvasive post-transplantation graft monitoring is discussed.

Influence of scaffold design on 3D printed cell constructs.

Additive manufacturing is currently receiving significant attention in the field of tissue engineering and biomaterial science. The development of precise, affordable 3D printing technologies has provided a new platform for novel research to be undertaken in 3D scaffold design and fabrication. In the past, a number of 3D scaffold designs have been fabricated to investigate the potential of a 3D printed scaffold as a construct which could support cellular life. These studies have shown promising results; however, few studies have utilized a low-cost desktop 3D printing technology as a potential rapid manufacturing route for different scaffold designs. Here six scaffold designs were manufactured using a Fused deposition modeling, a "bottom-up" solid freeform fabrication approach, to determine optimal scaffold architecture for three-dimensional cell growth. The scaffolds, produced from PLA, are coated using pullulan and hyaluronic acid to assess the coating influence on cell proliferation and metabolic rate. Scaffolds are characterized both pre- and postprocessing using water uptake analysis, mechanical testing, and morphological evaluation to study the inter-relationships between the printing process, scaffold design, and scaffold properties. It was found that there were key differences between each scaffold design in terms of porosity, diffusivity, swellability, and compressive strength. An optimal design was chosen based on these physical measurements which were then weighted in accordance to design importance based on literature and utilizing a design matrix technique. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 533-545, 2018.

Enhanced cell viability in hyaluronic acid coated poly(lactic-co-glycolic acid) porous scaffolds within microfluidic channels.

The concept of the present work is to produce porous optimised scaffolds of poly(lactic-co-glycolic acid) (PLGA) coated with hyaluronic acid (HA), to provide a suitable microenvironment for cellular proliferation. Freeze dried scaffolds were produced from PLGA with varying lactic acid and glycolic acid ratios along the polymer backbone, as follows: 50:50 ester terminated, 50:50 carboxylate end-group and 85:15 ester terminated. Subsequently, these scaffolds were immersed in crosslinked HA in order for the coating to enhance biological performance. Scaffolds were fully characterized with respect to surface morphology, physical and chemical properties. The biocompatibility of the scaffolds was firstly evaluated using standard L929 fibroblast cells in static culture and subsequently MCF-7 breast cancer cells were seeded on scaffolds which were incorporated within a microfluidic device. The results show that cells were attracted to and adhered to the scaffolds, with a higher affinity for HA coated scaffolds. In our system, cell viability was maintained up to 48h.

Cell based therapeutics in type 1 diabetes mellitus.

This review focuses on Type 1 diabetes mellitus (T1DM) and the role of bioengineering, nanotechnology and cell therapy in its treatment. T1DM is discussed in terms of its prevalence as well as the role of the extra cellular matrix (ECM) of the pancreas in its development and mode of action. Surface engineering strategies and the chemistries behind important cell encapsulation techniques, which are emerging from recent research in immunosuppression, are described. Key enabling technologies such as therapeutic agent immobilization on cells, oxygen releasing systems, gene delivery and bio imaging are assessed with respect to T1DM. These latest cell surface technologies provide unlimited possibilities for control of cell/cell and cell/ECM interactions, allowing the ability to confer "immune camouflage". Finally, we provide an outlook to the future of cell-based technologies for T1DM treatment and their likely deployment in clinical trials.

Association of electrospinning with electrospraying: a strategy to produce 3D scaffolds with incorporated stem cells for use in tissue engineering.

In tissue engineering, a uniform cell occupation of scaffolds is crucial to ensure the success of tissue regeneration. However, this point remains an unsolved problem in 3D scaffolds. In this study, a direct method to integrate cells into fiber scaffolds was investigated by combining the methods of electrospinning of fibers and bioelectrospraying of cells. With the associating of these methods, the cells were incorporated into the 3D scaffolds while the fibers were being produced. The scaffolds containing cells (SCCs) were produced using 20% poly(lactide-co-glycolide) solution for electrospinning and mesenchymal stem cells from deciduous teeth as a suspension for bioelectrospraying. After their production, the SCCs were cultivated for 15 days at 37°C with an atmosphere of 5% CO2. The 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide test demonstrated that the cells remained viable and were able to grow between the fibers. Scanning electron microscopy showed the presence of a high number of cells in the structure of the scaffolds and confocal images demonstrated that the cells were able to adapt and spread between the fibers. Histological analysis of the SCCs after 1 day of cultivation showed that the cells were uniformly distributed throughout the thickness of the scaffolds. Some physicochemical properties of the scaffolds were also investigated. SCCs exhibited good mechanical properties, compatible with their handling and further implantation. The results obtained in the present study suggest that the association of electrospinning and bioelectrospraying provides an interesting tool for forming 3D cell-integrated scaffolds, making it a viable alternative for use in tissue engineering.

Signaling mechanisms downstream of quinolinic acid targeting the cytoskeleton of rat striatal neurons and astrocytes.

The studies of signaling mechanisms involved in the disruption of the cytoskeleton homeostasis were performed in a model of quinolinic acid (QUIN) neurotoxicity in vitro. This investigation focused on the phosphorylation level of intermediate filament (IF) subunits of astrocytes (glial fibrillary acidic protein - GFAP) and neurons (low, medium and high molecular weight neurofilament subunits - NFL, NFM and NFH, respectively). The activity of the phosphorylating system associated with the IFs was investigated in striatal slices of rat exposed to QUIN or treated simultaneously with QUIN plus glutamate receptor antagonists, calcium channel blockers or kinase inhibitors. Results showed that in astrocytes, the action of 100 µM QUIN was mainly due to increased Ca(2+) influx through NMDA and L-type voltage-dependent Ca(2+) channels (L-VDCC). In neuronal cells QUIN acted through metabotropic glutamate receptor (mGluR) activation and influx of Ca(2+) through NMDA receptors and L-VDCC, as well as Ca(2+) release from intracellular stores. These mechanisms then set off a cascade of events including activation of PKA, PKCaMII and PKC, which phosphorylate head domain sites on GFAP and NFL. Also, Cdk5 was activated downstream of mGluR5, phosphorylating the KSP repeats on NFM and NFH. mGluR1 was upstream of phospholipase C (PLC) which, in turn, produced diacylglycerol (DAG) and inositol 3,4,5 triphosphate (IP3). DAG is important to activate PKC and phosphorylate NFL, while IP(3) contributed to Ca(2+) release from internal stores promoting hyperphosphorylation of KSP repeats on the tail domain of NFM and NFH. The present study supports the concept of glutamate and Ca(2+) contribution in excitotoxic neuronal damage provoked by QUIN associated to dysfunction of the cytoskeleton homeostasis and highlights the differential signaling mechanisms elicited in striatal astrocytes and neurons.

Diphenyl ditelluride induces hypophosphorylation of intermediate filaments through modulation of DARPP-32-dependent pathways in cerebral cortex of young rats.

We studied the effect of different concentrations of diphenyl ditelluride (PhTe)(2) on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and neurofilament (NF) subunits from cerebral cortex and hippocampus of rats during development. (PhTe)(2)-induced hypophosphorylation of GFAP and NF subunits only in cerebral cortex of 9- and 15-day-old animals but not in hippocampus. Hypophosphorylation was dependent on ionotropic glutamate receptors, as demonstrated by the specific inhibitors 10 µM DL-AP5 and 50 µM MK801, 100 µM CNQX and 100 µM DNQX. Also, 10 µM verapamil and 10 µM nifedipine, two L-voltage-dependent Ca(2+) channels (L-VDCC) blockers; 50 µM dantrolene, a ryanodine channel blocker, and the intracellular Ca(2+) chelator Bapta-AM (50 µM) totally prevented this effect. Results obtained with 0.2 µM calyculin A (PP1 and PP2A inhibitor), 1 µM Fostriecin a potent protein phosphatase 2A (PP2A) inhibitor, 100 µM FK-506 or 100 µM cyclosporine A, specific protein phosphatase 2B inhibitors, pointed to PP1 as the protein phosphatase directly involved in the hypophosphorylating effect of (PhTe)(2). Finally, we examined the activity of DARPP-32, an important endogenous Ca(2+)-mediated inhibitor of PP1 activity. Western blot assay using anti-DARPP-32, anti-pThr34DARPP-32, and anti-pThr75DARPP-32 antibodies showed a decreased phosphorylation level of the inhibitor at Thr34, compatible with inactivation of protein kinase A (PKA) by pThr75 DARPP-32. Decreased cAMP and catalytic subunit of PKA support that (PhTe)(2) acted on neuron and astrocyte cytoskeletal proteins through PKA-mediated inactivation of DARPP-32, promoting PP1 release and hypophosphorylation of IF proteins of those neural cells. Moreover, in the presence of Bapta, the level of the PKA catalytic subunit was not decreased by (PhTe)(2), suggesting that intracellular Ca(2+) levels could be upstream the signaling pathway elicited by this neurotoxicant and targeting the cytoskeleton.

Cross-talk among intracellular signaling pathways mediates the diphenyl ditelluride actions on the hippocampal cytoskeleton of young rats.

In the present report, we showed that diphenyl ditelluride (PhTe)(2) induced in vitro hyperphosphorylation of glial fibrillary acidic protein (GFAP), vimentin and neurofilament (NF) subunits in hippocampus of 21 day-old rats. Hyperphosphorylation was dependent on L-voltage dependent Ca(2+) channels (L-VDCC), N-methyl-d-aspartate (NMDA) and metabotropic glutamate receptors, as demonstrated by the specific inhibitors verapamil, DL-AP5 and MCPG, respectively. Also, dantrolene, a ryanodine channel blocker, EGTA and Bapta-AM, extra and intracellular Ca(2+) chelators respectively, totally prevented this effect. Activation of metabotropic glutamate receptors by (PhTe)(2) upregulates phospholipase C (PLC), producing inositol 1, 4, 5-trisphosphate (IP(3)) and diacylglycerol (DAG). Therefore, high Ca(2+) levels and DAG directly activate Ca(2+)/calmodulin-dependent protein kinase (PKCaMII) and protein kinase C (PCK), resulting in the hyperphosphorylation of Ser-57 in the carboxyl-terminal tail domain of the low molecular weight NF subunit (NF-L). Also, the activation of Erk1/2, and p38MAPK resulted in hyperphosphorylation of KSP repeats of the medium molecular weight NF subunit (NF-M). It is noteworthy that PKCaMII and PKC inhibitors prevented (PhTe)(2)-induced Erk1/2MAPK and p38MAPK activation as well as hyperphosphorylation of KSP repeats on NF-M, suggesting that PKCaMII and PKC could be upstream of this activation. Taken together, our results highlight the role of Ca(2+) as a mediator of the (PhTe)(2)-elicited signaling targeting specific phosphorylation sites on IF proteins of neural cells of rat hippocampus. Interestingly, this action shows a significant cross-talk among signaling pathways elicited by (PhTe)(2), connecting glutamate metabotropic cascade with activation of Ca(2+) channels. The extensively phosphorylated amino- and carboxyl- terminal sites could explain, at least in part, the neural dysfunction associated with (PhTe)(2) exposure.

Insucessos na implantologia oral / Failures in oral implantology

Os autores tecem alguns comentários sobre os insucessos na implantologia oral, evidenciando que o principal deles é a má formação do profissional nessa nobre àrea da Odontologia