Epidermal melanocytes metabolize extracellular nucleotides by purinergic enzymes.

The human epidermal melanocyte (hEM) are melanin-producing cells that provide skin pigmentation and protection against ultraviolet radiation. Although purinergic signaling is involved in skin biology and pathology, the presence of NTPDase members, as well as the rate of nucleotides degradation by melanocytes were not described yet. Therefore, in this study, we analyzed the expression of ectonucleotidases in hEM derived from discarded foreskin of male patients. The expression of purinergic enzymes was confirmed by mRNA and flow cytometry. Among the ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5´-nucleotidase were the ectoenzymes with higher expressions. The hydrolysis rate for ATP, ADP, and AMP was low in comparison to other primary cells already investigated. The amount of ATP in the culture medium was increased after a scratch wound and decreased to basal levels in 48 h, while the NTPDase1 and P2X7 expressions increased. Therefore, it is possible to suggest that after cell injury, the ATP released by hEM into the extracellular space will be hydrolyzed by ectonucleotidases as the NTPDase1 that will control the levels of nucleotides in the skin micro-environment.

Xanthan gum-based hydrogel containing nanocapsules for cutaneous diphenyl diselenide delivery in melanoma therapy.

The aim of this study was to further evaluate the antitumoral effect of (PhSe)2-loaded polymeric nanocapsules (NC (PhSe)2) against a resistant melanoma cell line (SK-Mel-103) and develop a xanthan gum-based hydrogel intending the NC (PhSe)2 cutaneous application. For the in vitro evaluation, cells were incubated with free (PhSe)2 or NC (PhSe)2 (0.7-200 µM) and after 48 h the MTT assay, propidium iodide uptake (necrosis marker) and nitrite levels were assessed. The hydrogels were developed by thickening of the NC (PhSe)2 suspension or (PhSe)2 solution with xanthan gum and characterized in terms of average diameter, polydispersity index, pH, drug content, spreadability, rheological profiles and in vitro permeation in human skin. The results showed that NC (PhSe)2 provided a superior antitumoral effect in comparison to free (PhSe)2 (IC50 value of 47.43 µM and 65.05 µM, respectively) and increased the nitrite content. Both compound forms induced propidium iodide uptake, suggesting a necrosis-related pathway could be involved in the cytotoxic action of (PhSe)2. All hydrogels showed pH values around 7, drug content close to the theoretical values (5 mg/g) and mean diameter in the nanometric range. Besides, formulations were classified as non-Newtonian flow with pseudoplastic behavior and suitable spreadability factor. Skin permeation studies revealed that the compound content was higher for the nano-based hydrogel in the dermis layer, demonstrating its superior permeation, achieved by the compound encapsulation. It is the first report on an adequate formulation development for cutaneous application of NC (PhSe)2 that could be used as an adjuvant treatment in melanoma therapy.

Bioglass 45S5: Structural characterization of short range order and analysis of biocompatibility with adipose-derived mesenchymal stromal cells in vitro and in vivo.

Bioactive glasses have potential applications in the field of regenerative medicine due to their bioactivity that permits interaction with both hard and soft tissues. In the same way, mesenchymal stromal cells (MSCs) have been experimentally tested as part of engineered constructs considering their self-renewal and multipotent capacities. However, to design an association, it is crucial to investigate the physical properties of bioglass 45S5, as well as its biocompatibility. Therefore, we investigated the structural short range order of the stoichiometric 45S5, by obtaining its total structure factors (S(K)) and total pair distribution function G(r). The in vitro compatibility of human MSCs with 45S5 was verified by viability, morphometry and osteoinduction assays, F-actin staining and scanning electron (SEM) analysis. The compatibility outcome was verified through a subcutaneous implantation in a murine model by grafting the 45S5 as a scaffold for allogeneic MSCs. The cell-substrate modulation includes the maintenance of the MSC viability and osteoinduction potential after being exposed to the 45S5 extract. A low spreading during cell adhesion was detected. Both normal actin cytoskeleton organization and nuclei irregularities were observed, besides an increase of hydroxyapatite (HA) depositions around cells. Cells showed satisfactory compatibility patterns when growing over 45S5 for 7, 30 and 90 days. The implant did not show any apparent toxicity for organs, or strong immunogenic reactions, and it was accompanied by a dense capsule formation around the graft. Our results indicate that MSCs can grow in the long term on the 45S5 while maintaining their characteristics. This fact, together with a non-toxicity to animals means that the 45S5 can be implemented in pre-clinical trials aiming MSC's transplantation leading to further bone and tissue repair.

Comparison of Human Denuded Amniotic Membrane and Porcine Small Intestine Submucosa as Scaffolds for Limbal Mesenchymal Stem Cells.

Blinding corneal scarring is usually treated with allogeneic graft tissue. Nevertheless, the global shortage of donors leaves millions of patients in need of therapy. Traditional tissue engineering strategies involves the combination of cells, growth factors, and scaffolds that can supply cellular biological components allowing to restore the tissue function. The mesenchymal stem cells found in the limbal stroma (L-MSCs) have a self-renewal potential for multilineage differentiation. Thus, in this work we compared the potential of human amniotic membrane (hAM) and porcine small intestine submucosa (SIS) as scaffolds for L-MSCs, aiming at potential applications in corneal regeneration. For that, L-MSCs were seeded on hAM and SIS and we analyzed their viability, actin cytoskeleton, nuclei morphology, cell density, adhesion and surface markers. Our results showed that cells adhered and integrated into both membranes with a high cell density, an important characteristic for cell therapy. However, due to its transparency, the hAM allowed a better observation of L-MSCs. In addition, the analysis of surface markers expression on L-MSCs after two weeks showed a slight increase in the percentages of negative markers for MSCs grown on SIS membrane. Thus, considering a long-term culture, the hAM was considered better in maintaining the MSCs phenotype. Regarding the function as scaffolds, SIS was as efficient as the amniotic membrane, considering that these two types of biological matrices maintained the cell viability, actin cytoskeleton, nuclei morphology and mesenchymal phenotype, without causing cell death. Therefore, our data in vitro provides evidence for future pre-clinical studies were these membranes can be used as a support to transport mesenchymal stem cells to the injured area, creating a kind of temporary curative, allowing the release of bioactive molecules, such as cytokines and growth factors and then promoting the tissue regeneration, both in human and veterinary medicine.