Silvana Gaiba

Characterization of human adipose-derived stem cells

PURPOSE There is a growing scientific interest in the plasticity and therapeutic potential of adipose-derived stem cells (ASCs), which are multipotent and abundant in adipose tissue and can differentiate in vitro into multiple lineages, including adipocytes, chondrocytes, osteoblasts, neural cells, endothelial cells and cardiomyocytes. The aim of this study was to isolate, cultivate and identify ASCs. METHODS Human adipose precursor cells were obtained from subcutaneous abdominal tissue. Recently dispersed cells were separated by density centrifugation gradient, cultured and then analyzed. RESULTS Human ASCs were able to replicate in our culture conditions. The cells maintained their phenotypes throughout the studied period on different passages confirming they suitability for in vitro cultivation. We also induced their adipogenic, osteogenic and chondrogenic differentiation, verifying their mesenchymal stem cells potentiality in vitro. Flow cytometry results showed that these cells expressed CD73, CD90 and CD105, (mesenchymal stem-cells markers), contrasting with the lack of expression of CD16, CD34 and CD45 (hematopoietic cells markers). CONCLUSION It was possible to isolate human adipose-derived stem cells by in vitro cultivation without adipogenic induction, maintaining their functional integrity and high proliferation levels. The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro.

Adipose-derived stem cells (ADSC) in the viability of a random pattern dorsal skin flap in rats

PURPOSE To evaluate the viability of random pattern dorsal skin flaps in rats after injection of adipose-derived stem cells (ADSC). METHODS Thirty five adult male Wistar EPM rats (weight 250-300 g) were distributed, at random, in two groups. I- Control (flap elevation with injection of saline solution) with fifteen animals and II- Experimental (flap elevation with injection of ADSC ) with fifteen animal. The ADSC were isolated from others five adult male rats. A dorsal skin flap measuring 10x4 cm was raised and a plastic barrier was placed between the flap and its bed in both groups and the injection (cells or saline solution) were perfomed immediately after the surgery. The percentage of flap necrosis was measured on the seventh postoperative day. RESULTS The ADSC were able to replicate in our culture conditions. We also induced their adipogenic, osteogenic and chondrogenic differentiation to verify their mesenchymal stem cells potentiality in vitro. The results were statistically significant showing that the ADSC decreased the area of necrosis (p<0.05). CONCLUSIONS The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro. The administration of adipose-derived stem cells was effective to increase the viability of the random random pattern dorsal skin flaps in rats.

Adipose-derived stem cells (ADSC) in the viability of random skin flap in rats

PURPOSE To evaluate the effects of the adipose-derived stem cells (ADSC) in the viability of random skin flap in rats. METHODS Thirty five adult male Wistar rats (weight 250-300 g) were used. ADSC were isolated from adult male rats (n=5). ADSC were separated, cultured and then analyzed. A dorsal skin flap measuring 10 x 4 cm was raised and a plastic barrier was placed between the flap and its bed. After the surgical procedure, the animals were randomized into two groups (n=15 each group), group control and group ADSC. In all groups the procedures were performed immediately after the surgery. The percentage of flap necrosis was measured on the seventh postoperative day. RESULTS The ADSC were able to replicate in our culture conditions. We also induced their adipogenic, osteogenic and chondrogenic differentiation, verifying their mesenchymal stem cells potentiality in vitro. The results were statistically significant showing that the ADSC decreased the area of necrosis (p<0.05). CONCLUSION The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro. The administration of adipose-derived stem cells was effective to increase the viability of the random skin flaps in rats.

Evaluation of antitumoral and antimicrobial activity of Morinda lcitrifolia L. grown in Southeast Brazil

PURPOSE To evaluate the antitumor and antimicrobial activity of ethanolic extract of Morinda citrifolia L. fruit cultivated in southeastern Brazil. METHODS Preparation ethanolic extract of the fruit of Morinda citrifolia L. Culture of melanoma cells B16-F10 for treatment with ethanolic extract of Morinda citrifolia L. fruit to determine cell viability by MTT and determination temporal effect of ethanolic extract fruit on the cell growth B16-F10 for 8 days. Evaluation of antimicrobial activity of ethanolic extract fruit against Staphylococcus aureus and Escherichia coli by determination of Minimum Inhibitory Concentration (MIC). RESULTS The ethanolic extract of Morinda citrifolia L. fruit (10mg/mL) decreased cellular activity and inhibited 45% the rate of cell proliferation of B16-F10 melanoma treated during period studied. The ethanolic extract of Morinda citrifolia L. fruit demonstrated antimicrobial activity inhibiting the growth of both microorganisms studied. Staphylococcus aureus was less resistant to ethanolic extract of Morinda citrifolia L. fruit than Escherichia coli, 1 mg/mL and 10 mg/mL, respectively. CONCLUSION What these results indicate that the ethanolic extract of the fruit of Morinda citrifolia L. showed antitumor activity with inhibition of viability and growth of B16-F10 cells and also showed antibacterial activity as induced inhibition of growth of Staphylococcus aureus and Escherichia coli.

The action of CGRP and SP on cultured skin fibroblasts

Background/purposeCalcitonin gene-related peptide (CGRP) is the most abundant neuropeptide in the skin, followed by substance P (SP), vasoactive intestinal peptide (VIP), and other neuropeptides in smaller amounts. The proliferative effect of neuropeptides on fibroblasts may affect wound healing and may be associated with hyperproliferative skin and mesenchymal disorders. Understanding the neuropeptidergic action on fibroblasts may provide relevant information to a deeper comprehension of the healing process. This study reviews the action of the main neuropeptides, CGRP and SP, on cultured human skin fibroblasts.MethodsA systematic literature search was conducted on Medline and Web of Science databases on December 21, 2013.ResultsA total of 74 articles were retrieved using the proposed search strategies and 3 were found in the references section of the selected articles. Thirteen of the retrieved articles studied the action of CGRP and SP on cultured human skin fibroblasts, 12 of which related to SP and 1 related to both CGRP and SP.ConclusionOnly one study was retrieved about the action of both CGRP and SP on cultured human skin fibroblasts. Further studies are necessary to investigate CGRP on skin fibroblasts and its role in the fibroplasia phase of wound healing.

Evaluation of antimicrobial and antitumoral activity of Garcinia mangostana L. (mangosteen) grown in Southeast Brazil

PURPOSE To characterize the anatomy of the fruit and leaf and the presence of phytocompounds. To evaluate the antitumor and antimicrobial activity of ethanolic extract of Garcinia mangostana L. (mangosteen) cultivated in southeastern Brazil. METHODS Anatomical characterization and histochemical reactions were performed for structural identification and the presence of phytocompounds. Preparation of ethanolic extract of the fruit, leaf and resin of mangosteen. Culture B16-F10 melanoma cells for treatment with mangosteen ethanolic extract to determine cell viability by MTT and genotoxic effect by comet assay. Evaluation by antimicrobial activity against Staphylococcus aureus and Escherichia coli by agar diffusion test and by determination of Minimum Inhibitory Concentration (MIC). RESULTS Our results showed many secretory canals in resin fruit and leaf; identifying lipids, starch, lignin and phenolic compounds. The leaf extract induced genotoxicity and apoptosis in B16-F10 cells, since the fragmentation of DNA in the comet assay. The ethanolic extract of mangosteen obtained in the resin, leaf and fruit showed antimicrobial activity against Staphylococcus aureus and Escherichia coli with a MIC at 0.1 mg/mL. CONCLUSION In conclusion, we have demonstrated both antimicrobial and antitumor activity of ethanol extract of mangosteen emphasizing its therapeutic potential in infectious diseases and in cancer, such as melanoma.

Development of experimental in vitro burn model

PURPOSE To propose an experimental burn model in NIH-3T3 cell line. METHODS Induction of thermal injury in cultures of mouse fibroblast - NIH-3T3- cell line and determination of cell viability by MTT and immunofluorescence. RESULTS The heating of the Petri dish increased proportionally to the temperature of the base and the time of exposure to microwave. In this in vitro burn model, using the cell line NIH-3T3 was observed drastic cellular injury with significant changes in cell viability and activity. It showed drastically modified cell morphology with altered membrane, cytoskeleton and nucleus, and low cellularity compared to the control group. CONCLUSION The burn model in vitro using the cell line NIH-3T3 was reproductive and efficient. This burn model was possible to determine significant changes in cell activity and decreased viability, with drastic change in morphology, cell lysis and death.

Gamma Radiation Induces p53-Mediated Cell Cycle Arrest in Bone Marrow Cells

The hematopoietic system is organized in a hierarchical manner in which rare hematopoietic stem cells initiate the hierarchy and have the ability to self-renew, proliferate and differentiate into different lineages of peripheral blood cells as well as to intermediate hematopoietic progenitor cells. Most hematopoietic stem cells are quiescent under steadystate conditions and function as a stock population to protect the hematopoietic system from exhaustion due to various stressful conditions. In contrast, hematopoietic progenitor cells are rapidly proliferating cells with limited self-renewal ability. The proliferation and differentiation of hematopoietic progenitor cells fulfills the requirements of normal hematopoiesis allowing the hematopoietic system to react promptly and effectively to meet the demand for increasing the output of mature cells during hematopoietic crisis such as loss of blood, hemolysis, infection, the depletion of HPCs by chemotherapy and/or radiotherapy (Reya, 2003; Weissman et al., 2001; Walkley et al., 2005).

Biological Effects Induced by Ultraviolet Radiation in Human Fibroblasts

As the most superficial body organ, skin plays an important role in protecting the body from environmental damage. The skin is composed of three layers: the epidermis, dermis and subcutaneous tissue. The epidermis, the outermost layer, has as main functions to protect the body against harmful environmental stimuli and to reduce fluid loss. It is a stratified squamous epithelium with several layers and its major cell type is the keratinocyte. This tissue is constantly being renewed by keratinization, a process of detachment of cornified cells (Blumenberg & Tomic-Canic, 1997). Located under the epidermis are the dermis and the dermal connective tissue, with extracellular matrix proteins such as collagen, elastic fibers, fibronectin, glycosaminoglycans and proteoglycans, which are produced and secreted into the extracellular space by fibroblasts, the major cell type found in this tissue (Makrantonaki & Zouboulis, 2007). The extracellular matrix proteins in the dermal connective tissue contribute for maintaining skin preservation and integrity (Hwang et al., 2011). Stromal fibroblasts play an important role in tissue homeostasis regulation and wound repair via protein synthesis and secretion of growth factors or cytokines of paracrine action with direct effect on proliferation and differentiation of adjacent epithelial tissues (Andriani et al., 2011). Solar ultraviolet (UV) radiation is a predictable epidemiologic risk factor for melanoma and non-melanoma skin cancers. (Katiyar et al., 2011). UV irradiation can impair cellular functions by directly damaging DNA to induce apoptosis (Waster & Ollinger, 2009). Among other things, longer UV wavelengths (UVB, UVA) induce oxidative stress and protein denaturation whereas short wavelength UV radiation (UVC) causes predominantly DNA damage to cells in the form of pyrimidine dimers, 6-4 photoproducts and apoptosis (Armstrong & Kricker, 2001; Gruijl et al., 2001). UVB irradiation damages skin cells by the formation of ROS (Reactive Oxygen Species) resulting in oxidative stress, an important mediator of damage to cell structures, including lipids and membranes, proteins, and DNA (Waster & Ollinger, 2009). However, it has less penetrating power than UVA and acts mainly on the epidermal basal layer of the skin. UVC, on the other hand, is extremely damaging to the skin because its wavelengths have enormous energy and induce genotoxic