Human serum is a suitable supplement for the osteogenic differentiation of human adipose-derived stem cells seeded on poly-3-hydroxibutyrate-co-3-hydroxyvalerate scaffolds.

Human adipose-derived stem cells (hASCs) are currently a point of focus for bone tissue engineering applications. However, the ex vivo expansion of stem cells before clinical application remains a challenge. Fetal bovine serum (FBS) is largely used as a medium supplement and exposes the recipient to infections and immunological reactions. In this study, we evaluated the osteogenic differentiation process of hASCs in poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) scaffolds with the osteogenic medium supplemented with pooled allogeneic human serum (aHS). The hASCs grown in the presence of FBS or aHS did not show remarkable differences in morphology or immunophenotype. The PHB-HV scaffolds, which were developed by the freeze-drying technique, showed an adequate porous structure and mechanical performance as observed by micro-computed tomography, scanning electron microscopy (SEM), and compression test. The three-dimensional structure was suitable for allowing cell colonization, which was revealed by SEM micrographs. Moreover, these scaffolds were not toxic to cells as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The differentiation capacity of hASCs seeded on scaffolds was confirmed by the reduction of the proliferation, the alkaline phosphatase (AP) activity, expression of osteogenic gene markers (AP, collagen type I, Runx2, and osteocalcin), and the expression of bone markers, such as osteopontin, osteocalcin, and collagen type I. The osteogenic capacity of hASCs seeded on PHB-HV scaffolds indicates that this scaffold is adequate for cell growth and differentiation and that aHS is a promising supplement for the in vitro expansion of hASCs. In conclusion, this strategy seems to be useful and safe for application in bone tissue engineering.

Genotype and expression of the enhanced gree fluorescent protein in LEW-Tg (EGFP) F455.5/Rrrc rats / Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

The Green fluorescent protein (GFP) was first described after being extracted from Aequorea victoria in 1987; Since then, GFP and its derivatives have been widely used in several experiments as cell and protein marker. In the present study it was verified the genotype of the offspring from crosses between heterozygote Lewis LEW-Tg (EGFP) F455.5/Rrrc rats and analyzed the expression of the enhanced green fluorescent protein (EGFP) in different cell types and genotypes. The genotype of the offspring was assessed by PCR and analysis of EGFP expression in different cells and genotypes, including mesenchymal stem cells (MSC) derived from adipose tissue and calvarial osteoblast cells. Expression of EGFP was verified by flow cytometry, fluorescence microscopy, and immunostaining. Through these methods, it was identified the genotypes of the offspring and determined the levels of expression of EGFP in two cell types. A difference in expression between the (EGFP +/+) and (EGFP +/-) genotypes was also observed in addition to the presence of autofluorescence. Further studies on the natural fluorescence of cells with the (EGFP +/-) genotype and that induced by presence of the EGFP are necessary.

A proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP.