No abstract

Mesenchymal stem cells (MSCs) are capable of differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and differentiation factors. It is widely acknowledged that basic fibroblast growth factor (bFGF) modulates chondrogenic and osteogenic differentiation of MSCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate on the cellular and molecular level that supplementation of bFGF in different phases of cell culture leads to a strong enhancement of adipogenesis of MSCs, as induced by an adipogenic hormonal cocktail. In cultures receiving bFGF, mRNA expression of peroxisome proliferator-activated receptor c2 (PPARc2), a key transcription factor in adipogenesis, was upregulated even prior to adipogenic induction. In order to investigate the effects of bFGF on PPARc ligand-induced adipogenic differentiation, the thiazolidinedione troglitazone was administered as a single adipogenic inducer. Basic FGF was demonstrated to also strongly increase adipogenesis induced by troglitazone, that is, bFGF clearly increased the responsiveness of MSCs to a PPARc ligand.

Despite the clinical need for reconstructive and plastic surgery, the supply of engineered adipose tissue equivalents still remains a challenge. As yet, only preadipocytes have been applied as a cell material for the in vitro tissue engineering of fat. Herein, we report the establishment of a three-dimensional (3-D) long-term cell culture, using bone marrow-derived mesenchymal stem cells (MSCs) as an alternative cell source and custom-made poly(lactic-co-glycolic acid) (PLGA) scaffolds as a cell carrier. Cell-polymer constructs were cultivated for 4 weeks in both the absence and presence of basic fibroblast growth factor (bFGF), which was previously shown to strongly enhance the adipogenesis of MSCs in conventional 2-D short-term culture. A striking enhancement of the adipogenic differentiation of MSCs and tissue development caused by bFGF in the 3-D culture was observed by osmium tetroxide histology and scanning electron microscopy. At the molecular level, reflecting the increased accumulation of lipids, bFGF increased the enzymatic activity of glycerol-3-phosphate dehydrogenase, a late marker of adipogenesis, and the expression of adipocyte-specific genes peroxisome proliferator activated receptor-gamma2 (PPARgamma2) and glucose transporter-4 (GLUT4), as assessed by reverse transcription-polymerase chain reaction. This study demonstrates that the use of bone marrow-derived MSCs, especially in combination with bFGF, may represent a promising approach to adipose tissue engineering.

Because nanoparticles are finding uses in myriad biomedical applications, including the delivery of nucleic acids, a detailed knowledge of their interaction with the biological system is of utmost importance. Here the size-dependent uptake of gold nanoparticles (AuNPs) (20, 30, 50 and 80 nm), coated with a layer-by-layer approach with nucleic acid and poly(ethylene imine) (PEI), into a variety of mammalian cell lines is studied. In contrast to other studies, the optimal particle diameter for cellular uptake is determined but also the number of therapeutic cargo molecules per cell. It is found that 20 nm AuNPs, with diameters of about 32 nm after the coating process and about 88 nm including the protein corona after incubation in cell culture medium, yield the highest number of nanoparticles and therapeutic DNA molecules per cell. Interestingly, PEI, which is known for its toxicity, can be applied at significantly higher concentrations than its IC(50) value, most likely because it is tightly bound to the AuNP surface and/or covered by a protein corona. These results are important for the future design of nanomaterials for the delivery of nucleic acids in two ways. They demonstrate that changes in the nanoparticle size can lead to significant differences in the number of therapeutic molecules delivered per cell, and they reveal that the toxicity of polyelectrolytes can be modulated by an appropriate binding to the nanoparticle surface.