The promotion of chondrogenesis, osteogenesis, and adipogenesis of human mesenchymal stem cells by multiple growth factors incorporated into nanosphere-coated microspheres.

The induction of stem cell differentiation by drugs and growth factors has been the objective of many studies designed to develop methods for the formation of new tissues or the repair of degenerated tissues via transplantation. In this study, drugs and growth factors with high potential for use in tissue repair were embedded in human mesenchymal stem cells (hMSCs), which were then induced to differentiate into chondrogenic, osteogenic, and adipogenic lineages. Additionally, microspheres coated and loaded with the drugs and growth factors successfully proliferated and, as expected, induced the differentiation of transplanted hMSCs into the desired specific cell types. Furthermore, hMSCs transplanted in micro-typed scaffolds prevented changes in differentiation. RT-PCR and western blot analysis of the resultant cartilage, bone, and adipose tissues showed that a combination of drugs and different growth factor types induced the differentiation of transplanted hMSCs. Additionally, histology and immunohistochemistry showed that specific ECMs and proteins released from transplanted hMSCs were present within the nanosphere-coated microspheres. The results of this study show that the regulation of stem cell differentiation by drugs and growth factors might enable the fabrication of therapeutic materials for the delivery of stem cells that are simpler to use, less expensive, and more easily controlled than the delivery systems currently available.

Chondrogenesis of human mesenchymal stem cells in fibrin constructs evaluated in vitro and in nude mouse and rabbit defects models.

In this study, hMSCs encapsulated in a fibrin hydrogel containing heparinized NPs loaded with TGF-ß3 (100 ng/ml), or TGF-ß3 (100 ng/ml) alone, were subjected to growth factor release and denaturation tests at one, two and four weeks in in vitro culture systems. Additionally, stem cell differentiation was assessed via RT-PCR, real-time quantitative PCR (qPCR), histology, and immunohistochemical assays. In the in vivo studies with nude mouse, when transplanted into nude mice, hMSCs embedded in fibrin hydrogels survived and proliferated more readily in those samples containing TGF-ß3-loaded NPs, or TGF-ß3 alone, compared to those containing only NPs or the fibrin hydrogel alone. Additionally, RT-PCR, real-time qPCR, histology, Western blotting, and immunohistochemistry analyses revealed that chondrocyte-specific extracellular matrix (ECM) genes and their proteins were expressed at high levels by hMSCs embedded in hydrogels containing TGF-ß3-loaded NPs. Finally, the results observed in the rabbit animal model treated with hMSCs embedded in a fibrin hydrogel containing TGF-ß3-loaded NPs were also evaluated by the RT-PCR, real-time qPCR, histology, Western blotting, and immunohistochemistry analyses. The in vitro and in vivo results indicated that transplanted hMSCs together with TGF-ß3 may constitute a clinically efficient method for the regeneration of hyaline articular cartilage.

Auxiliary role for D-alanylated wall teichoic acid in Toll-like receptor 2-mediated survival of Staphylococcus aureus in macrophages.

We previously reported that Staphylococcus aureus avoids killing within macrophages by exploiting the action of Toll-like receptor 2 (TLR2), which leads to the c-Jun N-terminal kinase (JNK)-mediated inhibition of superoxide production. To search for bacterial components responsible for this event, a series of S. aureus mutants, in which the synthesis of the cell wall was interrupted, were screened for the level of JNK activation in macrophages. In addition to a mutant lacking the lipoproteins that have been suggested to act as a TLR2 ligand, two mutant strains were found to activate the phosphorylation of JNK to a lesser extent than the parental strain, and this defect was recovered by acquisition of the corresponding wild-type genes. Macrophages that had phagocytosed the mutant strains produced more superoxide than those engulfing the parental strain, and the mutant bacteria were more efficiently killed in macrophages than the parent. The genes mutated, dltA and tagO, encoded proteins involved in the synthesis of D-alanylated wall teichoic acid. Unlike a cell wall fraction rich in lipoproteins, D-alanine-bound wall teichoic acid purified from the parent strain by itself did not activate JNK phosphorylation in macrophages. These results suggest that the d-alanylated wall teichoic acid of S. aureus modulates the cell wall milieu for lipoproteins so that they effectively serve as a ligand for TLR2.