Morphogenetic signals from chondrocytes promote chondrogenic and osteogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are potentially useful cells for musculoskeletal tissue engineering. However, controlling MSC differentiation and tissue formation in vivo remains a challenge. There is a significant need for well-defined and efficient protocols for directing MSC behaviors in vivo. We hypothesize that morphogenetic signals from chondrocytes may regulate MSC differentiation. In micromass culture of MSCs, incubation with chondrocyte-conditioned medium (CCM) significantly enhanced the production of cartilage specific matrix including type II collagen. In addition, incubation of MSCs with conditioned medium supplemented with osteogenic factors induced more osteogenesis and accumulation of calcium and increased ALP activity. These findings reveal that chondrocyte-secreted factors promote chondrogenesis as well as osteogenesis of MSCs during in vitro micromass culture. Moreover, when MSCs expanded with chondrocyte-conditioned medium were encapsulated in hydrogels and subsequently implanted into athymic mice, basophilic extracellular matrix deposition characteristic of neocartilage was evident. These results indicate that articular chondrocytes produce suitable morphogenetic factors that induce the differentiation program of MSCs in vitro and in vivo.

A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin.

The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1-DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC50 of DOX on the dissociation of Sp1-DNA complex is estimated to be 0.55 microM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques.