Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells.

Human adipose tissue represents an abundant reservoir of stromal cells with potential utility for tissue engineering. The current study demonstrates the ability of human adipose tissue-derived stromal cells to display some of the hallmarks of osteoblast differentiation in vitro. Following treatment with ascorbate, beta-glycerophosphate, dexamethasone, and 1,25 dihydroxy vitamin D(3), adipose tissue-derived stromal cells mineralize their extracellular matrix based on detection of calcium phosphate deposits using Alizarin Red and von Kossa histochemical stains. Fourier transform infrared analysis demonstrates the apatitic nature of these crystals. Mineralization is accompanied by increased expression or activity of the osteoblast-associated proteins osteocalcin and alkaline phosphatase. These and other osteoblast-associated gene markers are detected based on polymerase chain reaction. In contrast, the adipocyte gene markers--leptin, lipoprotein lipase, and peroxisome proliferator activated receptor gamma2--are reduced under mineralization conditions, consistent with the reciprocal relationship postulated to exist between adipocytes and osteoblasts. The current work supports the presence of a multipotent stromal cell population within human extramedullary adipose tissue. These findings have potential implications for human bone tissue bioengineering.

Therapeutic treatment of DMBA-induced mammary tumors with PPAR ligands.

The objective of this study was to evaluate the ability of troglitazone (a thiazolidinedione) and Wy-14,643 (a clofibrate) to inhibit progression of non-detectable and detectable mammary tumors in rats induced by 7,12 dimethylbenz(a)anthracene (DMBA) when compared to those receiving no treatment or tamoxifen. Although not as effective as tamoxifen in decreasing overall tumor incidence, Wy-14,643 reduced the percentage and number of malignant tumors that developed when compared to both troglitazone and control. Treatment of detectable tumors with either Wy-14,643 or troglitazone induced regression or stasis of total tumor volume in 40-50% of the animals, compared to only 10% in control and 65% in tamoxifen treated animals. Moreover, each PPAR ligand was as effective as tamoxifen in preventing additional tumor development. In summary, both PPAR ligands were more effective than no treatment in preventing tumor progression once detected. However, only the PPAR-alpha activator, Wy-14,643 was able to reduce the development of malignant tumors when administered prior to detection.

A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein.

As the use of adenoviral vectors in gene therapy protocols increases, there is a corresponding need for rapid, accurate, and reproducible titer methods. Multiple methods currently exist for determining titers of recombinant adenoviral vector, including optical absorbance, electron microscopy, fluorescent focus assay, and the "gold standard" plaque assay. This paper introduces a novel flow cytometric method for direct titer determination that relies on the expression of the green fluorescent protein (GFP), a tracking marker incorporated into several adenoviral vectors. This approach was compared to the plaque assay using 10(-4)- to 10(-6)-fold dilutions of a cesium-chloride-purified, GFP expressing adenovirus (AdEasy + GFP + GAL). The two approaches yielded similar titers: 3.25 +/- 1.85 x 10(9) PFU/mL versus 3.46 +/- 0.76 x 10(9) green fluorescent units/(gfu/mL). The flow cytometric method is complete within 24 h in contrast to the 7 x 10 days required by the plaque assay. These results indicate that the GFU/mL is an alternative functional titer method for fluorescent-tagged adenoviral vectors.

The teratogenic effects of valproic acid in human chondrogenesis in vitro.

The anticonvulsant drug valproic acid (VPA) is a known teratogen in humans. In general, anticonvulsants effect major systems in the embryo causing craniofacial, cardiovascular, neurological, urogenital, and major and minor skeletal defects. The limb defects associated with in utero VPA exposure include digital hypoplasia, ectrodactyly, radial ray aplasia, and proximal phocomelia. Human studies are limited to case reports and to retrospective and/or prospective studies. Although animal studies have demonstrated a teratogenic effect of VPA on skeletogenesis, these doses were well above the human therapeutic dose which makes extrapolation from these studies to humans difficult. The purpose of this research was to evaluate the potential deleterious effects of VPA on chondrogenesis, a process that occurs in human limb formation. To accomplish this goal, human chondrocytes were cultured in a three dimensional agarose gel and treated with VPA. The use of this model system was a novel approach to evaluate the teratogenic potential of VPA during chondrogenesis. The influence of VPA on human chondrocytes was monitored using histochemical, immunocytochemical, and morphological techniques. There was a decrease in mitotic activity and the extracellular matrix was modified. At human therapeutic doses, immunofluorescence revealed that type II collagen was reduced, while type I collagen increased. In addition, the alcian blue-staining matrices (i.e., sulfated proteoglycans) were reduced. Moreover, the Golgi apparatus had swelling in the trans-face cisternae suggesting that proteoglycan synthesis may be altered.