Characterization of dense artificial connective tissues generated in a newly designed bioreactor.

Dense connective tissues were generated simultaneously by accumulating collagen fibrils and fibroblasts on stainless steel mesh using a bioreactor system that we designed. The advantage of our system is that the artificial connective tissues can be generated within 24 hr in the absence of inhibitors against matrix metalloproteinases. The fibroblasts were suspended in 200 mL of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.5 mg/mL type I collagen. The mixed solution was circulated in two types of bioreactors with cylindrical or vertical configurations to generate luminal or parenchymal tissues, respectively. The gelatin zymography showed that MMPs were first detected in the media after 8 hr from the start of circulation and reached the highest levels on day 3. Glossy white aggregates, 1-3 mm in thickness, depending on the circulation period, accumulated on mesh grids. Fibroblasts were embedded in the network of collagen fibrils and possessed oval nuclei with or without prominent cell processes to form a bipolar shape. We could not observe distended cisternae of the endoplasmic reticula, the Golgi apparatus, or exploded mitochondria, showing hypoxic degenerative alterations of fibroblasts in dense connective tissues. The artificial tissues generated by our system will be useful for biological studies and transplantation therapy.

Mammalian Sox15 gene: promoter analysis and implications for placental evolution.

Sox15 belongs to the Sox (Sry-type HMG box) protein family, which is involved in placental development and muscle regeneration. Previously, we showed that the Sox15 gene is highly expressed in the trophoblast giant cells of the mouse placenta. To elucidate the molecular mechanisms of the tissue-dependent transcription of the gene, we isolated approximately 2.2 kb of the 5'-flanking sequence upstream of the transcription initiation site and used it to construct luciferase reporter plasmids. A variety of cell lines, including trophoblast stem (TS) cells, placenta-derived Rcho-1 cells, and myoblast C2C12 cells, required the same 5'-flanking sequence, from -109 to -8, for basal promoter activity. In contrast, the sequences from -297 to -149 and from -148 to -110 were required for cell-type-specific promoter activity in myoblast-derived C2C12 cells and placenta-derived Rcho-1 and TS cells, respectively. These results suggest that the region from -297 to -8 of the Sox15 gene contains three distinct cis-elements that respectively control placenta-specific, myoblast-specific, and common basal expression. We also searched for Sox15 ortholog(s) in the genome databases of various vertebrate species. The results indicated that the three regulatory promoter sequences of the Sox15 genes were conserved among eutherian mammals during vertebrate evolution. Interestingly, the marsupial opossum gene that is closest to Sox15 appeared to be a pseudogene. These findings indicate that Sox15 may have been involved in placental evolution.