Analysis of TGF-beta production by fusing and nonfusing mouse cranial sutures in vitro

The role of transforming growth factor beta (TGF-beta) in the regulation of cranial suture fusion has been studied by various qualitative techniques s uch as in situ hybridization and immunohistochemistry. Although the relativ e expression of TGF-beta isoforms has been assessed in these studies, incre ased expression of TGF-beta has not been demonstrated in a quantitative fas hion. Therefore, the purpose of this study was to quantify TGF-beta product ion by fusing (posterofrontal [PF]) and nonfusing (sagittal) mouse sutures using two different quantitative TGF-beta assays. The PF and sagittal sutur es of 25-day-old mice were harvested and cultured separately in vitro. Cult ure media conditioned for 48 hours were collected after 3, 6, 9, 12, 15, 18 , 21, 24, 27, and 30 days of culture, and total TGF-beta production was ass essed using a TGF-beta bioassay. For a quantitative TGF-beta 1 immunoassay, media conditioned for 48 hours were collected after 3, 5, 7, 9, 14, 22, an d 28 days of culture. The TGF-beta bioassay revealed large amounts of total TGF-beta activity in both PF and sagittal sutures during the first week of culture, with decreasing amounts thereafter. Absolute TGF-beta activity in conditioned media collected from PF sutures at several early time points w as higher than those obtained from sagittal sutures; however, these differe nces were not statistically significant. The results of the TGF-beta 1 immu noassay (enzyme-linked immunosorbent assay) were similar to the bioassay in that the highest TGF-beta 1 levels were noted during the first week of cul ture period and decreased thereafter. Analysis of variance of these samples , however, revealed significantly more TGF-beta 1 protein in samples collec ted from the PF suture compared with the sagittal suture on days 3 and 5 of culture (p < 0.05). TGF-beta 1 levels in the conditioned media obtained fr om PF sutures remained elevated compared with the sagittal suture on days 7 and 9; however, these differences were not statistically significant. Incr eased production of TGF-beta in the conditioned media of fusing PF sutures is the first such quantitative demonstration of growth factor upregulation during suture fusion and supports the hypothesis that IGF-beta expression m ay be important in cranial suture fusion.