The specific aims of this study are 1) to isolate and purify microvessels from the periosteum of the bones of bovine fetuses and 2) to compare the responses of endothelial cells grown from these microvessels, and of cultured bovine osteoblasts to parathyroid hormone, insulin, dexamethazone, prostaglandin E1, ascorbic acid, 1,25 dihydroxyvitamin D, platelet-derived fibroblast and endothelial cell growth factors and beta glycerol phosphate and 3) to determine how much and under what specific culture conditions periosteal endothelial cells modulate toward the osteoblastic phenotype. These differences will be measured by using: 3H-thymidine labeling for DNA synthesis, 3H-Proline labeling for collagen synthesis, 3H-methionine of 35SO4 labeling for non-collagenous proteins synthesis, cleavage of phenylphosphate for alkaline phosphatase activity, cyclic AMP accumulation as a response to parathyroid hormone and prostaglandin E1, alizarin red staining and electron microscopy to confirm matrix mineralization, and immuno-cytochemistry, to monitor modulations in phenotypic response. Periostea, 1"""""""" x 3"""""""" pieces, will be stripped from metatarsals of 6-9 month old fetal calves. The periosteal strips will be incubated in collagenase, homogenized and filtered through nylon mesh of decreasing pore size ranging from 200 to 100 Mum. Microvessels will be separated from free cells by a Percoll gradient, the microvessel layer being collected, washed, treated with collagenase and dipase and cultured on fibronectin or collagen-coated glass coverslips in 35 mm dishes. Factor VIII antibodies and antibodies to laminin and Type IV collagen will be used to test growing cultures for purity. Osteoblasts will be isolated by collagenase digestion from the outer cortical bone of 3-6 month old tibias and femurs. Cultures of skin fibroblasts and umbilical cord endothelial cells will serve as control cultures. All cells will be grown in M-199 with 4.5 g/1 glucose or MEM media, supplemented with 20% serum. Cultures of these cell types will be tested either at 5 days of culture, 24-48 hours subsequent to confluency (9-12 days) or after 15-18 days of culture. The results of this comparative study will clarify the responses of endothelial cells and osteoblasts to similar physiological stimuli and may provide evidence for or against the modulation of endothelial cells to osteoblasts. With this information, therapies for metabolic and genetic bone disease can be more clearly defined.
