GROWTH FACTOR REGULATION OF CRANIAL SUTURE MORPHOGENESIS

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Many severe craniofacial anomalies are associated with abnormal suture morphogenesis, and involve premature obliteration of sutures. Currently, the mechanisms underlying normal and abnormal suture morphogenesis are not known. TGF-beta3 was found to be essential for maintaining sutural integrity. The primary goal of these studies is to establish the mechanisms by which TGF-beta3 maintains calvarial sutures in their unossified state, yet allows them to function as bone growth centers. Since high concentrations of TGF-beta3 stimulate bone cell proliferation and collagen synthesis, important in the initial period of progression of cells to an osteoblast phenotype, the following hypothesis was formulated. For sutures to remain unossified, yet function as bone growth centers, a population of cells within the suture must remain mitogenic, replenishing cells maturing into osteoblasts. TGF-beta3 regulates cranial suture morphogenesis and maintenance of cranial sutures as bone growth centers by altering rates of cell proliferation within the suture. At high concentrations, TGF-beta3 stimulates cell proliferation and matrix production within the suture, while declining concentrations of TGF-beta3 promote maturation to an osteoblastic genotype, with matrix organization and mineralization. The following specific aims were designed to test this hypothesis: 1) test the mitogenic response of cells within the suture to TGF-beta3, 2) determine rates of extracellular matrix production by calvarial suture and bone treated with TGF-beta3, 3) test whether removal of TGF-beta3 promotes progression of cells to osteoblasts during bony obliteration of sutures and 4) confirm whether TGF-beta3 prevents osseous obliteration of rat calvarial sutures in vivo. A culture system in which fetal rat calvarial sutures undergo normal suture morphogenesis in the presence of intact dura mater, but undergo osseous obliteration in the absence of dura mater, will be used. Rates of mitogenesis and proliferating cell populations will be established by tritiated thymidine uptake. Rates of matrix production will be established by tritiated proline incorporation into collagen and non-collagen proteins. Alkaline phosphatase, Msx2 and osteocalcin production will be examined by northern blot analysis, in situ hybridization and PCR. Establishing the mechanisms by which TGF-beta3 regulates suture cell function will lead to an understanding of the role growth factors play in both normal and abnormal suture morphogenesis.