Bone defects arising due to trauma, disease, cancer, and congenital deformity constitute significant impairments to function in the axial and craniofacial skeletons. The goal of this project is to improve management of massive bone defects by developing methods to provide osteogenic bone grafts composed of purified extracellular matrix (ECM) alone and seeded with bone cells. Bone repair will be evaluated in critical size defects (CSD) in the calvaria and in long bones (femur) of aged rats. The extent of repair, relative to nontreated controls, will be evaluated by planimerty following three dimensional reconstruction of computed tomography (CT) and by histological analysis. In studies of long bones, strength of regenerated bone will be determined by torsion testing. The following specific aims are proposed: (10 Characterization and optimization of repair of calvarial CSD by determining: the mechanism of repair (intramembranous vs endochondral), the percent survival and transplanted osteoblasts, athe optimal collagen concentration, and whether transplanted bone chips (auto graft) enhance repair by collagen gels. (2) Evaluation of the utility of this method of repair in long bones by determining if these grafts mediate repair and by what mechanism, and by testing gel grafts within bio-degradable barriers to repair extensive long bone defects, and (3) Assessment of potential applicability to humans by testing grafts composed of human placental type I collagen containing human bone cells in repair of defects in athymic rats. The successful completion of these experiments will provide important insights into cellular interactions with ECM in vivo and may lead to new alternatives for treating bone defects. GRANTS=R01HD29457 The ultimate objective of this work is to understand how receptors and other DNA-binding proteins interact to regulate the transcription of tissue and hormone-specific genes. The aim of this proposal is to understand how prolactin (PRL) augments the progesterone-dependent transcription of the UG gene by regulating as many as four proteins that bind to the UG promoter. This goal will be achieved by 1) cloning and characterizing the cDNAs for the PRL/progesterone-dependent proteins that bond to the UG promoter; 2) determining whether PRL, in combination with progesterone, regulates the sequence-binding activity of the UG promoter-binding (UGPB) proteins; 3) quantifying hormone-dependent changes in the expression of mRNA for UGPB proteins by specific uterine cell types; 4) characterizing the interactions of PRL and progesterone in the regulation of the UG promoter in an in vitro cell transfection system. As many as four UGPB proteins will be cloned from lambdagt11 expression libraries. Phage clones will be subcloned into expression vectors and their recombinant proteins will be used as antigens to generate polyclonal antibodies. Identity of recombinant protein(s) with authentic UGPB proteins will be confirmed by gel mobility shift assays, DNase-! footprinting, and western blots with appropriate antibodies. Sequence information from purified UGPB proteins will provide the conclusive test of identity. The interaction(s) of PRL and progesterone in the uterus will be defined using hormone-treated estrous and long-term ovariectomized (LTOVX) rabbits. In gel mobility shift assays, PRL + progesterone increased the primary gel shift 60% over progesterone alone in estrous animals. PRL+progesterone treatment of LTOVX rabbits increased th primary gel shift 60% over progesterone alone and 30-fold over similarly treated estrous rabbits. Therefore, gel mobility shift assays will be used to determine whether the hormonal status of the animal alters the relative affinity, association rate constants and dissociation rate constants for UGPB proteins. Changes in binding site specificities will be defined by methylation interference. Hormone-dependent changes in the amount of UGPB mRNAs and proteins will be determined by Northern and Western analyses, respectively. In situ hybridization and immunocytochemistry will be used to determine whether hormone-dependent changes in UGPB mRNA distinguish certain subpopulations of epithelial cells in the uterine endometrium. If this is the case, then UGPB proteins likely play a unique role in the regulation of UG gene expression. Finally, a transient expression system and the hormone-responsive endometrial-like cell line, RBE-H9, will be used to show that sequences upstream of the transcription initiation site of the UG gene modulate the action of PRL. Understanding how PRL regulates the action of progesterone is critical to good reproductive health. Its manipulation in a clinical context holds potential for regulating fertility.