The proposed project aims to develop injectable, in situ hardening cell-polymer constructs for the repair of craniofacial bone defects. In addition to developing biomaterials well suited to this application, the project will also investigate the effect on bone regeneration of a number of critical material and cellular parameters, such that knowledge elucidated from the described studies can be broadly applied to other areas of tissue engineering. The first specific aim of the project is to synthesize and characterize novel injectable hydrogels that undergo physical gelation and chemical crosslinking at body temperature. These hydrogels will also contain calcium-binding domains that are hypothesized to both harden the material after injection and induce osteodifferentiation of encapsulated marrow stromal cells following matrix mineralization. A variety of methods to assess the physicochemical characteristics of the hydrogels will be used, and both in vitro and in vivo testing will be performed to evaluate cytocompatibility, stability, and degradation of the hydrogels. The second specific aim involves investigations of the effect different hydrogel formulations and the resultant physical parameters have on both encapsulated cell viability and bone regeneration within the well-established critical size rat calvarial defect model. Finally, the third specific aim is to investigate the effects that varying cellular parameters such as initial seeding density and the stage of osteodifferentiation of encapsulated marrow stromal cells have on the bone regenerative potential of the hydrogel constructs. In vivo studies are incorporated into each aim of the project such that, in keeping with the long-term goal of developing materials appropriate for clinical applications, efficacy with respect to bone regeneration and construct biocompatibility is assessed at each stage of the study.