PROJECT SUMMARY/ABSTRACT Bone regeneration occurs by either primary bone formation (intramembranous ossification) or through a tem- porary cartilage template (endochondral ossification). Failure of bone regeneration to proceed to completion represents a significant source of clinical burden. The gold standard treatment in these circumstances is bone autografting, which involves surgical procedures to harvest and transplant bone to the site of impaired regen- eration. This therapy produces additional morbidity and is significantly limited by tissue availability. Bone form- ing growth factors hold tremendous potential as an alternative to autografting procedures. Among them, bone morphogenetic protein 2 (BMP2) is an osteogenic morphogen that has been clinically approved to promote bone growth. However, very large amounts of BMP2 are required to form bone in vivo, a limitation which con- tributes to the substantial costs and multiple side-effects, some serious, associated with its use. To address these shortcomings, our long-term goal is to improve the clinical performance of BMP2. The overall objective of this application is to understand the effect of inflammation on bone formation stimulated by BMP2. Interleukin-1 (IL-1) is an inflammatory cytokine induced by BMP2 that inhibits the chondrogenic differentiation of skeletal progenitor cells. The central hypothesis of this proposal is that BMP2-stimulated IL-1 expression blocks endo- chondral bone formation. Excessive amounts of BMP2 are required to overcome this barrier and result in in- tramembranous ossification which produces morphologically and mechanically inferior bone. The central hy- pothesis will be tested by pursuing two specific aims: 1) Define pathways of in vivo bone formation using non- invasive longitudinal imaging 2) Determine the effect of IL-1 receptor antagonism on bone healing stimulated by either low- or high-dose BMP2. The first aim will incorporate two complementary sets of transgenic rats en- abling in vivo visualization of collagen I and collagen II expression during bone regeneration to discriminate intramembranous and endochondral bone formation, respectively. Femoral critical size defects (CSDs) in par- allel groups of transgenic animals will be treated with clinically-relevant amounts of BMP2 and imaged throughout the course of healing. In the second aim, femoral CSDs in wild-type rats will be treated with sub- therapeutic or clinically-relevant amounts of BMP2 in the presence or absence of the IL-1 receptor antagonist. These studies will determine whether targeted anti-inflammatory strategies can promote bone formation and improve bone quality with reduced amounts of BMP2. The proposed research is innovative as it explores the novel hypothesis that intramembranous ossification is the primary regenerative pathway engaged by high-dose BMP2. Furthermore, these studies will redirect healing towards endochondral ossification by inhibiting IL-1, thus permitting bone regenera...