Osteoclastic Regulation of Local Bone Formation

Va Loma Linda Healthcare System, Loma Linda CA

Investigators

Abstract

The healing of ~10% of several million traumatic or nontraumatic fractures occurring annually in the US is delayed or impaired resulting in nonunion or delayed union. The annual cost for their treatment and management exceeds $20 billion, leading to considerable healthcare costs and human suffering. A good understanding of the mechanism of fracture healing is critical for the development and proper use of novel and effective therapies. Fracture healing is a complex and dynamic process with osteoclasts and osteoblasts playing indispensable roles. The critical core of fracture healing is the callus remodeling, which is initiated with resorption of the cartilaginous callus followed by the coupled formation of bony callus that subsequently is remodeled to cortical bone indistinguishable from the native bone. The osteoclastic regulation of the local coupled bone formation is in part mediated through local release of osteoclastic soluble osteoanabolic factors and is an integral regulatory mechanism of callus remodeling. This study tests two specific hypotheses that are relevant to the osteoclastic regulation of local bone formation/regeneration: Hypothesis 1. Local injections of osteoclastic microvesicles (MV) into fractures accelerates the healing of skeletal fractures by accelerating fracture callus formation and remodeling. This hypothesis is supported by the following preliminary findings: 1) osteoclasts released soluble MV that have pro-osteogenic, pro-chondrogenic, and pro-angiogenic activities, 2) injections of osteoclastic MV at the surface of calvaria stimulated local bone formation, and 3) injections of MV into fractures promoted neo- angiogenesis in fracture callus and accelerated fracture healing. Hypothesis 2. EfnB2 is a key osteoanabolic component of osteoclastic MV and is in part responsible for the osteoanabolic actions of osteoclastic MV. Accordingly, a review of current literature and the following preliminary data suggest that EfnB2 is an attractive candidate of an osteoanabolic component of osteoclastic MV: 1) osteoclastic MV contains appreciable amounts of EfnB2, 2) pretreatment of MV with anti-EfnB2 antibody abrogated its osteogenic activity, 3) EfnB2-deficient MV had reduced osteogenic and fracture healing activities, whereas MV from osteoclasts overexpressing EfnB2 showed greater osteoanabolic activity, 4) pretreatment of osteoprogenitors with anti-EphB4 antibody prevented osteoclastic MV to exert its osteogenic activity. Aim 1 tests Hypothesis 1 by using a simple transverse femoral fracture model to determine: a) an optimal injection regimen for osteoclastic MV by determining the duration-of- stay of the MV after local injection at fracture site, optimal injection frequency, duration between injections, and optimal dose range; b) if an optimized injection regimen of osteoclastic MV would accelerate bony bridging of fracture gaps and increase torsional mechanical strength of fractured bones in male as well as female mice; and c) the cellular mechanism by which osteoclastic MV accelerates fracture healing through histological and immunohistochemical analyses of time-dependent effects of the MV treatment on osteoclasts, osteoblasts, and chondrocytes during the various fracture healing phases. Aim 2 tests Hypothesis 2 by determining: a) if treatment of osteoblasts, chondrocytes, and endothelial cells with osteoclastic MV in vitro upregulates their EphB4 forward signaling; b) if MV of EfnB2-deficient osteoclasts would show diminished osteogenic, chondrogenic, and angiogenic activities of osteoclastic MV in vitro; c) if MV of osteoclasts with EfnB2 overexpression exhibited enhanced chondrogenic, and angiogenic activities in vitro; d) if osteoblasts, chondrocytes, and endothelial cells isolated from EphB4-deficient mice show reduced osteogenic, chondrogenic, and angiogenic in response to osteoclastic MV in vitro; and e) if local injections of MV deficient in EfnB2 impedes, and local injections of MV with EfnB2 overexpression accelerates, fracture healing. If successful, this study could offer key mechanistic insights into how osteoclasts regulate local bone regeneration. It may also help to develop novel osteoclastic MV-based therapies for fracture healing and local bone regeneration.