Role of Hypoxia in Bone Formation

Growth plate injuries are a unique type of fracture where healing with cartilage instead of bone is desirable to avoid growth disturbance and resulting deformity. Mesenchymal stem cells (MSCs) that reside in the marrow spaces adjacent to the physis are responsible for healing the injuries. MSCs are pluriopotent cells that can differentiate to cartilage, bone or fat tissue based on conditions. Recent evidence has shown that local oxygen availability alters the differentiation of MSCs with hypoxic conditions favoring chondrogenesis. Hypoxia Inducible Factor 1 (HIF-1) is a key mechanism for sensing and responding to changes in oxygen. Therefore, we hypothesize that local oxygen tension alters MSC differentiation via the HIF-1 pathway. In Aim 1, we will test this hypothesis by determining the effects of hypoxia and altered HIF-1 on MSC differentiation in vitro. Primary mesenchymal stromal cells (MSCs) from murine bone marrow will be grown in conditions favoring bone or cartilage differentiation and exposed to normoxia or hypoxia. Differentiation will be assessed by gene expression (real time PCR) and by phenotypic expression of bone (mineralization) or cartilage (proteoglycans). Similarly, MSCs from mice with conditional mutations to increase HIF-1 activity (Von Hippel Lindau deletion) or decrease HIF-1 activity (HIF-1 deletion) will then be grown in osteogenic or chondrogenic conditions. To test whether the HIF-1 pathway impinges on differentiation to bone or cartilage, the cells will be exposed to normoxia or hypoxia and genotypic and phenotypic expression of bone or cartilage markers will be examined. In Aim 2, we will use an in vivo mouse model to evaluate MSC differentiation in healing of a surgically created defect across the physis that connects the epiphyseal and metaphyseal marrow spaces, altering local nutrient availability. The injury results in healing with a bony bridge formed by intramembranous ossification. Injuries will be imaged by CT and SPECT, detailed histology will be performed, and gene expression associated with hypoxia, chondrogenesis, and osteogenesis will be evaluated by real time PCR of the zone of injury. In Aim 3, we propose a future direction for development of an inducible mutation targeted to MSC's driven by the dermol or prxl promoter. This will allow manipulation of the HIF-1 pathway (or other desired target) in MSC's prior to differentiation in order to alter the healing response with the goal of preventing bone formation and the resulting growth disturbance.