Growth plate injuries are a unique type of fracture where healing with cartilage instead of bone is desirableto avoid growth disturbance and resulting deformity. Mesenchymal stem cells (MSCs) that reside in themarrow spaces adjacent to the physis are responsible for healing the injuries. MSCs are pluriopotent cellsthat can differentiate to cartilage, bone or fat tissue based on conditions. Recent evidence has shown thatlocal 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 MSCdifferentiation in vitro. Primary mesenchymal stromal cells (MSCs) from murine bone marrow will be grownin conditions favoring bone or cartilage differentiation and exposed to normoxia or hypoxia. Differentiationwill 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 toincrease HIF-1 activity (Von Hippel Lindau deletion) or decrease HIF-1 activity (HIF-1 deletion) will then begrown in osteogenic or chondrogenic conditions. To test whether the HIF-1 pathway impinges ondifferentiation to bone or cartilage, the cells will be exposed to normoxia or hypoxia and genotypic andphenotypic 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 surgicallycreated defect across the physis that connects the epiphyseal and metaphyseal marrow spaces, alteringlocal nutrient availability. The injury results in healing with a bony bridge formed by intramembranousossification. Injuries will be imaged by CT and SPECT, detailed histology will be performed, and geneexpression associated with hypoxia, chondrogenesis, and osteogenesis will be evaluated by real time PCRof the zone of injury.
In Aim 3, we propose a future direction for development of an inducible mutation targeted to MSC's drivenby 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 boneformation and the resulting growth disturbance.
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