Approximately 2% of Canadians sustain a bone fracture every year. The majority of these fractures heal well; however, about 5 to 10% of fractures develop delayed or incomplete healing requiring additional medical intervention. While we understand some of the mechanisms controlling bone healing, an improved and in-depth understanding of the precise molecular as well as cellular mechanisms involved in correct bone formation during fracture healing is of great importance. The processes that occur during bone healing recapitulate many of the steps involved in normal embryonic bone development [1, 5]. The cascade of synchronized cellular events involve three phases: 1) the inflammatory phase involving hematoma formation at the fracture site
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A number of studies have demonstrated that manipulation of G proteins, which transmit signals by activating diverse downstream effector molecules, including cAMP via Gαs and IP3 via Gαq/11, can profoundly affect bone mass [2]. This suggests that modifications of skeletal GPCR signaling through changes in the activity of G proteins can potentially have significant effects on bone healing and remodeling. Our lab has generated two transgenic mouse models with osteoblast-specific overexpression of Gαs (Gαs-Tg) and Gα11 (Gα11-Tg) to characterize the functional roles of Gαs and Gα11 in bone formation and remodeling during fracture …show more content…
Stabilized fractures of the tibia will be performed under anesthesia and the bones will be examined by X-ray and microCT at 14 or 21 days after fracture (n=6 per group) to obtain bone morphometric parameters including total callus volume, callus bone volume, callus mineralized volume fracture, callus mineral density, callus mineral content, and polar moment of inertia, . Histomorphometric analysis will be performed on the decalcified tibias stained with Safranin O/Fast Green for the assessment of bone and cartilage volumes as a percentage of total callus volume. For the selective visualization of osteoclast activity in vivo, fractured tibias (n=6 per group) will be paraffin-embedded and stained with tartrate-resistant acid phosphatase (TRAP). In addition, RNA will be extracted from the callus of the fracture site (n=6 for each genotype) to determine the expression levels of genes related to osteogenesis (Runx2, osterix, osteocalcin, Dkk1, β-catenin), chondrogenesis (Sox9, Col2a1, Col10) and osteoclastogenesis (TRAP, Opg,
A number of studies have demonstrated that manipulation of G proteins, which transmit signals by activating diverse downstream effector molecules, including cAMP via Gαs and IP3 via Gαq/11, can profoundly affect bone mass [2]. This suggests that modifications of skeletal GPCR signaling through changes in the activity of G proteins can potentially have significant effects on bone healing and remodeling. Our lab has generated two transgenic mouse models with osteoblast-specific overexpression of Gαs (Gαs-Tg) and Gα11 (Gα11-Tg) to characterize the functional roles of Gαs and Gα11 in bone formation and remodeling during fracture …show more content…
Stabilized fractures of the tibia will be performed under anesthesia and the bones will be examined by X-ray and microCT at 14 or 21 days after fracture (n=6 per group) to obtain bone morphometric parameters including total callus volume, callus bone volume, callus mineralized volume fracture, callus mineral density, callus mineral content, and polar moment of inertia, . Histomorphometric analysis will be performed on the decalcified tibias stained with Safranin O/Fast Green for the assessment of bone and cartilage volumes as a percentage of total callus volume. For the selective visualization of osteoclast activity in vivo, fractured tibias (n=6 per group) will be paraffin-embedded and stained with tartrate-resistant acid phosphatase (TRAP). In addition, RNA will be extracted from the callus of the fracture site (n=6 for each genotype) to determine the expression levels of genes related to osteogenesis (Runx2, osterix, osteocalcin, Dkk1, β-catenin), chondrogenesis (Sox9, Col2a1, Col10) and osteoclastogenesis (TRAP, Opg,