Background and Hypothesis: Ca2+/calmo-dulin-dependent protein kinase kinase 2 (CaMKK2) is a serine-threonine protein kinase that plays a significant role in both anabolic and catabolic pathways of bone remodeling. Mechanical loading of bone translates an external force into both biochemical and structural changes. It has been shown that deletion or inhibition of CaMKK2 results in increased bone density in male and female mice. We hypothesize that the lack of CaMKK2 in bone cells will result in loading-induced bone mass accrual with no difference between male and female mice. Experimental Design or Project Methods: The right tibia of anesthetized 16-week-old wild-type (WT) and CaMKK2 knockout (KO) mice were loaded at 2 Hz for 220 cycles and with peak forces specific to both sex and genotype. Loading was accomplished using an electro actuator (Bose ElectroForce 3200; EnduraTEC, Minnetonka, MN, USA). This was repeated 3, 5, 8 and 10 days after initial loading. The non-loaded left tibia served as an internal control. Calcein and alizarin red were administered intraperitoneally on days 9 and 16, respectively to metabolically label newly formed bone. Nineteen days after initial loading, mice were sacrificed. Blood and long bones of the lower limbs were collected for analysis. Results: Using microcomputer tomography; dynamic histomorphometry; histology, immunohistochemistry, enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction, we will assess bone volume, bone formation rate, and underlying mechanisms at the cellular and molecular level. These data are forthcoming. Conclusion and Potential Impact: With expanded knowledge on how bone growth is augmented, clinical outcomes related to osteoporosis and fracture healing, for example, may be improved. This may be accomplished through novel therapy related to these pathways that increases bone density or decreases fracture healing time.
Background and Hypothesis: Mechanical stimulation of bone results in the translation of external forces into a cascade of structural and biochemical changes which work to increase bone density and decrease fracture healing time. The specific mechanisms contributing to these processes are areas of active investigation. Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a serine-threonine protein kinase with key roles in both the anabolic and catabolic pathways of bone remodeling. We hypothesize that the absence of CaMKK2 potentiates an increase in bone density as a response to mechanical stimulation. Experimental Design or Project Methods: The right ulna of anesthetized C57BL/6 mice were loaded for 220 cycles at 2 Hz and with peak forces specific to both sex and genotype. Loading was completed using an electro actuator (Bose ElectroForce 3200; EnduraTEC, Minnetonka, MN, USA) and was repeated on days 3, 5, 8 and 10 after the initial procedure. The non-loaded left ulna served as an internal control. Calcein and alizarin red were administered intraperitoneally on days 9 and 16 respectively. Mice were sacrificed on day 19 after the initial load; blood and long bones of the lower limbs were collected for analysis. Results: Bone volumetric analyses will be measured using microcomputed tomography, bone formation rate will be assessed using dynamic histomorphometry measurements of double fluorochrome labeling, and cellular and molecular mechanisms will be assessed using histology, immunohistochemistry and real-time reverse transcription-polymerase chain reaction. These data are currently forthcoming. Conclusion and Potential Impact: Clinical outcomes of conditions ranging from stress fractures to osteoporosis may be improved by an increased understanding of the mechanisms through which bone growth is augmented. Expanded knowledge of these pathways may provide opportunities for the development of novel therapies which decrease healing times in the event of injury and increase bone density to combat degenerative disease states.
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