Bone Density Assessment

It is important to evaluate and compare bone mineral densities in various aging mouse cohorts and for any study that attempts to show the effect of any type of treatment on bone. Trabecular bone density can be measured by three established methods. These three methods include trabecular bone volume by histomorphometry (BV/TV%), trabecular bone density by peripheral quantitative computerized tomography (pQCT), and areal bone density of trabecular bone by duel-energy x-ray absorptiometry (DEXA). DEXA can be used to measure bone mineral content (BMC) and areal bone mineral density (BMD) (BMC/cm2).37

Osteoporosis is a common bony disease of aging women. This disease process involves loss of bone mass with subsequent skeletal fragility. A mouse model has been developed and specific phenotyping techniques can be employed to study this model. Conventional radiography as well as microradiography can be performed to evaluate bone density. Biomechanical studies such as whole-bone three- or four-point bending tests to failure is conducted with a servohydraulic testing system, and microcomputed tomography can be used to determine cortical volumetric BMD. The bending strength is measured at mid-diaphysis. The bone is placed horizontally with the anterior surface upwards. A pressing force is directed vertically to the midshaft of the bone, and the bone is compressed with a constant speed until failure. Breaking force or maximal load is defined as bending load at failure.38

Histologic analysis starts with fixed tissues that are then decalcified and stained for H&E or prepared and histomorphometrically analyzed. Growth plate measurements can be made on tibial samples stained with alcian blue/Van Gieson stain and sectioned to 4 um thick. An image-processing system is used coupled to a microscope. Histologic analysis can help distinguish osteoporosis from osteomalacia. Osteomalacia is a failure to mineralize versus osteoporosis which is a reduction in bone mass. Bone histomorphometry is measured as the ratio of trabecular bone volume to total volume. Areas of trabecular bone within a reference area are stained with H&E and measured in sections. Measurements are made on printed copies by point counting with a square lattice.38 Enzyme-histochemical staining can be done by staining for alkaline phosphatase activity or tartrate-resistant acid phosphatase. Serum assays are used to determine total protein, calcium, phosphorus, and creatinine. Serum osteocalcin, a marker of bone formation, is also helpful to determine. All these are important to distinguish osteoporosis from other osteopenic diseases such as primary hyperparathyroidism and renal osteodystrophy.39

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