PTH(1-34) reduces microdamage in the mouse femur under fatigue loading
Rostami N, Kohn DH, and Gardinier JD. PTH(1-34) reduces microdamage in the mouse femur under fatigue loading. J Orthop Res 2017; 35
Journal of orthopaedic research
INTRODUCTION: Parathyroid hormone (PTH) is a calciotropic hormone that plays a critical role in maintaining bone quality . The PTH(1-34) analog, is commonly used to increase bone mass and reduce fracture risk associated with skeletal disorders, such as osteoporosis. Osteoporotic fractures in particular generally occur under two modes of failure: traumatic and fatigue [1-3]. Microdamage generated under cyclic loading during daily activities contributes to fatigue failure. However, the effect of PTH(1-34) treatment on microdamage has been less studied in the literature. Therefore, the primary objective of this study was to determine how PTH(1-34) influences the fatigue properties of cortical bone. We hypothesize that increased bone formation following PTH(1-34) treatment will increase the resistance to microdamage. METHODS: 16 week old male C57B1/6J mice were weight-matched and divided into two groups: vehicle and PTH (1-34). For 21 consecutive days, subcutaneous injections were given to administer 50 μL of a saline solution (0.9% NaCl) as a vehicle control or 40 μg/kg of hPTH (1-34) (Bachem) in saline solution. On day 22, 5 mice from each group were sacrificed and femurs were removed and stripped of the soft tissue. Micro-Ct scans were taken of the left femur and then subjected to cyclic loading under four-point bending (BOSE, Electroforce 32000 series), with the anterior side under tension. The femur was first loaded for 20 cycles at 5N to determine the initial stiffness and force needed to generate 10,000 microstrain along the anterior side. This estimated force was then applied for 40,000 cycles at 2 Hz under load control. Testing was conducted in calcium chloride buffered saline solution at 37°C. After loading, both left and right (non-loaded control) samples were dehydrated in graded Ethanol with 1% Basic Fuchsin, infiltrated in a liquid methyl methacrylate monomer (Koldmount Cold Mounting Liquid), embedded in polymethylmethacrylate, sectioned and polished to 150μm thickness. Sections were imaged using confocal microscopy (Zeiss 100 Axiovert inverted microscope) with 559 nm laser and UPLSAPO 40X 2 objective lens. Z-stack images were obtained along the anterior side of each sample. In each image, crack density (Cr. Dn = # of cracks / cortical area), diffuse damage (Df.Dm.Dn = diffuse damage area / cortical area), and crack length (Cr.Ln) were quantified using ImageJ. The mean and standard deviations of each metric is reported. Statistical interactions between groups were determined using a student T-test with a p-value of <0.05 considered significant. RESULTS: In vehicle treated mice, crack density was significantly greater following fatigue loading compared to the non-fatigued limb (374.53 ± 115.39 #/mm2 vs. 132.52 ± 59.78 #/mm2), while PTH(1-34) treated mice exhibited no significant difference in crack density between fatigue and non-fatigued limbs (270.21 ± 69.59 #/mm2 vs. 151.69±66.72 #/mm2) (Fig. 1A). The density of diffuse damage in the fatigued limb was significantly greater in vehicle treated mice than the fatigued limb of PTH(1-34) treated mice (0.00703 ± 0.0016 mm2/mm2 vs. 0.0055 ± 0.0015 mm2/mm2) (Fig. 1B). Similarly, crack length in the fatigue limb was also significantly greater in vehicle treated mice than the fatigued limb of PTH(1-34) treated mice (35.067 ± 5.95 μm vs. 26.02±4.062 μm) (Fig. 1C). During fatigue loading, the loss in stiffness was smaller in the PTH(1-34) treated samples compared to vehicle (19.88 ± 6.15 % vs. 24.578 ± 7.55 %). No significant differences were observed between vehicle and PTH(1-34) treated groups for cross-sectional area (0.89 ± 0.06 mm vs. 0.90 ± 0.03) and moment of inertia (0.168 ± 0.03 mm4 vs. 0.173 ± 0.04 mm4) at the femur. DISCUSSION: Daily treatment with PTH(1-34) over a short period of time was sufficient to decrease the presence of microdamage following fatigue loading conditions. This increased resistance to microdamage due to PTH(1-34) treatment corresponded with minimal stiffness loss during fatigue loading. This reduction in microda age and stiffness loss during fatigue loading would also explain the increased fatigue life observed in the bovine femur following similar treatment with teriparatide .Contrary to our expectations, PTH(1-34) had minimal effect on cortical area and moment of inertia at the femur, while a significant increase in tibial cortical area and bone formation occurred . Studies using a similar dose of PTH(1-34) required only 4 weeks before changes in cortical area of the tibia occurred , while displaying minimal effects at the femur, even after 8 weeks of treatment . Thus, our results demonstrate PTH(1-34) can provide improvements in fracture resistance that do not have to be superceded by an increase in bone mass. (Figure Presented).