In vivo axial loading of the mouse tibia

Noninvasive methods to apply controlled, cyclic loads to the living skeleton are used as anabolic procedures to stimulate new bone formation in adults and enhance bone mass accrual in growing animals. These methods are also invaluable for understanding bone signaling pathways. Our focus here is on a particular loading model: in vivo axial compression of the mouse tibia. An advantage of loading the tibia is that changes are present in both the cancellous envelope of the proximal tibia and the cortical bone of the tibial diaphysis. To load the tibia of the mouse axially in vivo, a cyclic compressive load is applied up to five times a week to a single tibia per mouse for a duration lasting from 1 day to 6 weeks. With the contralateral limb as an internal control, the anabolic response of the skeleton to mechanical stimuli can be studied in a pairwise experimental design. Here, we describe the key parameters that must be considered before beginning an in vivo mouse tibial loading experiment, including methods for in vivo strain gauging of the tibial midshaft, and then we describe general methods for loading the mouse tibia for an experiment lasting multiple days

Tibial Loading Increases Osteogenic Gene Expression and Cortical Bone Volume in Mature and Middle-Aged Mice

There are conflicting data on whether age reduces the response of the skeleton to mechanical stimuli. We examined this question in female BALB/c mice of different ages, ranging from young to middle-aged (2, 4, 7, 12 months). We first assessed markers of bone turnover in control (non-loaded) mice. Serum osteocalcin and CTX declined significantly from 2 to 4 months (p<0.001). There were similar age-related declines in tibial mRNA expression of osteoblast- and osteoclast-related genes, most notably in late osteoblast/matrix genes. For example, Col1a1 expression declined 90% from 2 to 7 months (p<0.001). We then assessed tibial responses to mechanical loading using age-specific forces to produce similar peak strains (−1300 µε endocortical; −2350 µε periosteal). Axial tibial compression was applied to the right leg for 60 cycles/day on alternate days for 1 or 6 weeks. qPCR after 1 week revealed no effect of loading in young (2-month) mice, but significant increases in osteoblast/matrix genes in older mice. For example, in 12-month old mice Col1a1 was increased 6-fold in loaded tibias vs. controls (p = 0.001). In vivo microCT after 6 weeks revealed that loaded tibias in each age group had greater cortical bone volume (BV) than contralateral control tibias (p<0.05), due to relative periosteal expansion. The loading-induced increase in cortical BV was greatest in 4-month old mice (+13%; p<0.05 vs. other ages). In summary, non-loaded female BALB/c mice exhibit an age-related decline in measures related to bone formation. Yet when subjected to tibial compression, mice from 2–12 months have an increase in cortical bone volume. Older mice respond with an upregulation of osteoblast/matrix genes, which increase to levels comparable to young mice. We conclude that mechanical loading of the tibia is anabolic for cortical bone in young and middle-aged female BALB/c mice

Primary Cilia Exist in a Small Fraction of Cells in Trabecular Bone and Marrow

Primary cilia are potent mechanical and chemical sensory organelles in cells of bone lineage in tissue culture. Cell culture experiments suggest that primary cilia sense fluid flow and this stimulus is translated through biochemical signaling into an osteogenic response in bone cells. Moreover, in vivo, primary cilia knockout in bone cells attenuates bone formation in response to loading. However, understanding the role of the primary cilium in bone mechanotransduction requires knowledge of its incidence and location in vivo. We used immunohistochemistry to quantify the number of cells with primary cilia within the trabecular bone tissue and the enclosed marrow of ovine cervical vertebrae. Primary cilia were identified in osteocytes, bone lining cells, and in cells within the marrow, but were present in only a small fraction of cells. Approximately 4 % of osteocytes and 4.6 % of bone lining cells expressed primary cilia. Within the marrow space, only approximately 1 % of cells presented primary cilia. The low incidence of primary cilia may indicate that cilia either function as mechanosensors in a selected number of cells, function in concert with other mechanosensing mechanisms, or that the role of primary cilia in mechanosensing is secondary to its role in chemosensing or cellular attachment

Whole-body vibration slows the acquisition of fat in mature female rats.

OBJECTIVE: To evaluate the effects of whole-body vibration on fat, bone, leptin and muscle mass. METHODS/DESIGN: Thirty 7-month-old female 344 Fischer rats were randomized by weight into three groups (baseline, vibration or control; n=8-10 per group). Rats in the vibration group were placed inside individual compartments attached to a Pneu-Vibe vibration platform (Pneumex, Sandpoint, ID, USA) and vibrated at 30-50 Hz (6 mm peak to peak) for 30 min per day, 5 days per week, for 12 weeks. The vibration intervention consisted of six 5-min cycles with a 1-min break between cycles. RESULTS: There were significant body composition differences between the whole-body vibration and the control group. The whole-body vibration group weighed approximately 10% less (mean+/-s.d.; 207+/-10 vs 222+/-15 g, P\u3c0.03) and had less body fat (20.8+/-3.8 vs 26.8+/-5.9 g, P\u3c0.05), a lower percentage of body fat (10.2+/-1.7 vs 12+/-2.0%, P\u3c0.05), and lower serum leptin levels (1.06+/-0.45 vs 2.27+/-0.57 ng ml(-1), P\u3c0.01) than the age-matched controls. No differences were observed for total lean mass, bone mineral content (BMC), bone mineral density (BMD), insulin-like growth factor-I (IGF-I) or soleus (SOL) and extensor digitorum longus (EDL) mass or function. Regional high-resolution dual-energy X-ray absoptiometry scans of the lumbar spine (L1-4) revealed that the whole-body vibration group had significantly greater BMC (0.33+/-0.05 vs 0.26+/-0.03 g, P\u3c0.01) and BMD (0.21+/-0.01 vs 0.19+/-0.01 g cm(-2), P\u3c0.01) than the control group. No differences between the groups were observed in the amount of food consumed. CONCLUSION: These findings show that whole-body vibration reduced body fat accumulation and serum leptin without affecting whole body BMC, BMD or lean mass. However, the increase in vertebral BMC and BMD suggests that vibration may have resulted in local increases in bone mass and density. Also, whole-body vibration did not affect muscle function or food consumption