Faculty Bibliography

During spaceflight, astronauts will be exposed to a complex mixture of ionizing radiation that poses a risk to their health. Exposure of rodents to ionizing radiation on Earth causes bone loss and increases osteoclasts in cancellous tissue, but also may cause persistent damage to stem cells and osteoprogenitors. We hypothesized that ionizing radiation damages skeletal tissue despite a prolonged recovery period, and depletes the ability of cells in the osteoblast lineage to respond at a later time. The goal of the current study was to test if irradiation prevents bone accrual and bone formation induced by an anabolic mechanical stimulus. Tibial axial compression was used as an anabolic stimulus after irradiation with heavy ions. Mice (male, C57BL/6J, 16weeks) were exposed to high atomic number, high energy (HZE) iron ions (56Fe, 2Gy, 600MeV/ion) (IR, n=5) or sham-irradiated (Sham, n=5). In vivo axial loading was initiated 5months post-irradiation; right tibiae in anesthetized mice were subjected to an established protocol known to stimulate bone formation (cyclic 9N compressive pulse, 60cycles/day, 3day/wk for 4weeks). In vivo data showed no difference due to irradiation in the apparent stiffness of the lower limb at the initiation of the axial loading regimen. Axial loading increased cancellous bone volume by microcomputed tomography and bone formation rate by histomorphometry in both sham and irradiated animals, with a main effect of axial loading determined by two-factor ANOVA with repeated measure. There were no effects of radiation in cancellous bone microarchitecture and indices of bone formation. At the tibia diaphysis, results also revealed a main effect of axial loading on structure. Furthermore, irradiation prevented axial loading-induced stimulation of bone formation rate at the periosteal surface of cortical tissue. In summary, axial loading stimulated the net accrual of cancellous and cortical mass and increased cancellous bone formation rate despite prior exposure to ionizing radiation, in this case, HZE particles. Our findings suggest that mechanical stimuli may prove an effective treatment to improve skeletal structure following exposure to ionizing radiation.

Mechanical loading is a potent anabolic regulator of bone mass, and the first line of defense for bone loss is weight-bearing exercise. Likewise, protected weight bearing is the first prescribed physical therapy following orthopedic reconstructive surgery. In both cases, enhancement of new bone formation is the goal. Our understanding of the physical cues, mechanisms of force sensation, and the subsequent cellular response will help identify novel physical and therapeutic treatments for age- and disuse-related bone loss, delayed- and nonunion fractures, and significant bony defects. This review highlights important new insights into the principles and mechanisms governing mechanical adaptation of the skeleton during homeostasis and repair and ends with a summary of clinical implications stemming from our current understanding of how bone adapts to biophysical force.

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1-3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic-osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic-osteogenic coupling.

Axial compression of mouse limbs is commonly used to induce bone formation in a controlled, non-invasive manner. Determination of peak strains caused by loading is central to interpreting results. Load-strain calibration is typically performed using uniaxial strain gauges attached to the diaphyseal, periosteal surface of a small number of sacrificed animals. Strain is measured as the limb is loaded to a range of physiological loads known to be anabolic to bone. The load-strain relationship determined by this subgroup is then extrapolated to a larger group of experimental mice. This method of strain calculation requires the challenging process of strain gauging very small bones which is subject to variability in placement of the strain gauge. We previously developed a method to estimate animal-specific periosteal strain during axial ulnar loading using an image-based computational approach that does not require strain gauges. The purpose of this study was to compare the relationship between load-induced bone formation rates and periosteal strain at ulnar midshaft using three different methods to estimate strain: (A) Nominal strain values based solely on load-strain calibration; (B) Strains calculated from load-strain calibration, but scaled for differences in mid-shaft cross-sectional geometry among animals; and (C) An alternative image-based computational method for calculating strains based on beam theory and animal-specific bone geometry. Our results show that the alternative method (C) provides comparable correlation between strain and bone formation rates in the mouse ulna relative to the strain gauge-dependent methods (A and B), while avoiding the need to use strain gauges.

Mechanical loading is an important regulator in skeletal growth, maintenance, and aging. Estrogen receptors have a regulatory role in mechanically induced bone adaptation. Estrogen receptor-alpha (ERalpha) is known to enhance load-induced bone formation, whereas ERbeta negatively regulates this process. We hypothesized that ERbeta regulates mechanical signaling in osteoblasts. We tested this hypothesis by subjecting primary calvarial cells isolated from wild-type and ERbeta-knockout mice (BERKO) to oscillatory fluid flow in the absence or presence of estradiol (E2). We found that the known responses to fluid shear stress, i.e., phosphorylation of the mitogen-activated protein kinase ERK and upregulation of COX-2 expression, were inhibited in BERKO cells in the absence of E2. Flow-induced increase in prostaglandin E2 (PGE2) release was not altered in BERKO cells in the absence of E2, but was increased when E2 was present. Additionally, immunofluorescence analysis and estrogen response element luciferase assays revealed increased ERalpha expression and flow- and ligand-induced nuclear translocation as well as transcriptional activity in BERKO cells in both the presence and absence of E2. Taken together, these data suggest that ERbeta plays both ligand-dependent and ligand-independent roles in mechanical signaling in osteoblasts. Furthermore, our data suggest that one mechanism by which ERbeta regulates mechanotransduction in osteoblasts may result from its inhibitory effect on ERalpha expression and function. Targeting estrogen receptors (e.g., inhibiting ERbeta) may represent an effective approach for prevention and treatment of age-related bone loss.

BACKGROUND: Restoration of biomechanical strength following surgical reconstruction of tendon or ligament insertion tears is challenging because these injuries typically heal as fibrous scars. The authors hypothesize that injuries at the tendon-bone interface would benefit from reconstruction with decellularized composite tendon-bone grafts. METHODS: Tendon-bone grafts were harvested from Sprague-Dawley rats. Grafts subjected to decellularization were compared histologically and biomechanically with untreated grafts ex vivo and in a new in vivo model. Wistar rats underwent Sprague-Dawley allograft reconstruction using a pair-matched design. The rats were killed at 2 or 4 weeks. B-cell and macrophage infiltration was determined using immunohistochemistry, and explants were tested biomechanically. RESULTS: Decellularization resulted in a decrease in cells from 164 +/- 61 (untreated graft) to 13 +/- 7 cells per high-power field cells (p < 0.005) and a corresponding significant decrease in DNA content, and preserved scaffold architecture of the tendon-bone interface. Biomechanical comparison revealed no difference in failure load (p = 0.32), ultimate tensile stress (p = 0.76), or stiffness (p = 0.22) between decellularized grafts and untreated controls. Following in vivo reconstruction with tendon-bone interface grafts, decellularized grafts were stronger than untreated grafts at 2 weeks (p = 0.047) and at 4 weeks (p < 0.005). A persistent increase in B-cell and macrophage infiltration was observed in both the capsule surrounding the tendon-bone interface and the tendon substance in untreated controls. CONCLUSION: Decellularized tendon-bone grafts display better biomechanical properties at early healing time points and a decreased immune response compared with untreated grafts in vivo.