Low Intensity Pulsed Ultrasound: countermeasure for Microgravity-Induced Bone Loss
Uddin, Sardar Muhammad Zia
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Microgravity (MG) during space flight is known to have adverse effect on bone quality and quantity. Data collected from studies conducted on astronauts show a loss of 1-1.6% bone mineral density (BMD) per month of space-flight. This decreased BMD has been recorded in the load-bearing region of the legs and spine. The reduction in bone quality can be due to decreased osteoblast and/or increased osteoclast activity when exposed to microgravity. During space-flights, rigorous exercise has been used to reduce to bone loss due to microgravity, but thus far it has proven inadequate to produce significant results. Some studies have considered using drugs and various growth factors to maintain bone mass in a MG environment, but it can become too expensive to maintain over longer periods of time besides the systemic effects of such treatments. The effects of MG are partially attributed to the lack of mechanical force on bone tissue, which alters osteogenic gene expression, reduces rate of cell growth, proliferation, differentiation, cytoskeleton polymerization and cellular morphology. Thus, to reverse these adverse effects on bone physiology, it is important to provide cells with mechanical stimuli that can provide essential signals for cells to counter the adverse effects of microgravity. Low intensity pulsed ultrasound (LIPUS) can be readily applied in vivo; human studies have shown anabolic effects on osteopenic bone tissue. Furthermore, LIPUS has the potential to be an inexpensive and non-invasive targeted therapy for disuse induced bone loss. The objective of this study was to examine effects of ultrasound on a disuse bone model and osteoblast cell cultures in simulated microgravity. This will help us understand the effects of ultrasound on microgravity induced osteoporosis and provide a non-invasive and more targeted approach to reduce bone loss during space-flight. Ultrasound may provide an option towards future human space explorations. It is hypothesized that LIPUS stimulation will reverse the detrimental effects of microgravity on bone strength. The overall hypothesis will be tested by studying the effects of LIPUS on osteoblasts and MSCs in simulated microgravity in hind limb suspended mice models. The hypothesis was tested with in vivo and in vitro disuse models. Three-month old C57BL/6 mice were randomized to age match (AM), non-suspended sham (NS), non-suspended -LIPUS (NU), suspended sham (SS), and suspended-LIPUS (SU) groups. The results from the in vivo after four weeks of suspension, micro CT analyses showed significant decreases in trabecular bone volume/tissue volume (BV/TV) (36%, P<0.005),bone mineral density ( BMD) (3%, P<0.05), Trabecular thickness (Tb.Th) (12.5%, p<0.005), and increased in bone surface/bone volume (BS/BV) (16%,p<0.005) relative to age match (AM). Application of LIPUS for 20 min/day for 5 days/week, significantly increased, BMD (3%, p<0.05), Tb.Th (6%, p<0.05), and increased BS/BV (10%, p<0.005) relative to AM mice. Histomorphometric analyses showed increased bone formation at metaphysis endosteal and trabecular surfaces (0.104??0.07 vs. 0.031??0.30 ??m3/(??m2)/d) in SU mice relative to SS. Four-point bending tests of SS femurs showed reduced elastic modulus (53%, p<0.05), yield (33%, P<0.05), and ultimate strength (45%,p <0.05) at the femoral diaphysis relative to AM samples. LIPUS stimulation mitigated the adverse effects of disuse on bone elastic modulus (42%, p<0.05), yield strength (29%, p<0.05), and ultimate strength (39%, p<0.05) relative to SS femurs. Analyses of contralateral control limbs from SU or NU showed that LIPUS had no systemic effects, supporting the hypothesis that LIPUS provided targeted stimulation. The in vitro studies were conducted with MSCs and Osteoblast cells in Simulated Microgravity (SMG) conditions. MSCs were cultured in a 1D clinostat to simulate microgravity (SMG) and treated with LIPUS at 30mW/cm2 for 20 min/day. The results showed significant increases in ALP, OST, RANKL, RUNX2, and decreases in OPG in LIPUS treated SMG cultures compared to non-treated cultures. SMG significantly reduced ALP positive cells by 70%, p<0.01and ALP activity by 22%, p<0.05), while LIPUS treatment restored ALP positive cell number and activity to equivalence with normal gravity controls. Extracellular matrix (ECM) collagen and mineralization was assessed by Sirius Red and Alizarin Red staining, respectively. SMG cultures showed little or no collagen or mineralization, but LIPUS treatment restored collagen content to 50 %,( p<0.05) and mineralization by 45% (p<0.05) in SMG cultures relative to SMG samples. The data from the osteoblast study showed that osteoblast exposure to SMG results in significant decreases in proliferation (38% and 44% at day 4 and 6, respectively, p<0.01), collagen content (22%, p<0.05) and mineralization (37%, p < 0.05) and actin stress fibers. In contrast, LIPUS stimulation under SMG condition significantly increases the rate of proliferation (24% by day 6, p<0.05), collagen content (52%, p < 0.05) and matrix mineralization (25%, p<0.001) along with restoring formation of actin stress fibers in the SMG-exposed osteoblasts. The gene expression analysis showed significant increase in expression of RUNX2 and OST and reduced RANKL expression after LIPUS exposure. Collectively, the data suggest LIPUS has the potential to provide essential mechanotransductive anabolic stimulus to counter measure disuse-induced bone loss while showing no adverse effect on healthy bone. It also showed increased structural and mechanical integrity in LIPUS treated disuse bones. Furthermore, LIPUS increased MSCs osteogenic differentiation and osteoblastic activity in SMG. The gene expression data indicates that LIPUS has anabolic and anti-resorptive effects on osteoblast cells.