In the first phase, investigators used a clustered factorial design based on the interdependence of time, intensity and frequency to determine if short periods (10, 20 & 80 minutes) of low level mechanical stimuli (0.25 & 0.5g), induced non-invasively at distinct frequencies (22, 45 & 90Hz), were effective in stimulating bone formation in the normal weight-bearing rat. When animals were subject to the mechanical stimulus, they were not removed from their cage, but instead the cage was simply placed on the oscillating plate.
In phase two of the protocol, the goal was to identify both known and novel genes up regulated in bone tissue by disuse. The data reported here reflect two distinct experiments; the transcriptional activity was examined at 10 days (to determine the early response genes to disuse), and the histochemistry evaluated at both 10 and 28 days (to allow correlation to the long-term mechanical studies described above; only the 28-day histomorphometry are described here). Animals were individually housed and suspended by their tails, following the published routines and personal communication with Dr. Morey-Holton. Following sacrifice, the right tibia was prepared for histomorphometry of the proximal metaphysis. The left tibia was immediately frozen in liquid nitrogen to be used for the extraction of ribonucleic acid (RNA), which was isolated using the acid-guanidinium thiocynate-phenol-chloroform extraction method. The differential expression of genes was analyzed using the differential messenger RNA (mRNA) display method (DD-PCR) utilizing polymerase chain reaction (PCR) and RNA image kits. Any bands clearly present under one condition (i.e., disuse vs. control) and absent in the other, was subsequently removed from the gel and reamplified, cloned and sequenced using standard procedures in order to determine its identity. Northern analysis was used to confirm that such copy deoxyribonucleic acid (cDNA) fragments were differentially expressed and their relative expression determined using densitometry (normalized to glyseraldehyde-3-phosphate dehydrogenase (GAPDH)). For the gene identification protocols performed at 10 days, six animals were evaluated in each group, and each animal was evaluated independently (RNA was not pooled).
In phase three of this study, signals demonstrated as osteogenic in phase one were evaluated in terms of their ability to inhibit the remodeling changes caused by the tail-suspension.
Phase One: No visible changes in animal behavior were noticed during the stimulus periods. In essence, the goal was to identify several stimulus regimens that were anabolic, as well as several that failed to influence skeletal remodeling/modeling activity.
A single element strain gage, attached longitudinally to the tibial diaphysis of rats used for calibration showed that, similar to the sheep and turkey studies previously described, the mechanical intervention, at around 0.3g, generated strains less than 5µe. After 28 days of ten minutes per day, five days per week, exposure to a 0.25g mechanical stimulus, both of the higher frequency signals (45 Hz & 90 Hz) stimulated significant increases in both labeled surface and mineral apposition rate.
Phase Two: At 28 days, as compared to long-term controls, the tail-suspension caused Labeled Surface (LS) to drop 26% (LTC: 11.7%±1.6%; Disuse (DIS): 8.6%±2.4%; p<0.05), and Bone Formation Rate per Bone Volume (BFR/BV) (%/y) to drop - 52% in the tibia (LTC: 90%±18%; DIS: 42.9%±17.4%; p<0.05). These data are similar, but not identical, to that observed at 10 days. DD-PCR of RNA from the tibiae of the tail suspended animals successfully identified several gene candidates with increased expression following 10 days of disuse, as compared to long-term controls. Discussed here are two specific genes, one known, and one unknown. S-14 was first isolated from liver, and is believed to participate in adipose synthesis. S-14 has not been shown to be expressed in bone. The second, OPO-1 (Osteoporosis-1), is a novel gene in which the full-length clone has been isolated and sequenced (1.8 Kb). It is important to note that, while DD-PCR is and effective way of identifying both known and novel genes, it is inherently limited by the small scale in which it can be used, a potential limitation in the undoubtedly complex biological processes of formation and resorption. To more comprehensively address molecular basis of bone remodeling, we recently began using cDNA microarrays, discussed in detail in the approach section.
Using RNA extracted from the phase one animals, the relative transcriptional activity following the osteogenic activity relative to control, was examined. Interestingly, while MS-1 (Mechanical stimulus 1 = 28 days of disuse plus 10 minutes per day of a 0.25g mechanical intervention at 90 hertz (Hz)) failed to influence the activity of S-14 relative to control, MS-2 (Mechanical stimulus 2 = 28 days of disuse plus 10 minutes per day of a 0.25g mechanical intervention at 45 Hz) down regulated expression by 45%. Conversely, MS-1 down regulated the activity of OPO-1 by 74%, but MS-2 failed to influence expression. MS-3 (Mechanical stimulus 3 = 28 days of disuse plus 10 minutes per day of a 0.25g mechanical intervention at 22 Hz), which failed to influence parameters of bone remodeling relative to long-term control, failed to influence transcriptional activity of OPO-1 relative to control, while S-14 expression was increased by 33%, thus trending towards disuse activity.
Phase Three: As described in Phase Two, 28 days of tail suspension (DIS) caused both LS and BFR/BV to drop sharply, as compared to LTC. This suppression was not significantly different from the animals subject to disuse for most of the day (23h, 50m) and then allowed to freely bear weight for 10 minutes per day (D+WB). However, 10 minutes per day of either 90 Hz (D+MS1) or 45 Hz (D+MS2) loading normalized LS (+9%; +3%) and BFR/BV (+4%; +11%) to normal weight bearing levels as shown by LTC. Transcriptional activity from the bones of these animals is not yet evaluated. It should be noted that no significant weight differences from LTC were measured in any of the experimental groups.
Conclusion: In addition to the large amplitude strains typically associated with functional activity, smaller magnitude strain signals are evident in bone. These small strain signals persist over long durations, including passive actions, such as standing, and therefore represent a dominant component of the bone's functional strain history. Not surprisingly, therefore, animal models, such as rat-tail suspension, remove this regulatory stimulus, and are therefore permissive to the uncoupling of formation and resorption, and facilitate resorptive activity. In the studies reported here, the investigators have shown the anabolic potential of extremely low level, high frequency mechanical stimuli in several distinct animal models, as well as the first demonstration that these low level biomechanical signals effectively prevent osteoporosis from occurring, even when subject to 23 hours and 50 minutes of a strong stimulus for resorption. As these signals are induced non-invasively, and are so small in magnitude, their role in the prevention and/or treatment of skeletal disorders is promising.
The anabolic potential of extremely low level, high frequency mechanical stimuli is evident even when applied for a very short daily duration. Importantly, from a bioastronautics perspective, these signals effectively prevented disuse osteopenia from occurring, even when the bone was subjected to 23 hours and 50 minutes per day of this strong stimulus for resorption. This mechanical intervention has evolved from efforts to define the osteogenic components within functional activity, and then establishing a non-invasive way of re-introducing these anabolic signals to a skeleton deprived of them due to disuse or aging. Long-term animal studies have shown that both trabecular bone density and strength are actually augmented by such a prophylaxis. Further, preliminary clinical studies on a post-menopausal population are also encouraging; in a randomized double-blind study, short exposure (20 minutes per day) to such mechanical loads inhibits site-specific bone loss (femur and spine) in postmenopausal women. The strong inverse relation that we found between mechanically altered bone formation rates and the expression of a gene involved in osteoclastogenesis expression suggests that mechanical stimulation directly or indirectly influences the expression of this gene.
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