Recovery periods restore mechanosensitivity to dynamically loaded bone
Alexander G. Robling1,2,*,
David B. Burr1,2 and
Charles H. Turner2
1 Department of Anatomy and Cell Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA and
2 Department of Orthopaedic Surgery and Biomechanics and Biomaterials Research Center, Indiana University School of Medicine, Indianapolis, IN 46202, USA
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Fig. 1. Diagram of the rat tibia four-point bending apparatus with the rat in situ. (A) A mediolateral bending moment is produced in the portion of the tibial shaft between the two upper (padded) load points when a force is applied to the upper platen of the device. (B) By moving the lower load points inwards, so that they directly oppose the upper points, a force applied to the upper platen will squeeze the soft tissues intervening between the bone and the load points, as does the configuration depicted in A, but negligible bending of the shaft occurs. This sham configuration allows assessment of the effect of soft tissue irritation (and consequent inflammatory response) on bone formation, as distinct from a coordinated mechanically adaptive response (Robling et al., 2000).
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Fig. 2. Overview of the experimental design for the long-term (A) and short-term (B) recovery experiments. (A) The upper part outlines the experimental period; rats were loaded on days 1, 3 and 5 and then killed on day 16. The lower panel details a single loading day. Time of day appears across the top of the diagram. Each filled box within the diagram indicates a loading bout; the number inside the box indicates the number of cycles applied during that bout. The 0 h recovery group received a single bout of 360 cycles, which is equivalent to administering four bouts of 90 cycles with no time between each of the four bouts. Note that, at the end of each loading day, all rats (excluding those in the control group) had been given 360 load cycles. (B) The top panel outlines the experimental period; rats were loaded on days 1–5 and 8–12 and then killed on day 16. Rats were administered 36 load cycles per day with one of the following recovery periods introduced between cycles: 0.5 s (back-to-back cycles, i), 3.5 s (ii), 7 s (iii) or 14 s (iv). The 0.5 s group was loaded for 18 s (36 cycles at 2 Hz), the 3.5 s group was loaded for 2 min, the 7 s group was loaded for 4 min and 14 s group was loaded for 8 min (the total duration of the loading session is not shown on the figure for the 3.5, 7 and 14 s groups).
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Fig. 3. (A) Fluorescence photomicrographs of the endocortical surface of a right (loaded in bending) and left (nonloaded control) tibia from an animal in the 14 s recovery group. Sparse single and double labeling is present in the control limb, but the loaded limb shows long stretches of well-separated double labeling, particularly on the medial and lateral surfaces where strains were greatest. A, anterior; L, lateral; M, medial; P, posterior. (B) Higher-power photomicrographs of the boxed area from the loaded limb in A. The left and right panels illustrate the same field under polarized white light (left panel) and fluorescence (right panel). Taken together, the photomicrographs show that the bone formed during the experiment, i.e. the bone between labels, exhibits lamellar organization.
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Fig. 4. (A) Relative mineralizing surface, (B) relative mineral apposition rate and (C) relative bone formation rate in the long-term recovery experiment. The relative (right minus left) bone formation rate (rBFR/BS; C) and relative mineralizing surface (rMS/BS; A) were positively associated with the recovery period duration among the bending groups (open columns). Relative mineral apposition rate (rMAR; B) exhibited a bending effect, but no trends were apparent among the bending groups. With the exception of the 8 h sham group for relative mineralizing surface, sham bending (filled columns) elicited a response similar to that observed in the nonloaded group (filled column). For comparison among bending groups, an asterisk indicates a significant difference from the 0 h bending group and a double dagger indicates a significant difference from the 0.5 h group, based on Fisher’s protected LSD at  =0.05. The significance of right (loaded) versus left (nonloaded) comparisons is indicated in Table 1. Values are means + S.E.M. For values of N, see Table 1.
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Fig. 5. Relative bone formation rate (rBFR/BS) shows essentially the same response to 8 h of recovery as would be expected from extrapolation (grey line) to 12 h of recovery, which is the maximum recovery period possible for this experimental design (spacing the bouts more than 12 h apart would have extended the first group of four bouts (load ‘day’ 1) into the second group of four bouts (load ‘day’ 2), which began 48 h after the first group of bouts began; see Fig. 2). The curve is described by the equation: rBFR/BS=272+44.5log2(recovery time). Values are means ± S.E.M., N=7–9 per data point.
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Fig. 6. (A) Relative mineralizing surface, (B) relative mineral apposition rates and (C) relative bone formation rate in the short-term recovery experiment. The relative (right minus left) mineralizing surface (rMS/BS), relative mineral apposition rate (rMAR) and relative bone formation rate (rBFR/BS) were enhanced in the bending groups (open columns) but not in the sham-bending or control groups (filled columns). The 14 s recovery group exhibited a significantly greater relative bone formation rate and relative mineralizing surface than the other three bending groups, which were not significantly different from one another, but were significantly different from each of the three controls. For comparison among bending groups, an asterisk indicates a significant difference from the 0.5, 3.5 and 7 s bending groups, based on Fisher’s protected LSD at =0.05. The significance of right (loaded) versus left (nonloaded) comparisons is indicated in Table 2. Values are means + S.E.M. For values of N, see Table 2.
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Fig. 7. (A) Bone cell mechanosensitivity declines soon after a loading bout (black line) is initiated, until a mechanosensory saturated state is reached where further mechanical stimulation produces no further osteogenic response. A recovery period, during which mechanical stimulation is drastically reduced or withheld, is required to return sensitivity to its preload value (100 % mechanosensitivity is represented by the broken line at the top). (B) When bone cells are stimulated, then allowed to recover (grey line) for a period sufficient to restore full sensitivity (approximately 8 h on the basis of the data presented) before the next bout is applied, the succeeding loading bout will stimulate fully sensitive cells. Consequently, a robust anabolic response can be generated from each bout. (C) The same initial loading bout as applied in B will stimulate the cells, but if subsequent bouts are initiated before full recovery has been achieved, the loading bouts will be applied to cells experiencing (temporary) mechanosensory impairment. Consequently, a compromised osteogenic response will result. Note that bouts 2–4 begin when the cells are at approximately 50–60 % of maximal sensitivity. B is representative of the 8 h recovery group; C is representative of the 0.5 h group.
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© The Company of Biologists Ltd 2001
