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Keywords:

  • bone adaptation;
  • mechanical loading;
  • bone modeling;
  • rat tibia;
  • osteoporosis;
  • histomorphometry

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

A single 3-minute bout of mechanical loading increases bone formation in the rat tibia. We hypothesized that more frequent, shorter loading bouts would elicit a greater osteogenic response than a single 3-minute bout. The right tibias of 36 adult female Sprague-Dawley rats were subjected to 360 bending cycles per day of a 54N force delivered in 1, 2, 4, or 6 bouts on each of the 3 loading days. Rats in the 6-bouts/day group received 60 bending cycles per bout (60 × 6); rats in the 4-bouts/day group received 90 bending cycles per bout (90 × 4); the 2- and 1-bouts/day groups received 180 and 360 bending cycles per bout, respectively (180 × 2 and 360 × 1). A nonloaded, age-matched control group (0 × 0) and two sham-bending groups (60 × 6 and 360 × 1) also were included. Fluorochrome labeling revealed a 10-fold increase in endocortical lamellar bone formation rate (BFR/bone surface [BS]) in the right tibia versus the left (nonloaded) side in the 60 × 6 bending group. Endocortical BFR/BS in the right tibia of the 4-, 2-, and 1-bout bending groups exhibited 8-, 4-, and 4-fold increases, respectively, over the control side. Relative (right minus left) values for endocortical BFR/BS, mineralizing surface (MS/BS), and mineral apposition rate (MAR) were 65–94% greater in the 90 × 4 and 60 × 6 bending groups compared to the 360 × 1 bending group. Sham-bending tibias exhibited relative endocortical bone formation values similar to those collected from the control (0 × 0) group. The data show that 360 daily loading cycles applied at intervals of 60 × 6 or 90 × 4 represent a more osteogenic stimulus than 360 cycles applied all at once, and that mechanical loading is more osteogenic when divided into discrete loading bouts. Presumably, bone cells become increasingly “deaf” to the mechanical stimulus as loading cycles persist uninterrupted, and by allowing a rest period between loading bouts, the osteogenic effectiveness of subsequent cycles can be increased.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

THE VERTEBRATE appendicular skeleton comprises an assembly of rigid levers, on which muscle and substrate reaction forces must act to produce movement. To ensure mechanical integrity during functional use, bone tissue has evolved the ability to adapt its mass,(1,2) architecture,(3,4) and mechanical properties(5) to meet the prevailing dynamic mechanical loading environment. The cyclic deformation of bone matrix, which occurs during normal functional use, is the net result of a number of individual strain components. To gain an understanding of the exact component(s) that control bone adaptation, considerable effort has been spent on deconstructing the dynamic loading cycle into its constituent elements and investigating their effects individually on bone tissue kinetics. Results from a number of animal experiments have suggested that the rate,(6–8) magnitude,(9–11)distribution,(12,13) frequency,(14,15) and duration(12,16) of the dynamic strain stimulus are among the important components in bone adaptation, though their precise roles and interactions are poorly understood.

As a consequence of the recent emphasis on identifying the exact mechanical signal to which bone adapts, several other important characteristics of bone's modus operandi for adaptation have been overlooked. Among them is the effect of loading session (bout) frequency on bone formation. Several authors have suggested that the volume of newly formed bone observed at the end of a period (weeks or months) of increased loading stimulus represents the sum of discrete bone packets formed by individual bone-forming cellular units, each of which is activated by a bout of loading.(17,18) Thus, according to the model, by increasing the number of mechanical loading bouts, the number of active bone-forming cellular units is increased, which in turn results in a greater number of newly formed bone packets by the end of the experimental period. This model is supported by the positive, linear association between the number of loading days (where 1 bout is applied per day) and bone formation on both endocortical(18) and trabecular(19) surfaces. If this is true, it should be possible to increase bone formation by applying more than one mechanical loading bout per day on each loading day.

The osteogenic response to dynamic loading appears to saturate after relatively few cycles.(12,20,21) A second bout of mechanical loading, if administered before the bone cells have had time to regain mechanosensitivity from a first bout, might go undetected, given the saturated state of the cellular network. Consequently, the second bout would be incapable of activating a bone-forming cellular unit.

Based on the linear association between bone formation and the number of loading days (at 1 bout/day), 24 h appears to be a sufficient amount of time for the bone cell network to recover fully mechanosensitivity. Using the rat tibia 4-point bending model, we investigated whether the osteogenic response to mechanical loading could be increased by applying multiple bouts of 4-point bending within a 24-h period, while holding the total number of load cycles administered per day (360 cycles) constant. Specifically, we tested the hypothesis that a greater osteogenic response can be elicited from 360 daily load cycles if those cycles are partitioned into discrete loading bouts (separated by recovery periods) than if they are applied during a single loading session.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Sixty-three adult female Sprague-Dawley rats were selected for the experiment. The rats were housed two per cage at Indiana University's Laboratory Animal Resource Center (LARC) for 2 weeks before the experiment began and were provided standard rat chow and water ad libitum during the acclimation and experimental periods. All procedures performed in this experiment were in accordance with the Indiana University Animal Care and Use Committee guidelines. Under ether-induced anesthesia, the right tibia was subjected to either medial-lateral bending or sham bending using a 4-point bending apparatus described previously (Fig. 1).(15) When a downward-directed force is applied to the upper platen of this device, a bending moment is produced in the region between the upper loading points,(22) resulting in compression of the lateral tibial surface and tension on the medial surface.

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Figure Figure 1. (A) The 4-point bending apparatus used to apply mediolateral bending to the rat tibia. The limb is maintained in proper position during the loading bout by the foot stirrup and by pressure from the padded load points. Note that the upper proximal load point is beveled to accommodate fibular morphology. (B) Lateral view of the right tibia. The upper (U) and lower (L) load points are shown (shaded) in their actual position relative to the tibia during loading. The scale (left) is in millimeters. (C) The stippled region in B, from which histomorphometric data were collected, is illustrated in the transverse plane. The neutral plane (N.P.) and the strain distributions (relative shading) created by the 4-point bending apparatus are shown. The perforation in the posterior cortex, which is normally present at that cross-sectional level, houses the nutrient artery.

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Experimental design

The rats were divided randomly into four bending groups, two sham-bending groups, and one nonloaded control group (group n = 9) and were weighed each day. To each of the bending and sham-bending groups, 360 load cycles were applied to the right tibia on days 1, 3, and 5 of the experimental period (Fig. 2A).(23) Force was applied to the upper platen of the 4-point bending apparatus using an open loop, stepper motor-driven spring linkage. In all loading groups, load was applied as a haversine wave at a frequency of 2 Hz. The peak magnitude of the applied force was 54N, with a net change (peak-to-trough) in force during loading of 52N (a 2N static component was included). In the bending groups, the upper load points were spaced 11 mm apart and were centered between the lower load points, which were positioned 23 mm apart. This load point configuration imposes peak strains of approximately −2400 μϵ on the lateral periosteal surface and approximately −1300 μϵ on the lateral endocortical surface for a peak load of 54N.(22,24)

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Figure Figure 2. (A) Overview of the experimental design. Rats were loaded on days 1,3, and 5 and then killed on day 16. Fluorochrome labels were given on days 5 and 12. (B) The loading days in A are shown in greater detail (broken arrows). Time of day is indicated across the top of the figure. Each filled box within the diagram indicates a loading bout; the number inside the box indicates the number of cycles applied during that bout. Sham-bending bouts are indicated by black boxes; bending bouts are indicated by gray boxes. Note that at the end of each loading day, all rats (excluding those in the control group) had been given 360 load cycles.

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Bending groups differed from one another only in the temporal distribution of the 360 load cycles received throughout the day (Fig. 2B). On each loading day, one group of rats (60 × 6) received 60 cycles of bending in each of 6 discrete loading bouts, each bout separated by 2 h. A second group (90 × 4) received 90 cycles of bending in each of 4 discrete loading bouts, each bout separated by 3 h. A third group (180 × 2) was administered 180 cycles of bending twice per day, with 6 h between the first and second bout. The fourth bending group received all 360 bending cycles in one single bout (360 × 1).

Because force is applied to the tibia through the soft tissues (skin, muscle, fascia, vessels, and periosteum), it is also necessary to separate the potential effects of local irritation/inflammation on bone formation from those initiated by mechanical deformation of the bone tissue. To reveal the effects of irritation/inflammation, sham bending was applied to the right tibias in two groups, administered according to the 60 × 6 or 360 × 1 schedules. Sham bending was accomplished by aligning the lower loading points with the fixed upper points (both sets 11 mm apart). This arrangement can be used to apply the same load—and consequently the same irritation stimulus—to the soft tissues without bending of the diaphysis. Previous studies have shown that strain induced on the lateral periosteal surface using the sham configuration at 31N is negligible (∼140 μϵ).(25) An age-matched control group received neither loading nor ether. All rats were allowed normal cage activity between loading bouts.(26)

Bone labeling, processing, and histomorphometry

All rats were given an intraperitoneal (ip) injection of calcein (7 mg/kg body mass; Sigma Chemical Co., St. Louis, MO, U.S.A.) on days 5 and 12, and were killed on day 16. The right and left tibias were removed, cleaned of soft tissue, cleaved at the proximal and distal ends to allow proper infiltration of the marrow cavity, and immersed in 10% neutral buffered formalin for 48 h. The diaphyses were dehydrated in graded alcohols, cleared in xylene, and embedded in methylmethacrylate. Using a diamond-embedded wire saw (Histo-saw; Delaware Diamond Knives, Wilmington, DE, U.S.A.), transverse thick sections (∼70 μm) were removed from the tibial diaphysis 6 mm proximal to the tibia-fibula junction (Fig. 1B), and mounted unstained on standard microscope slides.

One slide per limb was read on a Nikon Optiphot fluorescence microscope (Nikon, Inc., Garden City, NY, U.S.A.). Ultrastructural organization of the newly formed tissue (woven-fibered vs. lamellar) was determined from polarized light microscopy. Using the Bioquant digitizing system (R & M Biometrics, Nashville, TN, U.S.A.), the following primary data were collected from the endocortical surface at ×125 magnification: total bone perimeter (B.Pm), single-label perimeter (sL.Pm), double-label perimeter (dL.Pm), and double-label area (dL.Ar).(27) At the same magnification, woven bone perimeter (Wo.B.Pm)—defined as those portions of the original periosteal surface overlain with new, labeled woven bone—was measured on the periosteal surface. Cortical bone area (Ct.B.Ar) and supraperiosteal woven bone area (Wo.B.Ar) were measured at ×25 magnification.

From these primary data, the following first- and second-order-derived quantities were calculated for the endocortical surface: mineralizing surface (MS/bone surface [BS] = [½ sL.Pm + dL.Pm]/B.Pm; %), mineral apposition rate (MAR = dL.Ar/dL.Pm per 7 days; μm/day), lamellar bone formation rate (BFR/BS = MAR × MS/BS × 3.65; μm3/μm2 per year), and from the periosteal envelope, percent woven BS (Wo.BS/BS = Wo.B.Pm/B.Pm; %) and percent woven bone area (Wo.B.Ar/Ct.B.Ar; %).

To control for individual differences in systemic factors, left tibia (nonloaded control) values were subtracted from right tibia values; this procedure results in a new set of relative (r) values for each variable (e.g., rBFR/BS). Differences between the loaded (right) and nonloaded (left) tibias were tested using Student's t-test for paired variates. Differences among group means were tested for significance by analysis of variance (ANOVA), followed by Fisher's protected least significant difference (PLSD) for all pairwise comparisons. Dunnett's method (two-tailed) was used to test for differences between the loaded groups and the control group (0 × 0).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Three of the 63 rats died during the loading period from anesthesia complications. Over the 5-day loading period, rats in the bending and sham-bending groups exhibited a slight but significant decline in body mass (pooled groups, p < 0.01). The degree of loss was positively associated with the number of anesthesia sessions per day (r2 = 0.82; p < 0.01), though none of the animals fell below 90% of initial body mass. After the loading period (days 5–16), body mass increased slightly but nonsignificantly in most groups. Left tibia bone formation rates in each of the loaded groups were not significantly different from those in the control group (Dunnett's post hoc, p > 0.05), which suggests that the different number of anesthesia sessions per day had no discernible effect on baseline bone formation.

A woven bone response was observed on the periosteal surface of all loaded tibias. None of the left (nonloaded) tibias exhibited any periosteal woven bone. There were no significant differences in percent woven bone surface or percent woven bone area between the sham-bending groups and their bout-matched bending counterparts. The similar periosteal response among bending and sham-bending animals indicates that this response is caused by periosteal pressure rather than bending strain, and, therefore, these data are uninformative. Consequently, the periosteal data will not be considered further.

On the endocortical surface of both loaded and nonloaded tibias, polarized light microscopy revealed that bone formation during the experimental period consisted of lamellar bone exclusively (Fig. 3). All bending groups exhibited significantly greater mineralizing surface, mineral apposition rate, and bone formation rate in the loaded tibias than in the nonloaded tibias (Table 1). Among the sham-bending groups, the 360 × 1 group exhibited no significant right vs. left differences for any of the derived quantities. The 60 × 6 sham-bending group exhibited no significant right vs. left difference for MAR; however, mineralizing surface and bone formation rate were slightly but significantly greater in the right tibia. Although significant, the right vs. left differences in mineralizing surface and bone formation rate in the 60 × 6 sham-bending group were minor compared with those calculated in the 60 × 6 bending group (Fig. 4). The control group (0 × 0) exhibited no significant side differences in any of the endocortical-derived quantities.

Table Table 1.. Summary of Endocortical Bone Formation After 1 Week of 4-Point Bending, Sham Bending, or No Loadinga
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Figure Figure 3. Photomicrographs of the left and right tibial endocortical surface from an animal in the 90 × 4 group. Among the high-bout bending groups, endocortical bone formation was markedly increased in the loaded tibia when compared with the nonloaded tibia from the same animal. New bone on the endocortical surface exhibited lamellar organization exclusively, confirmed by polarized light microscopy. Double labeling is easily identified in the loaded limb, particularly in the medial and lateral regions where the strains were greatest. The control limb exhibits rudimentary baseline bone formation.

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Figure Figure 4. On the endocortical surface, the (A) relative BFR/BS, (B) relative MS/BS, and (C) relative MAR were positively associated with the number of bouts per day among the bending groups (white bars). Sham bending (striped bars), when applied according to the high-bout (60 × 6) and low-bout (360 × 1) schedules, elicited a response similar to that observed in the nonloaded group (0 × 0). *Significantly different from 360 × 1 bending group; †significantly different from 180 × 2 group, based on Fisher's PLSD at α = 0.05.

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Relative (right minus left) mineralizing surface, mineral apposition rate, and bone formation rate were significantly greater among all bending groups when compared to the control (0 × 0) group (Dunnett's post hoc, p < 0.01), with the exception of mineral apposition rate in the 360 × 1 bending group. Conversely, both sham-bending groups exhibited no significant difference from the control group for any of the relative endocortical parameters (Table 1).

Each bending group exhibited significantly greater relative bone formation rate and relative mineral apposition rate than either of the two sham-bending groups PLSD, p < 0.05). Among the bending groups, increasing the number of bouts per day resulted in an increase in relative mineralizing surface, relative mineral apposition rate, and relative bone formation rate (Fig. 4); by applying the 360 bending cycles in 4 discrete bouts (90 × 4) rather than 1 large bout (360 × 1), a 71% increase in relative mineral apposition rate, an 80% increase in relative bone formation rate, and a 94% increase in mineralizing surface were achieved. As expected, the 180 × 2 group exhibited values between the 90 × 4 and 360 × 1 bending groups. The 180 × 2 group exhibited significantly lower relative mineral apposition rate than the 60 × 6 bending group and significantly lower mineralizing surface than the 90 × 4 group. No significant differences were found between the two greatest bout-per-day groups (60 × 6 bending and 90 × 4) for any of the derived endocortical values.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Our objective in this study was to determine whether the osteogenic response to a daily mechanical stimulus could be increased if the stimulus were applied in several discrete bouts rather than one longer bout. The results from the endocortical surface show that 360 bending cycles per day elicit a much greater osteogenic response—from 65 to 94% greater—if those cycles are partitioned into 4 or 6 bouts than if they are administered uninterrupted in 1 longer bout. These data suggest that the cellular and molecular events responsible for converting a mechanical signal into an osteogenic response experience a decay in function as a dynamic signal persists uninterrupted. By introducing a rest period between bouts (normal cage activity), the bone cells appear to regain some of their mechanosensitivity. For the same number of cycles, providing more “rest” periods, that is, administering the cycles in more bouts, results in a greater osteogenic response. A recovery period of approximately 2–3 h appears to be sufficient to reset the cells to their mechanosensitive state.

The increase in bone formation observed in the bending groups—particularly among the high bout (4-6 bouts/day) groups—was the result of an increase in mineralizing surface and an increase in mineral apposition rate. This suggests that the mechanically induced bone formation on the endocortical surface was caused by the combined effect of an increase in the number of osteoblasts activated and an increase in the synthetic activity of osteoblasts already on the bone surface.

These data should be considered in light of several limitations of the experiment. First, the positive association between bout frequency and bone formation raises the possibility that bone formation could be enhanced further by distributing the 360 daily cycles into more than 6 bouts/day. Extension of the experimental design (Fig. 2) beyond 6 bouts/day (e.g., 30 × 12) was not attempted because of technical limitations (it would require some rats to endure more than six separate anesthesia sessions per day). In addition, no significant differences were found between the 90 × 4 and 60 × 6 bending groups for any of the derived endocortical variables, suggesting that the peak response had been achieved. Second, the 60 × 6 sham-bending group exhibited slightly but significantly elevated mineralizing surface and bone formation rate in the loaded limb. However, relative values for these parameters were not significantly different from those in the no-load group (see Table 1). Furthermore, relative mineralizing surface and relative bone formation rate were three and seven times greater, respectively, among the 60 × 6 bending group compared to the 60 × 6 sham-bending group (Fig. 4). Thus, unlike the periosteal surface, the endocortical surface appears to have been largely unaffected by sham bending.

In conclusion, when 360 cycles of 4-point bending are applied to the rat tibia in several discrete bouts, separated by a few hours, the osteogenic response on the endocortical surface is greater than that observed if all 360 cycles are applied without interruption. We found that 4 to 6 brief loading bouts per day—each bout being separated by 2–3 h—improves the osteogenic response to 360 daily load cycles by nearly 100%. These data support the concept of a saturation curve for bone cell mechanosensitivity, and suggest that physical activity programs aimed at maintaining or improving bone mass can be optimized by scheduling mechanical loading bouts (exercise sessions) so that most of the load cycles occur during a time when the bone cell network is highly mechanosensitive. This probably would entail several shorter intervals of daily exercise, rather than a single sustained session.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We thank Mary Hooser, Diana Jacob, and Thurman Alvey for assistance with tissue processing. This work was supported by National Institutes of Health (NIH) grants R01 AR43730 and T32 AR07581.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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