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The effects of altered strain environments on bone tissue kinetics
Bone, 10, 215-221 (1989) Printed in the USA. All rights reserved.
8756-3282189 $3.00 + .OO Copyright 0 1989 Pergamon Press plc
The Effects of Altered Strain Environments on Bone Tissue Kinetics D. B. BURR,‘v2 M. B. SCHAFFLER,3 K. H. YANG,2 D. D. WU,2 M. LUKOSCHEK,2 N. SIVANERI,4 J. D. BLAHA and E. L. RADIN2
D. KANDZARI,’
’ Department of Anatomy, ’ Department of Orthopedic Surgery and Orthopedic Research Laboratory, West Virginia University, Morgantown, WV, uSA 3 Division of Anatomy, University of California at San Diego, La Jolla, CA, USA 4 Department of Mechanical Engineering, West Virginia University, Morgantown, WV, USA Address .for correspondence and reprints: David B. Burr, Ph.D., Department of Anatomy, West Virginia University Center, Morgantown, WV 26506, L&A.
1985) has demonstrated that manipulating the strain environment in well-controlled ways causes specific adaptive alterations. Those studies do not yield insight into the biological dynamics of the adaptive change. Aspects of the strain environment may affect the remodeling sequence at different stages and/or at different rates. An altered mechanical strain environment may affect activation frequency, the time required to form a new packet of bone (sigma), the balance between resorption and formation, or the time required to mineralize new bone. If mechanical factors are ever to be used as probes to reverse osteopenias (which can be caused by alterations of any of the above factors acting singly or in combination), then the way in which strain affects remodeling kinetics must be described. Moreover, these data provide the rate constants required for some mathematical models of bone adaptation (Cowin and van Buskirk 1978, 1979; Cowin and Firoozbakhsh 1981; Hart et al. 1984; Cowin et al. 1985; Frost 1988). Therefore, this kind of histomorphometric data provide necessary information for future mathematical predictions of bone response. We hypothesize that the adaptive response to elevated mechanical strain occurs by increasing the activation frequency for bone remodeling and by altering bone balance in favor of formation at each site, rather than through direct effects on the individual activity of already dtierentiated cells. The purpose of this work was to test this hypothesis by defining the alteration in bone tissue kinetics responsible for observed geometric changes in bone architecture in response to altered strain environments.
Abstract This study defines the alteration in bone tissue kinetics responsible for the “adaptive remodeling” response to altered strain environments. Adult beagle dogs were separated into three experimental groups: uhtar osteotomy, ulnar osteotomy with fracture fixation plate spanning the gap and sham surgery. Four sets of double fluorochrome labels were administered. Prior to sacrifice at 1, 3, and 6 months, strains were measured through rosette strain gages on the cranial and caudal surfaces of the intact radius. Histomorphometric analysis hulicated that the increased bone mass in response to elevated strain results tkom increased activation frequency of modeling with more sites undergoing formation processes than resorption processes on per&teal and endocortical surfaces. Increased remodeling activation did not lead to increased bone mass. There was no evidence that elevated strain changes the individual vigor of osteoclasts or osteoblasts, or that the sigma period was altered by elevated Strain.
Key Words: Strain-Bone
Health Sciences
kinetics-Remodeling-Dogs.
Introduction It is generally accepted that bone models and remodels in response to changes in its mechanical strain environment. Remodeling is a process in which the initiation of change (activation) is followed in sequence by the resorption and formation of bone (Activation-Resorption-Formation). Modeling, on the other hand, involves either the resorption or formation of bone following activation (Activation-Resorption or Activation-Formation), but resorption and formation do not occur in sequence at the same location. Both the nature of the mechanical stimuli which underlie the initiation of each process, and the tissue kinetics of the mechanically adaptive response, are still open to question. The work of Lanyon (Goodship et al. 1979; Lanyon and Bourn, 1979; Lanyon et al. 1979; O’Connor et al. 1982; Lanyon et al. 1982) and Rubin (Rubin and Lanyon 1984,
Materials and Methods Experimental
design
Seventeen skeletally mature 1 -2-year-old male beagle dogs weighing 11-18.5 kg were divided randomly into three experimental groups. Six dogs had a l-2 cm segment of the distal ulna removed (Group 0). Five additional animals (Group P) had ulnar osteotomies, but the osteotomy gap was spanned with a j-hole metal plate from the mini215
D. B. Burr et al.: Strain and bone kinetics
216
fragment fracture fixation set (Zimmer, Warsaw, IN). A sham operation was performed in six dogs: the periosteum of the ulna was incised in these animals, but no bone was removed (Group S). Animals were sacrificed at 1, 3, or 6 months after surgery. Five additional dogs were used to measure normal strain in the radius, and six dogs were used to measure strain changes 48 h following either an ulnar osteotomy alone, or after a plated osteotomy. These experimental groups are summarized in Fig. 1. All animals received food and water ad libitum during the experiment. For 2-4 weeks prior to surgery, each dog was acclimated to walk on the treadmill at 4 km/h for 20 minld, 5 dlwk. Following surgery and until sacrifice, dogs were housed in individual 4 ft x 8 ft pens, and each dog was exercised for 20 min daily on the treadmill using the protocol described. Animals in all groups were given four sets of double fluorochrome labels using a 1-7-1 scheme to monitor net bone formation and apposition rate throughout the experiment. Oxytetracycline (30 mg/kg; A. J. Buck, Pittsburgh, PA) was given orally prior to the osteotomy or sham surgery. Subsequently, DCAF (20 mg/kg; ICN, Cleveland, OH), alizarin red (25 mg/kg; Sigma, St. Louis, MO) and xylenol orange (90 mg/kg; Sigma, St. Louis, MO) were given intramuscularly at intervals outlined in Fig. 1. Two days prior to sacrifice strains were measured on the cranial and caudal surfaces of the intact right (treated) radius using 45” rosette strain gages. The magnitudes of the principal strains were calculated from the raw strain measurements using standard formulae.
Each animal was given general anesthesia of halothane, nitrous oxide and oxygen. Surgical approach to the ulna was made from the lateral surface of the dog’s forelimb. A 15 cm incision was made through the skin and fascia. Blunt dissection between the flexor carpi radialis and flexor carpi ulnaris was used to expose the ulna. Special care was taken to obtain hemostasis to limit any potentially dissecting hematoma. The periosteum of the ulna was split longitudinally over a 10 cm distance and elevated. The osteotomy was made under saline irrigation using a
2 1 PRE-OP
1
Strain gage application
Strains were measured using either stacked 45” rosettes (BLH Electronics No. FABR-12-12-S6) or a smaller set of unstacked 45” rosette gages (Micromeasurements, EA-06015-RJ-120). Under anesthesia, rosette strain gages were applied cranially and caudally to the midshaft of the radius. The strain gages were positioned so that one gage was aligned with the long axis of the radius, and position was radiographically verified postoperatively. Strain recording procedure
Prior to measuring strains during walking, each dog was held off the ground while the strain gages were zeroed. The dog was then walked at 4 km/h on the treadmill while strain recordings were made. The strain gage signal was amplified via bridge amplifiers (Measurement Group Model 2311, Micromeasurements, Raleigh, NC). The amplified strain data were collected from each gage of a rosette simultaneously using a 16 channel FM tape recorder (Bell and Howell) at a speed of I5 in/s. Data were monitored concurrently on two Gould brush chart recorders. Subsequently, raw strain data were digitized at 200 Hz using an IBM PC microcomputer, and principal strains and directions were calculated from this data. Histological procedures
Surgical procedures
WEEKS
Gigli saw. A 5-hole plate from the minifragment set was positioned on the lateral side of the ulna in Group P animals and fixed by four cortical bone screws (No. 8 or No. 10) in previously tapped drill holes.
2
4
6
6
10
12
14
16
16
20
22
24
POST-OP
Fig. 1. An outline of the experiment showing the number of animals in each experimental group, the period between surgery and sacrifice, and the label schedule. Group 0, ulnar osteotomy; Group P, ulnar osteotomy with fracture fixation plate spanning osteotomy gap; Group S, sham-operated group. The letters within each bar depict the label given: T, oxytetracycline; D, DCAF; A, alizarin red; X, xylenol orange.
and morphometric
measurements
Following sacrifice, the radius of each forelimb was removed for quantitative histological analysis of bone tissue kinetics. Undecalcified complete cross sections were cut at 25%, 50% and 75% of the length of the radius using a Buehler Isomet low speed saw with a circular blade, and preserved in 70% ETOH. Sections were also made through the screw holes in Group P and at the osteotomy site in Group 0 if different from the three levels described. Because strains were only measured at midshaft, only histological data from the midshaft sections are reported here. Because the site for histological analysis was removed from the distal surgical site, the chance for localized effects due to surgical procedures was reduced. Sections were ground in alcohol to 150 microns with 600 grit silicon carbide paper and polished with 0.30 grit alumina suspended in alcohol. The sections were stained using the tetrachrome method of Villanueva et al. (1964) and mounted. Up to three sections per radius were analyzed microscopically using reflected ultraviolet light at 212 x and 534 x magnification. Measurements listed in Table I were made by tracing sections onto a digitizing tablet through a drawing tube attached to the microscope. A Bioquant image analysis system (R & R Biometrics, Nashville, TN) interfaced to an IBM-PC XT microcomputer was used to collect the data. The experiments were designed to examine temporal differences in the entire adaptive process in response to altered strain environments, rather than to look for statistically significant differences among groups at predetermined time periods. For this reason, we used a smaller number of dogs, and, did more complete histomorphometric measurements on this sample. As can be seen from
D. B. Burr et al.: Strain and bone kinetics
217
Table I. Definition of measurements
Intracortical measurements 1. A, (#/mm2): The number of osteonal resorption spaces per unit area. 2. A, (#/mm2): The number of refilling osteons, as determined by stained osteoid seams. 3. Linear apposition rate o&d): The mean distance between fluorochrome labels in doubly labeled osteons, divided by the time between labels. 4. OSW (pm): The average width of osteonal osteoid seams. 5. MWT (pm): The mean wall thickness in completed osteons, that is, the distance from the cement line to the Haversian canal wall. 6. Sigma (d): The time required to initiate and complete a new osteon. 7. mu (#/y/mm*): The activation frequency, or “birth-rate” of new remodeling sites. Surface measurements 1. P; (%): The proportion of periosteal surface labeled with a particular fluorochrome. 2. E’. (%): The nronortion of endocortical surface labeled with a particular fluorochrome. 3. Linear apposition rate o.un/d): The mean distance between double fluorochrome labels on oeriosteal or endocortical surfaces, divided by the time
between labels. 4. Periosteal expansion rate (pm/y): The surface-based
bone
formation rate; the product of Pr’ and periosteal linear 5. Endocortical
contraction
apposition rate, x 365. rate (pm/y): The surface-based bone
formation rate; the product of E,’ and
endocortical linear apposition rate, x 365.
-1200
I
I
Results Strain measurements Figure 2 shows the change in principal strain magnitudes following a surgical osteotomy. The change in strain distribution in Groups 0 and P following surgery is most evi-
I
dent. While the cranial and caudal surfaces of the radius in beagle dogs are normally subject to compressive strain, removal of part of the ulna increases bending strains on the intact radius so that the cranial cortex is placed in tension. Caudal compressive strains are also increased following osteotomy. Caudal compressive strains remain elevated in Group 0 animals throughout the 6 month exoeriment. but those in Group P animals have returned to normal ‘by 3 months postoperatively. Six months after the osteotomy peak strain magnitudes on the radius in Group 0 animals are still elevated 2-3 times, but strains on the caudal surface of the radius of Group P animals have returned to normal. Adaptive changes on periosteal
Figures 3-10, comparing data at only 1, 3 and 6 months would provide a very different-and erroneous-view of the adaptive response. Standard deviations are presented where they are available or when differences between groups are large. We employed an analysis using 2 scores (Weinberg and Schumaker 1974) to determine whether measurements lie within the normal range of variation for each remodeling parameter. 2 scores measure the distance in standard deviation units a measurement lies from the presurgery population mean for a particular character (Weinberg and Schumaker 1974). The shaded area on the graphs represents two standard deviations about the mean presurgery data value. This can be considered the normal range of variation in adult beagle dogs. Values which lie outside this range have Z scores larger than 2.0 and can be considered, with 95% probability, to lie outside the range of variability for that particular characteristic. When variables have Z scores less than 2.0 we interpreted no differences from presurgery control values.
I
Fig. 2. Bar graph depicting mean principal strain magnitudes on the anterior and posterior surfaces of the radius for each group at three intervals following ulnar osteotomy or sham surgery. The first bar in each set represents mean strain on the anterior cortex while the second bar represents mean strain on the posterior cortex. Strains on the radius of the sham-operated animals are taken to be normal.
endocortical
and
surfaces
The surgical removal of a portion of the ulna produced an almost immediate response on the periosteal and endocortical envelopes of the radius. There was an early increase in periosteal woven bone apposition, resulting in an increased expansion rate by 2 weeks in Group 0 animals (Fig. 3a). Group P animals also showed the transient early increase in periosteal expansion of the radius, but this is reduced to within the normal range by 6 weeks. Although not reflected in the sham-operated animals, the transient nature of this reaction suggests it was a result of the surgical procedures. The transient response was followed by a more significant and sustained increase in periosteal new bone in Group 0, beginning between 4 and 6 weeks. This sustained increase was not observed in Group P. This response declined to normal by 12 weeks postsurgery and remained within the normal range through 24 weeks. Because the sustained response did not occur in Group P animals, it may be a reflection of the normal periosteal adaptive response to the altered strain environment. The greater proportion of labeled endocortical surface resulted in increased endocortical contraction rates, and demonstrates one reason for the smaller radial medullary cavities in Group 0 animals 6 months nostonerative (Fig. 4a). Again, a small early transient increase in endocor&l bone formation was observed in some, but not all, animals. After this, however, a more sustained formation drift oc-
D. B. Burr et al.: Strain and bone kinetics
218 PERIOSTEAL
EXPANSION
RATE
ENDOSTEAL
CONTRACTION
RATE
Oeteotomy n Plate A Sham * Pre-operative 0
ii
30 20 10 0
-10
- 10 -20 5
15
10
20
I
I
5
10
I
,
15 WEEKS
20
\r,I 25
25
PROPORTION
WEEKS
ENDOSTEAL
SURFACE
LABEL
l
PROPORTION
PERIOSTEAL
SURFACE
34 20
LABEL
a B
10
k E
5
7’ a
0
I
5
1
*Overlappingdata
T
w T - 15 g
Osteotomy . Plate A Sham * Pre-operative
l
Oateotomy . Plate A Sham
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I
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I
10
15
20
25
T
1
WEEKS -20
I 5
I
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15
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25
WEEKS
Fig. 3. Changes over the experimental period in the periosteal expansion rate (a) and in the proportion of the periosteal surface undergoing active mineralization (b). The gray region represents the normal range of variation. Points outside this range have 2 scores >2.0, and can be considered outside the normal range.
curred, beginning at about 4 weeks and ending at 16 weeks. The initiation of a second phase of increased formation was apparent by 24 weeks. In general, Groups P and S demonstrated little formation on the endocortical surface. However, one animal in Group P did show a marked response at about 16 weeks; because this was only represented by one animal at one time period, and did not constitute part of a recognizable pattern, this most likely does not indicate a sustained response to the strain regime but rather an aberration of the sampling procedure. Increased periosteal expansion and endocortical contraction rates were largely the result of increased total forming surface (Figs. 3b and 4b) rather than increased appositional rates (Fig. 5). The transient early response was caused by both increased forming surface and increased rate, but appositional rates on the periosteal and endocortical surfaces were normal throughout the rest of the experiment.
Fig. 4. Changes over the experimental period in the endocortical contraction rate (a) and in the proportion of the endocortical surface undergoing active mineralization (b). The gray region represents the normal range of variation. Points outside this range have Z scores >2.0 and can be considered outside the normal range.
entire experimental period. This reaction began earlier in Group 0 animals and was greater over the experimental period than in Group P animals, but these differences were small. No changes were detected in osteonal mean wall thickness (Fig. 7), suggesting that the increased resorption of bone was not accompanied by increased local activity of osteoclasts. The number of forming osteons was not increased within the first month after surgery in any group (Fig. 6a). This is expected and reasonable if the activation of new remodeling sites were caused by the surgical alteration of
WEEKS
Intracortical remodeling An altered strain environment increased activation frequency for new remodeling sites within the radial cortex
(Fig. 6b). There were 2-5 additional
remodeling
sites per
mm2 of bone cortex in the strain altered animals over the
mme
Fig. 5. Changes in linear appositional rates on the periosteal and endocortical bone surfaces. Within four weeks after surgery, linear apposition rates have returned to normal and remain within the normal range of variation for the duration of the experiment. This suggests that the activity of individual osteoblasts was not altered by the change in mechanical strain.
D. B. Burr et al.: Strain and bone kinetics f+ FORMING 151
OSTEONS/mm’,Af
219
# RESORPTION 101
,
# LABELED OSTEONS /mm*
SPACES/mm2,Ar
8 10 5 8
5
2
0
d -5 1
3
6
1
MONTHS
a)
3
6
MONTHS
bl Ar/Af
d
-1I 1 Cl
6
3
WEEKS
MONTHS
Fig. 6. Bar graphs depicting the change in number of formation sites (Ad, the change in number of resorption sites (A,), and the ratio of resorption to formation sites. These data show that there is an early increase in the number of sites undergoing active intracortical remodeling, suggesting an increased intracortical activation frequency.
the strain environment, because it would take this long for a new site to move from the resorption phase into the formation phase of remodeling. However, the number of forming osteons was increased by 3 months in both strain altered groups, and continued to be elevated (2 > 2.0) after 6 months. Again, Group 0 animals demonstrated slightly more active intracortical remodeling than Group P animals. The ratio of resorbing sites to forming sites (AJA,) provides a “map” of the remodeling process as new remodeling sites move from the resorption phase to the formation phase, and can indicate when the balance between these two processes has been restored. There was a large imbalance in favor of new resorption sites in all experimental groups 1 month after surgery (Fig. 6c). Because this imbalance occurred in Group S also, it must be considered a response to the surgery rather than to the alteration in strain. By the end of the first remodeling cycle at 3 months (1 sigma period), balance was restored, as we would expect it to be if the reaction were due to surgical intervention only. Equilibrium of resorption and formation was maintained in both Group P and Group S animals, but an imbalance in favor of formation occurred in the treated limb in Group 0 animals. This suggests that still elevated MEAN WALL THICKNESS
Fig. 8. The number of labeled osteons provides a measure of the number of new remodeling sites. The response was phasic in both osteotomized and plated animals and indicated that the sigma time for formation was about 10 weeks. The gray area represents the normal range of variation. Values outside this range have Z scores >2.0, indicating more sites of formation than normal.
strain magnitudes and altered distributions in these animals have the net long-term effect of initiating a second 12 week phase of remodeling, while the lower strain magnitudes in Group P animals do not have these effects. A more complete picture of osteonal remodeling is provided by Fig. 8. Here, no early transient response was detected, but maximum osteonal remodeling occurred 6 weeks and 16 weeks after surgery in Group 0 and P animals. Most of this response occurred within the periosteal new bone rather than in pre-existing cortex, which showed few new remodeling sites. The osteonal response lasted longer than did the surface adaptive responses, which were largely complete by 3 months. This is indicative of continuing adaptation within the rapidly formed new surface bone as it attempts to improve its material properties by remodeling woven bone into a lamellar organization. ‘lXvo remodeling sequences were observed during the 6 month experimental period. The sigma period for these responses was between 10 and 14 weeks. The second osteonal remodeling phase beginning about 12 weeks postosteotomy was less substantial than the initial one, perhaps suggesting a feedback system in which the magnitude of each subsequent remodeling cycle is diminished until complete adaptation is achieved. The increased intracortical remodeling does not reflect increased apposition rates (Fig. 9), but only an increased number of forming osteons, by which we may infer an increased intracortical activation frequency (Figs. 6 and 8). OSTEONAL LINEAR APPOSITION RATE 0 n
obtwtomy
Plate
Asham * Pro-operathfe
1
3
6
MONTHS DO
q P lYS
Fig. 7. There were no changes in osteonal mean wall thickness in response either to surgery or to the altered mechanical environment. This suggests that the activity of individual osteoclasts was not affected by the change in mechanical strain.
-1’
I 5
I
1
10
16
20
26
WEEKS
Fig. 9. The osteonal linear apposition rate increased in plated animals following surgery. However, it was never outside the normal range of variation (Z < 2.0).
D. B. Burr et al.: Strain and bone kinetics
220 The linear apposition rate increased briefly within the first 6 weeks postsurgery in Group P animals, but the standard deviation is quite large and this increase was never greater than two of the standard deviations from pre-operative values which we have defined as the normal physiological range. No increase was found in Group 0 or Group S animals. In the absence of any mineralization defect, normal linear apposition rates indicate normal cell-level osteoblast activity. Alteration of the strain environment had no effect on the time between osteoid deposition and its mineralization. (Fig. 10). Although it initially required five days less for mineralization to begin in the strain altered limb of Group 0 animals, this decreased lag time was not greater than that observed in some sham-operated animals at later time periods. Moreover, five days does not represent a signticant reduction of the mineralization lag time. Furthermore, no differences in osteoid seam width were detected between experimental and control limbs. Therefore, we conclude that alteration of the strain environment has little effect on the rate at which new osteoid becomes mineralized.
Discussion Transient versus steady-state
responses
To clearly demarcate the response of bone to altered strain environments, it is important to distinguish between transient responses and sustained responses. Alterations of tissue kinetics which are of short duration and which eventually subside without significantly altering the mechanical strain milieu of the bone are not truly mechanically adaptive. Such transient and nonspecific accelerations of modeling or remodeling processes can be indicative of a regional acceleratory phenomenon or RAP (Frost 1973a, 1973b). A RAP represents an acceleration of normally ongoing processes, and generally occurs in response to trauma, surgery or other stimulus which is out of the ordinary for a particular skeletal site. Many short-duration studies of adaptive change in bone observe this initial transient response to the experimental manipulation and interpret it as the longer-term adaptive response. The data from this experiment permitted the separation of transient effects from steady-state effects more directly related to the mechanically adapnve response. Groups 0 and P both demonstrated transient increases in bone forMINERALIZATION LAG TIME l Oateotomy S Plate A Sham * Pre-operative
5 P
t
T_
I
I
I
5
10
15 WEEKS
20
25
Fig. 10. There was no evidence of any sustained change in the time between osteoid deposition and mineralization of the new bone. The gray area represents the normal range of variation.
mation and in appositional rate along periosteal and endocortical surfaces within the first four weeks after surgery. This short-duration response was a RAP due to surgery, and was not adaptive; when it subsided, strain magnitudes and distributions in the radius were not different than they were immediately after surgery. Four to six weeks postoperatively, the early acceleration of modeling/remodeling processes slowed and was followed by a more sustained response lasting 2-3 months. This sustained response was adaptive, and altered the strain milieu. It is likely, however, that the effects of the RAP were still present, but were overwhelmed by the skeletal adaptation to altered strain. Because of this, the sustained response can be considered to reflect how altered strain affects bone tissue kinetics. The dynamics of skeletal adaptation
to altered strain
The gain or loss of bone depends on the birth-rate of new modeling of remodeling systems (activation frequency) and on the imbalance between resorption and formation (Pa&t 1979). Our data indicate that bone’s adaptive response to altered strain occurs primarily by increasing the activation frequency for new modeling and remodeling sites, but only altering the balance of resorption and formation processes on surfaces which are modeling. On periosteal and endocortical surfaces, there is more bone added to surfaces undergoing active formation than is being removed at those sites undergoing active resorption. The increased periosteal expansion and endosteal contraction rates are largely the result of greater surface areas undergoing active formation, rather than an increase in the linear appositional rate. Likewise, the activation frequency for Haversian remodeling is increased. The elevated activation frequency can be inferred from the increased number of resorption sites by 1 month and the subsequent increase in forming and labeled osteons by 6 months. However, the balance of resorption and formation is not altered within this skeletal envelope. There is no sustained change in porosity within the bone cortex as would be expected if the resorption-formation balance were altered. The tissue kinetic data support the hypothesis that periosteal and endocortical surfaces have reverted to a state of modeling in which resorption and formation are not coupled. Both resorption and formation are evident, but there is little evidence that these processes occurred in the same location. The scalloped cement lines which are evidence of the A-R-F sequence signifying remodeling were rare on these surfaces. There is no evidence that individual osteoclast or osteoblast activity is either enhanced or depressed by the increased strain or altered strain distribution alone. The mean wall thickness is one measure of the vigor of individual osteoclasts. Mean wall thickness was within the normal range of variation in all groups at all time periods. The linear apposition rate provides one measure of the vigor of individual osteoblasts. The early increase in appositional rates in Groups 0 and P on all bone envelopes indicates an increased local individual osteoblast vigor, but the transient nature of this increased vigor suggests it was related to the surgically induced RAP, rather than to the altered strain environment. Once this initial reaction had subsided. appositional rates remained within the normal range of variation (Z < 2.0) even in the presence of caudal compressive strains four times higher than normal. It is well known that there is a threshold of strain required for the proper differentiation (Roberts et al. 1981,
D. B. Burr et al.: Strain and
bone kinetics
221
1984) and function (Buckendahl et al. 1985; Doty 1985; Wronski et al. 1985; Simmons et al. 1986) of osteoblasts. However, it appears that once this threshold is reached, the individual activity of differentiated cells is not stimulated further, at least by the rather severe alteration in strain environment investigated in this experiment. As such, the sustained effects of increased loading appear to be mediated by recruiting more cells rather than by increasing individual cell activity. The phasic nature of the modeling and remodeling responses permits an estimate of the intracortical remodeling sigma period, and the sigma, which defines the period required to complete the A-F sequence for modeling on periosteal and endocortical surfaces. Our data show that the sigma period for intracortical remodeling following a change in strain magnitude and distribution is about 70-98 d. The sigmaf for the A-F sequence on the periosteal envelope was 56-70 d. These sigma periods correspond well to the normal sigma periods in dogs. T’akahashi et al. (1980) have shown that sigma, in beagle dogs is about 70 d, while the entire sigma period is 90- 100 d. There is no indication that the normal sigma periods for modeling and remodeling in beagle dogs are significantly altered by the magnitude of strain changes experienced by dogs in this study.
Conclusion There are several important conclusions drawn from the results of this experiment:
which may be
Modeling can be reactivated on the periosteal and even on the endocortical envelope in young adult animals when the mechanical environment of the bone is altered in special ways. The response to increased strain encompasses both modeling and remodeling responses. The increased bone mass which occurs in response to elevated strain is the result of the increased activation of modeling on the periosteal and endocortical bone surfaces. Although increased remodeling activation occurs intracortitally, there is no evidence that this leads to an alteration of bone mass. As such, increased osteonal activation frequency may represent the effects of the surgical RAP, even 3-6 months following surgery. There is no indication that the activity of individual osteoclasts or individual osteoblasts is heightened by increased mechanical strain. Moreover, there is no evidence that the sigma period is altered in any way by increased strain.
Acknowledgments: This research was supported by OREF grant 379-85, NIH grant AM 27127, the Orthopedic Education and Research Fund, and the West Virginia Orthopedic Research Fund. The authors appreciate the comments and criticism made of the original manuscript by Dr. Harold Frost, and also wish to thank Cary Johnson and Beth Edgar for tissue preparations; Dr. R. D. Boyd and Nina Clovis for assistance during surgical procedures; and especially Vince Kish for his help with instrumentation involved with bone strain recordings.
References Buckendahl, P. E.; Cann, C. E.; Grindeland, R. E.; Martin, R. B.; Mechanic, G.; Amaud, S. B. Osteocalcin as an indicator of bone metabolism during spaceflight. The Physiologist 28:S62-S63; 1985. Cowin, S. C.; van Buskirk, W. C. Internal bone remodeling induced by a medullary pin. J. Biomech. 11:269-275; 1978. Cowin, S. C.; van Buskirk, W. C. Surface bone remodeling induced by a medullary pin. J. Biomech. 12:269-276; 1979. Cowin, S. C.; Firoozbakhsh, K. Bone remodeling of diaphysial surfaces under constant load: theoretical predictions. J. Biomech. 7:471-484; 1981. Cowin, S. C.; Hart, R. T.; Balser, J. R.; Kohn, D. H. Functional adaptation in long bones: establishing in vivo values for surface remodeling rate coefficients. J. Biomech. 18:665-684; 1985. Doty, S. B. Morphologic and histochemical studies of bone cells from SL-3 rats. The Physiologist 28:S225-S226; 1985. Frost, H. M. Bone remodeling and its relation to metabolic bone disease. Springfield: Charles C. Thomas; 1973a. Frost, H. M. The origin and nature of transients in human bone remodeling dynamics. Frame, B.; Pa&t, A. M.; Duncan, H., eds. In: Clinical aspects of metabolic bone disease. Amsterdam: Excerpta Medica; 1973b:pp. 124-137. Frost, H. M. Structural adaptations to mechanical usage. A proposed “three-way rule” for bone modeling. Part I. V.C.O.T. l:7-17; 1988. Goodship, A. E.; Lanyon, L. E.; McFie, H. Functional adaptation of bone to increased stress. J. Bone Jt. Surg. 61A:539-546; 1979. Hart, R. T.; Davy, D. T.; Heiple, K. G. Mathematical modeling and numerical solutions for functionally dependent bone remodeling. Calcif. Tiss. Int. 36:SlO4-SlO9; 1984. Lanyon, L. E.; Bourn, S. The influence of mechanical function on the development and remodeling of the tibia. J. Bone Jt. Surg. 61A:263-273; 1979. Lanyon, L. E.; Magee, P. T.; Baggott, D. G. The relationship of functional stress and strain to the processes of bone remodeling. An experimental study on the sheep radius. J. Biomech. 12:593-600, 1979. Lanyon, L. E.; Goodship, A. E.; Pye, C. J.; MacFie, J. H. Mechanically adaptive bone remodeling. J. Biomech. IS: 141- 154; 1982. O’Connor, I. A.; Lanyon, L. E.; MacFie, H. The influence of strain rate on adaptive bone remodeling. J. Biomech. 15:767-781; 1982. Partitt, A. M. Quantum concept of bone remodeling and turnover: implications for the pathogenesis of osteoporosis. Calcif. Tiss. Int. 28:1-5; 1979. Roberts, W. E.; Mozsary, P. G.; Morey, E. R. Suppression of osteoblast differentiation during weightlessness. The Physiologist 24:S75-S76; 1981. Roberts, W. E.; Morey-Holton, E.; Gonsalves, M. Sensitivity of bone cell populations to weightlessness and simulated weightlessness. In: The gravity relevance in bone mineralisation processes. Paris: European Space Agency SP-203; 1984:~~. 67-72. Rubin, C. T.; Lanyon, L. E. Regulation of bone formation by applied dynamic loads. J. Bone Jt. Surg. 66A:397-402, 1984. Rubin, C. T.; Lanyon, L. E. Regulation of bone mass by mechanical strain magnitude. Calcif. Tiss. Int. 37:411-417; 1985. Simmons, D. J.; Russell, J. E.; Grynpas, M. D. Bone maturation and quality of bone material in rats flown on the space shuttle “SpacelabMission.” Bone and Mineral 1:485-493; 1986. Takahashi, H.; Norimatsu, H.; Watanabe, G.; Konno, T.; Inoue, J.; Fukada, M. The remodeling period (sigma) in canine and human cortical bone. In: Yoshitoshi, Y.; Fujita, T., eds. Calcium endocrinology. Tokyo: Chugai Igaku; 1980:~~. 13-31. Villaneuva, A. R.; Hattner, R. S.; Frost, H. M. A tetrachrome stain for fresh, mineralized bone sections, useful in the diagnosis of bone diseases. Stain Technol. 39:87-94; 1964. Weinberg, G. H.; Schumaker, J. A. Statistics: an intuitive approach. Monterey, CA: Brooks/Cole; 1974. Wronski, 1’. J.; Lowry, P. L.; Walsh, C. C.; Ignaszewski, L. A. Skeletal alterations in ovariectomized rats. Calcif. Tiss. Int. 37:324-328; 1985.
Received: Revised: Accepted:
May 17, 1988 October IO, 1988 January 31, 1989
8756-3282189 $3.00 + .OO Copyright 0 1989 Pergamon Press plc
The Effects of Altered Strain Environments on Bone Tissue Kinetics D. B. BURR,‘v2 M. B. SCHAFFLER,3 K. H. YANG,2 D. D. WU,2 M. LUKOSCHEK,2 N. SIVANERI,4 J. D. BLAHA and E. L. RADIN2
D. KANDZARI,’
’ Department of Anatomy, ’ Department of Orthopedic Surgery and Orthopedic Research Laboratory, West Virginia University, Morgantown, WV, uSA 3 Division of Anatomy, University of California at San Diego, La Jolla, CA, USA 4 Department of Mechanical Engineering, West Virginia University, Morgantown, WV, USA Address .for correspondence and reprints: David B. Burr, Ph.D., Department of Anatomy, West Virginia University Center, Morgantown, WV 26506, L&A.
1985) has demonstrated that manipulating the strain environment in well-controlled ways causes specific adaptive alterations. Those studies do not yield insight into the biological dynamics of the adaptive change. Aspects of the strain environment may affect the remodeling sequence at different stages and/or at different rates. An altered mechanical strain environment may affect activation frequency, the time required to form a new packet of bone (sigma), the balance between resorption and formation, or the time required to mineralize new bone. If mechanical factors are ever to be used as probes to reverse osteopenias (which can be caused by alterations of any of the above factors acting singly or in combination), then the way in which strain affects remodeling kinetics must be described. Moreover, these data provide the rate constants required for some mathematical models of bone adaptation (Cowin and van Buskirk 1978, 1979; Cowin and Firoozbakhsh 1981; Hart et al. 1984; Cowin et al. 1985; Frost 1988). Therefore, this kind of histomorphometric data provide necessary information for future mathematical predictions of bone response. We hypothesize that the adaptive response to elevated mechanical strain occurs by increasing the activation frequency for bone remodeling and by altering bone balance in favor of formation at each site, rather than through direct effects on the individual activity of already dtierentiated cells. The purpose of this work was to test this hypothesis by defining the alteration in bone tissue kinetics responsible for observed geometric changes in bone architecture in response to altered strain environments.
Abstract This study defines the alteration in bone tissue kinetics responsible for the “adaptive remodeling” response to altered strain environments. Adult beagle dogs were separated into three experimental groups: uhtar osteotomy, ulnar osteotomy with fracture fixation plate spanning the gap and sham surgery. Four sets of double fluorochrome labels were administered. Prior to sacrifice at 1, 3, and 6 months, strains were measured through rosette strain gages on the cranial and caudal surfaces of the intact radius. Histomorphometric analysis hulicated that the increased bone mass in response to elevated strain results tkom increased activation frequency of modeling with more sites undergoing formation processes than resorption processes on per&teal and endocortical surfaces. Increased remodeling activation did not lead to increased bone mass. There was no evidence that elevated strain changes the individual vigor of osteoclasts or osteoblasts, or that the sigma period was altered by elevated Strain.
Key Words: Strain-Bone
Health Sciences
kinetics-Remodeling-Dogs.
Introduction It is generally accepted that bone models and remodels in response to changes in its mechanical strain environment. Remodeling is a process in which the initiation of change (activation) is followed in sequence by the resorption and formation of bone (Activation-Resorption-Formation). Modeling, on the other hand, involves either the resorption or formation of bone following activation (Activation-Resorption or Activation-Formation), but resorption and formation do not occur in sequence at the same location. Both the nature of the mechanical stimuli which underlie the initiation of each process, and the tissue kinetics of the mechanically adaptive response, are still open to question. The work of Lanyon (Goodship et al. 1979; Lanyon and Bourn, 1979; Lanyon et al. 1979; O’Connor et al. 1982; Lanyon et al. 1982) and Rubin (Rubin and Lanyon 1984,
Materials and Methods Experimental
design
Seventeen skeletally mature 1 -2-year-old male beagle dogs weighing 11-18.5 kg were divided randomly into three experimental groups. Six dogs had a l-2 cm segment of the distal ulna removed (Group 0). Five additional animals (Group P) had ulnar osteotomies, but the osteotomy gap was spanned with a j-hole metal plate from the mini215
D. B. Burr et al.: Strain and bone kinetics
216
fragment fracture fixation set (Zimmer, Warsaw, IN). A sham operation was performed in six dogs: the periosteum of the ulna was incised in these animals, but no bone was removed (Group S). Animals were sacrificed at 1, 3, or 6 months after surgery. Five additional dogs were used to measure normal strain in the radius, and six dogs were used to measure strain changes 48 h following either an ulnar osteotomy alone, or after a plated osteotomy. These experimental groups are summarized in Fig. 1. All animals received food and water ad libitum during the experiment. For 2-4 weeks prior to surgery, each dog was acclimated to walk on the treadmill at 4 km/h for 20 minld, 5 dlwk. Following surgery and until sacrifice, dogs were housed in individual 4 ft x 8 ft pens, and each dog was exercised for 20 min daily on the treadmill using the protocol described. Animals in all groups were given four sets of double fluorochrome labels using a 1-7-1 scheme to monitor net bone formation and apposition rate throughout the experiment. Oxytetracycline (30 mg/kg; A. J. Buck, Pittsburgh, PA) was given orally prior to the osteotomy or sham surgery. Subsequently, DCAF (20 mg/kg; ICN, Cleveland, OH), alizarin red (25 mg/kg; Sigma, St. Louis, MO) and xylenol orange (90 mg/kg; Sigma, St. Louis, MO) were given intramuscularly at intervals outlined in Fig. 1. Two days prior to sacrifice strains were measured on the cranial and caudal surfaces of the intact right (treated) radius using 45” rosette strain gages. The magnitudes of the principal strains were calculated from the raw strain measurements using standard formulae.
Each animal was given general anesthesia of halothane, nitrous oxide and oxygen. Surgical approach to the ulna was made from the lateral surface of the dog’s forelimb. A 15 cm incision was made through the skin and fascia. Blunt dissection between the flexor carpi radialis and flexor carpi ulnaris was used to expose the ulna. Special care was taken to obtain hemostasis to limit any potentially dissecting hematoma. The periosteum of the ulna was split longitudinally over a 10 cm distance and elevated. The osteotomy was made under saline irrigation using a
2 1 PRE-OP
1
Strain gage application
Strains were measured using either stacked 45” rosettes (BLH Electronics No. FABR-12-12-S6) or a smaller set of unstacked 45” rosette gages (Micromeasurements, EA-06015-RJ-120). Under anesthesia, rosette strain gages were applied cranially and caudally to the midshaft of the radius. The strain gages were positioned so that one gage was aligned with the long axis of the radius, and position was radiographically verified postoperatively. Strain recording procedure
Prior to measuring strains during walking, each dog was held off the ground while the strain gages were zeroed. The dog was then walked at 4 km/h on the treadmill while strain recordings were made. The strain gage signal was amplified via bridge amplifiers (Measurement Group Model 2311, Micromeasurements, Raleigh, NC). The amplified strain data were collected from each gage of a rosette simultaneously using a 16 channel FM tape recorder (Bell and Howell) at a speed of I5 in/s. Data were monitored concurrently on two Gould brush chart recorders. Subsequently, raw strain data were digitized at 200 Hz using an IBM PC microcomputer, and principal strains and directions were calculated from this data. Histological procedures
Surgical procedures
WEEKS
Gigli saw. A 5-hole plate from the minifragment set was positioned on the lateral side of the ulna in Group P animals and fixed by four cortical bone screws (No. 8 or No. 10) in previously tapped drill holes.
2
4
6
6
10
12
14
16
16
20
22
24
POST-OP
Fig. 1. An outline of the experiment showing the number of animals in each experimental group, the period between surgery and sacrifice, and the label schedule. Group 0, ulnar osteotomy; Group P, ulnar osteotomy with fracture fixation plate spanning osteotomy gap; Group S, sham-operated group. The letters within each bar depict the label given: T, oxytetracycline; D, DCAF; A, alizarin red; X, xylenol orange.
and morphometric
measurements
Following sacrifice, the radius of each forelimb was removed for quantitative histological analysis of bone tissue kinetics. Undecalcified complete cross sections were cut at 25%, 50% and 75% of the length of the radius using a Buehler Isomet low speed saw with a circular blade, and preserved in 70% ETOH. Sections were also made through the screw holes in Group P and at the osteotomy site in Group 0 if different from the three levels described. Because strains were only measured at midshaft, only histological data from the midshaft sections are reported here. Because the site for histological analysis was removed from the distal surgical site, the chance for localized effects due to surgical procedures was reduced. Sections were ground in alcohol to 150 microns with 600 grit silicon carbide paper and polished with 0.30 grit alumina suspended in alcohol. The sections were stained using the tetrachrome method of Villanueva et al. (1964) and mounted. Up to three sections per radius were analyzed microscopically using reflected ultraviolet light at 212 x and 534 x magnification. Measurements listed in Table I were made by tracing sections onto a digitizing tablet through a drawing tube attached to the microscope. A Bioquant image analysis system (R & R Biometrics, Nashville, TN) interfaced to an IBM-PC XT microcomputer was used to collect the data. The experiments were designed to examine temporal differences in the entire adaptive process in response to altered strain environments, rather than to look for statistically significant differences among groups at predetermined time periods. For this reason, we used a smaller number of dogs, and, did more complete histomorphometric measurements on this sample. As can be seen from
D. B. Burr et al.: Strain and bone kinetics
217
Table I. Definition of measurements
Intracortical measurements 1. A, (#/mm2): The number of osteonal resorption spaces per unit area. 2. A, (#/mm2): The number of refilling osteons, as determined by stained osteoid seams. 3. Linear apposition rate o&d): The mean distance between fluorochrome labels in doubly labeled osteons, divided by the time between labels. 4. OSW (pm): The average width of osteonal osteoid seams. 5. MWT (pm): The mean wall thickness in completed osteons, that is, the distance from the cement line to the Haversian canal wall. 6. Sigma (d): The time required to initiate and complete a new osteon. 7. mu (#/y/mm*): The activation frequency, or “birth-rate” of new remodeling sites. Surface measurements 1. P; (%): The proportion of periosteal surface labeled with a particular fluorochrome. 2. E’. (%): The nronortion of endocortical surface labeled with a particular fluorochrome. 3. Linear apposition rate o.un/d): The mean distance between double fluorochrome labels on oeriosteal or endocortical surfaces, divided by the time
between labels. 4. Periosteal expansion rate (pm/y): The surface-based
bone
formation rate; the product of Pr’ and periosteal linear 5. Endocortical
contraction
apposition rate, x 365. rate (pm/y): The surface-based bone
formation rate; the product of E,’ and
endocortical linear apposition rate, x 365.
-1200
I
I
Results Strain measurements Figure 2 shows the change in principal strain magnitudes following a surgical osteotomy. The change in strain distribution in Groups 0 and P following surgery is most evi-
I
dent. While the cranial and caudal surfaces of the radius in beagle dogs are normally subject to compressive strain, removal of part of the ulna increases bending strains on the intact radius so that the cranial cortex is placed in tension. Caudal compressive strains are also increased following osteotomy. Caudal compressive strains remain elevated in Group 0 animals throughout the 6 month exoeriment. but those in Group P animals have returned to normal ‘by 3 months postoperatively. Six months after the osteotomy peak strain magnitudes on the radius in Group 0 animals are still elevated 2-3 times, but strains on the caudal surface of the radius of Group P animals have returned to normal. Adaptive changes on periosteal
Figures 3-10, comparing data at only 1, 3 and 6 months would provide a very different-and erroneous-view of the adaptive response. Standard deviations are presented where they are available or when differences between groups are large. We employed an analysis using 2 scores (Weinberg and Schumaker 1974) to determine whether measurements lie within the normal range of variation for each remodeling parameter. 2 scores measure the distance in standard deviation units a measurement lies from the presurgery population mean for a particular character (Weinberg and Schumaker 1974). The shaded area on the graphs represents two standard deviations about the mean presurgery data value. This can be considered the normal range of variation in adult beagle dogs. Values which lie outside this range have Z scores larger than 2.0 and can be considered, with 95% probability, to lie outside the range of variability for that particular characteristic. When variables have Z scores less than 2.0 we interpreted no differences from presurgery control values.
I
Fig. 2. Bar graph depicting mean principal strain magnitudes on the anterior and posterior surfaces of the radius for each group at three intervals following ulnar osteotomy or sham surgery. The first bar in each set represents mean strain on the anterior cortex while the second bar represents mean strain on the posterior cortex. Strains on the radius of the sham-operated animals are taken to be normal.
endocortical
and
surfaces
The surgical removal of a portion of the ulna produced an almost immediate response on the periosteal and endocortical envelopes of the radius. There was an early increase in periosteal woven bone apposition, resulting in an increased expansion rate by 2 weeks in Group 0 animals (Fig. 3a). Group P animals also showed the transient early increase in periosteal expansion of the radius, but this is reduced to within the normal range by 6 weeks. Although not reflected in the sham-operated animals, the transient nature of this reaction suggests it was a result of the surgical procedures. The transient response was followed by a more significant and sustained increase in periosteal new bone in Group 0, beginning between 4 and 6 weeks. This sustained increase was not observed in Group P. This response declined to normal by 12 weeks postsurgery and remained within the normal range through 24 weeks. Because the sustained response did not occur in Group P animals, it may be a reflection of the normal periosteal adaptive response to the altered strain environment. The greater proportion of labeled endocortical surface resulted in increased endocortical contraction rates, and demonstrates one reason for the smaller radial medullary cavities in Group 0 animals 6 months nostonerative (Fig. 4a). Again, a small early transient increase in endocor&l bone formation was observed in some, but not all, animals. After this, however, a more sustained formation drift oc-
D. B. Burr et al.: Strain and bone kinetics
218 PERIOSTEAL
EXPANSION
RATE
ENDOSTEAL
CONTRACTION
RATE
Oeteotomy n Plate A Sham * Pre-operative 0
ii
30 20 10 0
-10
- 10 -20 5
15
10
20
I
I
5
10
I
,
15 WEEKS
20
\r,I 25
25
PROPORTION
WEEKS
ENDOSTEAL
SURFACE
LABEL
l
PROPORTION
PERIOSTEAL
SURFACE
34 20
LABEL
a B
10
k E
5
7’ a
0
I
5
1
*Overlappingdata
T
w T - 15 g
Osteotomy . Plate A Sham * Pre-operative
l
Oateotomy . Plate A Sham
I
I
I
I
10
15
20
25
T
1
WEEKS -20
I 5
I
I
I
I
10
15
20
25
WEEKS
Fig. 3. Changes over the experimental period in the periosteal expansion rate (a) and in the proportion of the periosteal surface undergoing active mineralization (b). The gray region represents the normal range of variation. Points outside this range have 2 scores >2.0, and can be considered outside the normal range.
curred, beginning at about 4 weeks and ending at 16 weeks. The initiation of a second phase of increased formation was apparent by 24 weeks. In general, Groups P and S demonstrated little formation on the endocortical surface. However, one animal in Group P did show a marked response at about 16 weeks; because this was only represented by one animal at one time period, and did not constitute part of a recognizable pattern, this most likely does not indicate a sustained response to the strain regime but rather an aberration of the sampling procedure. Increased periosteal expansion and endocortical contraction rates were largely the result of increased total forming surface (Figs. 3b and 4b) rather than increased appositional rates (Fig. 5). The transient early response was caused by both increased forming surface and increased rate, but appositional rates on the periosteal and endocortical surfaces were normal throughout the rest of the experiment.
Fig. 4. Changes over the experimental period in the endocortical contraction rate (a) and in the proportion of the endocortical surface undergoing active mineralization (b). The gray region represents the normal range of variation. Points outside this range have Z scores >2.0 and can be considered outside the normal range.
entire experimental period. This reaction began earlier in Group 0 animals and was greater over the experimental period than in Group P animals, but these differences were small. No changes were detected in osteonal mean wall thickness (Fig. 7), suggesting that the increased resorption of bone was not accompanied by increased local activity of osteoclasts. The number of forming osteons was not increased within the first month after surgery in any group (Fig. 6a). This is expected and reasonable if the activation of new remodeling sites were caused by the surgical alteration of
WEEKS
Intracortical remodeling An altered strain environment increased activation frequency for new remodeling sites within the radial cortex
(Fig. 6b). There were 2-5 additional
remodeling
sites per
mm2 of bone cortex in the strain altered animals over the
mme
Fig. 5. Changes in linear appositional rates on the periosteal and endocortical bone surfaces. Within four weeks after surgery, linear apposition rates have returned to normal and remain within the normal range of variation for the duration of the experiment. This suggests that the activity of individual osteoblasts was not altered by the change in mechanical strain.
D. B. Burr et al.: Strain and bone kinetics f+ FORMING 151
OSTEONS/mm’,Af
219
# RESORPTION 101
,
# LABELED OSTEONS /mm*
SPACES/mm2,Ar
8 10 5 8
5
2
0
d -5 1
3
6
1
MONTHS
a)
3
6
MONTHS
bl Ar/Af
d
-1I 1 Cl
6
3
WEEKS
MONTHS
Fig. 6. Bar graphs depicting the change in number of formation sites (Ad, the change in number of resorption sites (A,), and the ratio of resorption to formation sites. These data show that there is an early increase in the number of sites undergoing active intracortical remodeling, suggesting an increased intracortical activation frequency.
the strain environment, because it would take this long for a new site to move from the resorption phase into the formation phase of remodeling. However, the number of forming osteons was increased by 3 months in both strain altered groups, and continued to be elevated (2 > 2.0) after 6 months. Again, Group 0 animals demonstrated slightly more active intracortical remodeling than Group P animals. The ratio of resorbing sites to forming sites (AJA,) provides a “map” of the remodeling process as new remodeling sites move from the resorption phase to the formation phase, and can indicate when the balance between these two processes has been restored. There was a large imbalance in favor of new resorption sites in all experimental groups 1 month after surgery (Fig. 6c). Because this imbalance occurred in Group S also, it must be considered a response to the surgery rather than to the alteration in strain. By the end of the first remodeling cycle at 3 months (1 sigma period), balance was restored, as we would expect it to be if the reaction were due to surgical intervention only. Equilibrium of resorption and formation was maintained in both Group P and Group S animals, but an imbalance in favor of formation occurred in the treated limb in Group 0 animals. This suggests that still elevated MEAN WALL THICKNESS
Fig. 8. The number of labeled osteons provides a measure of the number of new remodeling sites. The response was phasic in both osteotomized and plated animals and indicated that the sigma time for formation was about 10 weeks. The gray area represents the normal range of variation. Values outside this range have Z scores >2.0, indicating more sites of formation than normal.
strain magnitudes and altered distributions in these animals have the net long-term effect of initiating a second 12 week phase of remodeling, while the lower strain magnitudes in Group P animals do not have these effects. A more complete picture of osteonal remodeling is provided by Fig. 8. Here, no early transient response was detected, but maximum osteonal remodeling occurred 6 weeks and 16 weeks after surgery in Group 0 and P animals. Most of this response occurred within the periosteal new bone rather than in pre-existing cortex, which showed few new remodeling sites. The osteonal response lasted longer than did the surface adaptive responses, which were largely complete by 3 months. This is indicative of continuing adaptation within the rapidly formed new surface bone as it attempts to improve its material properties by remodeling woven bone into a lamellar organization. ‘lXvo remodeling sequences were observed during the 6 month experimental period. The sigma period for these responses was between 10 and 14 weeks. The second osteonal remodeling phase beginning about 12 weeks postosteotomy was less substantial than the initial one, perhaps suggesting a feedback system in which the magnitude of each subsequent remodeling cycle is diminished until complete adaptation is achieved. The increased intracortical remodeling does not reflect increased apposition rates (Fig. 9), but only an increased number of forming osteons, by which we may infer an increased intracortical activation frequency (Figs. 6 and 8). OSTEONAL LINEAR APPOSITION RATE 0 n
obtwtomy
Plate
Asham * Pro-operathfe
1
3
6
MONTHS DO
q P lYS
Fig. 7. There were no changes in osteonal mean wall thickness in response either to surgery or to the altered mechanical environment. This suggests that the activity of individual osteoclasts was not affected by the change in mechanical strain.
-1’
I 5
I
1
10
16
20
26
WEEKS
Fig. 9. The osteonal linear apposition rate increased in plated animals following surgery. However, it was never outside the normal range of variation (Z < 2.0).
D. B. Burr et al.: Strain and bone kinetics
220 The linear apposition rate increased briefly within the first 6 weeks postsurgery in Group P animals, but the standard deviation is quite large and this increase was never greater than two of the standard deviations from pre-operative values which we have defined as the normal physiological range. No increase was found in Group 0 or Group S animals. In the absence of any mineralization defect, normal linear apposition rates indicate normal cell-level osteoblast activity. Alteration of the strain environment had no effect on the time between osteoid deposition and its mineralization. (Fig. 10). Although it initially required five days less for mineralization to begin in the strain altered limb of Group 0 animals, this decreased lag time was not greater than that observed in some sham-operated animals at later time periods. Moreover, five days does not represent a signticant reduction of the mineralization lag time. Furthermore, no differences in osteoid seam width were detected between experimental and control limbs. Therefore, we conclude that alteration of the strain environment has little effect on the rate at which new osteoid becomes mineralized.
Discussion Transient versus steady-state
responses
To clearly demarcate the response of bone to altered strain environments, it is important to distinguish between transient responses and sustained responses. Alterations of tissue kinetics which are of short duration and which eventually subside without significantly altering the mechanical strain milieu of the bone are not truly mechanically adaptive. Such transient and nonspecific accelerations of modeling or remodeling processes can be indicative of a regional acceleratory phenomenon or RAP (Frost 1973a, 1973b). A RAP represents an acceleration of normally ongoing processes, and generally occurs in response to trauma, surgery or other stimulus which is out of the ordinary for a particular skeletal site. Many short-duration studies of adaptive change in bone observe this initial transient response to the experimental manipulation and interpret it as the longer-term adaptive response. The data from this experiment permitted the separation of transient effects from steady-state effects more directly related to the mechanically adapnve response. Groups 0 and P both demonstrated transient increases in bone forMINERALIZATION LAG TIME l Oateotomy S Plate A Sham * Pre-operative
5 P
t
T_
I
I
I
5
10
15 WEEKS
20
25
Fig. 10. There was no evidence of any sustained change in the time between osteoid deposition and mineralization of the new bone. The gray area represents the normal range of variation.
mation and in appositional rate along periosteal and endocortical surfaces within the first four weeks after surgery. This short-duration response was a RAP due to surgery, and was not adaptive; when it subsided, strain magnitudes and distributions in the radius were not different than they were immediately after surgery. Four to six weeks postoperatively, the early acceleration of modeling/remodeling processes slowed and was followed by a more sustained response lasting 2-3 months. This sustained response was adaptive, and altered the strain milieu. It is likely, however, that the effects of the RAP were still present, but were overwhelmed by the skeletal adaptation to altered strain. Because of this, the sustained response can be considered to reflect how altered strain affects bone tissue kinetics. The dynamics of skeletal adaptation
to altered strain
The gain or loss of bone depends on the birth-rate of new modeling of remodeling systems (activation frequency) and on the imbalance between resorption and formation (Pa&t 1979). Our data indicate that bone’s adaptive response to altered strain occurs primarily by increasing the activation frequency for new modeling and remodeling sites, but only altering the balance of resorption and formation processes on surfaces which are modeling. On periosteal and endocortical surfaces, there is more bone added to surfaces undergoing active formation than is being removed at those sites undergoing active resorption. The increased periosteal expansion and endosteal contraction rates are largely the result of greater surface areas undergoing active formation, rather than an increase in the linear appositional rate. Likewise, the activation frequency for Haversian remodeling is increased. The elevated activation frequency can be inferred from the increased number of resorption sites by 1 month and the subsequent increase in forming and labeled osteons by 6 months. However, the balance of resorption and formation is not altered within this skeletal envelope. There is no sustained change in porosity within the bone cortex as would be expected if the resorption-formation balance were altered. The tissue kinetic data support the hypothesis that periosteal and endocortical surfaces have reverted to a state of modeling in which resorption and formation are not coupled. Both resorption and formation are evident, but there is little evidence that these processes occurred in the same location. The scalloped cement lines which are evidence of the A-R-F sequence signifying remodeling were rare on these surfaces. There is no evidence that individual osteoclast or osteoblast activity is either enhanced or depressed by the increased strain or altered strain distribution alone. The mean wall thickness is one measure of the vigor of individual osteoclasts. Mean wall thickness was within the normal range of variation in all groups at all time periods. The linear apposition rate provides one measure of the vigor of individual osteoblasts. The early increase in appositional rates in Groups 0 and P on all bone envelopes indicates an increased local individual osteoblast vigor, but the transient nature of this increased vigor suggests it was related to the surgically induced RAP, rather than to the altered strain environment. Once this initial reaction had subsided. appositional rates remained within the normal range of variation (Z < 2.0) even in the presence of caudal compressive strains four times higher than normal. It is well known that there is a threshold of strain required for the proper differentiation (Roberts et al. 1981,
D. B. Burr et al.: Strain and
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1984) and function (Buckendahl et al. 1985; Doty 1985; Wronski et al. 1985; Simmons et al. 1986) of osteoblasts. However, it appears that once this threshold is reached, the individual activity of differentiated cells is not stimulated further, at least by the rather severe alteration in strain environment investigated in this experiment. As such, the sustained effects of increased loading appear to be mediated by recruiting more cells rather than by increasing individual cell activity. The phasic nature of the modeling and remodeling responses permits an estimate of the intracortical remodeling sigma period, and the sigma, which defines the period required to complete the A-F sequence for modeling on periosteal and endocortical surfaces. Our data show that the sigma period for intracortical remodeling following a change in strain magnitude and distribution is about 70-98 d. The sigmaf for the A-F sequence on the periosteal envelope was 56-70 d. These sigma periods correspond well to the normal sigma periods in dogs. T’akahashi et al. (1980) have shown that sigma, in beagle dogs is about 70 d, while the entire sigma period is 90- 100 d. There is no indication that the normal sigma periods for modeling and remodeling in beagle dogs are significantly altered by the magnitude of strain changes experienced by dogs in this study.
Conclusion There are several important conclusions drawn from the results of this experiment:
which may be
Modeling can be reactivated on the periosteal and even on the endocortical envelope in young adult animals when the mechanical environment of the bone is altered in special ways. The response to increased strain encompasses both modeling and remodeling responses. The increased bone mass which occurs in response to elevated strain is the result of the increased activation of modeling on the periosteal and endocortical bone surfaces. Although increased remodeling activation occurs intracortitally, there is no evidence that this leads to an alteration of bone mass. As such, increased osteonal activation frequency may represent the effects of the surgical RAP, even 3-6 months following surgery. There is no indication that the activity of individual osteoclasts or individual osteoblasts is heightened by increased mechanical strain. Moreover, there is no evidence that the sigma period is altered in any way by increased strain.
Acknowledgments: This research was supported by OREF grant 379-85, NIH grant AM 27127, the Orthopedic Education and Research Fund, and the West Virginia Orthopedic Research Fund. The authors appreciate the comments and criticism made of the original manuscript by Dr. Harold Frost, and also wish to thank Cary Johnson and Beth Edgar for tissue preparations; Dr. R. D. Boyd and Nina Clovis for assistance during surgical procedures; and especially Vince Kish for his help with instrumentation involved with bone strain recordings.
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Received: Revised: Accepted:
May 17, 1988 October IO, 1988 January 31, 1989