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Skeletal changes after tibial fracture in the rat
SKELETAL
CHANGES IN
JAMES
B.
WRAY,
M.D.,*
AFTER TIBIAL THE RAT
AND
THE REACTION of the entire organism to fracture has been the subject of intense investigation since the pioneer studies of Culbertson in 1932 [2]. It is now generally accepted that the soft tissues of the body take part in a generalized adjustment in which carbohydrate, fat, protein, and electrolyte metabolism are altered from the prefracture state. In contrast to the progress that has been made toward an understanding of post-traumatic metabolism in nonmineralized tissues, relatively little information has been gathered relative to the reaction of the skeleton to injury. It is known, however, that demineralization of varying severity develops in the injured bone and the adjacent skeleton of the involved extremity after a long-bone fracture [l, 51. Furthermore, it appears likely that some or most of the mineral mobilized from the injured extremity is excreted from the bdo y in view of the fact that previous investigators of postfracture metabolism have noted a temporarv elevation of urinary calcium excretion [ 61: From the departments of Orthopaedic Surgery and Pediatrics, State University of New York, Upstate Medical Center, Syracuse, New York. Supported by Grant AM 04868-06 from the National Institutes of Health. *Present address: Department of Orthopaedic Surgery, Indiana University Medical Center, 1100 West Michigan Street, Indianapolis, Indiana. Submitted for publication Dec. 16, 1968.
A.
J.
SCHNEIDER,
FRACTURE
M.D.,
PH.D.
The present investigation was designed to study two related but separate aspects of skeletal metabolism in the postfracture period; the compositional change of the fractured bone and the alterations, if any, that occur in the nonfractured bones from both injured and noninjured extremities. Two series of rats were subjected to a closed fracture of the tibia. At intervals after fracture the animals were killed and the tibias and femurs of both hind limbs, and the humeri from both fore limbs were excised, cleaned, and analyzed to determine their water, ash, and matrix content. The results of these analyses were subjected to statistical evaluation and form the basis of the following report.
MATERIALS
AND
METHODS
A total of 84 male rats of the SpragueDawley strain were employed in this study. Two separate experiments were performed. Forty-eight animals were used in the first experiment and 36 in the second. All animals were housed in metal cages in groups of four to five and were fed a diet of standard rat chow and water ad libitum. Animals in the first experiment averaged 400 gm. in weight while those in the second experiment ranged from 500 to 550 gm. Five days after arrival at the laboratory, each animal in the first experiment was weighed, as433
JOURNAL
OF
SURGICAL
RESEARCH
VOL.
9
NO.
signed a random number from a table of random numbers, and placed in one of eight groups of six animals each. Each animal was anesthetized with ether, weighed, and subjected to a closed fracture of one tibia by finger manipulation. Right and left tibias were alternatively fractured with the result that each group contained three right and three left tibia1 fractures. No effort was made to immobilize the fractures and the animals were allowed to run free in their cages after recovery from the anesthetic. Groups one through eight were killed with an overdose of ether immediately (zero time) and at 2%/,, 5, 7%, 10, 12yZ/,, 15, and 20 days after fracture, respectively, and were treated in the following manner. Each animal was weighed and both humeri, both femurs, and both tibiae were dissected free of soft tissue. The articular surfaces and epiphyses of the bones were left intact. Each bone was broken up into several fragments and placed in a tared bottle and weighed to determine its wet weight. The specimen was then dried in an oven at 160°F. for a period of 4 days and weighed to determine the dry weight. The calculated difference between wet and dry weight was considered to be the water content of the bone. The dry specimen was ashed in a muffle oven at 650°F. for 18 hours. The ash residue was then brought to room temperature in a desiccator and weighed to determine the ash weight. The difference between dry and ash weights was considered to be the weight of the matrix of the bone. The water content, matrix content, and ash content of each bone were expressed as grams per kilogram of total animal body weight (at the time of the initial anesthesia) for statistical analysis which employed a split-plot design. A separate analysis was performed for each component of bone. Data for the humeri and femurs were combined in one set of analyses. The data for both tibias were analyzed together for ash and matrix content. On the other hand, only the water content for the unfractured limb was subjected to statistical treatment. 434
7,
JULY
1969
RESULTS Meaningful changes in the skeleton of the animals in the first experiment are shown in graphic form in Figs. 1 and 2. Water, ash, and matrix values in grams per kilogram of body weight are plotted on the vertical axis, while time in days after fracture is plotted on the horizontal axis. The heavy lines in each graph represent the expected changes with time as determined by fitting linear regressions to the experimental data. All three bony constituents, except for water in the nonfractured bones, show significant differences from zero time. The water content of the nonfractured bones is significantly changed at 2% days only. Data derived from fractured and nonfractured tibias is shown in Fig. 1. Those changes which occurred in the fractured tibia are the most dramatic, It is obvious that there is a massive and almost immediate increase in the water content of the fractured bone. This is followed by substantial increases in mineral and matrix as the fracture heals. The ash and matrix content of the nonfractured tibia also increase linearly after the opposite tibia is fractured. It is not possible from these data to define precisely when the increase begins and it is entirely possible that there may be a slight decrease in the solid content of these bones during the first few days after fracture. The regression lines for both fractured and nonfractured tibias cross at % to 1 day after fracture, suggesting that bony accretion begins sometime during the first day after fracture. On the other hand, the errors in both slopes and levels of these lines do not rule out the possibility that accretion may begin as late as 5 days after fracture. Data obtained from both humeri and both femurs was combined and are shown in Fig. 2. As was the case with the tibias, the ash and matrix content of these bones increase linearly after one tibia has been fractured. Pertinent statistical data from these bones is presented in Table 1 where significant findings are summarized. It can be seen that there was a sig-
WRAY
I
I
AND
SCHNEIDER:
I
1
I
SKELETAL
I
CHAh-;GES
I
I
I
I
AFTER
TIBIAL
FRACTURE
I
1.5. FRACTURED 1.4 .
-------
TIBIA
UNFRACTURED
TIBIA
1.3 .
1.2
2 1.1 s bo . ’ * 2 m M .9, i 8 M .a
.7
.6
.5 .
.4
I 0
I
2.5
I
I
I
5.0 1.5 10.0 DAYS AFTER FRACTURE
I
12.5 OF
15.0 17.5 ONE TIBIA
I
20.0
Fig. 1. Changes of bony constituents of both tibias of rats subjected to fracture of one tibia. Changes in ash, water, and matrix in both fractured and nonfractured tibias are shown in this graph. Ash, water, and matrix values are expressed in grams per kilogram of body weight on the vertical axis, while time, in days after fracture, is shown on the horizontal axis. The heavy, unmarked lines seen in association with the data for each parameter and for both fractured and nonfractured tibias represent regression lines. Ash, water, and matrix increase in both fractured and nonfractured bones. On the other hand, the changes in the injured tibia are much more marked, and, in the case of the water content, quite dramatic. It is likely that the more marked changes in the fractured limb can be attributed to callus formation at the fracture site.
r&cant difference in ash weight between the humerus and femur on the fractured side and the same two bones from the nonfractured side. The nonfractured side bones were heavier in ash content. This difference between the two sides was unrelated to right or left handedness of the animal and was not observed relative to the matrix or water content. Other analyses not shown here revealed that the difference between the two sides was greater in the femurs than in the humeri. Although the discrepancy was not significant, the authors
are of the opinion that this observation is real rather than due to chance, and that the femur adjacent to the fractured tibia is subjected to a reduced rate of mineralization as compared to the femur from the nonfractured limb. A glance at the plots representing femur and humerus water content identifies a masked, but transient increase at 2% days after fracture. Reference to Table 1 shows that this increase (calculated for humeri and femurs combined) is significant. 435
JOURNAL
OF
SURGICAL
RESEARCH
VOL.
9
7,
NO.
JULY
1969
1.2
\ FEMUR
- MATRIX
.5
.4,
.3 Ly-_--~---~gEEi(
.2, A-
I
I
I
0
2.5
5.0 DAYS
AFTER
I
I
7.5
10.0 FRACTURE
I
I
12.5 OF
I
15.0 ONE
17.5
I
20.0
TIBIA
Fig. 2. Changes in bony constituents of femurs and humeri of rats subjected to fracture of one tibia. As in Fig. 1, water, matrix, and ash values are expressed in grams per kilogram of body weight and are plotted
on the vertical
axis, while time in days after fracture
is shown on the horizontal
axis. The
straight, uninterrupted Iine that is seen with each bony constituent on the graph is a regression line. As with the tibial data presented in Fig. 1, ash and matrix weight are seen to increase in a linear fashion. Pertinent calculations relative to these increases are given in Table I and indicate that they are significant ones (p < 0.05). In contrast to ash and matrix, the water content of the femurs and humeri does not increase significantly with time. On the other hand, a transient and significant increase in water content for both types of bones is seen at 2% days after fracture
The magnitude of the bony accretion that the nonfractured bones show after tibia1 fracture can be appreciated more readily by reference to Table 2. The starting and final weights of the constituents were calculated from the regression lines and the change is expressed as percentage of the starting weight. This change approximated 9% of the starting weight 20 days after fracture for both ash and matrix. Since the weight to kilogram of body weight values are expressed in terms of the initial weights of the animals, these increases with time can be considered to represent true accretion of bone whether the animals lost 436
(Table
1).
weight during the period prior to death or not. In actual fact, the animals lost weight during the first IO days reaching a low weight of 97% of their starting weight. At 20 days this loss was overcome when the animals weighed 0.7% more than their initial weights. The significant increases in ash and matrix weight that were observed to occur in the nonfractured bones raised the possibility that these changes could be due to normal growth processes. As the data gathered in the first experiment did not allow an answer to this question, a second experiment was carried out. Thirty-six rats, ranging from 500 to 550 gm. in
WRAY
Table
1.
Degrees of Freedom
Ash weight Individual regressions of ash weight on time for each type bone Residual error Fractured side vs. unfractured side (femur plus humerus weights combined) Residual error Matrix weight Individual regressions of matrix weight on time Residual error Water weight Weight at 2% days vs. remaining time periods Residual error
2.
Calculated
SCHNEIDER:
SKELETAL
CHANGES
AFTER
TIBIAL
FRACTURE
Summary of Analysis of Variance on Femur and Humerus Data
Source of Variation
Table
AND
Mean Squared Deviations
F
1
0.0138819
5.72
< 0.05
46 1
0.0024253 0.0022174
4.73
0.05
47
0.0004684
1
0.0082602
8.26
0.01
46
0.0009996
1
0.0503610
7.22
<0.025
41
0.0069739
P
Changes in Ash and Matrix Content of Unfractured Fracture of One Tibia
Tibia Ash (p./kg.)
Femur 5.25 Humerus 2.53
Femur 2.86 Humerus 0.76
Bones of Rats Subjected
Femur
( ;a;;;;) (p./kg.)
Slope of regressions (mg./kg./day)
Humerus
Matrix (gmv’kg.)
Ash (gdkg.)
to
Ash (gw’kg.)
Matrix (gwkg.)
Final weight (20 days)
0.9340
0.5363
1.2343
0.7018
0.5776
0.2921
Starting weight (0 days) Change in weight
0.8477
0.4908
1.11295
0.6445
0.5272
0.2778
0.0863
0.0455
0.1048
0.0573
0.0504
0.0143
9.27
9.27
8.89
9.55
5.14
y0 Change in weight with respect to starting weight
10.18
( and presumably skeletally mature), weight were divided into six groups of six animals each. All 36 animals were anesthetized and three of the six groups were subjected to a closed fracture of one tibia. One group of fractured animals and one group of nonfractured
animals were killed
at
zero time. Similar groups were killed at 10 and 20 days respectively. Analyses similar to those of the first experiment were carried out
and demonstrated that the bones of the nonfractured animals increased only in proportion to their
body
weight
while
the nonfrac-
tured bones in the fractured animals increased at twice
the rate seen in the nonfractured
ani-
mals in spite of a total weight loss at 10 days followed by recovery of the initial weight at 20 days.
Furthermore,
the
fractured
tibias
showed changes comparable to those seen in the first experiment
in that the ash; i.e., min437
JOURNAL
OF SURGICAL RESEARCH
VOL. g NO.
era1 content of the femur in the fractured limb was found to increase at a slower rate than that observed in the other nonfractured bones in the fractured animals. DISCUSSION The findings of this experiment suggest that postfracture skeletal change in the rat is generalized in extent and characterized by increased bone formation. Furthermore, it appears that both locaI and generalized factors are operative in producing these changes. The observations made in this study confirm the earlier radioisotope studies of Kolar and Babicky who found that Iong-bone fracture was followed by increased mineral exchange throughout the skeleton of the rat [8]. To the authors’ knowledge, however, the present experiment presents the first evidence that this increased mineral exchange results in a net increase in bone formation. Although the conclusions reached in this study appear to be a reasonable interpretation of the evidence, a minor word of caution is in order. Neither the present experiment, nor that of KoIar or Babicky, included bone samples from the spine or shoulder or pelvic girdles and it is possible, although unlikely, that such changes do not take place in those bones as observed in the peripheral skeleton. The cause of increased bone formation after fracture in the rat has not been determined but probably can be attributed to traumainduced endocrine activity. Of the several hormones; i.e., ACTH, growth hormone, epinephrine, cortisone, whose output is known to increase in the post-traumatic period [9], only growth hormone might be expected to stimulate bone formation. On the other hand, specific studies in this area will be necessary before this concept can be accepted. While the evidence indicates that the rat responds to a long-bone fracture with a generalized increase in bone formation, it is by no means certain that the human shows a similar response. Indeed, there are sound reasons for suspecting that the two species differ in their responses to trauma. It is known that the human with a long-bone fracture enters 438
7, JULY 1969 a period of negative calcium balance, a state which is hardly compatible with widespread new bone formation. Furthermore, of the three major determinants of the generalized response to injury; i.e., altered food intake, decreased physical activity, and the increased production of certain hormones, the rat and the human show gross differences in at least two. The activity of the rat, a four-legged animal, is affected to a limited degree by injury to a single hind limb whereas the human with a major fracture of a hind limb is usually confined to bed for a variable period of time. In addition, hind-limb fracture has little effect upon the diet of the rat while the human shows a lessened food intake in the immediate post-traumatic period. Aside from the generalized increase in bone formation observed in this study, it is clear that the fractured bone and its adjacent femur react differently from the remainder of the osseous system. In the tibia, this difference was both quantitative and qualitative in type. Quantitatively, the fractured tibia shows a much greater accretion of new bone than does the remainder of the skeleton. It may be presumed that this change is the result of callus formation at the fracture site. From a qualitative point of view, the tibia1 bone is less heavily mineralized than the other, nonfractured bones. Evidence to support this observation was obtained from analysis of the nonfractured tibias 20 days after fracture, which showed a mean matrix/ash ratio of 0.57, while the fractured tibias showed a mean matrix/ ash ratio of 0.71. Radiographs of the fractured tibias also suggested that the bones were mildly osteoporotic. On the other hand, these X-ray, changes are less marked than those seen in large mammals or in the human. It is most likely that the changes seen in the fractured tibia and its companion femur, in contrast to those in the uninjured extremities, are due to local factors rather than to endocrine influences. It is known that significant physiological adjustments occur in the limb with a long-bone fracture. These include an increased arterial inflow [7, lo], increased metabolic activity in the injured bone 131, and decreased metabolic activitv in the muscula-
WRAY
AND
SCHh-EIDER:
ture [4] of the injured extremity. All of these events appear to be associated with and may be the cause of a lessened bone mass. The failure to observe absolute bone loss in the fractured tibia may be due to a masking effect through callus formation at the fracture site, and partly, in the case of the femur on the fractured side, to the positive effects of the generalized skeletal reaction.
SKELETAL
CHANGES
AFTER
TIBIAL
FRACTURE
2. Culbertson,
D. P. Observations on the disturbance of metabolism produced by injury to the limbs. Quart. J. Med. 1:233, 1932.
3. DeRosa,
C. P., and Wray, J. B. Studies of metabolic activity in post-traumatic osteoporosis in the rabbit. (submitted for publication.)
4. DeRosa,
G. P., and Wray, J. B. Oxygen consumption of muscle following closed tibia1 fracture in the rat. Szrrg. Fowm 19:446, 1968.
5. Geiser, M., and Trueta,
J. Muscle action, bone and bone formation. J. Bone Jt. Surg. Amer. 40B:282, 1958.
rarefaction
SUMMARY Closed fracture of the tibia in the rat is followed by increased bone formation throughout the peripheral skeleton. On the other hand, new bone formation is less active in the femur of the injured limb and presumably in the fractured bone. Increased bone formation after tibia1 fracture in the rat is not the result of normal growth processes but rather, is postulated to be the result of a traumatically induced increase in hormone activity. REFERENCES 1. Allison, clinical
N., and Brooks, B. An experimental and study of changes in bone from nonuse.
Snrg. Gynec. O&et.
33:250, 1921.
6. Howard,
J. E., Parson, W., and Bigham, R. S., Jr. Studies on patients convalescent from fracture. III. Urinary excretion of calcium and phosphorus. Bull. Johns Hopkins Hosp. 77:291, 1945.
7. H&h,
A., and Glerud, S. Disuse of extremities. I. An arteriographic study in the rabbit. Actu Chir. Scud 120:220, 1960.
8. Kolar, J., and Babicky,
A. The influence of fracmetabolism of bone salts. Chir. Ortho. Truumn Cech. 31:92, 1964.
tures
on the
9. Ross, H., Johnston, I. D. A., Welborn,
T. A., and Wright, A. D. Effect of abdominal operation on glucose tolerance and serum levels of insulin, growth hormone, and hydrocortisone. Luncet 2: 563, 1966.
10. Wray,
J. B. Acute changes in femoral artery blood flow following closed tibia1 fracture in dogs. I. Bone Jt. Surg. 46A:1262, 1964.
439
CHANGES IN
JAMES
B.
WRAY,
M.D.,*
AFTER TIBIAL THE RAT
AND
THE REACTION of the entire organism to fracture has been the subject of intense investigation since the pioneer studies of Culbertson in 1932 [2]. It is now generally accepted that the soft tissues of the body take part in a generalized adjustment in which carbohydrate, fat, protein, and electrolyte metabolism are altered from the prefracture state. In contrast to the progress that has been made toward an understanding of post-traumatic metabolism in nonmineralized tissues, relatively little information has been gathered relative to the reaction of the skeleton to injury. It is known, however, that demineralization of varying severity develops in the injured bone and the adjacent skeleton of the involved extremity after a long-bone fracture [l, 51. Furthermore, it appears likely that some or most of the mineral mobilized from the injured extremity is excreted from the bdo y in view of the fact that previous investigators of postfracture metabolism have noted a temporarv elevation of urinary calcium excretion [ 61: From the departments of Orthopaedic Surgery and Pediatrics, State University of New York, Upstate Medical Center, Syracuse, New York. Supported by Grant AM 04868-06 from the National Institutes of Health. *Present address: Department of Orthopaedic Surgery, Indiana University Medical Center, 1100 West Michigan Street, Indianapolis, Indiana. Submitted for publication Dec. 16, 1968.
A.
J.
SCHNEIDER,
FRACTURE
M.D.,
PH.D.
The present investigation was designed to study two related but separate aspects of skeletal metabolism in the postfracture period; the compositional change of the fractured bone and the alterations, if any, that occur in the nonfractured bones from both injured and noninjured extremities. Two series of rats were subjected to a closed fracture of the tibia. At intervals after fracture the animals were killed and the tibias and femurs of both hind limbs, and the humeri from both fore limbs were excised, cleaned, and analyzed to determine their water, ash, and matrix content. The results of these analyses were subjected to statistical evaluation and form the basis of the following report.
MATERIALS
AND
METHODS
A total of 84 male rats of the SpragueDawley strain were employed in this study. Two separate experiments were performed. Forty-eight animals were used in the first experiment and 36 in the second. All animals were housed in metal cages in groups of four to five and were fed a diet of standard rat chow and water ad libitum. Animals in the first experiment averaged 400 gm. in weight while those in the second experiment ranged from 500 to 550 gm. Five days after arrival at the laboratory, each animal in the first experiment was weighed, as433
JOURNAL
OF
SURGICAL
RESEARCH
VOL.
9
NO.
signed a random number from a table of random numbers, and placed in one of eight groups of six animals each. Each animal was anesthetized with ether, weighed, and subjected to a closed fracture of one tibia by finger manipulation. Right and left tibias were alternatively fractured with the result that each group contained three right and three left tibia1 fractures. No effort was made to immobilize the fractures and the animals were allowed to run free in their cages after recovery from the anesthetic. Groups one through eight were killed with an overdose of ether immediately (zero time) and at 2%/,, 5, 7%, 10, 12yZ/,, 15, and 20 days after fracture, respectively, and were treated in the following manner. Each animal was weighed and both humeri, both femurs, and both tibiae were dissected free of soft tissue. The articular surfaces and epiphyses of the bones were left intact. Each bone was broken up into several fragments and placed in a tared bottle and weighed to determine its wet weight. The specimen was then dried in an oven at 160°F. for a period of 4 days and weighed to determine the dry weight. The calculated difference between wet and dry weight was considered to be the water content of the bone. The dry specimen was ashed in a muffle oven at 650°F. for 18 hours. The ash residue was then brought to room temperature in a desiccator and weighed to determine the ash weight. The difference between dry and ash weights was considered to be the weight of the matrix of the bone. The water content, matrix content, and ash content of each bone were expressed as grams per kilogram of total animal body weight (at the time of the initial anesthesia) for statistical analysis which employed a split-plot design. A separate analysis was performed for each component of bone. Data for the humeri and femurs were combined in one set of analyses. The data for both tibias were analyzed together for ash and matrix content. On the other hand, only the water content for the unfractured limb was subjected to statistical treatment. 434
7,
JULY
1969
RESULTS Meaningful changes in the skeleton of the animals in the first experiment are shown in graphic form in Figs. 1 and 2. Water, ash, and matrix values in grams per kilogram of body weight are plotted on the vertical axis, while time in days after fracture is plotted on the horizontal axis. The heavy lines in each graph represent the expected changes with time as determined by fitting linear regressions to the experimental data. All three bony constituents, except for water in the nonfractured bones, show significant differences from zero time. The water content of the nonfractured bones is significantly changed at 2% days only. Data derived from fractured and nonfractured tibias is shown in Fig. 1. Those changes which occurred in the fractured tibia are the most dramatic, It is obvious that there is a massive and almost immediate increase in the water content of the fractured bone. This is followed by substantial increases in mineral and matrix as the fracture heals. The ash and matrix content of the nonfractured tibia also increase linearly after the opposite tibia is fractured. It is not possible from these data to define precisely when the increase begins and it is entirely possible that there may be a slight decrease in the solid content of these bones during the first few days after fracture. The regression lines for both fractured and nonfractured tibias cross at % to 1 day after fracture, suggesting that bony accretion begins sometime during the first day after fracture. On the other hand, the errors in both slopes and levels of these lines do not rule out the possibility that accretion may begin as late as 5 days after fracture. Data obtained from both humeri and both femurs was combined and are shown in Fig. 2. As was the case with the tibias, the ash and matrix content of these bones increase linearly after one tibia has been fractured. Pertinent statistical data from these bones is presented in Table 1 where significant findings are summarized. It can be seen that there was a sig-
WRAY
I
I
AND
SCHNEIDER:
I
1
I
SKELETAL
I
CHAh-;GES
I
I
I
I
AFTER
TIBIAL
FRACTURE
I
1.5. FRACTURED 1.4 .
-------
TIBIA
UNFRACTURED
TIBIA
1.3 .
1.2
2 1.1 s bo . ’ * 2 m M .9, i 8 M .a
.7
.6
.5 .
.4
I 0
I
2.5
I
I
I
5.0 1.5 10.0 DAYS AFTER FRACTURE
I
12.5 OF
15.0 17.5 ONE TIBIA
I
20.0
Fig. 1. Changes of bony constituents of both tibias of rats subjected to fracture of one tibia. Changes in ash, water, and matrix in both fractured and nonfractured tibias are shown in this graph. Ash, water, and matrix values are expressed in grams per kilogram of body weight on the vertical axis, while time, in days after fracture, is shown on the horizontal axis. The heavy, unmarked lines seen in association with the data for each parameter and for both fractured and nonfractured tibias represent regression lines. Ash, water, and matrix increase in both fractured and nonfractured bones. On the other hand, the changes in the injured tibia are much more marked, and, in the case of the water content, quite dramatic. It is likely that the more marked changes in the fractured limb can be attributed to callus formation at the fracture site.
r&cant difference in ash weight between the humerus and femur on the fractured side and the same two bones from the nonfractured side. The nonfractured side bones were heavier in ash content. This difference between the two sides was unrelated to right or left handedness of the animal and was not observed relative to the matrix or water content. Other analyses not shown here revealed that the difference between the two sides was greater in the femurs than in the humeri. Although the discrepancy was not significant, the authors
are of the opinion that this observation is real rather than due to chance, and that the femur adjacent to the fractured tibia is subjected to a reduced rate of mineralization as compared to the femur from the nonfractured limb. A glance at the plots representing femur and humerus water content identifies a masked, but transient increase at 2% days after fracture. Reference to Table 1 shows that this increase (calculated for humeri and femurs combined) is significant. 435
JOURNAL
OF
SURGICAL
RESEARCH
VOL.
9
7,
NO.
JULY
1969
1.2
\ FEMUR
- MATRIX
.5
.4,
.3 Ly-_--~---~gEEi(
.2, A-
I
I
I
0
2.5
5.0 DAYS
AFTER
I
I
7.5
10.0 FRACTURE
I
I
12.5 OF
I
15.0 ONE
17.5
I
20.0
TIBIA
Fig. 2. Changes in bony constituents of femurs and humeri of rats subjected to fracture of one tibia. As in Fig. 1, water, matrix, and ash values are expressed in grams per kilogram of body weight and are plotted
on the vertical
axis, while time in days after fracture
is shown on the horizontal
axis. The
straight, uninterrupted Iine that is seen with each bony constituent on the graph is a regression line. As with the tibial data presented in Fig. 1, ash and matrix weight are seen to increase in a linear fashion. Pertinent calculations relative to these increases are given in Table I and indicate that they are significant ones (p < 0.05). In contrast to ash and matrix, the water content of the femurs and humeri does not increase significantly with time. On the other hand, a transient and significant increase in water content for both types of bones is seen at 2% days after fracture
The magnitude of the bony accretion that the nonfractured bones show after tibia1 fracture can be appreciated more readily by reference to Table 2. The starting and final weights of the constituents were calculated from the regression lines and the change is expressed as percentage of the starting weight. This change approximated 9% of the starting weight 20 days after fracture for both ash and matrix. Since the weight to kilogram of body weight values are expressed in terms of the initial weights of the animals, these increases with time can be considered to represent true accretion of bone whether the animals lost 436
(Table
1).
weight during the period prior to death or not. In actual fact, the animals lost weight during the first IO days reaching a low weight of 97% of their starting weight. At 20 days this loss was overcome when the animals weighed 0.7% more than their initial weights. The significant increases in ash and matrix weight that were observed to occur in the nonfractured bones raised the possibility that these changes could be due to normal growth processes. As the data gathered in the first experiment did not allow an answer to this question, a second experiment was carried out. Thirty-six rats, ranging from 500 to 550 gm. in
WRAY
Table
1.
Degrees of Freedom
Ash weight Individual regressions of ash weight on time for each type bone Residual error Fractured side vs. unfractured side (femur plus humerus weights combined) Residual error Matrix weight Individual regressions of matrix weight on time Residual error Water weight Weight at 2% days vs. remaining time periods Residual error
2.
Calculated
SCHNEIDER:
SKELETAL
CHANGES
AFTER
TIBIAL
FRACTURE
Summary of Analysis of Variance on Femur and Humerus Data
Source of Variation
Table
AND
Mean Squared Deviations
F
1
0.0138819
5.72
< 0.05
46 1
0.0024253 0.0022174
4.73
0.05
47
0.0004684
1
0.0082602
8.26
0.01
46
0.0009996
1
0.0503610
7.22
<0.025
41
0.0069739
P
Changes in Ash and Matrix Content of Unfractured Fracture of One Tibia
Tibia Ash (p./kg.)
Femur 5.25 Humerus 2.53
Femur 2.86 Humerus 0.76
Bones of Rats Subjected
Femur
( ;a;;;;) (p./kg.)
Slope of regressions (mg./kg./day)
Humerus
Matrix (gmv’kg.)
Ash (gdkg.)
to
Ash (gw’kg.)
Matrix (gwkg.)
Final weight (20 days)
0.9340
0.5363
1.2343
0.7018
0.5776
0.2921
Starting weight (0 days) Change in weight
0.8477
0.4908
1.11295
0.6445
0.5272
0.2778
0.0863
0.0455
0.1048
0.0573
0.0504
0.0143
9.27
9.27
8.89
9.55
5.14
y0 Change in weight with respect to starting weight
10.18
( and presumably skeletally mature), weight were divided into six groups of six animals each. All 36 animals were anesthetized and three of the six groups were subjected to a closed fracture of one tibia. One group of fractured animals and one group of nonfractured
animals were killed
at
zero time. Similar groups were killed at 10 and 20 days respectively. Analyses similar to those of the first experiment were carried out
and demonstrated that the bones of the nonfractured animals increased only in proportion to their
body
weight
while
the nonfrac-
tured bones in the fractured animals increased at twice
the rate seen in the nonfractured
ani-
mals in spite of a total weight loss at 10 days followed by recovery of the initial weight at 20 days.
Furthermore,
the
fractured
tibias
showed changes comparable to those seen in the first experiment
in that the ash; i.e., min437
JOURNAL
OF SURGICAL RESEARCH
VOL. g NO.
era1 content of the femur in the fractured limb was found to increase at a slower rate than that observed in the other nonfractured bones in the fractured animals. DISCUSSION The findings of this experiment suggest that postfracture skeletal change in the rat is generalized in extent and characterized by increased bone formation. Furthermore, it appears that both locaI and generalized factors are operative in producing these changes. The observations made in this study confirm the earlier radioisotope studies of Kolar and Babicky who found that Iong-bone fracture was followed by increased mineral exchange throughout the skeleton of the rat [8]. To the authors’ knowledge, however, the present experiment presents the first evidence that this increased mineral exchange results in a net increase in bone formation. Although the conclusions reached in this study appear to be a reasonable interpretation of the evidence, a minor word of caution is in order. Neither the present experiment, nor that of KoIar or Babicky, included bone samples from the spine or shoulder or pelvic girdles and it is possible, although unlikely, that such changes do not take place in those bones as observed in the peripheral skeleton. The cause of increased bone formation after fracture in the rat has not been determined but probably can be attributed to traumainduced endocrine activity. Of the several hormones; i.e., ACTH, growth hormone, epinephrine, cortisone, whose output is known to increase in the post-traumatic period [9], only growth hormone might be expected to stimulate bone formation. On the other hand, specific studies in this area will be necessary before this concept can be accepted. While the evidence indicates that the rat responds to a long-bone fracture with a generalized increase in bone formation, it is by no means certain that the human shows a similar response. Indeed, there are sound reasons for suspecting that the two species differ in their responses to trauma. It is known that the human with a long-bone fracture enters 438
7, JULY 1969 a period of negative calcium balance, a state which is hardly compatible with widespread new bone formation. Furthermore, of the three major determinants of the generalized response to injury; i.e., altered food intake, decreased physical activity, and the increased production of certain hormones, the rat and the human show gross differences in at least two. The activity of the rat, a four-legged animal, is affected to a limited degree by injury to a single hind limb whereas the human with a major fracture of a hind limb is usually confined to bed for a variable period of time. In addition, hind-limb fracture has little effect upon the diet of the rat while the human shows a lessened food intake in the immediate post-traumatic period. Aside from the generalized increase in bone formation observed in this study, it is clear that the fractured bone and its adjacent femur react differently from the remainder of the osseous system. In the tibia, this difference was both quantitative and qualitative in type. Quantitatively, the fractured tibia shows a much greater accretion of new bone than does the remainder of the skeleton. It may be presumed that this change is the result of callus formation at the fracture site. From a qualitative point of view, the tibia1 bone is less heavily mineralized than the other, nonfractured bones. Evidence to support this observation was obtained from analysis of the nonfractured tibias 20 days after fracture, which showed a mean matrix/ash ratio of 0.57, while the fractured tibias showed a mean matrix/ ash ratio of 0.71. Radiographs of the fractured tibias also suggested that the bones were mildly osteoporotic. On the other hand, these X-ray, changes are less marked than those seen in large mammals or in the human. It is most likely that the changes seen in the fractured tibia and its companion femur, in contrast to those in the uninjured extremities, are due to local factors rather than to endocrine influences. It is known that significant physiological adjustments occur in the limb with a long-bone fracture. These include an increased arterial inflow [7, lo], increased metabolic activity in the injured bone 131, and decreased metabolic activitv in the muscula-
WRAY
AND
SCHh-EIDER:
ture [4] of the injured extremity. All of these events appear to be associated with and may be the cause of a lessened bone mass. The failure to observe absolute bone loss in the fractured tibia may be due to a masking effect through callus formation at the fracture site, and partly, in the case of the femur on the fractured side, to the positive effects of the generalized skeletal reaction.
SKELETAL
CHANGES
AFTER
TIBIAL
FRACTURE
2. Culbertson,
D. P. Observations on the disturbance of metabolism produced by injury to the limbs. Quart. J. Med. 1:233, 1932.
3. DeRosa,
C. P., and Wray, J. B. Studies of metabolic activity in post-traumatic osteoporosis in the rabbit. (submitted for publication.)
4. DeRosa,
G. P., and Wray, J. B. Oxygen consumption of muscle following closed tibia1 fracture in the rat. Szrrg. Fowm 19:446, 1968.
5. Geiser, M., and Trueta,
J. Muscle action, bone and bone formation. J. Bone Jt. Surg. Amer. 40B:282, 1958.
rarefaction
SUMMARY Closed fracture of the tibia in the rat is followed by increased bone formation throughout the peripheral skeleton. On the other hand, new bone formation is less active in the femur of the injured limb and presumably in the fractured bone. Increased bone formation after tibia1 fracture in the rat is not the result of normal growth processes but rather, is postulated to be the result of a traumatically induced increase in hormone activity. REFERENCES 1. Allison, clinical
N., and Brooks, B. An experimental and study of changes in bone from nonuse.
Snrg. Gynec. O&et.
33:250, 1921.
6. Howard,
J. E., Parson, W., and Bigham, R. S., Jr. Studies on patients convalescent from fracture. III. Urinary excretion of calcium and phosphorus. Bull. Johns Hopkins Hosp. 77:291, 1945.
7. H&h,
A., and Glerud, S. Disuse of extremities. I. An arteriographic study in the rabbit. Actu Chir. Scud 120:220, 1960.
8. Kolar, J., and Babicky,
A. The influence of fracmetabolism of bone salts. Chir. Ortho. Truumn Cech. 31:92, 1964.
tures
on the
9. Ross, H., Johnston, I. D. A., Welborn,
T. A., and Wright, A. D. Effect of abdominal operation on glucose tolerance and serum levels of insulin, growth hormone, and hydrocortisone. Luncet 2: 563, 1966.
10. Wray,
J. B. Acute changes in femoral artery blood flow following closed tibia1 fracture in dogs. I. Bone Jt. Surg. 46A:1262, 1964.
439