- Email: [email protected]
The proliferative capacity and DNA synthesis of osteoblasts during fracture repair in normal and hypophysectomized rats
The proliferative capacity and DNA synthesis of osteoblastsduring fracture repair in normal and hypophysectomizedrats Joseph T. Nichols, D.D.S., M.S.,” Patrick D. Toto, D.D.S., M.S., and Nichok C. Chodms, D.D.S., MA’., Chicago, Ill. LOYOLA UNIVERSITY
SCHOOL OF DENTISTRY,
CHICAGO COLLEGE OF DENTAL
SURGERY
P
recursor cells preparing for mitosis may be radioactively labeled so that the subsequently differentiated cells can be identified. Tritiated thymidine specifically labels deoxyribonucleic acid during the synthesis period. Tonna,l in a study primarily concerned with aging, fractured the right femurs of albino Swiss mice of varying ages and killed the animals at intervals of 24 hours, 1 week, and 2 weeks. One hour prior to sacrifice, “flash” labeling of the cells was obtained by subcutaneous injection of tritiated thymidine. Tonna observed that osteogenic cells of the periosteum were most frequently labeled, although the number of labeled cells decreased with age. However, by 24 hours after fracture there was an increase in the population of labeled cells, even in mice 52 weeks old. Also, labeled cells were most numerous when osteogenic or chrondrogenic cells from the periosteum were actively converting the fracture callus into cartilage, Tonna concluded that the potential for fracture repair resides essentially in the proliferative capacity of the cells of the periosteum, which diminishes with age. This reduced capacity accounts for the reduction in the rate of fracture repair in older animals. According to Enoch and Coventry, 2 the periosteum consists of an outer layer of collagenous fibers, in which most of the cells are fibroblasts, and an inner layer composed of single fusiform cells in the resting state. The latter becomes a multicellular layer of plump osteoblasts when bone fracture occurs. This investigation was supported in part by the Brophy, Johnso?, Logan Endowment of Loyola University and also by United States Public Health Service General Research Support Grant, 5 SO1 FR-5310-05, General Research Support Branch, Division of Research Facilities and Resources. *Present address: 259 Meridian Rd., San JoNe, Calif.
418
Volume 25 Number 3
Yroliferatice
cupacity
of osteoblasts
419
Tonna and Cronkite3 studied the original cell types participating in fracture repair and also, by “flash” radioactive labeling, investigated common progenitor cells in the periosteum of intact and fractured femora of mice. They observed that pre-osteoblasts are “flash” labeled more frequently than osteoblasts or fiberoblasts but that osteocytes do not “flash” label. At 24 hours after fracture, the fraction of cells labeled in the periosteum was greater throughout the entire diaphysis than at the fracture site. One week a.fter fracture, however, the lab&d cells were most prevalent at the fracture site, although the number of lab&d cells was greatly decreased. They concluded further that it is unlikely that fibroblasts produce osteogenic cells. Leblond and Carriere4 have reported a decrease in the mitotic activity in the crypts of Lieberkiihn in hypophysectomized rats. However, the mitotic rstc is significantly increased upon the administration of growth hormone. Bois, Belanger, and Lebois5 in a similar study, found that hypophysectomized rats had a smaller cartilaginous plate and a lower mitotic index. They also noted that growth hormone significantly stimulated mitosis. The purpose of the present study was to demonstrate autoradiographically the proliferative capacity of the periosteum which developed following fracture of the fibula in normal and hypophysectomized rats. The frequency of labeled cells in the osteogenic layer of the periosteum following fracture in t.he control animal was used as a base line with which to compare DNA synthesis in the hypophysectomized animals. METHODS AND M.ATERIALS
Twenty-four young albino Sprague-Dawley rats, weighing between 80 and 90 grams each, were divided into two groups. Group I contained twelve normal animals, whereas Group II contained twelve hypophysectomized animals. The respective groups of animals were littermates and were housed in wire-bottom cages. The diet of the normal rats consisted of Purina Lab chow and drinking water ad libitum, whereas the hypophysectomized animals were maintained on 5 per cent dextrose in 0.25 per cent saline at a temperature of 80 to 83” I+‘.” The survival period lasted throughout the entire study.
Pig. 1. Representative
example
of fractured
fibula
confirmed
by radiographic
examination.
420
Nichols,
l’oto, and Chouhm
OX, 0.x & 02. March, 1968
The right fibula of each rat was fractured with digital pressure under inhalation ether anesthesia. The animals were killed at 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and 72 hours after fracture. One hour prior to sacrifice, each animal was injected intraperitoneally with 50 microcuries of tritiated thymidine specific a,ctivity, 1.9 c/m&I) at a rate of approximately 0.5 microcuries per gram of body weight. After each animal was killed, the right leg was severed at the proximal joint of the femur, the skin aad muscle were removed, and the specimen was fixed for 24 hours in 10 per cent buffered formalin. At this time all fractures were confirmed by x-ray (Fig. 1). The specimen was washed, decalcified in a 10 per cent solution of disodium versenate, embedded in paraffin, and sectioned at 3 to 6 microns. Autoradiograms were prepared and stained with hematoxylin and eosin.? The autoradiograms were examined microscopically with an oil-immersion lens at a magnification of x1,000. One thousand labeled and nonlabeled cells in the periosteum of the diaphysis of the fractured fibula were counted from each section made. The labeling index was established as the ratio of the number of labeled cells of the osteogenic layer of the periosteum per 1,000 per&teal cells.
Fig. 3. A, Normal periosteal thickness before injury. B, Thickened periosteum animal after fracture. (Hematoxylin and eosin stain. Magnification, x7.50.)
of 6 hour
ProZiferati,ve capacity of osteoblasts
Volume 25 Number 3
421
RESUtTS
The 6 and 12 hour animals exhibited the same reaction to the injury, namely, a thickening of the osteogenic layer of the periosteum along the entire diaphysis (Fig. 2). Labeled cells in the periosteum of the 6 and 12 hour specimens were rare ; only one labeled cell was seen in each (Table I, Figs. 3 and 4). Cell proliferation, as indicated by DNA synthesis, was stimulated between 12 and 18 hours after fracture as the labeling index in the control animals rose from 0.001 to 0.253. Table I. Labeled cell population of osteogenic layer of rat periosteum after fib&l
fracture Hours
Control
post-
fracture
Labeling
index
animals
Hypophysectomized per cent
1Labeling
Labeling
index
awim&
lLabe&ng
per oez
6
0.001
0.1
0.001
0.1
12 18 24 30 36 42 48
0.253 0.001 0.302 0.250 0.210 0.185 0.164
25.3 0.1 30.2 25.0 2 1.0 18.5 16.4
0.200 0.001 0.255 0.204 0.160 0.142 0.113
21i:X 25.5 30.4 16.0 14.2 11.3
60 54 66 72
0.106 0.118 0.090 0.081
10.6 11.8 9.0 8.1
0.088 0.095 0.074 0.067
2 7.4 6.7
r\1,
0.Y
HV~~VSECTOYIZEO
I
\
-
-
-
NllwLl-
0.m
LAIfllNG
Fig. index.
INDEX
3. Proliferative
rate
~ERlOSlEAl
CELL
NOB&f1
HYWPHYSEClOMlZfO
is plotted
All0
PROllFERLTtON
as a function
I” -.
“m.
of time after
fracture,
and labeling
422
Nichols,
Toto, and Choukas
A
R
Fig. 4. Labeling of 6 (a) and 12 (B) hour control animals. Arrows point to one labeled cell in each section. (Hematoxylin and eosin stain. Magnification, x750.)
The findings in the 18 hour hypophysectomized animals clearly showed a significantly less than normal proliferative capacity (Figs. 5 and 6), The osteogenic layer of the periosteum was greatly thickened, and the cells were round, plump, and highly labeled by tritiated thymidine. (Fig. 6). Maximum labeling occurred at 24 hours in the normal and hypophysectomized animals. Also interspersed between the labeled osteoblasts were groups of unlabeled osteoblasts (Fig. 7). The fracture segments were noted to be in good apposition (Fig. 8). The fibrous layer of the periosteum was well defined, with many spindle-shaped and fusiform nonlabeled fibroblasts. Progenitor cells of the osteogenic layer of the periosteum were rapidly synthesizing DNA in preparation for cell duplication (Fig. 3). From 18 hours through 48 hours, the distance between the respective curves exhibits a difference in the proliferative capacity, although both are observed to have decreased steadily. From 54 to 72 hours, however, the distance is insignificant, almost to the point of convergence. The first demonstration of a well-developed fibrous callus was observed in the 60 hour hypophysectomized animal. It had bridged the opposing fragments of the fracture and was twice the thickness of the fracture segment.
Volume 25 Number 3
Prolifemtive
cuywcity
of osteoblnsts
423
Fig. 5. Eighteen hour normal animal exhibits extensive labeling along cntirc length of diaphysis. (Hematoxylin and eosin stain. Magnification, x750.) Fig. 6. Labeling in 18 hour hypophysectomized animal can be observed to be decreased when compared to Fig. 5. (Hematoxylin and eosin stain. Magnification, x750.)
DISCUSSION
The proliferative capacity of the periosteum of normal and hypophyseetomized rats rapidly rises to a peak within 24 hours following fracture of the Akula. After this, the proliferative capacity decreases, as indicated by the number of cells in DNA synthesis. The close proximity of the limbs of the respective curves of cells in DNA synthesis (Fig. 3) shows that cell proliferation is stimulated by fracture of the fibula, in both normal and hypophysectomized rats. There are significantly more cells synthesizing DNA in normal animals, but differentiation occurs at approximately the sa,metime. This can be seen in the convergence of the curves. Although there are more proliferating cells in the normal rat periosteum than in the hypophysectomized rat, there is an equivalence in DNA synthesis after 54 hours. This suggests that maturation mf the cells in the periosteum occurs at about the same rate in both groups of animals. The catabolism of tritiated-thymidine-labeled cells results in the liberation of the thymidine label, which can be reutilized by tissue
424 Nichols, I’oto, und Choukas
OS, O.M. & 0.1’.
March, 1968
A
Pig. 7. Twenty four hour hypophysectomieed animals. A, Thickened periosteum. B, Labeled osteoblasts ; also noted are many spindle-shaped unlabeled fibroblasts. (Hematoxylin and eosin stain. Magnifications: A, x250; B, x750.)
cells preparing for duplication. According to Robinson,* this accounts for labeling for some time after the initial administration of tritiated thymidine. Reutilization is mentioned only to compare this process with the “flash” technique used in our study. The former method fa.cilitates the availability of the radiolabel for long periods of time, and consequently many generations of cells very possibly will become labeled. In the latter method, the isotope is available to duplicating cells for a period of one hour (maximum), after which time the animal is killed. Therefore, second-generation cells were not labeled. This explains the absence of labeled osteocytes. The hypophysectomized rats follow a pattern of cell proliferation similar to that of normal rats. Thisean be compared to the results of a study reported by Tonna and Cronkite,g who show’ that old mice with a lower proliferative rate of the periosteum follow a pattern of repair similar as do young mice. Moreover, both normal and old mice and rats and hypophysectomized rats show a peak of DNA synthesis at 24 hours. Furthermore, there exists a lag of approximately 12 to 18 hours during which little DNA synthesis occurs in normal and hypophysectomized animals.
Proliferative
Volume 25 Number 3
capacit2j of osteoblasts
425
Fig. 8. Numerous labeled cells can be seen in 24 hour control animals (A). Fracture segments are noted in good apposition (B). (Hematoxylin and eosin stain. Magnification,
x750.)
SUMMARY
AND CONCLUSIONS
The right fibulas of twelve normal and twelve hypophysectomized young albino rats of the Sprague-Dawley strain were fractured by digital pressure. One hour prior to sacrifice, each animal was given 50 microcuries of tritiated thymidine intraperitoneally. Animals were killed at 6 hour intervals, from 6 to 72 hours after fracture. Autoradiograms were prepa.red from tho experimental specimens and examined microscopically. Labeled cells of the osteogenic layer of the periosteum were found throughout the entire diaphysis, indicating that proliferation had been stimulated throughout the diaphyseal population and was not limited exclusively to the fracture site. The proliferative capacity of the periosteum was shown to increase to a maximum at 24 hours after fracture, in both normal and hypophysectomized rats. Although there was a significant difference in the number of such cells, they steadily decreased to a point of insignificant difference at 54 hours. This suggests that cells were undergoing differentiation from preosteoblasts to osteoblasts at about the same rate. The results of our study appear to support the hypothesis that the prolifera-
426
Nichols,
Toto, and Choukas
O.S.,O.M. &O.P. March, 1968
tive capacity of hypophysectomized animals is markedly less than that of the normal animals. No evidence was obtained to confirm studies favoring the osteogenic potential of the fibrous layer of the periosteum. REFERENCES
1. Tonna, E. A.: Tritiated Thymidine Study of the Cellular Contribution to Fracture Repair During Aging, Anat,. Rec. 126: 292, 1960. 2. Enoch, D. M., and Coventry, M. B.: Healing of Fractures, Minnesota M. J. 45: 278-84,
1962. 3. Tonna, E. A., and Cronkite, E. P.: Autoradiographic Studies of Cell Proliferation in the Periosteum of Intact and Fractured Femora of Mice Utilizing DNA Labeling With Tritiated Thymidine, Proc. Sot. Exper. Biol. & Med. 107: 719, 1961. 4. Leblond, C. P., and Carriere, R.: The Effect of Growth Hormone and Thyroxine on the Mitotic rate of the Intestinal Mucosa of the Rat, Endocrinology 56: 261, 1955. 5. Bois, P., Belanger, L. F., and Lebois, J.: Effect of Growth Hormone and Aminoacetonitrile on the Mitotic Rate of Epiphyseal Cartilage in Hypophysectomized Rats, Endocrinology 73: 507, 1963. 6. El Bolkainy, M. N.: Technic for Hypophysectomy of the Mouse, J. Nat. Cancer Inst. 30: 1077, 1963. 7. Joftes, D. L.: Radioautography, Principles and Procedures, J. Nuclear Med. 4: 143, 1963. 8. Robinson, S. H.: Delayed Incorporation of H-3 Thymidine Into DNA, Science 142: 392, 1963. 9. Tonna,. E. A., and Cronkite, E. P.: An Autoradiographic Study of Periosteal Cell Proliferatron With Tritiated Thymidine, Lab. Invest. 13: 161, 1964.
SCHOOL OF DENTISTRY,
CHICAGO COLLEGE OF DENTAL
SURGERY
P
recursor cells preparing for mitosis may be radioactively labeled so that the subsequently differentiated cells can be identified. Tritiated thymidine specifically labels deoxyribonucleic acid during the synthesis period. Tonna,l in a study primarily concerned with aging, fractured the right femurs of albino Swiss mice of varying ages and killed the animals at intervals of 24 hours, 1 week, and 2 weeks. One hour prior to sacrifice, “flash” labeling of the cells was obtained by subcutaneous injection of tritiated thymidine. Tonna observed that osteogenic cells of the periosteum were most frequently labeled, although the number of labeled cells decreased with age. However, by 24 hours after fracture there was an increase in the population of labeled cells, even in mice 52 weeks old. Also, labeled cells were most numerous when osteogenic or chrondrogenic cells from the periosteum were actively converting the fracture callus into cartilage, Tonna concluded that the potential for fracture repair resides essentially in the proliferative capacity of the cells of the periosteum, which diminishes with age. This reduced capacity accounts for the reduction in the rate of fracture repair in older animals. According to Enoch and Coventry, 2 the periosteum consists of an outer layer of collagenous fibers, in which most of the cells are fibroblasts, and an inner layer composed of single fusiform cells in the resting state. The latter becomes a multicellular layer of plump osteoblasts when bone fracture occurs. This investigation was supported in part by the Brophy, Johnso?, Logan Endowment of Loyola University and also by United States Public Health Service General Research Support Grant, 5 SO1 FR-5310-05, General Research Support Branch, Division of Research Facilities and Resources. *Present address: 259 Meridian Rd., San JoNe, Calif.
418
Volume 25 Number 3
Yroliferatice
cupacity
of osteoblasts
419
Tonna and Cronkite3 studied the original cell types participating in fracture repair and also, by “flash” radioactive labeling, investigated common progenitor cells in the periosteum of intact and fractured femora of mice. They observed that pre-osteoblasts are “flash” labeled more frequently than osteoblasts or fiberoblasts but that osteocytes do not “flash” label. At 24 hours after fracture, the fraction of cells labeled in the periosteum was greater throughout the entire diaphysis than at the fracture site. One week a.fter fracture, however, the lab&d cells were most prevalent at the fracture site, although the number of lab&d cells was greatly decreased. They concluded further that it is unlikely that fibroblasts produce osteogenic cells. Leblond and Carriere4 have reported a decrease in the mitotic activity in the crypts of Lieberkiihn in hypophysectomized rats. However, the mitotic rstc is significantly increased upon the administration of growth hormone. Bois, Belanger, and Lebois5 in a similar study, found that hypophysectomized rats had a smaller cartilaginous plate and a lower mitotic index. They also noted that growth hormone significantly stimulated mitosis. The purpose of the present study was to demonstrate autoradiographically the proliferative capacity of the periosteum which developed following fracture of the fibula in normal and hypophysectomized rats. The frequency of labeled cells in the osteogenic layer of the periosteum following fracture in t.he control animal was used as a base line with which to compare DNA synthesis in the hypophysectomized animals. METHODS AND M.ATERIALS
Twenty-four young albino Sprague-Dawley rats, weighing between 80 and 90 grams each, were divided into two groups. Group I contained twelve normal animals, whereas Group II contained twelve hypophysectomized animals. The respective groups of animals were littermates and were housed in wire-bottom cages. The diet of the normal rats consisted of Purina Lab chow and drinking water ad libitum, whereas the hypophysectomized animals were maintained on 5 per cent dextrose in 0.25 per cent saline at a temperature of 80 to 83” I+‘.” The survival period lasted throughout the entire study.
Pig. 1. Representative
example
of fractured
fibula
confirmed
by radiographic
examination.
420
Nichols,
l’oto, and Chouhm
OX, 0.x & 02. March, 1968
The right fibula of each rat was fractured with digital pressure under inhalation ether anesthesia. The animals were killed at 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and 72 hours after fracture. One hour prior to sacrifice, each animal was injected intraperitoneally with 50 microcuries of tritiated thymidine specific a,ctivity, 1.9 c/m&I) at a rate of approximately 0.5 microcuries per gram of body weight. After each animal was killed, the right leg was severed at the proximal joint of the femur, the skin aad muscle were removed, and the specimen was fixed for 24 hours in 10 per cent buffered formalin. At this time all fractures were confirmed by x-ray (Fig. 1). The specimen was washed, decalcified in a 10 per cent solution of disodium versenate, embedded in paraffin, and sectioned at 3 to 6 microns. Autoradiograms were prepared and stained with hematoxylin and eosin.? The autoradiograms were examined microscopically with an oil-immersion lens at a magnification of x1,000. One thousand labeled and nonlabeled cells in the periosteum of the diaphysis of the fractured fibula were counted from each section made. The labeling index was established as the ratio of the number of labeled cells of the osteogenic layer of the periosteum per 1,000 per&teal cells.
Fig. 3. A, Normal periosteal thickness before injury. B, Thickened periosteum animal after fracture. (Hematoxylin and eosin stain. Magnification, x7.50.)
of 6 hour
ProZiferati,ve capacity of osteoblasts
Volume 25 Number 3
421
RESUtTS
The 6 and 12 hour animals exhibited the same reaction to the injury, namely, a thickening of the osteogenic layer of the periosteum along the entire diaphysis (Fig. 2). Labeled cells in the periosteum of the 6 and 12 hour specimens were rare ; only one labeled cell was seen in each (Table I, Figs. 3 and 4). Cell proliferation, as indicated by DNA synthesis, was stimulated between 12 and 18 hours after fracture as the labeling index in the control animals rose from 0.001 to 0.253. Table I. Labeled cell population of osteogenic layer of rat periosteum after fib&l
fracture Hours
Control
post-
fracture
Labeling
index
animals
Hypophysectomized per cent
1Labeling
Labeling
index
awim&
lLabe&ng
per oez
6
0.001
0.1
0.001
0.1
12 18 24 30 36 42 48
0.253 0.001 0.302 0.250 0.210 0.185 0.164
25.3 0.1 30.2 25.0 2 1.0 18.5 16.4
0.200 0.001 0.255 0.204 0.160 0.142 0.113
21i:X 25.5 30.4 16.0 14.2 11.3
60 54 66 72
0.106 0.118 0.090 0.081
10.6 11.8 9.0 8.1
0.088 0.095 0.074 0.067
2 7.4 6.7
r\1,
0.Y
HV~~VSECTOYIZEO
I
\
-
-
-
NllwLl-
0.m
LAIfllNG
Fig. index.
INDEX
3. Proliferative
rate
~ERlOSlEAl
CELL
NOB&f1
HYWPHYSEClOMlZfO
is plotted
All0
PROllFERLTtON
as a function
I” -.
“m.
of time after
fracture,
and labeling
422
Nichols,
Toto, and Choukas
A
R
Fig. 4. Labeling of 6 (a) and 12 (B) hour control animals. Arrows point to one labeled cell in each section. (Hematoxylin and eosin stain. Magnification, x750.)
The findings in the 18 hour hypophysectomized animals clearly showed a significantly less than normal proliferative capacity (Figs. 5 and 6), The osteogenic layer of the periosteum was greatly thickened, and the cells were round, plump, and highly labeled by tritiated thymidine. (Fig. 6). Maximum labeling occurred at 24 hours in the normal and hypophysectomized animals. Also interspersed between the labeled osteoblasts were groups of unlabeled osteoblasts (Fig. 7). The fracture segments were noted to be in good apposition (Fig. 8). The fibrous layer of the periosteum was well defined, with many spindle-shaped and fusiform nonlabeled fibroblasts. Progenitor cells of the osteogenic layer of the periosteum were rapidly synthesizing DNA in preparation for cell duplication (Fig. 3). From 18 hours through 48 hours, the distance between the respective curves exhibits a difference in the proliferative capacity, although both are observed to have decreased steadily. From 54 to 72 hours, however, the distance is insignificant, almost to the point of convergence. The first demonstration of a well-developed fibrous callus was observed in the 60 hour hypophysectomized animal. It had bridged the opposing fragments of the fracture and was twice the thickness of the fracture segment.
Volume 25 Number 3
Prolifemtive
cuywcity
of osteoblnsts
423
Fig. 5. Eighteen hour normal animal exhibits extensive labeling along cntirc length of diaphysis. (Hematoxylin and eosin stain. Magnification, x750.) Fig. 6. Labeling in 18 hour hypophysectomized animal can be observed to be decreased when compared to Fig. 5. (Hematoxylin and eosin stain. Magnification, x750.)
DISCUSSION
The proliferative capacity of the periosteum of normal and hypophyseetomized rats rapidly rises to a peak within 24 hours following fracture of the Akula. After this, the proliferative capacity decreases, as indicated by the number of cells in DNA synthesis. The close proximity of the limbs of the respective curves of cells in DNA synthesis (Fig. 3) shows that cell proliferation is stimulated by fracture of the fibula, in both normal and hypophysectomized rats. There are significantly more cells synthesizing DNA in normal animals, but differentiation occurs at approximately the sa,metime. This can be seen in the convergence of the curves. Although there are more proliferating cells in the normal rat periosteum than in the hypophysectomized rat, there is an equivalence in DNA synthesis after 54 hours. This suggests that maturation mf the cells in the periosteum occurs at about the same rate in both groups of animals. The catabolism of tritiated-thymidine-labeled cells results in the liberation of the thymidine label, which can be reutilized by tissue
424 Nichols, I’oto, und Choukas
OS, O.M. & 0.1’.
March, 1968
A
Pig. 7. Twenty four hour hypophysectomieed animals. A, Thickened periosteum. B, Labeled osteoblasts ; also noted are many spindle-shaped unlabeled fibroblasts. (Hematoxylin and eosin stain. Magnifications: A, x250; B, x750.)
cells preparing for duplication. According to Robinson,* this accounts for labeling for some time after the initial administration of tritiated thymidine. Reutilization is mentioned only to compare this process with the “flash” technique used in our study. The former method fa.cilitates the availability of the radiolabel for long periods of time, and consequently many generations of cells very possibly will become labeled. In the latter method, the isotope is available to duplicating cells for a period of one hour (maximum), after which time the animal is killed. Therefore, second-generation cells were not labeled. This explains the absence of labeled osteocytes. The hypophysectomized rats follow a pattern of cell proliferation similar to that of normal rats. Thisean be compared to the results of a study reported by Tonna and Cronkite,g who show’ that old mice with a lower proliferative rate of the periosteum follow a pattern of repair similar as do young mice. Moreover, both normal and old mice and rats and hypophysectomized rats show a peak of DNA synthesis at 24 hours. Furthermore, there exists a lag of approximately 12 to 18 hours during which little DNA synthesis occurs in normal and hypophysectomized animals.
Proliferative
Volume 25 Number 3
capacit2j of osteoblasts
425
Fig. 8. Numerous labeled cells can be seen in 24 hour control animals (A). Fracture segments are noted in good apposition (B). (Hematoxylin and eosin stain. Magnification,
x750.)
SUMMARY
AND CONCLUSIONS
The right fibulas of twelve normal and twelve hypophysectomized young albino rats of the Sprague-Dawley strain were fractured by digital pressure. One hour prior to sacrifice, each animal was given 50 microcuries of tritiated thymidine intraperitoneally. Animals were killed at 6 hour intervals, from 6 to 72 hours after fracture. Autoradiograms were prepa.red from tho experimental specimens and examined microscopically. Labeled cells of the osteogenic layer of the periosteum were found throughout the entire diaphysis, indicating that proliferation had been stimulated throughout the diaphyseal population and was not limited exclusively to the fracture site. The proliferative capacity of the periosteum was shown to increase to a maximum at 24 hours after fracture, in both normal and hypophysectomized rats. Although there was a significant difference in the number of such cells, they steadily decreased to a point of insignificant difference at 54 hours. This suggests that cells were undergoing differentiation from preosteoblasts to osteoblasts at about the same rate. The results of our study appear to support the hypothesis that the prolifera-
426
Nichols,
Toto, and Choukas
O.S.,O.M. &O.P. March, 1968
tive capacity of hypophysectomized animals is markedly less than that of the normal animals. No evidence was obtained to confirm studies favoring the osteogenic potential of the fibrous layer of the periosteum. REFERENCES
1. Tonna, E. A.: Tritiated Thymidine Study of the Cellular Contribution to Fracture Repair During Aging, Anat,. Rec. 126: 292, 1960. 2. Enoch, D. M., and Coventry, M. B.: Healing of Fractures, Minnesota M. J. 45: 278-84,
1962. 3. Tonna, E. A., and Cronkite, E. P.: Autoradiographic Studies of Cell Proliferation in the Periosteum of Intact and Fractured Femora of Mice Utilizing DNA Labeling With Tritiated Thymidine, Proc. Sot. Exper. Biol. & Med. 107: 719, 1961. 4. Leblond, C. P., and Carriere, R.: The Effect of Growth Hormone and Thyroxine on the Mitotic rate of the Intestinal Mucosa of the Rat, Endocrinology 56: 261, 1955. 5. Bois, P., Belanger, L. F., and Lebois, J.: Effect of Growth Hormone and Aminoacetonitrile on the Mitotic Rate of Epiphyseal Cartilage in Hypophysectomized Rats, Endocrinology 73: 507, 1963. 6. El Bolkainy, M. N.: Technic for Hypophysectomy of the Mouse, J. Nat. Cancer Inst. 30: 1077, 1963. 7. Joftes, D. L.: Radioautography, Principles and Procedures, J. Nuclear Med. 4: 143, 1963. 8. Robinson, S. H.: Delayed Incorporation of H-3 Thymidine Into DNA, Science 142: 392, 1963. 9. Tonna,. E. A., and Cronkite, E. P.: An Autoradiographic Study of Periosteal Cell Proliferatron With Tritiated Thymidine, Lab. Invest. 13: 161, 1964.