Journal of Orthopaedic Research 8132-135 Raven Press, Ltd., New York 0 1990 Orthopaedic Research Society
Is Longitudinal Bone Growth Influenced by Diurnal Variation in the Mitotic Activity of Chondrocytes of the Growth Plate? Sharon Stevenson, *Ernst B. Hunziker, *Wolfgang Herrmann, and *Robert K. Schenk Department of Orthopaedics, Case Western Reserve University, Cleveland, Ohio, U.S.A.;and *The Institute of Anatomy, University of Berne, Berne, Switzerland
Summary: The diurnal variations in the mitotic index, height, and rate of linear bone growth were determined and correlations between these parameters examined. Young, unweaned, female Wistar rats were housed under standardized conditions, labeled with a fluorochrome 60 h before sacrifice, and killed at intervals throughout a 24-h period, specifically 0600, 1200, 1800, and 2400. The proximal tibia1 epiphyseal growth plates were collected and processed, and the mitotic index, growth plate height, and the rate of linear bone growth were measured. The mitotic index measured at 0600 was significantly higher than that measured at 1800 and 2400. Growth plates of rats sacrificed at 1200 were taller than those of rats sacrificed at 1800, but there was no difference between heights of growth plates from rats sacrificed at other times. Daily growth rate for all rats averaged 283.9 p d d a y and there were no statistically signifcant differences between daily growth rates measured at any time period. Our findings imply that in comparative, quantitative structural studies of animal groups, sacrifice should be carried out at identical times of the day, since, given a constant speed of vascular ingrowth and diurnal variation in width, relative diurnal accumulation and depletions of cells may take place. We also suggest that the daily growth rate and mitotic index be measured directly and not be considered a function of the height of the growth plate. Key Words: Mitotic index-Diurnal variation-Bone growth.
Endochondral ossification is an integrated and orderly process, comprising the formation, mineralization, and eventual replacement of cartilage by bone. This sequence is responsible for the linear growth of bone (4). Most of the work related to the diurnal variations in endochondral ossification has been carried out in laboratory rodents reared under strict environmental conditions, such as a photoperiod of 12 h alternating with 12 h of darkness and ad
lib feeding (6). Under such circumstances, and using bulk measurements such as isotopic labeling, researchers have shown that the most rapid matrix synthesis occurs during the photoperiod (9,ll) and that the most active mineralization occurs at night (7,IO). Circadian rhythms in peak mitotic activity have also been reported, but the data are conflicting (8,16) and the relationship between changes in mitotic index and linear bone growth has not been clarified. Accordingly, this experiment was designed to measure diurnal variations in the mitotic index. height. and rate of linear bone growth in the proximal tibia1 epiphysed growth plate of young feWistar rats and to examine the between these parameters.
Address correspondence and reprint rewests to Dr. s. Stevenson at Department of Orthopaedics, Case Western Reserve Universitv. 2074 Abinaton - Rd.. Cleveland. Ohio 44106. U.S.A.
Y
132
I
Y
BONE GROWTH AND DIURNAL MITOTIC VARIATIONS
133
MATERIALS AND METHODS
Young, unweaned female Wistar rats (Institute of Pathophysiology , Berne, Switzerland) were housed 12 to a cage in open rooms. They were conditioned to a 12 h/12 h light-dark cycle. The rooms were lighted from 0600 to 1800 and were dark from 1800 to 0600. The environmental temperature was 22" k 1°C. Laboratory chow (Altrumin 13/14) and water were available ad libitum. Calcein (15 g/kg), used to determine the rate of new bone formation and mineralization, was injected intraperitoneally 60 h before rats were killed. At the time of sacrifice, the rats were approximately 1 month old and weighed between 60 and 90 g. The proximal ends of the tibiae were removed under general anesthesia from groups of four rats at 0600, 1200, 1800, and 2400. The bones were cut parallel to the long axis into slices of about 1-2 mm thickness and immediately immersed in fixative. Slices destined for examination by fluorescent microscopy were fixed in 40% ethanol and embedded routinely in polymethyl methacrylate. Slices from which mitoses were to be counted were immersed in buffered 2% glutaraldehyde-0.7% ruthenium hexammine trichloride (RHT, Johnson Mathey Chemicals, Hertforshire, England). While continuously immersed in the fixation medium, these slices were further dissected under the stereomicroscope with razor blades into frontally and sagittally oriented slices comprising the growth cartilage and adjacent parts of the metaphysis and epiphysis (Fig. 1). After a 2-3 h prefixation in the glutaraldehyde-RHT solution, the slices were washed 3-5 min in the same buffer and then postfixed in 1% osmium tetraoxide. Additional processing was carried out as previously described (2). For orientation and light microscopic examination, 1 pm sections were cut with glass knives, always at a distance of 50-70 pm from the surface of the tissue blocks. Sections were stained with 1% toluidine blue 0. Six frontal and six sagittal sections from separate, randomly selected blocks (three from the left leg and three from the right leg) were examined from each rat. The height of the growth plate was estimated by point counting from immediately under the epiphyseal bone to the last transverse septum, and was expressed as a mean value from measurements taken at several locations across the growth plate width on each section (3). The proliferating zone was scanned, the number of mitotic figures was counted, and the number of fields examined
FIG. 1. To determine the mitotic index, mitotic figures were counted within the proliferating zone of the proximal tibia1 growth plate (PZ). One-micron vertical section, toluidine blue 0, x170.
was recorded. The mitotic index was calculated using the formula:
is the number of cells in mitosis in a where NVCm) given volume of tissue and Nv(t)is the total number of cells in the same volume of tissue. The proliferating zone is characterized by flat cells with similar heights arranged in distinct columns, and previously determined values for NV(,)and mean cellular volume were used in the calculation (3). Longitudinal growth rate was assessed by measuring the distance between the calcein label and the lower end of the growth plate (at the last intact transverse septum) using a Zeiss incident light fluorescence microscope equipped with a micrometric eyepiece. This distance provides an estimate of longitudinal growth during the 60 h between labeling and sacrifice. Division of this value by 2.5 (days) gives an
J Orthop Res, Vol. 8, N o . 1 , 1990
S . STEVENSON ET AL.
134
FIG. 2. This high-power (x750) photomicrograph illustrates two chondrocytes undergoing mitosis, one in prophase ( t )and one in telophase
(a).
index of daily growth rate. This calculation is based on the reasonable assumption that growth rate is constant during the 2.5-day period over which it was measured (5,17). At least six measurements were made on each section. The data were analyzed by one-way analysis of variance, the StudentNewman-Keuls test, and the Mann-Whitney U test. RESULTS The mitotic index measured at 0600 was significantly higher than that measured at 1800 (p < 0.05) and 2400 (p < 0.01) (Table 1). The mean daily growth rate for all rats at all time periods was 283.9 31.7 p d d a y . Owing perhaps in part to large standard deviations, there were no statistically significant differences between daily growth rates measured at any time period. No correlation between
*
variations in mitotic index and longitudinal growth rate was found under the conditions of this study. The growth plates of rats sacrificed at 1200 were taller (630.0 102.0 k) than those of rats sacrificed at 1800 (490.5 5 21.5 p) (p < 0.05), but there was no difference between heights of growth plates from rats sacrificed at other times. No correlation between growth plate height and mitotic index or daily growth rate (as measured over 60 h) was found.
*
DISCUSSION The mitotic index of epiphyseal growth plate chondrocytes was similar to that reported elsewhere (1) and did indeed vary according to a circadian rhythm. The greatest number of mitotic figures was noted at 0600, similar to the findings of Walker and Kember (16), but in contrast to the findings of
TABLE 1. Diurnal variations in growth parameters Time of sacrifice
Daily growth rate ( p d 2 4 h) Growth plate height (pm) Mitotic index
0600
1200
1800
2400
286.8 2 20.6 537.9 2 31.3 0.64 2 0.05
304.0 2 22.6 630.0 2 51.0" 0.53 2 0.07
271.8 2 7.2 490.5 10.8 0.37 2 0.03'
272.9 2 7.1 506.4 & 29.3 0.42 2 0.03'
*
Significantly taller than growth plates of rats killed at 1800 (p < 0.05). Significantly lower than mitotic index of rats killed at 0600 (p < 0.01). Significantly lower than mitotic index of rats killed at 0600 (p < 0.05). Four rats per time period; mitotic index was calculated from measurements on twelve sections (six frontal and six sagittal) per rat; growth plate height and daily growth rate were calculated from at least six measurements per rat. Data are expressed as the mean plus or minus the standard error of the mean. a
J Orthop Res, Vol. 8, No. I , 1990
BONE GROWTH AND DIURNAL MITOTIC VARIATIONS Simmons (8). Daily growth rate, on the other hand, varied only slightly among experimental groups. This is not surprising since this parameter may be affected more by chondrocyte hypertrophy, the rate of matrix mineralization, and vascular ingrowth than by cell proliferation (E. B. Hunziker, unpublished observations). The absence of significant growth rate differences may also be due to the resolution limits of the methods used, e.g., a 60-h study period, the inexactness of measuring small distances, the size of the measuring scale, and the width of the calcein band. The growth rate we measured is similar to that measured in the proximal tibial growth plate of male Wistar rats (14). The variation in growth plate height is interesting. The average height of 541.2 pm is comparable to that reported elsewhere (3). Previous reports indicate that most bone and cartilage is mineralized during the dark period (7,10,11). Given that cartilage can only be resorbed after it is mineralized, we expected that the growth plate might be shorter in the morning and taller in the evening. The wide growth plates seen in rats killed at noon may reflect not only the increased mitotic activity that would have occurred at 0600 but also the increase in matrix synthesis that occurs during the photoperiod (9,12,13). In addition, hypertrophic chondrocytes may accumulate when there is a lag period between matrix mineralization and vessel ingrowth. Thus, given a constant speed of vascular ingrowth and diurnal variation in width, relative diurnal cell accumulations and depletions may take place. Our findings imply that in comparative, quantitative structural studies of animal groups, sacrifice should be carried out at identical times of the day to reduce variation in several parameters and the effects of circadian rhythms. Data from one strain of rat cannot be extrapolated directly to other strains. Sprague-Dawley rats of a similar age have a smaller daily growth rate and thinner growth plates than these Wistar rats, the data from which compare very favorably to those derived from other Wistar rats (15). We suggest that the daily growth rate and mitotic index be measured directly-there is no proven correlation between these two parameters and the height of the growth plate, nor could we find a correlation between the diurnal variation in mitotic index and lon-
135
gitudinal bone growth under the conditions of this study. Acknowledgment: This work was supported by the Swiss National Science Foundation, Grant 3 158-0.84.
REFERENCES 1. Heeley JD, Dobeck JM, Derice RA: [3H]Thymidineuptake in cells of rat condylar cartilage. A m J Anat 167:451462, 1983 2. Hunziker EB, Herrmann W, Schenk RK: Improved cartilage fixation by ruthenium hexammine trichloride (RHT). J Ultrastruc Res 8l:l-12, 1982 3. Hunziker EB, Schenk RK, Cruz-Olive L-M: Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth. J Bone Joint Surg [Am] 69: 162173, 1987 4. Kember NF, Walker KVR: Control of bone growth in rats. Nature 229:428429, 1971 5. Nevo Z,Laron Z: Growth factors. A m J Dis Child 133:41% 428, 1979 6. Qudet C,Petrovic A: Nyctothemeral and seasonal variations in the number of tritiated labelled cells in the epiphyseal cartilage of the tibia in the growing rat. Effect of lighting duration and temperature. In: Biological Rhythms in Structure and Function. New York, Alan R. Liss, 1981, pp 187194 7. Russell JE, Grazman B, Simmons DJ: Mineralization in rat metaphyseal bone exhibits a circadian stage dependency. Proc SOCExp Biol Med 176:342-345, 1984 8. Simmons DJ: Circadian mitotic rhythm in epiphyseal cartilage. Nature 202:90&907, 1964 9. Simmons DJ: Chronobiology of endochondral ossification. Chronobiologia 1:97-109, 1974 10 Simmons DJ, Arsenis C, Whitson SW, Kahn SE, Boskey AL, Gollub N: Mineralization of rat epiphyseal cartilage: a circadian rhythm. Miner Electrolyte Metab 8:28-37, 1983 11. Simmons DJ, Lesker PA, Bratberg J, Sherman NE, Aub L: Circadian metabolic profiles in the rat skeleton. Trans Orthop Res SOC 1:22, 1976 12. Simmons DJ, Whiteside LA, Whitson SW: Biorhythmic profiles in the rat skeleton. Metab Bone Dis Relat Res 2:49-64, 1979 13. Simmons DJ: Experimental design and the implication of circadian skeletal rhythmicity. In: Skeletal Research. New York, Academic Press, 1979, pp 567-585 14. Taylor JF, Warrell E, Evans RA: Response of the growth plates to tibial osteotomy in rats. J Bone Joint Surg [Br] 69:664-669, August, 1987 15. Ueno K, Haba T, Woodbury D, Price P, Anderson R, Jee WSS: The effects of prostaglandin E, in rapidly growing rats: depressed longitudinal and radial growth and increased metaphyseal hard tissue mass. Bone 6:79-86, 1985 16. Walker KVR, Kember NF: Cell kinetics of growth cartilage in the rat tibia. I. Cell kinetics of growth cartilage in the rat tibia. Cell Tissue Kinet 5:401408, 1972 17. Walker KVR, Kember NF: Cell kinetics of growth cartilage in the rat tibia. 11. Measurements during aging. Cell Tissue Kinet 5:409419, 1972
J Orthop Res, Vol. 8, No. I , 1990
Is Longitudinal Bone Growth Influenced by Diurnal Variation in the Mitotic Activity of Chondrocytes of the Growth Plate? Sharon Stevenson, *Ernst B. Hunziker, *Wolfgang Herrmann, and *Robert K. Schenk Department of Orthopaedics, Case Western Reserve University, Cleveland, Ohio, U.S.A.;and *The Institute of Anatomy, University of Berne, Berne, Switzerland
Summary: The diurnal variations in the mitotic index, height, and rate of linear bone growth were determined and correlations between these parameters examined. Young, unweaned, female Wistar rats were housed under standardized conditions, labeled with a fluorochrome 60 h before sacrifice, and killed at intervals throughout a 24-h period, specifically 0600, 1200, 1800, and 2400. The proximal tibia1 epiphyseal growth plates were collected and processed, and the mitotic index, growth plate height, and the rate of linear bone growth were measured. The mitotic index measured at 0600 was significantly higher than that measured at 1800 and 2400. Growth plates of rats sacrificed at 1200 were taller than those of rats sacrificed at 1800, but there was no difference between heights of growth plates from rats sacrificed at other times. Daily growth rate for all rats averaged 283.9 p d d a y and there were no statistically signifcant differences between daily growth rates measured at any time period. Our findings imply that in comparative, quantitative structural studies of animal groups, sacrifice should be carried out at identical times of the day, since, given a constant speed of vascular ingrowth and diurnal variation in width, relative diurnal accumulation and depletions of cells may take place. We also suggest that the daily growth rate and mitotic index be measured directly and not be considered a function of the height of the growth plate. Key Words: Mitotic index-Diurnal variation-Bone growth.
Endochondral ossification is an integrated and orderly process, comprising the formation, mineralization, and eventual replacement of cartilage by bone. This sequence is responsible for the linear growth of bone (4). Most of the work related to the diurnal variations in endochondral ossification has been carried out in laboratory rodents reared under strict environmental conditions, such as a photoperiod of 12 h alternating with 12 h of darkness and ad
lib feeding (6). Under such circumstances, and using bulk measurements such as isotopic labeling, researchers have shown that the most rapid matrix synthesis occurs during the photoperiod (9,ll) and that the most active mineralization occurs at night (7,IO). Circadian rhythms in peak mitotic activity have also been reported, but the data are conflicting (8,16) and the relationship between changes in mitotic index and linear bone growth has not been clarified. Accordingly, this experiment was designed to measure diurnal variations in the mitotic index. height. and rate of linear bone growth in the proximal tibia1 epiphysed growth plate of young feWistar rats and to examine the between these parameters.
Address correspondence and reprint rewests to Dr. s. Stevenson at Department of Orthopaedics, Case Western Reserve Universitv. 2074 Abinaton - Rd.. Cleveland. Ohio 44106. U.S.A.
Y
132
I
Y
BONE GROWTH AND DIURNAL MITOTIC VARIATIONS
133
MATERIALS AND METHODS
Young, unweaned female Wistar rats (Institute of Pathophysiology , Berne, Switzerland) were housed 12 to a cage in open rooms. They were conditioned to a 12 h/12 h light-dark cycle. The rooms were lighted from 0600 to 1800 and were dark from 1800 to 0600. The environmental temperature was 22" k 1°C. Laboratory chow (Altrumin 13/14) and water were available ad libitum. Calcein (15 g/kg), used to determine the rate of new bone formation and mineralization, was injected intraperitoneally 60 h before rats were killed. At the time of sacrifice, the rats were approximately 1 month old and weighed between 60 and 90 g. The proximal ends of the tibiae were removed under general anesthesia from groups of four rats at 0600, 1200, 1800, and 2400. The bones were cut parallel to the long axis into slices of about 1-2 mm thickness and immediately immersed in fixative. Slices destined for examination by fluorescent microscopy were fixed in 40% ethanol and embedded routinely in polymethyl methacrylate. Slices from which mitoses were to be counted were immersed in buffered 2% glutaraldehyde-0.7% ruthenium hexammine trichloride (RHT, Johnson Mathey Chemicals, Hertforshire, England). While continuously immersed in the fixation medium, these slices were further dissected under the stereomicroscope with razor blades into frontally and sagittally oriented slices comprising the growth cartilage and adjacent parts of the metaphysis and epiphysis (Fig. 1). After a 2-3 h prefixation in the glutaraldehyde-RHT solution, the slices were washed 3-5 min in the same buffer and then postfixed in 1% osmium tetraoxide. Additional processing was carried out as previously described (2). For orientation and light microscopic examination, 1 pm sections were cut with glass knives, always at a distance of 50-70 pm from the surface of the tissue blocks. Sections were stained with 1% toluidine blue 0. Six frontal and six sagittal sections from separate, randomly selected blocks (three from the left leg and three from the right leg) were examined from each rat. The height of the growth plate was estimated by point counting from immediately under the epiphyseal bone to the last transverse septum, and was expressed as a mean value from measurements taken at several locations across the growth plate width on each section (3). The proliferating zone was scanned, the number of mitotic figures was counted, and the number of fields examined
FIG. 1. To determine the mitotic index, mitotic figures were counted within the proliferating zone of the proximal tibia1 growth plate (PZ). One-micron vertical section, toluidine blue 0, x170.
was recorded. The mitotic index was calculated using the formula:
is the number of cells in mitosis in a where NVCm) given volume of tissue and Nv(t)is the total number of cells in the same volume of tissue. The proliferating zone is characterized by flat cells with similar heights arranged in distinct columns, and previously determined values for NV(,)and mean cellular volume were used in the calculation (3). Longitudinal growth rate was assessed by measuring the distance between the calcein label and the lower end of the growth plate (at the last intact transverse septum) using a Zeiss incident light fluorescence microscope equipped with a micrometric eyepiece. This distance provides an estimate of longitudinal growth during the 60 h between labeling and sacrifice. Division of this value by 2.5 (days) gives an
J Orthop Res, Vol. 8, N o . 1 , 1990
S . STEVENSON ET AL.
134
FIG. 2. This high-power (x750) photomicrograph illustrates two chondrocytes undergoing mitosis, one in prophase ( t )and one in telophase
(a).
index of daily growth rate. This calculation is based on the reasonable assumption that growth rate is constant during the 2.5-day period over which it was measured (5,17). At least six measurements were made on each section. The data were analyzed by one-way analysis of variance, the StudentNewman-Keuls test, and the Mann-Whitney U test. RESULTS The mitotic index measured at 0600 was significantly higher than that measured at 1800 (p < 0.05) and 2400 (p < 0.01) (Table 1). The mean daily growth rate for all rats at all time periods was 283.9 31.7 p d d a y . Owing perhaps in part to large standard deviations, there were no statistically significant differences between daily growth rates measured at any time period. No correlation between
*
variations in mitotic index and longitudinal growth rate was found under the conditions of this study. The growth plates of rats sacrificed at 1200 were taller (630.0 102.0 k) than those of rats sacrificed at 1800 (490.5 5 21.5 p) (p < 0.05), but there was no difference between heights of growth plates from rats sacrificed at other times. No correlation between growth plate height and mitotic index or daily growth rate (as measured over 60 h) was found.
*
DISCUSSION The mitotic index of epiphyseal growth plate chondrocytes was similar to that reported elsewhere (1) and did indeed vary according to a circadian rhythm. The greatest number of mitotic figures was noted at 0600, similar to the findings of Walker and Kember (16), but in contrast to the findings of
TABLE 1. Diurnal variations in growth parameters Time of sacrifice
Daily growth rate ( p d 2 4 h) Growth plate height (pm) Mitotic index
0600
1200
1800
2400
286.8 2 20.6 537.9 2 31.3 0.64 2 0.05
304.0 2 22.6 630.0 2 51.0" 0.53 2 0.07
271.8 2 7.2 490.5 10.8 0.37 2 0.03'
272.9 2 7.1 506.4 & 29.3 0.42 2 0.03'
*
Significantly taller than growth plates of rats killed at 1800 (p < 0.05). Significantly lower than mitotic index of rats killed at 0600 (p < 0.01). Significantly lower than mitotic index of rats killed at 0600 (p < 0.05). Four rats per time period; mitotic index was calculated from measurements on twelve sections (six frontal and six sagittal) per rat; growth plate height and daily growth rate were calculated from at least six measurements per rat. Data are expressed as the mean plus or minus the standard error of the mean. a
J Orthop Res, Vol. 8, No. I , 1990
BONE GROWTH AND DIURNAL MITOTIC VARIATIONS Simmons (8). Daily growth rate, on the other hand, varied only slightly among experimental groups. This is not surprising since this parameter may be affected more by chondrocyte hypertrophy, the rate of matrix mineralization, and vascular ingrowth than by cell proliferation (E. B. Hunziker, unpublished observations). The absence of significant growth rate differences may also be due to the resolution limits of the methods used, e.g., a 60-h study period, the inexactness of measuring small distances, the size of the measuring scale, and the width of the calcein band. The growth rate we measured is similar to that measured in the proximal tibial growth plate of male Wistar rats (14). The variation in growth plate height is interesting. The average height of 541.2 pm is comparable to that reported elsewhere (3). Previous reports indicate that most bone and cartilage is mineralized during the dark period (7,10,11). Given that cartilage can only be resorbed after it is mineralized, we expected that the growth plate might be shorter in the morning and taller in the evening. The wide growth plates seen in rats killed at noon may reflect not only the increased mitotic activity that would have occurred at 0600 but also the increase in matrix synthesis that occurs during the photoperiod (9,12,13). In addition, hypertrophic chondrocytes may accumulate when there is a lag period between matrix mineralization and vessel ingrowth. Thus, given a constant speed of vascular ingrowth and diurnal variation in width, relative diurnal cell accumulations and depletions may take place. Our findings imply that in comparative, quantitative structural studies of animal groups, sacrifice should be carried out at identical times of the day to reduce variation in several parameters and the effects of circadian rhythms. Data from one strain of rat cannot be extrapolated directly to other strains. Sprague-Dawley rats of a similar age have a smaller daily growth rate and thinner growth plates than these Wistar rats, the data from which compare very favorably to those derived from other Wistar rats (15). We suggest that the daily growth rate and mitotic index be measured directly-there is no proven correlation between these two parameters and the height of the growth plate, nor could we find a correlation between the diurnal variation in mitotic index and lon-
135
gitudinal bone growth under the conditions of this study. Acknowledgment: This work was supported by the Swiss National Science Foundation, Grant 3 158-0.84.
REFERENCES 1. Heeley JD, Dobeck JM, Derice RA: [3H]Thymidineuptake in cells of rat condylar cartilage. A m J Anat 167:451462, 1983 2. Hunziker EB, Herrmann W, Schenk RK: Improved cartilage fixation by ruthenium hexammine trichloride (RHT). J Ultrastruc Res 8l:l-12, 1982 3. Hunziker EB, Schenk RK, Cruz-Olive L-M: Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth. J Bone Joint Surg [Am] 69: 162173, 1987 4. Kember NF, Walker KVR: Control of bone growth in rats. Nature 229:428429, 1971 5. Nevo Z,Laron Z: Growth factors. A m J Dis Child 133:41% 428, 1979 6. Qudet C,Petrovic A: Nyctothemeral and seasonal variations in the number of tritiated labelled cells in the epiphyseal cartilage of the tibia in the growing rat. Effect of lighting duration and temperature. In: Biological Rhythms in Structure and Function. New York, Alan R. Liss, 1981, pp 187194 7. Russell JE, Grazman B, Simmons DJ: Mineralization in rat metaphyseal bone exhibits a circadian stage dependency. Proc SOCExp Biol Med 176:342-345, 1984 8. Simmons DJ: Circadian mitotic rhythm in epiphyseal cartilage. Nature 202:90&907, 1964 9. Simmons DJ: Chronobiology of endochondral ossification. Chronobiologia 1:97-109, 1974 10 Simmons DJ, Arsenis C, Whitson SW, Kahn SE, Boskey AL, Gollub N: Mineralization of rat epiphyseal cartilage: a circadian rhythm. Miner Electrolyte Metab 8:28-37, 1983 11. Simmons DJ, Lesker PA, Bratberg J, Sherman NE, Aub L: Circadian metabolic profiles in the rat skeleton. Trans Orthop Res SOC 1:22, 1976 12. Simmons DJ, Whiteside LA, Whitson SW: Biorhythmic profiles in the rat skeleton. Metab Bone Dis Relat Res 2:49-64, 1979 13. Simmons DJ: Experimental design and the implication of circadian skeletal rhythmicity. In: Skeletal Research. New York, Academic Press, 1979, pp 567-585 14. Taylor JF, Warrell E, Evans RA: Response of the growth plates to tibial osteotomy in rats. J Bone Joint Surg [Br] 69:664-669, August, 1987 15. Ueno K, Haba T, Woodbury D, Price P, Anderson R, Jee WSS: The effects of prostaglandin E, in rapidly growing rats: depressed longitudinal and radial growth and increased metaphyseal hard tissue mass. Bone 6:79-86, 1985 16. Walker KVR, Kember NF: Cell kinetics of growth cartilage in the rat tibia. I. Cell kinetics of growth cartilage in the rat tibia. Cell Tissue Kinet 5:401408, 1972 17. Walker KVR, Kember NF: Cell kinetics of growth cartilage in the rat tibia. 11. Measurements during aging. Cell Tissue Kinet 5:409419, 1972
J Orthop Res, Vol. 8, No. I , 1990
