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Automated trabecular bone histomorphometry
Bone, 6, 357-359 (1985) Printed in USA. All rights reserved.
Copyright
8756-3282/85 $3.00 + .OO Q 1985 Pergamon Press Ltd.
Automated Trabecuiar Bone ~jstomorp~ometry E. POLIG’
and W.S.S.
JEEz
‘Kernforschungszenfrum Karlsruhe, lnstitut fur Genefik and Toxikologie von Spalfstoffen, Karlsruhe, Federal Republic of Germany. division of Radiobiology, Department of Pharmacology. University of Utah School of Medlcine, Salt Lake City, UT, USA.
Introduction
bone surfaces, and bone marrow are indicated by low, intermediate, and high light levels, respectively. The photomultiplier measures the light intensity, and its analog output is deposited in an A/D converter and stored in the core memory.
In a collaborative study involving Kernforschungszentrum Karlsruhe and the University of Utah, we are studying the toxicity of alpha-emitting bone-seeking radionuclides and the relationship between bone tumor incidence and the local dosimetry of radionuclides in bone. A key part of the study is determination of the microdistribution of alpha-emitting bone-seeking radionuciides in seiected trabecular bone sites, using a computer-controlled microscope photometer system. The microphotometric scanning routine permits measurement of morphometric parameters, including percentage of bone, perimeter, mean trabecular thickness, mean marrow cavity dimension, surface:volume ratio, and chord length distribution. This report summarizes the methodology and provides some examples of the data accumutated using the University of Utah Optical Track Scanner (UUOTS).
Computer software System control and data analysis were performed using a FORTRAN program. Details of the software WIII appear elsewhere.
Morphomefric
Materials and Methods
Five trabecular bone sites [proximal and distal humerus, proximal and distal femur, and proximal ulna) were analyzed In six young adult beagles that were admlnrstered an intravenous injection of ir’Am: three animals with 2.8 $t/kg and three with an 0.9 ii;Ct/ kg. The times from injection to sacrifice ranged between 7 and 20 days for Ihe 2.8 @t/kg dose level animals and between 1300 and 1569 days for the 0.9 pCi/kg dose level dogs (Table I) In another experiment, five beagle dogs about 418 days old were given a single intravenous injection of 8 6 &I ??YIa/kg and killed between 5 and 92 days postinJect{on The lumbar vertebral
calculation
S,,.i=$ori=~._s’ “b
Finally, the mean bone and marrow chord lengths were obtained from indtvidually measured 20 x 20 pm frames.
bodies of these dogs were analyzed to generate chord length dlstribution data Specimen
parameters
Several morphometric parameters were derived using automated Image analysis: (1) the area of trabecular bone, (2) the bone perimeter, (3) the surface:volume ratio (S,,), (4) the mean trabecular and marrow cavity thicknesses, and (5) the mean bone and marrow chord length. The area of trabecular bone is determined by addlng the indivrdual areas of frames located entirely over bone (B-frames) plus the coverage C of all the edge frames (E-frames) by relating the measured light extraction in an edge frame to the light extractton in neighboring marrow frames (Fig. 2). The total bone surface perimeter In a scanning area is the sum of the surface segment lengths (L) of each individual edge frame using the coverage (Cf. A relationship between L and C was established by a Monte Carlo simulation (Fig. 3). The surface:volume ratio was then calculated using the 4/s times perimeter/aLea formula (Ellas et al., 1971). The mean trabecular thickness (I ) from S,, was calculated using this formula.
Results and Discussion
preparation
Table I lists the morphometric parameters obtained by the microp~otometric scanning of trabecuiar bone from proximal humerus, distal humerus, proximal ulna, proximal femur, and distal femur from the animals in the two experiments, including percentage of bone, perimeter, mean thickness of trabecular bone, mean marrow cavity size, and surface:volume ratio. The animals injected with 0.9 $i 7”1Am/
qecimens were fixed in absolute acetone, embedded in polymethylmethacrylate plastic, and sawed longltudmally Into slices approxtmately 300 pm thick, which were then ground to 120 pm and stained with 1% alizann red S.
The University of Utah OptIcal Track Scanner conslsts of SIX basic hardware modules. MPV compact mlcroscope photometer with accessories, a minicomputer to control the microscope stage, hard disk, printer, and graphic display (Fig. 1). Light of a 540 nm wavelength wrth a band width of 11 nm scans a typical frame of 20 x 20 pm; an entlre scan area measures 4 x 6 mm. Stained bone,
kg show a distinctly increased percentage of bone, increased trabecular bone thickness with a correspondingly
reduced width of marrow spaces, and reduced surface:volume ratios. A similar thickening of trabeculae has been observed in other experiments with Z3gPu(Jee et al., 357
358
4th International
TABLE I. Morphometric
Bone Histomorphometry
Workshop
parameters
Parameters
Dose of Z41AM (puCi/kg)
% Bone
2.0b 0.9’ 2.8 0.9 2.8 0.9 2.8 0.9
Mean trabecular thickness (pm) Mean marrow cavity dimension (pm) Surface:volume ratio (1 /cm)
PH’”
DH
PU
PF
DF
28.9 41.1 161 216 396 306 213.4 164.8
47.3 67.3 271 331 310 162 134.1 104.6
50.0 64.6 300 408 297 226 122.6 84.5
40.0 42.0 200 265 330 381 173.0 132.9
35.3 45.3 172 237 330 292 200.7 145
at Ph, proximal humerus; DH, distal humerus; PU, proximal ulna; PF, proximal femur; DF, distal femur. b Time between injection and sacrifice, 7-20 days. c Time between injection and sacrifice, 1300- 1569 days
1972) and za1Am in rats (Polig, 1976). Therefore, radiation damage resulting in abnormal bone structure has occurred. The surface:volume ratios, which average 169/cm + 401 cm in the 2.8 PCi 241Am/kg dogs, is in good agreement with the average of 171/cm _t 39/cm observed by Kimmel and Jee (1982) and the 185/cm observed by Beddoe (1978). Knowledge of the relationship between the static and dynamic properties of the skeleton may be of value in understanding the occurrence or absence of bone dyscrasias, age-related bone loss, and osteoporosis in certain parts of the skeleton. We observed a strong relationship between high trabecular turnover rate (Kimmel and Jee, 1982) and lower percentage of trabecular bone, thinner trabeculae, and greater S,, ratio.
LSI I i/o2 Processor A/D 1,
Converter
5 a
Grophrc Display Fig. 1. Configuration photomultiplrer.
of microphotometnc
scannrng system
PM,
bone surface
06
0.1 Fig. 2. Pattern of scanning frames (20 x 20 pm) along a bone surface. 6, frames entirely over bone; M, frames entirely over marrow; E, frames over a bone-marrow Interface. The coverage C (fractron of an E-frame covered by bone) IS determined by analyzrng a 3 x 3 neighborhood (dashed lrne) usrng the average lrght transmission of marrow fields (M,, M, In the sample)
0'2
0'3
Coverage
0 L
0'5
(Cl
Fig. 3. Monte Carlo determination (calculatron) of relatronship between the coverage (C) and the surface segment length (L) in a unit frame. The vertical bars represent standard devratrons
4th International
Bone Histomorphometry
Workshop
T54A5 DENSITY
OF CHORD MEAN:
0
MARROW
MEAN
359
The morphometric determinations mentioned before are merely a byproduct of our bone-seeking radionuclide local dosimetry program. If efforts were concentrated on bone morphometry, the morphometric program could be readily improved and the scope of work expanded to include analysis of fluorescent-labeled bone specimens and distribution of trabecular bone density.
LVI-6A LENGTHS 238.0 ~535.4
This study was supported in part by NIH Grants No. AM-31844 and 27029, NASA Grant No. NAG-2-108, and US Department of Energy Contract DE-AC02-76EV-00119. We gratefu!ly acknowledge the skilled technical assistance of Floyd Johnson and Rebecca Dell. Acknowledgment:
0
I60
320 CHORD
LENGTH
480
640
800
ImIcrons)
Fig. 4. Relative frequency of chord length, mean bone length, and marrow chord length in a longitudinal section of a lumbar vertebral body from a beagle dog Injected with 2.8 ~LCIof 226Raand sacrificed 5 days postinjectlon. Many thick chord lengths are apparent in such longitudinal sections, due lo the preferential orientation of trabeculae along
the direction
of scannmg.
The scanner can also determine trabecular plate thickness. We can now readily measure and plot the distribution of trabecular bone chord lengths (Fig. 4). Such information may be invaluable in improving our understanding of the processes of age-related bone loss.
References Beddoe, A H.: A quantltatlve study of the structure of trabecular bone in man/ rhesus monkey, beagle and mlmature pig. Calcif. 7)~s. Res. 25. 273.281, 1978 Elias H., Henlng A. and Saharz D.E.: Stereology: Appkatlon to blomedlcal research. Phys/o/. Rev 51:158-200, 1971. Jee W S.S., Dell R.B. and Hashimoto E Quantltatwe morphology of vertebral trabecular bone In beaales lnrected with olutonium Urw of Utah Dtwslon of Radloblology Rep EOO-1’19-246. 193.217, 1972 Klmmel D.B. and Jee W S.S A quantltatrve hlstologlc study of bone turnover in young adult beagles Anal Ret 203.31-45, 1982 Pollg E. The Influence of 2d’Am and DPTA on morphomelnc parameters of the rat femurs. Rad. Enwon. Brophys 13 27-4 1, 1976
Copyright
8756-3282/85 $3.00 + .OO Q 1985 Pergamon Press Ltd.
Automated Trabecuiar Bone ~jstomorp~ometry E. POLIG’
and W.S.S.
JEEz
‘Kernforschungszenfrum Karlsruhe, lnstitut fur Genefik and Toxikologie von Spalfstoffen, Karlsruhe, Federal Republic of Germany. division of Radiobiology, Department of Pharmacology. University of Utah School of Medlcine, Salt Lake City, UT, USA.
Introduction
bone surfaces, and bone marrow are indicated by low, intermediate, and high light levels, respectively. The photomultiplier measures the light intensity, and its analog output is deposited in an A/D converter and stored in the core memory.
In a collaborative study involving Kernforschungszentrum Karlsruhe and the University of Utah, we are studying the toxicity of alpha-emitting bone-seeking radionuclides and the relationship between bone tumor incidence and the local dosimetry of radionuclides in bone. A key part of the study is determination of the microdistribution of alpha-emitting bone-seeking radionuciides in seiected trabecular bone sites, using a computer-controlled microscope photometer system. The microphotometric scanning routine permits measurement of morphometric parameters, including percentage of bone, perimeter, mean trabecular thickness, mean marrow cavity dimension, surface:volume ratio, and chord length distribution. This report summarizes the methodology and provides some examples of the data accumutated using the University of Utah Optical Track Scanner (UUOTS).
Computer software System control and data analysis were performed using a FORTRAN program. Details of the software WIII appear elsewhere.
Morphomefric
Materials and Methods
Five trabecular bone sites [proximal and distal humerus, proximal and distal femur, and proximal ulna) were analyzed In six young adult beagles that were admlnrstered an intravenous injection of ir’Am: three animals with 2.8 $t/kg and three with an 0.9 ii;Ct/ kg. The times from injection to sacrifice ranged between 7 and 20 days for Ihe 2.8 @t/kg dose level animals and between 1300 and 1569 days for the 0.9 pCi/kg dose level dogs (Table I) In another experiment, five beagle dogs about 418 days old were given a single intravenous injection of 8 6 &I ??YIa/kg and killed between 5 and 92 days postinJect{on The lumbar vertebral
calculation
S,,.i=$ori=~._s’ “b
Finally, the mean bone and marrow chord lengths were obtained from indtvidually measured 20 x 20 pm frames.
bodies of these dogs were analyzed to generate chord length dlstribution data Specimen
parameters
Several morphometric parameters were derived using automated Image analysis: (1) the area of trabecular bone, (2) the bone perimeter, (3) the surface:volume ratio (S,,), (4) the mean trabecular and marrow cavity thicknesses, and (5) the mean bone and marrow chord length. The area of trabecular bone is determined by addlng the indivrdual areas of frames located entirely over bone (B-frames) plus the coverage C of all the edge frames (E-frames) by relating the measured light extraction in an edge frame to the light extractton in neighboring marrow frames (Fig. 2). The total bone surface perimeter In a scanning area is the sum of the surface segment lengths (L) of each individual edge frame using the coverage (Cf. A relationship between L and C was established by a Monte Carlo simulation (Fig. 3). The surface:volume ratio was then calculated using the 4/s times perimeter/aLea formula (Ellas et al., 1971). The mean trabecular thickness (I ) from S,, was calculated using this formula.
Results and Discussion
preparation
Table I lists the morphometric parameters obtained by the microp~otometric scanning of trabecuiar bone from proximal humerus, distal humerus, proximal ulna, proximal femur, and distal femur from the animals in the two experiments, including percentage of bone, perimeter, mean thickness of trabecular bone, mean marrow cavity size, and surface:volume ratio. The animals injected with 0.9 $i 7”1Am/
qecimens were fixed in absolute acetone, embedded in polymethylmethacrylate plastic, and sawed longltudmally Into slices approxtmately 300 pm thick, which were then ground to 120 pm and stained with 1% alizann red S.
The University of Utah OptIcal Track Scanner conslsts of SIX basic hardware modules. MPV compact mlcroscope photometer with accessories, a minicomputer to control the microscope stage, hard disk, printer, and graphic display (Fig. 1). Light of a 540 nm wavelength wrth a band width of 11 nm scans a typical frame of 20 x 20 pm; an entlre scan area measures 4 x 6 mm. Stained bone,
kg show a distinctly increased percentage of bone, increased trabecular bone thickness with a correspondingly
reduced width of marrow spaces, and reduced surface:volume ratios. A similar thickening of trabeculae has been observed in other experiments with Z3gPu(Jee et al., 357
358
4th International
TABLE I. Morphometric
Bone Histomorphometry
Workshop
parameters
Parameters
Dose of Z41AM (puCi/kg)
% Bone
2.0b 0.9’ 2.8 0.9 2.8 0.9 2.8 0.9
Mean trabecular thickness (pm) Mean marrow cavity dimension (pm) Surface:volume ratio (1 /cm)
PH’”
DH
PU
PF
DF
28.9 41.1 161 216 396 306 213.4 164.8
47.3 67.3 271 331 310 162 134.1 104.6
50.0 64.6 300 408 297 226 122.6 84.5
40.0 42.0 200 265 330 381 173.0 132.9
35.3 45.3 172 237 330 292 200.7 145
at Ph, proximal humerus; DH, distal humerus; PU, proximal ulna; PF, proximal femur; DF, distal femur. b Time between injection and sacrifice, 7-20 days. c Time between injection and sacrifice, 1300- 1569 days
1972) and za1Am in rats (Polig, 1976). Therefore, radiation damage resulting in abnormal bone structure has occurred. The surface:volume ratios, which average 169/cm + 401 cm in the 2.8 PCi 241Am/kg dogs, is in good agreement with the average of 171/cm _t 39/cm observed by Kimmel and Jee (1982) and the 185/cm observed by Beddoe (1978). Knowledge of the relationship between the static and dynamic properties of the skeleton may be of value in understanding the occurrence or absence of bone dyscrasias, age-related bone loss, and osteoporosis in certain parts of the skeleton. We observed a strong relationship between high trabecular turnover rate (Kimmel and Jee, 1982) and lower percentage of trabecular bone, thinner trabeculae, and greater S,, ratio.
LSI I i/o2 Processor A/D 1,
Converter
5 a
Grophrc Display Fig. 1. Configuration photomultiplrer.
of microphotometnc
scannrng system
PM,
bone surface
06
0.1 Fig. 2. Pattern of scanning frames (20 x 20 pm) along a bone surface. 6, frames entirely over bone; M, frames entirely over marrow; E, frames over a bone-marrow Interface. The coverage C (fractron of an E-frame covered by bone) IS determined by analyzrng a 3 x 3 neighborhood (dashed lrne) usrng the average lrght transmission of marrow fields (M,, M, In the sample)
0'2
0'3
Coverage
0 L
0'5
(Cl
Fig. 3. Monte Carlo determination (calculatron) of relatronship between the coverage (C) and the surface segment length (L) in a unit frame. The vertical bars represent standard devratrons
4th International
Bone Histomorphometry
Workshop
T54A5 DENSITY
OF CHORD MEAN:
0
MARROW
MEAN
359
The morphometric determinations mentioned before are merely a byproduct of our bone-seeking radionuclide local dosimetry program. If efforts were concentrated on bone morphometry, the morphometric program could be readily improved and the scope of work expanded to include analysis of fluorescent-labeled bone specimens and distribution of trabecular bone density.
LVI-6A LENGTHS 238.0 ~535.4
This study was supported in part by NIH Grants No. AM-31844 and 27029, NASA Grant No. NAG-2-108, and US Department of Energy Contract DE-AC02-76EV-00119. We gratefu!ly acknowledge the skilled technical assistance of Floyd Johnson and Rebecca Dell. Acknowledgment:
0
I60
320 CHORD
LENGTH
480
640
800
ImIcrons)
Fig. 4. Relative frequency of chord length, mean bone length, and marrow chord length in a longitudinal section of a lumbar vertebral body from a beagle dog Injected with 2.8 ~LCIof 226Raand sacrificed 5 days postinjectlon. Many thick chord lengths are apparent in such longitudinal sections, due lo the preferential orientation of trabeculae along
the direction
of scannmg.
The scanner can also determine trabecular plate thickness. We can now readily measure and plot the distribution of trabecular bone chord lengths (Fig. 4). Such information may be invaluable in improving our understanding of the processes of age-related bone loss.
References Beddoe, A H.: A quantltatlve study of the structure of trabecular bone in man/ rhesus monkey, beagle and mlmature pig. Calcif. 7)~s. Res. 25. 273.281, 1978 Elias H., Henlng A. and Saharz D.E.: Stereology: Appkatlon to blomedlcal research. Phys/o/. Rev 51:158-200, 1971. Jee W S.S., Dell R.B. and Hashimoto E Quantltatwe morphology of vertebral trabecular bone In beaales lnrected with olutonium Urw of Utah Dtwslon of Radloblology Rep EOO-1’19-246. 193.217, 1972 Klmmel D.B. and Jee W S.S A quantltatrve hlstologlc study of bone turnover in young adult beagles Anal Ret 203.31-45, 1982 Pollg E. The Influence of 2d’Am and DPTA on morphomelnc parameters of the rat femurs. Rad. Enwon. Brophys 13 27-4 1, 1976