JOURNAL OF BONE AND MINERAL RESEARCH Volume 5, Number 6, 1990 Mary Ann Liebert, Inc., Publishers
Aluminum-Induced Neo-Osteogenesis: A Generalized Process Affecting Trabecular Networking in the Axial Skeleton L. DARRYL QUARLES,' GAYLE MURPHY,' JAMES B. VOGLER,' and MARC K. DREZNER'
ABSTRACT To determine if aluminum-induced neo-osteogenesis occurs in the axial skeleton, we compared spinal bone density and vertebral histology of beagles treated with aluminum for 8 and 16 weeks to that of untreated normals. Administration of aluminum (1.25 mg/kg) did not alter serum calcium, phosphorus, or creatinine but did result in a significant elevation of vertebral bone density, measured by quantitative computed tomography, after both 8 (286.7 f 12.4 mg/ml) and 16 (361.7 f 46.5 mg/ml) weeks of treatment compared with controls (212.2 f 4.5 mg/ml). In accord with the increased bone density, biopsies from the spine displayed evidence of neo-osteogenesis, including the presence of woven bone, both mineralized and unmineralized, within the marrow space. The genesis of such woven bone units resulted after 16 weeks in a significant increase in trabecular bone volume, woven and lamellar (51.2 f 4.4 versus 32.4 f 1.2%; p < 0.09, woven bone volume (9.1 f 3.6 versus 0 f 0%; p < 0.05), and trabecular number (4.5 f 0.3 versus 3.5 f 0.2 per mm; p < 0.05). In addition, scanning electron microscopic evaluation of the bone biopsies confirmed the existence of new trabecular plates that provided interconnections between existent units. These observations illustrate that aluminum-induced neo-osteogenesis positively influences trabecular networking in the axial skeleton. Such enhancement of bone histogenesis contrasts with the effects of other pharmacologic agents that solely alter the thickness of existing trabecular plates or rods within the vertebral spongiosa.
INTRODUCTION indicate that the normal loss of trabecular bone with age occurs predominantly by a process that reduces bone surface, thereby limiting new bone formation and resulting in a gradual loss of entire bone structural elements. Such attenuation of bone mass leaves that calcified tissue that remains more widely separated but only slightly reduced in thicknes~.''-~) The resultant increase in intertrabecular spacing transforms the essentially continuous trabecular network of a young person into the mainly discontinuous network characteristic of the elderly. Since the compressive strength of trabecular bone depends
R
ECENT STUDIES
more on the preservation of connections between the structural elements than on the amount of bone the ability to increase trabecular plates in the axial skeleton may be an essential requisite for effective therapy of osteopenic disorders. We recently reported that aluminum administration t o normal beagles stimulates new bone formation within the marrow cavity of the iliac crest.'') Thus in the present study we examined if aluminum similarly evokes neo-osteogenesis in thoracic and lumbar vertebrae. In beagles treated with aluminum for up to 16 weeks we examined spinal bone density by quantitative computed tomography and compared bone histology of vertebrae to that of the iliac crest. Our data indicate that aluminum
'Departments of Medicine, Pathology, Cell Biology and Radiology, Duke University Medical Center, Durham, NC. 'Department of Medicine, University of New Mexico, Albuquerque, NM. 625
QUARLES ET AL.
626
treatment induces neo-osteogenesis in the spine, which results in an increased trabecular plate number and concomitant enhancement of spinal bone density.
METHODS Study protocol A dozen 2-year-old beagles, weighing approximately 15 kg, were randomly divided into three groups. During the study all animals were housed under identical conditions in standard kennel runs and were fed a normal diet containing 1.2% calcium, 0.6% phosphorus, and 2200 U/kg (of diet) vitamin D, (Purina Lab Chow, Ralston-Purina Co., St. Louis, MO). Dogs in groups 1 and 2 received 1.25 mg elemental aluminum per kg as aluminum chloride intravenously three times per week for 8 and 16 weeks, respectively. At the completion of study we obtained blood for biochemical determinations as well as bone density measurements of the spine by quantitative computed tomography. In addition, we biopsied a lumbar vertebral body and the iliac crest in each animal. We employed bone density measurements and bone biopsies from age-matched beagles treated for 16 weeks with pH-matched sodium chloride as vehicle (group 3) as normative values for comparison in these studies. We also obtained blood for biochemical determinations from these animals at the completion of the study and after 8 and 16 weeks.
Biochemistries Serum calcium, phosphorus, and creatinine were measured by colorimetric techniques using an autoanalyzer (Technicon Instruments Corp., Tarrytown, NY). The ionized calcium concentration was determined using an 1C A1 analyzer (Radiometer, Inc., Copenhagen, Denmark), and values were corrected to a pH of 7.4.
Bone densitometry We measured vertebral mineral content in the dogs on a General Electric 9800 CT scanner using a mineral reference standard (calcium hydroxyapatite) for simultaneous calibration, a computed digitized scout image for localization, and single-energy techniques (8 kVp and variable Mineral content was determined in the midplane of six vertebrae, T12-L5, using scan planes parallel to the vertebral end plates and 1 cm thick slices, and the results are expressed in mineral equivalents of the standard (mg/ ml). The precision of the measurements was 2.5% (coefficient of variation). Values in each dog represent the average of the density measurements in the six vertebrae analyzed.
Bone histology Transcortical bone biopsies were obtained from the right anterior iliac crest in all animals under general anesthesia
as previously des~ribed.'~' Additional cores of bone were obtained from the second lumbar vertebra postmortem with an 8 mm trochar. Bone specimens were fixed in ethanol and embedded in methyl methacrylate. Sections (20 and 5 pm) from the specimens were prepared for histomorphologic examination according to published methodology.".8' Histomorphometric analysis of the biopsies was performed with a semiautomated system (Osteoplan, Carl Zeiss, Inc., Thornwood, NY) as detailed el~ewhere.'~) The following histologic functions were quantitated: (1) trabecular bone volume (Tb.BV/TV), the percentage of the sample composed of trabecular bone (woven and lamellar); (2) woven bone volume (Tb.WBV/TV), the percentage of the sample composed of woven trabecular bone; and (3) woven osteoid volume (WOV/TV), the percentage of the sample consisting of woven osteoid. In addition, since trabecular bone consists mainly of interconnecting pIates,''O~'') we partitioned the total trabecular bone volume into an index of trabecular plate thickness and number by calculating the following variables according to previously published methods"): (1) trabecular thickness (Tb.Th), an indirect estimate of mean trabecular thickness as determined in micrometers (2000/trabecular bone surface:volume ratio = trabecular thickness) that correlates well with values obtained by direct measurement of individual trabecular profile width('*'; (2) trabecular number (Tb.N), a derived index calculated (TBV x lO/Tb.Th) with dimensions length-' according to the parallel plate model of trabecular bone"' and representing the density of bone profiles per mm of tissue; and (3) trabecular separation (Tb.Sp), the average distance between edges of existent trabecular plates determined in accord with the parallel plate model of trabecular organization by the formula [(l/Tb.N) - Tb.Th]. In our analysis of the bone histology we discriminated woven from lamellar bone by polarized light microscopic examination of specimens according to previously described techniques. We did not analyze mineralization dynamics because the heterogeneous deposition of tetracycline within woven bone precluded quantitative assessment of calcification in the specimens from treated animals.
Scanning electron microscopy of bone After sections were obtained from representative vertebral biopsies for histomorphometric analysis, the methyl methacrylate surrounding the remainder of the biopsy was cut away and the sample prepared for viewing in the scanning electron microscope according to a modification of the techniques reported by Dempster et al.114)In brief, samples were deplasticized in chloroform for 7-10 days with daily changes of solvent and constant agitation. Subsequently, after air drying, a 50 nm thick conducting layer of gold was applied to the bones with a sputter coater and the biopsies examined in a Philips scanning electron microscope 501 at an accelerating voltage of 15 kV. The instrument is equipped with a standard secondary electron detector.
627
ALUMINUM-INDUCED NEO-OSTEOGENESIS IN THE AXIAL SKELETON
TABLE1. SERUMBIOCHEMISTRIES IN CONTROLS AND ALUMINUM-TREATED BEAGLES iCa
tCa
P
Cr
(rnEq/liter)
m/d4
(W/dO
OWdll
Controls
1.27 f 0.01a
10.7
f
0.14
3.5 f 0.20
0.90
Aluminum, 8 weeks Aluminum, 16 weeks
1.30
f
0.01
10.5
f
0.10
3.8 f 0.30
1.00 f 0.09
1.27
f
0.01
10.9
* 0.32
4.3 f 0.70
1.10
0.06
* 0.08
aValues in controls were unchanged over the duration of the study; values at 16 weeks are shown.
* **
450 7 400
T
:
350 1
300
U
*
Materials
-
Aluminum chloride (AICI, 6H,O) was obtained from Fisher Scientific (Pittsburgh, PA) and adult beagles from Boebel Veterinary Research Center (Mundelein, IL).
250 :
Y \
E"
previously described. ( " ) We performed all computations with the Statgraphic software package (Statistical Graphics Corp., Inc., Princeton, NJ) and a Compaq Deskpro 286 computer.
200: 150
RESULTS
100:
Biochemistries
50 7 OJ
CONTROLS
8W
16 W
ALUMINUW TREATED
FIG. 1. Vertebral bone densitometry in controls and aluminum-treated beagles. Vertebral mineral content was determined in T12-L5 o n a General Electric 9800 C T scanner. Results are expressed in mineral equivalents of a calcium hydroxyapatite standard (mg/ml). Aluminum treatment resulted in a progressive increase in bone density after 8 and 16 weeks of therapy. *Significantly different from controls at p < 0.05; **Significantly different from 8 weeks aluminum treatment at p < 0.05.
Over the 16 weeks of treatment, controls exhibited stable levels of serum total and ionized calcium, phosphorus, and creatinine. Moreover, administration of aluminum for both 8 and 16 weeks resulted in no significant change in any of these variables compared to those of controls (Table 1).
Bone densitometry In contrast, administration of aluminum to the beagles resulted in a significant elevation of vertebral bone density, measured by quantitative computed tomography, after both 8 and 16 weeks of treatment compared with that of controls (Fig. 1). Indeed the observed increments (35.1 and 70.4%, respectively) represented a progressive enhancement of bone density that proceeded at a similar rate throughout both periods of therapy (9.4 and 9.3 mg/ml per week).
Statistical analysis
*
Results are expressed as the mean SEM. Statistical evaluation for differences between groups was done using the Kruskal-Wallis procedure, a nonparametric analysis of variance, with a posteriori testing by the Tukey multiple comparison method. Regression analysis was completed as
Bone histology In accord with the increased bone density, biopsies from the spine displayed evidence of neo-osteogenesis (similar in magnitude to that in the iliac crest). In this regard, we observed woven bone, mineralized and unmineralized, within
628
QUARLES ET AL.
629
ALUMINUM-INDUCED NEO-OSTEOGENESIS IN THE AXIAL SKELETON TABLE 2. STATICINDICESOF TRABECULAR BONEIN CONTROL AND ALUMINUM-TREATED BEAGLES _ _ _ _ ~
~
BV/TV
(%l
* 1.2 * 1.7
_
_
~
_
WBV/TV
WOV / T V
(%I
(%)
o*o o*o
o*o o*o
Controls
Spine Iliac crest
32.4 27.0
Aluminum, 8 weeks
Spine Iliac crest
35.9 34.7
f
0.6 4.8
0.6a 2.0 6.0 f 4.6
Aluminum, 16 weeks
Spine Iliac crest
51.2 48.2
+ 4.4a.b + 2.9a
9.1 3.6a.b 13.4 f 2.la
f
~
*
*
*
0.4 0.2a 2.4 f 1.9 2.0 5.1
* 0.7a.b +
1.7a
Tb.N (no./mm)
*
3.5 0.2 2.7 f 0.1 3.5 3.4
f
f
0.1 0.3
4.5 f 0.3a.b 3.7 f O . l a
Tb.Th
Tb.Sp
(pm)
(pm)
93.6 95.8 101.2 112.4
+
*
7.9 10.1
197.5 280.0
f
6.4 6.4
191.3 194.5
115.0 zt 3.5a 126.7 f 2.Y
112.3 144.3
* 27.0 * 18.9 f f
7.1 26.4
* 17.0’ f
9.ga
asignificantly different from controls at p < 0.05. bSignificantly different from aluminum 8 weeks at p < 0.05.
FIG. 3. Neo-osteogenesis in the vertebral spongiosa of beagles treated for 16 weeks with aluminum. A 5 pm thick Villanueva-stained section reveals partially mineralized “new” trabecular structures (arrows) forming interconnections between previously existent plates. ( x 200) Dark lines (black arrows) define the “cement lines” at the trabecular bone surfaces over which the new foci of bone formed. The absence of a scalloped interface at these sites is consistent with the formation of new bone unrelated to previous resorptive activity.
FIG. 2. Microscopic appearance of a 5 pm thick Goldner-stained histologic section of vertebral bone obtained from a beagle treated with aluminum for 16 weeks (top) compared with that of a normal beagle (bottom). Mineralized trabecular bone from the dog receiving aluminum is marked by thickened trabecular plates as well as “arborization” within the marrow space. The newly formed structures are incompletely mineralized, with the majority of calcification occurring toward the center of the arborized structures. albeit heterogeneously. ( x 100)
QUARLES ET AL.
630 600
550 500
/
450 400 350 I-
Aluminum-Induced Neo-Osteogenesis: A Generalized Process Affecting Trabecular Networking in the Axial Skeleton L. DARRYL QUARLES,' GAYLE MURPHY,' JAMES B. VOGLER,' and MARC K. DREZNER'
ABSTRACT To determine if aluminum-induced neo-osteogenesis occurs in the axial skeleton, we compared spinal bone density and vertebral histology of beagles treated with aluminum for 8 and 16 weeks to that of untreated normals. Administration of aluminum (1.25 mg/kg) did not alter serum calcium, phosphorus, or creatinine but did result in a significant elevation of vertebral bone density, measured by quantitative computed tomography, after both 8 (286.7 f 12.4 mg/ml) and 16 (361.7 f 46.5 mg/ml) weeks of treatment compared with controls (212.2 f 4.5 mg/ml). In accord with the increased bone density, biopsies from the spine displayed evidence of neo-osteogenesis, including the presence of woven bone, both mineralized and unmineralized, within the marrow space. The genesis of such woven bone units resulted after 16 weeks in a significant increase in trabecular bone volume, woven and lamellar (51.2 f 4.4 versus 32.4 f 1.2%; p < 0.09, woven bone volume (9.1 f 3.6 versus 0 f 0%; p < 0.05), and trabecular number (4.5 f 0.3 versus 3.5 f 0.2 per mm; p < 0.05). In addition, scanning electron microscopic evaluation of the bone biopsies confirmed the existence of new trabecular plates that provided interconnections between existent units. These observations illustrate that aluminum-induced neo-osteogenesis positively influences trabecular networking in the axial skeleton. Such enhancement of bone histogenesis contrasts with the effects of other pharmacologic agents that solely alter the thickness of existing trabecular plates or rods within the vertebral spongiosa.
INTRODUCTION indicate that the normal loss of trabecular bone with age occurs predominantly by a process that reduces bone surface, thereby limiting new bone formation and resulting in a gradual loss of entire bone structural elements. Such attenuation of bone mass leaves that calcified tissue that remains more widely separated but only slightly reduced in thicknes~.''-~) The resultant increase in intertrabecular spacing transforms the essentially continuous trabecular network of a young person into the mainly discontinuous network characteristic of the elderly. Since the compressive strength of trabecular bone depends
R
ECENT STUDIES
more on the preservation of connections between the structural elements than on the amount of bone the ability to increase trabecular plates in the axial skeleton may be an essential requisite for effective therapy of osteopenic disorders. We recently reported that aluminum administration t o normal beagles stimulates new bone formation within the marrow cavity of the iliac crest.'') Thus in the present study we examined if aluminum similarly evokes neo-osteogenesis in thoracic and lumbar vertebrae. In beagles treated with aluminum for up to 16 weeks we examined spinal bone density by quantitative computed tomography and compared bone histology of vertebrae to that of the iliac crest. Our data indicate that aluminum
'Departments of Medicine, Pathology, Cell Biology and Radiology, Duke University Medical Center, Durham, NC. 'Department of Medicine, University of New Mexico, Albuquerque, NM. 625
QUARLES ET AL.
626
treatment induces neo-osteogenesis in the spine, which results in an increased trabecular plate number and concomitant enhancement of spinal bone density.
METHODS Study protocol A dozen 2-year-old beagles, weighing approximately 15 kg, were randomly divided into three groups. During the study all animals were housed under identical conditions in standard kennel runs and were fed a normal diet containing 1.2% calcium, 0.6% phosphorus, and 2200 U/kg (of diet) vitamin D, (Purina Lab Chow, Ralston-Purina Co., St. Louis, MO). Dogs in groups 1 and 2 received 1.25 mg elemental aluminum per kg as aluminum chloride intravenously three times per week for 8 and 16 weeks, respectively. At the completion of study we obtained blood for biochemical determinations as well as bone density measurements of the spine by quantitative computed tomography. In addition, we biopsied a lumbar vertebral body and the iliac crest in each animal. We employed bone density measurements and bone biopsies from age-matched beagles treated for 16 weeks with pH-matched sodium chloride as vehicle (group 3) as normative values for comparison in these studies. We also obtained blood for biochemical determinations from these animals at the completion of the study and after 8 and 16 weeks.
Biochemistries Serum calcium, phosphorus, and creatinine were measured by colorimetric techniques using an autoanalyzer (Technicon Instruments Corp., Tarrytown, NY). The ionized calcium concentration was determined using an 1C A1 analyzer (Radiometer, Inc., Copenhagen, Denmark), and values were corrected to a pH of 7.4.
Bone densitometry We measured vertebral mineral content in the dogs on a General Electric 9800 CT scanner using a mineral reference standard (calcium hydroxyapatite) for simultaneous calibration, a computed digitized scout image for localization, and single-energy techniques (8 kVp and variable Mineral content was determined in the midplane of six vertebrae, T12-L5, using scan planes parallel to the vertebral end plates and 1 cm thick slices, and the results are expressed in mineral equivalents of the standard (mg/ ml). The precision of the measurements was 2.5% (coefficient of variation). Values in each dog represent the average of the density measurements in the six vertebrae analyzed.
Bone histology Transcortical bone biopsies were obtained from the right anterior iliac crest in all animals under general anesthesia
as previously des~ribed.'~' Additional cores of bone were obtained from the second lumbar vertebra postmortem with an 8 mm trochar. Bone specimens were fixed in ethanol and embedded in methyl methacrylate. Sections (20 and 5 pm) from the specimens were prepared for histomorphologic examination according to published methodology.".8' Histomorphometric analysis of the biopsies was performed with a semiautomated system (Osteoplan, Carl Zeiss, Inc., Thornwood, NY) as detailed el~ewhere.'~) The following histologic functions were quantitated: (1) trabecular bone volume (Tb.BV/TV), the percentage of the sample composed of trabecular bone (woven and lamellar); (2) woven bone volume (Tb.WBV/TV), the percentage of the sample composed of woven trabecular bone; and (3) woven osteoid volume (WOV/TV), the percentage of the sample consisting of woven osteoid. In addition, since trabecular bone consists mainly of interconnecting pIates,''O~'') we partitioned the total trabecular bone volume into an index of trabecular plate thickness and number by calculating the following variables according to previously published methods"): (1) trabecular thickness (Tb.Th), an indirect estimate of mean trabecular thickness as determined in micrometers (2000/trabecular bone surface:volume ratio = trabecular thickness) that correlates well with values obtained by direct measurement of individual trabecular profile width('*'; (2) trabecular number (Tb.N), a derived index calculated (TBV x lO/Tb.Th) with dimensions length-' according to the parallel plate model of trabecular bone"' and representing the density of bone profiles per mm of tissue; and (3) trabecular separation (Tb.Sp), the average distance between edges of existent trabecular plates determined in accord with the parallel plate model of trabecular organization by the formula [(l/Tb.N) - Tb.Th]. In our analysis of the bone histology we discriminated woven from lamellar bone by polarized light microscopic examination of specimens according to previously described techniques. We did not analyze mineralization dynamics because the heterogeneous deposition of tetracycline within woven bone precluded quantitative assessment of calcification in the specimens from treated animals.
Scanning electron microscopy of bone After sections were obtained from representative vertebral biopsies for histomorphometric analysis, the methyl methacrylate surrounding the remainder of the biopsy was cut away and the sample prepared for viewing in the scanning electron microscope according to a modification of the techniques reported by Dempster et al.114)In brief, samples were deplasticized in chloroform for 7-10 days with daily changes of solvent and constant agitation. Subsequently, after air drying, a 50 nm thick conducting layer of gold was applied to the bones with a sputter coater and the biopsies examined in a Philips scanning electron microscope 501 at an accelerating voltage of 15 kV. The instrument is equipped with a standard secondary electron detector.
627
ALUMINUM-INDUCED NEO-OSTEOGENESIS IN THE AXIAL SKELETON
TABLE1. SERUMBIOCHEMISTRIES IN CONTROLS AND ALUMINUM-TREATED BEAGLES iCa
tCa
P
Cr
(rnEq/liter)
m/d4
(W/dO
OWdll
Controls
1.27 f 0.01a
10.7
f
0.14
3.5 f 0.20
0.90
Aluminum, 8 weeks Aluminum, 16 weeks
1.30
f
0.01
10.5
f
0.10
3.8 f 0.30
1.00 f 0.09
1.27
f
0.01
10.9
* 0.32
4.3 f 0.70
1.10
0.06
* 0.08
aValues in controls were unchanged over the duration of the study; values at 16 weeks are shown.
* **
450 7 400
T
:
350 1
300
U
*
Materials
-
Aluminum chloride (AICI, 6H,O) was obtained from Fisher Scientific (Pittsburgh, PA) and adult beagles from Boebel Veterinary Research Center (Mundelein, IL).
250 :
Y \
E"
previously described. ( " ) We performed all computations with the Statgraphic software package (Statistical Graphics Corp., Inc., Princeton, NJ) and a Compaq Deskpro 286 computer.
200: 150
RESULTS
100:
Biochemistries
50 7 OJ
CONTROLS
8W
16 W
ALUMINUW TREATED
FIG. 1. Vertebral bone densitometry in controls and aluminum-treated beagles. Vertebral mineral content was determined in T12-L5 o n a General Electric 9800 C T scanner. Results are expressed in mineral equivalents of a calcium hydroxyapatite standard (mg/ml). Aluminum treatment resulted in a progressive increase in bone density after 8 and 16 weeks of therapy. *Significantly different from controls at p < 0.05; **Significantly different from 8 weeks aluminum treatment at p < 0.05.
Over the 16 weeks of treatment, controls exhibited stable levels of serum total and ionized calcium, phosphorus, and creatinine. Moreover, administration of aluminum for both 8 and 16 weeks resulted in no significant change in any of these variables compared to those of controls (Table 1).
Bone densitometry In contrast, administration of aluminum to the beagles resulted in a significant elevation of vertebral bone density, measured by quantitative computed tomography, after both 8 and 16 weeks of treatment compared with that of controls (Fig. 1). Indeed the observed increments (35.1 and 70.4%, respectively) represented a progressive enhancement of bone density that proceeded at a similar rate throughout both periods of therapy (9.4 and 9.3 mg/ml per week).
Statistical analysis
*
Results are expressed as the mean SEM. Statistical evaluation for differences between groups was done using the Kruskal-Wallis procedure, a nonparametric analysis of variance, with a posteriori testing by the Tukey multiple comparison method. Regression analysis was completed as
Bone histology In accord with the increased bone density, biopsies from the spine displayed evidence of neo-osteogenesis (similar in magnitude to that in the iliac crest). In this regard, we observed woven bone, mineralized and unmineralized, within
628
QUARLES ET AL.
629
ALUMINUM-INDUCED NEO-OSTEOGENESIS IN THE AXIAL SKELETON TABLE 2. STATICINDICESOF TRABECULAR BONEIN CONTROL AND ALUMINUM-TREATED BEAGLES _ _ _ _ ~
~
BV/TV
(%l
* 1.2 * 1.7
_
_
~
_
WBV/TV
WOV / T V
(%I
(%)
o*o o*o
o*o o*o
Controls
Spine Iliac crest
32.4 27.0
Aluminum, 8 weeks
Spine Iliac crest
35.9 34.7
f
0.6 4.8
0.6a 2.0 6.0 f 4.6
Aluminum, 16 weeks
Spine Iliac crest
51.2 48.2
+ 4.4a.b + 2.9a
9.1 3.6a.b 13.4 f 2.la
f
~
*
*
*
0.4 0.2a 2.4 f 1.9 2.0 5.1
* 0.7a.b +
1.7a
Tb.N (no./mm)
*
3.5 0.2 2.7 f 0.1 3.5 3.4
f
f
0.1 0.3
4.5 f 0.3a.b 3.7 f O . l a
Tb.Th
Tb.Sp
(pm)
(pm)
93.6 95.8 101.2 112.4
+
*
7.9 10.1
197.5 280.0
f
6.4 6.4
191.3 194.5
115.0 zt 3.5a 126.7 f 2.Y
112.3 144.3
* 27.0 * 18.9 f f
7.1 26.4
* 17.0’ f
9.ga
asignificantly different from controls at p < 0.05. bSignificantly different from aluminum 8 weeks at p < 0.05.
FIG. 3. Neo-osteogenesis in the vertebral spongiosa of beagles treated for 16 weeks with aluminum. A 5 pm thick Villanueva-stained section reveals partially mineralized “new” trabecular structures (arrows) forming interconnections between previously existent plates. ( x 200) Dark lines (black arrows) define the “cement lines” at the trabecular bone surfaces over which the new foci of bone formed. The absence of a scalloped interface at these sites is consistent with the formation of new bone unrelated to previous resorptive activity.
FIG. 2. Microscopic appearance of a 5 pm thick Goldner-stained histologic section of vertebral bone obtained from a beagle treated with aluminum for 16 weeks (top) compared with that of a normal beagle (bottom). Mineralized trabecular bone from the dog receiving aluminum is marked by thickened trabecular plates as well as “arborization” within the marrow space. The newly formed structures are incompletely mineralized, with the majority of calcification occurring toward the center of the arborized structures. albeit heterogeneously. ( x 100)
QUARLES ET AL.
630 600
550 500
/
450 400 350 I-
