In vivo effects of high-dose methotrexate on bone remodeling in rats
Urszula Cegie³a, Leszek Œliwiñski, Ilona Kaczmarczyk-Sedlak, Joanna Folwarczna
Department of Pharmacology, Silesian Medical University, Jagielloñska 4, PL 41-200 Sosnowiec, Poland Correspondence: Urszula Cegie³a
Abstract:
Methotrexatae (MTX) is a folate antagonist. MTX osteopathy is well recognized to accompany a high-dose therapy with this drug for the treatment of childhood malignancy. Clinical tests also show that low-dose MTX used in the treatment of rheumatoid arthritis may impair bone formation in a population already predisposed to osteoporosis. However, results of clinical tests are hard to interpret, as it is necessary to take into account malignancy-induced changes in the osseous tissue, long-term immobility and concurrent administration of glucocorticosteroids.
We conductedin vivo tests to evaluate the effects of oral and intramuscular administration of high dose of MTX on bone remodeling processes in rats. Effects of MTX on the processes of bone remodeling were evaluated by assessing macrometric and histomorphometric parameters as well as mechanical properties of the femur.
The tests were carried out on male Wistar rats. Animals were divided into four groups, composed of 7 animals each: Control group (0.9% NaCl solution), MTX-1po group (MTX at the dose of 1 mg/kg po daily for 10 days: every day for the first five days, and after an 18-day interval, every day for five days), MTX-1im group (MTX at the dose of 1 mg/kg im daily for 10 days: every day for the first five days, and after an 18-day interval, every day for five days), MTX-5im group (MTX at the dose 5 mg/kg im daily for 2 days a week for the period of four weeks). Changes in bone remodeling were examined 4 weeks after the first MTX administration.
These results show that MTX administered intramuscularly at high doses inhibited the formation and mineralization of new osseous matrix and impaired mechanical properties of the femoral bone, whereas its oral administration had no effect on bone remodeling in rats.
Key words:
methotrexate, macrometric parameters, histomorphometric parameters, mechanical properties of the femur, bones, rats
Introduction
Methotrexate (MTX), an antimetabolite, is a folate antagonist. MTX is usually given orally but can also be given intramuscularly, intravenously or intrathe- cally [26]. The drug is commonly used at high doses (100–1000 mg/m2) in the treatment of acute lympho- blastic leukemia, lymphoma and osteosarcoma [20].
In autoimmune diseases and allografts, MTX is used
at low doses (5–25 mg/week) given orally or by intra- muscular injections [12, 20]. The main action of MTX consists of inhibition of dihydrofolate reductase, which reduces dietary folate to tetrahydrofolate, an essential cofactor in DNA and RNA synthesis. Intra- cellular MTX is converted by folylpolyglutamate syn- thase to polyglutamate forms that are not readily transported across the cell membrane. These polyglu- tamates inhibit dihydrofolate reductase, thereby de- creasing the amount of reduced folates, one-carbon
Pharmacological Reports 2005, 57, 504514 ISSN 1734-1140
Copyright © 2005 by Institute of Pharmacology Polish Academy of Sciences
donors for the purine ring formation inde novo purine synthesis. Methotrexate polyglutamates also directly inhibit phosphoribosylpyrophosphate amidotransfe- rase, glycinamide ribonucleotide transformylase, and 5-aminoimidazole-4-carboxamide ribonucleotide trans- formylase, key enzymes in thede novo purine synthe- sis pathway [1, 7, 9, 11].
The results of clinical tests of MTX effect on the osseous tissue are not clear. Methotrexate is known to cause osteopathy, which is characterized by bone pain, osteoporosis, and pathologic fractures. Osteopa- thy is recognized to accompany a high-dose therapy with this drug for the treatment of childhood malig- nancy [10, 29]. Several clinical studies have shown that a low-dose MTX therapy does not induce gener- alized bone loss [4, 15]. Buckley et al. proved that a long-term administration of MTX at low doses did not lead to reduced bone density, but in patients who were administered methotrexate with prednizolone, the bone loss was greater than after administering prednizolone alone [4]. Also Preston et al. reported two cases of osteopathy, including fractures, in post- menopausal women taking low-dose MTX for treat- ment of rheumatic disease [23], and the number of re- ports on osteopathy connected with a low-dose MTX administration is increasing [17, 34]. However, Minaur et al. did not find a negative effect of MTX on bone formation markers and histomorphometric pa- rameters connected with bone formation in patients treated with low-dose MTX. Clinical tests also show that low-dose MTX used in the treatment of rheuma- toid arthritis may impair bone formation in a popula- tion already predisposed to osteoporosis [21].
The results of clinical tests are hard to interpret, as it is necessary to take into account malignancy- induced changes in the osseous tissue, long-term im- mobility, e.g. after a graft, or a concurrent administra- tion of glucocorticosteroids [6, 13, 16].
Results obtained so far on the basis ofin vitro tests indicate that MTX reduces the number of human os- teoblasts [28]. However, Robson et al. did not reveal any effects of MTX, when used at similar concentra- tions [27], on chondrocyte proliferation, whereas Pre- ston et al. proved that MTX had a dose-dependent toxic effect on osteoblastic cells UMR 106 already at concentrations administered in the treatment of rheu- matoid arthritis [22].
We conductedin vivo tests to examine the effect of oral and intramuscular administration of high-dose MTX on bone remodeling in rats. We tested the MTX
effect on macrometric and histomorphometric parame- ters as well as mechanical properties of the femur.
Materials and Methods
The tests were carried out on male Wistar rats of ini- tial body mass between 225–240 g, from the Central Animal Farm of the Silesian Medical University. Dur- ing the experiment, the animals were housed in poly- styrene cages (590 × 385 × 200 mm), 7 in each cage.
The temperature and humidity were kept constant at 20 ± 3oC and 60–70%, respectively. The rats received water and feedad libitum. A standard laboratory ani- mal diet was used, containing 10.20 g/kg of calcium and 5.10 g/kg of phosphorus (pellet food GLM, Po- land). The permission for the animal tests and experi- ments was granted by the Local Ethics Commission, Katowice.
Animals were divided into four groups composed of 7 animals each (n = 7): Control – control group of rats, which were given 0.9% NaCl solution in the vol- ume of 2 ml/kgpo daily for 5 days, and after 18 days the volume of 1 ml/kgim daily for 5 days; MTX-1po group – rats, which were given MTX at the dose of 1 mg/kgpo daily for 10 days: every day for the first five days and after 18 days, every day for five days in the volume of 2 ml/kg (MTX was administered on days 2–6 and 25–29 of the experiment); MTX-1im group – rats, which were given MTX at the dose of 1 mg/kgim daily for 10 days: every day for the first five days and after 18 days, every day for five days in the volume of 1 ml/kg (MTX was administered on days 2–6 and 25–29 of the experiment); MTX-5im group – rats, which were given MTX at the dose 5 mg/kgim daily for 2 days a week for the period of 4 weeks in the volume of 1 ml/kg (MTX was adminis- tered on days 2–3, 9–10, 16–17 and 23–24 of the ex- periment). Administration of MTX or 0.9% NaCl so- lution started on day 2 of the experiment.
On day 1 of the experiment (24 h before starting to administer MTX or 0.9% NaCl solution) and on day 29 of the experiment (24 h before the animals were sacrificed), the rats were given tetracycline hydro- chloride at the dose of 20 mg/kgip. Tetracycline hy- drochloride was administered to all animals twice in order to establish the transverse growth of the tibial bone [5].
Changes in bone tissue were examined 4 weeks (28 days) after the first administration of MTX or 0.9%
NaCl solution, i.e. on day 30 of the experiment.
The animals were sacrificed by spinal cord dis- placement on day 30 of the experiment and then the right and left femoral bones, right and left tibial bones, and L-4 vertebra were isolated and cleaned of the adjacent soft tissues. The left femur was divided into epiphyses and diaphysis.
Changes in bone tissue caused by MTX were stud- ied by the evaluation of macrometric and histomor- phometric parameters as well as mechanical proper- ties of the femoral bone.
Macrometric parameters
The effect of MTX on macrometric parameters was assessed based on the determination of the mass of femoral epiphyses, femoral diaphysis, tibia and L-4 vertebra, the mineral and calcium content in these bones, the ratio of mineral content to the bone mass, and on the basis of determining the length and diame- ter of the femur and tibia.
The examined bone mass was determined immedi- ately after they were isolated and cleaned of soft tis- sues by weighing them using analytical balance type AS 200 made by Ohaus (accuracy to 0.0001 g). The measurements of the bone length and diameter (in the half-length of the bone) were performed using a slide caliper (accuracy to 0.01 cm). Bone mineral content was established by determining the mass of the exam- ined bones which had previously been mineralized at 640°C for 48 h. The mineralized bones were dis- solved in 6M HCl solution for 24 h and then calcium content was determined by means of a colometric method using a standard kit for calcium content deter- mination produced by Pointe Scientific. Absorption was measured by Pointe-180 plus biochemical ana- lyzer.
Histomorphometric parameters
The effect of MTX on histomorphometric parameters was assessed based on determining the transverse cross-section area of the cortical bone in the diaphysis and the transverse cross-section area of the marrow cavity in the tibia, as well as the ratio of the marrow cavity transverse cross-section area to the transverse cross-section area of the cortical bone, transverse growth of the tibia and the width of periosteal and en-
dosteal osteoid in the tibia, the width of trabeculae in the femoral epiphysis and metaphysis and the width of epiphysial cartilage in the femur. The examination of changes in histomorphometric parameters was car- ried out on histological preparations made of non- decalcified bone sections [6, 32].
Evaluation of the transverse cross-section area of the cortical bone and the transverse cross-section area of the marrow cavity in the tibia was performed on a preparation of the tibial transverse cross-section, us- ing Baylink’s planimetric method [3] and MP-3 la- nameter, with a magnifying power of 50.
The evaluation of changes in the remaining histo- morphometric parameters was performed by means of Nikon microscope of Optiphot-2 type interfaced with RGB camera, made by Cohu and with a computer equipped with Lucia G 4.51 software for digital histo- logical measurements. The tibial transverse growth was measured in UV light, whereas the remaining his- tomorphometric parameters were measured in visible light.
The tibial transverse growth on the side of perios- teum or endosteum, and periosteal and endosteal os- teoid width in each preparation was the arithmetic mean of 6–8 measurements taken along the entire bone circumference in a given preparation on the side of periosteum or on the side of endosteum, at 500×
magnification. The width of trabeculae in the epiphy- sis or metaphysis was presented as an arithmetic mean of 10–15 measurements taken for each preparation in the epiphysis or metaphysis, in one visual field, in the middle of a given preparation. Trabeculae width measurements in the epiphysis were performed about 600 µm under the cartilage, whereas the trabecular width in the metaphysis – about 600 µm above the cartilage, at 200× magnification. The width of the epiphysial cartilage in each preparation was presented as an arithmetic mean of 8–12 measurements per- formed alongside the length of the cartilage at 200×
magnification [5].
Mechanical properties of the femoral bone
The effect of MTX on mechanical properties of the femur was assessed on the basis of examining me- chanical properties of the femur and femoral neck, which included determination of the extrinsic stiff- ness of the femur, the ultimate load endured by the fe- mur, the load registered at the femoral diaphysis frac- ture (breaking load), the deformation of the femoral
diaphysis at the ultimate load and at the breaking load, and the load resulting in the femoral neck fracture.
Mechanical properties were tested using the left fe- mur and part of the right femur (proximal epiphysis and part of diaphysis) cut perpendicularly to the lon- gitudinal axis of the bone in its half-length.
The tests of mechanical properties of femoral dia- physis and femoral neck were carried out using a de- vice constructed at the Department of Pharmacology of Silesian Medical University in Sosnowiec, in coop- eration with Hottinger Baldwin Messtechnik in Poznañ.
The device comprises a mechanical system which applies load increasing linearly at the rate of 100 N/min, directed perpendicularly to the long bone axis and acting half-length of the femur (tests of mechani- cal properties of the whole femur) or directed parallel to the long bone axis and acting on the head of the fe- mur (a mechanical property test of the femoral neck).
The measurement of the load acting on the bone was performed by means of a PWZG strain gauge, whereas the deformation of the examined bone was measured using WZAPK inductive sensor. Amplified signals from the sensors were recorded as a function of load to deformation by means of XP recorder KP-6801A.
Body mass and the mass of internal organs
The effect of MTX on the body mass in rats was de- termined by weighing them using a GT 2100 type bal- ance made by Ohaus (accuracy to 0.01 g). The exam- ined mass of organs (liver, kidneys, testicles and epididymis) was determined immediately after they
were isolated and cleaned, by weighing them using an analytical AS 200 balance made by Ohaus (accuracy to 0.0001 g).
The results were presented as an arithmetic mean
± SEM. The results were statistically evaluated by Student’st-test for unpaired data.
Results
Changes in macrometric parameters after MTX administration
Changes in macrometric parameters are shown in Ta- ble 1. MTX administered at the dose of 1 mg/kgpo (MTX-1po group) caused no statistically significant changes of macrometric parameters, when compared to the results obtained for control rats.
Statistically significant changes in macrometric pa- rameters compared to the controls were observed after intramuscular administration of MTX. In MTX-1im group of rats (Fig. 1), we observed a decrease in fe- moral epiphyses mass (by 5.82%), the mass of tibia (by 5.06%) and L-4 vertebra (by 7.57%), a dimin- ished mineral content in the bones (by 9.77%, 9.67%
and 11.29%, respectively) and in femoral diaphysis (by 8.77%) and a diminished calcium content in the bones (by 6.20%, 5.81% and 6.19%, respectively).
MTX administered at the dose of 1 mg/kgim resulted also in a diminished femoral bone length (by 3.10%)
.
100 94.02* 9494* 92.43* .9023* 91.30* 90.33* 88.71** 93.80* 94.19* 93.81* 96.90** 97.46*
0 20 40 60 80 100
Bone mass
Mineral content
Calcium content
Percentofcontrol Control Tibia L-4vertebra
Femoraldiaphysis Femoraldiaphysis Tibia TibiaFemoralepiphyses L-4vertebra Femoralepiphyses L-4vertebra Femorallenght Tibiallenght
Fig. 1. Statistically significant changes in macrometric parameters in comparison with controls after MTX administration at the dose of 1 mg/kg im. Results are expressed as percentages of controls.
* p < 0.05, ** p < 0.01 – statistically significant differences in compari- son with controls
100 90.16** 88.48** 89.61** 85.58** 85.16** 86.46** 85.33* 84.21* 93.20** 95.16* 92.81** 92.68* 96.07** 95.68**
0 20 40 60 80 100
Bone mass
Mineral content
Calcium content
Percentofcontrol Control Tibiallenght
Tibia FemorallenghtL-4vertebra
Femoraldiaphysis Femoralepiphyses Tibia L-4vertebra Femoralepiphyses Femoraldiaphysis Tibia L-4vertebra Femoralepiphyses Femoraldiaphysis
Fig. 2. Statistically significant changes in macrometric parameters in comparison with controls after MTX administration at the dose of 5 mg/kg im. Results are expressed as percentages of controls.
* p < 0.05, ** p < 0.01 – statistically significant differences in compari- son with controls
and a diminished tibial bone length (by 2.54%). In the MTX-5im group of rats (Fig. 2), we observed a de- crease in the femoral epiphyses mass (by 9.84%), in the mass of femoral diaphysis (by 11.52%), tibia (by 10.39%) and L-4 vertebra (by 14.42%), a diminished mineral content in the bones (by 14.84%, 13.54%, 14.67% and 15.79%, respectively), a diminished cal- cium content in the bones (by 8.80%, 4.84%, 7.19%
and 7.32%, respectively) and a diminished femoral bone length (by 3.93%) as well as a diminished tibial bone length (by 4.32%).
Changes in histomorphometric parameters af- ter MTX administration
Changes in histomorphometric parameters are shown in Table 2. MTX administered at the dose of 1 mg/kg po (MTX-1po group) caused no statistically signifi- cant changes in histomorphometric parameters, in comparison with the results obtained for control rats.
Statistically significant changes in histomorpho- metric parameters compared with controls were ob- served after administering MTX at the dose of
Tab. 1. Changes in macrometric parameters after MTX administration
Groups
Control MTX-1po
MTX (1 mg/kg po)
MTX-1im
MTX (1 mg/kgim) MTX-5im MTX (5 mg/kgim)
Bone mass [mg] Femoral epiphyses 371.27 ± 8.07 369.79 ± 10.50 349.66 ± 8.67* 334.74 ± 7.79**
Femoral diaphysis 487.01 ± 10.95 496.54 ± 11.06 460.18 ± 7.04 430.92 ± 14.09**
Tibia 641.63 ± 10.59 650.50 ± 12.21 609.18 ± 7.68* 574.94 ± 20.89**
L-4 vertebra 295.01 ± 6.26 287.37 ± 4.69 272.68 ± 7.57* 252.48 ± 10.25**
Bone mineral content [mg] Femoral epiphyses 136.63 ± 6.94 126.26 ± 5.16 122.38 ± 4.02* 115.50 ± 3.32***
Femoral diaphysis 224.51 ± 5.91 224.34 ± 4.70 204.92 ± 3.51* 194.12 ± 7.04**
Tibia 262.17 ± 5.91 256.04 ± 6.21 36.82 ± 6.16* 223.70 ± 14.30* L-4 vertebra 90.23 ± 2.15 84.79 ± 1.70 80.04 ± 0.57** 75.98 ± 4.65**
Bone mineral content/bone mass ratio
Femoral epiphyses 0.366 ± 0.006 0.350 ± 0.005 0.350 ± 0.004 0.345 ± 0.004 Femoral diaphysis 0.461 ± 0.003 0.452 ± 0.003 0.445 ± 0.004 0.450 ± 0.006 Tibia 0.409 ± 0.006 0.393 ± 0.005 0.389 ± 0.006 0.388 ± 0.007 L-4 vertebra 0.306 ± 0.003 0.295 ± 0.006 0.294 ± 0.006 0.300 ± 0.008 Calcium content
[mg/g of bone minerals]
Femoral epiphyses 335.42 ± 5.16 327.51 ± 3.16 314.64 ± 4.05* 312.60 ± 3.16**
Femoral diaphysis 355.29 ± 5.70 349.51 ± 5.82 342.27 ± 3.99 338.07 ± 3.38*
Tibia 361.62 ± 5.17 358.48 ± 4.47 340.61 ± 4.90* 335.61 ± 7.52** L-4 vertebra 341.51 ± 4.66 329.06 ± 4.70 320.37 ± 4.28* 316.51 ± 1.82*
Bone length [cm] Femur 3.360 ± 0.015 3.333 ± 0.014 3.256 ± 0.031** 3.228 ± 0.033**
Tibia 3.813 ± 0.026 3.773 ± 0.017 3.716 ± 0.032* 3.648 ± 0.047** Bone diameter [cm] Femur 0.366 ± 0.009 0.368 ± 0.004 0.354 ± 0.003 0.350 ± 0.004
Tibia 0.289 ± 0.003 0.294 ± 0.003 0.281 ± 0.003 0.279 ± 0.005
Results are presented as the means ± SEM (n = 7). * p < 0.05, ** p < 0.01, *** p < 0.001 – statistically significant differences in comparison with controls;p < 0.05,p < 0.01 – statistically significant differences in comparison with results obtained in the MTX-1po group. Control – the control group of rats; MTX-1po group – MTX at the dose of 1 mg/kg po; MTX-1im group – MTX at the dose of 1 mg/kg im; MTX-5im group – MTX at the dose of 5 mg/kgim. Changes in macrometric parameters were examined 4 weeks after the first administration of MTX or 0.9% NaCl solution
1 mg/kg im (MTX-1im group) and at the dose of 5 mg/kg im (MTX-5im group). Statistically signifi- cant changes in histomorphometric parameters mani- festing as the reduced cortical tibia transverse cross- section area (by 5.73%), a decrease in osteoid width on the side of periosteum (by 11.11%), in tibial bone transverse growth on the side of periosteum (by 12.62%), epiphysial cartilage width (by 9.38%) and trabecular width in the femoral epiphysis (by 9.77%) and in the femoral metaphysis (by 6.81%) were ob-
served in MTX-1im group of rats (Fig. 3). The rats of MTX-5im group (Fig. 4) displayed statistically sig- nificant changes of histomorphometric parameters manifesting as reduced cortical tibia transverse cross-section area (by 9.68%), a decrease in osteoid width on the side of periosteum (by 17.02%) and on the side of endosteum (by 9.53%), in tibial bone trans- verse growth on the side of periosteum (by 12.62%) and on the side of endosteum (by 9.21%), a decreased epiphysial cartilage width (by 10.89%) and a de- creased trabecular width in the femoral epiphysis (by 14.80%) and in the femoral metaphysis (by 9.78%).
The rats of MTX-5im group also displayed a statisti- cally significant increase in the ratio of bone marrow transverse cross-section area to cortical tibial trans- verse cross-section area (by 5.10%).
Changes in mechanical properties of the femur after MTX administration
Changes in mechanical properties of the femoral bone are shown in Table 3. MTX administered at the dose of 1 mg/kg po (MTX-1po group) caused no statisti- cally significant changes in mechanical properties of the femoral bone, in comparison with the control rats.
Statistically significant changes in mechanical properties of the femoral bone when compared to the controls were observed after intramuscular admini- stration of MTX. The rats of MTX-1im group (Fig. 5) displayed changes in mechanical properties of the femoral bone manifesting as decreases in extrinsic stiffness (by 8.20%), in the ultimate load of the femo-
100 10091.80* 90.90* 88.95* 86.66** 90.62* 84.96** 84.17** 79.25*** 88.92*
0 20 40 60 80 100
Percentofcontrol
MTX-1 mg/kg im MTX-5 mg/kg im Control Extrinsicstiffness Ultimateloadof thefemoraldiaphysis Loadatfracture ofthefemoraldiaphysis Loadatfracture ofthefemoralneck Control Extrinsicstiffness Ultimateloadof thefemoraldiaphysis Loadatfracture ofthefemoraldiaphysis Loadatfracture ofthefemoralneck Deformation attheultimateload
Fig. 5. Statistically significant changes in mechanical properties of the femur in comparison with controls after MTX administration at the doses of 1 mg/kgim and 5 mg/kg im. Results are expressed as per- centages of controls. * p < 0.05, ** p < 0.01, *** p < 0.001 – statisti- cally significant differences in comparison with controls
100 90.32* 94.90* 83.02** 90.79* 82.98*** 90.47** 85.20*** 90.24*** 89.11*
0 20 40 60 80 100
Control Transversecross-sectionarea ofthetibialdiaphysis Transversecross-sectionareaofthe tibialmarrowcavity/tibialdiaphysisratio Transversegrowth onthesideofperiosteum Transversegrowth onthesideofendosteum Withofosteoid onthesideofperiosteum Withofosteoid onthesideofendosteum Withoftrabeculae infemoralepiphysis Withoftrabeculae infemoralmetaphysis Withofepiphysial cartilage
Percentofcontrol
Fig. 4. Statistically significant changes in histomorphometric pa- rameters in comparison with controls after MTX administration at the dose of 5 mg/kgim. Results are expressed as percentages of con- trols. * p < 0.05, ** p < 0.01, *** p < 0.001 – statistically significant dif- ferences in comparison with controls
88.89*
100 94.27* 87.38* 90.23** 93.19** 90.62*
0 20 40 60 80 100
Percentofcontrol Control Transversecross-sectionarea ofthetibialdiaphysis Transversegrowth onthesideofperiosteum Widthofosteoid onthesideofperiosteum Widthoftrabeculae infemoralepiphysis Widthoftrabeculae infemoralmetaphysis Widthofepiphysialcartilage
Fig. 3. Statistically significant changes in histomorphometric pa- rameters in comparison with controls after MTX administration at the dose of 1 mg/kgim. Results are expressed as percentages of con- trols. * p < 0.05, ** p < 0.01 – statistically significant differences in comparison with controls
Tab. 3. Changes in mechanical properties of the femur after MTX administration
Groups
Control MTX-1po
MTX (1 mg/kgpo) MTX-1im
MTX (1 mg/kgim) MTX-5im MTX (5 mg/kgim) Extrinsic stiffness [N/mm] 286.95 ± 7.25 289.11 ± 9.62 263.41 ± 4.22* 260.05 ± 4.71*
Deformation [mm] At ultimate load 0.334 ± 0.010 0.314 ± 0.009 0.302 ± 0.009 0.297 ± 0.002*
At breaking load 0.408 ± 0.005 0.382 ± 0.013 0.386 ± 0.008 0.368 ± 0.023
Load [N] Ultimate 88.42 ± 2.63 84.31 ± 0.99 80.38 ± 1.01* 75.12 ± 1.67**
Breaking 80.90 ± 1.47 77.56 ± 1.82 71.96 ± 1.90* 68.26 ± 2.22**
Load at fracture of the femoral neck [N] 83.43 ± 1.74 78.89 ± 0.92 72.30 ± 1.72** 66.12 ± 2.23***
Results are presented as the means ± SEM (n = 7). * p < 0.05, ** p < 0.01, *** p < 0.001 – statistically significant differences in comparison with controls; # p < 0.05, ## p < 0.01 – statistically significant differences in comparison with results obtained in the MTX-1po group. Control – con- trol group of rats; MTX-1po group – MTX at the dose of 1 mg/kg po; MTX-1im group – MTX at the dose of 1 mg/kg im; MTX-5im group – MTX at the dose of 5 mg/kgim. Changes in mechanical properties of the femur were examined 4 weeks after the first administration of MTX or 0.9%
NaCl solution
Tab. 2. Changes in histomorphometric parameters after MTX administration
Groups
Control MTX-1po
MTX (1 mg/kgpo) MTX-1im
MTX (1 mg/kgim) MTX-5im MTX (5 mg/kgim) Transverse cross-section area of the tibial diaphysis
[mm]
3.897 ± 0.068 3.956 ± 0.110 3.674 ± 0.056* 3.520 ± 0.133*
Transverse cross-section area of the tibial marrow cavity [mm]
1.221 ± 0.037 1.204 ± 0.027 1.167 ± 0.025 1.158 ± 0.033
Transverse cross-section area of the tibial marrow cavity/tibial diaphysis ratio
0.314 ± 0.006 0.305 ± 0.005 0.318 ± 0.007 0.330 ± 0.005*
Transverse growth of cortical bone in tibia [µm]
Periosteal 65.36 ± 2.57 59.35 ± 0.88 57.12 ± 1.98* 54.40 ± 0.97**
Endosteal 24.12 ± 0.47 23.34 ± 0.36 22.74 ± 0.99 21.90 ± 0.62*
Width of tibial osteoid [µm] Periosteal 20.37 ± 0.55 19.06 ± 0.41 18.11 ± 0.72* 16.90 ± 0.33***
Endosteal 14.34 ± 0.24 14.42 ± 0.40 13.43 ± 0.42 12.97 ± 0.26**
Width of epiphysial cartilage of femoral bone [µm] 95.18 ± 3.07 89.93 ± 1.32 86.24 ± 2.01* 84.81 ± 0.85* Width of trabeculae in the femur
[µm]
In epiphysis 89.89 ± 1.69 84.45 ± 2.32 81.11 ± 1.29** 76.59 ± 1.70*** In metaphysis 54.42 ± 0.49 51.90 ± 0.78 50.72 ± 1.05** 49.11 ± 1.15***
Results are presented as the means ± SEM (n = 7). * p < 0.05, ** p < 0.01, *** p < 0.001 – statistically significant differences in comparison with controls;p < 0.05,p < 0.01 – statistically significant differences in comparison with the results obtained in the MTX-1po group. Control – the control group of rats; MTX-1po group – MTX at the dose of 1 mg/kg po; MTX-1im group – MTX at the dose of 1 mg/kg im; MTX-5im group – MTX at the dose of 5 mg/kgim. Changes in histomorphometric parameters were examined 4 weeks after the first administration of MTX or 0.9% NaCl solution
ral diaphysis (by 9.10%), in the load at the femoral bone fracture (by 11.08%) and the load which caused femoral neck fracture (by 13.34%). The rats of MTX-5im group (Fig. 5) displayed statistically sig- nificant changes in mechanical properties of the femoral bone manifesting as decreases in extrinsic stiffness (by 9.38%), in the femoral diaphysis defor- mation in the point of the ultimate load endured by the femoral diaphysis (by 11.08%), in the ultimate load of the femoral diaphysis (by 15.04%), in the load at the femoral bone fracture (by 15.63%) and the load which caused femoral neck fracture (by 20.75%).
Changes in body mass and the mass of internal organs
Changes in body mass and the mass of internal organs after MTX administration are shown in Table 4. MTX administered at the dose of 1 mg/kg po (MTX-1po group) caused no statistically significant changes in the body mass growth and the growth of internal or- gan mass when compared to the controls (C group).
However, when administered at the dose of 1 mg/kg im (MTX-1im group), it caused a statistically signifi- cant reduction in body mass growth in rats compared to the controls: after 7 days by 181.83% and after 14 days by 58.76%, and administered at the dose of 5 mg/kg
im: (MTX-5im group) by 42.56% after 7 days, 69.81%
after 14 days, 58.99% after 21 days and by 65.41% af- ter 28 days.
In comparison with the controls, administration of MTX also resulted in a statistically significant reduc- tion in epididymis mass by 8.24% after administering it at the dose of 1 mg/kgim (MTX-1im group), and testicle mass by 9.30% as well as the mass of epidi- dymis by 12.75% after administering the dose of 5 mg/kgim (MTX-5im group).
Discussion
In these experiments, we assessed changes in the os- seous tissue in rats at 4 weeks following the starting date of MTX administration, due to the fact that to- date in vivo tests have shown that the period of 4 weeks was sufficient for changes in the osseous tis- sue in rats to develop. For example, experimental os- teopenia induced by ovariectomy or glucocorticoste- roid administration was acknowledged after 4 weeks following the bilateral ovariectomy or of prednioslone administration [6, 24]. Also after ethoposide admini- stration, changes in the osseous tissue in rats were
Tab. 4. Changes in body mass and internal organ mass after MTX administration
Groups
Control MTX-1po
MTX (1 mg/kgpo) MTX-1im
MTX (1 mg/kgim) MTX-5im MTX (5 mg/kgim) Initial body mass [g] 233.46 ± 3.29 232.34 ± 1.80 228.05 ± 3.19 233.90 ± 2.79 Body weight gain [g] after 7 days 35.31 ± 4.14 34.49 ± 2.92 –28.89 ± 4.49*** 20.28 ± 3.78*
after 14 days 51.00 ± 4.71 55.58 ± 4.36 21.03 ± 7.55** 15.40 ± 3.18**
after 21 days 67.06 ± 6.37 72.55 ± 3.93 54.16 ± 3.78 27.50 ± 9.23*
after 28 days 79.50 ± 5.65 83.67 ± 5.13 67.26 ± 1.43 27.50 ± 5.07***
Mass of organs [g] Liver 11.05 ± 0.63 11.44 ± 0.43 11.69 ± 0.60 11.02 ± 0.52
Kidneys 1.97 ± 0.07 2.04 ± 0.04 1.93 ± 0.05 1.85 ± 0.06
Testicles 2.93 ± 0.07 3.03 ± 0.06 2.93 ± 0.08 2.66 ± 0.11*
Epididymis 0.95 ± 0.03 0.97 ± 0.04 0.85 ± 0.01* 0.81 ± 0.02**
Results are presented as the means ± SEM (n = 7). * p < 0.05, ** p < 0.01, *** p < 0.001 – statistically significant differences in comparison with the controls;p < 0.05,p < 0.01,p < 0.001 – statistically significant differences in comparison with results obtained in the MTX-1po group.
Control – control group of rats; MTX-1po group – MTX at the dose of 1 mg/kg po; MTX-1im group – MTX at the dose of 1 mg/kg im; MTX-5im group – MTX at the dose of 5 mg/kgim. Changes in the mass of internal organs were examined 4 weeks after the first administration of MTX or 0.9% NaCl solution
found after this period [5], and since the process of bone remodeling in rats is faster, the results of such tests may be compared to drug administration in hu- mans for the period of even a few months [30].
Following the experiment, MTX was found not to have any negative effect on the osseous tissue when administered orally at 1 mg/kg, however, when ad- ministered intramuscularly, at 1 mg/kg or 5 mg/kg, it disturbed the process of bone remodeling in rats.
Bone remodeling is a normal process that allows the skeleton to adapt to local biomechanical changes and to repair microdamaged regions. The process re- quires coordination between osteoclasts, which resorb bone, and osteoblasts, which deposit minerals and matrix in previously resorbed areas. Bone multicellu- lar units (BMUs) are responsible for carrying out the bone remodeling process. A BMU exists as a result of an antagonistic, coupled action of osteoclasts and os- teoblasts that work together to replace old bone with new bone matrix [25].
The results obtained in this work indicate that MTX disturbs the process of bone remodeling in rats by inhibiting the synthesis and mineralization of new osseous matrix. The inhibition of osseous matrix syn- thesis is indicated in the first place by a reduced width of osteoid (non-mineralized bone matrix) on the side of periosteum, and following the administration of MTX at 5 mg/kgim, also on the side of endosteum, as well as a diminished width of bone trabeculae in the femoral epiphysis and metaphysis. The decreased synthesis of osseous matrix was also acknowledged by the results of determining the tibial transverse growth. A decrease in tibial transverse growth was found on the side of periosteum, and after MTX ad- ministration at 5 mg/kg im, also on the side of en- dosteum. The transverse cross section area of the cor- tical tibia was found to be decreased as well.
A decreased osseous matrix synthesis may result from the inhibitive effect of MTX on osteoblast dif- ferentiation. Osteoblasts responsible for bone forma- tion are derived from pluripotent mesenchymal stem cells in bone marrow, which are differentiated into os- teogenic cell precursors [2]. The inhibitive effect of MTX on the osteoblastic cells was acknowledged in in vitro tests conducted by Uehary et al., who demon- strated that MTX suppresses bone formation by inhib- iting the differentiation of early osteoblastic cells [33].
One cannot, however, exclude the possibility that MTX also inhibits the function of mature osteoblasts, especially taking into account that Davies et al.
showed inin vitro tests that MTX reduced the number of osteoblasts when administered at concentrations
£ 10–7 M [8]. Osteoblasts synthesize bone matrices and mineralize them, and our tests demonstrated that MTX inhibited osseous matrix mineralization. We found that the mineral and calcium contents in the femoral epiphyses, L-4 vertebra and the tibia were re- duced, and after administration of MTX at 5 mg/kgim the same occurred also in the femoral diaphysis. Such results indicate that MTX inhibits the function of ma- ture osteoblasts, thus impairing osseous tissue miner- alization. Inhibition of matrix mineralization by MTX was observed by May et al. using terminally differen- tiated osteoblasts [18].
A decreased synthesis and mineralization of the os- seous tissue following MTX administration were also acknowledged by the results of macrometric determi- nations. We observed a decrease in the tibial mass, in the masses of femoral and L-4 vertebra diaphyses, and after MTX administration at 5 mg/kg im also a decrease in the femoral diaphysis mass. MTX ad- ministered intramuscularly also caused a reduction in the length of the femoral and tibial bones.
It should, however, be emphasized that a reduced mineral content and a reduced width of trabeculae in the femoral epiphysis and metaphysis may also be a result of intensified bone resorption caused by MTX. Especially considering that May et al. found that prolonged administration of low dose MTX in fe- male rats caused significant osteopenia via suppres- sion of osteoblast activity and stimulation of osteo- clast recruitment, which resulted in increased bone re- sorption [19].
We did not observe an intensified process of re- sorption, either in the cortical bone, or in the cancel- lous bone after intramuscular MTX administration. In the cortical bone, the transverse cross-section area of the marrow cavity was not found to be increased, de- spite a decreased transverse cross-section area of the cortical tibia. No changes were found in the ratio of mineral content to the bone mass in the femoral dia- physis despite a decreased mineral content. In the cancellous bone, the ratio of mineral content to bone mass in the femoral epiphyses and in the L-4 vertebra was not found to be altered, despite a decrease in min- eral and calcium content in those bones and a de- creased width of trabeculae in the femoral epiphysis and metaphysis.
The fact that no changes occurred in the ratio of mineral content to the bone mass indicates that the
bone loss is proportional to the decrease in mineral content. Our results seemed to indicate that a decrease in mineral content after intramuscular administration of MTX was caused by the reduced mineralization following the inhibition of osseous matrix synthesis, and not by an intensified resorption process.
The above conclusion is also proved by the deter- minations of the ratio of marrow cavity transverse cross-section area to cortical tibia transverse cross- section area. After MTX administration at 5 mg/kg im, a statistically significant increase in this ratio was demonstrated, where the marrow cavity area did not display changes. Hence, one can conclude that the in- crease in the ratio is caused by a decrease in the area of the cortical tibia, not being the result of an intensi- fied resorption process (no increase in marrow cavity transverse cross-section area), but following a de- crease in bone matrix synthesis.
However, one cannot exclude the possibility that a long-term administration of MTX may affect the process of bone resorption, as demonstrated by May, et al., who administered MTX at 3 mg/kgip over the period of 16 weeks [19]. The discrepancy between our results and those obtained by May et al. may re- sult from the difference in the periods of MTX ad- ministration. This, however, calls for further in vivo research andin vitro tests with regard to MTX effects on osteoclasts.
The inhibition of bone matrix synthesis and miner- alization following intramuscular MTX administra- tion was also confirmed by determinations of me- chanical properties of the femur. It was demonstrated that MTX reduced mechanical endurance of both the cancellous and the cortical bone. The following was established: the load causing the femoral neck frac- ture, the ultimate load and the breaking load of the femoral diaphysis as well as extrinsic stiffness were found to be reduced, and following MTX administra- tion at 5 mg/kg, also the femoral diaphysis deforma- tion was found to be decreased at the point of ultimate load endured by the femoral diaphysis.
Our tests also demonstrated that MTX adminis- tered orally at 1 mg/kg did not have a negative effect on the osseous tissue, whereas its intramuscular ad- ministration at the same dose disturbed the osseous tissue synthesis and mineralization. Lack of osseous tissue changes in rats after oral administration of MTX may be a result of differences in its bioavail- ability. Bioavailability of methotrexatae preparations
in humans after intramuscular administration is 76%
and it is significantly higher than bioavailability of oral preparations, in particular after MTX administra- tion at doses exceeding 40 mg/m2[14, 31].
Conclusion
MTX administered intramuscularly disrupts bone re- modeling in rats by inhibiting the process of bone tis- sue synthesis and mineralization. Such disorder leads to the decreased mechanical properties of the femur.
No negative effects on bone remodeling in rats were found after oral administration of MTX.
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Received:
September 8, 2004; in revised form: March 7, 2005.
