EFFECTS OF NADROPARIN ON BONE
HISTOMORPHOMETRIC PARAMETERS IN RATS
Joanna Folwarczna
, Waldemar Janiec, Maria Barej, Urszula Cegie³a, Maria Pytlik, Ilona Kaczmarczyk-Sedlak
Department of Pharmacology, Medical University of Silesia, Jagielloñska 4, PL 41-200 Sosnowiec
Effects of nadroparin on bone histomorphometric parameters in rats.
J. FOLWARCZNA, W. JANIEC, M. BAREJ, U. CEGIE£A, M. PYTLIK, I. KACZMARCZYK-SEDLAK. Pol. J. Pharmacol., 2004, 56, 337–343.
Nadroparin calcium is a low-molecular-weight heparin. Low- molecular-weight heparins have a number of advantages over standard hepa- rin (heparin), but it is not clear if low-molecular-weight heparins have less effect on bones than heparin. Administration of heparin can lead to osteopo- rosis. The aim of the present study was to investigate the effects of nadroparin on the rat osseous system and compare them with those of hepa- rin. The experiments were carried out on female Wistar rats (13–15 weeks old at the beginning of the experiment), divided into 5 groups: I. Control, II.
Nadroparin (1000 anti-Xa IU/kg sc daily), III. Nadroparin (2000 anti-Xa IU/kg sc daily), IV. Heparin (1000 IU/kg sc daily), V. Heparin (2000 IU/kg sc daily). Nadroparin and heparin were administered for 4 weeks. Bone mass, mineral and calcium content, macrometric and histomorphometric pa- rameters (endosteal and periosteal transverse growth, width of endosteal and periosteal osteoid, transverse cross-section area of the cortical bone in the diaphysis and of the marrow cavity in the tibia, width of epiphyseal carti- lage, width of trabeculae in the epiphysis and metaphysis in the femur) were examined. The effect of heparin on the ratio of bone mineral content to bone mass was more pronounced than that of nadroparin. Nadroparin caused unfa- vorable changes in the investigated bone histomorphometric parameters, similar to those caused by heparin. Nadroparin and heparin caused disorder of bone formation and intensification of bone resorption in rats.
Key words: nadroparin, heparin, osteopenia, rat
INTRODUCTION
Nadroparin calcium is a low-molecular-weight heparin.
Long-term administration of standard heparin (heparin) can be associated with development of os- teoporosis. Heparin-induced osteoporosis is a rare but potentially serious complication of standard heparin and low-molecular-weight heparin therapy [36]. The absolute risk of symptomatic fracture with long-term administration of heparin has been estimated as less than 5% [8]. The real risk of de- velopment of heparin-induced osteoporosis re- mains unclear. Long-term administration of heparin is not prescribed frequently [13]. Currently, heparin is administered long-term mainly to prevent and treat venous thromboembolism during pregnancy, to prevent systemic embolism in pregnant women with mechanical heart valves, and to prevent preg- nancy loss in women with antiphospholipid anti- bodies, in some cases also in nonpregnant patients [15].
Low-molecular-weight heparins have a number of advantages over standard heparin. Low- molecular-weight heparins have a longer plasma half-life, better subcutaneous (sc) bioavailability, more predictable anticoagulant response, and re- quire less intense laboratory monitoring [19]. It is not clear if they have less effect on bones than stan- dard heparin.
Only few studies were conducted in order to es- tablish the risk of development of skeletal changes associated with administration of low-molecular- weight heparins in humans [1, 6, 21, 26, 32]. There are methodological and ethical difficulties associ- ated with conducting randomized, prospective, controlled studies in pregnant women [17], espe- cially concerning the effects on bones, requiring performing densitometric studies in spine. Due to the limited number of patients receiving low- molecular-weight heparins for long periods, in which the effect on the skeletal system was evalu- ated, the data concerning the effect of low- molecular-weight heparins on bones come mainly from the animal studies. Although there are reports indicating less influence of some low-molecular- weight heparins on animal bones than that of stan- dard heparin [23, 24, 27], there are also data indi- cating similar effects of low-molecular-weight heparins and standard heparin on the skeletal sys- tem [20].
Low-molecular-weight heparins differ in terms of their molecular weights, pharmacokinetics and potencies. They are not interchangeable on a unit- for-unit basis [17]. According to the US Food and Drug Administration, the low-molecular-weight heparins should be considered as individual drugs [10].
There are no reports on the effect of nadroparin on the skeletal system in humans and rats. The aim of the present study was to examine the effects of nadroparin on the histomorphometric parameters of bones in rats and compare them with those of stan- dard heparin.
MATERIALS and METHODS The experiments were carried out on female Wistar rats (13–15 weeks old at the beginning of the experiment), fed a standard diet ad libitum. The animals were divided into 5 groups (n = 8): I. Con- trol group, II. Nadroparin 1000 anti-Xa IU/kg sc daily, III. Nadroparin 2000 anti-Xa IU/kg sc daily, IV.
Heparin 1000 IU/kg sc daily, V. Heparin 2000 IU/kg sc daily.
Heparin sodium (Heparin “Biochemie”, Bio- chemie, Austria) or nadroparin calcium (Fraxipar- ine, Polfa, Poland) were administered sc once daily for 4 weeks. The control group received 0.9% sa- line in the same volume of 1 ml/kg sc daily. The animals were weighed every second day.
One day before the start and on the last day of administration of heparins or saline, the animals were given tetracycline hydrochloride (20 mg/kg ip) in order to mark the calcification front. The next day, animals were sacrificed and the right and left tibial and femoral bones and L-4 vertebra were ex- cised. In the isolated left bones, mass and mac- rometric parameters were determined (length, di- ameter of the diaphysis in the mid-length).
In order to determine the content of mineral substances in bones, the L-4 vertebra, left tibia and femur were mineralized at the temperature of 620°C for 48 h and weighed. In the mineralized bones, calcium content was determined by a color- imetric method.
The right femoral and tibial bones were used to prepare histological specimens, as described previ- ously [11, 29]. Briefly, transverse cross-sections were made from the tibial bone and a longitudinal section of the distal epiphysis was made from the femoral bone. The sections were ground on the tar-
nished glass. The first preparation from the tibia re- mained unstained. The rest of the preparations were stained using the Tripp and MacKay method (with- out decalcification) [34].
The histomorphometric measurements were made using a microscope Optiphot-2 (Nikon), con- nected through RGB camera (Cohu) to personal computer (program Lucia G 4.51, Laboratory Im- aging), with final magnifications of 200 and 500 times.
In the unstained preparation, the distance be- tween the tetracycline stripes was measured, on the periosteum side and on the marrow cavity side (pe- riosteal and endosteal transverse growth). In the stained preparation of the transverse cross-section of the tibia, the width of the endosteal and peri- osteal osteoid was determined. In the longitudinal preparation from the femur, the width of epiphyseal cartilage and the width of trabeculae in the epiphy- sis and metaphysis were measured.
The area of the transverse cross-section of the cortical bone in the tibial diaphysis and the area of the transverse cross-section of the marrow cavity in the tibia were measured in the stained preparation, with the use of a lanameter (magnification 50 times).
Results are presented as the mean ± SEM. Stu- dent’s t-test for unpaired observations was used for
evaluation of statistical significance of differences be- tween the control group and each heparin-treated group and between the groups treated with nadroparin and standard heparin at respective doses.
RESULTS
Although administration of nadroparin (1000 and 2000 anti-Xa IU/kg sc daily) or standard hepa- rin (1000 and 2000 IU/kg sc daily) for 4 weeks did not statistically significantly affect body mass gain in the investigated animals in comparison with the control group (Tab. 1), administration of the inves- tigated heparins affected bone parameters (Tab. 1 and 2).
Administration of nadroparin did not cause sig- nificant changes in the macrometric parameters of bones. Nadroparin slightly decreased the ratio of bone mineral content to bone mass in the investi- gated bones in comparison with the control group (a statistically significant decrease only in L-4 ver- tebra after the lower dose, by 6.96%). A tendency to decrease calcium content in the investigated bones was observed. There was a statistically sig- nificant decrease in the calcium content in the tibia after administration of the higher dose of nadroparin in comparison with the control group (by 5.73%).
Table 1. Effects of nadroparin (1000 and 2000 anti-Xa IU/kg sc daily) and heparin (1000 and 2000 IU/kg sc daily), administered for 4 weeks, on body mass gain and bone parameters in rats
Parameter I Control group II Nadroparin 1000 anti-Xa IU/kg sc daily
III Nadroparin 2000 anti-Xa IU/kg sc daily
IV Heparin 1000 IU/kg sc
daily
V Heparin 2000 IU/kg sc
daily Initial body mass [g] 234.98 ± 3.58 231.06 ± 5.87 229.58 ± 5.27 232.99 ± 5.22 232.21 ± 8.00 Body mass gain after 28 days [g] 25.84 ± 3.33 29.03 ± 3.18 31.60 ± 2.56 19.36 ± 3.56 30.20 ± 1.81 Bone mineral content /
bone mass ratio Tibia 0.449 ± 0.002 0.442 ± 0.003 0.447 ± 0.003 0.440 ± 0.005 0.437 ± 0.005*
Femur 0.442 ± 0.002 0.436 ± 0.004 0.446 ± 0.004hhh 0.439 ± 0.009 0.421 ± 0.003***
L-4 vertebra 0.364 ± 0.007 0.339 ± 0.006* 0.349 ± 0.008 0.352 ± 0.014 0.337 ± 0.010*
Calcium content
[mg/g ofbone mineral] Tibia 390.46 ± 8.39 374.84 ± 5.45 368.08 ± 6.14* 396.19 ± 11.09 378.49 ± 5.25 Femur 386.30 ± 12.59 359.75 ± 11.91 348. 81 ± 14.89 366.51 ± 16.70 382.13 ± 8.43 L-4 vertebra 395.67 ± 15.82 379.53 ± 8.64 404.52 ± 3.89hh 385.78 ± 18.74 373.80 ± 7.07 Bone length [mm] Tibia 35.86 ± 0.22 36.19 ± 0.16hh 35.88 ± 0.28 35.39 ± 0.14 36.30 ± 0.30
Femur 32.25 ± 0.29 32.36 ± 0.17 32.66 ± 0.33 31.93 ± 0.12 32.56 ± 0.33 Bone diameter [mm] Tibia 2.83 ± 0.08 2.71 ± 0.04 2.80 ± 0.05 2.78 ± 0.03 2.84 ± 0.08
Femur 3.40 ± 0.07 3.31 ± 0.04 3.39 ± 0.05 3.37 ± 0.05 3.46 ± 0.06 Results are presented as means ± SEM (n = 8). Students t-test for unpaired observations was used for estimation of statistical signifi- cance. * Significantly different from the control group (I); * p < 0.05, *** p < 0.001.hSignificant difference between the groups treated with nadroparin and standard heparin at the respective dose;hhp < 0.01,hhhp < 0.001
Administration of nadroparin affected histo- morphometric parameters of bones (Tab. 2). In the tibia, in comparison with the control group, nadroparin administered at a dose of 1000 anti-Xa IU/kg sc daily statistically significantly decreased the width of endosteal osteoid (by 8.66%) and en- dosteal transverse growth (by 16.44%). Nadroparin did not statistically significantly affect the peri- osteal transverse growth and the width of periosteal osteoid. In comparison with the control group, after administration of nadroparin at both doses, statisti- cally insignificant increases in the area of the trans- verse cross-section of the marrow cavity and the ra- tio of the area of the transverse cross-section of the marrow cavity to the area of the transverse cross- section of the tibial diaphysis were observed. In the femur, nadroparin caused decreases in the width of trabeculae in comparison with the control group, statistically significant in the epiphysis after both doses (by 13.31%) and in the metaphysis after the lower dose (by 10.84%). Administration of nad- roparin did not statistically significantly affect the width of epiphyseal cartilage.
The changes caused by nadroparin in the inves- tigated parameters of the rat bones were not always dose-dependent, contrary to those of standard hepa- rin.
After administration of heparin, there were no significant changes in the macrometric parameters of bones. Heparin decreased the ratio of bone min- eral content to bone mass in the investigated bones in comparison with the control group. The changes caused by heparin were dose-dependent (statisti- cally significant decreases in all the investigated bones after the higher dose, by 2.59–7.28%) and more pronounced than those caused by nadroparin.
Calcium content in most of the investigated bones was insignificantly decreased.
Heparin decreased the endosteal transverse growth in comparison with the control group, sta- tistically significantly at a dose of 2000 IU/kg sc daily (by 18.44%). Heparin did not statistically sig- nificantly affect the periosteal transverse growth and the width of endosteal and periosteal osteoid in comparison with the control group. Administration of heparin at both doses caused increases in the
Table 2. Effects of nadroparin (1000 and 2000 anti-Xa IU/kg sc daily) and heparin (1000 and 2000 IU/kg sc daily), administered for 4 weeks, on the histomorphometric parameters ofbones in rats
Parameter I Control group II Nadroparin 1000 anti-Xa IU/kg sc daily
III Nadroparin 2000 anti-Xa IU/kg sc daily
IV Heparin 1000 IU/kg sc daily
V Heparin 2000 IU/kg sc daily Width ofosteoid in the
tibia [mm] Periosteal 17.83 ± 0.91 16.89 ± 0.56 16.47 ± 0.37 17.01 ± 0.41 16.03 ± 0.42 Endosteal 12.00 ± 0.36 10.96 ± 0.22*,h 11.36 ± 0.35 11.74 ± 0.26 11.29 ± 0.28 Transverse growth of
the tibia [mm] Periosteal 55.39 ± 3.73 54.11 ± 3.15 55.99 ± 5.59 56.03 ± 5.23 59.44 ± 2.97 Endosteal 39.71 ± 1.60 33.18 ± 1.80* 39.50 ± 3.14 33.82 ± 2.72 32.39 ± 1.20**
Transverse cross-section area ofthe cortical bone in the tibial diaphysis [mm2]
3.09 ± 0.05 3.16 ± 0.14 3.12 ± 0.13 3.01 ± 0.09 3.09 ± 0.16
Transverse cross-section area ofthe
tibial marrow cavity [mm2] 1.01 ± 0.07 1.15 ± 0.08 1.12 ± 0.08 1.06 ± 0.08 1.17 ± 0.06 Transverse cross-section area ofthe
tibial marrow cavity/transverse cross-section area ofthe tibial diaphysis ratio
0.245 ± 0.011 0.273 ± 0.012 0.265 ± 0.014 0.259 ± 0.011 0.275 ± 0.008*
Width oftrabeculae in
the femur [mm] Epiphysis 80.13 ± 1.99 69.47 ± 2.40** 69.47 ± 1.02*** 73.23 ± 2.07* 68.91 ± 1.39***
Metaphysis 50.54 ± 1.32 45.06 ± 1.59* 47.60 ± 1.74 46.49 ± 1.64 44.45 ± 1.77*
Width ofepiphyseal cartilage in the
femur [mm] 60.03 ± 3.78 58.64 ± 9.39 57.16 ± 5.99 58.30 ± 5.86 66.21 ± 6.32
Results are presented as means ± SEM (n = 8). Students t-test for unpaired observations was used for estimation of statistical signifi- cance. * Significantly different from the control group (I); * p < 0.05, ** p < 0.01, *** p < 0.001.hSignificant difference between the groups treated with nadroparin and standard heparin at the respective dose;hp < 0.05
area of the transverse cross-section of the marrow cavity and the ratio of the area of the transverse cross-section of the marrow cavity to the area of the transverse cross-section of the tibial diaphysis.
The changes were not statistically significant in re- lation to the control group, with an exception of the ratio after administration of heparin at the higher dose (an increase by 12.43%). In the femur, heparin caused statistically significant decreases in the width of trabeculae in the epiphysis after the lower and higher doses (by 8.62% and 14.01%, respec- tively) and in the metaphysis after the higher dose (by 12.04%) in comparison with the control group.
Heparin did not statistically significantly affect the width of epiphyseal cartilage.
In most parameters, there were no statistically significant differences between the results in the groups treated with nadroparin and standard hepa- rin at respective doses.
DISCUSSION
Administration of nadroparin (1000 and 2000 anti-Xa IU/kg sc daily) and standard heparin (1000 and 2000 IU/kg sc daily) for 4 weeks caused fea- tures of osteopenia in the female rats. Although the exact mechanism of the damaging effect of heparin on the skeletal system remains unclear, numerous experimental data indicate that standard heparin disturbs bone formation and accelerates bone re- sorption [22, 25, 27].
Also in the present study, administration of standard heparin caused changes in bone histomor- phometric parameters, indicating the influence on processes of bone formation and bone resorption.
The effect of nadroparin on the histomorphometric parameters was similar to that of standard heparin.
In the cortical bone, both heparins caused disorder of bone formation (decreases in the endosteal trans- verse growth) and intensification of bone resorp- tion (increases in the area of the transverse cross- section of the marrow cavity and the ratio of the area of the transverse cross-section of the marrow cavity to the area of the transverse cross-section of the tibial diaphysis). The changes observed in the cancellous bone (decreases in the width of trabecu- lae) could be the effect of inhibition of bone forma- tion and/or intensification of bone resorption.
The histomorphometric changes were con- firmed by the decreases in the ratios of the bone mineral content to bone mass. There were reports
on the damaging effect of heparin mainly on the cancellous bone. Heparin was reported to signifi- cantly decrease bone mineral density in the cancel- lous, but not cortical bone, both in humans [7] and rats [25]. Here we report damaging effect of hepa- rin on the cancellous bone (L-4 vertebra) and corti- cal with cancellous bone (the tibia and femur).
Nadroparin, in the present study, markedly affected the ratio of the bone mineral content to bone mass only in the cancellous bone.
Results of the present study are at variance with those of Murray et al. [24], who did not demon- strate significant effects of nadroparin on the skele- tal system in rabbits.
The effects of standard heparin observed in the present study were dose-dependent, which is con- sistent with other reports [22, 25, 27]. On the other hand, the lack of dose-response relationship after administration of nadroparin is consistent with the results of clinical trials, in which, after administra- tion of heparin, no dose-response relationship could be demonstrated [2].
As it was mentioned before, low-molecular- weight heparins were reported to cause less damag- ing effect than standard heparin on the skeletal sys- tem in experimental animals and in vitro models.
According to Muir et al., unfractioned heparin decreased cancellous bone volume both by de- creasing the rate of bone formation and increasing the rate of bone resorption, whereas a low- molecular-weight heparin (tinzaparin) decreased only the rate of bone formation, causing less os- teopenia in rats [23]. On the other hand, Nishiyama et al., who compared the effect of dalteparin with that of heparin, concluded that the greatest differ- ence was in the lesser effect of dalteparin on osteo- blasts [27].
Unfractioned heparin was found in vitro to stimulate bone resorption from fetal rat calvaria at concentrations commonly used for prophylaxis and treatment of thromboembolism, reaching maxi- mum at 0.35 U/ml. In contrast, more than 50-fold higher concentrations of low-molecular-weight heparins (enoxaparin, dalteparin, tinzaparin and ar- deparin) than used clinically were required for an equivalent effect [15, 31]. Also a low-molecular- weight heparin (enoxaparin) was found to cause less effect than standard heparin on the activity of osteoblasts in a model of bone nodule formation in vitro [3]. However, low-molecular-weight heparins
(among others nadroparin) caused a significant in- hibition of osteoblast growth [16].
In the present study, a low-molecular-weight heparin, nadroparin, caused similar damaging ef- fect (only slightly weaker) on the rat skeletal sys- tem to that caused by heparin. The doses used in the present study, relatively high, were used in ex- perimental models of heparin-induced osteopenia [20, 25, 27]
Results of the present study are consistent with the results of Mätzsch et al. [20], who reported similar effect of standard heparin and a low- molecular-weight heparin (tinzaparin) on bones.
It is possible that different low-molecular-weight heparins may cause different effect on bones, not necessarily connected with their anticoagulant ac- tivity.
Based on their animal studies, Hirsh et al. [15]
hypothesize that heparin binds to bone matrix and interacts with a variety of cell types found in the bone environment, including cells of the osteoblast lineage. Such interactions may alter mesenchymal stem cell differentiation, reducing the number of mature osteoblasts, reduce collagen synthesis by osteoblasts, and release growth factors and/or cyto- kines capable of inducing the formation of osteo- clasts from pluripotent mononuclear precursors in the bone marrow [15].
The factors known to bind heparin are trans- forming growth factors TGF-b1, TGF-b2, TGF-b3, taking part in regulation of proliferation and differ- entiation of bone cells [28], bone morphogenetic protein 2 and osteogenic protein 2 (also belonging to the superfamily of transforming growth factors b) [18], acid and basic fibroblast growth factor [33], growth-promoting factors occurring in the cartilage matrix – chondromodulin I and II [14], insulin-like growth factors I and II [12], pleiotro- phin [9], osteoclastogenesis inhibitory factor [35].
Heparin is known to protect some growth factors characterized by their high affinity for heparin (heparin-binding growth factors) from proteolytic degradations and to potentiate their in vitro biologi- cal effects [4]. Heparin releases insulin-like growth factors from their binding proteins [5].
The differences between the effect of standard heparin and low-molecular-weight heparins, as well as the differences between the effects of different low-molecular-weight heparins, could be the result of their different capability to bind different pro-
teins, which can in different ways affect the pro- cesses of bone metabolism.
Results of the present study indicate that a low- molecular weight heparin, nadroparin, can exert unfavorable effects on the skeletal system, similar to those of standard heparin. As the changes caused by heparin in the skeletal system are not rapidly re- versible [30], one should be conscious that also low-molecular-weight heparins can unfavorably af- fect bones.
REFERENCES
1. Backos M, Rai R, Thomas E, Murphy M, Doré C, Re- gan L: Bone density changes in pregnant women treated with heparin: a prospective, longitudinal study. Hum Re- prod, 1999, 14, 2876–2880.
2. Barbour LA, Kick SD, Steiner JF, LoVerde ME, Hed- dleston LN, Lear JL, Barón AE, Barton PL: A pro- spective study of heparin-induced osteoporosis in preg- nancy using bone densitometry. Am J Obstet Gynecol, 1994, 170, 862–869.
3. Bhandari M, Hirsh J, Weitz JI, Young E, Venner TJ, Shaughnessy SG: The effects of standard and low mo- lecular weight heparin on bone nodule formation in vi- tro. Thromb Haemost, 1998, 80, 413–417.
4. Blanquaert F, Barritault D, Caruelle J-P: Effects of heparan-like polymers associated with growth factors on osteoblast proliferation and phenotype expression.
J Biomed Mater Res, 1999, 44, 63–72.
5. Campbell PG, Novak JF, Yanosick TB, McMaster JH:
Involvement of the plasmin system in dissociation of the insulin-like growth factor-binding protein com- plex. Endocrinology, 1992, 130, 1401–1412.
6. Casele HL, Laifer SA: Prospective evaluation of bone density in pregnant women receiving the low molecu- lar weight heparin enoxaparin sodium. J Matern-Fetal Med, 2000, 9, 122–125.
7. Dahlman TC, Sjöberg HE, Ringertz H: Bone mineral density during long-term prophylaxis with heparin in pregnancy. Am J Obstet Gynecol, 1994, 170, 1315–1320.
8. Douketis JD, Ginsberg JS, Burrows RF, Duku EK, Webber CE, Brill-Edwards P: The effects of long-term heparin therapy during pregnancy on bone density.
A prospective matched cohort study. Thromb Hae- most, 1996, 75, 254–257.
9. Dreyfus J, Brunet de Carvalho N, Duprez D, Raulais D, Vigny M: HB-GAM/pleiotrophin: localization of mRNA and protein in the chicken developing leg. Int J Dev Biol, 1998, 42, 189–198.
10. Eikelboom JW, Hankey GJ: Low molecular weight heparins and heparinoids. Med J Australia, 2002, 177, 379–383.
11. Folwarczna J, Pytlik M, Janiec W: Effects of doxycy- cline on development of changes in histomorphomet- ric parameters of bones induced by bilateral ovariec- tomy in rats. Pol J Pharmacol, 2003, 55, 433–441.
12. Fuller K, Chambers TJ, Gallagher AC: Heparin aug- ments osteoclast resorption-stimulating activity in se- rum. J Cell Physiol, 1991, 147, 208–214.
13. Ginsberg JS, Kowalchuk G, Hirsh J, Brill-Edwards P, Burrows R, Coates G, Webber C: Heparin effect on bone density. Thromb Haemost, 1990, 64, 286–289.
14. Hiraki Y, Inoue H, Kondo J, Kamizono A, Yoshitake Y, Shukunami C, Suzuki F: A novel growth-promoting factor derived from fetal bovine cartilage, chondro- modulin II. Purification and amino acid sequence.
J Biol Chem, 1996, 271, 22657–22662.
15. Hirsh J, Warkentin TE, Shaughnessy SG, Anand SS, Halperin JL, Raschke R, Granger C et al.: Heparin and low-molecular-weight heparin: mechanisms of action, pharmacokinetics, dosing, monitoring, efficacy, and safety. Chest, 2001, 119, Suppl. 1, 64S–94S.
16. Kock HJ, Handschin AE: Osteoblast growth inhibition by unfractionated heparin and by low molecular weight heparins: an in-vitro investigation. Clin Appl Thromb Hemost, 2002, 8, 251–255.
17. Laurent P, Dussarat GV, Bonal J, Jego C, Talard P, Bouchiat C, Cellarier G: Low molecular weight hepar- ins: a guide to their optimum use in pregnancy. Drugs, 2002, 62, 463–477.
18. Macias D, Ganan Y, Sampath TK, Piedra ME, Ros MA, Hurle JM: Role of BMP-2 and OP-1 (BMP-7) in programmed cell death and skeletogenesis during chick limb development. Development, 1997, 124, 1109–1117.
19. Martineau P, Tawil N: Low-molecular-weight hepar- ins in the treatment of deep-vein thrombosis. Ann Pharmacother, 1998, 32, 588–598, 601.
20. Mätzsch T, Bergqvist D, Hedner U, Nilsson B, Øster- gaard P: Effects of low molecular weight heparin and unfragmented heparin on induction of osteoporosis in rats. Thromb Haemost, 1990, 63, 505–509.
21. Monreal M, Lafoz E, Olive A, del Rio L, Vedia C:
Comparison of subcutaneous unfractionated heparin with a low molecular weight heparin (Fragmin) in pa- tients with venous thromboembolism and contraindica- tions to coumarin. Thromb Haemost, 1994, 71, 7–11.
22. Muir JM, Andrew M, Hirsh J, Weitz JI, Young E, De- schamps P, Shaughnessy SG: Histomorphometric analysis of the effects of standard heparin on trabecu- lar bone in vivo. Blood, 1996, 88, 1314–1320.
23. Muir JM, Hirsh J, Weitz JI, Andrew M, Young E, Shaughnessy SG: A histomorphometric comparison of the effects of heparin and low-molecular-weight hepa- rin on cancellous bone in rats. Blood, 1997, 89, 3236–3242.
24. Murray WJ, Lindo VS, Kakkar VV, Melissari E:
Long-term administration of heparin and heparin frac-
tions and osteoporosis in experimental animals. Blood Coagul Fibrinolysis, 1995, 6, 113–118.
25. Mutoh S, Takeshita N, Yoshino T, Yamaguchi I: Char- acterization of heparin-induced osteopenia in rats. En- docrinology, 1993, 133, 2743–2748.
26. Nelson-Piercy C, Letsky EA, de Swiet M: Low- molecular-weight heparin for obstetric thrombopro- phylaxis: experience of sixty-nine pregnancies in sixty-one women at high risk. Am J Obstet Gynecol, 1997, 176, 1062–1068.
27. Nishiyama M, Itoh F, Ujiie A: Low-molecular-weight heparin (dalteparin) demonstrated a weaker effect on rat bone metabolism compared with heparin. Jpn J Pharmacol, 1997, 74, 59–68.
28. Opperman LA, Nolen AA, Ogle RC: TGF-beta 1, TGF-beta 2, and TGF-beta 3 exhibit distinct patterns of expression during cranial suture formation and obliteration in vivo and in vitro. J Bone Miner Res, 1997, 12, 301–310.
29. Pytlik M, Janiec W, Misiarz-Myrta M, Guba³a I: Ef- fects of simvastatin on the development of osteopenia caused by ovariectomy in rats. Pol J Pharmacol, 2003, 55, 63–71.
30. Shaughnessy SG, Hirsh J, Bhandari M, Muir JM, Young E, Weitz JI: A histomorphometric evaluation of heparin-induced bone loss after discontinuation of heparin treatment in rats. Blood, 1999, 93, 1231–1236.
31. Shaughnessy SG, Young E, Deschamps P, Hirsh J:
The effects of low molecular weight and standard heparin on calcium loss from fetal rat calvaria. Blood, 1995, 86, 1368–1373.
32. Shefras J, Farquharson RG: Bone density studies in pregnant women receiving heparin. Eur J Obstet Gyn Reprod Biol, 1996, 65, 171–174.
33. Simmons HA, Raisz LG: Effects of acid and basic fi- broblast growth factor and heparin on resorption of cultured fetal rat long bones. J Bone Miner Res, 1991, 6, 1301–1305.
34. Tripp EJ, MacKay EH: Silver staining of bone prior to decalcification for quantitative determination of os- teoid in sections. Stain Technol, 1972, 47, 129–131.
35. Tsuda E, Goto M, Mochizuki S, Yano K, Kobayashi F, Morinaga T, Higashio K: Isolation of a novel cytokine from human fibroblasts that specifically inhibits os- teoclastogenesis. Biochem Biophys Res Commun, 1997, 234, 137–142.
36. Walenga JM, Bick RL: Heparin-induced thrombocy- topenia, paradoxical thromboembolism, and other side effects of heparin therapy. Med Clin N Am, 1998, 82, 635–658.
Received: January 5, 2004; in revised form: February 27, 2004.
