Hindlimb unloading in rat decreases preosteoblast proliferation assessed in vivo with BrdU incorporation

Odile Barou, Sabine Palle, Laurence Vico, Christian Alexandre, and Marie-Hélène Lafage-Proust

Laboratoire de Biologie du Tissu Osseux, Saint-Etienne University, 42023 Saint-Etienne Cedex 2, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Immobilization affects bone formation. However, the mechanisms regulating the decrease in osteoblast recruitment remain unclear. The aim of our study was to determine in vivo osteoblastic proliferation after short-term immobilization among the different bone compartments. Twelve Wistar 5-wk-old rats were assigned to two groups: six tail-suspended animals for 6 days and their six age-related controls. Osmotic minipumps, each containing 40 mg of bromodeoxyuridine (BrdU), were implanted intraperitoneally at day 4 until euthanasia. Histomorphometric measurements found a significantly lower bone volume in primary (ISP, -22%) and secondary spongiosa (IISP, -37%) in unloaded rats compared with their age-related controls. BrdU immunohistochemistry showed that the proliferation capacity of osteogenic precursors in ISP (-29%) and preosteoblasts in IISP (-80%) and in periosteum as well as bone marrow cells (-40%) was lowered by unloading. We demonstrated in vivo for the first time that 6-day tail suspension induced a significant decrease in proliferation of periosteal and trabecular preosteoblasts in ISP and IISP as well as in bone marrow cells.

immobilization; quantitative histomorphometry; rat tail suspension; osteoporosis

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

BONE MASS is the result of the balance between osteoblastic bone formation and osteoclastic resorption. Among the mutiple factors that regulate bone cell activities, mechanical environment plays a critical role, although it is poorly understood. It is now well evidenced that skeletal immobilization and microgravity (13, 28, 30) induce bone loss. Previous studies have shown that the decrease in bone mass could be related to various mechanisms. A rapid and transient increase in osteoclastic resorption was reported 3 days after tenotomy (29), which might be related to an increase in prostaglandin secretion since it was prevented with indomethacin (24). Furthermore, longer experiments (7- to 40-day unloading) showed a decrease in bone formation, which could be the result of two mechanisms: impairment of osteoblast activity at the cell level and/or lack of osteoblast recruitment at the tissue level. First, histodynamic studies in four immobilization models in the rat [tail suspension (27), tenotomy (24), sciatectomy (29), or cast (13)] showed a decrease in mineral apposition rate, which evaluates the amount of work performed by one osteoblast, and in tetracycline-labeled surface, which reflects the number of "working" osteoblasts. Second, ex vivo cultures of bone marrow and osteoblastic cells extracted from tail-suspended (14-16, 35) or sciatectomized rats (8) suggested that immobilization induced an alteration either in the number of stromal mesenchymal cells and/or a decrease in their osteogenic capacity due to a reduction in their proliferation or differentiation abilities. However, the discrepancies among the results of ex vivo cultures, probably related to different experimental conditions, did not allow any definitive conclusion.

The aim of our study was to quantitatively evaluate in vivo osteoblastic proliferation after a short-term tail-suspension unloading with a direct visualization of the mitotic response of cells from the osteoblast lineage, using a bromodeoxyuridine (BrdU) labeling. BrdU is incorporated into DNA during mitosis. It is visualized with immunohistochemistry, a more rapid and nonradioactive technique, compared with [3H]thymidine incorporation (6), and is compatible with histodynamic measurements (1). In addition, this histological technique allowed us to analyze cells in the four different bone compartments: osteogenic precursors in the primary spongiosae (ISP), preosteoblasts in the secondary spongiosae (IISP) and in the periosteum, and cells in the bone marrow. Moreover, we analyzed the relationship between tetracycline labeling parameters of mineralization with the actual preosteoblastic proliferation indexes. The adaptive bone response to the variations in mechanical strain seems to occur rapidly (31, 22). We therefore chose to evaluate short-term effects of unloading after a 6-day tail suspension in the rat.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Twelve male Wistar rats, aged 5 wk, (Iffa Credo, les Onçins, France) were acclimatized with standard conditions of temperature- (23°C ± 1°C) and light-controlled environment (12:12-h light-dark cycle). Rats were fed with a standard rodent chow and water ad libitum. After 1 wk (mean weight 192 ± 8 g), they were randomly divided in two groups: six rats with tail suspended and their six unsuspended controls kept in suspension cages. The rats were suspended without anesthesia using the tail traction method in a specially designed Plexiglas cage and according to a procedure previously described by Morey et al. (17) (two-thirds of the tail was wrapped with orthopedic tape) and Hargens et al. (7) (at an angle of 30° so that ~50% of the body weight is loaded onto the forelimbs).

The animals were double labeled with tetracycline (15 mg/kg body wt) at day 0 and calcein (20 mg/kg body wt) at day 5 by intraperitoneal injection. BrdU (Sigma, St. Quentin Fallavier, France) was diluted in dimethyl sulfoxide and distilled water (vol/vol) at a final concentration of 200 mg/ml. Two 1003D Azlet osmotic pumps (Charles River, St. Aubin les Elbeuf, France), each containing 100 µl of BrdU solution (40 mg BrdU/animal), were implanted intraperitoneally at day 4 (i.e., 3 days after the beginning of the suspension) under brief anesthesia by monoethylether inhalation. The animals were killed with anesthetic overdose (Nesdonal 0.1 mg/kg) at the end of day 6. The total duration of BrdU exposure was therefore 72 h. Left tibiae were dissected for conventional histomorphometry. Right tibiae were prepared for immunohistochemistry and histodynamic measurements. Moreover, a segment of bowel, which is a high-turnover tissue, was taken out from each animal in both groups and was used as a positive control to assess the correct function of the osmotic pumps.

Bones were processed as follows for bone histomorphometry. After animals were killed, left tibiae were excised, fixed, dehydrated in absolute acetone, embedded mineralized in methylmethacrylate, and sectioned sagittally at a thickness of 7 µm for subsequent measurements of bone histomorphometry. A Leitz-TAS+ automatic image analyzer equipped with a Bosch camera coupled with a Leitz Orthoplan microscope, was used to determine bone volume (bone volume/tissue volume, percentage of cancellous bone area) and structural indexes (trabecular bone thickness, trabecular bone number, and trabecular separation) in the ISP and IISP. These measurements were carried out on six modified Goldner sections. To compensate for possible differences in longitudinal growth between groups, region of interest representing IISP was delimited anatomically by the bottom of the ISP to the diaphysis-metaphysis frontier. We measured static bone cellular parameters in the IISP using a semiautomatic system composed of a digitizing tablet (Summasketch-Summagraphics) coupled with a microcomputer connected to a Reichert Polyvar microscope equipped with a drawing system (Camera Lucida). These measurements included the osteoid surfaces [osteoid surface/bone surface (BS), %] as the length of osteoid seams covering bone-forming surfaces on four Goldner's sections, osteoblastic surfaces (osteoblastic surfaces/BS, %) on four toluidine blue-stained sections, and osteoclast number (osteoclast number/bone perimeter, cells/mm of trabecular bone) corresponding to the number of osteoclastic cells covering bone-resorbing surfaces determined on four sections stained for tartrate-resistant acid phosphatase activity (2).

For immunohistochemistry and histodynamic measurements bones were processed as follows. Right tibiae were fixed in 70% ethanol for 24 h and then dehydrated in increasing concentrations of ethanol. Bones and bowel segments were then embedded in EPON plastic resin Embed 812-Araldite 502-DMP 30-DDSA medium (TAAB, France) at room temperature. Polymerization was performed under vacuum at 60°C for 5 days. Histodynamic parameters were measured on unstained 8-µm-thick sections under ultraviolet light: single- and double-labeled surface (SLS/BS and DLS/BS, %) mineral apposition rate (MAR, µm/day). Derived parameters were calculated: mineralizing surface (MS/BS = 1/2 SLS/BS + DLS/BS) and bone formation rate surface referent (BFR/BS = MS/BS × MAR, µm3 · µm-2 · day-1). The longitudinal growth rate was appreciated by measuring the mean distance between the second labeling in the ISP and the closest site at which the first labeling was visualized in the IISP.

Four-micrometer-thick sections were carried out on microtome. Sections were put on positively charged slides fitting in the Microprobe system (Polylabo, France) and dried at 37°C in an oven for 3 h. Sections were then deplastified in a 50% sodium ethoxide solution for 2 × 20 min and then rehydrated in decreasing ethanol concentrations and put in distilled water. Endogenous peroxidases were blocked with 3% H2O2 for 30 min. Nonspecific sites were blocked with tris(hydroxymethyl)aminomethane (Tris)/bovine serum albumin (BSA) 1%. Monoclonal primary antibody anti-BrdU (Sigma, France) was diluted at 1/500 with Tris/BSA 1% added for 1 h at room temperature. Revealment of the primary antibody was performed with a kit (Immunotech, France) using streptavidin conjugate and 3',3'-diaminobenzidine as chromogen. Each step included a 2 × 5-min wash in Tris, pH 7.6. The Microprobe system allowed the processing of two sections, one from a control rat and one from an unloaded rat, facing each other, leading to a good tissue preservation and an even deplastification. Three types of negative controls were prepared to evaluate background and nonspecific labeling. They consisted of sections from BrdU-treated rats omitting the primary antibody and sections from nontreated rats processed as the positive sections by following the full procedure or using a nonimmune immunoglobulin G as the primary antibody. Labeled cells were counted using an ocular integrator with a grid. Only dark brown nuclei were taken into account. In ISP, labeled cell number (regardless of cell type) was referenced to the bone-cartilage area (labeled cells/bone area) previously measured with the Leitz-TAS+ automatic image analyzer. In IISP, labeled juxtatrabecular preosteoblasts were counted and their number was referenced to the bone perimeter (labeled preosteoblasts/bone perimeter of IISP). Preosteoblasts are functionally defined as a transitional state between the osteoprogenitor cell and the differentiated osteoblast (24). They are morphologically defined, as described earlier by Kember (9), Young (33), and Kimmel and Jee (10), as ovoid cells located between the juxtatrabecular osteoblast layer and the bone marrow and are clearly illustrated in Fig. 2, A and B. Finally, the number of BrdU-labeled cells per square millimeter of marrow area was counted.

Humerus metaphyses from two unloaded animals were immunostained and compared with two controls to check that the effects observed in the tibia were related to unloading.

Statistics. Data were expressed as means ± SD for all parameters measured. Intergroup differences were analyzed by the one-way analysis of variance by ranks (Kruskal-Wallis test), and, when significant, the Mann-Whitney test was used to compare pairs of means.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tail-suspended rats weighed 12.5% less than their age-related controls at the end of the experiment (189 ± 33 vs. 216 ± 10 g, P < 0.05).

Histomorphometric results. Histomorphometric results are summarized in Tables 1 and 2. After 6 days of immobilization, tail-suspended rats exhibited a significant 37% lower bone volume in IISP, compared with their age-related controls (P < 0.005). In terms of architecture, this lower bone mass was characterized by fewer and less thick trabeculae (-22 and -24%, respectively). Suspension impaired bone formation at the cell and tissue levels. BFR was significantly lower (-20%, P < 0.05) in unloaded animals, and this was related to a lower MAR (-28%, P < 0.005), whereas DLS and MS did not show any change. Resorption parameters were similar in the two groups.

                              
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Table 1.   Bone mass and architecture parameters in ISP and IISP in unloaded rats vs. age-related controls

                              
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Table 2.   Bone remodeling and immunohistochemistry parameters in unloaded rats vs. age-related controls

Immunohistochemistry results. Immunohistochemistry results are summarized in Fig. 1. Sections from bowel were positive in every control and tail-suspended rat, demonstrating that all the osmotic pumps delivered BrdU correctly (Fig. 2C). We counted 29% fewer labeled cells (regardless of their type) per bone-cartilage area in the ISP and even fewer (-80%) labeled preosteoblasts in the IISP of unloaded rats, compared with their age-related controls. Few juxtatrabecular osteoblasts were BrdU labeled in control animals, whereas none were observed in suspended rats. In the IISP, tetracycline DLS correlated with the number of labeled preosteoblasts per bone perimeter in both groups but the slopes differed, suggesting that the osteoblast recruitment began to be altered in the unloaded animals (control: y = 0.03x + 0.95, r = 0.8, P < 0.01 vs. unloaded: y = 0.01x - 0.08, r = 0.8, P < 0.05). Moreover, whereas periosteal osteoblasts located at the junction of growth plate and cortices were the most highly stained in control rats, BrdU uptake in cells from the same area was faint or absent in suspended rats (Fig. 3, A and B). Finally, we found that the number of labeled cells was significantly lower (-40%, P < 0.01) in the unloaded rat bone marrow, compared with controls (Fig. 3, C and D). Interestingly, BrdU immunohistochemistry in the humerus showed that the pattern of labeled trabecular preosteoblasts and bone marrow cells appeared similar between controls and unloaded rats (Fig. 3, E and F).


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Fig. 1.   Quantitative evaluation of bromodeoxyuridine (BrdU)-labeled cells in three compartments [A: primary spongiosa (ISP), B: secondary spongiosa (IISP), C: bone marrow] in tibial metaphysis in control (filled bars) compared with unloaded rats (open bars). * P < 0.01; ** P < 0.0001.


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Fig. 2.   BrdU labeling of preosteoblasts in EPON plastic resin-embedded undecalcified IISP. B (×1,000): details of framed area seen in A (×250). Hematoxylin counterstaining: marrow cells (m), osteoid seam (o), calcified trabecula (t), marrow cells labeled (ml), osteoblastic cell (ob), preosteoblast (pob), BrdU-positive proliferative cells (*). C: representative images of BrdU-labeled cells (arrows) in bowel taken as control tissue with high proliferation rate for assessing correct function of osmotic minipumps (×250).


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Fig. 3.   Representative images of BrdU uptake showing lower proliferation capacity of periosteal osteoblastic cells in suspended rats (B) compared with age-related controls (A). c, Cortices; f, fibroblasts (×1,000). Representative images of BrdU uptake showing fewer labeled bone marrow cells (arrows) in suspended rats (D) compared with age-related controls (C; ×1,000). Representative images of BrdU uptake in humerus metaphyses (IISP) exhibiting a similar pattern of labeled cells between unloaded (F) and control (E) rats (×250), no counterstaining. Arrow, osteoblast; arrowhead, marrow cell; t, calcified trabecula.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our study was based on the effects of a 6-day hindlimb unloading on preosteoblast proliferation in different bone compartments of proximal tibia metaphysis. Few studies focused on the early adaptive bone response to changes in mechanical load. Raab-Cullen et al. (22) found that c-fos expression, which is strongly linked to osteoblast development and differentiation, was induced as early as 2 h after an increase in mechanical load in the rat tibia periosteum. Weinreb et al. (29) reported an increase in osteoclastic resorption 72 h after tenotomy and therefore demonstrated that immobilization was able to induce very early changes in bone remodeling at the tissue level. Taken together, these data led us to believe that, if the major consequence of unloading (i.e., bone loss) needed at least 7-8 days to appear, the events inducing the bone loss might occur much earlier. Moreover, we (27) and others (5) reported that the decrease in bone formation was the most dramatic after 8 days in the rat tail-suspension model, whereas trends to a rebound of bone formation parameters were shown at the end of the 2nd wk of tail suspension. Finally, in this model, the secretion of corticosteroids, the major hormones related to stress, is known to peak the 1st day of suspension and normalize within 3 days (3, 4, 18). Moreover, we observed a similar pattern of labeling in humerus metaphyses between tail-suspended and control rats, suggesting that the observed effects in tibia were actually related to unloading and not to a systemic effect of stress. Besides, we found in a preliminary study (1) that BrdU detection in rat trabecular undecalcified bone from IISP was optimized after a 72-h continuous exposure compared with sequential injections or a 48-h administration. Thus we chose in this study to evaluate preosteoblast proliferation as early as possible within the 1st wk of suspension, after the main effects of stress were no longer involved, i.e., between day 4 and day 6 of the suspension.

We found a significant 30% lower bone volume in IISP after a 6-day immobilization period compared with age-related controls. Given the longitudinal growth mechanisms in the rat tibia, the decrease in bone volume in IISP might be related to a decrease in bone and cartilage supplies from the ISP combined with an alteration of cellular activities in IISP. Our data confirmed that bone loss occurs earlier and in a greater extent in younger animals, as suggested by others (16, 20, 27).

There is a general agreement that immobilization depresses bone formation (13, 15, 18, 27, 29). The impairment of bone formation could be attributed to a decrease in osteoblastic recruitment and/or a decline in the amount of mineralized matrix synthetized by individual osteoblasts. It has been shown (15) that 14-day unloading induced a DLS 50% decrease in rats of similar age. In our experiment, the lower BFR found in the suspended rats was related to a lower MAR, suggesting that unloading primarily affected mineralization at the cell level, whereas the MSs, a "classic" parameter of osteoblast recruitment, were similar between the two groups. Interestingly, the evaluation of proliferation in the osteoblastic lineage with immunohistochemistry showed a lower number of BrdU-labeled cells in unloaded tibiae compared with controls. This apparent discrepancy between BrdU labeling and histodynamic measurements emphasizes the fact that, even in the rat model, osteoblastic proliferation, bone matrix synthesis, and matrix mineralization are three different processes, although linked and correlated in physiological conditions. Our findings support the fact that, for short-term experiments, histodynamic measurements might be less sensitive than the BrdU technique to detect early changes in osteoblast recruitment.

The decrease in osteoblast recruitment induced by immobilization might be the result of different mechanisms: a decline in the number of colony-forming units-fibroblast (CFU-F) at the origin of the osteoblastic lineage and/or a decrease in the proliferation capacity of preosteoblasts later in the osteoblastic lineage (25).

Ex vivo experiments, conducted by Machwate and co-workers (14-16), showed that the proliferation of adherent stromal cells extracted from tibiae of unloaded or control rats appeared to be similar, whereas the proliferation of alkaline phosphatase positive stromal cells (i.e., osteoprogenitor cells) was either decreased (15, 16) or unchanged (14) by unloading. Conversely, Keila et al. (8) showed in sciatectomized rats that the number of adherent marrow stromal cells as well as the number of mineralizing nodules was lowered in the unloaded limb, suggesting that the number of CFU-F was decreased by immobilization, their proliferation capacity remaining unchanged in culture. Zhang et al. (35) confirmed that the number of adherent stromal cells was decreased immediately after suspension. However, the authors found that stromal cell proliferation capacity was altered, whereas the percentage of alkaline phosphatase positive cells was similar between the two groups after 24 h of culture. These discrepancies between the results of the ex vivo experiments are probably related in part to methodological differences. In the present study, we showed in vivo that the number of BrdU-labeled cells was significantly lower not only in preosteoblasts of IISP of unloaded rats but also in cells from the ISP and in the bone marrow, compared with age-related controls. Unfortunately, in vivo methods for histologically identifying osteoblast precursors among bone marrow cells are not yet available. Recent data from studies using flow cytometry suggested that the number of osteoblast precursors selected with anti-sac-1 monoclonal antibody and wheat germ agglutinin is close to 0.006% in 5-fluoro-uridine-treated mice (26). That is a much smaller percentage than the number of cells that stopped dividing in the bone marrow of our suspended animals (-40%). This suggests that the decrease in marrow cellularity and increase in marrow fat described by Morey-Holton and Wronski (19) after tail suspension or spaceflight could be the result of a decrease in mesenchymal stromal cell proliferation.

The decrease in the number of labeled preosteoblasts could be the result of a decrease in the replication of CFU-F upstream and/or the decline in the proliferation capacity of preosteoblasts themselves. Previous well-conducted experiments on rat bone cell kinetics in longitudinal bone growth (10, 32, 33) using injections of tritiated thymidine estimated that cell cycle time of osteoprogenitors was around 36 h. In one of these studies, Kimmel and Jee (10) stressed that osteoblast precursors migrate along the trabeculum in the direction of bone elongation to supply new metaphyseal tissue with osteoprogenitor cells. Indeed, they found only a small number of labeled precursors in the IISP 1 and 24 h after [3H]thymidine injection. If we take this notion into account, the decrease in preosteoblasts from the IISP might be partly related to a decrease in precursors in the ISP. Given the schedule of BrdU administration in our animals (72 h), which represented two cell cycles for the dividing precursors labeled at the beginning of the experiment, we could not conclude whether the decrease of labeled preosteoblasts was only related to a decrease in CFU-F number or not. However, the absence of labeled mature osteoblasts in the suspended animals suggests that the preosteoblast proliferation capacity was also reduced.

It has been well demonstrated in tail-suspended rats (12) and in the cast immobilization model that cortical thickness of immobilized diaphysis was decreased. Histodynamic measurements suggested that the decrease in cross-sectional growth of diaphysis was the result of an early depressed periosteal modeling associated with an enhanced endocortical resorption. In periosteal preosteoblasts facing the ISP, we have shown that BrdU uptake was high in controls [confirming previous findings (21)] and faint after 6-day unloading. This is in agreement with Li and Jee (12), who reported that the decrease in BFR resulted from a combined diminution of labeled surface and MAR to a lesser extent. Our data suggest that periosteum, which was shown to respond to an increase in mechanical load (22), is highly sensitive to unloading as well. Unfortunately, periosteum from the lower part of tibiae lifted off during the immunostaining. Therefore, we could not state whether this decrease in BrdU uptake found in the upper periosteum was not related only to the decrease in growth rate found in unloaded animals.

In conclusion, we showed that 6-day immobilization induced a decrease in preosteoblast proliferation in vivo: dramatic in the upper part of the periosteum, severe in IISP, and moderate in the ISP. The longitudinal and transversal growth mechanisms in the rat might partly explain these differences. The 40% decrease in the number of BrdU-positive cells in the marrow, regardless of their type, demonstrated that stromal cell dividing rate was altered as well. Further in vivo studies, using sequentially euthanized animals after labeling of proliferating cells, are needed to evaluate the respective roles of the decrease in the different pools of dividing cells from the osteoblastic lineage in the loss of recruitment of working osteoblasts that lead to bone loss.

    ACKNOWLEDGEMENTS

This paper was presented as a poster at the September 1996 American Society for Bone and Mineral Research meeting in Seattle, WA, and as an oral presentation at the International Bone Morphometry meeting in Italy in October 1996.

    FOOTNOTES

Address for reprint requests: O. Barou, Laboratoire de Biologie du Tissu Osseux, Faculté de Médecine, 15 Rue A Paré, 42023 Saint-Etienne Cedex 2, France.

Received 13 June 1997; accepted in final form 7 October 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Barou, O., N. Laroche, S. Palle, C. Alexandre, and M. H. Lafage-Proust. Preosteoblastic proliferation assessed with BrdU in undecalcified rat trabecular bone. Correlation with histodynamic parameters. J. Histochem. Cytochem. 45: 1189-1195, 1997[Abstract/Free Full Text].

2.   Chappard, D., C. Alexandre, and G. Riffat. Histochemical identification of osteoclasts. Review of current method and reappraisal of a simple procedure for routine diagnosis on undecalcified human iliac crest bone biopsies. Basic. Appl. Histochem. 27: 75-85, 1983[Medline].

3.   Desplanches, D., M. H. Mayet, B. Sempore, J. Fusto, and R. Flandrois. Structural and functional responses to prolonged hindlimb suspension in rat muscle. J. Appl. Physiol. 63: 558-563, 1987[Abstract/Free Full Text].

4.   Feller, G. D., H. S. Ginoza, and E. R. Morey. Atrophy of rat skeletal muscles in simulated weightlessness. Physiologist 24: S9-S10, 1981.

5.   Globus, R. K., D. D. Bikle, and E. R. Morey-Holton. Effects of simulated weightlessness on bone mineral metabolism. Endocrinology 114: 2264-2270, 1984[Abstract].

6.   Grazner, H. G. Monoclonal antibody to 5-bromo and 5 iododeoxyuridine: a new reagent for detection of DNA replication. Science 218: 474-475, 1982[Medline].

7.   Hargens, A. R., J. Steskal, C. Johansson, and C. M. Tipton. Tissue fluid shift, forelimb loading and tail tension in tail-suspended rats. Physiologist 27: S37-S38, 1984.

8.   Keila, S., S. Pitaru, A. Grosskopf, and M. Weinreb. Bone marrow from mechanically unloaded rat bones expresses reduced osteogenic capacity in vitro. J. Bone Miner. Res. 9: 321-327, 1994[Medline].

9.   Kember, N. F. Cell division in endochondral ossification. A study of cell proliferation in rat bones by the method of tritiated thymidine autoradiography. J. Bone Joint Surg. Br. 42: 824-839, 1960.

10.   Kimmel, D. B., and W. S. S. Jee. Bone cell kinetics in longitudinal bone growth in the rat. Calcif. Tissue Int. 32: 123-133, 1980[Medline].

12.   Li, X. J., and W. S. S. Jee. Adaptation of diaphyseal structure to aging and decreased mechanical loading in the adult rat: a densitometric and histomorphometric study. Anat. Rec. 229: 291-297, 1991[Medline].

13.   Li, X. J., W. S. S. Jee, S. Y. Chow, and D. M. Woodbury. Adaptation of cancellous bone to aging and immobilization in the rat: a single photon absorptiometry and histomorphometry study. Anat. Rec. 227: 12-24, 1991.

14.   Machwate, M., E. Zerath, X. Holy, M. Hott, D. Godet, A. Lomri, and P. J. Marie. Systemic administration of transforming growth factor-b2 prevents the impaired bone formation in osteopenia induced by unloading in rats. J. Clin. Invest. 96: 1245-1253, 1995[Medline].

15.   Machwate, M., E. Zerath, X. Holy, M. Hott, D. Modrowski, A. Malouvier, and P. J. Marie. Skeletal unloading in rat decreases proliferation of rat bone marrow derived osteoblastic cells. Am. J. Physiol. 264 (Endocrinol. Metab. 27): E790-E799, 1993[Abstract/Free Full Text].

16.   Machwate, M., E. Zerath, X. Holy, P. Pastoureau, and P. J. Marie. Insulin-like growth factor-I increases trabecular bone formation and osteoblastic cell proliferation in unloaded rats. Endocrinology 134: 1031-1038, 1993[Abstract].

17.   Morey, E. R., E. E. Sabelman, R. T. Turner, and D. J. Baylink. A new rat model simulating some aspects of space flight. Physiologist 22: S23-S24, 1979[Medline].

18.   Morey-Holton, E. R., M. D. Bomalaski, M. R. Enayat-Gordan, M. R. Gonsalves, and T. J. Wronski. Is suppression of bone formation during simulated weightlessness gradual and related to glucorticoid levels. Physiologist 25: 145-146, 1982.

19.   Morey-Holton, E., and T. J. Wronski. Animal model for simulated weightlessness. Physiologist 24: S45-SS47, 1981.

20.   Novikov, V. E., and E. A. Ilyn. Age-related reactions of rat bones to their unloading. Aviat. Space Environ. Med. 12: 551-553, 1981.

21.   Owen, M. Cell population kinetics of an osteogenic tissue. J. Cell Biol. 19: 19-32, 1963[Abstract/Free Full Text].

22.   Raab-Cullen, D. M., M. A. Thiede, D. N. Petersen, D. B. Kimmel, and R. R. Recker. Mechanical loading stimulates rapid changes in periosteal gene expression. Calcif. Tissue Int. 55: 473-478, 1994[Medline].

23.   Rodan, G. A., and L. A. Raisz. Metabolic Bone Diseases, edited by L. V. Avioli, and S. M. Krane. Philadelphia, PA: Saunders, 1990, p. 1-7.

24.   Thompson, D. D., and G. A. Rodan. Indomethacin inhibition of tenotomy-induced bone resorption in rats. J. Bone Miner. Res. 3: 409-414, 1988[Medline].

25.   Urist, M. R., R. J. De Long, and G. A. M. Finnerman. Bone cell differentiation and growth factors. Science 20: 680-686, 1983.

26.   Van Vlasselaer, P., N. Falla, H. Snoeck, and E. Mathieu. Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-sac-1 monoclonal antibody and wheat germ agglutinin. Blood 84: 753-763, 1994[Abstract/Free Full Text].

27.   Vico, L., S. Bourrin, V. Novikov, J. M. Very, D. Chappard, and C. Alexandre. Adaptation of bone cellular activities to tail suspension in rats. Cells Mater. S1: 143-150, 1991.

28.   Vico, L., D. Chappard, S. Palle, A. V. Bakulin, V. E. Novikov, and C. Alexandre. Trabecular bone remodeling after seven days of weightlessness. Am. J. Physiol. 255 (Regulatory Integrative Comp. Physiol. 24): R243-R247, 1988[Abstract/Free Full Text].

29.   Weinreb, M., G. A. Rodan, and D. D. Thompson. Osteopenia in the immobilized rat hindlimb is associated with an increased bone resorption and decreased bone formation. Bone 10: 187-195, 1989[Medline].

30.   Wronski, J. J., E. R. Morey, S. Doty, A. C. Maese, and C. C. Walsh. Histomorphometric analysis of rat skeleton following space flight. Am. J. Physiol. 252 (Regulatory Integrative Comp. Physiol. 21): R252-R255, 1987[Abstract/Free Full Text].

31.   Yamaguchi, M., K. Ozaki, and T. Hoski. Simulated weightlessness and bone metabolism: decreases of protein and DNA syntheses in the femoral diaphysis of rats. Res. Exp. Med. (Berl.) 189: 331-337, 1989[Medline].

32.   Young, R. W. Cell proliferation and specialization during endochondral osteogenesis in young rats. J. Cell Biol. 14: 357-370, 1962[Abstract/Free Full Text].

33.   Young, R. W. Regional differences in cell generation time in growing rat tibiae. Exp. Cell Res. 26: 562-569, 1962.

35.   Zhang, R., S. C. Supowit, G. L. Klein, Z. Lu, M. D. Christensen, R. Lozano, and D. J. Simmons. Rat tail suspension reduces messenger RNA level for growth factors and osteopontin and decreases the osteoblastic differentiation of bone marrow stromal cells. J. Bone Miner. Res. 10: 415-423, 1995[Medline].


AJP Endocrinol Metab 274(1):E108-E114
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