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Division of Radiobiology, Department of Radiology, School of Medicine, University of Utah, Salt Lake City, Utah 84108
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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bone formation, cell proliferation, lactation, mechanisms of hormone action, osteoblasts, weaning
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After weaning, there is a rapid expansion of active bone mineralizing surface in the rat maternal skeleton, particularly on endosteal and endocortical surfaces [10, 11]. Bone formation and mineralization rates, as measured by histomorphometric methods, were substantially increased at 2 wk after weaning [10, 11], although earlier times were not studied. The first purpose of this study was to determine when increases in measures of bone formation began to occur in the maternal skeleton after weaning.
The osteoblast is the cell that synthesizes bone matrix and plays a role in its mineralization. The rapid expansion of bone surfaces that are forming and mineralizing new bone after lactation must be preceded by an expansion of the osteoblast population and production of new osteoblasts. Osteoblasts rarely divide in situ and thus new osteoblasts must be derived from precursor cells [17]. These may include inducible and determined progenitors, including preosteoblasts, bone-lining cells, stromal cells, or other mesenchymal-type cells [17]. All of the putative osteoblast progenitors appear capable of proliferation, however, proliferation does not appear to be necessary for the initial formation of osteoblasts in some models of induced osteogenesis [18–20]. Increases in osteoprogenitor cell proliferation are, however, associated with bone formation during endochondral osteogenesis [21, 22] and appear to correlate with the extent of active formation surface in normal and diseased humans [23]. Therefore, the second purpose of this study was to determine whether the formation of new osteoblasts in the rapidly expanding osteoblast population after weaning are derived from proliferating progenitors. This will provide fundamental new information on the physiological mechanisms of osteogenesis.
Substantial endocrine changes occur at weaning, including a rapid decrease in prolactin (PRL) [24] and, possibly, parathyroid hormone-like peptide (PTHLH), and an increase in estrogen associated with the reinitiation of the estrus or menstrual cycles. There is accumulating evidence that PRL and PTHLH are involved in the regulation of maternal skeletal metabolism during lactation [25, 26], and the reinitiation of menstrual cycles was associated with reconstitution of lactation-associated losses of bone mineral density in humans [1]. Therefore, the third purpose of this study was to correlate the events associated with the initiation of the postlactation skeletal anabolic phase with known endocrine changes that occur during and immediately after weaning. This information may provide new insights into the regulation of osteogenesis in the adult female skeleton and have implications for changes in skeletal functions after reproductive capacity ceases (e.g., postmenopausal osteoporosis).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Sprague-Dawley rats (Charles Rivers) were mated at 95–100 days of age and completed one reproductive cycle consisting of parturition, lactation for 20 days, and a 4-wk recovery period. Half the rats were mated a second time and the remainder were used for an age-matched, unmated, normal estrus-cycling comparison group. The average age at the second mating was 170–180 days and reflected the variations of mating and parturition in the first cycle. Litters in both the first and second reproductive cycles were normalized to 9 pups, and each lactation period was 20 days. There were 8–10 animals in each of the experimental groups. The rats were fed normal rodent chow (TekLab 8640, Harlan Diets, Madison, WI), given water ad libitum, and kept on a 12L:12D cycle. The protocol was approved by the Institutional Animal Care and Use Committee at the University of Utah.
Bromodeoxyuridine Labeling
Bromodeoxyuridine (BrdU) is a nucleoside analog that is incorporated into DNA during S-phase and is a convenient, nonradioactive method for detecting proliferating cells and their progeny [27]. To help determine the origin of the osteoblasts during the transition from lactation to weaning, the dams were given BrdU (Sigma Chemical Co., St. Louis, MO) i.p. injections beginning the day after weaning at the following intervals: 0800, 1600, 2400, and again at 0800 h. The BrdU was given at a dose of 25 mg/kg body weight. The nonlactating comparison groups consisted of age-matched, normal estrus cycling rats that had been through the first reproductive cycle but not the second. Necropsies were performed at 25, 48, 72, 96, 120, and 144 h after the first BrdU injection. This corresponds to Days 2, 3, 4, 5, 6, and 7 after weaning, respectively.
Fluorochrome Bone Markers for Histomorphometry
Calcein (Sigma) at 20 mg/kg body weight was given by s.c injection 6 days before necropsy and tetracycline-HCl (Sigma), 25 mg/kg body weight, was given 1 day before necropsy.
Histomorphometry
At necropsy, the fifth and sixth lumbar vertebral bodies were removed and fixed for 24 h in 10% phosphate-buffered formalin. The tissues were then dehydrated in ethanol and embedded undecalcified in methyl methacrylate. Longitudinal sections through the vertebral bodies were cut on a low-speed bone saw (Isomet; Buehler, Lake Bluff, IL), mounted on plastic slides, and ground to 
30 µm using a precision grinding machine (Exact, Norderstedt, Germany). These unstained sections were viewed using an image analysis system and a fluorescence microscope. Images were captured with a Q Imaging Retiga 1300 C digital camera and measurements calculated using Bioquant Nova Prime XP software (R&M Biometrics, Inc. Nashville, TN). The primary histomorphometric indices, single-labeled surface (sLS), double-labeled surface (dLS), bone area, and perimeter and interlabel width were measured in the center of the section, approximately 4.5 mm2, and used to calculate %sLS, %dLS, the percentage of mineralizing surface (%MS), mineral apposition rate (MAR), and bone formation rates (BFR). The MAR was corrected for obliquity [28]. Bone formation rates were calculated using MS from the dLS plus 0.5 of the sLS. Bone formation rates were calculated using the bone perimeter or surface-referent (BFRs, µm2 µm/d) and the bone area-referent (BFRa, µm2 µm2/d). After quantification of the fluorochrome markers, the sections were stained with a Geimsa stain and the percentage of bone surfaces covered by osteoblasts (osteoblast surface) was measured in the same cancellous region. The histomorphometric measurements and derivations followed accepted standards [29].
Histochemical Methods
Lumbar vertebrae were trimmed and fixed in 10% buffered formalin for 48 h. The bones were then decalcified in neutral 10% EDTA, embedded in paraffin, and sections prepared. Following removal of the paraffin with xylene the sections were stained using a strepavidin-biotin system for BrdU detection (Zymed Laboratories, San Francisco, CA). The stain was visualized using the diaminobenzidine reaction and counterstained with hematoxylin.
To quantify the appearance of BrdU-labeled cells in the osteoblast population, two sections of the cancellous bone in the lumbar vertebral body from each animal were used. The relative number of labeled osteoblast nuclei were counted in the cancellous bone beginning at 0.5 mm beneath each growth plate. The endocortical surfaces were not included. Osteoblasts were identified by the plump trapezoid shape and their location on the bone surface, and only those with a nucleus in the cell profile were counted. Stromal cells or bone-lining cells were not included in the counts but the appearance of labeled nuclei in these populations is described. The cancellous bone of the lumbar vertebral body was selected for these analyses because it has a lower turnover and is less influenced by endochondral growth compared with the long bones.
Statistics
The histomorphometric measurements in the age-matched, normal estrus cycling animals did not change over the 5-day experimental period and were combined into one group. The significance of differences with the histomorphometry data between the normal estrus cycling group and the postweaning groups were determined by an analysis of variance followed by a Tukey honestly significant difference comparison post hoc test to determine significance between groups. In the BrdU kinetic data, the nonmated groups were compared with their respective mated groups using a similar statistical analysis. A level of P < 0.05 was considered significant and the data are expressed as the mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bone formation indices with time after weaning are presented in Table 1 and Figure 1. The first group, 25 h after the first BrdU injection, corresponds to Day 2 postweaning. At this time, all bone formation indices were substantially greater than those measured in the age-matched, normal estrus cycling group. The percent of dLS, MS, and BFR continued to increase for the first week after lactation. Over a 5-day period, from Day 2 to Day 7 after lactation, these same indices more than doubled. By 7 days after the end of lactation, the bone formation rates were more than 7 times greater in the postweaning dams than in the age-matched, normal estrus cycling animals. The percent of surface covered by osteoblasts was significantly greater than the normal estrus cycling animals at all times. There is also a significantly greater osteoblast surface from Day 3 to Day 7 compared with that on Day 2. The percent of osteoblast surface seemed to plateau by Day 3 after lactation (Table 1).
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The extent and characteristics of the bone formation activity on the bone surface is perhaps best visualized in micrographs (Fig. 2). The first label (calcein) was given 6 days, and the second label (tetracycline) was given 1 day before necropsy. In bone sections taken at 7 days after weaning (Fig. 2A), there were extensive single- and double-labeled fluorochrome seams compared with unmated, age-matched normal estrus cycling rats (Fig. 2B). In this example from a postweaning rat, most of the surface is labeled with one or both of the fluorochrome markers, indicating active bone mineralization on most surfaces when the respective labels were given.
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Kinetics of BrdU-labeled Bone Surface Cells
Considering the extent of labeled surfaces and the bone formation indices observed during and immediately after weaning (Table 1 and Figs. 1 and 2), it was not surprising to see large populations of osteoblasts appearing on bone surfaces after weaning. By 25 h after the first BrdU injection (Day 2 after weaning), a number of BrdU-labeled cells were observed on or near bone surfaces (Fig. 3A). About 17% of the osteoblast population contained a BrdU label at this time (Fig. 4), but other cells near the surface that had incorporated a label included differentiating osteoblasts and adjacent stromal cells (Fig. 3, A and B). Although infrequent, mitotic figures were observed next to the bone surface (Fig. 3B). In some regions, bone-lining cells with typical flat morphology had also incorporated a BrdU label by 25 h after the first injection (Fig. 3C).
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From 48 to 144 h after the first BrdU injection, the percentage of labeled osteoblasts appeared to have reached a plateau at about 20%–24%. In some osteoblast domains, most of the cells were labeled (Fig. 3D), while in others, relatively fewer cells were labeled.
As early as 72 h after the first BrdU injection, labeled osteoblasts were beginning to become buried in matrix, thus becoming osteocytes. Labeled osteocytes were clearly evident in some bone formation domains by 120 h after the first BrdU injection (Fig. 3E).
In contrast with these results, the bone formation indices were considerably smaller in the lumbar cancellous bone in the rats that had completed and recovered from the first reproductive cycle but not mated the second time. These normal estrus cycling rats had relatively low rates of bone formation (Table 1 and Fig. 1). Because the active forming surface in these animals was a fraction of that in the postlactation animals, the osteoblast population was considerably smaller. There was some incorporation of labeled nuclei into bone surface cells (Fig. 3F), but they never exceeded 4% of the total population (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first purpose of this study was to determine how soon after weaning maternal indices of bone formation were observed to increase. The histomorphometric analyses showed that indices of cancellous bone formation were significantly increased within several days after weaning. The fraction of the bone surface covered by osteoblasts (osteoblast surface), for example, was significantly greater by the third day after weaning compared with the second day after weaning. Additionally, the osteoblast surface at this time was greater by about a factor of 10 than that observed in age-matched, normal estrus cycling, nonmated animals. Thus an acceleration of maternal skeletal osteogenesis and the initiation of the postlactation anabolic period occurs at or shortly after weaning.
The second purpose of this study was to determine whether the increase in the osteoblast population could be attributed to proliferation and differentiation of osteoprogenitor cells. As noted above, the osteoblast population rapidly expands after weaning, resulting in increases in bone formation rates. The results from the BrdU experiments clearly demonstrate that a considerable fraction of the osteoblasts that were appearing in this rapidly expanding population had incorporated a BrdU label. Thus, these BrdU-labeled cells had been derived from a progenitor that had presumably gone through a cell division. These proliferating progenitors could include stromal and other osteoprogenitor cells not directly adjacent to the bone surface, as well as bone-lining cells residing directly on the bone surface.
The derivation of osteoblasts from proliferating progenitors, as observed in the present study, is similar to the origin of osteoblasts during endochondral osteogenesis [21, 22, 30]. However, the development of osteoblasts during the natural physiological states of growth and postlactation appear to differ from bone induction by pharmacological administration of intermittent PTH. Dobnig and Turner [20] reported that the origin of new bone-forming cells following PTH administration did not require cell proliferation. They suggested that the source of the osteoblasts were from preexisting and postproliferative bone surface cells, including the bone-lining cell. In another study using mechanical stimulation as the osteogenic stimulus, the initial (48 h) expansion of the osteoblast population did not appear to be derived from proliferating progenitors but later (96 h), substantial BrdU-labeled osteoblasts appeared on bone surfaces [19]. In the present study, labeled osteoblasts were observed on bone surfaces by 25 h after the first BrdU label, and this is consistent with early 3H-thymidine studies in growing bone in which labeled osteoblasts began to appear within the same time frame [21, 22, 30].
There is some experimental evidence that the bone-lining cell retains osteogenic potential in the adult animal [31]. As noted above, the direct differentiation of bone-lining cells into osteoblasts, without a cell division, was reported to contribute to the osteogenic response to exogenously administered PTH [20]. We have previously presented evidence for the direct differentiation of bone-lining cells into osteoblasts as well as their apparent contribution to the proliferating progenitor pool during estrogen-induced bone formation in adult male birds [18, 32]. Domains of BrdU-labeled bone-lining cells were observed in this study, suggesting that these cells contributed to the proliferating pool of potential osteogenic precursors.
Osteocytes are formed by the burial of osteoblasts in matrix and are considered a terminally differentiated cell. Osteoblasts that were being buried, and thus developing into osteocytes, and which contained a BrdU label, were first observed in the postlactation animals by 72 h after BrdU administration. Completely buried osteocytes were readily evident by 120 h after BrdU administration. BrdU-labeled osteocytes were not observed in the normal estrus-cycling animals at these same time periods. This would indicate that the shortest life span of the osteoblasts in the postlactation animals was from 3 to 5 days, which is shorter than previously reported in other models. A transit time from administration of a DNA label to the appearance of osteocytes was reported to be about 9–10 days on endosteal and periosteal surfaces in mice [33] and on secondary haversian canals in dogs [34]. This suggests that the osteoblast life span may be shorter in the maternal skeleton at the beginning of the postlactation anabolic period, perhaps to accommodate the greatly increased bone deposition that occurs during this period.
The third purpose of this study was to correlate the events associated with the postlactation anabolic period with known changes in some potential endocrine mediators. Because of the rapid and dramatic expansion of the osteoblast population immediately after weaning, it seems that a large population of rapidly responsive osteoprogenitor cells must have been already present before weaning. It is possible that there was an expansion and priming of the osteoprogenitor pool during lactation, before weaning. This is supported by observations that labeled osteoblasts appeared at a more rapid rate in this study, consistent with the rapid kinetics of osteoblast development during normal growth [21, 22, 30], but it was different from exogenous induction through mechanical or pharmacological means [19, 20], as discussed above. Because the mineral requirements of the mammary gland are largely driving maternal mineral homeostasis during lactation, perhaps factors secreted from the mammary gland might have a role in the priming or expansion (or both) of the osteoprogenitor pool during lactation. In this regard, PTHLH is produced by the mammary gland during lactation [35] and is present in maternal serum [36, 37]. Accumulating evidence indicates an endocrine role for PTHLH in the regulation of bone resorption, turnover, and bone mass during lactation [26, 38, 39]. In vitro data suggest, interestingly, that PTHLH expands the osteoprogenitor pool without directly promoting osteoblast differentiation [40]. Thus is it possible that another endocrine function of PTHLH that is produced by the mammary gland during lactation is to either expand or prime the osteoprogenitor pool for the rapid phase of increased bone formation that follows weaning. Furthermore, because PTHLH may actually impede the differentiation of osteoblasts from osteoprogenitor cells [40], it is possible that the decreases in mammary gland production of the PTHLH at weaning may be a contributing endocrine initiator of the postlactation anabolic phase.
Prolactin is a primary endocrine regulator during lactation and PRL levels decrease rapidly upon separation of the pups from the dam [24]. There is evidence for a significant role for PRL in maternal skeletal metabolism. Short-term increases in PRL are believed to promote bone turnover (bone resorption and formation), perhaps during lactation [25], and changes in PRL during pregnancy may be associated with an increased intestinal absorption of calcium [41], a positive calcium balance, and skeletal mineral accrual [42]. Prolactin receptors have now been identified on osteoblasts, but not on osteoclasts [43]. Prolonged increases in PRL, however, are associated with an excess bone resorption (e.g., hyperprolactinemia) [44]. Because PRL may be associated with the maintenance of skeletal metabolism and bone turnover during lactation, the sudden decline in this hormone may also represent an important endocrine signal for the initiation or progression of the postlactation anabolic phase.
The return to estrus or initiation of menstrual cycles after weaning may also be associated with the maintenance and regulation of the postlactation anabolic period. Rats begin to return to estrus in late lactation and after weaning, although individuals vary, and have normal estrus cycles during the peak periods of bone formation for several months after lactation. In humans, the return of normal menstrual cycles has been associated with the restoration of bone mineral density and bone mass [12]. The precise roles of estrogen, progesterone, and other reproductive hormones in accrual of skeletal mass and strength during this period remain to be determined.
In summary, the postlactation recovery period appears to be the most anabolic phase in the life history of the adult female skeleton [5]. This study demonstrates that this phase is initiated immediately after weaning. Indices of bone formation rates in the maternal skeleton begin to increase within several days of weaning and, within the first week, are substantially elevated. The increases in bone formation at weaning can be attributed to a rapid expansion of the osteoblast population, resulting in a greater fraction of the total bone surface forming new bone. This study also demonstrates that proliferating progenitors are a substantial source of new osteoblast development in the postlactation recovery period in the mother. Incorporation of labeled nuclei into osteoblasts occurs within the first 24 h after BrdU label (2 days after weaning) and buried, labeled osteocytes are observed within 3–4 days. This represents a shorter life cycle of the osteoblast than previously reported during normal skeletal development. The endocrine regulation of these events is of considerable interest, but as of yet, and largely unknown, it may involve lactation hormones and their changes at weaning, perhaps including PTHLH, PRL, and resumption of cyclic gonadal hormone secretion with the reinitiation of estrus or menses.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Scott C. Miller, Division of Radiobiology, 729 Arapeen Drive, Suite 2334, Salt Lake City, UT 84108-1218. FAX: 801 581 7008; scott.miller{at}hsc.utah.edu
Received: 6 January 2005.
First decision: 4 February 2005.
Accepted: 17 March 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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