Evidence of sequential remodeling in rat trabecular boneMorphology dynamic histomorphometry and changes during skeletal maturation.код для вставкиСкачать
THE ANATOMICAL RECORD 208:137-145 (1984) Evidence of Sequential Remodeling in Rat Trabecular Bone: Morphology, Dynamic Histomorphometry, and Changes During Skeletal Maturation ROLAND BARON, ROBERT TROSS, AND AGNES VIGNERY Yale University School of Medicine, Departments of Medicine and Cell Biology, New Haven, CT 06510 ABSTRACT The occurrence of a sequential bone remodeling activity, similar to what is observed in human bone, is demonstrated in rat trabecular bone a t the level of the secondary spongiosa. A complete dynamic histomorphometric analysis of the remodeling activity, using undecalcified sections and double fluorescent labels, has consequently been performed in young adults (220 g, 8 weeks old) and in more mature animals (320 g, 12 weeks old). The results showed that, despite a similar trabecular bone volume, younger animals had a five times higher bone formation rate and five times more osteoclasts than more mature animals. The higher bone formation rate was due in part to a threefold higher extent of double-labeled trabecular bone surface and in part to a 1.5-fold faster mineralization rate. These results therefore demonstrate a marked slowing down of bone turnover during skeletal maturation in the rat. The values obtained in this study have been compared with measurements made in other parts of the skeleton in the same species (Vignery and Baron 1978, 1980b; Tran Van et al., 1982a) or in humans. This comparison indicated that 12-week-oldrats had a turnover rate very similar to values observed in iliac crest trabecular bone in adult humans. The rat is therefore a good experimental animal for the study of trabecular bone remodeling but since large variations occur during skeletal maturation, care should be taken in the selection of an age group relevant to the type of questions being asked. Although widely used for the assessment of bone remodeling activity in humans, dynamic histomorphometric techniques have seldom been applied to small laboratory animals like the rat. Partial studies have been published but they deal either only with cortical bone (Baylink et al., 1970; Tam and Anderson, 1980) or with limited measurements of trabecular bone activity (Schenk et al., 1973; Miller and Jee, 1975; Kimmel and Jee, 1980; Wronski and Morey, 1982) often made in the growth plate area. In addition, and because of the prolonged growth of this species, the fact that bone remodeling occurs in the rat similar to that in human trabecular bone, is still controversial (Frost, 1976). We have demonstrated in previous studies (Baron, 1973; Vignery and Baron, 1980b)that bone remodeling occurs in rat alveolar bone and follows the same sequence as that described in humans (Frost, 1964; Rasmussen and Bordier, 1974). We have also recently 0 1984 ALAN R. LISS, INC studied the cellular kinetics of a n induced bone remodeling sequence along the periosteum in the rat and showed that it was similar to the sequence observed in other species (Tran Van et al., 1982a). The aims of this study were therefore 1)to verify the occurence of such a sequential remodeling activity in adult rat trabecular bone; 2) t o measure this activity; 3) to compare it to the data we obtained in alveolar bone and along the periosteum of the same species and in humans; and 4) to evaluate the effect of skeletal maturation on bone remodeling activity in the rat. MATERIALS AND METHODS Twenty-four male Wistar-Lewis rats, 14 weighing 220 20 g (8 wk old) and 10 weighing 320 20 g (12 wk old) were used in this * Received January 14, 1983; accepted May 27, 1983. Address reprint requests to Dr. Roland Baron. 138 R. BARON, R. TROSS, AND A. VIGNERY study. They were fed a normal rodent diet (Purina) and tap water ad libitum for 14 days. They were housed in individual cages and on a 12 W12 h light-dark cycle. Double fluorescent labeling of their bones was performed by the intraperitoneal injection of calcein (DCAF, Merck) a t a concentration of 30 mg/ kg of body weight in a constant volume of 0.1 ml of a saline solution containing 2% NaHC03. The animals were injected a t days 7 and 13 and sacrificed a t day 14, 24 hours after the last calcein injection. Histology Two vertebrae in the proximal third of the tail were dissected out and fixed for 48 h in 40% ethanol a t 4°C. They were then dehydrated in increasing concentrations of ethanol and embedded in methyl-methacrylate without decalcification (Baron et al., 1983). Unserial undecalcified longitudinal sections, 4 pm thick, were performed in the central area of the vertebrae using a Jung K or a n Autocut microtome. These sections were stained with toluidine blue at pH 3.7 for morphometry. Sections 10 pm thick were also performed and kept unstained for observation and measurements using fluorescence microscopy. Morphometry All measurements were made in the trabecular bone with the exclusion of the area situated within 1 mm from the epiphyseal growth plates (Fig. 1)in order to exclude the primary spongiosa (Kimmel and Jee, 1980). Measurement of the following parameters were performed at a x 125 magnification using a MOP 3 Planimeter (Carl Zeiss): whole trabecular bone tissue (WTBT), trabecular bone volume (TBV), osteoid volume (OV), trabecular bone surface (TBS), osteoid surface (OS), osteoblastic surface (ObS), osteoclastic surface (OCS), number of osteoclasts (#OC), and reversal surface [Revs; i.e., Howship's lacunae not containing a n osteoclast (Baron, 197711. The mean wall thickness [MWT, i.e., the mean distance between the cement line and the bone surface in completed remodeling foci (Frost, 197711 was measured a t a using a micrometer, magnification of ~ 2 5 0 on sections that were decalcified and stained with toluidine blue at a basic pH (Fig. 2; Fig. 3b,c). The unstained sections were observed under ultraviolet (UV) light and measurement of TBS, double-labeled surface (2LS, Figs. 4b and 5), and single-labeled surface (ILS, Fig. 5) were made a t a magnification of x250. The mineralization rate (MR) was measured on the same sections a t a magnifi. calculations were made cation of ~ 4 0 0 All from these 14 measured parameters according to the method described by Frost (1977) and using the correction factor for the oblique) MWT and MR ness of the sections ( ~ 0 . 7 3for (Frost, 1977). Namely, TBV is expressed as percentage of WTBT; OV is expressed as percentage of WTBT [absolute osteoid volume (AOV)];OS, ObS, OCS, Revs, and total lacunar surface (TLS) are expressed as percentage of TBS, as well as the 1LS and 2 L S the mean osteoid thickness (OT) is calculated by dividing OV (pm3>by 0s (pm'); the #OC is divided by TBS (mm) and expressed a s the #OC/mm of TBS; the MR is calculated by dividing the mean interval between double labels (pm) by the number of days between the calcein injections and expressed a s p d day; the mineralization lag time (MLT) was calculated by dividing OT (pm) by MR ( p d day) and expressed in days. The bone formation rate (BFR) at the tissue level is calculated as a n index by multiplying the 2LS by the MR. BFR a t the cellular level (BFWOb) is calculated as a n index by dividing BFR by the extent of the ObS. The bone resorption rate (BRR) has been calculated as a n index a t the cellular level (BRWOC) by assuming that the bone volume remains constant and dividing the BFR by the #OC/mm TBS. The duration of the different steps of the remodeling sequence is estimated on the basis of the duration of the formation phase [Sigma F = MWT (pm)/MR (pdday)]; the duration of the other phases, namely Sigma R and Sigma reversal, are calculated as [Sigma F/ 0s (mm) x OCS (mm) or Revs (mm)] and expressed in days. The results are given as Fig. 1. Undecalcified section of a rat tail vertebra showing the primary spongiosa (ps) excluded from the measurements by eliminating 1 mm under the cartilaginous growth plate; the measured area consists of the secondary spongiosa (ss) excluding the midshaft and the cortices. Magnification, x 35. Fig. 2. Sections decalcified after sectioning and stained with basic toluidine blue allow a clear distinction between the primary spongiosa (a) in which the trabeculae contain cartilaginous remnants (cr, white arrows) and the secondary spongiosa (b) not showing cartilaginous remnants and where reversal lines (black arrows) delineate completed remodeling sites. Magnification, ~ 2 5 0 . TRABECULAR BONE REMODELING IN THE RAT 139 140 R. BARON, R. TROSS, AND A. VIGNERY Fig. 3. Morphologic evidence of bone remodeling in rat trabecular bone. Undecalcified sections (a) showing the various steps of cellular activity of the remodeling sequence with osteoclasts (OC), reversal sites (Rev, black arrow), and osteoblasts (Ob)lining osteoid (white arrow). After decalcification and basic toluidine blue staining, individual remodeling sites are demonstrated: 03)during the formation phase of remodeling (black arrows show reversal line); and (c) completed remodeling sites, one (white arrows) older than the other (black arrows). Magnification, ~ 4 0 0 . Fig. 4. (a) Bone-forming site viewed on serial undecalcified sections stained with toluidine blue (black arrow = osteoblasts; white arrow = osteoid) and under UV light (b)showing the double fluorescent label of the same site (black arrows). Magnification, ~400. Fig. 5. Fluorescent labels along a trabecula demonstrating a double-labeled area (2L) and a single-labeled area adjacent to it (1L). Magnification, ~400. 142 R. BARON, R. TROSS, AND A. VIGNERY the mean + 1SD. Statistical comparison between the two groups was made by the Student's t test and the difference considered significant when p < 0.05. RESULTS The distance between cartilaginous growth plates in the tail vertebrae used in this study is 8.7 f 0.1 mm in the 8-week-old rats and 9.2 & 0.2 mm in the 12 week-old-animals. An overall growth of 0.5 mm therefore occurred in 4 weeks a t the organ level (18 pdday). The overall size of the diaphysis marrow cavity is apparently not different in the two groups. Unlike the primary spongiosa, the trabeculae found in the secondary spongiosa do not have cartilaginous remnants in their center (Fig. 2). In addition to this lack of cartilage, direct evidence for the occurrence of sequential remodeling along the endosteum of trabecular bone in the secondary spongiosa is provided by the presence of completed remodeling sites outlined by scalloped reversal cement lines (Fig. 2b; Fig. 3b,c). Morphologic examination of bone cells along the trabeculae clearly show the occurrence of bone formation by osteoblasts (Figs. 3a, 4a), bone resorption by multinucleated osteoclasts (Fig. 3a), and reversal lacunae with active mononucleated cells within Howship lacunae (Fig. 3a), all occurring within well-delineated remodeling sites. The results of all measurements made on the trabecular bone of the tail vertebrae of the two groups of animals are shown in Table 1. Despite a similar TBV, there were marked differences in other parameters of bone remodeling between the two groups. In essence, the results show a much higher turnover rate in younger animals than in more mature rats. ObS is about twice as great and OCS about four times greater in 220-g rats; the specific density of trabecular surface being similar in the two groups for the area under study, this means that a higher amount of bone is turned over in the younger animals. Measurements of double fluorescent labels show the same trends: the total labeled surface is more than twice as great in the younger animals; this is essentially due to the double-labeled surface which is close to four times higher in the younger group. In addition, the daily MR is decreased by about 25% in the more mature animals. The rate a t which bone matrix is laid down TABLE 1. Dynamic morphometry of rat tail trabecular bone remodeling' 220 g (8 wk) (n = 14) TBV (% tissue) AOV (% tissue) 0s (% trab. S) ObS (9% trab. S) OCS (8 trab. S) Revs (% trab. S) TLS (% trab. S) OT (pm) MWT (pm corrected) 1Lab S (% trab. S) 2 Lab S (% trab. S) MR (pmiday, corrected) MLT (days) BFR index (tissue level) BFR/Ob index BRRiOC index 320 g (12 wk) (n = 10) 18.2 (1.6) 17.5 (1.7) 1.4 (0.3) 0.9 (0.3)' 41.1 (3.8) 22.0 (6.8)** 28.9 (4.7) 14.3 (6.9)** 3.4 (1.3) 0.9 (0.6)** 3.2 (1.0) 2.6 (1.0) 6.6 (1.4) 3.5 (1.3)** 8.2 (1.0) 5.8 (0.8)** 18.8 (3.8) 15.1 (4.5) 34.3 (9.7) 25.0 (13.2) 37.5 (8.9) 10.4 (5.5)** 0.61 (0.08) 0.45 (0.05)** 13.4 (1.2) 12.9 (0.9) 22.9 (3.2) 4.7 (4.1)*** 7.8 (3.6) 3.3 (3.3)* 5.9 (3.9) 6.7 (4.6) For abbreviations, see text, Materials and Methods. Lab S , labeled surface; trab. S, tTabecular surface. *Mean ? SD. Significant differences between the two groups: *p < 0.05; '*p < 0.01; ***p < 0.0001. by osteoblasts is, however, proportionally decreased since the mean osteoid seam thickness is reduced by about the same amount in older rats ( 30%), maintaining the MLT constant (MLT = 13 days). The additive effects of this higher MR, higher 2LS, and greater extent of active ObS are demonstrated by the calculated dynamic parameters: the BFR a t the tissue level is close to five times higher in the younger animals. The TBV being similar in the two groups, this is therefore a direct reflection of the turnover rate, and the bone resorption rate is most likely also five times higher in the younger group. No differences are, however, seen at the cellular level of osteoclastic bone resorption, each osteoclast resorbing about the same amount of bone in the two groups, with the TBV remaining constant. This also indicates that the BFR is decreased a t the cellular level in the older animals, since there is a five times lower BFR at the tissue level with only a 3 . 7 ~drop in 2LS. All these results clearly indicate a major change in the birth rate or size of bone remodeling units at the tissue level: the higher turnover rate in younger animals is almost entirely attributable to a higher number or larger size of remodeling units and only partially to a higher formation rate at the individual cell level. Calculated durations of the different phases of the bone remodeling sequence in the two groups of animals (Table 2) show that the reversal phase takes a longer time in older animals - TRABECULAR BONE REMODELING IN THE RAT 143 (formation of bone not preceded by resorption) in younger animals, our data show that this is not the case, at least in the secondary 8 wk old 12 wk old spongiosa. If the ratio of ObS to OCS is taken 2.5 (0.9) 1.4 (0.9) Sigma resorption as a reflection of the balance between formaSigma reversal 2.4 (1.0) 4.2 (0.8)* tion (F)and resorption (R), it is much higher 31 (4) 33 (6) Sigma formation (15.9) in older animals than in younger aniTotal (sigma BMU) 36 (7) 38 (7.5) mals (8.5); this, however, does not take into BMU, basic multicellular unit; see Frost (1964). account the rate a t which bone matrix is *Significant difference between groups: p < 0.05 formed and mineralized. A better indication of the balance between F and R is given by with a slower turnover rate, but that the calculating the ratio of the BFR, which takes active resorption phase is slightly shorter, into account the MR, to the OCS: this ratio leaving the total duration of each remodeling (6.7 VS. 5.2) shows that the balance between F and R is very similar in the two groups, cycle unchanged. therefore explaining the similarities in TBV DISCUSSION and TBS specific density. Three conclusions This study shows that despite the continu- can be drawn from this observation: 1)the ous growth of this animal, sequential bone secondary spongiosa of tail vertebrae is alremodeling occurs in the secondary spon- most entirely subjected to remodeling activgiosa of tail vertebrae trabecular bone in the ity in the rat, independently of the age of the rat, as demonstrated by the presence of typi- animals; 2) bone formation and resorption cal completed remodeling sites with reversal are coupled and balanced in both groups, only cement lines and by dynamic histomorphom- the turnover rate is different (about five etry. Trabecular bone therefore constitutes times higher in younger animals); and 3) the the third instance in which we have shown net positive balance at the organ level, rebone remodeling activity in this animal, the sulting in growth, is the result of the net two others being alveolar bone (Baron, 1973; formation of new bone tissue a t the growth Vignery and Baron, 1980b) and the man- plate and primary spongiosa, not of a positive dibular periosteal surface (Tran Van et al., balance and/or modeling activity in the sec1982a). The fact, however, remains that no ondary spongiosa. It is important to underHaversian remodeling of cortical bone can be line the fact that this might be true of tail demonstrated in this species and this is very vertebrae but less so for the metaphysis of likely the reason why it was felt that no growing long bones in the same animal. remodeling occurred in the rat (Frost, 1976). Kimmel and Jee (1980) have shown a growth One has, however, to exclude the growth of about 170 pmlday in 170g rat long bones, plates and primary spongiosa from the mea- a value 10 times higher than the growth rate sured areas in order to avoid mixing growth of the tail vertebrae in this study (220-320 g and remodeling activities (Kimmel and Jee, rats). In addition, the height of the meta1980).In addition, this study shows that, sim- physis in growing long bones is maintained ilar to humans, skeletal maturation profound- constant by a net resorption of trabecular ly affects the remodeling activity in the rat bone toward the diaphysis, allowing a n exand, according to the goal of each specific pansion of the marrow cavity, when such study, one should select the experimental an- phenomenon is much less apparent in these slowly growing tail vertebrae. Incidentally, imal at a specific age. The data obtained in our two groups of rats the data obtained in normal childrens’ iliac are strikingly similar to the data obtained in crest trabecular bones (Bulla et al., 1979; children and adult human trabecular bone Glorieux et al., 1980; Marie and Gloriew, remodeling, respectively. Here in the normal 1981; Marie et al., 1982; Baron et al., 1983) rat, as well as in normal humans (Marie et when compared to normal adults (Meunier et al., 1982; Baron et al., 19831, we see that al., 1973; Schenk, 1976; Schulz and Delling, despite a n approximately similar TBV, the 1976; Melsen and Mosekilde, 1978; Frost et extent of bone-forming surface (ObS and 2LS) al., 1981) suggest that the same situation as well as the BFR (MR, OT) are higher in occurs in humans. Similar to humans, we also observed in younger animals. Although this could be interpreted superficially as indicating a more this study a lower MR in adults than in positive balance or the presence of modeling younger animals. Since this decrease in MR TABLE 2. Duration (in days) of the different phases of the remodeling sequence 144 R. BARON, R. TROSS, AND A. VIGNERY is associated with a thinner osteoid seam and a constant MLT, it suggests that the BFR is lower a t the level of the osteoblast. With BRR a t the level of the osteoclast remaining constant, this should lead to a net bone loss. This, however, occurs neither in our two groups nor in humans between childhood and maturity, because of a more profound decrease in the number of resorbing osteoclasts ( - 4 X ) than in the osteoblast-covered trabecular surface ( - 2 x), therefore reestablishing a n equilibrium between bone formation and bone resorption a t the tissue level. The calculated durations of the different phases of the remodeling sequence in this study have to be compared between the two groups and also with data obtained on the same animal in three other instances (Vignery and Baron, 1978; Vignery and Baron, 1980a; Tran Van et al., 1982a) and in humans Wignery and Baron, 1980a). The most striking observation in all these instances is the similarity of the duration of the active resorption phase between the species (rat vs. human), the sites (alveolar bone, periosteum, trabecular bone), the circumstances (normal vs. induced remodeling), and the method of measurement (directly measured vs. calculated). In all these studies, osteoclastic resorption lasts from 1.5 to 2.5 days (2.2 0.7). This value differs markedly from values published by others for human bone resorption ( - 15 days, Frost, 1963, 1977; Meunier et al., 1973)because these authors use the TLS for calculation, without making the difference between OCS and Revs (Baron, 1977; Baron et al., 1981). These are, however, two very different steps in the remodeling sequence and should be considered separately (Baron, 1977; Vignery and Baron, 1980a; Baron et al., 1981; Tran Van et al., 1982a,b).The similarity in duration of osteoclastic resorption suggests that the normal active lifespan of osteoclasts at one site is somewhere around 2 days in these two species. The duration of the reversal phase seems, on the other hand, to vary more widely in all the studies mentioned above. The only instance in which it was directly measured in the rat showed a duration of 4 days (Tran Van et al., 1982a), a value similar to alveolar bone remodeling calculations (Vignery and Baron, 1980a). In the present study, this duration goes from 2.6 in the younger animals to 4.2 days in the older ones. The proportional duration of this phase, expressed as a ratio with the osteoclastic phase, is a reflec- + tion of the coupling F-R (Baron, 1977; Baron et al., 1981). This ratio is 3 in alveolar bone (Vignery and Baron, 1980a1, 2 in periosteal remodeling (Tran Van et al., (1982a1, and here 3 in older animals and 1 in younger ones where the turnover rate is higher. In humans, this ratio is 5 in normal adults and children but goes up to 20 in individuals with senile osteopenia and down to 1 or 2 in persons with balanced high-turnover states like hyperparathyroidism and Paget’s disease (Baron et al., 1981). This suggests that the time necessary for coupling during the reversal phase might be inversely proportional to the turnover rate. In conclusion, this study shows that sequential remodeling occurs in the secondary spongiosa of rat trabecular bone. As in humans, young adults have a higher turnover rate than more mature animals but both groups have a n equilibrated balance between bone formation and resorption. The continuous growth of this animal is therefore mostly attributable, at least in tail vertebrae, to events taking place at the growth plate and primary spongiosa, leading to a n increase in size and absolute bone mass a t the organ level. ACKNOWLEDGMENTS This work was supported by grant DE 04724 from the National Institutes of Health. The authors would like to express their thanks to Mrs. Lynn Neff and Mrs. Ann Silverglate for their expert technical help and to Mrs. Barbara Devlin for typing the manuscript. LITERATURE CITED Baron, R. (1973) Remaniement de 1’0s alveolaire et des fibres desmodontales au cours de la migration physiologique. J. Biol. Buccale, 1:E-170. Baron, R. (1977) Importance of the intermediate phase between resorption and formation in the understanding and measurement of the bone remodeling sequence. 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