THE ANATOMICAL RECORD 291:283–292 (2008) Ontogeny of Skeletal Maturation in the Juvenile Rat JASON A. HORTON,* JASON T. BARITEAU, RICHARD M. LOOMIS, JUDITH A. STRAUSS, AND TIMOTHY A. DAMRON SUNY Upstate Medical University, Department of Orthopedic Surgery, 3120 Institute for Human Performance, Musculoskeletal Sciences Research Center, Syracuse, New York ABSTRACT Systemic regulation of the cellular processes that produce endochondral elongation and endochondral mineralization during postnatal skeletal maturation are not completely understood. In particular, a mechanism coupling the decline of cellular activity in the bone microenvironment to the onset of sexual maturity remains elusive. The purpose of this study was to empirically integrate the dynamic progression of bone mineral accrual and endochondral elongation as a function of animal age in growing male and female Sprague-Dawley rats. We used serial dual-energy X-ray absorptiometry (DXA) and radiography to study the temporal progression of bone growth and mineral accrual from weaning to adulthood. We observed that skeletal maturation proceeds in a pattern adequately described by the Gompertz function. During this period of growth, we found that serum markers of osteoblastic bone formation declined with age, while osteoclastic bone resorption activity remained unchanged. We also report a slight lag in the age at inﬂection in the rate of bone mineral accrual relative to the rate of tibial elongation and that both endochondral processes eventually come to asymptotic equilibrium by approximately 20 weeks of age. In addition, we studied tibial growth plate histomorphometry at select time points through 1 year of age. We report that, despite the histologic persistence of physeal cartilage, very little proliferative or elongative activity was measured in this tissue beyond 20 weeks of age. Taken together, these data provide insight to the temporal coordination of postnatal endochondral growth processes. Anat Rec, 291:283–292, 2008. Ó 2008 Wiley-Liss, Inc. Key words: skeletal maturation; DXA; endochondral mineralization; endochondral elongation; osteocalcin; TRAP5b; bone density Conceptually, postnatal skeletal maturation in mammals may be considered an ex utero extension of a developmental process begun early in embryonic life (Tuan, 2004; Provot and Schipani, 2005). From the onset of chondrogenesis in the developing limb bud, the processes of endochondral elongation and endochondral ossiﬁcation proceed along a nearly exponential trajectory (Goldring et al., 2006). However, as the individual ages, this growth rate subsequently declines toward asymptote as sexual maturation is achieved (Kilborn et al., 2002). However, the mechanism coupling of these processes is not well understood (Engelbregt et al., 2004). The rapid progression of postnatal development in the Sprague-Dawley rat combined with recent developments Ó 2008 WILEY-LISS, INC. Grant sponsor: NIH/NCI-RO1; Grant number: CA83892; Grant sponsor: David G. Murray Endowment. *Correspondence to: Jason A. Horton, SUNY Upstate Medical University, Department of Orthopedic Surgery, 3120 Institute for Human Performance, Musculoskeletal Sciences Research Center, 505 Irving Avenue, Syracuse, NY 13210. Fax: 315-4646638. E-mail: email@example.com Received 7 February 2007; Accepted 18 November 2007 DOI 10.1002/ar.20650 Published online 29 January 2008 in Wiley InterScience (www. interscience.wiley.com). 284 HORTON ET AL. in various radiologic, biochemical, and histomorphometric methodologies make this organism an attractive model for the study of skeletal maturation. Radiographic technologies such as dual-energy X-ray absorptiometry (DXA) and quantitative computed tomography (QCT) are widely used in clinical bone densitometry. Recently, these imaging modalities have been developed for use with small animal laboratory models, providing the basic scientist with data that are clinically relevant and readily translated (Ammann et al., 1992; Horton et al., 2003). Similarly, histomorphometric, biochemical, and serological methods have been developed to determine the rate of osteoblastic bone synthesis, osteoclastic bone resorption, and growth plate chondrocyte proliferation occurring in the bone microenvironment. Although data regarding skeletal growth and mineral content have been available for almost a century (Donaldson, 1915), few investigators have endeavored to study the process of skeletal maturation by combining radiologic, histomorphometric, and serologic approaches as we have here in a commonly used laboratory small animal. The purpose of this study was to integrate, as a function of animal age, the dynamic processes of bone mineral accrual and endochondral elongation from weaning to growth equilibrium. We hypothesized that these processes would conform to the Gompertzian growth function and that there would be a strong association between the rate of bone elongation, mineralization, and the metabolic activity of the cells that are directly responsible for promotion of the aspects of skeletal maturation in rats. MATERIALS AND METHODS Animal Subjects and Husbandry Weanling 28-day-old male (n 5 6) and female (n 5 6) Sprague-Dawley rats (Taconic Farms, Germantown, NY) were selected for serial examination of bone density and longitudinal growth. Five additional groups of n 5 3 male rats each 42, 63, 112, 140, and 350 days old were obtained to provide tissue for histomorphometry. In the laboratory, this species has been reported to achieve sexual maturity at 45–55 days in males and 36–38 days in females (Ojeda and Urbanski, 1994; Engelbregt et al., 2004). All animals were housed three-per-cage under ambient conditions of 228C and 50% humidity. All animals had ad libitum access to standard rat chow and tap water. Daily photoperiods were 12-hr light/dark. Animals were allowed to acclimatize to the local environment for 1 week before study initiation. At the conclusion of the study, all animals were humanely euthanized by carbon dioxide inhalation. All experimental procedures described in this study were performed according to a protocol approved by our Institutional Animal Care and Use Committee. Radiography and DXA Bone Densitometry Beginning at 28 days of age, and on a semiweekly schedule thereafter, all animals in each of the two groups of six rats each were anesthetized by an intramuscular injection of a tiletamine/zolazepam cocktail (30 mg/kg Telazol, Fort Dodge Animal Health, Fort Dodge, IA). Upon sedation, animals were placed in a supine posture and immobilized in such a manner that the hip, knee, and ankle joints were held at 90 degrees ﬂexion (Horton et al., 2003). The animals were then positioned on the scanning bed of the PIXImus2 (GE-Lunar, Madison, WI) small animal research DXA bone densitometer. Unilateral images were obtained independently for each hindlimb inclusive of the respective tibia, femur, lumbar spine, and pelvic anatomy in each scan; animals were then repositioned, and scans were repeated for the contralateral limb. After image capture, regions of interest were placed around the entire femur and lumbar spine, and readings of bone mineral content and projected area were determined from the relative attenuation of highand low-energy X-ray beams. The expression areal bone mineral density (aBMD, mg/cm2) describes the distribution of bone mineral within the selected anatomy per unit projected area. Here, we have elected to present our data as aBMD, as this expression normalizes the amount of bone mineral to the rate of change in bone size (Horton et al., 2003). Densitometric values for both femurs were averaged for each animal, while the reported density of the lumbar spine reﬂects the average value of the two views taken for each of the hindlimbs. Before anesthetic recovery, animals were placed into a prone position, maintaining 90 degrees ﬂexion as described previously, and contact radiographs were taken with a calibration marker used to normalize images. Tibial length was measured from digitized ﬁlms as the axial distance from the tibial plateau distally to the apex of the tibio-talar articulation (Tamurian et al., 1999), using NIH image v1.62 (National Institutes of Health, Bethesda, MD). Data reported are the average of three determinations for each bone measured. Coefﬁcients of variation were less than 0.3% for repeated measures. Blood Collection and Metabolic Marker Assays Blood was obtained by tail venipuncture as described by Bober (1988) while animals were brieﬂy anesthetized by isoﬂuorane inhalation. Serum was then isolated and stored at 2808C until analysis. A two-site immunoradiometric assay was used to determine serum levels of osteocalcin (Immutopics, San Clemente, CA), a 50 amino acid peptide associated with osteoblastic bone formation activity (Morris et al., 1992). An enzyme-linked immunosorbent assay (RatTRAP ELISA Kit, SBA-IDS, Fountain Hills, AZ) was used to evaluate temporal patterns of bone resorptive activity (Halleen et al., 2000; Alatalo et al., 2003), as reﬂected by tartrate-resistant acid phosphatase isoform 5b (TRAP5b), which is secreted into the serum by osteoclast cells actively engaged in bone resorption. Assays were performed in duplicate for each sample following the kit manufacturer’s protocol. Histomorphometry and Immunohistochemistry Forty-eight hours before being euthanized, animals received an intraperitoneal injection of oxytetracycline (OTC, 50 mg/kg, Phoenix Pharmaceuticals, St. Joseph, MO) to pulse-label newly mineralizing tissue. Thirty minutes before being euthanized, animals received an intraperitoneal injection of 50 -bromo-2-deoxyuridine (BrdU, 25 mg/kg, Sigma, St. Louis, MO) to label cells engaged in DNA synthesis. Immediately after euthanasia, the proximal 1.5 cm of both tibiae were collected, bisected sagittally, and ﬁxed in 70% ethanol. Tissue SKELETAL MATURATION IN JUVENILE RATS 285 Fig. 1. Measurement of daily growth rate by oxytetracycline (OTC) migration. A: Corresponding digital images (310 magniﬁcation) of the proximal tibial metaphysis of a 42-day-old male rat captured by epiﬂuorescence microscopy with a UV-1a ﬁlter set (Ex. 365nm/Em. 400 bandpass) or by phase contrast microscopy. B: To facilitate measure- ment of the daily growth rate (see also Fig. 3a, inset), regions identiﬁed as displaying OTC ﬂuorescence in image A were superimposed (in red). Subsequently, the average distance between these contours were measured (arrow). hemispheres were subsequently embedded in methylmethacrylate resin (Erben, 1997), serially sectioned at 5 mm thickness, and mounted on aminopropyltrimethoxysilane subbed slides. Six sections were selected at uniform random 80 mm thickness intervals and prepared for either BrdU immunohistochemistry or OTC-ﬂuorescence microscopy. ImagePro Plus software (v4.0, Media Cybernetics, Silver Spring, MD), was used to perform all histologic analyses from digitally captured images taken at 310 magniﬁcation. For estimation of daily growth rate, three tissue sections were sampled as described earlier, de-plasticized in xylene, and cover-slipped. Two corresponding digital images of each unstained tissue section were obtained under brightﬁeld phase contrast or UV1a epiﬂuorescence (Ex. 350 nm/ Em. >400 nm bandpass) illumination, respectively (Fig. 1). Subsequently, ImagePro Plus was used to merge these images, and the distance between the metaphyseal chondro-osseous junction and the front of OTC-labeled mineralized matrix was measured as described previously (Farnum and Wilsman, 1989; Damron et al., 2006; Horton et al., 2006). For this technique, coefﬁcients of variation were less than 0.5% in repeated measurements of any given section (e.g., investigator error) and 3–8% between uniform random sections sampled from any tissue block (e.g., biological variability). Immunohistochemical labeling of BrdU incorporation was performed following antigen retrieval as previously described by Baroukh et al. (2000), using a mouse antiBrdU monoclonal antibody (#347580, 1:100, Becton Dickinson, San Jose, CA), and visualized through horseradish peroxidase–diaminobenzidine histochemistry (SK4100, Vector Laboratories, Burlingame, CA). An index of proliferative activity was then calculated as outlined in our earlier reports (Damron et al., 2006; Horton et al., 2006). In brief, we calculated the proportion of BrdU-immunoreactive cellular proﬁles per total cellular proﬁles in the selected sections. This proportion was then modiﬁed by application of a correction factor to account for the probability of observing a nucleated cellular proﬁle in any randomly selected cellular proﬁle observed in a tissue section less than one cell-diameter in thickness (Farnum and Wilsman, 1993). Statistical Analyses Data mean and standard deviation were calculated for each parameter measured at each time point and plotted against the animal age at the time of measurement using Excel Software (Microsoft Inc., Redmond, WA). All other statistical functions were performed using StatView v5.0.1 software (SAS Institute, Cary, NC). Analyses of var- 286 HORTON ET AL. iance with Bonferroni-Dunn post hoc tests were performed to identify successive periods of signiﬁcant growth accumulation. Unpaired Student’s t-tests were performed to demonstrate gender divergence at each time point, accepting differences as signiﬁcant when P 0.05. Simple and multiple regression analyses were used to assess the signiﬁcance and degree of association between variables. The measured data for body weight gain, femoral and lumbar bone mineral accretion, and tibial elongation were ﬁtted to a Gompertzian growth curve of the form described by Laird (1965a,b, 1967). This function temporally describes growth procession through successive phases of rapid, decaying, and asymptotic growth. Within this function, iterative regressions with exponential, logistic, and linear models were used to estimate the age at which transition between the respective periods would occur. RESULTS AND OBSERVATIONS Serial Assessment of Bone Mineral Accrual by DXA Measurements of bone mineral content aBMD by DXA are shown for the femur (Fig. 2) and lumbar (L1–L4) vertebral segments (Fig. 3). Initially, bone mineral density rapidly accrued on an exponential trajectory in both male and female rats before decay in growth became apparent at 78 days of age for the femur (r2 0.98; Fig. 2) or at 71 days in the lumbar spine (r2 0.97; Fig. 3). Beyond 78 days of age, femurs from male rats had accumulated signiﬁcantly greater aBMD (t-test; P 0.043) than age-matched females. The lumbar spine of male rats showed greater aBMD than female counterparts only at 113 and 284 days of age (P 0.018). No statistically signiﬁcant difference was demonstrated in lumbar aBMD between sexes at any other time point. decayed, and no statistically signiﬁcant growth was observed beyond 109 days of age in females (r2 5 0.84; P 5 0.189) and 144 days in males (r2 5 0.80; P 5 0.73) when measured radiographically. This ﬁnding may reﬂect an insensitivity to measured differences of less than approximately 0.05 mm by this method. Thus, a histomorphometric OTC pulse-migration study (Fig. 2a, inset) was performed at select time points to estimate daily growth rate with much greater resolving power than possible by radiography. Growth rate was measured in 42-day-old males during the mid-exponential growth phase and found to proceed at average rate of 240 6 5 mm/day by OTC migration (Figs. 5, 6). Temporally signiﬁcant declines in growth rate were also observed between initial measurement and the 71-day early- (101 6 5 mm/day) and 112-day late- (44 6 7 mm/day) decay phases (ANOVA, P 0.002) measurements. Tibial growth rate of animals sampled at either early- (16 6 2 mm/day at 140 days) or late- (7 6 4 mm/day at 350 days) time points during plateau phase were not signiﬁcantly different from each other (P 5 0.2), but were both signiﬁcantly reduced compared with all previous time points (ANOVA, P 0.0001). Furthermore, regression analysis showed a very strong inverse association (r2 > 0.99) between tibial length and growth rate. An index of proliferative activity of epiphyseal growth plate chondrocytes was calculated by BrdU immunohistochemistry (Fig. 6). Proliferative activity decreased signiﬁcantly with advancing age (P < 0.003). Additionally, the proliferative index calculated from 351 day old tissues (1.51 6 1.70%) was not signiﬁcantly different from 0. Regression analysis suggested an association between OTC growth rate (r2 5 0.81) and tibial length (r2 5 0.83). CONCLUSIONS AND DISCUSSION Serum Markers of Osteoblast and Osteoclast Activity Serum markers of bone metabolic activity were assayed at time points corresponding to the period of signiﬁcant of bone growth and mineral accrual (Fig. 4). Serum levels of osteocalcin suggest that the rate of bone formation decreases signiﬁcantly with increasing age in both males and females (analysis of variance [ANOVA], P 0.016). There were no signiﬁcant gender differences at any time point (t-test, P > 0.102). Serum TRAP5b levels did not ﬂuctuate signiﬁcantly during the period observed serially within either sex (ANOVA, P 0.182). Gender differences were generally unremarkable for both serum osteocalcin and TRAP5b (t-test, P > 0.206), although there was a statistically signiﬁcant difference between sexes in TRAP5b levels noted at 56 days of age (t-test, P 5 0.043). Regression analysis showed a positive association between serum osteocalcin levels and BMD (lumbar r2 5 0.74, femoral r2 5 0.71). However, TRAP5b levels were not as closely associated with BMD at either site (r2 < 0.41). Serial Studies of Endochondral Elongation Measurements taken from contact radiographs revealed that tibiae in both male and female rats grew at a nearly exponential rate (r2 0.91) through 64 days of age (Fig. 2). This rate of elongation subsequently Several growth functions have been proposed, which are intended to describe the temporal progression of ontogenetic processes (Kingsland, 1995). Common among several of these formulae is the ability to derive an inﬂection point from empirical data, which estimates the age at which some process alters the trajectory of growth from an exponential rate toward decay. We have imputed our experimental data to a Gompertzian function of the form derived by Laird (1965a,b, 1967) and report that the overall process of skeletal maturation proceeds along a trajectory adequately predicted by this function. Before the calculated inﬂection point, however, we found that our data regarding tibial elongation and bone mineral accrual ﬁt more closely to the trajectory of an exponential model (Figs. 2–4 dashed lines), than to the Gompertz. Similarly, subsequent growth rate decay and asymptotic phases were respectively found to ﬁt more closely to curves generated by logistic and linear models than to the Gompertz during these phases. Postnatal skeletal growth and maturation is a complex process that couples endochondral elongation and mineralization to osseous modeling and re-modeling. It has long been assumed that bone mass acquisition during childhood and early adulthood is of paramount importance in prevention of osteoporotic bone disease later in life (Loro et al., 2000). In humans, there appears to be a relatively short but crucial time period during which most signiﬁcant bone mineral accrual occurs (Gilsanz SKELETAL MATURATION IN JUVENILE RATS Fig. 2. Femoral bone mineral density (BMD). a,b: Data points show mean (6 1 SD) femoral BMD of male (a) and female (b) rats as determined by in vivo dual-energy X-ray absorptiometry (DXA) bone densitometry. Solid lines show Gompertzian best-ﬁt of data, while dashed lines show extended trajectory of the exponential growth phase. 287 288 HORTON ET AL. Fig. 3. Lumbar bone mineral density (BMD). a,b: Data points show mean (6 1 SD) lumbar BMD of male (a) and female (b) rats as determined by in vivo dual-energy X-ray absorptiometry (DXA) bone densitometry. Solid lines show Gompertzian best-ﬁt of data, whereas dashed lines show extended trajectory of the exponential growth phase. SKELETAL MATURATION IN JUVENILE RATS 289 Fig. 4. Serum markers of bone metabolic activity. Data points show mean (6 1 SD) serum level of TRAP5b (solid line, U/L) and osteocalcin (dashed line, ng/ml). and Nelson, 2003; Mora and Gilsanz, 2003). This period of peak bone mineral accrual lags slightly behind the 2year period of peak longitudinal growth during puberty (Fournier et al., 1997; Baroncelli and Saggese, 2000; Whiting et al., 2004). The inﬂection points derived from this model demonstrate that there is a temporal lag between the processes of endochondral elongation and mineralization and that these inﬂection points occur slightly after the age at which these animals have been reported to achieve sexual maturity. Studies of growing mice (Eckstein et al., 2004), rabbits (Gafni et al., 2002), and adolescent humans (Neu et al., 2001), are analogous to our ﬁndings in rats, suggesting a general pattern of decreased bone formation rate approaching asymptote. Our present experiment revealed that the decline n serum osteocalcin was positively associated with both femoral and lumbar bone mineral accrual during growth, while serum TRAP5b was not closely associated. Our observation of an age-associated decline in serum osteocalcin levels in growing rats was similar to that reported in mice by Eckstein and colleagues (2004). However, in the current study we did not observe a reduction of serum TRAP5b levels in aged rats that corresponded to the reported decline in urinary markers of bone resorption in the former study. Such disparity between data sets may be related to species differences between the animals used in the respective studies, or a difference in the assay method used. Humans experience a brief period of preadolescent growth quiescence preceding the pubertal growth spurt, which corresponds temporally to the initiation of sexual maturation (Arfai et al., 2002; Braillon, 2003). This speciﬁc pattern is not observed in rats. Rather, skeletal growth of the rat proceeds continuously through the juvenile and prepubertal phases along an exponential trajectory evident at birth (Laird, 1965b; Kilborn et al., 2002), which later decays as a function of animal age and endocrine activity. In this study, we have observed that signiﬁcant longitudinal growth enters a decay phase at approximately 64 days of age, lagging slightly behind the reported age at which sexual maturity is assumed by the appearance of mature spermatozoa in the vas deferens in male (45–55 days), or the ﬁrst ovulation in female (35–40 days) rats (Ojeda and Urbanski, 1994). Although our data appear to provide a compelling temporal correlate of skeletal and sexual maturation, our study was not designed to relate our radiographic assays of skeletal maturation to the processes of puberty and sexual maturation. Thus, our data do not speciﬁcally address the commonly held notion that skeletal and sexual maturation processes are mutually responsive to endocrine inﬂuences governed by the hypothalamo–pituitary axis (Ojeda and Urbanski, 1994; Robson et al., 2002). The sequential pattern of growth plate thinning and closure is generally considered to be a very sensitive correlate of skeletal maturity to endocrine status and 290 HORTON ET AL. Fig. 5. Tibial length and elongation. a,b: Data points show mean (6 1 SD) tibial length of male (a) and female (b) rats. Solid lines show Gompertzian best-ﬁt of data, whereas hatched lines show extended trajectory of the exponential growth phase. Inset a shows mean (6 1 SD) oxytetracycline (OTC) growth rate derived from histologic analysis of age-matched male rats. SKELETAL MATURATION IN JUVENILE RATS 291 Fig. 6. Growth plate chondrocyte proliferation. Data show the mean (6 1 SD) proliferative index derived from immunohistochemical labeling of bromodeoxyuridine (BrdU) incorporation by S-phase proliferative chondrocytes. Inset: Representative BrdU immunohistochemis- try at 42 (A), 63 (B), 112 (C), 140 (D), and 350 (E) days of age; original magniﬁcation, 310. A–D counterstained with hematoxylin; E counterstained with methyl green. Scale bar 5100 mm. chronologic age (Zerin and Hernandez, 1991). In some species, the epiphysis may remain ‘‘open’’ for some time beyond the achievement of sexual maturity (Kilborn et al., 2002). As previously demonstrated in rats by both Trudel et al. (2001) and Roach et al. (2003) and others, we observed histologically ‘open’ physes in rats at 1 year of age. In departure from the former and in agreement with the latter study, we measured no signiﬁcant proliferative activity or daily growth accumulation by the proximal tibial growth plate by our pulse labeling studies at this late time point. In addition, we have presented data obtained by both radiologic measurement of tibial length and histomorphometric measurement of OTC-based daily growth rate, indicating that beyond 20-weeks of age, tibial growth had declined to a rate that produced only statistically insigniﬁcant gains. Our inability to detect any signiﬁcant elongation in these older animals, as has been reported by others, may reﬂect a limitation of sensitivity inherent in the measurement approaches we have taken. However, that we fail to see substantial BrdU uptake in the growth plate and that there are few remaining chondrocytes of either proliferative or hypertrophic morphology in the growth plate of these older animals (Fig. 6E) suggests that whatever growth accrues at these advanced ages is not likely a product of chondrocytic duplication and hypertrophic enlargement (Wilsman et al., 1996). Rather, it is possible and indeed quite likely that this residual elongation and may be the product of some interstitial growth mechanism such as matrix secretion (Pavasant et al., 1996), rather than the product of a persistent primary endochondral process. It was our purpose in this study to examine skeletal maturation during the transition from preadolescentto-adult, rather than the preceding neonatal phase. As such, we are unable to comment on any gender or temporal patterns established during the earlier periods of growth or on whether patterns established during this period are analogous to those observed in humans (Gafni et al., 2002). Second, our histomorphometric observations on the decay of longitudinal growth rate as a function of age considered only the proximal tibial growth plate. It is widely known that various growth plates throughout the body display different growth rates (Wilsman et al., 1996) and that they close in a speciﬁc, reproducible sequence (Zerin and Hernandez, 1991). Finally, our experiment did not examine the role of endocrine inﬂuences on skeletal biology, so we are limited solely to discussion of the temporal correspondence of these developmental processes of maturation, rather than any speciﬁc mechanism coordinating their progression. In the long-term, we plan to use this model to 292 HORTON ET AL. study what role that insults affecting skeletal metabolism and growth during childhood and adolescence, may have on adult bone quality. The methodologies used to study this progression are quite analogous to those used in clinical practice, and thus are geared toward facilitating studies that are translational in nature. ACKNOWLEDGMENTS This research was conducted with support of the David G. Murray Professor of Orthopedic Surgery Endowment. 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