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Association between Craniofacial Growth and Urinary Bone Metabolic Markers (Pyridinoline, Deoxypyridinoline) in Growing Rats

Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, 1–2–3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan;

*corresponding author, tanne{at}hiroshima-u.ac.jp

	ABSTRACT

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Pyridinoline (Pyr) and deoxypyridinoline (Dpyr) are intermolecular cross-links of mature collagen and reflect the bone turnover. The purpose of this study was to elucidate the association between craniofacial growth and urinary Pyr and Dpyr levels. Lateral cephalograms and 24-hour urine were taken for 7 male rats from 5 to 20 wks old. The urinary Pyr and Dpyr were quantified by high-performance liquid chromatography. The neurocranium and upper viscerocranium exhibited significant increases in size, with the maximum rate at around 6 wks old. The mandible presented more substantial growth, with the maximum change at 8 wks old. The urinary Pyr and Dpyr levels gradually increased and reached the maximum at 8 wks old. No prominent association was found between neurocranial growth and urinary levels of pyridinium cross-links, whereas Pyr and Dpyr levels exhibited similar time-dependent metabolic changes to mandibular growth. In conclusion, it is shown that urinary pyridinium cross-links may be useful for the prediction of mandibular growth.

KEY WORDS: urinary bone marker • pyridinoline • deoxypyridinoline • HPLC • craniofacial growth

	INTRODUCTION

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Pyridinoline (Pyr) and its minor analog, deoxypyridinoline (Dpyr), are trifunctional 3-hydroxypyridinium cross-links between collagen molecules and contribute to stabilizing and reinforcing the whole structure of such collagenous tissues as bone and cartilage (Fujimoto, 1977; Ogawa et al., 1982). These intermolecular cross-links are discharged during the disintegration of mature collagen, and excreted into urine without metabolization. Therefore, the urinary levels of pyridinium cross-links have been recognized as useful markers for predicting the degradation of mature collagen in bone and cartilage (Robins et al., 1986; Hanson et al., 1992). Pyr and Dpyr have mainly been applied to clinical diagnosis for patients with such bone metabolic diseases as primary hyperparathyroidism (Uebelhart et al., 1990), hyperthyroidism (Harvey et al., 1991), Paget’s disease (Robins et al., 1991), osteoporosis (McLaren et al., 1993), and osteomalacia (Seibel et al., 1994). We have recently demonstrated that the urinary Pyr/Dpyr ratio can be used for the detection of condylar cartilage degradation in rats with experimentally induced osteoarthritis of the temporomandibular joint (Imada et al., in press).

Bone turnover and the consequent excretion of urinary collagen cross-links, in general, are low in adults, and relatively constant over time (Beardsworth et al., 1990). During growth, in contrast, the size and shape of bone experience substantial changes associated with active bone remodeling, and bone metabolic products are thus excreted into the urine and blood. A puberty-related increase in the urinary levels of pyridinium cross-links (Marowska et al., 1993; Blumsohn et al., 1994; Fujimoto et al., 1995; Mora et al., 1998), and other bone resorption markers such as type I collagen cross-linked N-telopeptides in urine (Bollen and Eyre, 1994) and C-telopeptides in serum (Tommasi et al., 1996) has been documented in the literature. From these findings, it would be reasonably assumed that the metabolic products result from active bone remodeling during pubertal growth and can be applied to the prediction of craniofacial growth in young patients.

However, there have been no longitudinal studies for the association between craniofacial growth and urinary levels of Pyr and Dpyr. The present study was thus designed to elucidate the association between craniofacial growth and urinary levels of Pyr and Dpyr in growing rats and to explore their clinical availability as bone metabolic markers for growth prediction.

	MATERIALS & METHODS

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Experimental Animals
Seven Wistar-strain male rats 4 wks old were used as the experimental animals. Permission for the animal experiment was granted by the Ethics Committee of the Hiroshima University Faculty of Dentistry. Since the body sizes of rats before 5 wks old were too small for the collection of sufficient amounts of urine for the quantification of pyridinium cross-links, the experimental period was defined as being from 5 to 20 wks after birth.

Morphometric Analyses with Lateral Cephalograms
Lateral cephalograms were taken of all the rats every 2 wks, from 5 to 17 wks, by use of a rat and mouse cephalometer (RM-50, Asahi Roentgen Industry Co., Kyoto, Japan) with dental occlusal film (DF-50, Eastman Kodak, Rochester, NY, USA), while the animals were under general anesthesia with sodium pentobarbital (Nembutal, Dainabot Co., Osaka, Japan).

The cephalograms were scanned with an image scanner (GT-9000, Epson, Tokyo, Japan) and magnified 5X on a personal computer (FM-V DESKPOWER T-III23, Fujitsu, Tokyo, Japan). Cephalometric analysis was performed according to the method developed by Ishizuka (1996). Twelve anatomic landmarks and two construction points, U1' and Co', were established for the present study, as shown in Figs. 1A and 1B. Nine measurement items are described in Fig. 1B.

View larger version (29K): [in this window] [in a new window]   Figure 1. Landmarks and measurement items for cephalometric analysis (A) The illustration of landmarks on the lateral cephalogram for morphometric analysis. (B) Definitions of the landmarks and measurement items for cephalometric analysis.

Each aliquot (0.5 mL) was hydrolyzed with an equal volume of 12 M HCl for 18 hrs at 107°C. The hydrolyses were centrifuged at 10,000 x g for 5 min. Purified fractions of 0.5 mL in a mixture (butanol/acetic acid, 4/1) of 2.5 mL were applied to disposable extraction columns (DECs), which we prepared by packing 4-mL Bond Elut reservoirs with 100 mg of CC31 microgranular cellulose powder (Whatman Biosystems, Maidstone, UK) contained between two polyethylene frits. After being washed with 8 mL of mobile phase (butanol/acetic acid/water, 4/1/1) and 0.5 mL of tetrahydrofuran, Pyr and Dpyr were eluted by 0.63 mL of 50 mM heptafluorobutyric acid (HFBA) and filtrated with a 0.45-µm Millipore filter (Gelman Sciences, Ann Arbor, MI, USA).

The solvents were analyzed by ion-paired and reverse-phase high-performance liquid chromatography (Waters 600E Multisolvent Delivery System, Waters, Milford, MA, USA). A 4.6 x 150 mm column (Symmetry 3.5 µm C18, Waters, Milford, MA, USA) was used. Water/10 mM of HFBA and acetonitrile (AcCN)/10 mM of HFBA (1/3) were used for the mobile phase, and a two-stage gradient with AcCN was used to separate Pyr and Dpyr. The column effluent was monitored by fluorescence spectroscopy (Waters 474 Scanning Fluorescence Detector, Waters, Milford, MA, USA) at excitation and emission wavelengths of 295 and 400 nm, respectively. The levels of Pyr or Dpyr were quantified according to the calibration curves established with pure Pyr and Dpyr (Wako, Osaka, Japan). The total amounts of Pyr and Dpyr in 24-hour urine samples were obtained, and the Pyr/Dpyr ratios were calculated from the results.

Statistical Treatment
We used a Student’s t test to compare growth increments for 2 wks in animals from 5 to 15 wks old with those from animals 15 to 17 wks old. Similarly, according to the Student’s t test, the urinary levels of Pyr and Dpyr and the Pyr/Dpyr ratio from animals 5 to 19 wks old were compared with those from animals at 20 wks old.

	RESULTS

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Changes in Body Weight
In the growth velocity curve, two peaks were found at 6 to 7 wks and 12 to 13 wks (Fig. 2). Since typical changes in body weight were observed in the present study, it is suggested that the rats experienced normal growth regardless of a series of experiments.

View larger version (14K): [in this window] [in a new window]   Figure 2. Changes in the body weights of rats. Upper line and left-side scale indicate the cumulative growth in the body weight of rats weekly from 4 to 20 wks (n = 7). Lower line and right-side scale denote the growth velocity. Significant difference by Student’s t test is indicated by one asterisk (p < 0.05) or two asterisks (p < 0.01).

View larger version (24K): [in this window] [in a new window]   Figure 3. Changes in the size of the craniofacial skeleton and the incremental changes for every 2 wks. Upper lines and left-side scales indicate the cumulative growth in the size of the craniofacial skeleton of rats twice weekly from 5 to 20 wks (n = 7). Lower lines and right-side scales indicate their growth velocity. Significant difference by Student’s t test is indicated by one asterisk (p < 0.05) or two asterisks (p < 0.01). (A) Size of anterior cranial base (S-E). (B) Size of posterior cranial base (S-Ba). (C) Total skull length (Po-A). (D) Nasal bone length (N-A). (E) Viscerocranial height at the first molar (U1-U1'). (F) Maxillary bone length (Mu-Bu). (G) Mandibular corpus length (Go-Pg). (H) Ramus height (Co-Co'). (I) Mandibular effective length (Co-Pg).

Increases in the mandibular corpus length (Go-Pg), ramus height (Co-Co'), and effective length (Co-Pg) reached the maximum during the period from 7 to 9 wks and thereafter reduced substantially with a lapse of time (Figs. 3G, 3H, 3I). Co-Co' and Co-Pg were significantly greater during the period from 5 to 9 wks (p < 0.05), and Go-Pg was significantly larger during the period from 7 to 9 wks (p < 0.05), whereas the increments were not significant at the later stages from 9 to 17 wks. On the whole, the increment was greater in the horizontal than in the vertical direction.

Change in the Amount of Pyridinium Cross-links
Changes in the urinary Pyr and Dpyr levels exhibited almost similar patterns in animals from 5 to 20 wks of age, with a single peak at 8 wks (Figs. 4A, 4B). The Pyr level was significantly higher from 7 to 11 wks than at 20 wks (8 wks, p < 0.01; 7 and 9 to 11 wks, p < 0.05), whereas the level of Dpyr was significantly greater from 7 to 9 wks than at 20 wks (8 wks, p < 0.01; 7 and 9 wks, p < 0.05). After the peak, urinary levels decreased, and no significant changes in the urinary levels of Pyr and Dpyr were found for the remaining periods of 13–20 wks and 10–20 wks, respectively.

View larger version (15K): [in this window] [in a new window]   Figure 4. Changes in urinary levels of pyridinium cross-links. The amounts of pyridinoline (Pyr) and deoxypyridinoline (Dpyr) in 24-hour urine collections from rats were measured weekly from 5 to 20 wks by use of high-performance liquid chromatography (n = 7). The Pyr/Dpyr ratio was calculated from the results. Significant difference by Student’s t test is indicated by one asterisk (p < 0.05) or two asterisks (p < 0.01). (A) Pyr, (B) Dpyr, (C) Pyr/Dpyr ratio.

	DISCUSSION

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Urinary excretion of bone metabolic markers exhibits a circadian rhythm (Bollen and Eyre, 1994). In previous studies, such a circadian variation was found in humans: The urinary levels of Pyr and Dpyr were low during the day and high at night (Eastell et al., 1992). These findings confirm that a 24-hour urine sample is most suitable for the quantification of pyridinium cross-links. In the present study, a metabolic cage was used for collecting the 24-hour urine samples from rats. Since the hereditary differences in skeletal growth are smallest among Wistar-strain rats (Asano, 1986), this animal seems the most appropriate experimental model for examination of the association between skeletal growth and urinary levels of bone metabolic markers.

A previous study reported that neurocranial growth is completed earlier than viscerocranial growth in rats (Baer, 1954). Growth of the calvaria and cranial base is achieved by 2 and 5 wks after birth, respectively (Hoyte, 1971). The viscerocranium is located below the neurocranium at birth, after which the viscerocranium and neurocranium become parallel, with anterior displacement of the viscerocranium. It is well-accepted that mandibular growth is small in juveniles and reaches the maximum during puberty (Hanada, 1967).

In the present study, changes in the neurocranium (S-E and S-Ba) and maxilla (Mu-Bu and U1-U1') were small after 5 wks. The upper viscerocranium (Po-A and N-A) presented considerable growth until 14 wks, with the obvious nasal projection in the anterior direction. On the other hand, the growth pattern of the mandible was different from that of the upper viscerocranium and neurocranium. Growth increments in the mandible (Go-Pg, Co-Co', and Co-Pg) were substantial during 7–9 wks after birth, and decreased gradually after the peak velocity at approximately 8 wks. The growth pattern of the neurocranium is classified as neural, and is closely related to enlargement of the brain (Hoyte, 1971). Based on human growth curves (Proffit, 1986), the maxilla shows a growth pattern intermediate between neural and general patterns, whereas the mandible exhibits a growth pattern of the general type. Growth acceleration in the general body at puberty thus coincides with that in the mandible. With these considerations, it would be a reasonable assumption that the nature of craniofacial growth in rats is almost equivalent to that in humans, although some differences in nasomaxillary growth surely exist between rats and humans. Further study is anticipated to demonstrate the relationship between urinary Pyr and Dpyr levels and craniofacial growth in humans.

In the present study, both urinary Pyr and Dpyr levels were high during the early experimental period from 5 to 10 wks, and highest at 8 wks. During this period, changes in the size of the upper viscerocranium and mandible were significantly large. The peak of change in mandibular growth was especially coincident with the peaks of the urinary pyridinium cross-links. Although no association was found between neurocranial or maxillary growth and urinary levels of pyridinium cross-links, the peak of mandibular growth was induced at exactly the same time as those of urinary Pyr and Dpyr levels. Furthermore, the period of significantly substantial mandibular growth was in agreement with that of the significantly enhanced urinary level of Dpyr. Mandibular growth is mainly achieved by endochondral bone formation in the condyle, whereas growth of the upper viscerocranium in the rat results mainly from membranous and sutural bone formation and cartilaginous growth of the nasal septum. The mandible and other long bones, following a general growth pattern, experience active bone remodeling and substantial growth at almost the same time in puberty. This may account for the highest excretion of cross-links into urine during maximum mandibular growth. However, the peak of the Pyr/Dpyr ratio was reduced at 10 wks, later than those of both cross-links. Since Dpyr is mostly specific to bone (Eyre et al., 1984), changes in the urinary Dpyr level are considered to reflect the overall bone metabolism. In contrast, Pyr is most abundant in cartilage (Eyre et al., 1984), and hence the level does not exclusively reflect the bone metabolism. This may explain why the period of the significantly higher Pyr level was longer than that of Dpyr, and the maximum Pyr/Dpyr ratio appeared later than that of urinary Pyr and Dpry levels. The Pyr/Dpyr ratio was reported to be higher at 12–18 yrs than at 3–11 yrs or adult in humans (Marowska et al., 1993), which is consistent with the present results. From these findings, the Pyr/Dpyr ratio in urine may be a useful marker of bone metabolism as well as urinary levels of Pyr and Dpyr. It is also considered that individual variations in their levels may be corrected when the bone metabolism is evaluated by the Pyr/Dpyr ratio.

From these results, it is emphasized that urinary Pyr and Dpyr levels exhibit a significantly high association with mandibular growth in terms of timing and magnitude, suggesting that the pyridinium cross-links would be a useful diagnostic determinant for predicting mandibular growth. In conclusion, a new diagnostic system with urinary pyridinium cross-links may be established in the near future for the prediction of maximum or pubertal mandibular growth.

	ACKNOWLEDGMENTS

 
This research was supported by Grants-in-Aid (Nos. 10877337, 11771322) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Received March 25, 2002; Last revision August 27, 2002; Accepted October 7, 2002

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