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Osteoclastogenic Activity during Mandibular Distraction Osteogenesis
L.C. Wang1,
I. Takahashi1,*,
Y. Sasano2,
J. Sugawara1 and
H. Mitani1
1 Division of Orthodontics and Dentofacial Orthopedics,
2 Division of Craniofacial Development and Regeneration, Tohoku University Graduate School of Dentistry, 4-1, Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan;
Correspondence: * corresponding author, takahasi{at}mail.tains.tohoku.ac.jp
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ABSTRACT
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Mandibular distraction osteogenesis is a well-developed clinical modality for the treatment of craniofacial deformities and dental arch discrepancies, in combination with orthodontic treatment. However, in our previous study, orthodontic tooth movement into the distraction gap caused severe root resorption. The present study aimed to clarify the osteoclastogenic activity of cells in the distraction gap. We hypothesized that the gene expression of osteoclastogenic- and osteoclast-supporting molecules in osteoblasts and stromal cells would increase at distraction sites during the consolidation period. An animal model experiment involving rabbits was designed for mandibular distraction osteogenesis and subjected to in situ hybridization analysis. The number of osteoclasts was larger in the distraction gap during the early consolidation period than in normal controls, due to an increase of gene expression for osteoclastogenic cytokines in osteoblasts. It was concluded that osteoclastogenic and osteoclastic activities are stimulated at distraction sites during the early consolidation period.
Key Words: mandibular distraction osteogenesis • cathepsin K • IL-1β • TNF- • RANKL.
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INTRODUCTION
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Mandibular distraction osteogenesis has been clinically applied in multiple scenarios, including craniofacial deformities, dental arch discrepancies, and temporomandibular joint reconstruction combined with prosthetic and/or orthodontic treatment (Snyder et al., 1973; McCarthy et al., 1992; Samchukov et al., 1998). Previous studies have indicated that tooth movement into distraction sites can be faster at the initial stage of consolidation, and can be achieved with lighter force (Cope et al., 1999, 2002; Liou et al., 2000). However, our previous study revealed that root resorption occurred when teeth moved into immature distraction regenerated bone (Nakamoto et al., 2002). Since the distraction regeneration site may be in a high-turnover phase of bone remodeling, we suspected that not only osteogenic activity but also osteoclastogenic, osteoclastic, and even odontoclastogenic activities could all be higher than in normal bone.
Osteogenic activity in the distraction gap has been demonstrated. Although bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) were found to be expressed and to contribute to osteogenic activity in distraction-regenerate bone (Okazaki et al., 1999; Rauch et al., 2000; Farhadieh et al., 2004), the expression level of osteoclastogenic factors at distraction sites remains unclear. Interleukin-1β (IL-1β), tumor necrosis factor (TNF-), and receptor activator for nuclear factor  B ligand (RANKL) are known to induce osteoclastogenesis by activating TRAF-2 and TRAF-6 pathways in osteoclast precursors under inflammatory or physiological conditions in bone (Lam et al., 2000; Gravallese et al., 2001; Udagawa, 2002; Huang et al., 2003; Kwan Tat et al., 2004).
It has been demonstrated that matrix metalloproteinase-9 (MMP-9) and cathepsin K are highly expressed in osteoclasts for proteolytic degradation of bone matrix (Littlewood-Evans et al., 1997; Dodds et al., 1998). MMP-9, a secreted enzyme, and cathepsin K, a lysosomal enzyme, are specifically expressed in osteoclasts (Uemura et al., 2000; Sasaki, 2003). In addition, osteopontin provides a scaffold for osteoclasts to attach to the bone surface through the vβ3 integrin (Teitelbaum, 2000; Kwan Tat et al., 2004). These 3 molecules are useful markers for osteoclastic activities and capabilities in bone tissues.
In the present study, we hypothesized that the expression of IL-1β, TNF-, RANKL, and osteopontin genes in osteoblasts at distraction sites in bone would increase as a consequence of an increased number of osteoclasts expressing cathepsin K and MMP-9, during consolidation in mandibular distraction osteogenesis.
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MATERIALS & METHODS
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Experimental Animals and Surgical Procedure
Twenty-one mature male Japanese white rabbits (aged 15–24 wks; weighing 2.7–3.3 kg each) were used in this experiment. All experimental animals were randomly selected and divided among 1 control and 6 experimental groups, each consisting of 3 animals. All experimental procedures were performed after authorization by the Animal Care and Ethics Committee of Tohoku University.
Surgical procedures were performed while the animals were under general anesthesia induced by intravenous injection of pentobarbital (25 mg/kg) with a prophylactic dose of Furmarin (Shionogi & Co., Osaka, Japan) and subcutaneous local anesthesia (1% Lidocaine). After a 4-cm incision was made over the inferior mandibular border, a periosteal flap was elevated. Once the mandibular body was osteotomized anterior to the first premolar unilaterally, a custom-made external distractor (Fig. 1A ) was fixed with 4 titanium mini-screws (Leibinger Co., Freiburg, Germany; Fig. 1B). The periosteal flap was repositioned and sutured after trial activation of the distractor. Trauma to nerves and vessels was minimized during surgery. The distraction direction and device capability were confirmed with trial activation.
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Figure 1. Design of the custom-made distractor (A), surgical procedure (B), radiographs before and after distraction (C,D), occlusal view of the experimental mandible (E), gross view of the distracted animal (F), and photomicrographs from H&E-stained sections from control (G,H), and groups at wk 0 (I,J), 1 wk (K,L), 2 wks (M,N), 4 wks (O,P), and 8 wks (Q,R) of the different consolidation periods are indicated. An arrow in C and solid lines in D, I, K, and M indicate the osteotomy line. An arrow in E indicates the asymmetrical occlusal attrition of the molars in experimental animals. An arrow in F indicates the midline deviation in experimental animals after distraction. OB, original bone; RB, regenerated bone; DG, distraction gap; Fi, fibrovascular tissue; Mar, marginal area; and Mid, central zone of the distraction gap. Original magnification in G, I, K, M, O, and Q, x1.6 (bar = 1.5 mm); and in H, J, L, N, P, and R, x20 (bar = 100 µm). All experiments were performed in triplicate (N = 3).
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Post-operative Care and Unilateral Mandibular Distraction Protocol
Lepetan (0.05 mg/kg) (Otsuka Pharmaceutical Co., Tokyo, Japan) was administered subcutaneously as analgesia. Post-operatively, the clinical condition and body weight of each experimental animal were monitored twice a day until the end of the experimental period.
After 3.5 days of latency, unilateral mandibular distraction was performed at a rate of 0.5 mm/12 hrs for 7 days, to produce a distraction gap of approximately 7 mm. At the end of each consolidation period (i.e., 0, 1, 2, 4, 6, or 8 wks), 3 animals were killed. Control animals were killed at ages equivalent to 4 wks of the experimental period. To confirm the amount of distraction, we took radiographs immediately before and after distraction.
Riboprobe Preparation
The distracted tissue specimens were taken prior to fixation, then underwent total RNA extraction for reverse-transcription polymerase chain-reaction (RT-PCR). The genes of interest were then amplified by PCR and subcloned into a pCR-II TOPO vector (Invitrogen, Carlsbad, CA, USA). The molecules selected for evaluation were cathepsin K, MMP-9, RANKL, IL-1β, TNF-, and osteopontin. The cDNA fragment for RANKL was cloned through nested RT-PCR with primers based on the consensus sequence aligning human, mouse, and rat RANKL. All subcloned cDNA fragments were verified by restriction enzyme digestion and confirmed by dideoxynucleotide sequencing. Digoxigenin (Dig)-labeled riboprobes were generated (Table 1).
Tissue Preparation and Histological Examination
All experimental animals were killed by perfusion with 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate-buffered saline (PBS), pH 7.4, under pentobarbital anesthesia. After the mandible was dissected, radiographs were taken with an aluminum step wedge for the evaluation of bone density. Then, the distracted specimen areas were excised with their surrounding tissues and further fixed in the same fixative for 12 hrs. Adjacent portions of the other sides of the mandibles in experimental animals and corresponding portions in control animals were also excised as control specimens and further processed. Specimens were decalcified in 10% ethylene diamine tetra acetic acid in 0.01 M PBS, pH 7.4, at 4°C for 9–12 mos. Samples were dehydrated in a graded series of ethanol and embedded in paraffin. Finally, 5-µm-thick serial sections were cut for histological evaluation and in situ hybridization.
The sections were deparaffinized and rehydrated in a graded series of ethanol. For histological evaluation, the sections were stained with hematoxylin and eosin (H&E).
In situ Hybridization (ISH)
The protocol for ISH used in the present study was described in previous studies (Ohtani et al., 1992; Sasano et al., 1996) and elsewhere. Briefly, sections were pre-treated in 0.2 N HCl, incubated in 20 µg/mL proteinase K (Roche Diagnostics, Indianapolis, IN, USA), dehydrated, then air-dried. Thereafter, they were incubated with antisense or sense probes in the hybridization mixture with various concentrations between 5 and 100 ng/mL for 16 hrs at 45°C, depending on the probe. After being washed with 2x sodium chloride-sodium citrate buffer, sections were treated with ribonuclease A (20 µg/mL) (Sigma, St Louis, MO, USA). Next, they were incubated with anti-Dig antibody overnight at 4°C before signals were visualized. Counterstaining with methyl green was performed, and sections were mounted.
Osteoclasts, Bone Density, and Statistics
The number of multinucleated cells expressing cathepsin K was counted as osteoclasts in each group. This analysis was performed in the central region of distraction sites in the bone containing a fibrous interzone. Five 1225 µm x 1715 µm areas were randomly selected in each consolidation period. Bone density of the distraction gap was evaluated on soft x-ray photographs by means of NIH image software (NIH, Bethesda, MD, USA). One 3 mm x 3 mm area in the distraction gap was randomly selected on each radiograph at each experimental period for all experimental animals. The level of bone density was quantified in terms of aluminum thickness per aluminum step wedge. Method errors in numbers of osteoclasts and bone density were tested by three repeated and independent measurements and were less than 2.3 and 0.05 mm aluminum unit, respectively. All quantified data underwent statistical analysis by Fisher’s test, with a P value of < 0.01 and < 0.05, respectively.
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RESULTS
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Gross Observation
On radiographic film taken before and after distraction, the amount of distraction in each experimental animal was confirmed to be approximately 7.0 mm at the inferior mandibular border (Figs. 1C , 1D). After distraction, midline deviation and occlusal plane canting were observed (Figs. 1E, 1F).
Histologic and Radiographic Observations
In control samples, thick cortical bone and well-developed bone marrow were observed anterior to the first molar (Figs. 1G, 1H). Bone-lining cells were identified over the inner and outer periosteum.
In the experimental group, the distraction sites had bone trabeculae oriented from the osteotomy line parallel to the distraction direction. Intra-bony remodeling was seen from the beginning of consolidation to the 2nd week of consolidation (Figs. 1I–1N). Before evaluating the cascade changes, we defined 2 areas for observation: (1) marginal area extremely close to the osteotomy line, and (2) the central zone right in the middle.
At the beginning of consolidation, the osteotomy line was identified as a clean-cut line (Figs. 1I, 1K). Although fibrovascular tissue mainly occupied the central zone (Fig. 1I), premature bone trabeculae extended into the distraction gap parallel to the distraction direction (Figs. 1I–1K). While parts of the central zone still contained fibrous tissue, it was obvious that the other part of bony tissue had already further matured and begun merging into the central region (Figs. 1K, 1L). Through 2 to 4 wks of consolidation, the distraction gap was filled with cancellous bone, and the ossification level increased (Figs. 1M to 1P). Bone trabeculae became more mature as the bone marrow matured, which replaced the fibrous tissue formed during distraction (Figs. 1O–1R). Finally, most of the newly formed bone trabeculae were replaced by marrow space and mature bone at 8 wks (Figs. 1Q, 1R).
The calcification progress of the distraction gap was monitored (Table 2). The bone density of the distraction gaps was significantly lower in the early consolidation periods than in the control, while it equaled the control level after 4 wks.
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Table 2. Calcification Rate, Number of Osteoclasts, and Gene Expression in the Distraction Gap during the Consolidation Period
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Number of Osteoclasts and Gene Expression Patterns
We found statistically significant increases in the number of osteoclasts at the end of 0, 1, and 2 wks (P < 0.01) of consolidation. A 25-fold increase in the number of osteoclasts was recorded at wk 0, and it gradually decreased to normal control levels at 4 wks of consolidation.
A positive signal for cathepsin K was completely restricted in multinucleated osteoclasts in all stages of consolidation (Figs. 2A, 2B), whereas MMP-9 not only was expressed primarily in osteoclasts, but also was found in some mononuclear cells (Figs. 2C, 2D). Expression patterns and time-course changes in gene expression of IL-1β, TNF-, RANKL, and osteopontin were summarized (Table 2).
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Figure 2. Photomicrographs from sections of in situ hybridization are indicated. Sections probed with Cathepsin K (A,B), MMP-9 (C,D), RANKL (E-H), IL-1β (I-L), TNF- (M-P), and osteopontin (Q-T). A, C, E, I, M, and Q: control specimens. B, D, F, J, N, and R: zero-week consolidation period group. G, K, O, and S: two-week consolidation group. H, L, P, and T: eight-week consolidation group. Black arrowheads indicate the positive signals. White arrowheads indicate the mononuclear cells positive for MMP-9. Arrows indicate the osteocytes in immature bone matrix. Original magnification: x20 (bar = 100 µm). All experiments were performed in triplicate (N = 3).
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Hybridization signals for RANKL were observed in osteoblasts at wk 0 of consolidation. The signals gradually increased and peaked at wk 2, and decreased by 8 wks of consolidation (Figs. 2E–2H). While cells in control samples showed almost no signal (Fig. 2E), bone-lining cells in wk 8 were still positive for RANKL (Fig. 2H).
IL-1β was weakly expressed in bone-lining cells of both control and eight-week consolidation groups (Figs. 2I, 2L). The expression of IL-1β in cuboidal osteoblasts increased and was maintained at a relatively constant level between 0 and 6 wks (Figs. 2J, 2K) of consolidation, with a slight increase in the 2nd and 4th wks. Osteocytes in premature bone trabeculae also showed positive signals to IL-1β at 0 to 2 wks of consolidation (Figs. 2J, 2K).
TNF- was expressed in bone-lining cells and osteoclasts in controls (Fig. 2M). The number of cells expressing TNF- increased during 0 to 8 wks of consolidation compared with controls (Figs. 2M–2P). Expression levels of TNF- in each cell were relatively constant.
Osteopontin was expressed in bone-lining cells of controls (Fig. 2Q). The numbers of osteoblasts and osteocytes expressing osteopontin increased in 0 to 2 wks of consolidation (Table 2C).
Negative Control Experiments
No hybridization signals were observed in any sections applied with sense riboprobes (data not shown).
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DISCUSSION
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Many of the osteogenic growth factors, such as BMPs, and FGFs are expressed in the cells at distraction sites in the bone (Okazaki et al., 1999; Rauch et al., 2000; Campisi et al., 2002), and have been experimentally proven to accelerate bone formation in the distraction gap (Rauch et al., 2000; Ashinoff et al., 2004). Bone formation in the distraction gap is regarded as both bone modeling and remodeling. Our previous study (Nakamoto et al., 2002) indicated that severe root resorption took place when teeth were moved through immature regenerated bone at distraction sites. Therefore, we hypothesized that not only osteogenic activity but also osteoclastogenic and/or odontoclastogenic activity could be induced. Since osteoclastogenesis is well-known to be regulated by molecules such as RANKL, TNF-, and IL-1β (Udagawa, 2002), we have attempted to verify their expression patterns at distraction sites. While RANKL can induce osteoclastogenesis under physiological conditions such as tooth movement (Ma et al., 2004), IL-1β and TNF- cooperatively induce osteoclastogenesis, whether under inflammatory conditions (Rifas, 1999) or in bone fracture healing (Kon et al., 2001). Thus, the increase in the number of osteoclasts demonstrated in bone at distraction sites could have resulted from the increase in gene expression as well as from the increase in numbers of osteoblasts expressing those molecules. In addition, osteopontin, which provided a scaffold to osteoclasts (Teitelbaum, 2000; Kwan Tat et al., 2004), was also highly expressed at distraction sites. Thus, regeneration was probably a feature of the bone-remodeling phase, with higher turnover rate under both inflammatory and physiological conditions.
Albeit its unclear origin, the mechanisms to recruit, differentiate, and maintain appear similar between odontoclasts and osteoclasts, with similarities in ultrastructural features and immunocytochemical properties (Sasaki, 2003). Consequently, the severe root resorption mentioned previously can be explained, given that odontoclasts were recruited from the same origin as osteoclasts, and their activity should be enhanced in the compression side of moving teeth by pro-inflammatory cytokines and RANKL expressed at distraction sites during the early consolidation period.
In conclusion, the osteoclastogenic and osteoclastic activities in the distraction gap were considered to be up-regulated and increasing in the early consolidation period, then nearly diminished in later stages. Consequent to the increase of osteoclastogenic and osteoclastic activities, the risk will increase for root resorption of orthodontic tooth movement through the distraction gap during the early consolidation period. Further experiments are needed to clarify the mechanism of how odontoclasts are recruited in bone at distraction sites.
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ACKNOWLEDGMENTS
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We are grateful to Professor Emeritus, Dr. Manabu Kagayama, Tohoku University Graduate School of Dentistry, for his instruction and valuable advice throughout the project. This research has been supported by Grants-in-Aid (#11771308) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
Received for publication November 30, 2004.
Revision received June 27, 2005.
Accepted for publication July 11, 2005.
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Journal of Dental Research, Vol. 84, No. 11,
1010-1015 (2005)
DOI: 10.1177/154405910508401108
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ABSTRACT
INTRODUCTION