Age-related osteogenesis on lateral force application to rat incisor – Part III: Periodontal and periosteal bone remodeling

INTRODUCTION

It has been known that tooth movement is fundamentally induced by bone resorption of the pressure side (the alveolar bone side of the periodontal ligament directly contacted with periodontal membrane), and bone formation of the tension side according to pressure-tension theory.^[1] However, orthodontic tooth movement may accompany several side effects including root resorption, bone dehiscence, and bone fenestration associated with bone modeling in the periosteal side of alveolar bone.^[2-4]

Lingual or buccolingual tooth movement requires substantial bone modeling not only in the alveolus but also in the periosteal side of alveolar bone, generally known as compensatory bone formation.^[5-7] Although some studies observed positive results regarding compensatory bone formation even with severe buccolingual tooth movement,^[5-8] controversies still exist.^[9,10] In the studies performed by Steiner et al.^[11] and Engelking and Zachrisson,^[12] when upper and lower incisor was moved forward, alveolar bone loss was observed in monkeys, and a significant amount of labial bone tissues was regenerated when the incisor was moved backward again. In addition, Garib et al. identified an average of 7.1 mm of bone dehiscence when maxillary expansion was carried out in young patients utilizing rapid maxillary expansion.^[13] Moreover, Sarikaya et al. observed a reduction in palatal bone thickness during the posterior retraction of incisors.^[14] Dorfman,^[15] Artun and Krogstad,^[16] and Yared^[17] also reported that bone dehiscence and gingival recession were induced in the anterior tipping of lower incisors. On the other hand, Ruf et al.^[18] and Artun and Grobéty^[19] illustrated that the anterior tipping of lower incisors did not cause bone dehiscence and gingival recession in young patients, implementing that age could attribute to the difference in the capacity of compensatory bone formation. Jäger exhibited lowered bone formation capacity and response against stimulation on aging^[20] whereas Bridges et al. found that low bone mineral density and anatomical resistance of alveolar bone would be advantageous for tooth movement in the young group.^[21]

To resolve the discrepancies, additional studies are needed to investigate the cellular behavior on the periodontal and periosteal side of alveolar bone on orthodontic force application. The objectives of this study were to investigate the differences of bone modeling around the alveolar bone depending on age and duration of lateral force application on the rat incisors through an immunohistochemical method.

MATERIAL AND METHODS

Experiment animals and device installation

This study was approved in advance by the Institutional Animal Care and Use Committee. A total of 50 male Sprague-Dawley rats included 25 rats in the adult group (52 weeks) and 25 rats in the young group (10 weeks) were involved in this study.

A double-helical spring used 0.014” stainless steel wire was applied to the left and right incisor with 40 g of lateral force. Then, five rats in each age group were allocated into the control, 3 days, 7 days, 14 days, and 21 days’ groups regarding the duration of experimental device application.

Anesthetics (0.05 ml of Rompun; Rompun, Bayer, Korea; and 0.45 ml of Zoletil; Zoletil 50, Virbac Lab, Carros, France) were intraperitoneally administered to the experimental groups for installation and activation of helical spring according to part I and II of this serial study,^[22,23] using a high speed 1/4 round bur [Figure 1a]. After the experimental period, the elasticity of the spring was confirmed.

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Figure 1:

(a) Spring activated on rat incisor, (b) tissue section direction, (c) region of interest (×40).

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Production and staining of tissue specimens

Hematoxylin-Eosin (H-E) staining

The control group and the experimental group were sacrificed at each time point by a strong dose of ether then the upper pre-maxilla was dissected. Isolated tissues were fixed with 4% paraformaldehyde for 4 days followed by clearing with Calci-Clear Rapid^™ solution (National Diagnostics, Atlanta, USA) for 24 h. After the clearing, the tissues were cut in the direction described in [Figure 1b] to examine the periosteal side of alveolar bone, then were subjected to paraffin embedding in 5 μm thicknesses. After the tissue sections on the slides were deparaffinized using xylene, they were hydrated by a sequentially low concentration of alcohol. H-E staining was performed based on general rules and histological examination was carried out on the areas of interest [Figures 1c, 2, and 3].

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Figure 2:

Hematoxylin-eosin stain in the young group in the crestal (i) and middle (ii) areas on the control (a and f), 3 days (b and g), 7 days (c and h), 14 days (d and i), 21 days (e and j) groups, respectively (×100).

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Figure 3:

Hematoxylin-eosin stain in the adult group in the crestal (i) and middle (ii) areas on the control (a and f), 3 days (b and g), 7 days (c and h), 14 days (d and i), 21 days (e and j) groups, respectively (×100).

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Proliferating cell nuclear antigen (PCNA) immunohistochemical staining

The tissue sections on the slides were deparaffinized using xylene, then hydrated by sequentially low concentration of alcohol followed by antigen retrieval through 0.5% Triton X-100 solution at room temperature for 10 min. To inactivate endogenous peroxidase, the tissue sections were incubated in 0.3% H₂O₂ for 5 min and the reaction was blocked by 5% bovine serum albumin for 30 min.

For PCNA staining, FL-261(Santa Cruz Co., USA) was diluted in 1:100 with a primary antibody and was subjected to incubation at room temperature for 1 h followed by overnight incubation at 4^°C. Envision kit (DAKO Co., Denmark) was utilized as 2^nd antibody-conjugated polymer. After DAB development for 2–3 min, contrast staining was performed using Gill’s hematoxylin, and dehydration was done with a gradual increase of alcohol concentration followed by mounting. Prepared slides were subjected to histological examination [Figures 4 and 5].

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Figure 4:

PCNA immunohistochemistry stain in the young group in the crestal (i) and middle (ii) areas on the control (a and f), 3 days (b and g), 7 days (c and h), 14 days (d and i), 21 days (e and j) groups, respectively (×400). PCNA: Proliferating cell nuclear antigen.

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Figure 5:

PCNA immunohistochemistry stain in the adult group in the crestal (i) and middle (ii) areas on the control (a and f), 3 days (b and g), 7 days (c and h), 14 days (d and i), 21 days (e and j) groups, respectively (×400). PCNA: Proliferating cell nuclear antigen.

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Fibroblast growth factor receptor 2 (FGFR2) immunohistochemical staining

The tissue sections on the slides were deparaffinized utilizing xylene and then hydrated by a sequentially low concentration of alcohol. Antigen retrieval was carried out using proteinase K at 36°C for 10 min. Endogenous peroxidase inactivation was performed by incubating the sections in 0.3% H₂O₂ for 5 min and the reaction was blocked by 5% bovine serum albumin for 30 min. To implement FGFR2 immunohistochemical staining, Beksc-122 (Santa Cruz Co., USA) was diluted in 1:100 with a primary antibody and then incubated at room temperature for 1 h followed by overnight incubation at 4°C. Envision kit (DAKO) was employed as 2^nd antibody-conjugated polymer. DAB development was performed for 2–3 min and then was subjected to contrast staining using Gill’s hematoxylin. The sections were then dehydrated with a gradual increase of alcohol concentration followed by mounting [Figures 6 and 7].

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Figure 6:

FGFR2 immunohistochemistry stain in the young group in the crestal (i) and middle (ii) areas on the control (a and f), 3 days (b and g), 7 days (c and h), 14 days (d and i), 21 days (e and j) groups, respectively (×400). FGFR2: Fibroblast growth factor receptor 2.

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Figure 7:

FGFR2 immunohistochemistry stain in the adult group in the crestal (i) and middle (ii) areas on the control (a and f), 3 days (b and g), 7 days (c and h), 14 days (d and i), 21 days (e and j) groups, respectively (×400). FGFR2: Fibroblast growth factor receptor 2.

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Masson trichrome staining

The tissue sections on the slides were subjected to deparaffinization using xylene followed by hydration utilizing a sequentially low concentration of alcohol. The nuclear stain was done by Weigert’s iron hematoxylin for 10 min and rinsed. The sections were distained by 1% HCl-alcohol solution and then rinsed. Ponceau-acid fuchsin solution was applied for background staining for 10 min and then rinsed with 0.2% acetic acid solution. Mordant staining was performed using phosphomolybdic acid-orange G solution for 5 min followed by rinsing with 0.2% acetic acid. Collagen was stained using a light green staining solution and then unbound light green was washed out by 0.2% acetic acid. The slides were dried and then subjected to histological examination [Figure 8].

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Figure 8:

Masson trichrome stain in (a) the control group, (b) at the day 3 in the young group, (c) at the day 7 in the adult group, (d) at the day 14 in the young group, (e) at the day 21 in the young group, (f) at the day 21 in the adult group (×100).

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RESULTS

The areas of interest were selected on the stained slides and then examined [Figure 1c].

DISCUSSION

Tooth movement beyond the alveolar bone necessitates bone modeling in the periosteal side of alveolar bone.^[2-4] However, there have been few studies on compensatory bone formation related to orthodontic tooth movement. This study aimed to compare the pattern of bone modeling histologically in different age groups and duration of force application when orthodontic force (40 g) was applied to the incisor of young (12 weeks) and adult (48–52 weeks) rats using double-helical spring.

Regarding the compensatory bone formation in tooth movement, Shimpo et al.^[5] demonstrated significant bone formation in lingual cortical bone regardless of age groups when rat molar was tipped to the lingual side, and Wingard^[8] found considerable reformation of buccolingual cortical bone in monkeys. However, many clinical studies have reported the loss of alveolar bone in the buccolingual movement.^[13-16] In the present study, compensatory bone formation has been observed in the crestal area of the periosteal side of alveolar bone on day 3 in the young group and day 7 in the adult group [Figures 2, 3 and 8]. Frost suggested a Mechanostat theory favoring the bone formation in the direction of bone tissue bending as bone tissues adapt to the external force.^[25] Mechanostat theory demonstrates compensatory bone formation in tooth movement sufficiently and it is in agreement with the histological results in this study. In the young group, following the lateral tooth movement, the alveolar bone surface of the periodontal ligament side was hyalinized in the crestal area and may have resulted in external bending of alveolar bone as an orthodontic force was transferred to the alveolar bone.^[6,7] This may explain the early bone formation in the crestal area in both groups. However, the indirect bone resorption of the hyalinized tissues on the crestal area progressed up to 21 days, therefore, the orthodontic force was not transferred to the alveolar bone, and the periodontal ligament space was increased due to direct bone resorption at the middle area. Therefore, there was no bone resorption progressed and bone modeling in the periosteal side of alveolar bone was reduced and became uniform. By the same mechanism, the compensatory bone formation was exhibited in the adult group of the 7 days group but bone modeling was not actively in the middle area due to the reduction of bone tissue turn-over by age-related tissue activity and bone mineral density.^[21,26,27]

In this study, the irregularity on the surface of cortical bone was observed in the periosteal side of alveolar bone induced by orthodontic force [Figures 2 and 3]. This phenomenon had been reported in the study of Storey,^[7] in which the different shapes of irregularity were explained based on the strength of force applied. In addition, Petrtýl et al. reported that the bone formation toward the external force was a general bone remodeling aspect occurring in the cortical bone when external force was applied.^[28] They also explained the remodeling of internal cortical bone based on the bone tissue formation as osteoclasts absorb the surface of cortical bone in depth toward the external force followed by a tremendous number of osteoblasts.

In this study, PCNA and FGFR2 immunohistochemical staining were performed to investigate the reaction of bone tissues by orthodontic force. PCNA shows a positive reaction of the cells differentiating in a non-specific manner so that greater the positive reaction indicates more active tissue reaction against stimulation. FGFR2 immunohistochemical staining has specificity for osteoblast differentiating osteoprogenitor cells.^[29,30] In this study, most of the PCNA positives were matched with the FGFR2 positives, indicating that bone modeling occurred actively in the periosteal side of alveolar bone in the reaction to orthodontic force [Figures 4-7]. Regarding the response in the periosteal side of alveolar bone against external force, Skerry et al.,^[31] KleinNulend et al.,^[32] and Cowin et al.^[33] addressed that osteocytes presented in the bone tissues play a role as mechanosensors that transduced the force through the bone. This can explain the osteoblastic activity in the middle portion of the alveolar bone, especially in the young group [Figures 2, 4, and 8]. Considering the age-related changes in cell compositions of bone tissues, Roholl reported that the number of osteoblasts increased with an increase of age while the number of osteoclasts was not influenced.^[34] In this study, the initial positive reaction period was not different between the young and adult groups. However, differences were observed in the age-related tissue activity such as the lower level of expression and relatively slower maximum positive reaction period in the adult group. A decrease of PCNA and FGFR2 reaction in the adult group represents a decrease of bone formation capacity according to an increase of age [Figures 5 and 7]. It would play a significant role in the homeostasis of alveolar bone along with an increase of bone mineral density in alveolar bone on aging,^[21,26,27] reduction of periodontal ligament space, and a decrease of tooth movement speed in the adult group due to changes in the physical properties of periodontal ligament.^[35]

To the best of our knowledge, this is the first report on the histologic mechanism of compensatory bone formation by the lateral force. Interestingly summary, we observed osteoblastic activity on the periosteal side preceded the bone resorption and tooth displacement within the “pressure side” of periodontal ligament, especially in the crestal and middle areas. However, further studies are needed regard to biological reactions on aging under different sizes and durations of orthodontic force with long-term observation.

CONCLUSION

Taken together, we confirmed that orthodontic tooth movement facilitated cell proliferation, osteocyte differentiation, and bone formation on the surface of the periosteal side of alveolar bone, especially in the young group. The reduced bone modeling reaction in the adult group is considered a factor that restricts excessive tooth movement toward the buccolingual side of the alveolar bone during orthodontic treatment.