Effects of Selective Alveolar Decortication on the Periodontal Complex

Abstract


Background
Selective alveolar bone decortication has been performed as a means to accelerate tooth movement. 1e theory behind this procedure is that decortication can induce demineralization of the alveolar bone that surrounds the tooth root, leading to a softening of the mineralized matrix that offers less resistance to tooth movement.This demineralized condition accelerates tooth movement until the alveolar bone remineralizes, and thus reduces treatment time by 4-6 months. 2 During this demineralization and reminerlization, it is postulated that osteoblast and osteoclast populations alter in numbers and cause an osteopenic effect, which leads to a regional acceleratory phenomenon (RAP). 3,4RAP consists of four phases: activation, resorption, reversal, and formation. 5In the activation phase, receptor activator of nuclear factor kappa-B ligand (RANKL) cooperates with receptor activator of nuclear factor kappa-B (RANK), leading to the differentiation of preosteoclasts into mature osteoclasts. 6Bone resorption continues for about 2 weeks.In the reversal phase, secretion of osteoid by osteoblasts predominates.
Performing a localized alveolar decortication without tooth movement can provide insights into the biologic mechanisms taking place in the periodontal ligament space (PDL).Decortication of both buccal and lingual bone plates results in increased bone resorption and bone formation in rats with peaks at 3 weeks 7 .While alteration in bone turnover has been reported, the underlying response in the PDL following the localized alveolar decortication remains unclear.The aim of this study was to investigate the biologic reactions in the PDL space and the alveolar bone after selective alveolar decortication as the function of time.We combined this procedure with a genetic approach to modulate Wnt signaling: we used Lrp5G171V (Lrp5 ACT )mice, where bone mass is markedly increased due to reduced bone resorption 8 and adenovirus Dkk1 (Ad-Dkk1) treated mice that have reduced bone mass due to increased resorption. 9

Generation of Selective Decortication
Twenty-ve 10 weeks-old male mice (C57BL/6) were used for this study.A regular hard diet and water ad libitum were supplied.Following anesthesia with ketamine/xylazine (87.5/12.5 mg/kg, respectively), the right cheek was shaved and an incision was made along the mandibular ramus, and the buccinators muscle was elevated until the molar alveolus was found.About 10.0 mm up from the mandibular base (the deepest point of the mandibular base concavity), a hole with a diameter of 1.0 mm was created through the surrounding tissues on the buccal surface of mandibular alveolar ridge on the distal side of the mandibular third molar.A handpiece with #331 bur (diameter 1.0mm, head length 1.6mm) was used to form the hole that does not penetrate the underlying trabecular bone (Fig. 1).The region was irrigated, and the skin was closed with use of a suture.Mice were sacri ced at multiple time points including 0, 3, 7, 14, 28 days after decortication (PD0, PD3, PD7, PD14, PD28: 5 mice for each group).

Generation of Mouse Strains
The generation of Lrp5G171V mice (here referred to as Lrp5 ACT mice) were described previously; 10 ve 2 month-old mice were analyzed 7 days after decortication.The generation of Ad-Dkk1 treated mice were described previously; 11 ve 2 month-old mice were analyzed 7 days after decortication.Selective decortication was performed and sacri ced at PD7.

Sample Preparation and Processing
Right mandibular molar areas in mice were harvested and placed in 4% paraformaldehyde for one night at 4°C.Demineralization of samples were processed in a microwave using 19% EDTA for fourteen days.After process of decalci cation, specimens were dehydrated using an ascending ethanol series and para n embedding was followed.Longitudinal sections were cut in an 18-micron thick and collected on Superfrost-plus slides for analysis.Equivalent levels and planes of section in the distal root of the mandibular third molar were selected from each of the mouse for analysis.

Ethics
All procedures and the housing and care of mice were performed in accordance with animal welfare based on an approval of Stanford University Institutional Animal Care and Use Committee (protocol #13146).The study was reported in accordance with ARRIVE guidelines.

Results
In uence of Decoration on the Tissue Architecture of PDL Histologic examination of the mandibular periodontal complex revealed that compared to PD0 (Fig. 2A,2A'), decortication did not noticeably alter the brillar structure of the PDL at PD3 (Fig. 2B,2B'), PD7 (Fig. 2C,2C'), PD14 (Fig. 2D,2D') and PD28 (Fig. 2E,2E') in the wild-type mice.Higher magni cation images of the periodontal complex showed that the periodontal ligament consists of numerous cells, probably broblasts within and between the ber bundles.We turned to analyses of the alveolar bone osteogenesis and homeostasis in the PDL for a deeper understanding of how decortication affected the alveolar bone and the PDL with increasing healing time.
In uence of Decoration on the Osteogenic factors in the PDL Cells of the periodontal ligament are osteogenic in nature. 14In PD0, Osterix expression was observed close to cementum in the PDL space.Compared to its expression in PD0 (Fig. 3A), Osterix expression on the decortication side was not altered in the PDL space of PD3 (Fig. 3B).With increasing healing time, Osterix expression on the decortication side maintained throughout the PDL space (Fig. 3C-E).Runx2 expression followed the same pattern: on the intact side, Runx2 expression was observed throughout the PDL space (Fig. 3F); on the decortication side, Runx2 expression was maintained with increasing healing time (Fig. 3G-J).Osteopontin was expressed in both cementoblasts and osteoblasts of PD0 (Fig. 3K).Similarily, Osteopontin expression was maintained at decortication sides with increasing healing time (Fig. 3L-O).Following decortication of the alveolar bone, osteogenic factors in the PDL space maintained the expression on the decortication side until PD28.
A previous report suggests that expression of osteogenic factors in the PDL space could be related with TGF-β. 15We investigated the possible relationship between the osteogenic factors and TGF-β in the PDL space after selective decortication in the surrounding alveolar bone.As reported, TGF-β was observed in the PDL space at PD0 (Fig. 3P), 16 and its expression was maintained during the healing period (Fig. 3Q-T).
In uence of Decoration on the Bone Turnover ALP activity in PD0 was nearly undetectable in the PDL (Fig. 4A), while ALP activity on the decortication side was gradually increased in the PDL space with increasing healing time (Fig. 4B-E).Using TRAP staining we visualized a level of osteoclast activity on PD0 (Fig. 4F).TRAP activity at both PD3 and PD7 was noticeably increased (Fig. 4G,4H) compared to that in the intact side (Fig. 4F).TRAP activity in the decortication side at PD28 returned to the level seen on PD0 (Fig. 4I,4J).
Osteoclast activity is regulated by the combined actions of the osteoprotegerin (OPG) receptor and its ligand, RANKL.Expression of RANKL on the decortication side of periodontal complex (Fig. 4K-O) followed the same trend observed with TRAP activity.Compared with PD0, particularly high levels of RANKL were observed in the PDL at PD3 (Fig. 4L).We turned to analyses animal models with gain-and loss-of-function of Wnt signaling to better understand how high bone mass and loss of bone mass phenotypes affected the periodontium at PD7.

In uence of Decoration on the PDL Homeostasis and Bone Turnover in animal models with gain-and loss-of-funtion of Wnt signaling
We observed differences in the expression of osteogenic transcription factors in the PDL of Lrp5 ACT and Ad-Dkk1 treated mice after creation of decortication.For Lrp5 ACT mice, expression of Osterix (Fig. 5A') on the decortication side of the PDL space was slightly increased throughout the PDL space compared to its expression on the intact side (Fig. 5A).For Lrp5 ACT mice, expression of Runx2 (Fig. 5C') on the decortication side of the PDL space was slightly elevated, close to cementum, compared to its expression on the intact side as well (Fig. 5C).For Lrp5 ACT mice, expression of OPN (Fig. 5E') on the decortication side of the PDL space was also elevated, close to alveolar bone, compared to its expression on the intact side (Fig. 5E).ALP activity in the PDL of Lrp5 ACT was slightly increased on the decortication side, close to alveolar bone (Fig. 5G, G').For Ad-Dkk1 treated mice, expression of Osterix was not changed on the decortication side of the PDL space compared to the intact side (Fig. 5B, B').Expression of Runx2 was equivalent between the intact and decortication sides of Ad-Dkk1 treated mice (Fig. 5D, D').Expression of Osteopontin in the decortication side was partly observed in the cementum and alveolar bone of Ad-Dkk1 treated mice (Fig. 5F, F').ALP level was not altered on the decortication side of Ad-Dkk1 treated mice compared to the intact side (Fig. 5H, H').
Compared to the intact side of Lrp5 ACT mice, osteoclast activity was signi cantly increased on the decortication side of Lrp5 ACT mice (Fig. 5I, I').Even in Lrp5 ACT mice where higher Wnt signal is associated with decreased osteoclast activity, creation of decortication in the surrounding bone caused dramatic increase of osteoclast activity.In Ad-Dkk1 treated mice, strong expression of osteoclast activity in the intact side was also observed in the decortication side (Fig. 5J, J').

Discussion
Little is known how decortication in alveolar bone causes any alteration of homeostasis in the PDL space.Decortication of alveolar bone did not alter the tissue architecture in the PDL space (Fig. 2).The expression of osteogenic factors was also maintained on the decortication side until PD 28 (Fig. 2).Stimulation by decortication in the alveolar bone may in uence cell signaling in the PDL space.One of the possible candidates for cell signaling is TGF-β, which involves in growth, proliferation and differentiation of osteoblasts. 17,18In the same context, PDL cells were more differentiated upon elevation of TGF-β-receptor III, 15 and TGF-β-receptor II knock-out mouse exhibited defects in proliferation and differentiation of osteogenic cells. 17TGF-β expression in the PDL cells was demonstrated, 16 and it was also expressed in the PDL space before creation of decortication (Fig. 3P).Here, we found expression of TGF-β in the PDL space was continuously maintained until PD 28 (Fig. 3Q-T).Along with the expression of osteogenic factors, the expression of TGF-β was maintained on the decortication side until PD 28.
Following decortication, ALP activity was gradually increased with increasing healing time (Fig. 4A-E).Osteoclast activity was highest at PD3 (Fig. 4G) and returned to baseline at PD 28 (Fig. 4J).Thus, we observed different phase of bone turnover: a resorption phase at PD 3 and a formation phase at PD 28.At the early time period, bone resorption mostly occurred and then bone formation exceeds bone resorption.Later, a phase of mineralization followed, which is consistent with a previous study where the widespread formation of alveolar bone occurred at PD 21 after decortication. 5,19An increase of bone formation in response to decortication has also been observed in the rabbit tibia at 4 weeks. 3,20e sought clues into alteration of molecular reaction in the PDL space of high bone mass and loss of bone mass phenotypes following creation of decortication.Previously reported, the expression of osteogenic markers is high in an environment where Wnt signaling is up-regulated. 21Expression of osteogenic factors in the PDL space of Lrp5 ACT mice was slightly elevated at PD 7 (Fig. 5).Upon decortication, ampli ed Wnt signal was positively associated with increase of osteogenic factors expression in the PDL space of Lrp5 ACT mice.One question that remains to be answered is whether upregulated Wnt signaling can signi cantly enhance the expression of osteogenic factors, especially ones related to mineralization, in the Lrp5 ACT mice later time period.Expressions of osteogenic factors in Ad-Dkk1 treated mice, however, were not altered following decortication in the surrounding alveolar bone at PD 7. 11 The Lrp5 ACT showed alveolar bone accumulation with either unaltered bone resorption 22 or decreased bone resorption. 23Although the physiologic mechanism is not fully understood, decreased bone resorption in the Lrp5 ACT is due to reduced number and/or function of osteoclasts by altering the OPG to RANKL ratio. 24Here, selective decortication caused a dramatic increase of osteoclast activity in Lrp5 ACT mice, which was also observed in Lrp5 ACT mice after creation of injury. 11Upon speculation of the reason for increased osteoclast activity, in ammatory reaction in the Lrp5 ACT mice after selective decortication could exceed a threshold tolerance for expression of Wnt signaling during osteoclastogenesis. 25

Abbreviations ligament space
Lrp5

Figures
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Figure 3 Expression
Figure 3