Roles of autophagy in orthodontic tooth movement

Abstract

Introduction: Orthodontic tooth movement (OTM) relies on efficient remodeling of alveolar bone. While a well-controlled inflammatory response is essential during OTM, the mechanism regulating inflammation is unknown. Autophagy, a conserved catabolic pathway, has been shown to protect cells from excess inflammation in disease states. We hypothesize that autophagy plays a role in regulating inflammation during OTM.

Methods: A split-mouth design was used to force load molars in adult male mice, carrying a GFP-LC3 transgene for in vivo detection of autophagy. Confocal microscopy, Western blot, and quantitative polymerase chain reaction analyses were used to evaluate autophagy activation in tissues of loaded and control molars at time points after force application. Rapamycin, a Food and Drug Administration-approved immunosuppressant, was injected to evaluate induction of autophagy.

Results: Autophagy activity increases shortly after loading, primarily on the compression side of the tooth, and is closely associated with inflammatory cytokine expression and osteoclast recruitment. Daily administration of rapamycin, an autophagy activator, led to reduced tooth movement and osteoclast recruitment, suggesting that autophagy downregulates the inflammatory response and bone turnover during OTM.

Conclusions: This is the first demonstration that shows that autophagy is induced by orthodontic loading and plays a role during OTM, likely via negative regulation of inflammatory response and bone turnover. Exploring roles of autophagy in OTM holds great promise, as aberrant autophagy is associated with periodontal disease and its related systemic inflammatory disorders.

Figure 1.. Schematic representation of the autophagy pathway.

The schema summarizes the main steps of autophagy activation and flux, with labels for key proteins. Autophagy is an intracellular survival mechanism and modulator of inflammation. Under stressful conditions, AMPK inhibits mTOR and phosphorylates ULK1, initiating autophagy.^, Then activated ULK1-protein complex targets Beclin-1/VPS34 class III PI3K complex, which recruits downstream ATG proteins and promotes formation of the autophagosome, a double-membraned organelle that sequesters cytosolic components. During autophagosome maturation, cytoplasmic LC3I is translocated to autophagosomes, where LC3I is conjugated with phosphatidylethanolaminie (PE) to form lipidated LC3II. Lipidated LC3II represents a dynamic marker for autophagy. Finally, mature autophagosomes fuse with lysosomes, where the engulfed components marked with autophagy receptor p62, are degraded and recycled.^,

Figure 2.. Orthodontic loading activates autophagy.

A-J’’, Zeiss LSM710 confocal microscope imaged sections of first molar distal roots from GFP-LC3 mice post-loading. A-J: Magnification x40, scale bar: 150 μm; A’- J”: Magnification x60, scale bar: 100 μm; A’-J’: Mesial PDL zoom-in (left yellow box in A). A’’-J’’: Distal PDL zoom-in (right yellow box in A). F-J’’: Experimental: Mesial, compression-left. Distal, tension-right. F, Large yellow arrow indicates direction of force application with compression/mesial on the right and tension/distal on the left. F’, F’’: Arrowheads: zoomed-in cells with puncta. Green: GFP-LC3; Blue: DAPI nuclei. K, Quantification of autophagosome puncta versus days post-loading in PDL of control and loaded molars on the mesial side of the molars. Fluorescent puncta quantified in a uniform (100 μm x 150 μm) area using Image J. L, Quantification of autophagosome puncta versus days post-loading in PDL of control and loaded molars on the distal side of the molars. M, Western blot analysis of phospho-Ulk1 and p62.

Figure 3.. Expression qPCR analysis of autophagy, inflammatory and bone turnover markers.

Expression of autophagy (A-C, BECN1, LC3, ATG5), bone turnover (D-F, RANKL, OPG, MMP9), and inflammatory markers (G-H, NFATC1, TFNα, IL-1β) increases in peri-dental tissues after orthodontic loading at time points days 1, 3, 5, and 10 after loading. Inflammatory marker Il-6 was not transcriptionally altered in peri-dental tissues after loading.

Figure 4.. OTM is drastically reduced by autophagy gain of function with rapamycin.

[A-C] Schematic of orthodontic force application in mice with a split-mouth design. Occlusal view of the maxilla before [A] and after [C] placement of a NiTi coil spring on the experimental (E) side. The control (C) side has no spring placed and the molar remains unloaded. [B] 30 g of force measured by a force gauge prior to cementing the spring with light-cured composite resin. [D-I] 2D CT radiographs of control [D-F] and experimental [G-I] side molars. Left: mesial, compression side. Right: distal, tension side. Scale bar: 2 mm. [J-M] 2D CT radiographs of control [J-K] and experimental [L-M] side molars of mice injected with either saline vehicle or rapamycin. [N] OTM quantification at days post-loading in mice injected with saline vehicle or rapamycin. [O] Graph comparing OTM measured in μm of uninjected mice, saline vehicle injected mice and rapamycin injected mice.

Figure 5.. Osteoclasts increase after orthodontic loading, but numbers are suppressed with rapamycin-mediated autophagy activation.

A-P, TRAP-stained sections. A-H, Saline vehicle injection. I-P, Rapamycin injection. Control: no loading. Experimental: loaded, 30g of force. Scale bar: 150 μm. Mesial, compression: left. Distal, tension: right. Q, Quantification of TRAP+ cells versus days post-loading (p<0.05).