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3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model

Published: April 15th, 2021



1Department of Oral Medicine, Infection and Immunity, School of Dental Medicine, Harvard University

We present a protocol for generating orthodontic tooth movement in mice and methods for 3D visualization of the collagen fibers and blood vessels of periodontal ligament without sectioning.

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to understand these complex remodeling processes, it is critical to study the tooth and periodontal tissues within their 3D context and therefore minimize any sectioning and tissue artefacts. Mouse models are often utilized in developmental and structural biology, as well as in biomechanics due to their small size, high metabolic rate, genetics and ease of handling. In principle this also makes them excellent models for dental related studies. However, a major impediment is their small tooth size, the molars in particular. This paper is aimed at providing a step by step protocol for generating orthodontic tooth movement and two methods for 3D imaging of the periodontal ligament fibrous component of a mouse mandibular molar. The first method presented is based on a micro-CT setup enabling phase enhancement imaging of fresh collagen tissues. The second method is a bone clearing method using ethyl cinnamate that enables imaging through the bone without sectioning and preserves endogenous fluorescence. Combining this clearing method with reporter mice like Flk1-Cre;TdTomato provided a first of its kind opportunity to image the 3D vasculature in the PDL and alveolar bone.

The basic underlying biological process in orthodontic tooth movement (OTM) is bone remodeling. The trigger for this remodeling process is attributed to changes in the structure of the periodontal ligament (PDL) such as extracellular matrix (ECM) stress, necrosis as well as blood vessel destruction and formation1,2,3. Other possible triggers for alveolar bone remodeling are related to force sensing by osteocytes in the bone, as well as mechanical deformation of the alveolar bone itself; however their role in OTM is still not fully elucidated4,

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All animal experiments were performed in compliance with NIH's Guidelines for the Care and Use of Laboratory Animals and guidelines from the Harvard University Institutional Animal Care and Use Committee (Protocol no. 01840).

1. Orthodontic Tooth Movement

  1. To generate a mouse bed, use a flat plastic platform with a wedge shaped, 45° angled headrest. The headrest can be generated by cutting a plastic box.
    1. Elevate the head end of the platform to generate an approx.......

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This paper presents a method to produce OTM as well as two methods for 3D imaging of collagen fibers inside the PDL without any sectioning. For animal research purposes, when alignment of the teeth is not necessary, a tooth movement is considered orthodontic if it generates remodeling of the alveolar bone at all root levels. Constant force level applied on teeth is required in order to generate a reliable OTM. Here, an activated shape-memory NiTi coil is used to generate a consistent force of 10 g throughout the experime.......

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Generating OTM in mice is highly desired due to the size, genetics and handling advantages. Using the mandible provides an easy handling both in terms of tissue dissection as well as sample preparation and imaging. Here we presented a method to generate OTM with translational movement of the tooth inside the bone within 7 days of OTM. Using this protocol, the overall duration of the tooth movement can be extended, since the activated coil delivers a constant force level for movement of up to approximately 1 mm. Howe.......

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This study was supported by the NIH (NIDCR R00- DE025053, PI:Naveh). We would like to thank Harvard Center for Biological Imaging for infrastructure and support. All figures are generated with


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Name Company Catalog Number Comments
1-mL BD Luer-Lok syringe BD 309628
1X phosphate buffered saline VWR Life Sciences 0780-10L
200 proof ethanol VWR Life Sciences V1016
Aluminum alloy 5019 wire Sigma-aldrich GF15828813 0.08 mm diameter wire, length 100th, temper hard. Used as wire ligature around molar.
Avizo 9.7 Thermo Fisher Scientific N/A Used to analyze microCT scans
Castroviejo Micro Needle Holders Fine Science Tools 12060-01
Clr Plan-Apochromat 20x/1.0,CorrVIS-IR M27 85mm Zeiss N/A Used for second harmonic generation imaging
Cone socket handle, single ended, hand-form G.Hartzell and son 126-CSH3 Handle of the inspection mirror
EC Plan-Neofluar 5x/0.16 Zeiss 440321-9902 Used for light-sheet imaging
Elipar DeepCure-S LED curing light 3M ESPE 76985
Eppendorf safe-lock tubes, 1.5mL Eppendorf 22363204
Ethyl cinnamate, >= 98% Sigma-aldrich W243000-1KG-K
Hypodermic Needle, 27G x 1/2'' BD 305109
Ketathesia 100mg/ml Henry Schein Animal Health NDC:11695-0702-1
KIMWIPES delicate task wipers Kimberly-Clark 21905-026 (VWR Catalog number) Purchased from VWR
LightSheet Z.1 dual illumination microscope system Zeiss LightSheet Z.1/LightSheet 7 Used for lightsheet imaging
LSM 880 NLO multi-photon microscope Zeiss LSM 880 NLO Used for two-photon imaging
MEGAmicro, plane, 5mm dia, SS-Thread Hahnenkratt 6220 Front surface inspectrio mirror
MicroCT machine, MicroXCT-200 Xradia MICRO XCT-200
Mini-Colibri Fine Science Tools 17000-01
PermaFlo Flowable Composite Ultradent 948
Procedure platform N/A N/A Custom-made from lab materials
Routine stereo micscope M80 Leica Micosystems M80
Sentalloy NiTi open coil spring TOMY Inc. A 0.15mm diameter closed NiTi coil with an inner coil diameter of 0.9mm delivers a force of 10g. Similar products can be purchased from Dentsply Sirona. 
T-304 stainless steel ligature wire, 0.009'' diameter Orthodontics SBLW109 0.009''(.23mm) diameter, Soft temper
X-Ject E (Xylazine) 100mg/ml Henry Schein Animal Health NDC:11695-7085-1
Z100 Restorative, A2 shade 3M ESPE 5904A2

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