Accepted Manuscript A histological study of deformation of the mandibular condyle caused by distraction in a rat model Naoko Sakagami , DDS, PhD Tadaharu Kobayashi , DDS, PhD Kayoko NozawaInoue , DDS, PhD Kimimitsu Oda , DDS, PhD Taku Kojima , DDS, PhD Takeyasu Maeda , DDS, PhD Chikara Saito , DDS, PhD PII:

S2212-4403(14)00468-4

DOI:

10.1016/j.oooo.2014.05.003

Reference:

OOOO 917

To appear in:

Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology

Received Date: 30 November 2013 Revised Date:

3 April 2014

Accepted Date: 6 May 2014

Please cite this article as: Sakagami N, Kobayashi T, Nozawa-Inoue K, Oda K, Kojima T, Maeda T, Saito C, A histological study of deformation of the mandibular condyle caused by distraction in a rat model, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology (2014), doi: 10.1016/ j.oooo.2014.05.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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A histological study of deformation of the mandibular condyle caused by distraction in a rat model Naoko Sakagami, DDS, PhD,a Tadaharu Kobayashi, DDS, PhD,b Kayoko Nozawa-Inoue, DDS, PhD,c Kimimitsu Oda, DDS, PhD,d Taku Kojima, DDS, PhD,e Takeyasu Maeda, DDS,

a

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PhD,f and Chikara Saito, DDS, PhDg Graduate student, Division of Reconstructive Surgery for Oral and Maxillofacial Region,

Niigata University Graduate School of Medical and Dental Sciences. Reserch Fellow of the Japan Society for the Promotion of Science

Professor, Division of Reconstructive Surgery for Oral and Maxillofacial Region, Niigata

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b

University Graduate School of Medical and Dental Sciences.

Associate Professor, Division of Oral Anatomy, Niigata University Graduate School of

Medical and Dental Sciences. d

Professor, Division of Biochemistry, Niigata University Graduate School of Medical and

Dental Sciences. e

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c

Assistant Professor, Division of Reconstructive Surgery for Oral and Maxillofacial Region,

Niigata University Graduate School of Medical and Dental Sciences. f

Professor, Division of Oral Anatomy, Niigata University Graduate School of Medical and

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Dental Sciences.

Emeritus Professor, Division of Reconstructive Surgery for Oral & Maxillofacial Region,

Niigata University Graduate School of Medical and Dental Sciences. Address all correspondence to:

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Dr. Naoko Sakagami

Division of Reconstructive Surgery for Oral and Maxillofacial Region

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Niigata University Graduate School of Medical and Dental Sciences 2-5274 Gakkocho-dori, Chuo-ku, Niigata 951-8514 JAPAN [email protected] This study was supported by a scientific grant (#22592208) from the Ministry of Education, Science, Culture and Sports (MEXT) and Grant-in-Aid for JSPS Fellows (#233854) from Japan Society for the Promotion of Science (JSPS). The abstract is 143 words. The complete manuscript is 4540 words. The number of reference is 32. The numbe of figure is 8, and table is 1.

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Abstract Objective: This study investigated the bone resorption process of the rat mandibular condyle following mandibular distraction.

mandibular distraction at 0.175 mm/12 h for 10 days.

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Study design: Male Wistar rats at 10 weeks of age underwent unilateral Histologic and

histochemical analyses were employed at postoperative Day 1 and Weeks 1 and

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3.

Results: Micro CT observations showed that deformation of the condyle in the

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anterior region where a discontinuity of the condylar cartilage layer was found in histologic sections. This destroyed area gathered many osteoclasts.

In the

central region, disorganization with a thin hypertrophic cell layer was recognizable by Day1, but later thickened. Morphological recovery of the

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mandibular condyle could be attained by Week 3 in this animal model. Conclusion: These morphological findings indicate that the rapid deformation of the condyle with destruction of the cartilage layer and bone resorption by

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artificial distraction.

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Introduction Adequate mechanical loading is essential to maintain bone morphology, but excessive degrees can cause progressive deformity.1 Although previous

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reports have shown that loading is an important factor for normal bone growth and metabolism, it is well known that inadequate overloading by sustained

pathological conditions in bone tissue. 2-4

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gravitational and muscular loads cause skeletal deformities or progressive

Progressive condylar resorption (PCR), an irreversible complication,

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leads to the development of late skeletal relapse after orthognathic surgery, including bilateral sagittal split osteotomies, Le Fort I osteotomies, and bimaxillary osteotomies.

PCR brings about severe morphological changes in

condylar configurations with a reduction of volume and decrease in ramus height.

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Its etiology, however, remains not to be fully understood. To date, previous clinical and radiological studies have indicated some factors leading to PCR. For example, mandibular advancement following orthognathic surgery stretches

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the surrounding soft-tissue components. Such a tension force makes the condyle retrude into the glenoid fossa which generates pressure on the condylar

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head, resulting in condylar resorption when mechanical loading exceeds the adaptive capacity of the condyle.5,6-12 However, animal studies with mandibular distraction have engendered some controversy over the precise nature of the pressure--which is probably related to morphological alterations in the temporomandibular joint (TMJ), due to different distraction protocols and varying animal models.13-19 Our recent study reported on an animal model with mandibular

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distraction which is advantageous for the easy observation of tissue and/or cellular reactions.20

Although this animal model was able to demonstrate the

cellular events during bone formation with stable lengthening following

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mandibular distraction, it failed to show the bone resorption process which is

closely related to the etiology of PCR. This study was therefore undertaken to examine the effects of mechanical forces on the morphology of the mandibular

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condyle following distraction by micro CT analysis and histologic methods in a rat experimental model we previously described.20 Particular attention is given

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to characteristic histologic changes in two regions -- the anterior and central

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regions -- of the mandibular condyle.

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Materials and Methods All experiments were performed under the guidelines of the Niigata University

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Intramural Animal Use and Care Committee (approval number: Sindaiken-27)

Experimental animals

Thirty male Wistar rats, aged 10 weeks, were used in this experimental study.

other an untreated control (n=15).

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They were randomly divided into two groups: one experimental (n=15) and the Animals in both groups were further

Week 3 after distraction).

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subdivided into three groups based on observation periods (Day 1, Week 1, and The animals were allowed free access to powder

food and water throughout the study. The health of the animals was monitored,

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and body weight, food, and water intake were measured daily.

Device design

The distraction device was assembled using an orthodontic jackscrew

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(Dentaurum, Tokyo, Japan), with each end embedded in acrylic resin, as previously described.20 The devices were attached in specific positions with

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four self-tapping titanium bone screws (Stryker Leibinger and Co., Freiburg, Germany) (Fig. 1A).

Activation of each device enabled a precise and gradual

distraction because each 180-degree turn separated the osteotomized bone edges by 0.175 mm.

Surgical procedures The protocol for the surgical procedures appeared in our previous report.20

To

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secure the reproducibility on the surgical procedures, we put the distraction device to the right side of the mandible in the experimental group. Briefly, under an intraperitoneal injection of 8% chloral hydrate (400mg/kg b.w.) and a local

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injection of 1% lidocaine (0.5mg/100g b.w.), the buccal cortical bone at the body of the right hemimandible was cut between the second and third molars with a double-sided diamond disk (Horicco, Hopf, Ringleb and Co, Berlin, Germany) After drilling two bicortical holes, two

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and a fissure bur (Shofu, Kyoto, Japan).

self-tapping titanium bone screws were inserted into the holes. Two more

stability of the distraction device.

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titanium screws were threaded near the previous screws to maintain further the The distraction device was then attached to

the exposed screws with acrylic resin.

Then the lingual cortical bone was cut

with a fissure bur and fractured artificially.

Penicillin G (2000 U/100g, b.w.) was

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head with a distraction device.

Figure 1B shows the lateral view of a

administrated subcutaneously for five days following.

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Distraction protocol

The distraction protocol (Fig. 2) included a latency phase of five days after

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surgery, a distraction phase of 10 days during which each distraction was gradually performed at a rate of 0.175 mm every 12h (for a total lengthening of 3.5 mm), and a consolidation phase of up to 3 weeks during which each device maintained 3.5 mm of distraction lengthening. The actual distraction distances were calculated to confirm that the distraction process was successful.

Gaps between the inner surfaces of the

screws which fixed the external distraction device to the right hemimandible

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were measured using digital calipers (Digimatic Caliper, Mitutoyo, Japan) before and after distraction. Measurements were taken three times, and the average gaps were calculated at all stages. The actual distraction distance was defined

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as the difference between the gaps before and after distraction (see Table 1). Since a severe lateral cross-bite and an overgrowth of incisors were observed

were cut to allow easier access to feeding.

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Tissue preparation

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during and after the unilateral mandibular lengthening (Fig. 1C), these incisors

At appointed time, the animals were perfused with 4% paraformaldehyde in a 0.1M phosphate buffer (pH 7.4) under deep anesthesia in the same manner described above.

Tissue samples of mandibles with the surrounding soft

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tissues were immersed in the same fixative at 4ºC for 12 hours, with the external distraction device in situ.

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Micro CT analysis

The mandibles with a device were scanned by a Micro CT (Elescan; Nittetsu

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Elex, Osaka, Japan) at 71 kV and 100 mA (magnification 1.39, voxel pitch 62 µm, pixel size 62 µm and projection number 900). Three-dimensional images were reconstructed from the micro CT data, using a TRI/3D-BON software (Ratoc, Tokyo, Japan)

Histologic and immunohistochemical analysis After a micro CT analysis, tissue blocks including the TMJ on the lengthened

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side were decalcified with a 10% ethylene diamine tetra-acetic acid disodium (EDTA-2Na) solution for 6 weeks at 4 ºC and finally embedded in paraffin. Serial paraffin sections were sagittally cut at a thickness of 4.5 µm using a Some sections

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Retratome (REM-700; Yamato Koki Industrial, Asaka, Japan).

were processed for staining with either hematoxylin and eosin (H-E), or AZAN. Enzymatic histochemistry for tartrate-resistant acid phosphatase

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(TRAPase) was also employed for the demonstration of osteoclasts. For a double staining, before TRAPase histochemistry, dewaxed paraffin sections

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were incubated with a rabbit polyclonal antiserum against tissue non-specific alkaline phosphatase (ALPase; provided by Prof. Oda, Division of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan).

They were then reacted with a horseradish peroxidase-conjugated

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anti-rabbit IgG (Vector Laboratories, Inc., Burlingame, CA). An enzymatic reaction was developed with 0.002% 3, 3’-diaminobenzidine tetrahydrochloride (DAB) and 0.002% H2O2 in 0.05 M Tris-HCl buffer (pH 7.6).

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All the sections were counterstained with methyl green and photographed with an AxioCam HRc using a Zeiss microscope (AxioVision, Carl

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Zeiss Co. Ltd, Jena, Germany). Although we first established three areas for observation at the anterior,

central and posterior regions, a micro CT analysis could not demonstrate any specific changes at the posterior region in our preliminary observations. We therefore selected the anterior and central regions of the mandibular condyle for areas of histological observation in this study. In addition, micro CT observations showed contralateral condyle exhibited only rotation without any

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change in its shape, position, and radiolucency.

In this study, therefore we

focused on the changes in the condyle of the ipsilateral side as an animal model of the mandibular advancement.

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A highly skilled researcher performed the surgical procedures and the others analyzed micro CT, histological and immunohistochemical findings.

The

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analyses on the experimental and control groups were not blinded.

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Results

Animal conditions and distraction distance

In the experimental group, no remarkable complications--including wound infection--were found throughout the time period.

Grooming behavior Furthermore, there was no

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deteriorated on Day 1, but gradually improved later.

obvious change in jaw movement. Animals in the control group did not show any complications. Animals of both groups consistently gained weight.

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Although the body weights of the control group were greater than those of experimental animals at all stages, there was no significant difference between

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them except for Day 1 (Fig. 3). All distraction devices were stabilized on the osteotomy area until

fixation, as confirmed by measurement of the distraction distance which indicated 3.35 ± 0.03 mm (Table 1) as the average distance throughout the experimental period. No significant differences in distraction distance appeared between Day 1 and Week 1, or between Weeks 1 and 3, indicating that the distraction devices used in this study maintained a constant distraction distance

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(Table1; single-factor ANOVA).

Macroscopic observation with micro CT analysis

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Micro CT observations showed that the mandibular condyles in the control group had a smooth surface, with neither any irregularities nor erosion (Fig. 4A).

In

the experimental group, mandibular distraction caused the mandibular condyles The anterior area of the condyles on Day 1 exhibited a

slightly irregular and rough surface (Fig. 4B).

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to move posteriorly.

This morphological change in the

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surface became more apparent at Weeks 1 and 3, when a remarkable defect was recognizable in the anterior region by micro CT observations (Fig. 4C, D), suggesting that bone resorption proceeded daily.

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Histologic and histochemical findings Control group

The articular cavity between the temporal bone and mandibular condyle was

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completely divided into upper and lower cavities by the presence of the articular disk which consisted of a dense, collagenous tissue. The periphery of the

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articular disk was thick while its center was thinner.

A few folds of the synovial

membrane protruded into the articular cavity, particularly in the posterior region of the upper articular cavity (Fig. 5A).

The mandibular condyle had a smooth

surface (Fig. 5A) which was covered with a fibrous tissue colored blue with AZAN staining (Fig. 5B). The condylar cartilage layer was clearly divided into four layers from the condylar surface: fibrous, proliferative, maturative, and hypertrophic cell layers

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(Fig. 5A, D, F).

However, the thickness of the cartilage layer varied between

anterior and central regions (Fig. 5D, F) while the central region--including the posterior region--had a clear four-layer structure (Fig. 5F).

The cartilage layer

contact with compact bone and a fibrous capsule (Fig. 5D).

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at the anterior region gradually disappeared, ultimately coming into direct

However, this thin

cartilage layer was not located throughout the anterior region (Fig. 5D).

The

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densely-arranged trabecular bone beneath the cartilage layer multiply

interconnected at the central region (Fig. 5A, F), and comparatively thick

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trabeculae ran parallel to the longitudinal axis of the ramus of the mandible. However, the anterior part of the condylar neck did not develop trabeculae, appearing as a mass of compact bone (Fig. 5D).

A whole view of the TMJ (Fig. 5C) showed numerous osteoclasts with

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TRAPase reaction located in the chondro-osseous junction as well as the bone marrow space throughout the condyle while TRAPase reactive osteoclasts were scattered in the bone marrow of the temporal bone. The arrangement of the

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osteoclasts with TRAPase reaction formed a single line on the condylar bone at the anterior region where the cartilage layer appeared thin or none. TRAPase

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reaction was not recognizable in either the articular disk or the synovial membrane.

Another accumulation of osteoclasts was found in the anterior part

of the mandibular ramus. Double staining with TRAPase reaction and ALPase immunoreaction more clearly demonstrated the distribution of osteoclasts colored red and osteoblasts colored brown; the surface of the bone marrow space was lined with osteoclasts and osteoblasts in both regions (Fig. 5E, G). The chondrocytes in

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the maturative cell layer were immunoreactive to ALPase (Fig. 5G).

Experimental group at Day 1

by application of a distraction device (Fig. 6A).

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Observation of histologic sections also confirmed a translocation of the condyle The mandibular condyle

showed a deformation of the surface with an irregular outline of the condyle (Fig. This irregularly surfaced area was lined with a thicker fibrous tissue (Fig.

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6A).

referred to as the destroyed area.

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6B) and had completely lost the cartilage layer (Fig. 6D).

This area shall be

The cartilage layer in the central region also exhibited disorganization with a disappearance of the hypertrophic cell layer (Fig. 6F).

In addition,

expansion of the bone marrow space was observed in this region (Fig. 6A, F).

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Histochemistry for TRAPase reaction demonstrated the arrangement of osteoclasts at the chondro-osseous junction in the center of the condyle, but a very few osteoclasts were located along the surface of the bone marrow space A noticeable accumulation of osteoclasts reactive to TRAPase lay in

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(Fig. 6C).

the destroyed area (Fig. 6C), as confirmed with a combined staining with

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TRAPase and ALPase demonstrating a concentration of osteoclasts and osteoblasts around the area lined with a fibrous tissue in the anterior region (Fig. 6E).

In the central region, a chondro-osseous junction beneath the cartilage

layer also contained many osteoclasts, indicating erosion of the cartilage from the lower cartilage layer by TRAP-positive cells (Fig. 6G).

Experimental group at Weeks 1 and 3

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At Week 1, the deformation of the mandibular condyle proceeded severer compared with the previous stage (Fig. 7A).

The bone surface of the

destroyed area -- which came to be replaced by a fibrous tissue (Fig. 7B) -In the central region, on the other

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displayed a more irregular outline (Fig. 7D).

hand, the cartilage layer became as readily apparent as the control; the

thickness of the cartilage layer appeared to increase with recovery of the

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hypertrophic cell layer (Fig. 7F).

Histochemistry for TRAPase exhibited the occurrence of many

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comparatively large osteoclasts in the destroyed area as well as the entire length of the anterior part of the condylar neck in the anterior region (Fig. 7C).

The

chondro-osseous junction in the central region also contained many osteoclasts exhibiting TRAPase reaction (Fig. 7C).

In double staining, a predominant

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distribution of large osteoclasts reactive to TRAPase was found at the bone surface of the destroyed area (Fig. 7E).

However, it was difficult to identify the

osteoclasts at the bone marrow wall where they were exclusively lined with Immature bone, whose outline was lined with TRAPase

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ALPase (Fig. 7E).

reactive materials, was discernible at the chondro-osseous junction of the

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central region, indicating bone deposition there (Fig. 7G).

Furthermore, the

maturative cell layer immunopositive for ALPase appeared to increase in thickness (Fig. 7G).

At Week 3, the destroyed area in the anterior region expanded in depth and antero-posterior direction compared with Week 1 (Fig. 8A). The formation of a fibrous tissue was prominent in the destroyed area (Fig. 8A, C). Organization of the cartilage layer in the central region recovered to that of the

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control (Fig. 8E).

Many osteoclasts remained at the bone surface of the

destroyed area as shown by TRAPase-histochemistry (Fig. 8B).

Double

staining demonstrated an intense ALPase immunoreaction for a fibrous tissue of

osteoclasts (Fig. 8D).

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the destroyed area near the condylar bone in addition to TRAPase reactive

Formation of immature newly-formed bone with clear

cement lines reactive to TRAPase was confirmed at the chondro-osseous

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junction in the central region, indicating the continuous bone deposition there to

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serve to increase the thickness of the central region of the condyle (Fig. 8F).

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Discussion Physiological stress to the condyle--if adequate--is essential for condylar remodeling in mature rats.21,22 In contrast, non-physiological, often inadequate,

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stress that exceeds bone tolerance is harmful for tissue structures. In fact, a previous report demonstrated that biomechanical forces exposed to the

condyles caused morphologic alterations in the articular cartilage and underlying

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condylar bone in rats.13 Compressive force on the condyle using appliances such as coil springs and shift plates reduces the thickness of the cartilage: that is,

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it inhibits the proliferation of chondrocytes and the amount of extracellular matrix in rats.23,24 The risks and severity of degenerative changes in the TMJ such as PCR after orthognathic surgery have been documented,5-8, 25-27 but the mechanisms remain unclear. We therefore evaluated morphological and

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histological changes in the TMJ after mandibular lengthening by distraction of the mandibular body in a rat experimental model which we have previously described.20 The structure and histology of the rat articular cartilage is known to

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be similar to those of humans, some differences in morphology notwithstanding.28 In general, rats exhibit fewer differences in genetic factors

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between individuals,29,30 and it is easier to obtain data from a number of rats for studies.

The vector of mandibular movement due to the distraction in this study

resulted in a deviation of the mandible toward the contralateral side with the development of a lateral cross bite. The changes in mandibular morphology caused a posterior displacement of the right condyle relative to the glenoid fossa, and the anterior region of the condyle was compressed to the protrusion of the

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temporal bone as observed in micro CT analysis.

This finding readily suggests

that these changes produce excessive mechanical loading on the condyles. We confirmed that each rat was able to intake food as well as to open and close

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the mandible normally while wearing the distraction device and avoid a

possibility of atrophy from disuse in the TMJ. This is supported by the changes in body weight; there was no significant difference between control and

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experimental groups except on the first day after distraction.

Numerous other experimental studies have reported hyperplasia of the

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condylar cartilage in regions under conditions of elevated pressure.14, 15 In mandibular distraction, however, there is controversy over the nature of pressure related alterations in the TMJ with a distracted mandible.16-19 Although many researchers agree with the notion that mandibular distraction influences

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condylar morphology, opinions have varied with regard to the extent of these changes and to what extent normalization of the TMJ occurs with time.

This

lack of consistency in the literature might be a result of differences in

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experimental designs, distraction protocols, and types of animal models.16-19 The present study revealed deformation of the mandibular condyle in two

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regions: one at the chondro-osseous junction of the central region and the other in the anterior area with a thin cartilage layer or without one at all.

In the central

region, disorganization of the cartilage cell layer and expansion of the bone marrow space were observed at early stage of mechanical loading.

This area

showed the organization of cartilage layer and bone deposition at the chondro-osseous junction to suggest the occurrence of immature newly formed bone, resulting in an increase in height in the central region. The mechanism

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for these tissue reactions in the central region may reflect both adaptation and remodeling.

On the other hand, a part of the anterior region which was defined

as the destroyed area in this study underwent a remarkable bone resorption.

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This critical area, which exhibits a thin cartilage layer under normal conditions, has histologic features that easily indicate it to be a weak point of the mandibular condyle against mechanical stress.

In addition to the histological differences

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between anterior and central regions, another possible explanation for these

findings is that the mechanical loading in the anterior region of the condyle is Therefore, the anterior region

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larger than in the central and posterior regions.

of the condyle is more likely to be affected by compressive force to induce bone resorption than other regions.

Mechanical loading that exceeds the capacity for

adaptation may cause cellular damage and the disruption of remodeling in

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regions where pressure is greatest, leading to degenerative changes.15 Accordingly, it seems that the mechanical loading to the condyle in the present animal model exceeded the bone tolerance.

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There have been many experimental studies related to the effect of compressive forces on cartilage.

It has been reported that mandibular condylar

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cartilage is maintained by the balance between proliferation and erosion.22 Teramoto et al.23 noted a reduced proliferative activity on condyles under compressive force.

An in vitro study using explanted chondrocytes showed

that continuous compressive force reduced glycosaminoglycan chains and collagen synthesis and that intermittent force stimulated the synthesis of these matrix components31. Moffett et al.32 advocated a mechanism called regressive modeling, in which the first recognizable changes of regressive modeling occur

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at the junction of cartilage and the subchondral bone plate and a defect of the bone is subsequently produced. The present study similarly demonstrated the initial morphologic alterations in the condylar cartilage and subchondral bone Accordingly, the cartilage tissue at the condyle

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marrow at the central region.

and subchondral bone marrow both seem to be highly sensitive to mechanical

degenerative changes in the condyle.

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loading and are key elements in determining subsequent remodeling or

Because our experimental design displayed short-term results, the

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long-term alteration from these compression forces remains unclear. In our research, degenerative alteration such as bone resorption and fibrillation were found in the destroyed area at the anterior region of the condyle.

More

TRAP-positive osteoclasts in the anterior region of the condyle remained in the

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experimental group. Therefore, these changes might continue as irreversible alterations during a long-term follow-up. In conclusion, the present study revealed that our rat distraction model

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resulted in two major changes: condylar resorption in the anterior region and bone formation at the chondro-osseous junction at the central region.

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Mechanical loading by mandibular distraction caused various histological changes, most notably, bone resorption in the anterior region of the condyle, suggesting that overloading to the condyle is a key factor for PCR. Further investigations using this animal model will be necessary for clarifying the pathogenetic mechanism of PCR.

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Acknowledgements The authors cordially thank the staffs of the Divisions of Reconstructive Surgery

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School of Medical and Dental Sciences, Niigata, Japan.

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for Oral and Maxillofacial Region and Oral Anatomy, Niigata University Graduate

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19. Kim SG, Park JC, Kang DW, Kim BO, Yoon JH, Cho SI, et al. Correlation of immunohistochemical characteristics of the craniomandibular joint with the

2003;61:1189-97.

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degree of mandibular lengthening in rabbits. J Oral Maxillofac Surg

20. Ali MN, Ejiri S, Kobayashi T, Anwar RB, Oda K, Ohshima H, et al. Histologic

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study of the cellular events during rat mandibular distraction osteogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:325-35.

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21. Sugiyama H, Lee K, Imoto S, Sasaki A, Kawata T, Yamaguchi K, et al. Influences of vertical occlusal discrepancies on condylar responses and craniofacial growth in growing rats. Angle Orthod 1999;69:356-64. 22. Luder HU, Leblond CP, von der Mark K. Cellular stages in cartilage formation as revealed by morphometry, radioautography and type II collagen immunostaining of the mandibular condyle from weanling rats. Am J Anat 1988;182:197-214.

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23. Teramoto M, Kaneko S, Shibata S, Yanagishita M, Soma K. Effect of compressive forces on extracellular matrix in rat mandibular condylar cartilage. J Bone Miner Metab 2003;21:276-86.

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24. Watanabe A, Yamaguchi M, Utsunomiya T, Yamamoto H, Kasai K.

Histopathological changes in collagen and matrix metalloproteinase levels in articular condyle of experimental model rats with jaw deformity. Orthod

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Craniofac Res 2008;11:105-18.

25. Schellhas KP, Wilkes CH, Fritts HM, Omlie MR, Lagrotteria LB. MR of

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osteochondritis dissecans and avascular necrosis of the mandibular condyle. AJR Am J Roentgenol 1989;152:551-60.

26. De Clercq CA, Neyt LF, Mommaerts MY, Abeloos JV, De Mot BM. Condylar resorption in orthognathic surgery: a retrospective study. Int J Adult

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Orthodon Orthognath Surg 1994;9:233-40.

27. Merkx MA, Van Damme PA. Condylar resorption after orthognathic surgery. Evaluation of treatment in 8 patients. J Craniomaxillofac Surg 1994;22:53-8.

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28. Ingervall B, Freden H, Heyden G. Histochemical study of mandibular joint adaptation in experimental posterior mandibular displacement in the rat.

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Arch Oral Biol 1972;17:661-71. 29. Asano T. The effects of mandibular retractive force on the growing rat mandible. Am J Orthod Dentofacial Orthop 1986 ;90(6):464-74. 30. Yamada K. [A histological study on the changes in the attachment of the deep layer of the masseter muscle on the rat mandible during bite raising]. Nihon Kyosei Shika Gakkai Zasshi 1985;44:611-26. 31. Copray JC, Jansen HW, Duterloo HS. Effects of compressive forces on

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proliferation and matrix synthesis in mandibular condylar cartilage of the rat in vitro. Arch Oral Biol 1985;30:299-304. 32. Moffett BC Jr., Johnson LC, McCabe JB, Askew HC. Articular remodeling in

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the adult human temporomandibular joint. Am J Anat 1964;115:119-41.

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Fig. 1. A distraction device and animal heads with a distraction device. (A) A custom-made distraction device.

a: The body of the device made from

acrylic resin, b: titanium bone screws for fixation of the device, c: an expansion

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screw for the active distraction, d: a stick for turning the expansion screw. (B) A lateral view of an animal head with a distraction device.

(C) A lower view of an animal head. The animal exhibits an obvious lateral

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cross bite after distraction using a distraction device.

Fig. 2. The mandibular distraction protocol in this study.

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the appointed time for sacrifice.

Thick arrows indicate

Fig. 3. Changes in body weight during the experimental period. *P < 0.05 as compared with control animals (Student’s t-test).

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experimental group.

Cont: control group, Exp:

Fig. 4. Photographs showing reconstructed three-dimensional images obtained from microscopic computerized tomography (Micro CT) data for the control group (A), at Day 1 (B), Week 1 (C), and Week 3 (D).

The morphological

changes in the condyle occur in its anterior regions as shown by arrows.

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Fig. 5. Photomicrographs showing histologic and histochemical analyses in the control group. Sagittal central sections of temporomandibular joints. arrow indicates the anterior direction.

A thick

(A) The temporomandibular joint consists

Stained with hematoxylin and eosin.

(B) The temporomandibular joint with

Note how the mandibular condyle is entirely covered with a

fibrous tissue colored blue (arrows). condyle reactive to TRAPase.

The TRAPase reaction is distributed throughout

Note the smaller degree of TRAPase reaction in the

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the mandibular condyle.

(C) A whole view of a rat mandibular

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AZAN staining.

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of the mandibular condyle (MC), articular disk (AD), and temporal bone (TB).

anterior region. (D, E) The anterior region of the condyle. Hematoxylin and eosin staining (D), and double-staining with TRAPase and ALPase (E). Note differences in the thickness of the cartilage layer between the right (arrows) and A TRAPase reaction, colored red, is observed at

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left sides (arrowheads) (D).

the chondro-osseous junction as well as the bone marrow wall.

ALPase

immunoreaction is found at the half of the cartilage layer and at the bone surface

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(E). (F, G) The central region of the condyle. Hematoxylin and eosin staining (F), and double-staining with TRAPase and ALPase (G).

(F) The cartilage layer

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is clearly divided into four layers: fibrous (Fb), proliferative (P), maturative (M), and hypertrophic cell layers (Hy).

A TRAPase reaction and ALPase

immunopositivity are found at the half of the cartilage layer and osteoclasts, and at the bone surface, respectively.

Scale bars: 500µm (A-C), 200µm (D-G)

Fig. 6. Photomicrographs of the experimental group at Day 1. (A) The

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temporomandibular joint shifts posteriorly (compared to Fig. 5A).

A part of the

anterior region (arrows) shows deformation, and the subchondral bone marrow at the central region has expanded.

Stained with hematoxylin and eosin.

condyle designated as a destroyed area.

AZAN staining.

a rat mandibular condyle reactive to TRAPase.

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(B) A fibrous layer (asterisks) at the anterior region is observed invading into the (C) A whole view of

TRAPase reactive osteoclasts (D-G) Higher

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are observed to concentrate in the destroyed area (arrows).

magnifications of the destroyed area in the anterior region (D, E) and the central Hematoxylin and eosin staining (D, F), and

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region (F, G) of the condyle.

double-staining with TRAPase and ALPase (E, G). The cartilage layer in the destroyed area has diminished (D), but the cartilage layer remains albeit with a thinner appearance compared with the previous stage at the central region (F).

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Comparatively large osteoclasts with TRAPase reactions colored red occur at the bone surface of the destroyed area (E). The hypertrophic layer is difficult to Scale bars: 500µm (A-C), 200µm (D-G)

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identify (G).

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Fig. 7. Photomicrographs of the experimental group at Week 1.

(A, B) The

deformation of the mandibular condyle has proceeded (compared with Figs. 5A and 6A). The destroyed area is observed to spread (asterisk), and the cartilage layer appears to increase in thickness (arrows) (A). (arrows) is occupied by a fibrous tissue. (A) and AZAN staining (B).

The wide destroyed area

Stained with hematoxylin and eosin

(C) Osteoclasts with TRAPase reaction gather at

the widely-expanded destroyed area.

(D-G) Higher magnifications of the

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destroyed area in the anterior region (D, E) and of the central region (F, G). Hematoxylin and eosin staining (D, F), and double-staining with TRAPase and ALPase (E, G).

The thick fibrous tissue is covered with the condylar bone with

irregular bone surface at this destroyed area (D).

Note the

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active bone resorption (C), as demonstrated by a double staining (E).

In the central region, the

cartilage layer has repaired itself to be identifiable as four clear layers (F).

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Clear cement lines with TRAPase reactions (arrows), suggesting the formation of newly-formed bone (asterisks), are recognized at the chondro-osseous Scale bars: 500µm (A-C), 200µm (D-G)

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junction (G).

Fig. 8. Photomicrographs of the experimental group at Week 3. The active

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bone resorption and fibrillation have occurred as the destroyed area (arrows) has advanced (A, C), confirmed with an accumulation of TRAPase osteoclasts remains at the widely-expanded destroyed area (arrows in B).

However, the

region (E).

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four layers of the cartilage cell layer have become more visible in the central Stained with hematoxylin and eosin (C, E) and AZAN staining (A).

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Enzymatic histochemistry for TRAPase reaction (B).

(D, F) Double staining of

the destroyed area in the anterior region (D) and the central region (F). Numerous osteoclasts with TRAPase colored red remain on the bone surface in the destroyed area (D).

In the central region, newly-formed bone (asterisks)

with clear cement lines reactive to TRAPase has increased in volume at the chondro-osseous junction (F).

Scale bars: 500µm (A, B, G), 200µm (C, D, E, F)

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Final distraction distance

(n=5)

mm (mean ± SD)

Day 1

3.35 ± 0.02

Week 1

3.36 ± 0.03

Week 3

3.34 ± 0.03

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Specimen group

Distraction distances in different group

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Table 1.

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Average of final distraction distance = 3.35 ± 0.03

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Our study revealed that various histological changes caused by mandibular distraction,

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suggesting that overloading to the condyle is a key factor for PCR.

A histologic study of deformation of the mandibular condyle caused by distraction in a rat model.

This study investigated the bone resorption process of the rat mandibular condyle after mandibular distraction...
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