Development of the distraction osteogenesis model for rat mandibular

inferior border and alveolar bone

HYUN SEUNG MOON

The Graduate School Yonsei University

Department of Dental Science

Development of the distraction osteogenesis model for rat mandibular

inferior border and alveolar bone

Directed by Professor JUNG-YUL CHA

The Master's Thesis

submitted to the Department of Dental science

and the Graduate School of Yonsei University

in partial fulfillment of the requirements for the degree of

Master of Dental Science

HYUN SEUNG MOON

June 2010

This certifies that the dissertation of HYUN-

SEUNG MOON is approved.

Thesis supervisor

The Graduate School

Yonsei University June 2010

감사의 글

논문이 완성되기까지 항상 세심한 지도와 격려로 이끌어주시고 부족한

논문을 살펴주신 차정열 지도 교수님께 진심으로 감사 드리며, 귀중한

시간을 내주시어 관심과 조언을 아끼지 않으신 황충주 교수님, 유형석

교수님께 깊이 감사 드립니다. 또한 교정학을 공부할 수 있도록 기회를

주시고 늘 따뜻한 조언과 가르침을 주신 박영철 교수님, 백형선 교수님,

김경호 교수님, 이기준 교수님, 정주령 교수님께도 깊은 감사를 드립니다.

수련 생활 동안 큰 힘이 되어주고 함께 고생한 의국 동기들과 (박정진,

백인규, 윤정원, 이원제, 최태현), 의국 선배님들, 의국 후배들에게도

감사의 마음을 전합니다. 또한 실험을 비롯해 논문을 위해 수고해 준

몽고에서 온 모노 선생에게도 깊은 감사의 말을 전합니다.

항상 아낌 없는 지원과 믿음으로 저를 지켜봐 주신 부모님과 동생

그리고 바쁜 가운데서도 언제나 믿고 따라준 사랑하는 아내 정지은과 이

작고 소중한 기쁨을 함께 나누고자 합니다. 마지막으로 하나님께 감사

드리며 이 모든 영광을 돌립니다.

2010년 6 월 저자

i

Table of contents

List of tables ····································································································· ii List of figures ·································································································· iii ABSTRACT ····································································································· v I. INTRODUCTION ························································································ 1 II. MATERIALS AND METHODS ································································· 4

A. Animals ································································································· 4 B. Methods ································································································· 4

a. Device design···················································································· 4 b. Anesthesia and surgical procedure ···················································· 4

c. Post-operative care ············································································ 6 d. Distraction protocol ·········································································· 7

e. Tissue preparation ············································································· 8 f. Micro CT analysis ············································································· 9

III. RESULTS ································································································ 10

A. Histologic analysis ··············································································· 12 B. Micro CT analysis ················································································ 14

IV. DISCUSSION ·························································································· 18 V. CONCLUSION ························································································· 22 REFERENCES ······························································································· 23 ABSTRACT (IN KOREAN) ·········································································· 25

ii

List of tables

Table 1. Preset values of micro computated tomography scanning ·················· 9

Table 2. Intraoperative and postoperative complications and their rates ········· 11

Table 3. Average distances between two middle bone screws on the stages ······· 12

Table 4. Average bucco-lingual thickness of rat mandible ····························· 16

iii

List of figures

Fig. 1. Distraction device designs

A. Device assembled by jackscrew and surgical plate without bend.

B. Device embedding jackscrew into acrylic resin.

C. Final device assembled by jackscrew and bent surgical plate and

ready made Bioplast block to reinforce mandibular angle area ·········· 4

Fig. 2.

A. Schematic illustraction of osteotomy location and position of holes

for four miniscrews securing the device on the mandible.

B. The distraction device applied to the left hemimandible secured by

bone screws ························································································ 6

Fig. 3. Schematic diagram showing the operation procedures of rat mandibular

distraction osteogenesis. ········································································ 6

Fig. 4. Rat wearing neck collar for immobilizing of distraction device. ··········· 7

Fig. 5. The experimental protocol of sequential stages of rat mandibular

distraction osteogenesis. ········································································ 8

Fig. 6. Mandibular bone fracture is observed at the proximal segment. ·········· 10

Fig. 7. Diagram showing the change of average weight of all subjects. ·········· 11

Fig. 8. Histologic and immunohistochemical analysis on the distraction

osteogenesis of rat mandibular inferior border. ··································· 13

Fig. 9. Histologic analysis on the alveolar bone between M2 and M3 of rat

mandibular distraction osteogenesis at the 4 weeks of consolidation. ······ 14

Fig. 10. The distance between the anterior border of alveolar bone and the

furcation of second molar. ································································· 15 Fig. 11.

A, Transaxial views of right hemimandible numbered to correspond to axial view.

iv

B, Axial view showing rat right hemimandible is marked with

transaxial reference lines. ······························································· 16

Fig. 12. Periapical views and reconstructed 3-dimensional micro CT images

at the sequential stages of rat distraction osteogenesis ······················· 17

v

ABSTRACT

Development of the distraction osteogenesis model for rat mandibular inferior border and alveolar bone

HYUN-SEUNG MOON

Department of Dental Science

The Graduate School, Yonsei University

(Directed by Professor JUNG-YUL CHA)

Osteogenic distraction is defined as surgical procedure which creates new bone and adjacent

other tissues including gingiva, skin, muscle, tendon, blood vessels and nerves by incremental

traction of osteomized bone edges. By virtue of it’s advantages, treatment effects of distraction

osteogenesis on patients have been reported. In recent, even the mechanism of distraction

osteogenesis has been extensively applied to alveolar bone area as an alternative to the

traditional treatment. Tremendous development of the technique has been achieved, but much

of molecular events involved in the process remained still questionable.

The aim of this study is to establish an experimental rat model for distraction osteogenesis on

mandible and alveolar bone concurrently for investigating histologic and radiographic analysis

and to develop the device for various conditions of distraction. The results of this study were

as follows.

1. Micro CT analysis was performed for the anatomical structure of rat mandible and

provided the guides for the location and design of osteotomy and positioning the

bone screws stabilizing the distraction device.

2. The rat distraction osteogenesis model for mandibular inferior border and alveolar

bone was successfully established on the basis of the protocols used in this study.

The protocols contain 5 days of latency period, distraction rate of 0.20 mm/day, at a

single session, and 4 weeks of consolidation phase.

3. New bone formation of the rat model occurred by combination of two modes.

vi

Intramembranous ossification was predominant at the end of distraction period,

while endochondral ossification was observed at the center of distraction gap at the

end of 4 weeks of consolidation period.

The mandibular inferior border and alveolar distraction model for rats was successfully

established in this study. This model can be applied to various conditions related to the

distraction osteogenesis and provide knowledge for improvements of distraction

osteogenesis treatment for patients.

Key words: distraction osteogenesis, micro CT, alveolar distraction, rat model

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Development of the distraction osteogenesis model for rat mandibular inferior border and alveolar bone

HYUN-SEUNG MOON, D. D. S.

Department of Dental Science

Graduate School of Yonsei University

(Directed by Prof. JUNG-YUL CHA, D. D. S., M.S.D., Ph. D.)

I. Introduction

Osteogenic distraction is defined as surgical procedure which creates new bone and adjacent

other tissues including gingiva, skin, muscle, tendon, blood vessels and nerves by incremental

traction of osteomized bone edges. (Cope, Samchukov, and Cherkashin, 1999). By

considerable effort of researchers and it's evolution, the technique of craniofacial distraction

osteogenesis (DO) has been applied to many clinical situations which are previously unable to

approach with conventional treatment methods during past couple of decades. Distraction

osteogenesis is indicated for following cases; severe deficiency on the growth of jaws such as

Pierre Robin syndrome, severe mandibular growth deficiency requiring more than 10-15 mm

lengthening, and short mandibular ramus.

Research for osteogenic distraction initiated in the field of the orthopedics and traumatology.

Although Codivilla (1994) first introduced limb lengthening on femurs by distraction

osteogenesis, it was Russian traumatologist, Gavril Ilizarov (1989a, 1989b) who made the

technique popular with extenssive canine tibiae experiments in the 1950s. He developed new

distraction device, studied mechanical, physiologic conditions for bony regeneration. The

procedure is based on the principle, "law of tension-stress" established by him, whereby

gradual traction of the tissues constructs the environment that stimulates tissue growth and

regeneration. However, most of studies on this subject based on old paradigm of bone healing

in which bone effector cells function independently and mechanical strain has little effect on

the characteristics of bone (Teitelbaum, 2000). Thus new concept of bone healing process is

required to appreciate how the distraction osteogenesis functions. The Utah paradigm

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describes that specific strain dependent signals control the adaptive mechanism of bone and

strain history of bone (Frost, 2000, 2001), not effector cell alone, determines whole responses

on the procedures. As Ilizarov suggested "law of tension-stress", the Utah paradigm can

provide a better understanding of how bone works in distraction osteogenesis.

It is reported that success of the technique depends on biologic and biomechanical factors

equally. Biomechanical factors include extrinsic or fixator-related factors and intrinsic or

tissue-related factors (Cope et al., 1999; Samchukov et al., 1998). Extrinsic parameters are

related to the dimension of securing screws and material properties of the distraction device.

Intrinsic parameters are related to the properties of distracted bone segments, the length of

consolidation phase, and tensile force on soft tissue. It is needed to consider the mechanical

factors mentioned above for the success of distraction osteogenesis.

Tremendous development of the technique has been achieved, but much of molecular events

involved in the process remained still questionable. Thus, it is inevitable to perform animal

experiments to clarify the responses at the molecular level resulting from sequential

distraction stages and to apply findings from that to clinical situations. Snyder et al. (1973)

using a canine model, were the first to adapt the limb DO principle to the craniofacial skeleton.

Their success was a considerable technical accomplishment that ignited the field of

craniofacial DO, and created the momentum for numerous experimental surgical models.

Mehrara Babak et al. (1999) reported that TGF-b 1 expression showed the similar pattern to

the progress of osteoblast development and is related to the control of angiogenesis on rat

mandibular distraction model. These animal studies refined the technical principles of

craniofacial distraction. In addition, it is reported that rat models have advantages over large

animals, including lower cost, the availability of numerous molecular reagents, and the

statistical power as it is possible to use a large number of subjects (Connolly et al., 2002).

In recent, the mechanism of DO has been extensively applied to alveolar bone area as an

alternative to the traditional treatment methods such as alveolar ridge augmentation. Alveolar

distraction osteogenesis overcame drawbacks of traditional methods like morbidity of the

donor site, graft resorption, additional operative procedure, and soft tissue scar due to prior

trauma or surgery. In their animal study, Block and the colleagues (1996) reported that

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distraction osteogenesis can augment an alveolar ridge vertically and offer support for

implants. Moreover, horizontal lengthening of alveolar bone can be achieved by premaxilla

distraction osteogenesis (Iida et al., 2007).

Despite the prevalence of this technique in craniofacial complex, the protocol including

latency period, distraction rate, consolidation period, is largely based on long bone distraction.

It is demonstrated that there would be several limitations if the principles of long bone

distraction osteogenesis is directly applied to craniofacial bones since craniofacial bones have

more complicated structure and attachment of muscles than that of long bone so that it needs

more sophisticated control of 3-dimensional force vector. Moreover, it has different functional

and histologic characteristics. In comparison to Ilizarov's recommendations for long bones in

which corticotomy is used to separate long bone to maintain blood supply, distraction

ostegenesis of facial bone begins with osteotomy as it has rich blood supply. Djasim et al.

(2007) reported that it is optimal to have a 5-day latency period, a distraction rate of 0.2-

0.6mm/day with a continuous rhythm, and a 16-42 day consolidation period for rat mandibular

model in the review article. In case of alveolar distraction, Cano et al. (2006) recommended 4-

7 day of latency period to prevent the exposure of bone to oral cavity. The protocol can be

different depending on what kinds of animals are used as an experimental subjects, their size

and status of growth stage.

The aim of this study is to establish a experimental rat model for distraction osteogenesis on

mandible and alveolar bone concurrently for investigating histologic and radiographic analysis,

to establish optimal protocol for sequential stages and to develop the device for various

conditions of distraction.

- 4 -

II. Materials and Methods

Animals

20 male Sprawl-Darley rats aged 16 weeks (450-500 g) were used in this study. All animals

were housed in separate cages, with a 12-hour light/dark schedule and had free access to

kibbles and water. All experiments were reviewed by the Committee on the Guidelines for

Animal Experimentation of Yonsei University and were performed according to the

recommendations or conditions proposed by the review Committee (Y 09-120).

Device design

Several different device were designed in this study. First, conventional one was made using

an orthodontic hygienic type jackscrew (Dentaurum, Tokyo, Japan) at which L-shaped surgical

plate (KLS martin, Tuttlingen, Germany) was connected by composite resin. This device

caused radiographic blurring when taking micro CT because part of surgical plate was exposed

to region of interest. To avoid this, the second device was assembled by embedding

orthodontic jackscrew into acrylic resin instead of metal plate. Because this model needed

long miniscrews, they prohibited the wound from healing. Finally, This study chose modified

L-shaped surgical plate which was given bend and jackscrew. Especially, to minimize metal

exposure of jackscrew during CT scanning, 45 degree distal bend is given at the point where

horizontal and vertical elements meet (Fig. 1).

Fig. 1. Distraction device designs.

A. Device assembled by jackscrew and surgical plate without bend.

B. Device embedding jackscrew into acrylic resin.

C. Final device assembled by jackscrew and bent surgical plate and ready made

Anesthesia and surgical procedures

The rats were anesthetized using intraperitoneal injections of Rumpun (Bayer, Korea) and

A B C

- 5 -

Zoletil (Virbac Lab, Carros, France) to achieve general anesthesia and 1 % lidocaine

(0.5mg/100g body weight) for local anesthesia.

After shaving, a 2-cm incision was made along the inferior border of the left hemimandible

skin. The masseter muscle was reflected transversely and the body of the left hemimandible

was exposed. The design of osteotomy and location of bone screws were decided on the

basis of the information by micro CT data. First, the cortical bone was cut 2 mm vertically

starting between the second and third molars (Fig. 2). Then, 1cm long horizontal ostetomy

was made. All osteotomy was performed with diamond point bur at 10,000 rpm in an

irrigated field. Two bicortical holes were drilled anterior and posterior to the second vertical

osteotomy line, respectively, at 10,000 rpm on an irrigated field. The custom made

distraction device was stabilized to the bone by threading two self tapping titanium bone

screws, diameter of 1.5 mm and 5.0 mm long (BMK, Seoul, Korea) to the anterior holes.

The muscle on the lingual side of mandibular border was also reflected and preformed

Bioplast block (Scheu dental technology, Germany) on that area for providing the

stabilizaition of bone screws. Two more screws are drilled into the mandibular angle. From

the posterior end of the horizontal ostetomy line, another vertical cut was made. The second

vertical osteotomy is performed after securing the device to stabilize bone fragments in their

own position without displacement.

A complete osteotomy was confirmed by expanding the distractor several turns and

observing that bone edges were actually widened. The distractor was then returned to the

initial position. After the wound was cleaned with saline irrigation, the muscle layer was

returned to their normal position and sutured with absorbable silk, Vicryl (Ethicon, U. S. A.)

and the skin was closed around holding screws (Fig. 3).

The animals were allowed to recover and penicillin G (2000 U/100 g body weight) was

administered intramuscularly immediately after the operation. For the rest of the experiment

time, the subjects were housed separately, fed a soft diet, and their teeth were clipped.

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Fig. 2. A. Schematic illustraction of osteotomy location (blue lines) and position of holes for

four miniscrews securing the device on the mandible (blue circles). B. The distraction device

applied to the left hemimandible secured by miniscrews. C. Localization of ostetomy lines in

previous studies.

Fig. 3. Schematic diagram showing the operation procedures of rat mandibular distraction

osteogenesis.

Post-operation care

20 mL Lactated Ringer's solution was injected subcutaneously for 10 days following surgery.

Post surgical managements included injection of antibiotics, and analgesics, trimming of

incisors to prevent impingement of palatal soft tissue by overgrowing teeth and daily weighing.

All animals had free access to water and soft diet made by mixing powdered kibbles and water.

Neck collar was placed to all animals to immobilize the distraction device (Fig. 4).

CBA

- 7 -

Fig. 4. Rat wearing neck collar for immobilizing of distraction device.

Distraction protocol (Fig. 5)

There were latency phase of 5 days after osteotomy, followed by a distraction phase of 12

days, during which gradual distractions were performed at a rate of 0.20 mm every 24 hours

(for a total lengthening of 2.40 mm), and a consolidation phase of 4 weeks, during which the

external distraction device maintained in place with no distractions. 20 rats were randomized

to each of two groups. Rats in distraction group were sacrificed immediately after distraction

period and ones in consolidation group after finishing 4 weeks of consolidation period.

Measurement of distraction distance was taken immediately at the time after latency period

(T0), after distraction period (T1) and after consolidation period (T2) on the CT images to

confirm how long bone edges were actually separated and that distracted gap is successfully

maintained during consolidation. The distance measured was the length between the center

points of the two middle screws securing external distraction device to the left hemimandible.

Measurements were repeated two times on each stages. The actual distraction distance was

calculated by the mean difference between the average postdistraction and the average

predistraction distances.

- 8 -

Fig. 5. The experimental protocol of sequential stages of rat mandibular distraction

osteogenesis.

Tissue preparation

The specimens were fixed on 4% paraformaldehyde for 24 hours, decalcfied with 0.5mol/L

ethylenediamine tetraacetate (EDTA) 7.5% for 5 weeks. The superior and inferior part of the

mandible was embedded in paraffin. Nine micron sections were mounted on SP 1600

microtome (LEICA, Germany), and haematoxylin and eosin (HE) staining was performed for

histological observation and histomorphometric analysis.

- 9 -

Micro CT analysis

In vivo micro CT scannings of hemimandibles were obtained using micro CT scanner

(SkyScan micro CT 1076, Skyscan, Kontich, Belgium) at a voltage of 100 kV and a current of

100 mA. CT scannings were taken at the end of latency period, at the end of distraction and at

the end of consolidation phase. Examinations included scout views, selection of distraction

area, reconstruction of image data and analysis. Serial transverse scan images were taken at a

resolution of 18 μm. 2-dimensional and 3-dimensional reconstructed by DataViewer Version

1.3.2 (Skyscan, Kontich, Belgium), Nrecon Ver 1.5 (Skyscan, Aartselaar, Belgium) program. A

3-dimensional region of interest was generated by interpolation between 2 dimensional free-

hand selections of the distracted area and excluded the metal part of distraction device to avoid

image blurring.

Table 1. Preset values of micro computated tomography scanning

Conditions Pre-set values

Filter Al 0.5mm

Resolution 18 ㎛

Voltage 100kV

Current 100mA

Exposure time 1180mS

Rotation step 0.5°

Calibrate pixel density Hydroxyapatite phantom - density 0.25-0.75 g·cm³ CaHA

- 10 -

III. RESULTS

Totally 20 rats were operated and of 20, 18 rats survived till the completion of our study

(mortality rate 10 %). 1 case of intraoperative death occurred resulting from anesthetic

complication. One rat died for progressive weight loss and malnutrition. To supplement the

nutrition, feeding solution was provided after surgery. No subject died by severe weight loss or

malnutrition after adding this solution. The rate of mandibular fracture was 5 % and this

occurred during threading of miniscrews (Fig. 6). It is necessary to be careful handling with

rat mandible since it is so thin and delicate that slight impact can cause fracture. There was

device dislodgments in the early time of our experiments. Application of custom made neck

collar could prevent rats from touching the device and prevent device dislodgment. Custom

made block, Bioplast could rigidly stabilize miniscrews to the bone as well as reinforce the

weak angle area. However, use of custom-made block resulted in slight underlying bone

resorption at which the block is applicated locally (Fig. 6).

Fig. 6. Mandibular bone fracture (white arrow) is observed at the proximal segment.

Rectangular custom made block resulted in resorption of bone at the proximal segment.

- 11 -

Table 2. Intraoperative and postoperative complications and their rates

Complication Percentage

Intraoperative death 5 % (1/20) Mandibular Fracture 5 % (1/20) Device dislodgment 5 % (1/20) Death by Malnutrition 5 % (1/20) Infection 5 % (1/20)

Total 25 % (5/20)

Weight loss was found for all animals immediately after the operation. It took 20 days to

recover their preoperative weight (Fig. 7). After distraction, all rats developed deviated

anterior crossbite.

Fig. 7. Diagram showing the change of average weight of all subjects .

The jackscrew used in this study expands 0.20 mm by every 90 degree turn. Totally 2.40 mm

lengthening was expected following the distraction procotol. However, the actual lengthening

of reference points, the center of two middle miniscrews, calculated on CT images was 2.02

mm and reduced to 1.88 mm during consolidation period.

- 12 -

Table 3. Average distances between two middle miniscrews on the stages (unit; mm)

Stages Distance (Mean ± SD)

T1-T0 2.02 ± 0.22

T2-T0 1.88 ± 0.19

Histologic analysis

After 12 days distraction period

At the completion of distraction period, the distraction gap is filled with fibrous tissue and

collagen fibers are found to align along the axis of distraction vector on the slides of inferior

border. Newly formed bone trabeculae appeared to project form the margin of osteotomy to

the center of the distraction gap. Around this trabecular bone, the emergence of osteoblasts

was noted (Fig. 8, A-B). The slide of the rat inferior border having infection demonstrated that

the fibrous tissue intervened the gap between bone fragements (Fig. 8, E-F).

After 4 weeks of consolidation

The center of distraction gap displayed the cartilage tissue (Fig. 9, A-C). Trabecular bone at

the time of 4 weeks of consolidation became thicker compared to that of distraction period

(Fig. 8 C-D). Osteoblasts and osteoclasts were found around the area of angiogenesis. On the

other hand, failure subject by infection and device immobilization revealed the infiltration of

inflammatory cells and granulation tissue at the center of distraction gap.

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Fig. 8. Histologic and immunohistochemical analysis on the inferior border of rat mandibular

distraction osteogenesis. A-B, after 12 days of distraction; C-F after 4 weeks of consolidation.

Magnification: 4 x (A, C, E), 10 x (B, D, F)

A B

C D

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- 14 -

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e

- 15 -

initial stability of miniscrews, considerations for these factors are required to the success of

distraction osteogenesis.

Fig. 10. . The starting point of vertical osteotomy line passing through alveolar bone was

decided according to the distance (d) between the furcation of second molar (f) and the

anterior border of alveolar bone (a).This distance was measured on sagittal views of micro CT

images. The distance (d) between the anterior border of alveolar bone (a) and the furcation of

second molar (f) for precise positioning of vertical osteotomy.

The CT scanned image at the end of latency period showed distinct gap between bone edges.

The gap has been enlarged during distraction phase with securing the device to the bone. After

finishing 4 weeks of consolidation the gap is filled with radiopaque material demonstrating the

formation of new bone (Fig. 12).

d

fa

- 16 -

Fig. 11. The bucco-lingual thickness of the rat mandible was measured on the traansaxial

views at the level miniscrews inserted. A, Axial view showing rat right hemimandible is

marked with transaxial reference lines . B, Transaxial views of right hemimandible numbered

to correspond to axial view. The transaxial views are a bucco-lingual cross sectional view at

every 0.5 mm from the first molar to mandibular angle area. Red lines indicate the thickness

of bone at which miniscrews are inserted.

Table 4. Average bucco-lingual thickness of rat mandible, mm

TS Mean ± SD TS Mean ± SD TS Mean ± SD

1 2.73 ± 0.12 6 2.36 ± 0.13 11 0.73 ± 0.08

2 2.77 ± 0.11 7 1.79 ± 0.14 12 0.69 ± 0.09

3 2.81 ± 0.20 8 1.19 ± 0.11 13 0.58 ± 0.09

4 2.60 ± 0.13 9 1.21 ± 0.12 14 0.47 ± 0.03

5 2.48 ± 0.21 10 0.84 ± 0.10 15 0.43 ± 0.05

* Numbers corresponding to the axial view of Fig. 11 above

* TS: Serial number of transaxial view

A B

- 17 -

Fig. 12. Periapical views (A, C, E) and reconstructed 3-dimensional micro CT images (B, D, F)

at the sequential stages of rat distraction osteogenesis. A and B, Latency of 5 days; C and D,

distraction of 12 days; E and F; consolidation of 4 weeks.

A B

F E

D C

- 18 -

IV. DISCUSSION

Various custom-made distraction devices were described to reproduce distraction

osteogenesis minimizing complications. The purpose of this study is to create rat model for

alveolar bone as well as mandible to evaluate histological and radiographic analysis. This

study established the model which is reproducible and has low complication rates. The

mortality rate was similar to that of others (Connolly et al., 2002; Eski et al., 2005).

Intraoperative death was largely resulted from inexperienced anesthetic skill. The problem

disappeared after improvement of the technique.

In the early time of this study, post operative death occurred after progressive weight loss. To

avoid this, rats were injected with Ringer’s solution peritonealy for 5 days after surgery and

fed with enteral feeding solution made of nutrition drinks containing proteins and water.

Uptaking of liquid food was also helpful to stabilize the device as use of chewing action,

resulting in repeated jaw movements, is reduced. After providing enteral feeding solution, the

death by dramatic weight loss was not observed.

Another problem for the development of distraction model was dislodgment of the device

after operation. As Samchukov (1998) described the importance of device fixation for success

of distraction osteogenesis, it is fundamental to maintain the device till the completion of

consolidation period. Device fail results from two main reasons, screw loosening or fracture of

bone. Rats touch the device with their foot constantly after waking up from anesthesia. This

stimuli trigger mobility of threaded screws and eventual failure. The problem was figured out

by wearing custom made neck collar prohibiting them from nudging the device. At the

beginning of the study, there was bone fractures near the margin of bone, at which miniscrews

were inserted. Thus, at least, 2 mm of spare distance was left from the margin of the bone to

avoid weakening of bone. In addition, application of preformed block underlying lingual

surface of mandibular angle, reinforced weakest part of mandible and tightly stabilized bone

screws to the bone. However, it was inevitable to have slight localized resorption of bone by

the pressure resulted from the contact of block to bone as previously reported by Connolly

(2002). After finding this limitation, thinner, thickness of 0.5mm block was used for the

smaller area covering only one of four miniscrews, most posterior one.

- 19 -

To include mandibular body and alveolar bone, designing of osteotomy was main concern

of this study. The past studies can be classified mainly two groups according to the location

of osteotomy. Rowe and colleagues (1998) localized the osteotomy line posterior to the

molar teeth placing the device posterior area. Because the posterior region of rat mandible is

extremely thin and weak, the device dislodgment rate has been reported up to 90%.

Furthermore, even small impact can cause bone fracture and device loosening. Due to

device dislodgement, they moved it to the anterior so that the osteotomy is located between

two molar teeth. However, the second one also criticized for two points of view, proximity

to the oral cavity susceptible to infection and irritation of dental precursor cells (Buchman et

al., 2002). While osteotomy made on intermolars is better for securing device, when it

closes to oral cavity, the risk of infection increases. In this study, modified N-shaped

osteotomy, combination of two vertical and one horizontal osteotomy lines, was developed.

The transaxial views of rat mandible were analyzed using CT scans. First vertical line which

starts from between the second and third molar passes through alveolar bone. The starting

point of vertical osteotomy was guided by ‘L’ shape indicating needle. To reduce the risk of

infection performed was not a complete osteotomy but indentation with diamond point bur

near the teeth. Completion of osteotomy was made with intentional fracture induced by

activating the distraction device. The second osteotomy split mandibular body transversely.

During the early time of our experiments, the horizontal osteotomy was 7mm long. This

located posterior holes where miniscrews are threaded around the thin part of mandubular

ramus having increased risks of bone fracture and device dislodgment. The limitations were

overwhelmed by shortening of horizontal line to 4mm and using custom made block

underlying the lingual surface of the ramus. This allowed the two holes to move to the

anterior. However, localized slight bone resorption occurred as the result of pressure.

Another vertical osteotomy which is parallel to the first vertical osteotomy, initiates from

the end of horizontal osteotomy to the margin of mandible. Although this complex

osteotomy penetrates teeth and has the risk of infection, compared to others the method has

several advantages such as the inclusion of alveolar and mandibular bone together and

reduction of the device dislodgment rate by locating second vertical line as mesially as

possible.

- 20 -

Several different devices were designed in this study. Firstly conventional one was made using

an orthodontic hygienic type jackscrew (Dentaurum, Tokyo, Japan) at which L-shaped surgical

plate was connected by composite resin. This device caused radiographic blurring when taking

micro CT scanning because part of surgical plate was exposed to region of interest. To solve this

problem, the second device was assembled by embedding orthodontic jackscrew into acrylic

resin instead of metal plate. Because this model needed long miniscrews to secure the device to

the mandible, they irritated wound healing and are susceptible to loosen. The final device was

made by modifying first device with bending the surgical plate. Despite the interference of metal

part for CT scanning, the first device was superior to the second one in biocompatibility. The

metal blurring was reduced by distally bending the surgical plate at the connection part of

horizontal and vertical elements.

After osteotomy procedure, distraction osteogenesis consists of 3 fundamental sequential

phases in which different biologic phenomena occur. These have been experimentally studied

in bones of alveolar, endochondral or intramembranous origin. A latency phase of 4 to 7 days

is indicated for facial bone and alveolar bone respectively. Even though no latency period is

required for large animals, It was reported that 5 days of latency was optimal for small animals

such as rats and mice. This study followed the protocol, 5 days of latency period (Djasim et al.,

2007).

In animal experimental studies, 1 mm/day seems to be the optimal rate of distraction for most

animals, except for the rat model. Due to small size comapring to other animal models, 0.2-

0.6mm/day is appropriate for rats. It was demonstrated that distraction rates up to 0.50 mm per

day produced excellent responses (Paccione et al., 2001). According to the report by Illizarov,

continuous distraction is optimal condition of rhythm for the best outcome. To reduce the

inconvenience and the stress of rats, 0.20 mm/day was applied at a single session in this study.

Consolidation phase is the period between the stopping of traction force and removal of

distraction device. In craniofacial bones, a 3-5 week phase is recommended for growing patients

and a 6-12 week phase for non growing patients. However, no study reported that neoformed

bone gained the bone quantity and quality of normal bone in a year of consolidation time. This

study allowed 4 weeks for the consolidation of divided bone segments.

- 21 -

Micro CT images were taken preliminarily for analyzing anatomy of the rat mandible and

deciding the position of the bone screws securing the distraction device. The distance

between the anterior border of alveolar bone and the second molar, measured on the micro CT

images, provided the guide to locate vertical osteotomy line. The serial views of coronal

section demonstrated that thickness of bucco-lingual bone was considerably thin especially

ramal area for stabilizing the distraction device. This information changed the length of

horizontal osteotomy line to be shorter, 4mm to locate bone screws and distraction device to

the anterior area. In vivo micro CT scanning were obtained three times at the time after 5 day

latency period, 12 day distraction and 4 week of consolidation for each subject under general

anesthesia. The images scanned after latency demonstrated the clear osteotomy line. The

specimens after distraction showed increased gap between bone segments. It is observed that 4

weeks of consolidation filled the gap with radiopaque material in the CT images.

Rat long bone had three modes of ossification including transchondral bone formation not

found on the rat mandibular distraction model (Yasui et al., 1997). Ali et al.(2009) reported

that the new bone tissue was produced through intramembranous ossification during the early

and the late consolidation phase whereas only during the early consolidation period,

endochondral bone formation was shown only at the center of distraction gap. Histologic

analysis of this study revealed that collagen fibers were aligned along the distraction vector

and trabecular bone structure was formed by osteoblasts. This supports the fact that

intramembranous ossification occurs during the distraction phase by tensile force. At the time

of 4 weeks of consolidation, the gap between bone segments was filled with cartilage tissue

demonstrating the chondrogenesis. Considering the results of histologic analysis for this study

proving those of the previous studies, new bone formation occurred by combination of two

modes.

Although this study had the drawbacks such as the need of animal sacrifice for the

qualitative analysis of the newly formed bone through micro CT scans and limited application

of distraction conditions, the results of this study will be the basis of clinical improvements of

distraction osteogenesis treatment.

- 22 -

V. CONCLUSION

During the last several decades, the rat model has been used as reliable tools for studying

distraction osteogenesis and various osteotomy and distraction device designs have been

developed. However, existing designs have some limitations. Our study created the new rat

distraction model and designed distraction device with modification. The features of our

model are as follows.

1. Micro CT analysis was performed for the anatomical structure of rat mandible and

provided the guides for the location and design of osteotomy and positioning the

bone screws stabilizing the distraction device.

2. The rat model for mandibular inferior border and alveolar bone was successfully

established on the basis of the protocols used in this study. The protocols contain 5

days of latency period, distraction rate of 0.20 mm/day, at a single session, and 4

weeks of consolidation phase.

3. New bone formation of the rat model occurred by combination of two modes.

Intramembranous ossification was predominant at the end of distraction period,

while endochondral ossification was also observed with intramembranous one at the

end of 4 weeks of consolidation period.

To analyze new bone formation quantitatively, the improvement of device design and the

consideration for device materials are required. With this model, the application of various

conditions related to the distraction osteogenesis will be available. The results of this study

will be the basis of clinical improvements of distraction osteogenesis treatment.

- 23 -

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Codivilla, A. 1994. "On the means of lengthening, in the lower limbs, the muscles and tissues

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Connolly, J. P., Liu, Z. J., Wang, L., Whelan, M. F., Huang, G. J., Williams, J. K., King, G. J.

2002. "A custom mandibular distraction device for the rat". J Craniofac Surg 13(3):

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Cope, J. B., Samchukov, M. L., Cherkashin, A. M. 1999. "Mandibular distraction osteogenesis:

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Djasim, U. M., Wolvius, E. B., van Neck, J. W., Weinans, H., van der Wal, K. G. 2007.

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877-883.

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Frost, H. M. 2001. "The Utah paradigm on animal models of skeletal disorders: quo vadis?". J

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Iida, S., Yagi, T., Yamashiro, T., Okura, M., Takada, K., Kogo, M. 2007. "Maxillary anterior

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국 문 요 약

백서 하악 하연과 치조골의 골신장 모델 개발

연세대학교 대학원 치의학과 문현승

(지도교수 차정열)

골신장술은 골절개를 통해 분리된 절편을 점진적으로 변위 시킴으로써 신생

골을 생성하는 외과적 술식이며 골을 비롯하여 주변의 치은, 피부, 근육 등의 조

직이 동반되어 신장되므로 일반적인 악교정 수술에 비해 다양한 장점을 지닌다.

이러한 배경 하에 두개안면 기형증 환자의 치료 효과가 보고되고 있으며, 최근 임

플란트 보철을 위한 치조골 증강술에도 적용되고 있다. 임상적인 사용의 보편화에

도 불구하고 골신장술에 대한 기초적인 연구는 충분히 이뤄지지 않았으며 골신장

술의 과정들은 대부분 장골의 연구를 바탕으로 하는 한계를 가지고 있다. 따라서

본 연구는 16 주의 수컷 백서 20마리를 대상으로 하악 하연과 치조골 부위에 분

자생물학적 연구와 방사선학적 분석을 위한 골신장술을 모델을 확립하고자 하였

으며 다음과 같은 결과를 얻었다.

1. Micro CT 분석을 통해 백서 하악골의 해부학적 구조에 대한 연구를 시행

하였으며 이 결과를 바탕으로 골절개선의 위치 및 형태, 장치 고정을 위

한 bone screw의 식립 위치를 결정하였다.

2. 본 연구에서 설정한 새로운 골절단 방법과 함께 사용한 골절단 후 휴지기,

골확장기의 활성화 속도와 빈도, 골경화 기간을 통해 성공적으로 하악 치

조골과 하악골 하연의 동시적인 골신장술 모델을 확립하였다.

3. 조직학적 분석결과 백서 하악골의 치조골과 하악골 하연은 두 종류의 골

화 과정을 통해 신생골을 형성하였다. 골확장기 동안에는 주로 막성골 골

화를 통해 신생골이 형성되었고, 경화기 동안에는 분절된 골편 사이 중심

에서 연골내 골화도 함께 관찰되었다.

본 연구에서 백서 하악골 하연과 치조골에 대한 골신장술 모델을 성공적으로 구

축하였다. 이 모델은 다양한 골신장 관련 조건의 연구를 위하여 유용하게 사용할

수 있을 것이며 골신장술의 임상 적용에 대한 기초가 될 것이다.

핵심 되는 말: 골 신장술, 치조골 신장술, Micro CT, 백서 모델