THE EFFECT OF VERTICAL MANDIBULAR DISTRACTION OSTEOGENESIS ON … · 2010-10-22 · investigation...

INVESTIGATION OF VERTICAL MANDIBULAR DISTRACTION OSTEOGENESIS ON THE MASTICATORY MUSCLES IN A ‘UNILATERAL HEMIFACIAL MICROSOMIA LIKE’ DEFECT

IN THE SHEEP MODEL

RUMAIZI SHAARI D.V.M (UPM), M.V.M (UPM)

Thesis submitted for the degree of DOCTOR OF PHILOSOPHY (PhD)

Oral and Maxillofacial Surgery Unit Dental School Faculty of Health Sciences

The University of Adelaide Adelaide, South Australia, 5005

October, 2005 Revision September, 2008

Results Chapter 4

Investigation of Vertical Mandibular Distraction Osteogenesis on Masticatory Muscles in ‘Unilateral Hemifacial Microsomia Like’ Defect in the Sheep Model

111

HAPTER 4: RESULTS

.0 Animals itially, thirty lambs were involved in the first procedure to create a “hemifacial microsomia like

efect”. Five of the thirty lambs (17%) died and were excluded from the experiment (Table 4.1).

nother five lambs (17%) had infected wounds and showed signs of swelling and reddening of

kin at the surgical site. Upon palpation the swollen area was firm and warm. Pus was also

resent and a thick yellow discharge exuded from the wounds on pressure. The infected

ounds were treated under general anaesthesia. The wounds were incised and the pus and

ecrotic tissues were removed, debrided and irrigated with copious normal saline and Betadine

n packed with the ribbon gauze soaked with Betadine solution.

e ribbon gauze was exposed through one small incision on the ventral inferior

C

4In

d

A

s

p

w

n

solution. The wound was the

One end of th

surface of the mandibular ramus. The ribbon gauze was progressively pulled out over a 3-5 day

period until the whole gauze came out. The five lambs with infected wounds were treated with

antibiotics for an extra 7 days. Of these three lambs responded well to this treatment and they

were included in these experiments. However, two failed to respond and were sacrificed.

No Problems No. of animals Stages Remarks 1. General

health 2 lambs

Latency period

Intestinal perforation

(Lambs died) 1 lamb Post 1st operation Pulpy kidney

2. Technical 1 lamb

1 lamb

During 2nd operation

During 1st operation

Accidental

anaesthesia

extubation

Not tolerated

(Lambs died) 3. Infection 5 lambs Pre-distraction(n=1)

Distraction (n=4) Lambs were treated and used

Table 4. 1: Number of sheep with problems; general health and technical problems.

Results Chapter 4


112

Altog r u nd of experimen tion 3.3.5). The

lambs were randomly assigned to 6 experimental h

group except group 1 contains only 3 animals.

All surviving sheep tolerated the procedure well and gained we ly throughout

experimental period (Graph 4.1) heep gained an average 5.5% weight at the end of

distraction process. The weight gain es ave 5%) during the

onsolidation period and started to slow down to average 7% before animals were killed. The

eight gain of the current experiment was within the normal growth curve for Merino sheep

ethe , twenty-five sheep survived ntil the e t (see Sec

groups (Group 1 – 6) with 4 animals in eac

ight constant

. The s

increased 3 tim rage (16.

c

w

(Butterfield, 1988) .

0

5

45

10

15

20

25

35

Wei

ght(k

g)

30

40

Pre-dis Post-dist Removal Sacrifice

Stages of experiment

Weight (kg)

Graph 4. 1: The weight gain of sheep (kg) of sheep at different stages throughout experimental period.

Results Chapter 4


113

4.1 Anaesthesia Induction was with 3-5% halothane inhalation, which was well tolerated by the experimental

lambs. The recovery was much smoother with the inhalation method compared to the

intravenous induction with 5% thiopentone sodium at dosage of 12.5 mg/kg body weight for 10

week old lambs. The lambs with intravenous induction showed either light anaesthesia or very

deep anaesthesia and some lambs showed sign of apnoea and/or lack of muscle relaxation.

Two lambs died due to technical problems with the anaesthesia. Lambs aged 6 months of age

for the second procedure tolerated intravenous induction much better.

4.2 Post Mortem Three lambs were observed to have general health problems and showed signs of depression,

ematuria and became progressively moribund and died. At post mortem, they showed some

egree of lung congestion, mild to severe congestion of the kidneys and the most interesting

loss of appetite, weakness and later showed signs of diarrhoea, tooth grinding and one had

h

d

finding was the sign of intestinal perforation with evidence of intestinal contents in the

abdominal cavity in two lambs. The perforation happened at the pyloric-duodenal junction

through a small hole about 0.5 cm in diameter (Figure 4.1). The hematuric lamb showed signs

of pulpy kidney. Based on the post mortem findings, the death of the animals may be

suggested due to multiple vital organ failure from septicaemia. While the intestinal perforation

might be due to the use of analgesia and/or underlining diseases such as enterotoxemic

bacteria.

Results Chapter 4


114

Figure 4. 1: The post mortem finding, the intestinal perforation at duodenum, which

contaminated abdominal cavity.

There was yellowish necrotic tissue surrounding the distracted gap in one of the sheep that had

been treated previously (Figure 4.2). The stump of the right superficial masseter could be

identified with evidence that the edge of the previous myectomy cut had healed. Whitish fibrous

tissue was present on the surface of the right middle masseter muscle (Figure 4.3). The right

masseter muscle, which had the device in place at sacrifice, was firmer when compared to

other non-infected animals after device removal. The inner side of the right masseter muscle

was lined with a thin whitish fibrous tissue with some areas thicker than other and tight

adhesion to the mandibular ramus. The medial pterygoid, generally looked normal but in some

animals the muscle looked and felt softer, similar to a muscle with disuse atrophy.

Abomasum (True stomach)

Pyloric sphincter

Duodenum

Duodenum Perforation

Contaminated with intestinal contents

Results Chapter 4


115

Presence of yellow necrotic tissueFigure 4. 2: s and pus surrounding the distracted gap at post

mortem.

Figure 4. 3: Presence of whitish fibrous tissues on the right distal surface of masseter muscle.

The edge of the superficial masseter stump (point of forceps).

Results Chapter 4


116

4.3 ‘Unilateral Hemifacial Microsomia-like Defect’ model Twenty-three lambs showed midline shifting (about 3.1 mm) to the operated side 3 months

after right condylectomy and myectomy of superficial masseter muscle. Based on the Kaban

classification, the lambs showed type 1 and type O0 M1 E0 N0 S1 O.M.E.N.S classification

(Kaban et al., 1988; Vento et al., 1991), which is characterised by a smaller mandible and

temporomandibular joint with unilateral loss of mandibular mass.

4.4 Mandibular Ramus (Bone) Measurements Results of the bone component were from the same experiment, which involved the same

animals and experimental protocols as for the soft tissues. The summary of the bone results

4.4.1 Radiology 4.4.1.1 Distraction gap

It was reported that there were differences in distraction gaps achieved in different groups

(Table 4.2). The mean distance of the distracted gap was stated as 8.8 mm and the standard

deviation was 0.16 mm.

4.4.1.2 Measurement of vertical and horizontal distance

In Dr Syed Zainal’s experiment, the distances of AB, BC and AC were measured at pre-

distraction, post distraction, at removal of the device and at sacrifice. Double measurement was

done on the s ce was calculated

the nearest 0.5 mm. Only one operator carried out the measurements.

was obtained from Syed Zainal (2005).

ame distances by using the metal ruler and the average distan

to

Results Chapter 4


117

vement from the targeted gap 10

erved the distraction gaps at

0 mm (Syed Zainal, 2005). The Mathys device, which was designed for human may not be

tory system.

Mean distraction gap (mm ±(SD))

The mean distracted gap was 8.8 mm, which was 88% achie

mm. Three faulty devices were noted, one was for the first time used and the other two were

noted as second time used. Other devices were stable as the first and second attempts did not

show any problem. The distraction gaps ranged between 8–11 mm.

The current study showed that 50% of the time the distractors pres

1

suitable for the biomechanics of the sheep mastica

Group at sacrifice/ removal of device 1a. Pre-distraction 0 (0) (N=1) 1. Immediately Post distraction 7.7 (3.51)

9 (0)

2. Consolidation 2 months & 10 (0)

(N=4)

(N= 4) 10.3 (0.79)

(N=4) 5. Consolidation 3 months 10 (0)

(N= 3) 2a. Consolidation 2 months (N=1)

remodelling 1 month

3. Consolidation 3 months 7.4 (3.09)

4. Consolidation 3 months remodelling 1 month

remodelling 2 months (N=4) 6. Consolidation 4 months 7.36 (0.82) (N=4) Mean 8.8 (0.16)

Table 4. 2: The distraction gaps in the different groups.

Results Chapter 4


118

at sacrifice was recorded

ith the device in place. Interestingly, the vertical height was noted to have slightly increased

nth consolidation period with

ne month after device removal was also reported to show greater reduction at sacrifice.

4.4.1.3 Vertical height distance (AB)

The vertical height of the experimental group was reported to show a small percentage of

reduction at sacrifice with the device in place for 3 months or more. Group 3, 4 and 5 had the

device in place for 3 months (Table 4.3). A 3% reduction in height

w

after one month of device removal (Group 4) and during second month post device removal

(Group 5) the vertical height remained stable. The 2 month neutral fixation (Group 2a) with

distraction showed higher percentages of reduction. The two mo

o

Table 4. 3: The vertical distance of AB changes at post distraction (Post-D) and sacrifice (Sac). Percentage (%) of the increase (�); decrease (�) and remain the same (�).

(Adapted from Syed Zainal, 2005).

NOTE: This table is included on page 118 of the print copy of the thesis held in the University of Adelaide Library.

Results Chapter 4


119

ncrease the vertical height of the experimental side by

.4.1.4 Horizontal distance (BC)

The horizontal distance (BC) wa r ow ny chang distraction

process and at sacrifice (Syed a a 2005). Th increased distance BC on the experimental

side o have a ran 14% with an average of 3.5%. The height

incre to an of ang ion of X-ray. The mean growth rate of the

control side was approximately 2%.

4.4.1.5 Oblique distance (AC)

The orted ranging from 1.6% - 14.8% (average 6.5%) increase (Syed

Zainal, 2005). The error was noted in Group 6 and was due to an increased angulation of the

X-ray cone to the film. Increase in angle caused increase in the distance. The mean growth

rate as 3.44 .

The distraction process was found to i

24.9% and the growth rate of the control side was at 2.4%. Therefore the true increase of the

vertical ramus on the experimental side from distraction was approximately by 22.5%. Groups

1a and 2a were not included in the statistical analysis because of the small number (n=1) for

each group and those in Group 1a were not involved in distraction. Based on paired sample t-

test, the vertical height was generally not significant except in groups where the device was in

place for 3 months, was subjected to device removal and sacrificed one month (Group 4) and

two months (Group 5) later.

4

s eported not to sh a es after the

Z in l, e

was observed t ge between 0.5-

ase was stated to be due error ulat

distance of AC was rep

of AC on control side w %

Results Chapter 4


120

ported to be consistently stable where the value of the difference

as almost the same for groups 4, 5, and 6. Group 1 was reported to show a minimum value

consolidation period. Group 2 was reported to have small

4.6 Bone Histology

.6.1 Pre-distraction (Group1a)

s reported to show active fibroblasts, active osteoblasts,

oup 1)

he distracted gap of immediately post distraction was reported to have thick trabeculae, thin

eriosteum bridging the two segments and formation of woven bone. Woven bone was

presented on the periphery of the medial part of the gap. The centre of the gap contained

cartilage cells. The medial part contained active osteoblasts and fibroblasts. The inferior part

4.5 Direct Measurement 4.5.1 Vertical Distance (AB)

The measurements were conducted using the hand-held vernier calliper (Leibinger, Tokyo,

Japan) (accurate to the nearest 0.5 mm) (Syed Zainal, 2005). The measurement was

conducted at pre-distraction and sacrifice. The measurement was compared and the

differences were then calculated.

The experimental site was re

w

because this group did not have

differences, and was suggested to have relapsed. Two animals in Group 3 were reported to

have 45-60% reduction in distraction gap (based on radiological examination). It was

suggested this related to a mechanical fault of the device.

4

The pre-distraction section wa

endothelial cells and inflammatory cells in the thick fibroblast connective tissues within the

osteotomy gap (see appendix (Figure 8.1 and 8.2)).

4.6.2 Immediately post distraction (Gr

T

p

Results Chapter 4


121

alised trabecular bone and thick periosteum laterally. The mature

ed at the peripheral edge of the distracted gap (see appendix

riod of 2 months was reported to show signs of incomplete

one union on the mid ramus sections. The distracted gap was found to be full of inflammatory

The ratio of the bone surface to the

tal tissue volume was reported to decrease in this group after showing the highest ratio

mediately post distraction.

with a developed Haversian system and

ore compact bone had developed. It was also reported that there were more osteoblast than

oven bone was presented on the periphery of

was presented with gener

trabecular bone was also not

(Figure 8.3, Figure 8.4, Figure 8.5, Figure 8.6 and Figure 8.7)).

4.6.3 Consolidation for 2 months (Group 2a)

The animal with a consolidation pe

b

cells, fibrous tissues and thick periosteum. Furthermore, the active bone remodelling continued

as it was noted the presence of fibroblasts and osteoblasts at periphery (see appendix (Figure

8.8 and Figure 8.9)).

The ratio of bone surface (mm²) to the total tissue volume was reported to be small. This was

suggested due to fibrous healing within the distracted gap.

to

im

4.6.4 Consolidation 2 months and remodelling 1 month (Group 2)

The animals with consolidation for 2 months and sacrifice one month after device removal were

reported to have generalised lamellar bone formation

m

osteoclast cells with many reversal lines. The w

posterior part, which suggested less mature bone in this area (see appendix (Figure 8.10,

Figure 8.11, Figure 8.12, Figure 8.13 and Figure 8.14)). The formation of bone was reported to

be the same both Group 2a and Group 2.

Results Chapter 4


122

Haversian canals were more

rominent and contained a lot of blood vessels. The activities of the osteoblasts and some

ities of these cells persist during remodelling

t and osteoclast activities were reported in distracted bone of animals

ith a consolidation period of 3 months and sacrifice one month after device removal (see

compact bone with reversal lines, large numbers of osteoclasts

ithin Howship’s lacunae and active osteoblasts were reported on the distracted bone

ths after device removal

4.6.5 Consolidation 3 months (Group 3)

The osteocytes were noted to be present between the compact mature bone tissues after a 3

month consolidation period. The Haversian systems with

p

osteoclasts were observed suggesting the activ

(see appendix (Figure 8.15 and Figure 8.16)).


There was compact bone with prominent reversal lines, thick periosteum medially and

presence of osteoblas

w

appendix (Figure 8.17, Figure 8.18 and Figure 8.19)).


There was generalised dense

w

belonging to animals with consolidation 3 months and sacrifice 2 mon

(see appendix (Figure 8.20)).

The ratio of total bone to total tissue volume was reported to increase 20% in middle and

posterior ramus sections when compared to the other groups. Bone volume was noted to

increase in longer consolidation and remodelling periods.

Results Chapter 4


123

this group. The ratio of bone surface to total

s and was suggested to

Trabecular thickness was reported to increase as the consolidation and remodelling increased.

The highest value was reported in Group 6 and the lowest value was in Group 2. Trabecular

separation (distance between edges of the trabeculae) was reported to be small in more

mature bone. In mature bone, trabeculae moved closer and reduced the trabecular distance.

The number of trabeculae stabilised starting from two months of consolidation. Trabecular

number was counted per millimetre area. The trabeculae number was reported to increase with

time and stabilised in Group 2-6.


Dense compact bone and prominent reversal lines were reported in all histological sections of

the sheep having consolidation of 4 months. It was also noted that bone formation and

resorption persisted which suggests that remodelling activities continued (see appendix (Figure

8.21, Figure 8.22, Figure 8.23, Figure 8.24, Figure 8.25, Figure 8.26 and Figure 8.27)).

Bone volume was reported to increase by 30% in

tissue volume was reported to decline with longer consolidation period

be stable after 3 months of consolidation.

4.7 Bone Structure

Results Chapter 4


124

4.8.1.1 Weight of masseter muscles

The reduction of weight of the masseter muscles experimental side was estimated at post

mortem. The superficial masseter of the left side was dissected and weighed separately and

then as a whole with the middle and deep layer. Overall, it showed that the superficial masseter

myectomy reduced the muscle bulk on the experimental side approximately by 26.5-32.6%.

(Table 4.4)

___________________________________________________________________ mated percentage (%) of reduction Average

. Immediately post distraction 33 28.6 28.9 25

3. Consolidation 3 months 33.3

4. Consolidation 3 months 33.3

36.4

Consolidation 3 months 25 remodelling 2 months 30 26.5

22.2 28.6

Consolidation 4 months 27.3 30 31 33.3 33.3

4.8 Gross Masticatory Muscle Changes 4.8.1 Weight of masticatory muscles

Groups Esti________________________________________________________________________________ 1 2. Consolidation 2 months 20 remodelling 1 month 33.3 30.0 33.3 33.3

25 31.5 37.5 30

remodelling 1 month 27.3 32.6 33.3 5. 6.

able 4. 4: Estimated reduction of the masseter muscle by superficial masseter myectomy.

T

Results Chapter 4


125

the experimental side (right) and control side (Left)

nt difference (p � 0.05) in all groups (Table 4.5) (Graph

4.2). The weight of the masseter muscles on both sides showed an increase as the animals

grew. There was no point where the weight of the distracted muscle increased more than the

weight of contralateral side.

Side Experiment Control Paired t-test

Statistically, the weight difference between

of masseter muscles showed a significa

Group Mean ± SD (gm) Mean ± SD (gm) p� 0.05

1. Imm

ediately Post distraction 20(5) 28.33(7.64) 0.038 (N= 3)

C nsolidatio 3 months

0.006

2. Consolidation 2 months & 20(4.1) 41.3(7.50) 0.003 remodelling 1 month (N=4) 3. Consolidation 3 months 28.75(4.8) 43.8(4.8) 0.006 (N= 4) 4. o n 31.25(6.3) 53.8(6.3) 0.001 remodelling 1 month (N=4) 5. Consolidation 3 months 33.75(4.8) 47.5(10.4) 0.022

remodelling 2 months (N=4) 6. Consolidation 4 months 31.25(6.3) 48.75(4.79) (N=4)

NS: Not significant

Table 4. 5: Paired t-test for weight of masseter muscle between control and experimental sides

s. for six group

Results Chapter 4


126

30

40

50

60

Wei

gh (g

m)

0

10

20

70

Co ons3R1 Co on

lidation odelling pe

t Experiment

Control

Imm P-DO Cons2R1

onso

ns3 C

nd rem

ns3R2 C

ds

s4

C a rio

Graph 4. 2: The weight (gm) of masseter muscles of the experimental and control sides in six

There ences bet een groups. The tests revealed that the animal with

immediately post distraction (Group 1) showed statistica nt differen muscle

weight when compared with animals in Group 2 and Group 4. The last 2 groups of animals

control and as the consolidation period is prolonged the percentage reduction in weight on the

distracted side increases. It was also noted that the higher percentages of reduction in weight

were noted 1 month after device removal with 2 month consolidation periods (Group 2) and

three month (Group 4) neutral fixation with the device. Group 2 showed up to 52% less

masseter weight and Group 4 was 42% lighter than the control side respectively.

groups.

were significant differ w

lly significa ces in

showed no differences (Table 4.6) (Graph 4.3). Group 1 presented with 29% less weight than

Results Chapter 4


127

Net Different Group (Mean ± SD(gm)) 1. Immediate Post distraction 8.33 (2.887) (N= 3) 2. Consolidation 2 months & 21.25 (0) remodelling 1 month (N=4) 3. Consolidation 3 months 15 (5) (N= 4) 4. Consolidation 3 months 22.5 (4.787) remodelling 1 month (N=4) 5. Consolidation 3 months 13.75 (2.887) remodelling 2 months (N=4) 6. Consolidation 4 months 17.5 (6.292) (N=4)

Table 4. 6: The net change in weight (gm) of masseter muscles between the control and

experimental sides in six groups.

0

5

15

20

25

Consolidation and remodelling periods

Wig

htgm

)

10

30

Imm P-DO Cons2R1 Cons3 Cons3R1 Cons3R2 Cons4

e (

raph 4. 3: The net change in weight (gm) of masseter muscles between the control and experimental sides in six groups.

G

Results Chapter 4


128

4.8.1.2 Weigh goid M scle

In general, the id muscl perimental side was slightly lighter

compared to t ugh sta alysis using Paired t-test (p� 0.05)

showed there ce (Ta le 4.7 and 4.8) (Graph 4.4 and 4.5). The lighter weight

may have bee f the side trophy al ough this could not be

confirmed. At sc of the ex erimental side looked thinner and felt softer.

The muscle on both sides increased in weight as the animal grew.

Side Experiment Control

t of Medial Ptery u

weight of medial pterygo e on the ex

he contralateral side altho tistical an

was no significan b

n due to a reduced use o that led to a th

post mortem the mu le p

Paired t-test Group Mean ± SD (gm) Mean ± SD (gm) p� 0.05

1. Immed 8.33(2.9) NS

iately Post distraction 8.33(2.9) (N= 3 2. Consolidation 2 months & 12.5(5) 15(4.1) NS remodelling 1 month (N=4) 3. Consolidation 3 months 16.25(2.5) 18.75(2.5) NS (N= 4) 4. Consolidation 3 months 15(4.1) 20(0) NS remodelling 1 month (N=4) 5. Consolidation 3 months 12.5(2.9) 15(0) NS remodelling 2 months (N=4) 6. Consolidation 4 months 15(4.1) 16.25(2.5) NS (N=4)

)

NS: Not significant

Table 4. 7: Paired t-test for weight of medial pterygoid muscle between control and experimental sides in six groups.

Results Chapter 4


129

0

5

10

15

20

25

Imm

P-DO

Cons2

R1on

s3

Cons3

R1

Cons3

R2Con

s4

Wei

ght (

gm)

C


Experiment

Control

Graph he weight (gm o e us r e trol sides in six groups.

4. 4: T ) f m dial pterygoid m cles of the expe im ntal and con

Net Different Group

ate P (Mean ± SD(gm))

ost tra on 0 (0) 1. Immedi dis cti (N= 3)

olidation 2

s &

ont 2.5 2 th

3 months 1.67 (2.5)

month 5 ( 4 o th

month 2.5 4

odelling 2 m ths =4)

4 months 1.25 (2.887)

2. Cons mn

h ( .887) remodelling 1 mo

(N=4) solidation3. Con

(N= 4) 3

4. Consolidation

delling 1 ms 6. 55)

remo n (N=4)

solidation 3 5. Con rem

s ( .083) on

(N6. Consolidation

(N=4)

Table 4. 8: The net change in weight (gm) of medial pterygoid muscles between the control and

experimental sides in six groups.

Results Chapter 4


130

-4

-2

0

2

4

6

8

10

12

14


Consolidation and remodeling periods

Wei

ght (

gm)

Graph 4. 5: The net change in weight (gm) of medial pterygoid muscles between the control and experimental sides in six groups.

The net chan edial pterygoid muscles in Group 4 (consolidation 3 months and

remodelling 1 ercentag %), which indicated the experimental side

muscle weigh d vice However, w months after device

removal, there was an increased in weight on th ental side in

. ge of weight of m

month) showed highest p e (25

ed less one month after e removal. t o

e experim Group 5 (Graph 4.5).

Results Chapter 4


131

4.8.2 Length of Four Planes on Masseter Muscles.

Repeatability coefficient was calculated for each plane on experimental and control side for

each group (Table 4.9) (APPENDIX 5). The calculation is based on this formula:

Repeatability coefficient = / �df² x 100 � 2n df = the different between the second and first readings n = the number of double measurements ___________________________________________________________________ Groups Experimental sides Control sides ___________________________________________________________________ Anterior plane (AB)

1 29 29

3 18 4 43 00

6 18 18 osterior plane (EF)

1 29 20 2 35 00 3 31 00 4 31 18 5 43 18 6 31 25

1 29 41 2 25 00 3 25 18 4 18 18 5 18 18 6 43 31

2 31 18 35

5 31 18 6 43 25

Middle plane (CD) 1 20 20 2 31 18 3 25 18 4 25 18 5 31 00

P

Oblique plane (AD)

Table 4. 9: Repeatability coefficient for four planes and groups.

Results Chapter 4


132

ich can be considered an acceptable

of A B)

ength o nterior lane of between

raph 4.6)

S

On average, the repeatability coefficient is about 22, wh

level for measurement but higher readings were recorded for some of the measurements and

this could be due to small sample size.

4.8.2.1 Length nterior Plane of Masseter Muscle (A

L f a p masseter muscle (AB) showed no significant difference

experimental side and control sides in all groups except in Group 6 (Table 4.10) (G

and (Table 4.11)(Graph 4.7).

ide Experiment Control Paired t-test G 1.di

roup Mean ± SD (mm) Mean ± SD (mm) p � 0.05

Immediately Post straction (N= 3) 72(9.64) 68.33(7.23) NS

ation 2 months & 62.5(8.66) 68.5(4.66) NS

3. 4.

6. N

2. Consolid

remodelling 1 month (N=4) Consolidation 3 months 63.5(6.12) 73(6.22) NS (N= 4) Consolidation 3 months 71.25(5.91) 77(4.69) NS remodelling 1 month

4) (N=5. Consolidation 3 months 76.5(1.73) 75.5(4.20) NS

remodelling 2 months (N=4) Consolidation 4 months 71(7.62) 79(4.69) 0.049 (N=4)

S: Not significant

Table 4. 10: al

Paired t-test for length AB (mm) (anterior plane) between control and experimentsides in six groups.

Results Chapter 4


133

0

Imm

DO

ConR1

Cos3

ConR1

ConR2

Cs4

10203040

90

P-

s2 n s3 s3 on


leng

th ( 50

607080

mm

)

Experimental

Control

raph 4. 6: The length of anterior plane (AB) (mm) of masseter muscles for the experimental

and control sides in six groups.

Net Different

G

Group (Mean ± SD(mm))

t distraction -3.667 (3.055) 1. Immediate Pos (N= 3)

(6.856

5 (4.96

75 (6.0

(5.31 s

(5.716

2. Consolidation 2 months & remodelling 1 month

6 )

(N=4) 3. Consolidation 3 months (N= 4)

9. 7)

4. Consolidation 3 months remodelling 1 month

5. 55)

(N=4) 5. Consolidation 3 months remodelling 2 month

-1 5)

(N=4) 6. Consolidation 4 months

=4)

8 ) (N

Table 4. 1 e in length of anterior plane (AB) (mm) of masseter muscle between the control and experiment sides in six groups.

1: The net chang

Results Chapter 4


134

-10

-5

0

5

10

15

20



Leng

th (m

m)

Graph 4. 7: The net change in length of anterior plane (AB) (mm) of masseter muscle between

ased on the ty, the distra

length by 5.4 side ro onsolidation period, length

of AB on cont n, G oup ); Group 3 ( % ; Group 4 (7.5%) and

Group 6 (10.1%) with Group 5, the distracted side was 1.3% longer than control side.

4.8.2.2 Leng f masse r m scle (CD)

There was a tween he tal side and control side in groups 4,

5 and 6. The middle plane of t ly shorter on the experimental

de when the the neutral fixat f 3 months and were killed one or two months

(Table 4.12) (Graph 4.8). In general, the experimental side was shorter in length as compared

to the control sides. Both sides continued to increase in length but the distracted side showed a

significantly shorter length in longer neutral fixation and after device removal as in group 4, 5

and 6.

the control and experimental sides in six groups.

B basis of relativi ction process, however, managed to increase the

% more than the control (G up 1). During the c

rol side was much longer i r 2 (8.9% 13 )

th of middle plane o te u

significant difference be t experimen

he masseter muscle was significant

si animals had ion or

after device removal and also in Group 6 where the device was in place for 4 months (p � 0.05)

Results Chapter 4


135

Side Experiment Control Paired t-test Group Mean ± SD (mm) Mean ± SD (mm) p � 0.05 1. Immediately Post distraction 86.33(10.12) 91.67(7.1) NS (N= 3) 2. Consolidation 2 months & 82.38(5.12) 91.5(5.32) NS remodelling 1 month (N=4) 3. Consolidation 3 months 87.25(2.63) 95.75(3.30) NS (N= 3) 4. Consolidation 3 months 97.25(1.71) 107.25(1.5) 0.003 remodelling 1 month (N=4)

6. Consolidation 4 months 98(2.45) 107.25(4.35) 0.007 (N=4)

5. Consolidation 3 months 100(2.16) 108.5(1.92) 0.006 remodelling 2 months

(N=4)

NS: Not significant Table 4. 12: Paired t-test for length CD (mm) (middle plane) between control and experimental

sides in six groups.

0

40

60

80

Imm P-DO

Cons2R1 Cons3 Cons3R1 Cons3R2 Cons4


Legt

hm

)

20

120

n (m

100

Experiment

Control

Graph 4. 8: The length of middle plane (CD) (mm) of masseter muscle for the experimental and control sides in six groups.

Results Chapter 4


136

here was no difference in the net differences between control and experimental sides. There

were no s increases in th le erime dle lane r muscle

between groups (Table 4.13) (Graph 4.9).

T

obviou e ngth of exp ntal mid p of massete

Net Different Group

ost d (Mean ±

istr tio 33 (6. 1) SD(mm))

1. Immediate P ac n 5. 80 (N= 3)

tion 2 months &

125 (5

on s 5 (2.7 )

ths (9.34 h

8.5 (2.16)

)

6. Consolidation 4 months 9.25 (2.38) (N=4)

2. Consolida 9.

.447) remodelling 1 month

(N=4) ation 3 m

3. Consolid th 8. 54 (N= 4)

on 4. Consolidation 3 m

ng 1 mont10 9)

remodelli (N=4)

tion 3 months 5. Consolida

remodelling 2 months (N=4

Table 4. 13: The net change in length of middle plane (CD) (mm) of masseter muscles between

the control and experimental sides in six groups.

-5

0

5

10

15

20

25



Leng

th (m

m)

Graph 4. 9: The net change in length of middle plane (CD) (mm) of masseter muscles between the control and experimental sides in six groups.

Results Chapter 4


137

was 5.8% shorter than the control side. The higher

the percentage difference, the shorter the mi e.

Group 2 and Group 4 showed the highest percentage of 10% and 9.3% respectively. Animal

with two mon owed s the distracted muscle one month

after removal lthough Group 4 showed a higher percentage, two months after

the removal entag w r (7.8%) i G oup 5. A pattern of

improvement was found from one month to 2 months after device removal with 3 months

consolidation of the ev a high percentage of reduction

(Group 3 and

There was no difference in length of the posterior plane of masseter muscle between the

experimental and control sides, except in Group 6. The length was significantly shorter on the

experimental side as compared to the control side after 4 months neutral fixation (p � 0.05)

(Table 4.14) (Graph: 4.10). There was an increase in the length of the posterior plane of the

experimental side immediately after completion of distraction but the length was reduced as in

Group 2.

Generally, the basis of relative of the middle plane of the masseter muscle indicated that the

control side was longer than the distracted side. The distraction process did not increase the

length of this plane, the experimental side

ddle plane of experimental masseter muscl

th consolidation period sh hortening of

of the device. A

of the device the perc e as smalle n r

periods. The presence d ice also showed

Group 6).

4.8.2.3 Length of Posterior Plane of Masseter Muscle (EF)

Results Chapter 4


138

Side Experiment Control Paired t-test Group Mean ± SD (mm) Mean ± SD (mm) p � 0.05

1. Immediately Post

distraction 45.33(10.26) 35.67(6.03) NS (N= 3) 2. Consolidation 2 months & 29.5(13.33) 34(10.55) NS remodelling 1 month (N=4) 3. Consolidation 3 months 36.25(7.81) 34(5.35) NS

4. Consolidation 3 months 35.25(7.5) 40(8.98) NS

(N=4)

remodelling 2 months (N=4) 6. Consolidation 4 months 34.25(4.57) 42.25(2.22) 0.021 (N=4)

(N= 4)

remodelling 1 month

5. Consolidation 3 months 34(4.97) 35.25(4.99) NS

NS: Not significant

m) (posterior plane) between control and

Table 4. 14: Paired t-test for length EF (mexperimental sides in six groups.

0

10

20

30

40

50

Len

th (m

)

60



gm

Experiment

Control

or plane (EF) (mm) of masseter muscles for the experimental and control sides in six groups.

Graph 4. 10: The length of posteri

Results Chapter 4


139

here were significant differences (p � 0.05) between groups; Group 1 (immediately post

distraction) as compared to Groups 2, 4 and 6. The length was long ate post

distraction (Table 4.15) (Graph 4.11). Group 1 showed increased length of posterior plane of

masseter muscle on the experimental side after achieving the desired degree

this was .

T

er in immedi

of distraction but

not statistically significant

Net Different Group (Mean ± SD(mm))

ate Post di ract n -9.667(4.933)

1. Immedi st io (N= 3)

month 4.5 (3.202) lling 1 mon

dation 3 months -2.25(3.559)

(N= 4) ion 3 m ths 4.75(4.359)

remodelling 1 month

(N=4) 6. Consolidation 4 months 8(3.403) (N=4)

2. Consolidation 2 s & remode th (N=4) 3. Consoli

4. Consolidat on

(N=4) 5. Consolidation 3 months 1.25 (7.676) remodelling 2 months

Table 4. 15: The net change in length of posterior plane (EF) (mm) of masseter muscles between the control and experimental sides in six groups.

Results Chapter 4


140

-20

-15

-10

15


lidation a g periods

h)

-5

0

5

10

Leng

t (m

m

Conso nd remodellin

Graph 4. 11: The net change in length of posterior plane (EF) (mm) of masseter muscles d exper e six groups.

The distracte ost distrac on, presented 27% longer than the control side.

Group 3 showed 6.6% longer on the distracted si distracted side was

13.2% shorter than the control side and group 4 rter by 11.9 . roup 5 showed 3.6%

horter which indicated improvement in the second month after 3 month consolidation. The

have caused c

.8.2.4 Length of Oblique plane of Masseter Muscle (AD)

here was a significant difference (p � 0.05) between the experimental and control sides in

nimals that were subjected to neutral fixation for 3 months and sacrifice 1 month after device

moval (Group 4) and neutral fixation for 4 months (Group 6) (Table 4.16) (Graph 4.12). The 3

onth neutral fixation and sacrifice 1 month after device removal group showed significant

shortening of the oblique plane of the distracted side but not at 2 months after device removal.

between the control an im ntal sides in

d side, immediately p ti

de. Group 2 showed the

was sho % G

s

highest percentage (19%) in Group 6 suggested the prolonged presence of the device might

ontracture of the muscle at the posterior plane.

4

T

a

re

m

Results Chapter 4


141

If the neutral fixation was continued for 4 months the oblique plane of distracted side was also

found to be significantly shorter when compared to the control side.

Side Experiment Control Paired t-test Group Mean ± SD (mm) Mean ± SD (mm) p � 0.05 1. Immediately Post distraction 84.33(9.29) 91.33(6.67) NS (N= 3) 2. Consolidation 2 months & 78.88(6.79) 90.5(3.32) NS remodelling 1 month (N=4) 3. Consolidation 3 months 85.25(8.66) 94(4.16) NS (N= 4) 4. Consolidation 3 months 92(2.45) 103.75(3.1) 0.002 remodelling 1 month (N=4) 5. Consolidation 3 months 98(4.24) 99.5(3.32) NS

6. Consoli 0.005 (N=4)

remodelling 2 months (N=4)

dation 4 months 95(0.82) 104.25(2.36)

NS: Not significant

sides in six groups. Table 4. 16: Paired t-test for length AD (mm) (oblique plane) between control and experimental

0

Imm P

DO

Cons

R1

C

3

ConR1

ConR2

C

4

20

40

60

120

- 2 ons

s3 s3 ons


Leng

th (

80

100

mm

)

Experiment

Control

Graph 4. 12: The length of oblique plane (AD) (mm) of masseter muscles for the experimental and control sides in six groups.

Results Chapter 4


142

Net Differe

There was no significant difference between groups (for differences between control and

experimental sides) (Table 4.17) (Graph 4.13).

nt Group S

(Mean ± D(mm))

1. Immediate Post distraction 7 (5.568) (N= 3)

solidation 11.625 (8.77) th

) idation 8.75 (2.5)

11.75 (8.615)

onth )

1.5 (2.217) months

9.25 (3.697)

2. Con 2 months & remodelling 1 mon (N=4 3. Consol 3 months (N= 4) 4. Consolidation remodelling 1 m

3 months

(N=4 5. Consolidation remodelling 2

3 months

(N=4) 6. Consolidation

(N=4) 4 months

able t chan e in leng of oblique plane (AD) (mm) of masseter muscles

between the control and experimental sides in six groups. T 4. 17: The ne g th

-5

0

5

10

15

20

25



Leng

th (m

m)

Graph 4. 13: The net change in length of oblique plane (AD) (mm) of masseter muscles between the control and experimental sides in six groups.

Results Chapter 4


143

control side. T oup 5 (1.5%). This was also

another indica more stable on the second month after device removal

with a 3 mont

Generally, the four planes; anterio F) and oblique (AD) in Group

2 showed that the distracted side was obviously horter than the control. lthough the length of

the distracted p 4), it as this was a temporary adjustment

as Group 5 ller pe en as also shown that reduction in

xperimental lengths in all planes 1 month after device removal with a 2 month consolidation

andibular ramus. The height of the mandibular ramus on the experimental side was reported

to have reduced, at that period (Syed Zainal, 2005). This was also supported by the presence

of woven bone and this condition was not stable and this was suggested to contribute to

relapse.

The oblique plane showed that Groups 2 and 4 have the highest percentage difference (12.8%

and 11.3% respectively), which indicates the shortness of the distracted side compared to the

he smallest percentage difference was found in Gr

tion that the muscle was

h consolidation period.

r (AB), middle (CD), posterior (E

s A

side was shorter (Grou w believed that

presented with a sma rc tage. It w

e

period (Group 2) can be suggested to contribute to a reduction in the vertical height of the

m

Results Chapter 4


144

4.8.3.1 Cross Section (mm²) of Masseter Muscle

Repeatability coefficient = / �d²

4.8.3 Ultrasonography

A repeatability coefficient was calculated for cross section scanned images for experimental

and control side for each group (Table 4.18) (APPENDIX 6). The calculation is based on this

formula:

x 100

3 18 21 4 34 14 5 16 31 6 38 27

istal masseter 1 17 12 2 21 09 3 17 14 4 29 17 5 51 19 6 09 31

� 2n d = the different between the second and first readings n = the number of double measurements ___________________________________________________________________ Group Experimental sides Control sides ___________________________________________________________________ Proximal masseter

1 18 26 2 12 19 3 21 35 4 34 36 5 16 15 6 21 21

Middle masseter 1 31 29 2 13 18

D

Table 4. 18: Repeatability coefficient for cross section (mm²) of image scans at three masseter

levels for control and experimental sides in six groups.

Results Chapter 4


145

bility coefficient for tracing to get the cross sectional area of the

asseter muscle was approximately 22. The reading was low and thus the method of

it

n

sted to have contributed to some of the high reading of repeatability coefficient.

Se tion (mm ) of pro uscle

here wa a signif ant diffe l sides in

er muscle

val of the

raph 4.14). The proxim ter al side was

much smaller when a

as the animals gre al

d n

and remodelling pe

On average the repeata

m

measurement was acceptable. There were higher reading in some of the measurements and

can be suggested to be due to the small sample size. The lack of experience and training ca

be sugge

4.8.3.1.1 Cross c ² ximal Masseter M

T s ic rence (p � 0.05), between the experimental and contro

Group 4. There was a significantly smaller cross-sectional area of proximal masset

when animals had 3 months of neutral fixation and were sacrificed 1 month after remo

device Table( 4.19) (G al masse muscle on the experiment

compared to the control side. There were increases in cross sectional are

w on both experimental and control sides. The cross section of proxim

masseter muscle id not show major changes during the distraction or during the consolidatio

riods (Table 4.20) (Graph 4.15).

Results Chapter 4


146

Side Experiment Control Paired t-test Group (Mean ± SD(mm²)) (Mean ± SD (mm²)) p � 0.05

distraction 551.7(83.3) 571.7(113) NS 1. Immediately Post

(N= 3) 2. Consolidation 2 months & 587.5(62.4) 632.5(113) NS remodelling 1 month (N=4) 3. Consolidation 3 months 655(61.5) 723.8(62.9) NS (N= 4)

0.022

(N=4)

remodelling 2 months

4. Consolidation 3 months 707.5(92.2) 787.5(58.7) remodelling 1 month

5. Consolidation 3 months 786.3(114.9) 771.3(176.3) NS

(N=4) 6. Consolidation 4 months 817.5(42.5) 840(91.9) NS (N=4) NS: Not significant

masseter muscles between control and experimental sides in six groups. Table 4. 19: Paired t-test for the cross sectional area (mm²) of scanned images of the proximal

0

100200

300

400500

600

700

800900

1000

Imm P-DO



Cro

ss-s

ectio

n (m

m²)

Experiment

Control

raph 4. 14: The cross section (mm²) of the scan images from the proximal masseter muscles on the experimental and control sides in six groups.

G

Results Chapter 4


147

Net Dif en

fer t Group (M

ediate Post distraction 20 (35) ean ± SD(mm))

1. Imm (N= 32. C

) 2 months & 45 (102.6 m

=4) i n 3 months 68.75 (67)

) n 3 months 80 (87.6

1 m N=4)

i n 3 months -15 (36.7) g 2 m

=4) i n 4 months 22.5 (104.5) )

onsolidati n remodelling 1

oonth

(N3. Consolidat (N=

o4o4. Consolidati

remodelling onth (5. Consolidat remodellin

oonths

(N6. Consolidat

(N=4o

a t change in cress sec images from proximal masseter

muscle between the control and experimental sides in six groups. T ble 4. 20: The ne tion (mm²) of the scan

-100

-50

0

50

100

150

200



Cro

ss s

ectio

n (m

m²)

Graph 4. 15: The net change in cross section (mm²) of the scan images from proximal masseter muscle between the control and experimental sides in six groups.

Results Chapter 4


148

ased on the basis of relativity, the cross section of the proximal masseter muscle was 3.5 %

smaller than diately after d tr tion (Group 1). In consolidation of 2 and 3

months, the cross section of the distraction side was reduced as seen in Group 2, 3 and 4

(7.1%, 9.5% and 10.2% respectively). Interest second mo th after device removal,

with a 3 month consolidation period (Group 5) showed a much smaller percentage, which

indicated exp ction of uscle. Figure 4.4 shows the

cross sectiona ages of the proximal masseter muscles.

B

the control imme is ac

ingly, the n

ansion of the cross se experimental side m

l ultrasonography im

A B Figure 4. 4: The scan images of the proximal masseter muscle. Control side (A) and

experimental side (B).

Results Chapter 4


149

4.8.3.1.2 Cross Section (mm²) of Middle Masseter Muscle

There was no significant difference (p � 0.05) in the cross section of the middle masseter

muscle between experimental and control sides in all groups (Table: 4.21), (Graph: 4.16) and

(Table 4.22) (Graph 4.17).

Side Experiment Control Paired t-test Group (Mean ± SD (mm²)) (Mean ± SD (mm²)) p � 0.05

NS 1. Immediately Post distraction 645.(182.5) 580(45.8) (N= 3) 2. Consolidation 2 months & 627.5(148.2) 666.3(142.9) NS remodelling 1 month (N=4) 3. Consolidation 3 months 821.3(92.7) 785(140.8) NS (N= 4) 4. Consolidation 3 months 722.5(49.9) 821.3(108.5) NS remodelling 1 month (N=4) 5. Consolidation 3 months 810(153.5) 842.5(188.8) NS remodelling 2 months (N=4) 6. Consolidation 4 months 850(38.7) 888.8(132.9) NS (N=4)

NS: Not significant

able 4. 21: Paired t-test for the cross sectional area (mm²) of scanned images of the middle

masseter muscle between control and experimental sides in six groups. T

Results Chapter 4


150

0

200

400

600

800

1000

ectio

nm

m²)

1200

Imm P- Cons2 Cons3 Cons3R1 Cons3R2 Cons4

oli modelling

Cro

ss s

( Experimental

Control

DO R1

Cons dation and re periods

Graph 4. 16: The cross section (m can imag dle mas eter muscles on perimen l an control sid s in six g ups.

ren

m²) of the s es of the mid sthe ex ta d e ro

Net Diffe t Group

s

on (Mean ± S

t distra -65 (138.6 D(mm))

1. Immediate Po cti ) (N= 3)

mont 38.75 (12 onth

(N=4) ation 3 months -36.25 (127.1)

(N= 4) 4. Consolidation 3 months 98.75(68)

remodelling 2 months (N=4) 6. Consolidation 4 months 38.75 (141) (N=4)

2. Consolidation 2delling 1 m

hs & 2.4) remo

3. Consolid

remodelling 1 month (N=4) 5. Consolidation 3 months 32.5 (68)

Table 4. 22: The net change in cross section (mm²) of the scan images from the middle

masseter muscles between the control and experimental sides in six groups.

Results Chapter 4


151

-250

-200

-150

-100

-50

0

50

100

150

200

250



Cro

ss s

ectio

n (m

m²)

Graph 4. 17: can images from the middle masseter muscles between the control and experimental sides in six groups.

Generally the both side r as the anim ls grew. The basis of

relative showed that the distraction caused an in ss section of the middle masseter

muscle, on the distracted side, of side. A 2 month consolidation

and sacrifice e remo al ) resulted .8% smaller in cross

section of the thermore G m ll in cross section than

control. The longer consolidation period showed lower percentages (G

6 (4.4%)), wh he distr cte ss section was closer to the control

ide.

The net change in cross section (mm²) of the s

cross sectional areas on s got large a

crease in cro

11.2% greater than the control

one month after devic v (Group 2 in 5

distracted side. Fur roup 4 showed 12 % s a er

roup 5 (3.9%) and Group

ich can suggests that t a d side cro

s

Results Chapter 4


152

A B

Figure 4. 5: The scan images of the middle masseter muscles and example of tracing (white tracing). Control side (A) and experimental side (B).

There were si the

cross section of the distal masseter muscle in animals with neutral fixation for 3 months and

sacrifice 1 month later (Group 4), and animals with neutral fixation for 4 months (Group 6).

Neutral fixation for 3 months, caused statistically significant reduction in cross section of the

distracted muscle 1 month after device removal (Group 4) but was not statistically smaller after

2 months (Group 5). Presence of the device at neutral fixation for a 4 month consolidation may

result in shrinkage of the distracted muscle (Group 6) (Table 4.23) (Graph 4.18) and (Table

4.24) (Graph 4.19)

4.8.3.1.3 Cross Section (mm²) of Distal Masseter Muscle

gnificant differences (p � 0.05) between the experimental and control sides of

Results Chapter 4


153

Side Experiment Control Paired t-test

Group (Mean ± SD(mm²)) (Mean ± SD (mm²)) p � 0.05 1. Immediately Post distraction 315(139.4) 373.3(111.5) NS (N= 3) 2. Consolidation 2 months & 330(174.8) 407.5(83.1) NS remodelling 1 month (N=4) 3. Consolidation 3 months 301.3(36.4) 340(57.9) NS (N= 4)

remod (N=4) 5. Consolidation 3 months 277.5(15) 427.5(143.9) NS remodelling 2 months (N=4)

0.013

4. Consolidation 3 months 298.8(48.7) 395(58.2) 0.029 elling 1 month

6. Consolidation 4 months 340(29.7) 430(23.5) (N=4)

NS: Not significant Table 4. 23: Paired t-test for cross sectional area (mm²) of scanned images at distal masseter

muscles between control and experimental sides in six groups.

0

100

200

300

400

500

600

ross

sec

tion

mm

²)

Imm P-DO Cons2 R1 Cons3 Cons3R1 Cons3R2 Cons4


C (

Experimental

Control

Graph 4. 18: The cross section (mm²) of the scan images from the distal masseter muscles on the experimental and control sides in six groups.

There were no differences between the 6 groups (Graph 4.19).

Results Chapter 4


154

Net Different

Group (Mean 1. Immediate P t trac (

± SD(mm)) os dis tion 58.33 62.5)

(N= 3) 77.5 (82.6)

onths 38.75 (33.4)

lidation 3 onths 96.25 (99.1) nth

onths 150 (48.9) onths

onths 90 (139.3)

2. Consolidation 2 months & remodelling 1 month (N=4) 3. Consolidation 3 (N= 4)

m

4. Conso m remodelling 1 mo (N=4) 5. Consolidation 3 remodelling 2 m

m

(N=4) 6. Consolidation 4 (N=4)

m

able chang in oss section (mm²) of the scan images from the distal

masseter muscles between the control and experimental sides in six groups. T 4. 24: The net e cr

-100

-50

0

50

100

150

200

250



Cro

ss s

ectio

n (m

m²)

Graph 4. 19: The net change in cross section (mm²) of the scan images from the distal

masseter muscles between the control and experimental sides in six groups.

Results Chapter 4


155

he cross section of the distal distracted muscle became smaller with longer consolidation and

remodelling period (Group 5). Group 3, 4 and 5 had the device fixed in place for 3 months but

after device removal the cross section becam aller in the irst month and 35%

smaller in the second month compared to control side. Group 6 als presented with high

percentage (2 uggested that is henomenon may b d e to less muscle bulk

at the distal level that leads to fibrous healing.

A

T

e 24.4% sm f

o

1%). It can be s th p e u

B

Figure 4. 6: The scan images of the distal masseter muscles. Control side (A) and experimental side (B).

Results Chapter 4


156

4.8.3.2 Thickness (mm) of Masseter Muscle Repeatability coefficient was calculated for each thickness of scanned images for experimental

and control side for each groups (Table 4.25). The calculation is based on this formula:

Repeatability coefficient = / �d² x 100 � 2n d = the different be n = the number of

tween the second and first readings double measurements

__________________________________________________________________ Group Experimental sides Control sides ___________________________________________________________________ Proximal masseter

1 09 06 2 09 06 3 17 09 4 09 36

6 08 06 iddle masseter

1 21 17 2 00 11 3 11 05 4 21 09 5 13 12 6 09 14

istal masseter 1 04 08 2 08 04 3 08 04 4 05 06 5 04 06 6 05 12

_

5 05 09

M

D

Table 4. 25: Repeatability coefficient for thickness (mm) of scan images at three levels of masseter muscle for control and experimental sides in six groups.

he lower reading of the repeatability coefficient can be considered acceptable for method of

easurement.

T

m

Results Chapter 4


157

er Muscle

(Table

4.26) (Graph 4.20) and ggest that the proximal level

ed b increas d muscle ld be the adaptation or compensation

thickness

onth after

s) (Group

r distrac

Side t

4.8.3.2.1 Thickness (mm) of Proximal Masset

The thickness of the experimental side was significantly wider (p � 0.05) compared to the

control side in Group 2 with neutral fixation for 2 months and sacrifice 1 month later

(Table 4.27) (Graph 4.21). This may su

result was caus y e contraction. It cou

activity after removal of the device. Other groups were not statistically different in

from the control side. Generally, the distracted muscle was thicker within the first m

device removal as in Groups 2, 4 and 5. The longer the device was in place (4 month

6) result in a thicked e ted muscle than at 3 months (Group 3).

Experiment Control Paired t-tesGroup (Mean ± SD (mm)) (Mean ± SD (mm)) p � 0.05 1. Immediately Post distraction 15.5(0.5) 14(3.04) NS 2. Cons remo 3. Cons

5. Cons remo 6. Cons

(N= 3) olidation 2 months & 18.75(1.85) 16.13(2.06) 0.012 delling 1 month (N=4) olidation 3 months 17.75(1.94) 19.25(0.65) NS (N= 4)

months 20.5(1.47) 19.75(1.32) NS 4. Consolidation 3 remodelling 1 month

(N=4) olidation 3 months 17.5(2.12) 17.38(2.32) NS delling 2 months (N=4) olidation 4 months 20.63(1.11) 19.88(2.5) NS (N=4)

NS: Not significant

scan images at the proximal level of masseterTable 4. 26: Paired t-test for thickness (mm) of muscles between control and experimental sides in six groups.

Results Chapter 4


158

0

5

10

15

Imm P-DO Cons2 R1 Cons3 Cons3R1 Cons3R2 Cons4


Thi

ess

(mm

)

20

25

ckn

Experimental

Control

Graph 4. 20: The thickness (mm) of the scanned images from the proximal masseter muscles on experimental and control sides in six groups.

T as no statisti l ffere 6 gr w

nt

here w ca di nce between the oups when tested ith ANOVA.

Net Differe Group (Mean ± SD(mm)

st -1.5 )

1. Immediate Po distraction (2.6) (N= 3)

onsolida n 2 months & -2.625 (1.92) g 1 mo

(N= tion 3 1.5

(N= 4) . Consolida n 3 months -0.75 (0.95)

g 1 mo =4)

. Consolida n 3 months -0.125 (1.55) ng 2 m

(N=4) 6. Consolidation 4 months -0.75 (2.29)

(N )

2. C tio remodellin nth 4) 3. Consolida months (1.94) 4 tio remodellin nth (N5 tio remodelli onths

=4

Table 4. 27: The net change in thickness (mm) of the scan images from the proximal masseter

muscles between the control and experimental sides in six groups.

Results Chapter 4


159

-5

-4

-3

-2

-1

0

1

2

3

4



Thic

knes

s (m

m)

Graph 4. 21: The net change in thickness (mm) of the scan images from the proximal masseter muscles between the control and experimental sides in six groups.

ept Group

3 (experimen ner than control). The distraction process caused the distracted

muscle to inc the 1). G up 2, the distracted

muscle was 1 trol si e nger con ion and remodelling

groups, the percentage was smaller, Group 4

4.8.3.2.2 Thic le Masseter Muscle

There was a � 0.05) between the experimental and control sides, for

animals in gr aph 4.22) and (Table 4.29) (Graph 4.23). Group 3

(three months with neutral fixation) showed t perimental side mu c was thicker than the

Group 6 reduced in thick ide

was inconsistent because the experimental side was thicker in Groups 1, 2, 3, and 5 but not in

Groups 4 and 6.

Based on the basis of relativity, the experimental sides were thicker in all groups exc

tal side 7.8% thin

rease 10.7% thicker than control side (Group ro

6.3 % thicker than the con d but in the lo solidat

(3.8%), Group 5 (0.7%) and Group 6 (3.8%).

kness (mm) of Midd

significant difference (p

oups 3 and 6 (Table 4.28), (Gr

he ex s le

control side. If the device remained in place for another month, the experimental muscle in

ness. In general the thickness of the experimental and control s

Results Chapter 4


160

Side Experiment Control Paired t-test Group (Mean ± SD (mm)) (Mean ± SD (mm)) p � 0.05 1. Immediately Post distraction 20(3.5) 16.83(2.02) NS (N= 3) 2. Consolidation 2 months & 18.25(2.75) 18(3.14) NS remodelling 1 month (N=4) 3. Consolidation 3 months 22.5(1.87) 19.375(2.5) 0.025 (N= 4) 4. Consolidation 3 months 20(2.27) 21.625(2.32) NS remodelling 1 month (N=4) 5. Consolidation 3 months 21(2.42) 18.75(2.22) NS remodelling 2 months

(N=4) 6. Consolidation 4 months 17.88(2.36) 21.88(1.89) 0.002

(N=4)

NS: Not significant

Table 4. 28: Paired t-test for thickness (mm) of scan images for middle masseter muscles between control and experimental sides in six groups.

There were significant differences (p � 0.05) between the groups. For animals with four months

neutral fixation (Group 6) the muscles were significantly thicker than the muscle which

belonged to animals in immediate post distraction (Group 1), neutral fixation for 3 mon

ths

roup 3) and combination of 3 months device in place and sacrifice after 1month (Group 5) (G

but the last 3 groups showed no differences.

Results Chapter 4


161

0

5

10ickn

15ess

(20

mm

25

30

2 R1 ons3 Cons3R1 ons3R2 C 4

ida remodelling pe

Imm P-DO Cons C C ons

Consol tion and riods

Th)

Experimental

Control

Graph m) of the scan images from le masseter le on the mental and control sides in six groups.

4. 22: The thickness (m the midd muscexperi

Net Different Group (Mean ± SD(mm)) 1. Immediate Post distraction -3.167 (4.04) (N= 3)

remodelling 1 month (N=4) 3. Consolidation 3 months -3.125 (0.82) (N= 4) 4. Consolidation 3 months 1.625 (2.9) remodelling 1 month (N=4) 5. Consolidation 3 months

2. Consolidation 2 months & -0.25 (1.5)

-2.25 (3.43)

remodelling 2 months (N=4) 6. Consolidation 4 months 4 (2.4) (N=4)

Table 4. 29: The net change in thickness (mm) of the scan images from the middle masseter

muscles betweeen the control and experimental sides in six groups.

Results Chapter 4


162

-8

-6

-4

-2

0

2

4

6

8

Imm P-DO



Thic

knes

s (m

m)

Graph 4. 23: The net change in thickness (mm) of the scan images from the middle masseter muscles between the control and experimental sides in six groups.

Immediately post distraction, the experimental side r 8% thicker middle masseter

muscle than the control side (Group 1). Intere p 2 sho e the middle masseter

thickness alm Group 3 and 5 showed that the

thicker than the control side, by 16.1% and 12% respectively. Group 4 and 6 showed the

reverse findin al side as 18.3% thi e than the control side.

It can be suggested that a reduction on the vertical height did not influence the thickness in

Group 2.

.8.3.2.3 Thickness (mm) of Distal Masseter Muscle

f the distal masseter muscle except in Group 1(Table 4.30), (Graph 4.24) and (Table 4.31)

raph 4.25). Generally, it was found that the thickness of distal masseter muscles in the

experimental side was much thinner in all groups. This was the result of the superficial

masseter myectomy in the first operation.

esulted in 18.

stingly, Grou w d

ost similar to control side. experimental side was

g where the experiment w 7.5% and nn r

4

There was no significant difference between the experimental and control side in the thickness

o

(G

Results Chapter 4


163

Side Experiment Control Paired t-test Group (Mean ± SD (mm)) (Mean ± SD (mm)) p � 0.05 1. Immediately Post distraction 7.83(2.36) 9(2.65) 0.02 (N= 3) 2. Consolidation 2 months & 9.13(3.28) 9.88(0.85) NS remodelling 1 month (N=4) 3. Consolidation 3 months 8.63(1.70) 8.63(0.48) NS (N= 4) 4. Consolidation 3 months 8.5(1.68) 8.88(1.25) NS remodelling 1 month (N=4) 5. Consolidation 3 months 8.13(0.85) 9.63(1.65) NS remodelling 2 months (N=4)

NS: Not significant

6. Consolidation 4 months 9.5(0.71) 11.13(2.93) NS (N=4)

Table 4.30: Paired t-test for thickness (mm) of scan images for distal masseter muscles between control and experimental sides in six groups.

0

2

4

6

8

10

14

16

12

ns3R2 Cons4

io d s

ExperimentalControl

Imm P-DO Cons2 R1 Cons3 Cons3R1 Co

C o nso lid at io n and remo d ell ing p er

Graph 4. 24: The thickness (mm) of the scan images from the distal masseter muscles on the experimental and control sides in six groups.

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164

Net Different

Group ±

1.167 (0.29) (Mean SD(mm))

1. Immediate Post distraction (N= 3)

0.17 (1.78

0 (3.35)

0.375 (2.63)

th 1.5 (2.29)

nths 1.625 (2.35)

2. Consolidation 2 months & remodelling 1 month

)

(N=4) 3. Consolidation 3 months (N= 4) 4. Consolidation 3 months remodelling 1 mon (N=4) 5. Consolidation 3 months remodelling 2 mo (N=4) 6. Consolidation 4 months

(N=4)

of the scan images from the distal masseter

muscles between the control and experimental sides in six groups. Table 4. 31: The net change in thickness (mm)

-4-3-2-1012345

Imm P-D

O

Cons2

R1

Cons3

Cons3

R1

Cons3

R2

Cons4


Thic

knes

s (m

m)

Graph 4. 25: The net change in the thickness (mm) of the scan images from the distal masseter muscles between the control and experimental sides in six groups.

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165

he distal thicknesses of the experimental masseter muscle as a percentage basis relative

were thinner de but similar in Group 3. The

experimental st distraction. It was also

noted that di ner than n longer consolidation periods and

remodelling G nd Group 6 (1 6 ).

4.9 Histopathology Quantitative Score A semi-quantitative evaluation was perform d blindly and independently by one observer.

Repeat deter oncorda c duplicate histological evaluation at

month intervals (Table 4.20) and (Appendix 7). The scoring for this calculation involved the

__________________________________________________________________ Sides Number of pairs Different reading Same reading (First and second) (First and second) ___________________________________________________________________ Experimental side 828 46 782 Control side 828 0 828 ___________________________________________________________________ 1656 46 1610 ___________________________________________________________________ Table 4. 32: Double determination for the histological scoring.

T

in Group 1, 2, 4, 5 and 6 compared to the control si

muscle was 13% thinner than the control immediate po

stracted muscle was thin normal i

roup 5 (15.6%) a 4. %

e

mination showed 97% c n e, based on

2

experimental and control sides.

_

Results Chapter 4


166

0

2

4

6

8

10

12

Po nD C n n Co

Tota

l sco

re ProxMiddDis

st Do

Co2S

1on

D3

CoD3S

1

CoD3S

2nD

4


muscles on the eGraph 4. 26: Histopathological activity at the proximal, middle and distal levels of the masseter

xperimental side in six groups.

6

5

4

3

0

1

2

Post D

o

ConD2S

1

ConD3

ConD3S

1

ConD3S

2

ConD4


Tota

l sco

re

ProxMiddDis

Graph 4. 27: Histopathological activity at proximal, middle and distal levels of the medial

pterygoid muscles on the experimental side in six groups.

Results Chapter 4


167

4.9.1 Immediately post distraction (Group 1)

Histopathologically the distracted middle masseter muscles after completion of the distraction

displayed the highest activity followed by the distal and then the proximal level (Graph 4.26).

The medial pterygoid muscle showed that the distal medial pterygoid muscle had the highest

activity followed by the middle and the proximal levels (Graph 4.27). The findings were

reversed between the masseter and medial pterygoid muscles (Table 4.21). There were

significant differences (p � 0.1) between the control and experimental sides in 6 parameters.

There were differences in regeneration of the middle masseter muscle (p = 0.083) and necrosis

in the medial pterygoid muscle. (p = 0.083). Generalised atrophy was observed in all of the

animals although this was not statistically significant (Figure 4.9 and Figure 4.10) compared to


The distal level of the masseter muscle predominantly showed higher histopathological activity,

followed by the middle and proximal levels (Graph 4.26). The proximal level of medial pterygoid

muscles showed higher cellular activity followed by the middle and distal levels (Graph 4.27).

The distal masseter muscles and proximal medial pterygoid muscles showed obvious atrophy

(Table 4.22) when compared to the control side. There were statistical differences (p � 0.1) in 2

parameters (atrophy and proliferation of fibroblasts). There was generalised muscle fibre

atrophy on the experimental side of the distal masseter (p � 0.059) and proximal pterygoid (p �

0.083). There was also increased fibroblast proliferation (p � 0.063), as there was the obvious

presence of th paces (Figure 4.13).

here was repair of the damaged muscle in the proximal medial pterygoid muscles (p� 0.083).

the control side (Figure 6, 7 and 8).

e connective tissue in the perimysium and endomysium s

T

Results Chapter 4


168

proximal levels (Graph 4.26). The proximal and middle

vels of the medial pterygoid muscle on the experimental side showed a slight increase in

histopathological score


The masseter and medial pterygoid muscles at all three levels showed very minimal

histopathological activity (Graph 4.26 and Graph 4.27) (Table 4.23). The presence of the

device, as neutral fixation for the masseter and medial pterygoid muscles in 3 months, showed

no significant difference in muscles activity between the experimental and control side (Table

4.22).


One month after removal of device, with 3 month neutral fixation, it was found that the distal

level of the masseter muscle on the experimental side, had the highest histopathological

activity followed by the middle and

le

histopathological activity compared to the distal level (Graph 4.27). The

for this group is reported (refer Table 4.24). The Wilcoxon test showed there are significant

differences between the control and experimental sides (p � 0.1) in regeneration of the middle

masseter. The cellular activity of the masseter muscles were mainly at the middle and distal

levels. There was statistically significant evidence that the middle masseter muscles on the

experimental side were undergoing necrosis (p � 0.083). The distal masseter levels on the

experimental side suggested an increase in connective tissue deposition in the perimysium and

endomysium areas (p � 0.063) that resulted in sclerosis (p � 0.063).

Results Chapter 4


169

elling 2 months (Group 5)

raph 4.26 and Graph 4.27). The histopathological score for the masseter and medial

terygoid muscles are reported (refer Table 4.24). The Wilcoxon test showed there was a

tal sides for fibroblast

4.9.5 Consolidation 3 months and remod

The distal level of the masseter muscle predominantly showed higher histopathological activity.

At this point in time the middle and proximal levels of masseter muscle showed minimal

histopathological activity. The proximal level of the medial pterygoid muscles showed slightly

higher histopathological activity followed by the middle and distal levels but still at a minimal

level (G

p

significant difference (p � 0.1) between the control and experimen

proliferation of the distal masseter muscle. The fibroblast proliferation was observed to continue

during the second month after device removal with 3 months consolidation periods (p � 0.059).


Histopathological activity was highest in the proximal level of masseter muscles followed by the

distal and middle levels (Graph 4.26). The medial pterygoid muscles showed minimal

histopathological activities at all 3 levels (Graph 4.27). The histopathological score for the

masseter and medial pterygoid muscles in animals with the device in place for 4 months after

cessation of the distraction process indicated that the muscle was in a stable condition with the

absence of histopathological activity (Table 4.25). A Wilcoxon test showed there was no

significant difference (p � 0.1) between the control and experimental sides for the

histopathology score.

Results Chapter 4


170

Experimental side Control side

Dys Atr Nec Pro Scl Reg Dys Atr Nec Pro Scl Reg

1 1 1 1 0 1 Middle 0 0 0 0 0 0

Proximal

Pterygoid

Proximal 0 0 0 0 0 0 Middle 0 0 0 0 0 0

Gr.34 Masseter

1 3 0 1 2 1 Middle 0 0 1 0 0 0

Nec: Necrosis lasts

Scl: Sclerosis Reg: Regeneration

Table 4. 33: Histopathological scores for Group 1, sacrifice immediately post distraction.

SITES Group IPD Gr. 04 Masseter 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 3 2 1 1 3 1 Middle 0 0 0 0 0 0 0 0 0 0 2 0 Distal 0 0 0 0 0 0

Pterygoid 1 0 1 0 0 0 Proximal 0 0 0 0 0 0

1 1 1 0 0 0 Distal 0 0 0 0 0 0

Gr. 18 Masseter 0 3 1 2 2 1 0 0 0 0 0 0 1 3 1 2 3 1 Middle 0 0 0 0 0 0 1 1 1 1 1 1 Distal 0 0 0 0 0 0

0 0 1 0 0 0 0 0 1 0 0 1 1 0 1 1 2 2 Distal 0 0 0 0 0 0

0 2 0 1 3 1 Proximal 0 0 0 0 0 0

0 0 1 3 3 3 Distal 0 0 1 0 0 0

Pterygoid 0 0 1 0 0 1 Proximal 0 0 0 0 0 0 0 3 0 0 1 0 Middle 0 0 1 0 0 0 0 3 0 0 3 0 Distal 0 0 0 0 0 0 Dys: Dystrophy Atr: Atrophy

Pro: Proliferation of Fibrob

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171

able 4. 34: Histopathological scores for Group 2, consolidation 2 months and remodelling 1

E roup D A N P Scl Reg ys ec o eg

0 2 1 1 1 0 0 3 1 0 0 0 0 2 1 1 1 0

0 1 0 0 1 1 0 0 0 0 1 0 0 0 0 0 1 0

3 3 1 3 3 2 3 2 0 3 3 1 3 3 0 3 3 2

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 1 0 1 1 0 3 3 0 3 3 1 3 3 1 3 3 3

0 1 0 1 1 1 0 1 0 1 1 0 0 1 1 0 0 0

r.33

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 0 0 0 0 0 0

d 1 1 0 0 0 0 0 0 0 0 1 1 0 Middle 0 0 0 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0

Dystrophy Atrophy Necrosis

Sclerosis

Reg: Regeneration

Tmonth.

xperimental side SITES Control side G ys tr ec ro D Atr N Pr Scl R Cons2 R1 Gr. 06 Masseter Proximal 0 0 0 0 0 0 Middle 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Pterygoid Proximal 0 0 0 0 0 0 Middle 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Gr. 28 Masseter Proximal 0 0 0 0 0 0 Middle 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Pterygoid Proximal 0 0 0 0 0 0 Middle 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Gr.30 Masseter Proximal 0 0 0 0 0 0 Middle 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Pterygoid Proximal 0 0 0 0 0 0 Middle 0 0 0 0 0 0 Distal 0 0 0 0 0 0 G Masseter 0 0 0 0 Proximal 0 0 1 1 Middle 1 3 0 1 2 Distal Pterygoi 1 1 1 1 Proximal 0 0 Dys: Atr: Nec: Pro: Proliferation of Fibroblasts Scl:

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172

Table 4. 35: Histopathological scores for Group 3, consolidation period for 3 months.

ITES Dys Atr Nec Pro Scl Reg Dys Atr Nec Pro Scl Reg

s3

Experimental side Control side S Group Con Gr. 09 Masseter 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 1 Middle 0 0 0 0 0 0 0 0 0 0 0 1 Distal 0 0 0 0 0 0 Pterygoid 0 0 0 0 0 1 Proximal 0 0 0 0 0 0 0 0 0 0 0 1 Middle 0 0 0 0 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Gr. 25 Masseter 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 2 0 Middle 0 0 0 0 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Pterygoid 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Gr.29 Masseter 0 0 0 0 1 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Pterygoid 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 0 0 0 0 1 0 Distal 0 0 0 0 0 0 Gr.35 Masseter 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Pterygoid 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Dys: Dystrophy Atr: Atrophy Nec: Necrosis Pro: Proliferation of Fibroblasts Scl: Sclerosis Reg: Regeneration

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173

4, consolidation period for 3 months and

up ro cl eg o eg

erygoid

erygoid

erygoid

erygoid

Table 4. 36: Histopathological scores for Group remodelling 1 month.

Experimental side ys tr ec

SITES Control side

Gro D A N P S R Dys Atr Nec Pr Scl R Cons3 R1 Gr. 03 Masseter 0

0 0 2

0 0

0 2

0 3

0 Proximal 0 0 0 0

0 0

0 0

0 0

1 Middle 0 0 2 3 0 2 3 0 Distal 0 0 0 0 0 0

0

0 0

0

Pt

0 0 0

1

0 0

0

Proximal 0 0 0 0

0 0

0 0

0 0

1 Middle 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Gr. 24

0

0

0

0

Masseter

0 0 0

0

0 1

1

Proximal 0 0 0 0

0 0

0 0

0 0

1 Middle 0 0 0 0 0 1 1 0 Distal 0 0 0 0 0 0

Pt 0

0 0 0

0 0

0 0

0 0

0 Proximal 0 0 0 0

0 0

0 0

0 0

0 Middle 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0

Gr.24

Masseter 0 0

0 0

0 1

0 0

0 0

1 Proximal 0 0 0 0

0 0

0 0

0 0

1 Middle 0 0 2 2 1 2 3 2 Distal 0 0 0 0 0 0

Pt 0

0 0 0

0 0

0 0

0 0

0 Proximal 0 0 0 0

0 0

0 0

0 0

0 Middle 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0

Gr.32

Masseter 0 0 1 0

0 0 1 1

Proximal 0 0 0 0

0 0

0 0

0 0

0 1 0 0 Middle 0 0 0 0 0 1 1 0 Distal 0 0 0 0 0 0

Pt 0

0 0

0 0 0

1 0

1 0

0 Proximal 0 0 0 0

0 0

0 0

0 0

0 Middle 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Dys: DystropAtr: Atrophy

hy

Nec: NecrosisPro: Proliferati

on o ibro asts

Scl: Sclerosis f F bl

Reg: Regeneration

Results Chapter 4


174

Table 4. 37: Histopathological scores for Group 5, consolidation period 3 months and remodelling 2 months.

Experimental side SITES Control side Group Dys Atr Nec Pro Scl Reg Dys Atr Nec Pro Scl Reg Cons3 R2 Gr. 01 Masseter 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 1 1 0 1 1 0 Distal 0 0 0 0 0 0 Pterygoid 0 0 0 0 0 1 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Gr. 16 Masseter 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 0 0 0 1 1 0 Distal 0 0 0 0 0 0 Pterygoid 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Gr.19 Masseter 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 2 3 0 2 2 0 Distal 0 0 0 0 0 0 Pterygoid 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Gr.21 Masseter 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 1 2 0 1 1 1 Distal 0 0 0 0 0 0 Pterygoid 0 0 0 0 0 0 Proximal 0 0 0 0 0 0 0 0 0 0 0 0 Middle 0 0 0 0 0 0 0 0 0 0 0 0 Distal 0 0 0 0 0 0 Dys: Dystrophy Atr: Atrophy Nec: Necrosis Pro: Proliferation of Fibroblasts Scl: Sclerosis Reg: Regeneration

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175

Table 4. 38: Histopathological scores for Group 6, consolidation period 4 months. C SITES

Dys Atr o R g Dys Atr Nec Pro Scl Reg

M 3 0 Pro 0 0 0 0 0 1 Mi 0 0 0 0

0 0 0 Distal 0 0 0 0 0

Pt 0 0 Pro 0 0 0 0 0 0 Mi 0 0 0 0

0 0 0 Distal 0 0 0 0 0

M 0 0 Pro 0 0 0 0 2 0 Mi 0 0 0 0

0 Distal 0 0 0 0 0

Pt 0 0 Pro 0 0 0 0 0 0 Mi 0 0 0 0

0 Distal 0 0 0 0 0

M 0 0 Pro 0 0 0 0 0 0 Mi 0 0 0 0

0 Distal 0 0 0 0 0

Pt 0 0 Pro 0 0 0 0 0 0 Mi 0 0 0 0

0 Distal 0 0 0 0 0

M 0 0 Pro 0 0 0 0 0 0 Mi 0 0 0 0

0 Distal 0 0 0 0 0

Pt 0 0 Pro 0 0 0 0 0 3 Mi 0 0 0 0

0 Distal 0 0 0 0 0

Reg:

Experimental side

ontrol side

Group

Nec Pr Scl e Cons4

08

Gr. asseter 0 0 0 0 ximal 0 0 0 0 0 0 ddle 0 0 0 0 0 0

erygoid 0 0 0 0 ximal 0 0 0 0 0 0 ddle 0 0 0 0 0 0

20

Gr. asseter 3 3 0 0 ximal 0 0 3

3 3 0 3 0 0 ddle 0 0

0 2 3

erygoid 0 0 0 0 ximal 0 0 0

0 0 0 0 0 0 ddle 0 0

0 0 0

Gr.22 asseter 0 0 0 0 ximal 0 0 0

0 0 0 0 0 0 ddle 0 0

0 0 0

erygoid 0 0 0 0 ximal 0 0 0

0 0 0 0 0 0 ddle 0 0

0 0 0

Gr.23 asseter 0 0 0 0 ximal 0 0 0

0 0 0 0 0 0 ddle 0 0

0 0 0

erygoid 0 0 0 0 ximal 0 0 0 0

00 0

0 ddle 0 00

0

y 0 0

Dys: Dystroph Atr: Atrophy Nec: Necrosis Pro: Proliferation of Fibroblasts Scl: Sclerosis

Regeneration

Results Chapter 4


176

Figure 4. 7: Normal muscle (control side). orm uscle re M) eri sium (P), artery (A),

vein (V) and collagen (C). H&E (100x).

N al m fib s ( , p my

Figure 4. 8: Normal muscle (control side). No l mu le re M) u le in e (S),

eri siu (P ei V n artery (A). H&E (100x).

rma sc fib s ( , m sc sp dlp my m ), v n ( ) a d

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177

usculotendinous junction (MTJ) normaFigure 4. 9: M l muscle (control side). Normal muscle

fibres (M), tendon (T) and perimysium (P). H&E (100x).

Figure 4. 10: Dystrophy in several muscle fascicles. Normal muscle fibres (M), dystrophic

muscles (Ds), perimysium (P) and fat tissues (F). H&E (40x).

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178

Figure 4. 11: Muscular dystrophy. Dystrophic muscle (Ds), normal muscle (M), perimysium (P)

and fat tissue (F). H&E (100x).

Figure 4. 12: Generalised muscle atrophy and increase in perimysial (P) and endomysial (e)

spaces. Normal muscle fibre size (M). (Group 1). H&E (100x).

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179

Figure 4. 13: Generalised muscle atrophy (m), increased perimysial (P) and endomysial (E)

spaces. Normal muscle fibres (M) and fat tissues (F). (Group 1) H&E (40x).

Figure 4. 14: Generalised muscle atrophy. Atrophic muscle (m), increased perimysial spaces.

Artery (A). (Group 2). H&E (100x).

Results Chapter 4


180

ecrosis of muscle fibres. Necrotic muscle (N), normal muscle (M), regenera Figure 4. 15: N tion

(R) and fat tissue (F). H&E (400x).

crosis of muscle fibres. Necrotic mFigure 4. 16: Ne uscle (N), normal muscle (M), regeneration

(R) and fat tissue (F). H&E (400x).

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181

Figure 4. 17: Necrosis of muscle fibre (N). Normal muscle (M), musculotendinous junction

(MTJ) and tendon (T). H&E (400x).

Figure 4. 18: Increased connective tissues (green) in the perimysium (P) and endomysium

(E). Red striping (arrows) on the collagen. Normal muscle (M) and fat tissues (F). One step Gomori's trichrome (100x).

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182

Figure 4. 19: Presence of the red striping amongst the collagen (green). One step Gomori's

trichrome (400x).

Figure 4. 20: Increase of the connective tissues at perimysium and proliferation of fibroblasts

(Fb), collagen (C), normal muscle (M) and perimysium (P). H&E (400x).

Results Chapter 4


183

Figure 4. 21: Presence of new muscle cells within the damaged muscles. Regeneration (R),

necrotic muscle (N), normal muscle (M) and fat tissues (F). H&E (250x).

Figure 4. 22: Presence of regeneration (R) within damage muscle (N). Normal muscle (M).

H&E (400x).

Results Chapter 4


184

4.10 Other Findings The current study observed the presence of a parasite within the muscle cells. The parasite

was known as Sarcocystis (Figure 4.23 and Figure 4.24). The presence of the parasite does

not interfere with the study as its presence did not induce inflammation or any histopathological

changes in the surrounding muscle fibers.

4.10.1 Sarcocystis Species

Sarcocystis is a larger genus of intracellular, cyst-forming coccidian parasites belonging to the

protozoan (protistan) phylum Sporozoa (Apicomplexa) (Dubey et al., 1989). The organisms

have an obligatory two-host life cycle, involving a sexual generation in entero-epithelial cells of

the intermediate host, usually a herbivore (cow and sheep). Ingestion of the infective

sporocysts originated from the definitive host in faecal form. The parasite will proliferate in the

tissues of the intermediate host and finally establish itself as sarcocysts. They include

cystozoites or bradyzoites and are present in the muscle and nervous tissues of the

intermediate host, and if the definitive hosts eat the infected immediate host, the cycle of this

parasite will continue and complete (Figure 4.25).

the definitive host such as carnivores (cat and dog) and asexual development in the tissues of

Results Chapter 4


185

Figure 4. 23: The cross section of the Sarcocystis sp (arrow), within the muscle fibre. Normal muscle (M), nucleus of muscle (n) and endomysium (E). H&E (400x).

Figure 4. 24: The longitudinal section of Sarcocystis sp (arrow) within the muscle fibre of the

sheep. Normal muscle (M), nucleus of muscle (n) and endomysium (E). H&E (400x).

Results Chapter 4


186

Figure 4. 25: Life cycle of the genus Sarcocystis (Adapted from (Tenter, 1995)) 4.10.2 Prevalence of Sarcocystis species infection in sheep

Sheep in Australia have a high prevalence rate of infection with Sarcocystis spp. Munday

conducted a serological study on sheep, cattle and pigs in Tasmania (Munday, 1975).

Serological results indicate that over 90% of sheep are infected. This study also showed that

percentage of infestation increase as the sheep get older with macroscopic sarcocysts; 0.6% of

lambs, 8.8% young adult sheep and 66% in 4 years and older sheep (Munday, 1975).

O’Donoghue and Ford conducted a prevalence study of Sarcocystis spp, infections in sheep for

slaughter in South Australia (O'Donoghue & Ford, 1986). Their study showed that the

macroscopic c

arger ovoid cysts) and S. medusiformis (small slender cysts).

ysts were presence on 6.7% of the sheep, which was identified as S. gigantean

(l

Discussion Chapter 5


187

CHAPTER 5: DISCUSSION This study was the first comprehensive experiment on vertical mandibular ramus distraction

osteogenesis, which comprised the surgical creation of the defect, correction of the defect with

the distraction procedure and experimentation on the appropriate consolidation periods before

device removal. The phenomenon of relapse was studied on the bone and masticatory muscle.

There was a clear lack of animal studies specifically done in the field of relapse. This reflects

the published clinical studies which generally only include very brief mention regarding this key

parameter (Cho et al., 2001; McCarthy, 1994; McCarthy et al., 2002; McCarthy et al., 1992;

Molina & Ortiz Monasterio, 1995)

The current animal experiment shows that it is possible to develop a ‘unilateral hemifacial

gically in a sheep model. The mandibular bone deformity was then

and masticatory muscle showed an unstable

ondition for the first 2 months post distraction of the device but continued to become more

stable after 3 months post distraction.

5.1 Selection of Sheep as Animal Model

d on animal models.

microsomia like’ defect sur

corrected with distraction osteogenesis. The bone

c

Sheep were selected as the animal experimental model because of their suitability for surgical

modification and their size is expedient and to some degree comparable to humans (Hecker,

1983). The sheep have a major advantage of calmness and adapt well to confinement (Hecker,

1983). Their similar body size to humans allows serial sampling and multiple experimental

procedures (Martini et al., 2001). Martini and associates did a literature review on sheep as an

animal model for orthopaedic research (Martini et al., 2001). They noted that for the past 30

years, there were a total of 21,500 orthopaedic studies conducte



188

hey stated that rats were used most often (36%) and sheep contributed only 2%. Their review

h as canines were used for fracture studies (42%) and

ory system (Bosanquet & Goss,

987). The anatomical location of the mandibular condyle is higher than the tooth row and

It was estimated that the current research cost

pproximately AUD40,000.00.

type B

T

also elaborated that large animals suc

osteoporosis (41%); rabbits for osteoarthritis (44%); rabbit and dogs were also used for

distraction studies (37%); and sheep involved 9-12% for the conditions of fracture,

osteoporosis, osteoarthritis and lengthening. Phylogenetically, sheep are the right model for

the craniofacial research (Swennen et al., 2002). The anatomy and the growth of the sheep

have some characteristics close to human development.

The relative similarity of the sheep temporomandibular joint to humans’ give rise to

opportunities of conducting experiments related to the masticat

1

occlusal plane, and lateral movement during grinding resulting from posterior inclination of

condyle head is another feature that similarly exists in humans (Finn, 1994). Sheep have the

advantage of being readily available in Australia, with less ethical problems and they are easily

handled and adapt well with the long confinement. On the other hand, conducting large animal

research is much more expensive.

a

5.2 Mortality and Morbidity The mortality rate for the current study was five out of 30 lambs (17%). Two of the five lambs

died due to intestinal perforation during the latency period and one lamb died of pulpy kidney

after the first operation. Generally, the death of the 3 animals was due to an underlying disease

state, which had been induced after stress, such as long anaesthesia and surgical time. It is

strongly recommended that the sheep for laboratory research should be vaccinated against

enterotoxemia, tetanus and contagious ecthyma (zoonotic). Clostridium perfringens



189

e pulse oximeter was not in place to

is animal during the procedure, so the respiration and oxygen saturation was not monitored.

is review.

ut of thirty sheep (17%) was also high, despite strict aseptic

infects the lambs up to 3 weeks of age. The post mortem finding such as pulpy kidney

suggested occurrence of acute onset of the disease and contributed to death in the three

lambs. Another two of the five lambs died due to anaesthetic complications. One lamb died is

caused by accidental detachment of the connection between the anaesthetic machine and the

endotracheal tube. The accident happened towards the end of surgery where the focus of the

surgeons was on the surgery and there was no separate anaesthetist. The extensive

manipulations of the head contributed to the accident. Th

th

Another lamb died shortly after induction and intubation. This animal was one of the smallest

animals in the group and may have been secondary to an overdose of an anaesthetic drug. No

post mortem was performed for this lamb.

The number of animal deaths in this experiment is high. Review of the craniofacial distraction

osteogenesis in animal models stated that there were 13 sheep experiments. Out of 213 sheep

involved only 2 animal losses were reported due to anaesthetic complications (Swennen et al.,

2002). The losses due to other complications or whether the animals with complications were

excluded were not reported in th

The infection rate was five o

surgical technique and antibiotic prophylaxis. Most of the infected cases were observed after

placement of the device and were localised to the osteotomy sites. The infections were

managed by open wound treatment. The infected wounds were explored and examined. Full

debridement, irrigation with copious saline and Betadine solution and packing with ribbon

gauze soaked with Betadine solution to create an aseptic environment, is the way to manage



190

age (Karaharju-Suvanto, 1994).

this type of wound in sheep. The three lambs healed well and were included in the current

experiment. The device fixed in these lambs was the reused device, therefore, it is possible the

remaining bone tissues attached to the inner side of the device induced an infection.

Twenty-five sheep in the current study tolerated the surgery and management protocol

throughout the duration of experiment. The weight gain of the animals was within the normal

weight gain curve (Butterfield, 1988). There was intended to be four sheep in Group 2a;

unfortunately three developed infection. Therefore the three sheep were placed in Group 1 for

sacrifice immediately post distraction. The one sheep that remained in Group 2a also had an

infection and the distracted site developed a fibrous union. Therefore, the results of Group 2a

are atypical.

5.3 Mandibular Growth The lambs first had surgery at about 10 weeks of age and the surgical correction with the

distraction was done at approximately 22 weeks of age. The growth of the sheep is at an

accelerating stage during this period of time. The growth in sheep continues to increase until

puberty which is at around 50-60 weeks of

The mandibular growth rate of the experimental and control sides in the vertical and antero-

posterior dimensions was reported to remain the same and there were no signs of any

compensatory growth observed in the deformity and the distraction side. This finding has been

supported by Karaharju-Suvanto in his study on analysed cephalometric changes after

distraction, where he stated that there was no compensatory growth on the control side

(Karaharju-Suvanto, 1994).



191

own masticate on both sides of the jaws randomly and with no preferential side.

pproximately 560 cycles for mastication and 654 cycles for rumination occur and changing

De Jongh et al., 1989). To reduce the masticatory forces, the

educe the force on

e surgical side.

o consume food immediately on full recovery from anaesthesia.

This functional loading was applied during the consolidation period.

Based on Syed Zainal (2005), the growth rate of the vertical distance on the control side was at

2.4%. The distracted side the vertical distance growth rate was about 22.5%, being a

combination of normal growth plus distraction.

5.4 Issues relating to the model 5.4.1 Biomechanics of masticatory forces, effect on position and fixation

Ruminants like sheep constantly masticate. The current study showed that sheep recovering

from the anaesthesia immediately started masticating using both sides of the jaws.

Experiments on the biomechanical force on the sheep masticatory apparatus found that the

maximum TMJ reaction forces were 914 N as compared to 500 N in humans (Finn, 1994)

Sheep are kn

A

side every 20 cycles (

coronoidotomy was performed in this study but it is still believed that prolonged high force may

have contributed to the failure of some of the distractors. Furthermore, myectomy of the

superficial masseter muscle reduced the muscle mass by 26 .5-32.6%. Besides reducing the

muscle mass to mimic hemifacial microsomia condition, it also helped to r

th

In human patients, bone remodelling starts after removal of the device and the bone slowly

returns to full functional loading with a normal diet over some months. This experiment allowed

the animals to consume a normal diet such as lucerne, hay and sheep pellets almost

immediately after surgical placement of the device. Based on the Animal Ethics committee, the

animal should be allowed t



192

rocess. Therefore, the current device, which is

ppropriate for humans, may not be suitable for sheep.

gap in a range of 8 – 11mm even with multiple usages. Based on the

urrent study the device was 50% (12 out of 25) likely to maintain a stable distraction gap. The

The selection of the device should be able to cope with the total mechanical force produced by

sheep during mastication. The device should be able to withstand the constant high mechanical

force during mastication and the rumination p

a

5.4.2 Device design and stability

The manufacturer has proven the mechanical instability in one of the devices. The device was

new and first time used, the gap between the proximal and distal plate was not properly closed

and unstable. In total, eleven devices were been provided for this experiment and most of them

have been used more than once in order to compensate for the large cost involved in this

study. This is against the recommendation from the manufacturer but because of budget

constraints multiple usage of the device was done. Therefore, this may affect the findings of the

current experiment.

In general, the distraction process managed to create the distraction gap 88% (8.8mm) from

the targeted 10mm. Failure of the devices was observed in 3 cases. One device was the first

time used and the other 2 for the second time. The other devices worked well and produced the

range of distracted

c

design of the current device by Mathys is suitable for human use but less so for the

experimental sheep.

The high mechanical forces and high frequency during mastication in sheep need a more solid

support and robustness to withstand the constant masticatory forces. Furthermore the design



193

late with a double rod, to have better

upport to withstand the strong masticatory force.

The current studies showed that interference of the mandibular condyle growth centre in a very

young and growing animal resulted in abnormality of the mandible. The mandibular symphysis

was shifted approximately 3.1 mm toward the operated side, three months after condylectomy

and superficial masseter muscle myectomy. Miyamoto et al. supported this finding in their

study on the effect of unilateral condylectomy on mandibular growth in 10 week old lambs

(Miyamoto et al., 2001a). They observed a 3.5 mm midline shift toward the surgical side after 3

months unilateral condylectomy. They noted that the operated mandibular ramus was shorter

vertically and laterally wider. Miyamoto and co-workers also conducted a separate study to

look at the effect of unilateral myectomy of the superficial masseter muscle on the

temporomandibular joints on 10 week olds lambs (Miyamoto et al., 2001c). They concluded

that reduction of masseter muscle mass resulted in alteration of biomechanical activities based

on changes in articular cartilage thickness, but no facial asymmetry.

The current study induced a ‘unilateral hemifacial microsomia like’ defect in the sheep model.

Based on classification by Kaban, these lambs are categorised as Type I hemifacial

of footplate was relatively small and sometime difficult to fix to both segments. The inferior

screw was fixed very close to the osteotomy cut. Continuous production of highly mechanical

force during mastication may cause microfracture and instability. In future experiments a

customised device should be designed bigger with a p

s

5.5 Vertical Mandibular Distraction Osteogenesis on ‘Unilateral Hemifacial Microsomia Like’ Defect.



194

ion. The creation of a

efect allowed us to perform a correction on the deficient bone and soft tissue instead of on a

nd Castano with pigs (Castano et al., 2001). The transgenic mouse (Hfm strain)

is an interesting model for further understanding the development of the defect but it has

limitations in terms of small size, expensive cost and need for special handling facilities.

an also be suggested due

small sample size. Tracing of the scan image also needed skill and experience. The

measurement.

microsomia and a type O0 M1 E0 N0 S1 O.M.E.N.S classification (Kaban et al., 1988; Vento et

al., 1991).

The current experiment surgically created the facial defect and it was not congenitally

developed. Basically this animal was normal initially before the first operat

d

normal bone which is usually what occurs in other studies, for example Fisher with dogs (Fisher

et al., 1997) a

5.6 Repeatability Coefficient

The average repeatability coefficient for the measurement of the planes on the masseter

muscle is approximately 22. This can be considered as an acceptable method of measurement

and the presence of higher percentages of the repeatability coefficient for some of the

measurements can be suggested due to small sample size.

The average repeatability coefficient for tracing to get the cross sectional area of masseter

muscle was also approximately 22. The value was low and the method of measurement was

acceptable. There were higher values in some of the groups and it c

to

repeatability coefficient for the measurement of the thickness of the masseter muscle has a low

value and the current measurement can be considered as an acceptable method of



195

sing ultrasonography was an acceptable method of assessing the condition of the

istracted muscle in clinical situation.

.7.1 Direct measurement

at the achieved vertical height by distraction was stable in the

nger consolidation period. The longer consolidation periods are 3 and 4 months with neutral

model, the relapse phenomenon

ccurred when the device was removed two months after the end of distraction.

Generally the method of measurement of the cross sectional area and thickness of the scan

images by u

d

5.7 Distracted bone Before discussion on the soft tissue, it is worth appreciating the discussion of the bone

component which was adapted from Syed Zainal (2005) (Sections 5.7.1 -. 5.7.3) She observed

changes of bone components in 4 different ways; direct measurement, radiological

measurement, histological description and histomorphometry evaluation.

5

Direct measurement showed th

lo

fixation after distraction ended. Furthermore, one and two months after device removal with 3

months consolidation periods also showed a stable vertical height. It was also noted that the

control side did not show compensation activity when the experimental side was distracted. Dr

Syed Zainal found that in vertical ramus distraction in sheep

o

5.7.2 Radiological measurement

The radiological measurement showed that the distraction increased the vertical height up to

22.5%. Relapse occurred in groups with 2-month consolidation (Group 2a and 2) and 3 months

consolidations (Group 3). Group 2 and group 3 was reported to show evidence of relapse of

5% and 3% respectively. It was also noted that two devices in Group 3 were mechanically



196

ven though the 3 month consolidation was sufficient time for bone to consolidate and

ical finding of the current experiment showed this was statistically

.7.3 Bone histology

structure was reported in the process of adaptation and healing of

also support

e role of periosteum in osteogenesis (Williams et al., 2005).

faulty and that may have caused the reduction of the distracted gap by 45-60% after it reached

10mm initially. The faulty device could not withstand the relapse force.

E

stabilise, the radiolog

insignificant in Group 4 and 6. This might be due to a Type 2 error, which was associated with

a small sample size. The vertical ramus distraction was reported not to affect the horizontal

component of the mandible.

5

The component of the bone

bone in different consolidation and remodelling periods by histological evaluation (Syed Zainal,

2005). The role of periosteum as source of new bone supply was suggested and the thickness

was measured. The current study reported that the thickness of periosteum was not

predisposed to one particular side and in some instances was bilaterally similar. A previous

pilot study stated that the periosteum was thicker laterally (Varughese, 2002). The periosteum

supplied new bone for further bone formation and ossification was of intramembranous type in

mandibular distraction osteogenesis in pigs (Glowacki et al., 2004). Other studies

th

Post distraction bones have been shown to have increased blood vessel formation, early

woven bone and fibrous connective tissue formation. Fibroblast formation was stated to form

parallel to the distraction vector within the distracted gaps and the new bone proliferated from

the periphery of host bone towards the centre (Karaharju et al., 1993; Karp et al., 1992).



197

one formation (Fang et

l., 2004). Cartilage formation was reported to increase immediately post distraction (Syed

als with poor fixation did not show any cartilage formation

istologically.

one remodelling was reported to persist in the longest consolidation period (4 months). Bone

Intramembranous ossification was observed to be associated with inflammation, the trabecular

arrangement within the distracted gap and trabecular bone to lamellar b

a

Zainal, 2005) and continued through the two month consolidation period (Varughese, 2002). In

his study there were no reported cases of wound infection which strongly suggested true bone

activity during that period of time. The increase in cartilage formation in the current study was

suggested to be due to infection. The poor fixation of the device may not influence the cartilage

formation as the other anim

h

The anterior segment was reported to heal faster than the posterior side (Syed Zainal, 2005).

The posterior segment presented with woven bone peripherally. This finding suggested that the

masticatory force caused more functional loading on the anterior part, leading to earlier

remodelling of this segment.

Complete bone maturation in animals with consolidation for 3 months and longer can be used

as an indication of the optimal time to remove the device. The distracted bone was reported to

have same bony quality as the original bone (Syed Zainal, 2005).

B

remodelling reflected the activity of osteoblastic and osteoclastic cells. In this study

consolidation for 3 months and sacrifice 2 months after device removal (total 5 months),

showed full bone maturation but remodelling was still persisting, though with less osteoclastic

as compared to osteoblastic activities. Remodelling activities continued up to a year or more



198

achieved 100% bone stiffness the neutral fixation should be for approximately

6 days or about three months consolidation.

showed the muscle increased in weight during the distraction

rocess but ceased as the distraction ended (Schumacher et al., 1994). Other studies showed

.8.1 Weight of Masticatory Muscle

before the distracted bone structure was comparable to that of pre-existing bone (Schenk &

Gachter, 1994). Histomorphometric analysis on the distracted bone showed that the bone

quality of the experimental side was similar to the control side after 3 month consolidation

periods. The current finding was supported by a study by Kaban and associates conducted a

correlation study of the biomechanical stiffness with radiological density and ultrasound of

mandibular distraction in Yucatan minipigs (Kaban et al., 2003). They showed that following 24

days of neutral fixation during the consolidation period they achieved high clinical stability and

increased bone density but only showed 25.5% stiffness as compared to control. Based on

their findings, to

9

5.8 Effect of Mandibular Distraction Osteogenesis on Masticatory Muscles in sheep limb muscle distraction.

Distraction on the lower limb

p

that there was no difference in distracted limb muscles between operated and control limbs

(Sun et al., 1994).

5

5.8.1.1 Weight of Masseter Muscle

In normal condition, De Jongh and associates stated that the wet weight of the superficial

masseter was 34.2 grams and the deep masseter was 77.5 grams (De Jongh et al., 1989). This

figure was higher compared to the current study, but that related to adult sheep. Experimentally

the muscle bulk can be reduced in creating the defect which mimics the hemifacial microsomia



199

the experimental side continued to grow as the

nimal grew but at a slower rate.

e did not reach the weight level of the control side at any time of the

iu et al., 2003). Although in their study, both masseter muscles were normal

ithout creation of defect, but the distraction resulted in a lighter weight. The reduction in

atrophy. The perpendicular orientation of the masseter

have also caused the atrophy.

in animal model. Miyamoto and associates, showed that there was a slight reduction (30 mg) in

the weight of the masseter muscle compared to the control (33 mg) after unilateral

condylectomy in 10 week old lambs at 3 months post surgery but this was not statistically

different (Miyamoto et al., 2001a). The current study showed that the masseter muscle on the

experimental side was always lighter in weight as compared to the control side. This is the

experimental result as the muscle was partly removed surgically to reduce muscle mass and

the masticatory mechanical loading to mimic hemifacial microsomia. The removal of part of the

superficial masseter reduced the total weight by approximately 26.5-32.6% on the experimental

side. Both sides of the experimental and control masseter muscle showed an increase in

weight as the animals grew. It indicated that

a

The distracted muscl

consolidation and remodelling periods in this study. The current study showed there was

minimal weight gain at the end of distraction. Liu and associates supported the finding of the

current study; the rat mandibular distraction osteogenesis resulted in a lighter weight of the

masseter muscle (L

w

weight might have been due to disuse

muscle to the vector of distraction may

There is a tendency that the study muscles gain weight in the longer consolidation period and 2

months after device removal. Increased in muscle weight is one of indicator of stability of

muscle and if relate to the status of bone healing in current study which present with high



200

the distracted side responded with an increase of 29% from

e original volume at the end of the distraction process but the control side only increased by

Generally, the distraction osteogenesis did not increase the muscle mass in the experimental

side. The experimental side was lighter in weight when compared to the contralateral side. The

lighter weight also might be due to reduction in function of the experimental side, which caused

by some degree, disuse atrophy. The gross assessment of the muscle at post mortem in the

current study showed that the muscles appeared softer and much thinner compared to the

control side. The distracted muscle was smaller in weight during the first month after removal of

device for both 2 months and 3 months consolidation periods. The unstable bone and muscle

healing one month after device removal with 2 months consolidation period may have

lamellar bone. The stronger and stable lamellar bone and the weight gain of the surrounding

muscle may prevent the relapse.

5.8.1.2 Weight of Medial Pterygoid Muscle

Normally the medial pterygoid muscle weighed about 30.5 grams in adult sheep (De Jongh et

al., 1989). The current study showed that the weight of the medial pterygoid muscle was much

lower than that reported by De Jongh as this study used lambs and young adult sheep.

However, Mackool and associates conducted a comparative study of the medial pterygoid

muscle pre and post-distraction using computer tomographic scanning in human patients with

Pierre Robin Syndrome and unilateral hemifacial microsomia, after mandibular distraction

osteogenesis. They reported that

th

10% (Mackool et al., 2003). The current study showed that the distracted muscle did not have

any incremental change; the mean weights on both sides are similar immediately after

distraction.



201

period was a temporary episode. In the second month after

evice removal with a 3 months consolidation periods, the muscles on the experimental side

d the distracted bone was reported to be more

5.8.2.1 Length of Anterior Plane of Masseter Muscle (AB)

The distraction process influenced the anterior plane by increasing the length on experimental

side basic relatively by 5.4% over the control side (Group 1). The presence of the device and

early removal of the device (less than 3 month consolidation period) resulted in a shortening on

the experimental side of AB compared to the control side as basic relatively, Group 2 (8.9%);

roup 3 (13%); Group 4 (7.5%) and Group 6 (10.1%). Interestingly two months after device

contributed to the relapse. On the other hand, weight changes after one month device removal

following a 3 months consolidation

d

started to show some increase in weight an

stable and had consolidated well at this point. This phenomenon is an important finding as it

may prevent the chances of relapse. Therefore, it can be suggested that a 3 month

consolidation period is the optimum time as the bone is consolidated and stable, and changes

of muscle weight are a more temporary condition.

5.8.2 Length of Landmarks of Masseter Muscles

G

removal, following 3 months consolidation period (Group 5) the experimental side showed an

increased in length with a minimum difference (1.3%) from control side.

Prolonged placement of the device with longer consolidation (4 months) showed a significant

shortening of length in the experimental side. On the other hand, the length of the anterior

plane was almost similar after 2 months device removal with 3 month consolidation period.

Alternatively, it may be that the presence of the molar row creates a more stable position. The

current study showed that the animals used the operated side for mastication immediately after



202

th) and also during 1 - 2 months after device

moval with a 3 month consolidation period.

.8.2.2 Length of Middle Plane of Masseter Muscle (CD)

The middle masseter muscle has the most bulk of the masseter muscle and it was speculated

that this plane has the most muscle force and is more adaptable to changes. Generally, the

middle plane of the masseter muscle showed the control side was longer than the distracted

side. The distraction process did not increase the length of this plane; the experimental side

was 5.8% smaller than the control side. The higher percentage indicated a shorter middle plane

of the experimental side masseter muscle. Surgical removal of the device caused a reduction in

length after one month with 2 months or 3 months device in place Group 2 (10%) and Group 4

(9.3%). Although Group 4 showed a higher percentage, the following month the percentage

was smaller (7.8%) in Group 5. This also showed the pattern of improvement from one month

to 2 months after device removal following a 3 month consolidation periods. The presence of

the device in the longer consolidation period (4 months) also showed a high percentage of

reduction (Group 3 and Group 6). It can be suggested that the more muscle bulk across the

operation and throughout the consolidation periods. The loading on the occlusal row on the

operated side was functional almost immediately after surgical placement of the device. The

presence of the occlusal plane may suggest the restriction of the deviation of the jaw at this

plane. The growing and moulding of the new distracted bone during the consolidation period

had less effect on the length of the anterior plane of the masseter muscle. Prolonged

placement of the device such as 4 months might cause some degree of muscle contracture at

the insertion area or restriction of muscle development. The distracted bone was reported to be

stable in the longer consolidation period (3 - 4 mon

re

5



203

5.8.2.3 Length of Posterior Plane of Masseter Muscle (EF)

The length of the posterior plane of the masseter muscle on the experimental side was

generally shorter than the control side except in the immediate post distraction and in animals

with neutral fixation for 3 months. Again at 4 months, consolidation showed a significantly

shorter experimental side when compared to the control side. It can be suggested the

prolonged placement of the device may have caused restriction of development and

adjustment. This plane was close to the surgical site and may have had been subjected to

major stretching and lifting during surgery. The posterior plane was closer to the vertical vector

of distraction. The distraction increased the length up to 27% more than control. The muscles

became shorter one month after device removal with 2 and 3 month consolidation periods. The

muscles became shorter in response to device removal with 2 and 3 month consolidation

periods. Furthermore, in the two months after device removal following a 3 month consolidation

period, the length of muscle on the experimental side almost reached the length of the control

side. Therefore, it can be suggested that a 3 month consolidation period was sufficient and the

muscle was shown to readjust its length in the second month after device removal.

upward and dorsally from the original insertion. It also suggested that the placement of the

plane the better the adaptation, adjustment and more stability. In relation to bone consolidation,

the middle plane of masseter muscle is suggested to be stable in all groups.

5.8.2.4 Length of Oblique Plane of Masseter Muscle (AD)

Generally the oblique plane of masseter muscle showed that the experimental side was shorter

compared to the control side. Group 4 and Group 6 showed significant differences. It can be

suggested that the masseter insertion at the angle of mandible has some degree of shifting



204

onth following a 3

onth consolidation period.

The 3 month consolidation period is a sufficient period. The distracted bone was reported to be

stable and completed consolidation in both 3 and 4 month consolidation periods. The length of

the anterior plane (AB) and posterior plane (EF) was directly affected by the distraction and

surgical removal of the device. The muscle showed transitional changes in length during the

first month after device removal but showed more in the second month following a 3 month

consolidation period. The middle plane (CD) was quite stable and the changes were almost the

same in all groups. The oblique plane showed the experimental length was almost the same as

the control side, 2 months after removal of device following a 3 month consolidation period.

Prolonged placement of the device in the consolidation period resulted in shortening of the

experimental side muscle. Based on these findings the removal of device is advised after a 3

month consolidation period.

5.8.3 Ultrasonography

.8.3.1.1 Cross Section (mm²) at the Proximal of the Masseter Muscle

he cross section of proximal masseter muscles on the experimental side is smaller in the early

consolidation periods (Group 1 and 2) and the first month after device removal with 3 months

consolidation periods (Group 3). The cross section of experimental side was significantly

smaller than control (p � 0.05) (Group 3). Interestingly, the cross section started to become

broader in the longest consolidation and remodelling periods (Group 5). The proximal level was

the origin of the masseter muscle. Both sides showed an increase in cross section as the

distractor for 3 months is sufficient. It should be noted that there will be some muscle changes

in the first month after device removal and it will be resolved in the second m

m

5.8.3.1 Cross Section (mm²) of Masseter Muscle 5

T



205

l sides to become smaller than control. It was also noted that

rolonged fixation of the device (Group 6) resulted in reduction of the cross section on the

ter chance to regenerate. Beside that, the distraction resulted in a

roader cross section immediately post distraction (Group 1) and consolidation 3 months

oval resulted in the cross section becoming smaller in the first month

ion period of 3 months, the cross section

of the experimental side broadly returned back during the second month after device removal.

The percentage of differences was reduced from 11.2% (Group 4) to 3.9% (Group 5), which

means the reduction in the cross section was a temporary adjustment although there was no

statistical significance.

animals grew. The current study also showed that usually the cross section of the distracted

side was smaller than the control side. The device removal at early consolidation resulted in the

cross section of experimenta

p

experimental side.

5.8.3.1.2 Cross Section (mm²) of Middle Masseter Muscle

The middle level of the masseter is the belly of the masseter muscles. This section is

comprised of a lot of muscle as compared to fascia. Statistical analysis did not detect any

significant differences between experimental and control sides in groups and between groups.

Though there were reductions of muscle from the superficial masseter myectomy, but this did

not affect the cross section of this level. The muscle area of the middle masseter can be

suggested to adjust and compensate well with the distraction process and with either the

presence or absence of the device. The middle level of masseter muscle also consists of a lot

of muscle bulk that has a bet

b

(Group 3). The device rem

(Group 2 and Group 4). Furthermore, with a consolidat



206

resence of the fibrous tissue as a reaction to the device or surgical lifting

uring placement and removal of the device. There was also present, a whitish fibrous tissue at

elevation of the distal masseter

oval the muscle may be in a stage of adjustment from the surgical

moval of the device. In the second month the muscle was more stable and became much

thicker.

5.8.3.1.3 Cross Section (mm²) of Distal Masseter Muscle

The cross section of the masseter muscle at the distal level in Group 4 and Group 6 was

significantly smaller (p � 0.05) when compared to the control side. The smaller cross section in

longer consolidation periods might be due to contracture of the muscle. The contracture may

be caused by the p

d

the distal level observed at post mortem. The process of

muscle during placement of device; presence of device through the distraction and

consolidation periods; and also surgical removal of the device in some groups probably caused

some of the muscle damage and readjustment of the position of the insertion of the masseter

muscle.

The net measurement of the cross section between control and experimental sides showed the

experimental side tends to become smaller in the longer consolidation period, Group 4, 5 and

6. The cross section on the experimental side showed a higher percentage (35%) reduction

than the control side, which indicated a flattening or shrinkage of muscle. This may be related

to the formation of fibrous tissue. The distal of the masseter muscle comprises less muscle,

and this reduces the regeneration of the contractile component but increases the proliferation

of the connective tissue. The histological examination confirmed this.

Three months neutral fixation is the optimal duration for the consolidation period and the first

month after device rem

re



207

ent study showed that with 3 months of neutral fixation, the distracted muscle was

icker than the control side. As the device remained in place for another month the thickness

The current study showed that the distal of the masseter on the affected side showed

a reduction in thickness after 1 and 2 months after removal of the device following 3 months

5.8.3.2 Thickness (mm) of Masseter Muscle 5.8.3.2.1 Thickness (mm) of Proximal of Masseter Muscle

The proximal of the masseter muscle showed inconsistency in thickness at different times of

consolidation and remodelling. Group 2 showed the distracted muscle was statistically thicker

than the control side. There were no statistical differences in the other groups. However, the

mean of the measurements of experimental side were thicker, one month after removal of the

device than the control. This may be because of the muscle manipulation related to the surgical

removal of the device. Prolonged neutral fixation also caused the affected side to be thicker.

5.8.3.2.2 Thickness of Middle of Masseter Muscle

The curr

th

became less than the control side. Generally in the current study the thickness of the

experimental and control side was not consistent throughout the different consolidation periods

and after removal of the device. Although there was no statistically significant difference, it can

be suggested that the thickness of middle part of masseter muscle was in constant change in

relation to the presence of the neutral fixation and after removal of device. This tendency also

can be an indication that this was the level at which the majority of the contractile components

adapted to change.

5.8.3.2.3 Thickness (mm) of Distal of Masseter muscle

The distal of the masseter on the distracted side was only statistically thinner immediately after

distraction. Generally, the distracted sides were thinner compared to the control sides in all

groups.



208

e muscle contracture due to scar tissue created

ggested that the presence of the

he observation procedure using the grid method is reliable as the current study showed 98%

al changes in muscle cells in injury and

tracted masseter showed greater muscle cell changes as compared to the

istal and proximal but the distal of the medial pterygoid muscle of the same side showed the

ere are opposite reactions of the

neutral fixation. This showed that there might b

during lifting of muscle to remove the device. It also can be su

activator at minimum muscle bulk such as at the insertion level will lead to the deposition of

connective tissue. The development of the connective tissue may be due to the contractile

tissues not readily regenerating. The reduction in the contractile component may lead to

shrinkage of muscle.

5.9 Histopathological Evaluation T

concordance. The observation of the histopathologic

traction will give an idea of reversibility or permanence of damage to the muscle. There are

very few studies on the effect of distraction osteogenesis on the craniofacial musculature as

compared to the lower limb muscles. Most of the studies on the effect of distraction on the

facial muscles focused on the distraction process and early consolidation. The current study

was conducted to look at the histopathological activities on neutral fixation of 2, 3, and 4

months and 1 or 2 months after device removal.

5.9.1 Immediately post distraction

The middle of the dis

d

highest activity followed by middle and origin level. Th

masseter as compared to the medial pterygoid muscle. The masseter and medial pterygoid

muscles may have a direct effect at different levels and be based on its relation to the

osteotomy and distraction vector. Regeneration occurred predominantly at the middle



209

nd proximal level for the medial pterygoid muscle after 2 months neutral

xation and 1 month without external support. The distal of the experimental masseter was

phy. The muscle cells looked smaller as compared to the

control side. There were also increased inter-fibre spaces indicated by the presence of

connective tissues. The current study also showed that the fibroblasts were active in this period

where there was evidence of increased numbers of fibroblast cells.

Distraction osteogenesis of the lower extremities showed that the weakness of the muscle and

the over-stretching of muscles resulted from failure of the contractile component to proliferate

(Williams et al., 2001). The deposition of collagen type III in the perimysial area was noted in

this condition (Williams et al., 1998). Excessive presence of perimysial collagen caused over-

stretching of lower limb muscles and led to muscle contracture and reduced range of joint

movement (Williams et al., 1999). There were different capacities of adjustment and adaptation

between the contractile and connective components of the muscles of the lower extremities

(Williams et al., 1998). They also stated that if the contractile components were not able to

adjust to the new position, connective tissue proliferated to repair the injuries. The current

study involved the masseter and medial pterygoid muscles which act on a single joint. The

tension is greater on the point where the pressure is created, such as at the distal of

masticatory muscle, occlusal plane and temporomandibular joint.

distracted masseter muscle but necrosis was more in the medial pterygoid muscle. The

generalised atrophy may be due to the traction process, which may exceed muscle tolerance.

5.9.2 Consolidation 2 months and remodelling 1 month

There were changes in histopathological activity, there was more activity at the distal level of

the masseter muscle a

fi

statistically significant regarding atro



210

ere no statistically significant differences at the proximal, middle and distal of both muscles.

t as the distracted muscle did not

the middle and proximal

vel of the masseter muscles. On the other hand, the proximal and middle of the distracted

d

5.9.3 Consolidation 3 months

Both masseter and medial pterygoid muscles showed minimal histopathological activity. There

w

Neutral fixation, inactive stage for 3 months was sufficien

show major cellular pathological activity. This shows that the distracted muscle adjusted itself

to the new position. The minimal histological activities are an indication of stability of the

muscle or the muscle is back to normal function.

5.9.4 Consolidation 3 months and remodelling 1 month

The first month after removal of device, with 3 months neutral fixation, showed that the distal of

the distracted muscle increased histopathological activity more than

le

medial pterygoid muscle showed higher muscle histopathological activity than the insertion

level, this was statistically significant (p � 0.1). The distracted masseter muscle was stable with

minimal muscle histopathological activity as in Group 3, but the surgical removal of the device

induced histopathological activity. Furthermore, one month after removal of the device showe

degenerative activities such as necrosis (middle level), increased connective tissues deposition

at the perimysial and endomysial levels that lead to sclerosis (distal level) and at the same time

the development of new muscle cells as regeneration occurred at middle level. The proximal of

the distracted masseter was more occupied by connective tissues where there was more

proliferation of fibroblast and deposition connective tissue (sclerosis) in the muscle.



211

nd remodelling 2 months

masseter during the first month after device

moval is a temporary adjustment. On the other hand, the distal of the distracted masseter

is an irreversible histopathological

t for all 3

vels at this time.

5.9.5 Consolidation 3 months a

Two months after removal of the device with 3 months neutral fixation, the distal of the

distracted masseter still showed the higher level of histopathological activity. The middle and

proximal showed very minimal pathological activity. This is an indication that the proximal and

the middle level of the distracted masseter returned to normal activity in the second month after

device removal following a 3 month consolidation period. It also indicated that the

histopathological activity of the middle distracted

re

showed statistically significant fibroblast proliferation that

change.

The proximal of the distracted medial pterygoid muscle showed slightly higher activity followed

by the middle and distal levels. There were no statistically significant changes at the 3 levels in

the pterygoid muscle indicating reversible cellular changes.

5.9.6 Consolidation 4 months

Prolonged placement of the device for 4 months, showed a shifting of the histopathological

activity to the proximal of the distracted masseter muscle, followed by the distal and middle

levels. On the other hand, the distracted medial pterygoid muscle showed very minimal

histopathological activity or it can be suggested to have gone back to normal activity. Both the

distracted muscles (masseter and medial pterygoid) were statistically not significan

le



212

ted bone on the experimental side

onsolidation and also during the first month after removal of the device. By

omparison with 3 month or longer consolidation the distracted bone reached the hard callus

the proximal and middle levels of

asseter, which was the majority of the muscle bulk, but was not stable in the insertion. The

n the connective tissues deposition. Hence from the current

Van Sickels et al., 1988). Relapse is common in orthognathic surgery and

sults in dental malocclusion, extended or repeated treatments and unaesthetic results.

Relapse in orthognathic surgery is more comon with surgery which results in soft tissue

The current study showed that the bone healing of the distrac

was stable and had consolidated well by 3 months or more after neutral fixation. The muscle

had a tendency to respond to the surgical removal of the device by reduction in weight,

shortening of length, changes in cross section and thickness. This adaptive behaviour of the

muscle was observed during the first month after device removal both 2 and 3 months

consolidation periods. The histopathological activity was also observed to have the same

pattern of changes at this period of time.

The healing of the distracted bone was still at fibrous formation and soft callus stage at 2

months c

c

healing stage. In terms of muscle adaptation, the second month after device removal, the

muscle showed minimal muscle weight, length, cross section and thickness changes. The

histopathological changes of muscle were also stable at

m

healing of the distal was more o

study, it can be suggested that the 3 months or longer consolidation is the sufficient time to

allow the device to be in place.

5.10 Comparison of the findings of this study to the literature Relapse is the phenomena where surgically lengthened hard and soft tissues return to their

presurgical status (

re



213

1995). Other studies have reported a relatively low

te of relapse. A questionnaire study reported an overall rate of 64.8% of cases did relapse,

ith midface distraction osteogenesis in sheep, some relapse occurred early but none after 3

ction of an osteotomy in the

stretching, such as in the vertical mandibular ramus osteotomy in high angle cases (Van

Sickels et al., 1988).

It has been suggested that distraction osteogenesis allows for adaptation of the hard and soft

tissues over time and thus minimising relapse (Swennen et al., 2001). In a large study of

distraction osteogenesis, Molina & Ortez Monasterio reported no relapse at either 3 months or

up to 3 years (Molina & Ortiz Monasterio,

ra

usually occurring in the first six months (Mofid et al., 2001). Maxilliary distraction was reported

to have relapse occur in 22.2% of cases in the first six months (Cho et al., 2001). Mandibular

distraction was reported as having 50% of cases of relapse at one year post distraction

(Huisinga-Fischer et al., 2003). Ko et al. reported that relapse occurred in 30% of cases one

year after mandibular distraction with a multidirectional device (Ko et al., 2004). A greater

degree of relapse was reported between high mandibular cases as compared to low angle

cases (van Strijen et al., 2004). Thus, although the initial distraction osteogenesis cases were

reported as having no relapse, more recent studies show relapse occurs. Some indicate that

this is mainly in the first few months, although some relapse occurs at up to one year.

W

months of consolidation (Rachmiel et al., 1995). Mandibular distra

anterior mandibular diastema area showed no relapse at 12 months after distraction (McTavish

et al., 2000). It is important to note that both the midface and the anterior mandible are not

subjected to the direct effects of masticatory muscles. Current study is the first study involving

distraction of the vertical ramus with stretching of the masticatory muscles in an experimental



214

hs consolidation period and the masticatory muscles healing were stable

ow to non histopatholigal changes) which might give an indication of stability of these tissues.

e

ium and

erimysial fibrosis (Simpson et al., 1995). However, another study suggested that there were

animal. Results in this thesis showed that the distracted bone was pack with stable lamelar

structure after 3 mont

(l

Therefore, chances of relapse to occur will be reduced.

Masticatory muscles are considered to be a major factor in orthognathic surgery stability and

relapse (Ellis & Carlson, 1983; Van Sickels et al., 1986; Gassman et al., 1990; and Douma et

al., 1991). In hypoplastic mandibles the outcome of surgical correction was influenced by th

anatomical interactions of function, degree of deformity, location of the associated structures

and magnitude of movement. The stability of fixation and the distraction vector related to the

muscle forces are all important variables (Trauner & Obwegeser, 1957; Schendel et al., 1978

and Van Sickels & Richardson, 1996).

In distraction osteogenesis of the limbs the muscles gained weight during distraction but not in

the consolidation phase (Schumacher et al., 1994). They also suggested that muscle oedema

and increased proliferation of new cells contributed to this weight gain. Another suggestion was

that the limb muscles gain weight as there was increased formation of endomys

p

no muscle changes during the distraction process (Sun et al., 1994).

With distraction osteogenesis, masticatory muscle forces have an influence on the distraction

vectors; this was was shown by a 3D CT reconstruction and simulation of multisite osteotomies

for correction of a hemifacial microsomia patient. This showed that the position of the

distraction vector was influenced by the muscle forces (Demann & Haug, 2002). Gonzalez et



215

ss caused a series of changes in the

uscles during the multiple surgical procedures during the whole process.

f both the bone and the soft tissues was most in the first

months. The current study also shows no weight gain and indeed the presence of muscle

al. reported that with mandibular distraction osteogenesis in the baboon, the rotation and

movement of the osteotomy segments was related to muscle forces (Gonzalez et al., 2001).

Distraction osteogenesis has also been shown to involve both detachment of the muscles and

elongation (Liu et al., 2003). The gradual distraction proce

m

The current study showed that there was no weight gain after distraction. This was the greatest

in the proximal and middle part of the masseter muscle as compared to the thinner anterior and

posterior planes. Histopathological changes were shown to increase in activity after every

surgical event. The sensitivity of the muscle tissue towards the surgical process probably

contributes to the instability of the muscle that may cause some parts of the masticatory

muscles to be weaker. This instability o

2

atrophy after distraction. This indicates that distraction does not stimulate muscle growth or

proliferation. Thus our findings with the muscles agrees with those of Liu et al., who was using

the rat showed that distraction did not stimulate weight gain (Liu et al., 2003). We also agree

with Marquez et al., who had shown that the distraction process reduces the volume of the

surrounding soft tissues of the affected side of patients with hemifacial microsomia (Marquez et

al. 2000).

Conclusions and Future Directions Chapter 6


216

CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTIONS

To assess the development of the surgically created ‘unilateral hemifacial microsomia

like’ defect as a model for mandibular distraction osteogenesis in the sheep model.

The sheep is an appropriate animal model for a ‘unilateral hemifacial microsomia like’ defect.

Surgery performed on the 10th week old lambs showed mandibular ramus shortening and

decreases in muscle bulk. Sheep are also suitable to be used for multiple surgical procedures.

Objective 2:

d poor distractor rigidity.

The anterior part of the distracted ramus healed faster than the posterior part as the bone

matured earlier. It was also observed that the periosteum was thicker on the lateral side than

the medial side but it also depended on the degree of surgical trauma.

6.1 Conclusions General Objectives

Objective 1:

The effect of distraction of the bony vertical mandibular ramus of the mandibular in this

experiment has been previously reported (Syed Zainal, 2005)

The surgically created ‘unilateral hemifacial microsomia like’ defect in the bone has been

successfully corrected by distraction osteogenesis. The distraction osteogenesis had increased

(22%) the vertical ramus height. The process of distraction is to lengthen the mandibular ramus

resulting in angiogenesis in the early stage of distraction. The bone healed by means of

intramembranous ossification but occasionally endochondral ossification occurs in cases of

infection an



217

he distracted ramus bone matures at three months after completion of distraction in the sheep

odel. It is safe to remove the distractor after a 3 months neutral fixation (consolidation period)

distraction. The distracted mandibular ramus bone formation continues in a

period and bone remodelling continues throughout the 4 months

eriod.

The Mathys vertical distractor is appropriate for humans but may not be a suitable device to

withstand the high masticatory force of sheep. Distractor designs, for the sheep vertical

mandibular ramus, should be more robust with larger footplates and double metal bars (Syed

Zainal, 2005).

To study adaptation of the masticatory muscles (masseter and medial pterygoid) during

the vertical mandibular ramus distraction osteogenesis.

There was no increase in the muscle mass after distraction. The anterior and posterior plane

was altered by an increased in length after distraction but not the middle and oblique planes.

The ultrasound investigation showed there were changes in the cross section and thickness of

the middle level of masseter muscles and this resulted from the regeneration process.

Objective 4: o evaluate the masticatory muscles adaptation during neutral fixation.

T

m

for vertical ramus

longer consolidation

consolidation p

Objective 3

T

Generally there was no change in the masticatory muscle during this period.



218

To evaluate the masticatory muscles adaptation after device removal during the

remodelling period.

One month after device removal the muscle reduced in weight, reduction in the length of

anterior and posterior planes as well as the reduction of the cross section of the proximal and

middle level of masseter muscles. The proliferation of fibroblasts was the predominant

histopathological change after the device removal.

Objective 6

endation for the vertical mandibular distraction osteogenesis protocol.

enerally, there was gross and histological adaptation and adjustment in the masseter and

oid muscles during distraction, neutral fixation and after the removal of the device

between experimental and control sides and between different time groups. The sensitivity of

muscles toward the surgical procedures can be suggested to induce instability of the

surrounding area and was strongly predicted as the cause of relapse in bone at the 2 months

neutral fixation. Based on the current study, the recommended neutral fixation or placement of

device after distraction was 3 months, as the distracted bone had maturated, strongly

consolidated and was able to withstand the unstable condition during the first month after

device removal. This unstable condition was a temporary condition as in the second month

Objective 5.

To investigate the optimum time for both the neutral fixation and removal of device as

part of recomm

G

medial pteryg

after device removal the muscle had normalised.



219

ctives

e in the vertical mandibular height by distraction osteogenesis

ncrease in the vertical mandibular height by distraction osteogenesis alter

e

also increased following distraction but the

istal thickness was unchanged. As a result the middle and proximal of masseter muscle

creased in cross sectional area and thickness following distraction but not at the distal level.

Specific Obje

1) Does an increas

increase the mass of the masseter and medial pterygoid muscles?

The distraction osteogenesis of the vertical ramus height did not resulting in weight gain on the

experimental side to equalise the weight gain on both of the control side masseter and medial

pterygoid muscles. Hence these muscles did not grow or increase in size during distraction.

2) Does an i

the length of the anterior, middle, posterior and oblique of the masseter muscles?

The anterior and posterior planes increased their length corresponding to the increase in th

vertical mandibular ramus height by distraction, but the distraction process did not influence the

middle and oblique plane. Therefore, there is a differential effect on the masseter muscles with

changes in length at the anterior and posterior edges.

3) Does an increase in the vertical mandibular height by distraction osteogenesis alter

the cross sectional area and thickness of the proximal and middle and distal of the

masseter muscle?

Ultrasound investigation showed that increases occurred in the cross sectional area of the

middle masseter muscle but had no effect at the proximal and distal levels. The thickness of

the proximal and middle masseter muscles was

d

in



220

in the vertical mandibular height by distraction osteogenesis induce

eralised atrophy of the

lso

duced histological changes in the distal medial pterygoid muscles.

ult in

) Does neutral fixation, following distraction with different consolidation periods (2, 3

e anterior and posterior planes reduced in length during this period but

4) Does an increase

changes in the histopathology of the masseter and medial pterygoid muscles?

The increase in the vertical mandibular ramus by distraction induced gen

muscle fibres and most activity occurred predominately in the middle masseter muscle with

regeneration of this muscle. Thus it can be stated that regeneration increased the size of the

cross sectional area and thickness of the middle masseter muscle. The distraction process a

in

5) Does the process of detachment of the muscle during the surgical placement res

the repositioning of the masseter muscles?

The lifting of the muscles, especially at the distal level of masseter muscles, did result in minor,

partial repositioning of muscle outline especially where the activator was protruding.

6

and 4 months) give greater stability to the masseter and medial pterygoid muscles?

The muscles were stable and no weight changes were recorded with the device in place

(neutral fixation). Th

there was no change in middle and oblique planes during neutral fixation. Neutral fixation

caused a more stable condition in the cross sectional area and thickness in the three levels of

masseter muscles during this period. The histology of the experimental muscles was also

normal as well as the control side.



221

tral fixations. The length

as stabilised during the second month with the 3 month neutral fixation. The middle and

ion. These

uscles were stable during the second month. The cross section of the distal of masseter

l of the device resulted in histopathological changes within

e first month, which is related to the changes in the cross section and thickness.

7) Does removal of the device after distraction and neutral fixation cause changes to the

masseter and medial pterygoid muscles?

One month after removal of the device a relative reduction in weight of the masseter and

medial pterygoid muscles occurred after both 2 and 3 months of neutral fixation. There was an

increased in weight and an improvement in condition initially but the initial benefit from

distraction had lost with time. There was also a reduction in length of anterior and posterior

planes a month after removal of the device within 2 and 3 month neu

w

oblique planes were stable throughout the experiment.

The removal of the device induced reduction in the proximal and middle cross section of the

masseter muscles one month after removal of device in 2 and 3 month neutral fixat

m

muscle was consistently smaller in cross section with a longer consolidation periods.

Device removal induced an increase of the thickness of the proximal and middle level of the

masseter muscles but the distal level became thinner with longer consolidation periods. Device

removal also induced more proliferation of fibroblasts that may have caused sclerosis of the

muscle especially in the distal masseter muscle. It is suggested that this scarring may have

resulted in the thinning at the distal level of the masseter muscle, during the second month

after removal. The surgical remova

th



222

tions were similar one month after the removal in

ferent to an

bnormally short mandible which is then lengthened to the normal dimensions.

The muscle changes one month after removal of the device of a 3 month neutral fixation were a

temporary condition. The changes and adapta

a 2 months neutral fixation. The muscle revealed to return to a normal condition after two

months the removal of device within a 3 month neutral fixation. Therefore a 3 month neutral

fixation is a safe and optimal period before device removal as the bone is more stable and fully

consolidated.

8) Is ultrasound a clinically effective assessment tool to evaluate the condition of the

distracted muscles?

The ultrasound was a beneficial tool when used to assess the in vivo condition of the distracted

muscle. Ultrasound machine operators need to be fully skilled and experienced in its operation

to optimise results. It is recommended that clinicians use ultrasound to assess the progress

and stability of the muscles in distraction osteogenesis patients.

6.2 Future Directions The study of treatment methods of craniofacial deformity is difficult in that these human

conditions are rare and individually widely variable. Animal models can be useful as they allow

for standardisation. A common mistake in animal research is that treatment methods are

applied to normal animals. Thus for example a normal animal which has a mandibular

lengthening procedure which takes the mandible to an abnormal length is quite dif

a



223

al Surgery Research Group at the University of Adelaide. It has the benefit that

asonably identical hemifacial microsomic sheep can be produced. It does however require

o exist in rodents but not in larger mammals. The problem with rodents

that they are considerably smaller than humans and thus make subsequent surgical

hening process. There were

owever some problems in the study which in future could be addressed. This would

f the study:

e number of

nimals in each group should be increased by at least six and probably to ten. This larger

The animal also needs to have some similarity to humans. In this regard the sheep mandible

with its similar size, broad ramus, high mandibular plane in relationship to the

temporomandibular joint and with a multidirectional joint movement is relatively similar to

humans. Sheep are also robust animals which can withstand multiple operations and heal at a

similar rate to humans. A sheep model of hemifacial microsomia was developed by the Oral

and Maxillofaci

re

surgical manipulation, thus if it were possible a genetically abnormal animal model would be

better. Such models d

is

manipulation, particularly using implant devices, more difficult.

The current findings of this thesis on the distracted bone and muscle healing do confirm the

adaptability of the sheep bone and muscle towards the lengt

h

strengthen the results o

1. Number of animals vs control groups

There are many compounding factors that contribute to the variation and response and thus the

outcome of the experiment. In the current experiment there were differences in the response of

the sheep to anaesthesia and surgery and some unexpected drug responses. Thus the low

sample number per group has contributed to the higher probability of Type II error. To achieve

a satisfactorily statistical result and reduce the extent of the Type II errors, th

a



224

istractor on the healing process

f the muscles and would act as a control for the histological profile of the muscle without

n the skull of an anaesthetised but non-recovered animal.

is would allow measurement of landmarks between the bones and the muscle. It can also

ges of the repositioning of the muscles.

number of animals would allow a more normal distribution of mean measurements and

minimise the skewed distribution of the data. It would also decrease the high standard

deviation in the changes in the measurements of both control and experimental groups. It

would also allow a more strict policy so that where for example animals suffer sub-lethal

infections they can be discarded rather than needing to be used as in this study.

2. Control.

The number of animals used for control groups in this thesis experiment was kept to a

minimum. An important addition would be a sham pre-distraction control group. In these the

device would be placed without activation of the distracter into the fully characterised

microsomia like defect. This would show the influence of the d

o

including the effect of distraction.

Another important control group to be considered is the simulation measurement whereby

10mm distraction was performed o

Th

be used as a reference for the predicted chan

3. The phenomenon of relapse.

The current study showed that after three months of consolidation then the muscle and bones

were largely stable. In humans following orthognathic surgery it has been well documented

that subtle changes in position of the bones continue for at least a year and commonly longer.

Hence it would be useful to investigate the changes over a much longer remodelling period.



225

mechanical with a screw projecting through the skin. This is

a potential route for infection and this did occur in some animals in the study. Hence a smaller

One could reasonably predict that with the muscle scarring which is induced by the procedure

then this would slowly but progressively act on the bones.

One could further characterise the activities of the distracted muscles by use of

electromyography during the consolidation and remodelling periods.

4. Device

The distracter used in the current study was designed and customised for use in humans. The

sheep have a much greater mechanical force and chewing repetition than humans. Thus in

some instances this did create instability of the device and forces against the activation. Hence

it would be useful if a distracter specific to the sheep was developed which could take into

consideration the much higher mechanical forces of mastication in sheep. The other issue with

the current device is that they are

but stronger device which could be progressively expanded remotely would be a major

advance.

Summary If the above issues were addressed then it would make this study more scientifically robust. It

already considerable time and cost requirements of this would however greatly increase the

study. Perhaps the better alternative would be the development of a genetically mediated

hemifacial microsomia treated by a miniaturised remotely activated distraction device in

sufficient numbers on a sheep or primate model.

THE EFFECT OF VERTICAL MANDIBULAR DISTRACTION OSTEOGENESIS ON … · 2010-10-22 · investigation...

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