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The physiology of habitual bone strains

de Jong, W.C.

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Citation for published version (APA):de Jong, W. C. (2011). The physiology of habitual bone strains.

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Download date: 17 Aug 2019

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CHAPTER 5

THE ROLE OF

MASTICATORY MUSCLES

IN THE CONTINUOUS LOADING

OF THE MANDIBLE

Chapter 5

~ 86 ~

§ 5.1 Abstract

Muscles are considered to play an important role in the ongoing daily loading of bone,

especially in the masticatory apparatus. Currently, there are no measurements describing

this role over longer periods of time. We made simultaneous and wireless in-vivo recordings

of habitual strains of the rabbit mandible and masseter muscle and digastric muscle activity

up to ~25 hours. The extent to which habitually occurring bone strains were related to

muscle-activity bursts in time and in amplitude is described.

The data reveal the masseter muscle to load the mandible almost continuously

throughout the day, either within cyclic activity bouts or with thousands of isolated muscle

bursts. Mandibular strain events rarely took place without simultaneous masseter activity,

whereas the digastric muscle only played a small role in loading the mandible. The average

intensity of the masseter-muscle activity bouts was strongly linked to the average amplitude

of the concomitant bone-strain events. However, within cyclic loading bouts, individual

pairs of muscle bursts and strain events showed no relation in amplitude. Larger bone-strain

events, presumably related to larger muscle-activity levels, had more constant principal-

strain directions. Finally, muscle-to-bone force transmissions were detected to take place at

frequencies up to 15 Hz.

We conclude that in the ongoing habitual loading of the rabbit mandible, the

masseter muscle plays an almost non-stop role. In addition, our results support the

possibility that muscle activity is a source of low-amplitude, high-frequency bone loading.

Masticatory Muscles and Bone Strain

~ 87 ~

§ 5.2 Introduction

A relationship exists between skeletal muscle activity and the morphology and composition

of bone (Shaw and Stock, 2009). Contractions of skeletal muscles are believed to have the

most prominent and dynamic role in the habitual loading environment of bone tissue (Burr,

1997; Schoenau and Fricke, 2006). Profound changes in skeletal-muscle recruitment illustrate

this muscle-bone relationship. For example, not only does the playing arm of tennis players

have hypertrophied muscles, its humerus also has a higher bone-mineral density (Kannus et

al., 1994). In addition, the increase in playing-arm muscle size is correlated with the increase

in several bone-strength-indicating parameters (Daly et al., 2004). Conversely, muscle

paralysis has been shown to lead to cortical and trabecular bone mass decrease (Warner et

al., 2006; Poliachik et al., 2010). The muscle-bone relationship might also exist on a more

delicate level; the radius of the arm dominant in habitual everyday activities has a greater

mass and volume than that of the non-dominant arm (MacIntyre et al., 1999). It is unclear,

however, to what extent in time and in amplitude the daily activity of skeletal muscles is

related to the daily loading of bone.

A suitable musculoskeletal system to study the muscle-bone relationship is the

masticatory apparatus. As the mandible is not weight bearing and the gravitational forces

working on it are small, the origin of the daily loads mandibular bone experiences can be

assumed to lie mainly in muscle contractions and the resulting reaction forces. Several

muscle groups insert on the mandible, but the masticatory muscles—due to their strength

and functional relation to the jaw bones—may be considered the most important loaders.

Paralysis of the masseter muscle, for example, results in both its own atrophy and in the

subsequent atrophy, or growth retardation, of the mandible it inserts on (Matic et al., 2007;

Kim et al., 2008).

The role of masticatory muscles in the mechanical loading of the mandible has been

studied predominantly within the context of chewing behaviour using electromyography

together with bone-load sensors (Weijs and De Jongh, 1977; Hylander et al., 1987; Teng and

Herring, 1998). Extensive analyses have been performed on the relation of masticatory-

muscle biopotentials with mandibular strain amplitudes within chewing cycles (Hylander

and Johnson, 1989, 1993; Liu et al., 2004). Outside of these bouts of chewing behaviour the

role of masticatory muscles in the everyday habitual loading of the mandible has been

neglected mostly. Here, we hypothesise that the role of masticatory muscles in the habitual

Chapter 5

~ 88 ~

loading of the mandible might be near-continuous; expanding beyond the boundaries of

mastication. Previous long-term measurements of habitual bone strain in the mandible

revealed the existence of numerous isolated bone-strain events, occurring between bouts of

more cyclic bone loading, which also contribute to the daily bone strain history (De Jong et

al., 2010a, 2010c).

Here we describe the habitual relationship between masticatory muscle activity

and mandibular bone strain, without confining to chewing behaviour. The hypothesis that

daily bone strains are associated mainly with the activity of muscles—in terms of both time

and amplitude—is tested. To this end, co-appearance of muscle activity and bone strain is

analysed from long-term electromyograms of masseter and digastric muscle activity and

simultaneously recorded mandibular bone strain. Also, amplitude distributions of habitual

masseter and digastric activity bursts and bone-strain events are compared. The masseter

muscles are the rabbit’s main jaw closers, whereas the digastric muscles function as jaw

openers (Schumacher and Rehmer, 1960; Weijs and Dantuma, 1981). We, therefore, expect a

relation in time and in amplitude for masseter activity and mandibular strains. Habitual

masseter-burst amplitudes and counts are compared to the co-appearing compressive

principal-strain amplitudes and counts. Digastric activity is expected to elicit measurable

bone strains, but without a relation in amplitude, as jaw opening will not result in reaction

forces as large as during jaw closing.

§ 5.3 Materials and methods

Laboratory animals

Ten adult (~4 months old) male New Zealand white rabbits (Oryctolagus cuniculus) weighing

3.7 ± 0.2 kilograms were used for the experiments. Rabbits were used as they are large

enough to house both a bone-strain and an electromyography transmitter. The animals were

kept in 73 x 73 x 46 cm cages, received food and water ad libitum, daily portions of hay, and a

wooden block that served as an extra gnawing object. Lights were dimmed between 18.00 h

and 6.00 h. The rabbits were allowed at least two weeks of acclimatisation before

implantation of the transmitters. The experiments were approved by the Animal Ethics


~ 89 ~

Committee of the Academic Medical Centre of the University of Amsterdam and executed

in accordance with Dutch legislation.

Wireless electromyography (EMG) and bone-surface strain measurement

Masticatory-muscle biopotentials and bone-surface strain were recorded wirelessly using

implantable transmitters (Langenbach et al., 2002; Van Wessel et al., 2006; De Jong et al.,

2010b). An overview of their main features follows below.

Two-channel TL11M2-F20-EET implants from Data Sciences International (DSI, St.

Paul, Minnesota, USA) recorded masseter and digastric muscle biopotentials with two

indwelling electrodes per channel. Each electrode consisted of a silicone-tubed stainless steel

double-helix wire with a diameter of 0.2 mm. The inter-electrode distance once placed in the

sampled muscle was approximately 6 mm and the effective electrode length was 4 mm. The

bipolar recordings were transmitted on a carrier frequency of 455 kHz to nearby DSI

receivers (RMC-1). A DSI Data Exchange Matrix collected the data from the receivers and

stored them onto a computer. The sample frequency was 250 Hz per channel. In five animals

only masseter activity was recorded, in two others only digastric activity, and in three

animals both masseter and digastric activity were recorded. All electrodes were placed

unilaterally, at the left side. In the masseter muscle, electrodes were placed at the antero-

ventral side of the superficial masseter, near the motor end plates (Widmer et al., 1997). In

the digastric muscle, electrodes were placed in the middle of its muscle belly (Figure 5.1).

Variability in the electrode location was kept at a minimum.

A customised MicroStrain V-Link (Williston, Vermont, USA) connected to a

stacked triple-gauge rosette (L2A-06-031WW-350, Vishay, Malvern, Pennsylvania, USA) was

used for the wireless bone-strain measurements. Strain measurements were transmitted on a

carrier frequency of 2.4 GHz to a nearby MicroStrain USB Base Station, which stored the

data on a desktop computer. Bone strain was sampled at a frequency of 617 Hz and

measured in all ten rabbits. The gauge rosette was positioned on the left lateral surface of

the mandibular corpus, anteriorly of the masseter insertion and inferiorly of the molars. The

orientations of the three gauges were rostroventral (A), vertical (B), and caudoventral (C),

with an angle of 45° between gauges A and B and between gauges B and C (Figure 5.1).

Chapter 5

~ 90 ~

Figure 5.1 Schematic drawings of the rabbit skull with some skeletal muscles (top and middle

drawings) and a detail depicting the orientations of the strain gauges (A, B, and C) in the gauge rosette

(bottom drawing). The circles in the top and middle drawings indicate the locations of the pairs of wire

electrodes in the masseter and digastric muscles. The rectangle in the lateral view depicts the location of

the strain-gauge rosette on the mandibular corpus, which in vivo is partly covered by the bulging

masseter muscle.

superficial temporal

posterior deep masseter

posterior superficial masseter

superficial masseter

superficial masseter

medial pterygoid

mylohyoid

stylohyoid

digastric

lateral view

ventral view

stacked triple-gauge rosette detail

A

B

C45° 45°


~ 91 ~

Surgical procedure and medications

The day before and the day after the aseptic surgical placement of the two implants, rabbits

received 5.0 mg/kg enrofloxacin (Baytril, Bayer, Mijdrecht, the Netherlands) to suppress

infections. Before surgery, rabbits were administered a subcutaneous dose of 0.03 mg/kg

buprenorphine (Temgesic, Schering-Plough, Utrecht, the Netherlands), an analgesic.

General anaesthesia was started with a subcutaneous dose of 15 mg/kg ketamine (Nimatek,

EuroVet Animal Health, Bladel, the Netherlands) combined with 0.40 mg/kg

dexmedetomidine (Dexdomitor, Orion Pharma, Espoo, Finland). The rabbit’s eyes were

covered with Oculentum Simplex (Pharmachemie B.V., Haarlem, the Netherlands) to

prevent them from dehydrating. Anaesthesia was maintained through intratracheal dosage

of 0.8-1.2 % isoflurane in a 1:1 mixture of oxygen and air. Spirometry and oxymetry

monitored breathing frequency and oxygen saturation of the blood, respectively. Body

temperature was kept at 37 °C using heating pads.

An incision was made in the rabbit’s neck fold through which subcutaneous

pockets were made to house the transmitters. The EMG electrodes and the wired strain-

gauge rosette were led subcutaneously to a second incision in the mandibular region. Here,

the electrodes were placed in either the masseter or the digastric or both muscles, using a

longitudinally-ground hypodermic needle as a slide (Nuijens et al., 1997). Electrodes were

anchored at the muscle surface with a single suture. To attach the strain-gauge rosette, a

mandibular bone-surface area of about 1 cm2 was cleared of the surrounding soft tissues and

periosteum. The bone surface was cleaned with sterile swabs, degreased with an 80 %

ethanol solution, and dried to the air. The gauge rosette was glued to the bone with

Histoacryl (B. Braun, Tuttlingen, Germany) and pressed firmly onto it for several minutes

(Cochran, 1972).

Following surgery, the animal was given a subcutaneous dose of 2 mg/kg

carprofen (Rimadyl, Pfizer Animal Health B.V., Capelle aan den IJssel, the Netherlands) to

suppress inflammation and pain. After collection of the measurements, rabbits were

sacrificed with an overdose of pentobarbital sodium (Euthesate, CEVA Santé Animale,

Maassluis, the Netherlands).

Chapter 5

~ 92 ~

Data analysis

Bone-strain recordings and electromyograms were analysed using the software program

Spike2 (version 5.21, Cambridge Electronic Design Limited, Cambridge, UK). Masseter and

digastric electromyograms were rectified and smoothed by calculating the moving root-

mean-square value over ∆t = 0.040 s. In each recording, the highest biopotential peak served

as the 100 % activity level of the sampled muscle as rabbits cannot be ordered to give a

maximum muscle contraction. All other peaks were expressed as a percentage of that

biopotential. Distribution histograms were made of the EMG-peak amplitudes, or burst

amplitudes, that crossed the 5 % activity level of the recording. Histograms of muscle-burst

amplitudes had a bin width of 2.5 %.

Bone-strain recordings were rid of drift by subtracting the average of all data

points from t - 3600 s to t + 3600 s from each data point at time t. The three drift-free strain-

gauge recordings were used to calculate the compressive and tensile principal-strain signals.

In the sections of the Results describing associations in time between masseter and digastric

muscle bursts and mandibular strain, the tensile principal strain is used in the figures for

clarity purposes (deviations from zero pointing upwards). Distribution histograms were

made of the principal-strain amplitudes of all strain events of which the base of the peak lay

below the threshold of 20 microstrain (µε) and the peak maximum crossed the threshold of

30 µε, safely above noise. These histograms had a bin width of 12.5 µε.

For further analysis of the relation between masseter muscle activity and

mandibular deformations, the compressive principal strain was used as compression at the

site of measurement was expected to have a more clear relation to contractions of the

masseter than tension. For six amplitude levels of compressive principal strain (10 ± 3 µε, 35

± 3 µε, 75 ± 10 µε, 150 ± 10 µε, 300 ± 10 µε, and 500 ± 10 µε) accompanying masseter-burst

amplitudes were collected. Amplitudes of compressive principal strain greater than 500 µε

were very rare and could not be included in the analysis. For each of the strain levels 11

masseter bursts were collected by searching a strain event with the specific amplitude (e.g., -

10 ± 3 µε), writing down the accompanying muscle-burst amplitude, and doing so at 11 time

points divided evenly over the length of the entire measurement per rabbit.

To evaluate whether the masseter muscle plays different roles in more intense

(including feeding-behaviour-related) versus less intense loading of the mandible, the

average number of masseter-muscle bursts per hour was compared to the average number


~ 93 ~

of bone-strain events per hour. This comparison was made for two amplitude domains

which represented the more intense and less intense parts of loading. Chewing is a more

intense cyclic masticatory behaviour eliciting compressive principal bone strains with

amplitudes of 149 - 320 µε on the working side of the mandible (Weijs and De Jongh, 1977).

Using the masseter activity levels found with compressive principal strains of 150 ± 10 µε, a

division was made in the total count of masseter bursts above and below the mV level

associated with this compressive principal-strain magnitude for each rabbit. The number of

masseter bursts per strain event was assumed to be an indication of the role of the masseter

in these two ranges of mandibular loading.

To quantify variation in the direction of the principal strains, the angle φ between

the direction of the first principal strain and the orientation of gauge A of the strain-gauge

rosette was calculated for 70 bone-strain events per rabbit. These strain events were

collected from seven sites evenly divided over each mandibular bone-strain recording—

including 10 successive strain events at each site. In this strain-event collection some care

was taken to include larger strain events (i.e., compressive principal strain > 150 µε), which

in practice meant ‘scrolling’ through the strain recordings away from a site to nearby large

strain peaks. The direction of the first principal strain is oriented at an angle of 90° from the

direction of the second principal strain. However, for eight rabbits, we simply used the

angle ϕ (without adding or subtracting 90°) to plot against the second principal-strain

amplitude in scatter distributions. The following formulas were used for the calculation of

angle ϕ and the second principal strain:

� � 12 tan� �2 � � � � � � � � �

� � 12 � � � �� 12 �� 2 � � � � ��

in which εA, εB, and εC were the drift-removed strain values measured by the individual

gauges A, B, and C from the gauge rosette (Figure 5.1) and ε2 is the second principal strain,

i.e, the compressive principal strain.

The relation between digastric muscle activity and bone strain was too weak and

therefore no such analyses were done for this muscle.

Chapter 5

~ 94 ~

Statistics

The EMG amplitudes co-appearing with the various levels of compressive strain were tested

for differences using two-tailed paired t-tests. Differences in mean EMG amplitude per

compressive-strain level were considered significant when P ≤ 0.05.

§ 5.4 Results

The rabbits recovered quickly from the placement of the two transmitters and started

feeding and exploring as soon as three hours after surgery. Simultaneous telemetric

recordings of masticatory muscle activity and mandibular bone strain were started on the

day of surgery. The average simultaneous recording length of muscle activity and bone

strain was 16.6 ± 6.6 h (n = 10).

Throughout all recordings, longer and shorter bouts of activity of the masseter

muscle coincided clearly with bouts of mandibular bone strain. The time between these

bouts was filled with multitudes of isolated muscle bursts and co-appearing strain events

(Figure 5.2A). The co-appearance was especially pronounced for periods of higher muscle-

activity levels and larger bone-strain amplitudes (> 150 µε), which included chewing

(personal observation, W.C. de Jong). On a smaller time scale the co-appearance of muscle-

activity bouts and strain-event bouts, as well as of the isolated bursts and events, is

illustrated even more clearly (Figure 5.2B). Within bouts there was no correspondence

between the individual muscle-burst amplitudes—in raw or root-mean-squared form—and

the bone strain-event amplitudes (Figure 5.2C). Occasionally, the level of an entire masseter-

activity bout did not correspond at all with the amplitude of the accompanying strain-event

series (Figure 5.3). Between the bouts of more intense repetitive and rhythmic mandibular

loading thousands of isolated strain events occurred, which were almost always

accompanied by masseter muscle bursts (Figure 5.4).

The relation in time between activity of the digastric muscle and mandibular bone

strain was not as pronounced as between the masseter muscle and mandibular bone strain.

There were no clearly delineated bouts of digastric activity and bone strain (Figure 5.5A and

B). A one-on-one co-appearance of digastric bursts and strain events was present only


~ 95 ~

Figure 5.2 Representative stretches of unprocessed masseter electromyogram (EMG, upper graphs in

each time window) and tensile principal bone strain (lower graphs). mV = millivolt, µε = microstrain.

Panels B and C have identical y-axes. Note the co-appearance throughout the recordings of masseter-

activity bouts and strain-event episodes (A and B). This co-appearance persists to the level of individual

muscle bursts and strain events (C). Negative values in the tensile principal strain graphs are an artefact

of the drift-removal procedure which was applied before calculation of the principal strains.

occasionally (Figure 5.5B). A relationship in the amplitudes of co-appearing digastric muscle

bursts and bone-strain events was absent (Figure 5.5C).

In some instances, neither masseter nor digastric activity seemed to have a clear

relation to the simultaneously recorded mandibular strain events (Figure 5.6).

Passages of co-appearing rhythmic masseter muscle bursts and strain events were

manifold for the frequency of 5 Hz (Figure 5.2C), well known as the frequency at which food

is chewed and possibly a frequency used for all sorts of oral behaviours in the rabbit.

Incidentally, masseter muscle bursts were found to coincide with the strain events they

elicited up to frequencies of 15 Hz (Figure 5.7). This illustrates that high-frequency muscular

contractions may cause bone deformations with the same frequency.

6 hrs

8 min 7 s

250 µε

1.5 mV

1.5 mV

A

B C

0

0

0

250 µε

0

creo

Chapter 5

~ 96 ~

Figure 5.3 Two 25-minute examples that include discordances in amplitude between co-appearing

episodes of masseter activity and bone-strain events. The examples in A and B were taken from two

different rabbits. In both A and B the upper graph is the electromyogram (processed with a root-mean-

square function over ∆t = 0.040 s) and the lower graph is the tensile principal bone strain. Horizontal

braces indicate where strain amplitudes are lower compared to those co-appearing with muscle-activity

bouts of the same burst activity level. Negative values in the tensile principal strain graphs are an

artefact of the drift-removal procedure which was applied before calculation of the principal strains.

Note that panels A and B have different y-axes.

Figure 5.4 Representative 5-min stretches of root-mean-squared (∆t = 0.040 s) masseter

electromyograms (upper graphs in A and B) and the simultaneously recorded mandibular tensile

principal strain. Panels A and B were taken from different rabbits. Mandibular bone-strain events,

although outside of cyclic loading bouts, are primarily the result of masseter activity.

A

B

25 min

200 µε

0.4 mV

400 µε

0.3 mV

0

0

0

0

5 min

0.15 mV

0.20 mV

75 µε

75 µε

0

0

0

0

A

B

creo

Fig


Panels B and C have identical y

activity and mandibular bone strain can be detected. On smaller time scales (B and C), co

digastric bursts and strain events can also be seen, but there is no relation in amplitude.

Figure

recorded tensile and compressive principal strains. The EMGs are root

Here, neither masseter nor digastric activity corresponds clearly with the bon

although some co

minutes.

Figure





Figure



although some co

minutes.

5.5





5.6



although some co

minutes.

Representative stretches of unprocessed digastric electromyograms (EMG, upper graphs in





Two 17



although some co






Two 17-



although some co-






-min stretches of masseter and digastric electromyograms with the simultaneously



-appearance of muscle bursts and bone






in stretches of masseter and digastric electromyograms with the simultaneously



appearance of muscle bursts and bone












Panels B and C have identical y-axes. On a larger time scale (A)









axes. On a larger time scale (A)

























~










~ 97










97 ~











~









appearance of muscle bursts and bone-strain events can be seen in the first six










strain events can be seen in the first six




axes. On a larger time scale (A) some relation in time between digastric










some relation in time between digastric




recorded tensile and compressive principal strains. The EMGs are root-mean










mean










mean-squared (∆t = 0.040 s).

Here, neither masseter nor digastric activity corresponds clearly with the bone-









squared (∆t = 0.040 s).

-strain recording,









squared (∆t = 0.040 s).

strain recording,






activity and mandibular bone strain can be detected. On smaller time scales (B and C), co-appearance of



squared (∆t = 0.040 s).

strain recording,






appearance of


squared (∆t = 0.040 s).

strain recording,






appearance of


squared (∆t = 0.040 s).

strain recording,






appearance of


squared (∆t = 0.040 s).




appearance of

A

B

150

0.7

A

B

20

0.6

150 µε

0.7 mV

20 µε

0.6 mV

0

0

0

0

0

0

0

0

9 min9 min

CC

10 s

3 hrs

10 s

3 hrs

creo

Chapter 5

~ 98 ~

Figure 5.7 Two 5-s example recordings of high-frequency force transmission. Note that the amplitude scales differ between panels A and B. The examples were taken from two different rabbits. In both A and B the upper graphs are raw masseter electromyograms, used here as the root-mean-square function mostly removes high-frequency content, and the lower graphs are the tensile principal strain. In A the masseter muscle strains the mandible at a frequency of 8 Hz and in B the masseter muscle strains the mandible at a frequency of 15 Hz. mV = millivolt, µε = microstrain.

In most rabbits, significantly increasing masseter-burst amplitudes were found with larger

compressive principal-strain amplitudes (Table 5.1). However, masseter bursts eliciting

compressive principal strains of 500 µε were not larger than those co-appearing with

compressive principal strains of 300 µε (Table 5.1). Above and below the masseter muscle

activity level associated with the principal-strain amplitude of -150 µε, the burst-number-to-

strain-event ratio was the same (Table 5.2). On average, per hour, three times more masseter

bursts were detected above the 5 % activity level than strain events above the 30 µε level.

The interindividual variation was large, however, and in one rabbit 10 times more masseter

bursts than strain events were registered, both above and below the -150 µε level.

5 s

masseter EMG

masseter EMG

bone strain

bone strain

f = 8 Hz

f = 15 Hz

70 µε

0.3 mVA

B0

0

60 µε

0.2 mV

0

0


~ 99 ~

Figure 5.8 Average amplitude distributions of habitual masseter muscle bursts (n = 8), digastric muscle

bursts (n = 5), and bone-strain events (n = 10). µε = microstrain. Note that the y-axes have log scales. The

thin bars indicate the standard deviations. Per hour, there are more masseter bursts above the 20 %

activity level than digastric bursts. Negative strain amplitudes refer to compressive strain.

Table 5.1 Masseter burst amplitudes, from EMGs root-mean-squared over ∆t = 0.040 s, co-appearing

with amplitudes of compressive principal bone strain. Masseter activity is expressed as a percentage of

the peak burst voltage. Shown are means ± standard deviations (SD) of 11 muscle bursts per bone-strain

amplitude per rabbit. A ‘-‘ indicates that not enough strain events were found for that specific

amplitude level. µε = microstrain.

individual compressive principal-strain amplitude level [µε]

10 35 75 150 300 500

mas

seter muscle activity ±

SD

[% of pea

k voltag

e]

1 5.5 ± 2.7 6.1 ± 1.1 16.9 ± 7.5* 29.3 ± 6.8* 41.0 ± 8.4* 44.3 ± 10.1

2 3.8 ± 2.0 11.1 ± 5.8* 19.4 ± 6.9* 14.9 ± 3.8 33.4 ± 5.5* 27.9 ± 6.2

3 2.0 ± 1.6 6.5 ± 2.5* 5.4 ± 3.3 - - -

4 3.2 ± 1.5 9.4 ± 5.6* 19.8 ± 11.0* 40.9 ± 12.1* 48.9 ± 14.0 -

5 4.3 ± 1.3 14.2 ± 3.1* 13.3 ± 4.6 18.2 ± 6.6* 27.2 ± 7.5* 32.3 ± 5.1

6 7.1 ± 4.6 32.8 ± 10.1* 54.5 ± 21.8* 50.6 ± 9.6 - -

7 5.3 ± 3.7 12.8 ± 5.4* 37.5 ± 14.8* 75.0 ± 12.3* - -

8 5.3 ± 2.8 9.0 ± 3.5* 15.6 ± 3.7* 28.0 ± 6.5* - -

mean ± SD 4.5 ± 1.6 12.7 ± 8.6 22.8 ± 15.7 36.7 ± 20.9 37.6 ± 9.4 34.8 ± 8.5

* Significantly larger than the EMG amplitudes from that rabbit accompanying the first lower bone

compression level; P < 0.05.

1

10

100

1000

10000

8 20 33 45 58 70 83 95

bursts/ hour

% of peak voltage

1

10

100

1000

10000

8 20 33 45 58 70 83 95

bursts/ hour

% of peak voltage

1

10

100

1000

-500 -375 -250 -125 0 125 250 375 500

events/ hour

principal strain amplitude [µε]

masseter activity

n = 8

digastric activity

n = 5

bone-strain events

n = 10

Chapter 5

~ 100 ~

Distribution histograms of the amplitudes of masseter and digastric activity bursts and of

bone-strain events displayed an exponential decrease in occurrence of larger amplitudes,

but there was no clear resemblance between the muscle-burst and bone-strain amplitude

distributions (Figure 5.8). Compared to the digastric muscle, masseter muscle activity

contained more bursts for amplitudes above 20 % of the peak voltage. Using the

compressive principal bone strain, more strain events could be detected above the 30 µε

level, as evidenced by the larger area occupied by the event counts on the negative, i.e.,

compression amplitude half of the histogram compared to the positive, i.e., tension

amplitude half.

Greater amplitudes of compression were related to more constant values for the

principal-strain orientation (Figure 5.9). In most rabbits there was a tendency for angle ϕ to have values between 20 - 30° for second principal-strain amplitudes below -150 µε.

Therefore, the direction of the second principal strain was more horizontal than the

orientation of gauge C when compression amplitudes exceeded the 150 µε level.

Table 5.2 Counts per hour of compressive strain events below and above the level of 150 µε, and

masseter-muscle bursts below and above the mV level causing a compressive principal strain of 150 µε.

Only strain events with amplitudes greater than 30 µε and muscle bursts above the 5 % activity level are

included in the counts (see Material and methods section). In individual 3, no distinction could be made

in the masseter bursts as there were not enough large strain events to measure the masseter burst level

causing a compressive principal strain of 150 µε (see Table 5.1). µε = microstrain, mV = millivolt.

individual events/ h

≤ 150 µε

bursts/ h

≤ mV150 µε

bursts/

events

events/ h

> 150 µε

bursts/ h

> mV150 µε

bursts/

events

1 1462 1440 1.0 213 277 1.3

2 981 496 0.5 130 257 2.0

3 159 - - 4 - -

4 512 757 1.5 103 207 2.0

5 586 1117 1.9 254 541 2.1

6 322 3094 9.6 8 81 10.1

7 769 4771 6.2 6 8 1.3

8 665 656 1.0 22 41 1.9

mean ± SD 3.1 ± 3.5 3.0 ± 3.2


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Figure 5.9 Scatter distributions of the angle ϕ plotted against the second principal-strain amplitude.

Each dot indicates one bone-strain event. Positive angles are oriented counter clockwise from the

rostroventrally orientated gauge A, negative angles clockwise. The numbers in the lower left corners of

each graph indicate the rabbit individuals. Note the tendency of ϕ to have more constant values with

increasing amplitude of bone compression.

-40

-20

0

20

40

-400 -300 -200 -100 0

-40

-20

0

20

40

-400 -300 -200 -100 0

-40

-20

0

20

40

-400 -300 -200 -100 0

-40

-20

0

20

40

-400 -300 -200 -100 0

-40

-20

0

20

40

-400 -300 -200 -100 0

-40

-20

0

20

40

-400 -300 -200 -100 0

-40

-20

0

20

40

-400 -300 -200 -100 0

-40

-20

0

20

40

-400 -300 -200 -100 0

second principal strain ε2 [µε]

angle Φ

[ °]

# 8# 7

# 1 # 2

# 3 # 4

# 5 # 6

Chapter 5

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§ 5.5 Discussion

Our measurements reveal masseter muscle contractions to be a primary contributor to the

ongoing loading history of the rabbit mandible. The role of the masseter muscle was not

confined to bouts of more intense cyclic loading, which include chewing, but also comprised

thousands of isolated loading events with amplitudes mostly smaller than those of the bouts

of cyclic loading. Masseter electromyograms greatly resembled mandibular bone-strain

recordings in terms of co-appearing muscle bursts and strain events throughout entire

recordings. However, the average number of masseter bursts per hour was greater than the

average number of bone strain events per hour (Table 5.2). One explanation is the exclusion

from our analysis of strain events with principal amplitudes smaller than 30 µε, although

this does not explain the larger number of more intense masseter bursts compared to the

number of strain events with principal amplitudes greater than 150 µε. It is possible that not

all registered masseter bursts resulted in a measurable mandibular bone strain.

Occasions where bone-strain events were not accompanied by a masseter activity

burst were rare. Both larger and smaller bone-strain events featured co-appearing masseter

bursts, almost without exception, suggesting equal roles of the masseter muscle in both

high-intensity and low-intensity loading. Greater amplitudes of mandibular bone strain

were related to both greater amplitudes of masseter activity and to a more constant

orientation of the principal-strain directions. During large-amplitude loading events the

direction of the second principal strain in the surface of the mandibular corpus was aligned

more horizontally. This could mean that the working line of the masseter muscle becomes

more constant when the muscle exerts larger forces. This, however, is disputable as no other

jaw-closing muscles were sampled and their contribution to larger strain events is unknown.

Other masticatory muscles than the masseter and the digastric will load the

mandible as well, but no electromyograms were made of these muscles. Although the loads

they exert on the mandible may cause strain events not accompanied by a registered

masseter burst, the majority of their contractions are likely paralleled by the activity of the

sampled masseter muscle. Especially during chewing, masticatory muscles primarily work

in triplets: the masseter and medial pterygoid muscles of one side and the temporal muscle

of the contralateral side (Langenbach and Van Eijden, 2001). Although chewing is the main

function of the masticatory apparatus, the daily loading history of the mandible includes

much more than the short bouts of this behaviour. In all these oral behaviours the masseter


~ 103 ~

muscle seems to play a main role as indicated by the close fit between its activity and the

mandibular bone strain throughout the day.

An amplitude relation between masseter activity and mandibular strain was

present only on a coarse scale, evidenced by the visually matching amplitudes of complete

bouts of muscle activity and bone strain and the increasing EMG amplitudes found for

increasing compressive bone-strain levels. On a fine amplitude scale, amplitudes of

individual masseter bursts were unrelated to those of their strain-event counterparts. This

can be explained firstly by bone deformation being the result of several loads, of which

masseter activity is only one. The activity of the other masticatory muscles, the facial

muscles, the suprahyoid muscles, as well as additional reaction forces from incisors, molars,

and temporomandibular joints will load the mandible also. Secondly, concerning chewing

bouts, the level of activation of each of the masticatory muscles and their exact activation

pattern in time will vary from cycle to cycle, as the position and mechanical properties of the

masticated food will change continuously (Morimoto et al., 1985). In Figure 5.3, the

discordance of masseter EMG amplitudes and bone-strain amplitudes of complete activity

bouts might simply be the result of a switch of working side and balancing side during

mastication. Thirdly, we sampled only the superficial part of the masseter muscle. As the

rabbit masseter is extremely compartmentalised by aponeuroses (Schumacher and Rehmer,

1960; Weijs and Dantuma, 1981; Widmer et al., 1997), the magnitudes and directions of its

contractions cannot always be captured by one or two EMG channels. Obviously, reaction

forces on the mandible from outside the rabbit—which would occur when the animal were

to, e.g., scratch its head—might also have caused bone-strain events. Video registrations of

the laboratory animal might have explained the behaviour behind some of those events.

However, earlier attempts to film rabbit behaviours made clear that many activities of the

masticatory muscles were simply invisible from the outside of the animal.

Amplitudes of digastric muscle activity resembled very weakly or not at all those

of mandibular bone strain. Digastric muscles are jaw openers and jaw opening generally

does not need large muscle forces (Weijs and Muhl, 1987), nor does it bring about large

reaction forces. The reaction forces that are present during opening arise from passive

tensions in the soft tissues that cover the mandible and from the loads in the jaw joints.

These loads are small and far away from the site of strain measurement and will therefore

only elicit small bone strains near and underneath the gauge rosette. In contrast, the

masseter is active mainly when the mandible cannot close any further, which results in

Chapter 5

~ 104 ~

larger bone-strain amplitudes. In addition, digastric muscles have small cross-sectional

areas and lower weights compared to the masseter and pterygoid muscles (Weijs and

Dantuma, 1981; Langenbach and Weijs, 1990) and, consequently, can exert only smaller

maximal forces on the mandible. Situations in which the digastric muscles are more active

than the masseter muscles, such as during grooming and limb licking (Yamada et al., 1993),

could have given rise to instances of better resemblance between digastric activity and

mandibular bone strain.

The data presented in this paper illustrate that the mechanical link between jaw-

muscle activity and loading of the mandibular bone is strong. This strong mechanical link

might explain the known functional relation between jaw-muscle activity and mandibular

bone growth, modelling, and maintenance. During growth in utero the presence of active

masticatory muscles is known to be essential to a proper shaping of the mandible (Rot-

Nikcevic et al., 2006). Impaired force output of masticatory muscles during post-natal

growth results in a retarded mandibular bone growth as well (Kwon et al., 2007; Matic et al.,

2007). In adulthood, the functionality of the mechanical relation between masticatory muscle

activity and mandibular bone morphology remains. Botulinum-toxin treatment of the

masseter muscle in adult rats, e.g., induces architectural changes in the mandible (Tsai et al.,

2010). As injection of botulinum toxin type A into the masseter muscle is a procedure

performed more and more, either for aesthetic (Kim et al., 2010; Wu, 2010) or medical

purposes (Daelen et al., 1997; Tan and Jankovic, 2000), this might be of some consideration

to the clinic.

Low-magnitude, high-frequency mechanical loads have been associated with

anabolic effects on bone (Rubin et al., 2001; Midura et al., 2005; Goodship et al., 2009). Our

data unveil that masticatory muscle bursts at 5 % of peak activity are associated with small

strains in the mandibular bone (Table 5.1). We found that masseter and digastric bursts at

that activity level may take place at least 1000 times per hour (Figure 5.8 and Table 5.2),

which is in accordance with earlier publications (Van Wessel et al., 2005). Also, the

frequency component of muscle activity is still very strong above 5 Hz and even 15 Hz force

transmissions to bone were detected (Figure 5.7). Therefore, the present study supports the

possibility that part of all habitually occurring high-frequency bone strains might have their

origin in muscle activity and that these strains quite possibly stimulate homeostatic bone

turnover (Rubin et al., 1990; Turner et al., 1995; Fritton et al., 2000; Judex and Rubin, 2010).


~ 105 ~

This study demonstrates that wireless and simultaneous measurement of muscle activity

and bone-surface strain in vivo is feasible for middle-sized and larger animals up to about

one day. However, our methodology has limitations. Firstly, muscle activity and bone strain

were recorded with two separate systems. Due to minute periods of signal dropout and

variation in hardware clock rates the two signals could not always be synchronised.

Secondly, the synchronisation of the strain recordings and electromyograms was performed

visually. Although a matching between a series of muscle-activity bursts and bone-strain

events can be attained fairly easily, the exact relation in time of an individual pair of one

muscle burst and the subsequent bone-strain event could not be studied objectively.

Excitation-contraction lag times are known to be short for jaw-closing muscles, though,

commonly featuring values from ~13 ms to peak twitch tension in the cat masseter (Taylor

et al., 1973), to ~30 ms in the macaque masseter (Hylander and Johnson, 1993), to ~43 ms in

the pig temporal muscle (Teng and Herring, 1998). An implantable transmitter with sensors

able to capture both muscle biopotentials and bone deformation would be more effective in

quantifying the role of muscle activity in the daily habitual loading of bone.

To conclude, our results reveal that in the masticatory apparatus of the rabbit, jaw muscles

play the main role in the ongoing habitual loading of the mandible. Not only is the mandible

loaded by jaw muscles during repetitive behaviours, like chewing, but also almost

continuously throughout the day outside of these cyclic bouts. Most bone-strain events are

accompanied by masseter-muscle activity. Activity of the digastric muscle corresponds only

weakly with the occurrence of mandibular strain events. A relation in masseter

electromyogram amplitudes and bone-strain amplitudes exists on a coarse scale.

Furthermore, muscle forces can dynamically strain the mandible up to frequencies of ~15

Hz. The orientation of the principal strains becomes more constant with increasing strain

amplitude. This might indicate that the muscles loading the mandible during such high-

intensity events have more constant working-line directions during larger force output.

Chapter 5

~ 106 ~

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