A NEW INSTRUMENTED BIOLOGICAL DEVICE DESIGNED TO

APPLY MECHANICAL SHOCKS TO BONE CELLS

Laurent Navarro

, Jean-Charles Pinoli

, Henri Besset

, Ren´e Guyonnet

Ecole Nationale Sup´erieure des Mines de Saint-Etienne

Centre Ing´enierie et Sant´e (CIS) and LPMG-UMR CNRS 5148

Sciences des Processus Industriels et Naturels (SPIN)

158 cours Fauriel, 42023 Saint- Etienne cedex 2, France

Laurence Vico, Alain Guignandon

Universit´e Jean Monnet de Saint Etienne, Laboratoire de Biologie du Tissu Osseux (LBTO) and INSERM U890

15 rue Ambroise Par´e, 42023 Saint-Etienne Cedex 2, France

Keywords:

Mechanical shocks, biomechanical device, energy, acceleration forces, Fourier analysis.

Abstract:

A new device called biomechanical stimulation device (BSD) has been recently developped and is under

patenting process. This BSD allows to apply shocks to a biomaterial disc, on which bone cells have been

seeded. To observe the real behaviour of the biomaterial under shock loading, the BSD is instrumented with

an impact hammer and an accelerometer. Force and acceleration signals are recorded, and signal analysis can

be performed, in particular Fourier analysis. The results obtained lead to a better understanding of the stimulus

that the cells can perceive at the top surface of the biomaterial disc. It appears that mechanical shocks applied

at 1 Hps (Hit per second) or 10 Hps generate a frequency content up to 35 kHz. The main further objective

will be to characterize the inﬂuence of mechanical shocks on bone cells proliferation.

1 INTRODUCTION

Bone cells activity deals with several medical stakes

like osteoporosis and osteogenesis imperfectae, but

also with biocompatibility in the case of bone pros-

thesis implantation. Bone cells activity is related to

mechanical stimulation. Actually, the right term for

”bone cells activity” is osteogenesis. Osteogenesis

consists in a balance between bone synthesis (ensured

by osteoblastic cells) and bone resorption (ensured by

osteoclastic cells) (Bilezikian JP, 2002). Osteoblasts

and osteoclasts do not act at the same time : it is a

continuous looped process. First, the osteoclasts de-

stroy the bone and make holes. Then, the osteoclasts

withdraw and the osteoblasts take their place to form

the bone. After bone creation, osteoblasts leave and

osteoclasts come again to destroy the bone.

H. M. Frost showed with his Mechanostat (Frost,

1987) that the osteogenic process is strongly inﬂu-

enced by mechanical stimuli. Previous studies have

reported different kinds of mechanical stimuli that

are efﬁcient for osteogenesis: ultrasounds, hydro-

static pressure, ﬂuid shear stress, biaxial and uniaxial

stretch, bending, nanostimulation with atomic force

microscopy and acceleration forces (Tjandrawinata

et al., 1997; Kacena et al., 2003; Hatton et al., 2003).

The effect of acceleration forces on osteogenesis has

already been analysed, but the exact stimulation that

the cells could perceive remains not entirely known.

Recently, a new biomechanical stimulation device

(BSD) has been developed and patented. It has been

built in the Ecole Nationale Superieure des Mines de

Saint Etienne (ENSMSE). This device aims at apply-

ing shocks to cells withoutdirect contact (acceleration

forces) between the cells and a mechanical impactor.

Shocks are applied to a biomaterial disc on which

cells are seeded. This allows to use different biomate-

rials in order to test their biocompatibility. Adhesion

of the cells on the biomaterial depends on the physic-

ochemical properties of this biomaterial. This adhe-

sion is a very important factor for the cellular activity

(Anselme et al., 2000; Ignatius et al., 2005; Jayara-

man et al., 2004).

In order to better understand the mechanical strain

and vibrational content involved, a signal acquisition

and signal processing study have been performed on

the BSD. More precisely, the purpose of this study

was to analyse the vibrational content resulting from a

272

Navarro L., Pinoli J., Besset H., Guyonnet R., Vico L. and Guignandon A. (2008).

A NEW INSTRUMENTED BIOLOGICAL DEVICE DESIGNED TO APPLY MECHANICAL SHOCKS TO BONE CELLS.

In Proceedings of the First International Conference on Biomedical Electronics and Devices, pages 272-278

DOI: 10.5220/0001047002720278

 SciTePress

shock on a biomaterial. Biomaterial biocompatibility

can be tested with the BSD, and the size of the bio-

material enables imaging and force investigations by

using Atomic Force Microscopy (AFM) (with 10 mm

diameter and 2 mm thickness disc). The BSD will be

presented in this paper in section 2. Then, the exper-

imental setup allowing the signals to be recorded is

detailed in section 3. Next, in section 4, results of the

signal acquisition will be analyzed. Furthermore, a

synthesis on this study and a conclusion will be given.

2 EXPERIMENTAL DEVICE

(BSD)

A schematic diagram of the BSD is shown in Fig. 1.

Four different biomaterial discs (Titanium (Ti6Al4V),

Hydroxy-apatite (HAP), cortical bone and trabecular

bone) have been used. The discs are normally sealed

into a culture chamber ﬁlled with culture medium,

and cells are seeded on the top of the disc. However,

all signal acquisitions have been performed without

medium and cells in order to only characterise the me-

chanical behaviour of the disc. This is a fundamental

step that is necessary to better understand what kind

of vibratory stimuli the cells can perceive at the top

of the biomaterial disc. During the mechanical stim-

ulations, a vertical actuator is activated with a small

and short stroke, high force solenoid, and a titanium

hammer head is adapted to the solenoid stalk.

The mechanical impacts are directly driven

through a current ampliﬁer. Square type stimulation

signals are used according to the current supply mode

of the solenoid (DC current). Since the input signal

waveform is not sinuso¨ıdal, the signal frequencyis de-

ﬁned in terms of hits per second (Hps) rather than in

Hz. This notation is used to avoid misunderstanding

between shocks application and resulting frequency

content. Some authors have shown that in vitro cul-

tured osteoblasts respond to mechanical strain at fre-

quency values between 1 Hz and 10 Hz (Lanyon,

1984; Neidlinger-Wilke et al., 1994; Kaspar et al.,

2000). The 1 Hz stimulation frequency corresponds

to the human locomotor behaviour. In this study, me-

chanical shocks are applied at these two stimulation

rates : 1 Hps and 10 Hps.

3 SIGNAL ACQUISITION

Signal acquisition is performed using two sensors

coupled on the BSD, as shown in Fig. 2.

An Integrated Circuit Piezoelectric (ICP



) ac-

Figure 1: Schematic diagram of the BSD culture chamber

used for in vitro experiments. Impact hammer is activated

by a solenoid, the head of the impact hammer strikes the

external surface of the biomaterial disc. Bone cells are cul-

tured on the surface of biomaterial and the culture cham-

ber is ﬁlled with medium. The impact hammer strikes the

biomaterial with predeﬁned frequency and duration. The

different components of culture chamber and hammer head

represented in the ﬁgure : (1) Macrolon



top lid, (2)

teﬂon cork,(3) Macrolon



main part of culture chamber,

(4) culture medium, (5) cell culture, (6) biomaterial disc,

(7) Macrolon



bottom lid, (8) seal.

celerometer (model 352C23, PCB Piezotronics, Inc.,

NY, USA) is ﬁxed on the discs top surfaces by direct

adhesive mounting and an Impulse Force Test Ham-

mer (model 086D80, PCB Piezotronics, Inc., NY,

USA) is adapted to the solenoid stalk and used as me-

chanical impactor. These sensors are connected to a

signal conditioner (model 442B104, PCB Piezotron-

ics) and acquired signals are recorded in a PC by

means of an acquisition card (NI DAQ Pad-6015, Na-

tional Instruments) with a sampling rate of 100 kHz

using a program written with LabVIEW



software.

A functional diagram of sensors monitoring, and main

sensors characteristics are given in Fig. 3, 4 respec-

tively. Accelerometer and Impulse force Test Ham-

mer signals are recorded simultaneously during the

impact. Acceleration and Force signals samples are

interlaced to ensure synchronized acquisition for fu-

ture joint analysis.

4 RESULTS

4.1 Signal Analysis

The experimental BSD produces two different types

of signal : force signals from the instrumented impact

hammer and acceleration signal from the accelerom-

eter. Ten discs of each biomaterial have been pro-

A NEW INSTRUMENTED BIOLOGICAL DEVICE DESIGNED TO APPLY MECHANICAL SHOCKS TO BONE

CELLS

273

Figure 2: Schematic diagram of the culture chamber used

for signal acquisition in air : (1) Biomaterial disc, (2) ICP



accelerometer, (3) ICP



Impulse Force Test Hammer.

Figure 3: BSD is driven by ampliﬁed voltage current sig-

nals from the computer. During mechanical shocks, signals

of ICP



. accelerometer and ICP



impulse hammer con-

nected to BSD are simulteanously transmitted to the com-

puter.

Accelerometer : 5 mV/g ( 15%) sensitivity

50 kHz frequency range

Impact hammer : 22.5 mV/N sensitivity

12 kHz Frequency Range ( 5%)

Figure 4: Sensitivity and frequency range given by the man-

ufacturer for the two sensors.

cessed. Standard mean deviation have been calcu-

lated for each acceleration signals: Ti6Al4V, 783±31

g; HAP, 1197±87 g; Cortical bone, 1215±21 g; Tra-

becular bone, 225±24 g; and for each force signals:

Ti6Al4V, 34±0.6 N; HAP, 28±0.2 N; Cortical bone,

23±0.2 N; Trabecular bone, 8±0.6 N. These values

exhibit that Trabecular bone is softer than the other

biomaterials.

The ﬁrst step of the signal analysis consists in a

zoom on the force and acceleration signals (Fig. 5) of

the four materials : Ti6Al4V, Hydroxy-apatite(HAP),

cortical bone and trabecular bone. This analysis en-

ables to show important characteristics that can not be

seen easily. For example, several rebounds between

the impactor and the disc can be seen for only one

hit. The delay between two rebounds is shortening

as their amplitude decreases. A ﬂat part can also be

seen at the end of all the signals : it corresponds to the

sustain time of the solenoid due to the square driving

signal.

The second step of the signal analysis is the calcu-

lation of the Fourier Transform (FT) and the represen-

tation of the magnitude (Flandrin, 1993). The Fourier

Transform (FT) X( f) of a signal x(t) is expressed as :

( f) =

+∞

−∞

x(t)e

−i2π ft

where t denotes the time and f the frequency.

The FT has the property of expressing a signal in

a frequency space.

The magnitude of the Fourier transform gives the

global frequency content of a signal (Fig. 5). Usually,

only the positive frequencies of the Fourier Transform

magnitude are shown in Fig. 6, since they possess a

physical meaning. In addition, the Shannon principle

is taken into account to avoid aliasing effect. This

principle is respected by the BSD signal acquisition

process.

The frequency spectrums of the signals are multi-

modal and different for the four discs. The Ti6Al4V

force signal frequencyspectrum (Fig. 6a) shows that a

slightly higher amplitude occurs for the Ti6Al4V than

for the other materials. It is interesting to notice that

HAP and cortical bone force frequency spectrums are

regular (Fig. 6b,c), it might be due to the fact that they

have almost the same chemical composition. Never-

theless, the cortical bone force frequency spectrum’s

pattern is smooth, whereas the HAP force frequency

spectrum pattern is sharper. The frequency spectrum

of the trabecular bone is very low, up to 1500 Hz, be-

cause the trabecular bone is softer than the cortical

bone. The acceleration frequency spectrums are not

regular and present several peaks, but relevant peaks

can not be observed on these spectrums. However, it

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0.52 0.54 0.56 0.58 0.6 0.62

Ti6Al4V

Time (s)

Force (N)

0.52 0.54 0.56 0.58 0.6 0.62

HAP

Time (s)

Force (N)

(a) (b)

0.52 0.54 0.56 0.58 0.6 0.62

Cortical bone

Time (s)

Force (N)

0.48 0.5 0.52 0.54 0.56 0.58

Trabecular bone

Time (s)

Force (N)

0.51 0.52 0.53 0.54

500

1000

1500

Ti6Al4V

Time (s)

Acceleration (g)

0.51 0.52 0.53 0.54

500

1000

1500

2000

2500

HAP

Time (s)

Acceleration (g)

(e) (f)

0.51 0.52 0.53 0.54

500

1000

1500

2000

Cortical bone

Time (s)

Acceleration (g)

0.48 0.49 0.5 0.51

100

150

200

250

300

350

400

Trabecular bone

Time (s)

Acceleration (g)

(g) (h)

Figure 5: Force and Acceleration signals, recorded at 100 kHz sampling frequency with 12 bit amplitude resolution. (a) :

Ti6Al4V acceleration signal. (b) : HAP acceleration signal. (c) : Cortical Bone acceleration signal. (d) : Trabecular bone

acceleration signal. (e) : Ti6Al4V force signal. (f) : HAP force signal. (g) : Cortical Bone force signal. (h) : Trabecular bone

force signal.

A NEW INSTRUMENTED BIOLOGICAL DEVICE DESIGNED TO APPLY MECHANICAL SHOCKS TO BONE

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275

1000 2000 3000 4000 5000

500

1000

1500

Ti6Al4V

Frequency (Hz)

Amplitude

1000 2000 3000 4000 5000

200

400

600

800

1000

1200

HAP

Frequency (Hz)

Amplitude

(a) (b)

1000 2000 3000 4000 5000

200

400

600

800

1000

1200

Cortical bone

Frequency (Hz)

Amplitude

1000 2000 3000 4000 5000

500

1000

1500

Trabecular bone

Frequency (Hz)

Amplitude

1 2 3 4 5

x 10

5000

10000

15000

Ti6Al4V

Frequency (Hz)

Amplitude

1 2 3 4 5

x 10

5000

10000

15000

HAP

Frequency (Hz)

Amplitude

(e) (f)

1 2 3 4 5

x 10

5000

10000

15000

Cortical bone

Frequency (Hz)

Amplitude

1 2 3 4 5

x 10

5000

10000

15000

Trabecular bone

Frequency (Hz)

Amplitude

(g) (h)

Figure 6: Fourier transform (FT) spectrums of the acquired force and acceleration signals. (a) : magnitude of Ti6Al4V force

FT. (b) : magnitude of HAP force FT. (c) : magnitude of cortical bone force FT. (d) : magnitude of trabecular bone force

FT. (e) : magnitude of Ti6Al4V acceleration FT. (f) : magnitude of HAP acceleration FT. (g) : magnitude of cortical bone

acceleration FT. (h) : magnitude of trabecular bone acceleration FT.

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is noticeable that the cortical acceleration frequency

spectrum is higher than the others.

4.2 Energy

From the acceleration signal recorded, the kinetic en-

ergy of the Ti6Al4V disc/accelerometer unit during

the mechanical shock is computed with the classical

formula

v(t)

dt.

(with E

: kinetic energy, m: mass , v: speed, given for

each disc-accelerometer unit.)

The speeds of the different disc-accelerometer

units are computed by integration of the acceleration

signals, which occurs in the data processing sequence

after application of 30 Hz Butterworth high-pass ﬁl-

ter to remove continuous component. The frequency

response curve of the Butterworth ﬁlter is practically

ﬂat in the passband. However, the use of this kind

of ﬁlter introduces a non linear frequency dephasing.

In this study, only the frequency content is expected

and not the signal phase, thus the application of But-

terworth ﬁlter is well adapted. The kinetic energy is

calculated per surface unit: Ti6Al4V, 4.8±0.1 pJ/µm

for 1 Hps and 50.2±1.1 pJ/µm

for 10 Hps; HAP,

2.0±0.04 pJ/µm

for 1 Hps and 20.6±0.6 pJ/µm

for

10 Hps; Cortical bone, 8.5±0.2 pJ/µm

for 1 Hps

and 65.1±3.6 pJ/µm

for 10 Hps; Trabecular bone,

0.9±0.2 pJ/µm

for 1 Hps and 8.8±3.2 pJ/µm

for 10

Hps. Notice that values of kinetic energy at 10 Hps

are 10 times higher than those at 1 Hps.

5 SYNTHESIS

This new BSD was initially developed to simulate the

effects of mechanical impacts on bone cells cultured

on biomaterials to compare them with the effects of

impacts during walking or running activities. A pre-

vious study on Ground Reaction Forces (GRF) (Gi-

akas G, 2001) has shown that frequency content dur-

ing impact phase of running is limited to 100 Hz, ac-

celeration magnitude is ∼10 g. During shocks ap-

plied with the BSD, a frequency range comprised be-

tween 100 Hz and 4 kHz, an acceleration magnitude

of 783,1± 32,5 g at 1 Hps and an acceleration time of

∼1 ms have been found on Ti6Al4V for example. So

it is quite difﬁcult to directly put in relation the results

obtained during this kind of mechanical impact with

those obtained by GRF studies.

In this study, recording conditions for acceleration

and force signals were slightly different from condi-

tions used in case of in vitro experiments, particularly

concerning the sealing of the culture chamber.

The disc should be excited by a frequency higher

than his resonnant frequency to enter in resonnance.

To verify this assumption, a numerical simulation was

perfomed and the ﬁrst resonant frequency mode of

the Ti6Al4V disc was found to be at 144 kHz, which

is much higher than the frequencies observed on the

force signal Fourier transform. Consequently, the disc

can not enter in resonance and induces some self-

frequencies. The results found during signal record-

ing can be extrapolated to in vitro conditions.

When mechanical shocks were applied to the

discs, whatever the signal stimulation frequency was

(1 Hps and 10 Hps), one could observe that the shape

and the mean maximal value of the force and accel-

eration signals were identical. Analysis of accelera-

tion signals leads to an estimation of the kinetic en-

ergy of the tested disc during one impact. It has been

found that the amount of kinetic energy at 10 Hps is

10 times greater than that calculated at 1 Hps. Consid-

ering these results, in the case of mechanical shock,

it appears that it is more interesting to analyse more

accurately the frequency content of force signals, to

compare kinetic energy derived from accelerations

signals, and to focus on the force perceived by cells

when they are subjected to acceleration phase. By ap-

plying the fundamental law of dynamic, the acceler-

ation force during impact has been calculated by es-

timating cellular mass (1 picogramm): a force of ∼1

nN per cell has been found. This acceleration force

is comparable to forces for which biological events

have been previously observed. Consequently, it can

be expected that this new kind of mechanical stimula-

tion would have biological effects on bone cells.

A critical analysis can be done concerning the

reproducibility of the acceleration and force peaks’

measurements. The values and their errors are for

each material: Ti6Al4V, 783±31 g; HAP, 1197±87 g;

Cortical bone, 1215±21 g; Trabecular bone, 225±24

g; and for each force signals: Ti6Al4V, 34±0.6 N;

HAP, 28±0.2 N; Cortical bone, 23±0.2 N; Trabecu-

lar bone, 8±0.6 N. The error is less important in the

case of hard materials (cortical bone acceleration er-

ror is < 2% for example). However, a 10% error oc-

curs on the measurements of acceleration and force

peaks on the trabecular bone. This can be due to the

non-heterogeneity of the material and probably to the

non-ﬂatness of the surface. In fact, the guidance of

the hammer is not perfect so the head of the hammer

does not hit in the same place every time. This is an

improvement to ensure to the BSD for future work.

An other improvement has been already done, it con-

sists in a screw that allows to ajust the stroke of the

A NEW INSTRUMENTED BIOLOGICAL DEVICE DESIGNED TO APPLY MECHANICAL SHOCKS TO BONE

CELLS

277

hammer. This leads to the possibility of controlling

the peak value and it reduces drastically the errors be-

tween different discs (The error for one disc is about

10 times smaller than that between discs).

6 CONCLUSIONS

A new device designed to apply mechanical shocks

to bone cells cultured on biomaterials has been devel-

oped. It allows to measure and compute shock pa-

rameters during impact : value and frequency content

of force impact, acceleration and kinetic energy for

each disc. When signals’ characteristics during im-

pact at different stimulation frequencies (1 Hps and 10

Hps) are compared, similar characteristics are found

for force signals but acceleration signals and kinetic

energy are different. Moreover, the computed value

of the acceleration force should lead to the observa-

tion of cellular responses. In conclusion, this new me-

chanical stimulator could be used for in vitro studies

to better understand bone cells mechanotransduction

during impacts.

Further studies, particularly concerning the bio-

logical effects of mechanical shocks on bone cells,

will be presented in future papers. Studies concern-

ing other signal analysis tools like time-frequency or

time-scale representations will also be held, since it

seems interesting to know if the BSD is a new way

to characterize biomaterials. Other biomaterial will

be tested, and Atomic Force Microscopy (AFM) will

be used in order to observe the bone cells behaviour

before and after mechanical shocks loading.

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