Noris-Surez, et al.

Acta Microscopica Vol. 18, No. 3, 2009, pp. 278 - 286

BONE COLLAGEN ROLE IN PIEZOELECTRIC MEDIATED REMINERALIZATION

A. M. Ferreira a, G. Gonzlez b, R.J. Gonzlez-Paz c, J. L. Feijoo c, J. Lira-Olivares c, K. Noris-Surez a,*
a

Laboratorio Ingeniera de tejidos, Dpto. de Biologa Celular, Universidad Simn Bolvar, Caracas, Venezuela.
Laboratorio de Ciencia e Ingeniera de Materiales, Departamento de Ingeniera, Instituto Venezolano de Investigaciones
Cientficas (IVIC)
c
Dpto. de Ciencia de los Materiales, GPUSB Universidad Simn Bolvar /, Caracas, Venezuela
* Corresponding author, E-mail: knoris@usb.ve, phone: +58 212 9064218, Fax: +58 212 9063064

Recibido: Julio 2009.

Aprobado: Noviembre 2009.
Publicado: Noviembre 2009.

ABSTRACT
Bone tissue is characterized by its mineral structure, showing unique properties as mechanical resistance, toughness and
elasticity. These are due to its composite nature of collagen (the most important constituent protein in the bone matrix), with
hydroxyapatite crystals. The place of collagen in the organization and growth promotion of mineral bone tissue is still under
study. In this paper an evaluation of the piezoelectric effect of collagen, using the biomimetic method and scanning electron
microscopy, is presented. It was found that after 3.5 weeks of exposure, deformed collagen initiate the precipitation of , at
the collagen fiber holes, but only on the compressed side. This precipitation process, completely cover the surface in 5
weeks of exposure to the Simulated Body Fluid (SBF). This confirms that the piezoelectric effect is a probable mechanism
for apatite nucleation and that the collagen fiber holes are preferential sites, however they dont promote nucleation by
themselves. Thus Wolfs Law seems to be dictated by piezoelectricity alone.
Keywords: bone collagen; intelligent biomaterial; SEM, bone piezoelectricity; biomimetic method.
PAPEL DEL COLAGENO OSEO EN LA REMINERALIZACION MEDIADA POR PIEZOELECTRICIDAD
RESUMEN
El tejido seo se caracteriza por ser un tejido mineralizado que muestra propiedades nicas de resistencia y elasticidad las
cuales se deben a la combinacin del colgeno (principal protena constituyente de la matriz sea) con los cristales de
hidroxiapatita. El papel del colgeno en la organizacin y promocin de la mineralizacin de este tejido sigue siendo objeto
de estudio. En el presente trabajo nos propusimos evaluar, mediante microscopia electrnica de barrido, la deposicin del
mineral inducido por el efecto piezoelctrico sobre el colgeno seo empleando el mtodo biomimtico. Encontrado que al
cabo de 3 semanas de tratar a las muestras de colgeno (deformadas) se inicia la nucleacin de apatita principalmente en
las regiones de la fibra de colgeno que corresponden a los espaciamientos, solo en la superficie deformada en comprensin.
Y que dicho proceso alcanza a recubrir de mineral totalmente esta superficie al cabo de 5 semanas de tratamiento,
confirmando que los espaciamientos favorecen la nucleacin mas no son nucleantes por s mismos, siendo el efecto
piezoelctrico un posible mecanismo que regula la orientacin de la deposicin del mineral sobre las superficies seas,
favoreciendo la deposicin sobre la superficie deformada en compresin.
Palabras claves: colgeno seo; biomaterial inteligente; MEB; piezoelectricidad sea; mtodo biomimtico.
INTRODUCTION

mineral phase called biological hydroxyapatite, and about

Bone is a connective tissue that is characterized by its

30% what of

mineralization, comprising about 70% calcium phosphate

(about 90%) of collagen type I. The combination of

organic phase, which consists mostly

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collagen with the mineral phase helps to create a

Hodges [6] described the presence of crystal in the hole

substance that has two properties of great relevance, such

and superficially, in turkey tendon treated samples,

as stiffness and strength, when this tissue is subjected to

suggesting that mineral nucleation can also occur on the

mechanical stress. Many intrinsic and extrinsic factors

surface, and that is not an exclusive process of the hole.

affect the mechanical properties of bone in response to

Marino [7] proposed that bone collagen is piezoelectric

load.

of

and Noris-Suarez et al [8] shows that in a cell free system

mineralization (high mineral density increases the Young

in the presence of a simulated body fluid, mineral

modulus) and the organization of tissues (i.e. porosity,

deposition on the surface is promoted when deformed in

orientation

the

compression (convex). Understanding the process of

histological structure), while extrinsic factors include the

mineralization is still today one of the research areas for

type of deformation (the bone is stronger in compression

further development, its study is critical in order to be

than in tension), the time elapsed during the deformation

able to develop materials and increasingly functional

event and magnitude of deforming strain. These factors

prosthesis. In the present study we aimed to the process

are of particular interest because bone tissue (and other

of demineralization by treatment with EDTA (a chelator

materials) is particularly viscoelastic [1]. Bones are

of calcium) and remineralization using the biomimetic

constantly remodeling in order to adapt to the mechanical

method to assess whether the remineralization of

requirements to which they are subjected. The role of

collagen in this process originates mainly in the spacing

collagen in the organization of the mineral component is

or occurs in a superficial way

Intrinsic

of

factors

collagen

include

fibers,

the

density,

degree

and

quite complex, as demonstrated by Takano and

colleagues [2], who found that bone crystals are
deposited at random over an array of randomly oriented

MATERIALS AND METHODS

collagen in bone newly formed, initially no differences in

stiffness (or anisotropy), but then gradually become
anisotropic and mineralization is consolidated after 16
weeks. These changes seem to increase because the
crystals reach a more regular basis after this period of
time without submitting changes in the orientation of
collagen [3]. Glimcher in 1959 [4] proposes a theory
about the calcification of bones based on three concepts:
1) bone matrix is composed mostly of collagen fibers, 2)
the initiation of calcification occurs in the hole or gaps
that occur in the collagen fibrils, 3) the generation of the
earliest mineral aggregates (nuclei) occurs with a
heterogeneous

nucleation

process.

According

to

Glimcher and Krane [5] approximately 50% of the

inorganic substance is contained in the hole collagen,
during the final stage of calcification, the crystals

COLLAGEN DEMINERALIZATION
The collagen was obtained from New Zeland rabbits
bones, demineralized as previously described by NorisSurez et al [8]. Briefly, bones were demineralized by
dissolving the mineral phase with a solution of 0.5M
EDTA, pH 7.4 under agitation at 4 C, changing this
solution for a fresh solution each 3 or 4 days. In a
previous paper [9] showed that the loss of the mineral
phase (hydroxyapatite) helps to reduce the thermal
stability of the collagen macromolecule due to the loss of
interaction between. Each sample was retired from de
solution and washed gently with distilled water to assure
that all EDTA was removed from the sample. The
demineralized collagen obtained in this form is referred
subsequently as a control sample.

develop in regions outside of these areas and eventually

among the interfibrillar spaces. However, Landis and
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DEFORMATION PROCESS

was replaced in the system weekly by fresh solution.

The used procedure to deform the collagen substrates

Also, the samples of deformed and undeformed bone

consists basically on bending the substrate inside a

cortical collagen were retired from the system weekly

plastic tube, maintaining it constrained, thus deformed,

and preserved at 20 C for analysis.

forming an arc coincident with the internal radius of the

tube that is 0.6 cm. This deformation, assured a

RESULTS AND DISCUSION

considerable stress, sufficient to separate elastically

In a previous paper [9] it was shown that the loss of the

molecules of the collagen thus generating the polarizing

mineral phase (hydroxyapatite) helps to reduce the

piezoelectric effect. The dipoles thus formed, could

thermal stability of the collagen macromolecule due to

induce the nucleation and deposition of the apatite. On

the loss of interaction between hydroxyapatite crystals

the constraining tubes, relatively wide rectangular

and collagen matrix of bone. In this same study shows

windows were opened, to provide direct contact of the

that bone samples lost up to 52% of the mineral phase in

sample with the SBF. Altogether, collagen substrates

just the first 72 hours once is subjected to treatment with

were deformed and exposed to the SBF; a similar number

EDTA (confirmed by FTIR), and found that the

of undeformed samples were used for calcification

temperatures of denaturation and degradation of bone

control. The samples were exposed for five weeks, on the

collagen (determined by DSC) decreased proportionally

average.

to the extent that the bone loses its mineral phase, finding
an endotherm at 166 C in bone and 113C in the samples

REMINERALIZATION. BIOMIMETIC METHOD

treated with EDTA.

In order to simulate the blood plasma, a physiological

fluid, following the specifications presented by Kokubo
in the preparation of one Simulated Body Fluid (SBF)
#39, was prepared using the biomimetic method, to
simulate the conditions for bone growth. The fluid
temperature was kept at 36.5 0.5 C and completely
free from microorganism growth using sodium azide 1%.
An acrylic column on a universal support was used,
inside of which, the substrates of collagen with and
without deformation (that is, inside and outside
constraining tubes) were submerged in the SBF. With the
purpose of simulating the blood circulation in the body,
an outside beaker that contained fresh SBF fluid was
placed inside a stove to control the temperature to 37 C
and by a peristaltic pump; the SBF was impelled to the
column

guaranteeing

that

it

circulated

cyclically

throughout the column. The sample-containing column

was duly thermally isolated to avoid heat exchange with
the atmosphere, trying to maintain the temperature of the

This work was aimed at assessing the appearance of

decalcified

samples

at

intermediate

times

of

decalcification treatment using EDTA and analyzed by

scanning electron microscopy. Figure 1 shows a
micrograph for a sample of cortical bone after 96 h of
decalcification. It can be noted that after this period of
time of treatment, traces of mineral phase are not
removed,

which

coincides

with

the

dark

bands

characteristic of collagen fibers (corresponding to

spaces). This suggests that these crystals are the most
difficult to remove when bone is treated with EDTA.
However, removal is achieved almost completely when
the treatment is extended for a total of 45 days (data not
shown). It also stresses the fact that the mineral is not
evenly spaced along the fiber, suggesting that nucleation
does not occur on a regular basis throughout all spaces in
the living organism and therefore they do not appear to
be nucleating.

system nearest to body temperature. The solution of SBF

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Moreover, as already mentioned in the introduction,

deformed. To do this, using the biomimetic method,

previous studies [8] showed that by deforming the bone

samples were treated for 3 , 4 and 5 weeks.

collagen, which possesses piezoelectric properties [10],

is generated, according to Wolf's Law, a charge
separation that can stimulate mineral deposition in the
area under compression (convex) but not in the area
subjected to tension (concave). Figure 2 shows the
micrographs obtained in a recent paper [8], of deform
samples of bone collagen and immersed for 4 weeks in
BSF following the biomimetic method. Proving that the
deformed surface in compression, due to the phenomenon

Figure 3 shows the micrographs of the collagen surface

under compression, after 3 weeks of treatment with the
biomimetic method (using a tuning ambient electronic
microscope). It can be seen in Figures 3a and 3b the
growth of clusters of mineral on the surface. It is seen
that although some nuclei appear to be initiated from the
spacing (indicated by arrowheads), others seem to grow
superficially.

of piezoelectricity, polarizes the area negatively (as

illustrated in Figure 2) and promotes the deposition of
calcium phosphates, in contrast to deform material in
tension, where some points of mineralized material after
4 weeks are hardly noticeable.

Fig. 1. SEM micrograph obtained by demineralized bone

treated with EDTA during 96 hours. Mineral fragments
were observed which correspond to the spacing of the
collagen fiber (indicated by white arrows).

Given the findings obtained above [8], we proposed in

this paper evaluate what could be the role of the spacing
or hole of collagen in the induction of mineralization
mediated by the piezoelectric phenomenon when it is

Fig. 2. SEM micrographs of demineralized bone collagen

deformed after 4 weeks immersed in the SFB: upper
micrograph correspond to compression side and down
micrograph correspond to tension side. The image was
modified from the reference [8].
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While still is a subject of discussion and analysis, how

collagen participates in the process of nucleation and
crystal growth, Glincher and Krane [5] suggests that this
process is carried out in two stages: first, the nuclei form
in the spaces within the collagen fibrils, and secondly,
these nuclei crystallize and gradually fill and complete
the spaces so that at the end of the process, their shape
and size are shown to be identical with those of the holes.
According to Glimcher and Krane [5] approximately
50% of the inorganic substance can be contained in this
space during the final stage of calcification. The crystals
grow in regions outside of these areas and eventually
among the interfibrillar spaces, as seen in Figures 3 and 4
(see white arrows).
Moreover, Wuthier [11] concluded that repetitive holes
observed in the collagen fibers (as seen in Figures 3 and
4) are poor apatite nucleating agents. These holes require
long periods of incubation, with a high concentration of
ionic products to induce the deposition of inorganic
structures. It is conceivable, moreover, that the holes act
primarily as recipients and propagators of nucleation,
rather than as initiators of the process of calcification.
In Figure 4b can be seen the details of how these mineral
nuclei increase their growth and begin to cover the
collagen fibers after 5 weeks of treatment. On the
contrary it can be seen in the area subjected to tension
(Figure 4c) than for the same treatment time, it just
begins to see some nucleation points on the collagen
fiber. As seen in the micrograph, those early mineral
aggregates

(nuclei)

occur

under

a heterogeneous

Fig. 3: SEM micrograph of demineralized bone collagen

deformed with compression forces and immersed in SBF
for 31/2 weeks. In figure a) the mineral nucleation points
are appreciated between the bands of the collagen fibers
(arrow heads), b) the mineral is growing above the fiber.

nucleation process described by Glimcher [4], who

reported that collagen type I has holes of 64 nm, which
seem to favor the nucleation process while tropocollagen
by themselves cannot [12].

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this would induce a physiological effect translated or
mediated by a piezoelectric effect, which in turn would
benefit the deposition of mineral at the surface under
compression.

Deforming

forces

in

tension

or

compression generate modifications or changes that

result in substantial changes and with specific orientation
[13]. This explains the results shown in Figure 4, which
highlights an important mineralization in the area
subjected to compression after 5 weeks of treatment
(figure 5 show EDS results, of 4 weeks of treatment),
whereas the surface tension shows a very poor
mineralization in comparison.

Fig. 5. EDS analyses of cortical bone collagen,

compression side at 4th weeks of biomimetic treatment,
shown a Ca/P = 1.69, similar to that found in HAP in
bone (with Ca/P= 1.67)
Hodge (reviewed in [12]) showed the presence of black
Fig. 4: SEM micrographs of demineralized bone collagen
immersed in SBF for 5 weeks: a) Compression side:
Mineral growth on collagen surface is shown, b)
Compression side: Mineral deposition detailed on
collagen fibers, c) Tension side: collagen fibbers

holes between neighboring collagen and suggested that

the mineralization was initiated in these areas. This has
been demonstrated in a later work where it has been
observed, as in the micrographs of remineralization in
Figure 3, how small crystals nucleate and grow in these

The cortical bone collagen is ideally designed to carry

out a piezoelectric process, unlike the cartilage, bone
collagen is highly oriented and follows a pattern of order,
so that the collagen structure provides a collective
cohesive response against a mechanical load [13], and

areas, which form continuous channels open to the

surface of collagen. Lees and Prostak [14], meanwhile ,
have demonstrated that the size of the hydroxyapatite
crystals exceed the intermolecular spacing in collagen
fibrils and describe most of the crystals in bone are
located in interfibrillar spaces, coinciding with the results
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obtained by us (Figures 3 and 4). The micrographs,

collagen type I, only are able to mineralize in specific

figures 3 and 4, which correspond to the remineralization

pathological situations.

process obtained by submitting samples of deformed

collagen to the biomimetic method, are of special interest
since they present similarities with corresponding images
obtained at the bone demineralization after 96h as
presented in Figure 1, which notes the presence of
whitish spherical structures on the microfibril (indicated
by white arrows). It is noted that these structures are
close to the dark bands on the collagen fibrils, suggesting
that these spaces are favoring the nucleation and

In a previous work [8] we suggested that the effect

produced

by

the

electrochemical

generation

of

piezoelectric dipoles can be the source of initial growth

of HA on collagen substrate. In fact, we proposed a
model in which the negative electrostatic charge on the
surface deformed in compression attract calcium ions,
which in turn attract the phosphate ions, which finally
allow the nucleation of apatite crystals

orientation of calcium phosphate crystals by electrostatic

CONCLUSIONS

interactions. Although poorly described as nucleating

Using SEM was possible to determine that the

sites [15], Landis and Silver [16] argue that in such

piezoelectric effect promotes apatite deposition not only

spaces favorable conditions are present not only for

in the "holes" of collagen but also on the surface. It was

nucleation but also influencing the size, shape,

also found that the mere presence of these structures or

orientation

the

spaces are not enough, but it is where the highest amount

stereochemical features presented by these areas of the

of mineral deposition it is found when the process is

collagen fibril. Also found in other studies that the

induced by deformation of the piezoelectric material.

and

alignment,

more

specifically

interaction of Ca and P ions with collagen (mediated

through specific amino acid residues that form a

ACKNOWLEDGEMENTS

structural part of the dark bands of collagen fibrils), alter

We thank the Venezuelan National Science Foundation

the protein's ability to store elastic energy, which is

FONACIT through grant G-200100900 and the Research

critical for the maintenance and function of bone [17].

and Development Unit (DID) of Simn Bolvar

According to the results found in this work we could

University, GPUSB (GID-02, GID-08), for the support

argue that the piezoelectricity that is generated by

given to accomplish this work. A.M. Ferreira is a

deforming

recipient of a graduate research assistantship from the

the

collagen

fibril

induces

spatial

rearrangements on the collagen fiber that would favor

DID, Universidad Simn Bolvar.

interactions on the dark bands (corresponding toholes)

and probably inducing the charged amino acids that are

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