alvina


29

ABSTRACT

Osteoporosis is characterized by lower bone mineral density (BMD) and
microarchitectural degeneration, which tends to increase bone fragility and
fracture risk. Bone microstructure depends on interactions between the mineral
atoms, which may perform substitution or incorporation into bone crystals, and
may dynamically take over the function of calcium or may become a
complementary part. The mineral atoms may also become a composite in the
hydroxyapatite crystals. The aim of this study was to find an association between
the bone microstructure and hydoxyapatite crystal structure in osteoporosis, in
comparison to normal bone. The subjects of this study were surgery patients at
the Department of Orthopedics of Ulin General Hospital in Banjarmasin and
other centers. Inclusion criteria consisted of the presence of fracture of trabecular
bone, normal or osteoporotic BMD values, and no past history of chronic disease.
Bone was obtained from fracture patients during surgery. The characteristics of
the hydroxyapatite crystals were analyzed by X-ray diffraction (XRD) and the
microarchitecture by scanning electron microscopy (SEM). SEM showed
degeneration of the microarchitecture of osteoporotic bone, in comparison with
normal bone. On XRD there was a peak of hydoxyapatite crystals only and no
other crystal phases. Diffraction patterns showed a larger crystal size in
osteoporotic bone as compared to normal bone, indicating higher porosity. It
may be concluded that there is a difference in crystal size and morphologic
distribution of hydoxyapatite in osteoporotic bone, determining bone
microarchitecture.

Keywords: Microarchitecture, hydroxyapatite, osteoporosis

*Department of Orthopedics,
Ulin Regional General
Hospital, Faculty of Medicine,
Lambung Mangkurat
University, Banjarmasin
**Department of Biology,
Faculty of Mathematics and
Natural Sciences,
Brawijaya University, Malang
***Department of Orthopedics,
Syaiful Anwar Regional
General Hospital,
Faculty of Medicine,
Brawijaya University, Malang
****Department of
Orthopedics, Hasan Sadikin
Hospital, Faculty of Medicine,
Padjadjaran University,
Bandung
*****X-ray Diffraction
Laboratory, Central Laboratory,
Faculty of Mathematics and
Natural Sciences,
Malang State University

Correspondence
dr. Zairin Noor, SpOT(K)
Department of Orthopedics,
Ulin Regional General Hospital
Jl. A. Yani Km 2 No.43
Banjarmasin 70233
Email: noorzairin@yahoo.com

Univ Med 2011;30:29-35.

Assessment of microarchitecture and crystal structure
of hydroxyapatite in osteoporosis

Zairin Noor*, Sutiman B Sumitro**, Mohammad Hidayat***,
Agus Hadian Rahim**** and Ahmad Taufiq*****

January-April, 2011January-April, 2011January-April, 2011January-April, 2011January-April, 2011             Vol.30 - No.1            Vol.30 - No.1            Vol.30 - No.1            Vol.30 - No.1            Vol.30 - No.1

UNIVERSA MEDICINA



30

Noor, Sumitro, Hidayat, et al                                                                                            Microarchitecture and crystal structure

INTRODUCTION

The characteristic features of osteoporosis
are low bone mineral density (BMD) and
m i c r o a r c h i t e c t u r a l  d e g e n e r a t i o n ,  w h i c h
increases the fragility of bone and the risk of
fractures.(1-3) In developing or less developed
countries the prevalence of osteoporosis is still
not known with certainty, due to a lack of
studies. In Asian population segments of those
over 50 years old, osteoporosis of the lumbar
spine has a prevalence of 11%-37% in women
and of 5.4%-37.4% in men; osteoporosis of the
femoral neck accounts for 2% of women and
6.3%-11.4% of men; and osteoporosis of the hip
affects 16% of women and 3.8%-24.3% of
men.(4-9) According to the white paper issued by
t h e  I n d o n e s i a n  O s t e o p o r o s i s  S o c i e t y
( P e rh i m p u n a n  O s t e o p o ro s i s  I n d o n e s i a ,
PEROSI), the prevalence of osteoporosis in
2007 was 28.8% in men and 32.3% in women.(10)
These percentages were consistent with the
results of an analysis of osteoporosis risk by
the Nutritional Research and Development
Center (Pusat Penelitian dan Pengembangan
Gizi, Puslitbang Gizi) of the Department of
Health (Depkes) in cooperation with Fonterra
Brands Indonesia, in 2005 2 in 5 Indonesians
were at risk for osteoporosis.(11)

The paradigm assumed in taking measures
for improving BMD is an adequate intake of
calcium, although field data show inconsistent
results. The study by Prentice et al.(12) revealed
that osteoporosis was rarely found in the
Gambian population, although having a low
daily dietary calcium intake. A similarly low
fracture rate is found in calcium-deficient Asian
populations. On the other hand, a high incidence
of fractures was found in Western populations,
which have a high calcium consumption.
Therefore it must be concluded that bone
strength and elasticity does not solely depend
on bone density but also on bone quality.(13)

Mammalian bone is a mineralized tissue
comprising solid phases of calcium phosphate
mineral and organic matrix, with a crystal

structure similar to that of the geological mineral
hydroxyapatite, Ca10(PO4)6(OH)2. Biological
hydroxyapatites have multiple substitutions and
deficiencies at all ionic sites.(14)

The microstructure of a material strongly
affects its physical characteristics, such as
s t r e n g t h ,  d u c t i l i t y,  h a r d n e s s ,  c o r r o s i o n
r e s i s t a n c e ,  a n d  b e h a v i o r  a t  l o w  o r  h i g h
temperatures. The microstructural development
of bone is determined by the role of the mineral
atoms. Each single atom may be substituted in
the bone crystals or may be incorporated into
them, and may dynamically replace the function
of calcium or may complement calcium and
simultaneously affect the elasticity and strength
of bone. Bone microstructure has frequently
been studied in experimental animals as well
as in humans,(15-17) but there have been few
studies of the microstructure of osteoporotic
bone.(18)

Substitution and incorporation determines
the pattern and quality of growth of the bony
matrix, which ultimately affects the atomic
configuration of the hydroxyapatite complex
and interaction of collagen with hydroxyapatite,
thus affecting the characteristics of the bone.
In the study of Ren et al.(19) it was found that
substitution of Ca2+ ions by Zn2+ ions in the
hydroxyapatite structure significantly decreased
the crystal size proportionally with increasing
zinc concentrations. The implication was that
zinc inhibited crystallization and crystal growth
in hydroxyapatite, which was consistent with
the findings of Miyaji et al.(20) This phenomenon
is believed to be due to the difference in size of
Ca2+ and Zn2+ ions. Zn2+ ions have an ionic radius
of 0.74 Å, which is significantly smaller than
the ionic radius of Ca2+ of 0.99 Å, thus the
substitution of Ca2+ by Zn2+ results in a decrease
in the lattice parameters and reduction in crystal
lattice volume, leading to impaired crystal
growth.(21)

The current study on the crystallization
profile of hydroxyapatite in osteoporotic bone,
in comparison with normal bone, is the first of
its kind in Indonesia. The aim of this study was



31

to evaluate the relationship between the
crystallization profile of hydroxyapatite and
the microstructural profile of osteoporotic
bone, compared to normal bone, leading to an
understanding of the role of mineral atoms as
the basic components of bone composite in the
course of osteoporosis.

METHODS

Research design
A laboratory observational study using

cross-sectional design was conducted to assess
the microstructure and crystal profile in
osteoporotic bone.

Study subjects
P a t i e n t s  u n d e r g o i n g  s u rg e r y  a t  t h e

D e p a r t m e n t  o f  O r t h o p e d i c s  o f  t h e  U l i n
Regional General Hospital in Banjarmasin and
at other centers. The inclusion criteria for this
study were: i) presence of fracture of trabecular
bone (in patients undergoing surgery); ii)
normal or osteoporotic BMD values; and iii)
no past history of chronic disease.

Tools for bone measurements
During the surgery bone samples were

t a k e n  f o r  t h e  f o l l o w i n g  i n v e s t i g a t i o n s /
assessments: i) bone microstructure by means
of scanning electron microscopy (SEM); ii)
characteristics of hydroxyapatite crystals in
bone by X-ray diffraction (XRD); and iii)
BMD. On the basis of the BMD scores, the
subjects were assigned to two groups, i.e. the
normal group and the osteoporosis group.
According to calculations performed with Epi
Info version 6, the mininum sample size was
32 for á =95% and â =80%. Assessment of BMD
was performed at Ulin Regional General
Hospital in Banjarmasin and Syaiful Anwar
Regional General Hospital in Malang. SEM
and XRD were conducted in the Physics
Laboratory and the Central Laboratory of the
Faculty of Mathematics and Natural Sciences
(FMIPA), Malang State Hospital. XRD was

also performed on standard hydroxyapatite
crystals.

Characterization of the X-ray diffraction
r e s u l t s  w a s  p e r f o r m e d  b y  m e a n s  o f
PA N a n a l y t i c a l  X ’ P e r t  P R O - M P D ,  f o r
osteoporotic and normal bone. Subsequent
a n a l y s i s  w a s  b y  m e a n s  o f  t h e  s o f t w a r e
programs High Score Plus, Crystal Maker and
DDVIEW, complemented with the latest
version of PDF2. Diffraction spectra were
recorded at an angle of 2θ, from 200 to 60o,
with a Cu-Kα radiation source (wave length =
1.54056 Å, 40 mA, 40 kV) and step size of
0.05o. Mean apatite crystal size in osteoporotic
and normal bone was evaluated by means of
the Scherrer equation.(22) Rietvield refinement
analysis was obtained from spectrum details
of osteoporotic and normal bone apatite
samples.

R i e t v e l d  r e f i n e m e n t  a n a l y s i s  w a s
performed by means of the High Score Plus
program. Space groups, cell parameters, atomic
p o s i t i o n s ,  a n d  h y d r o x y a p a t i t e  t h e r m a l
parameters were introduced in an initial
structural model. Rietveld refinement was
performed in several stages. In the first stage,
scale factors and background were refined,
followed by refinement of other parameters,
respectively comprising profile parameters,
z e r o  s h i f t ,  a s y m m e t r i c  p a r a m e t e r s ,  c e l l
parameters, preferential orientation, atomic
c o o r d i n a t e s ,  t h e r m a l  p a r a m e t e r s ,  a n d
occupancy factors.

Ethical clearance
The study was approved by the Health

R e s e a r c h  E t h i c s  C o m m i t t e e  F a c u l t y  o f
Medicine University of Brawijaya. All the
subjects were informed of the purpose of the
study and were requested to sign an informed
consent form.

Statistical analysis
D a t a  w e r e  a n a l y z e d  b y  a n a l y s i s  o f

variance followed by Tukey when appropriate.
A p value < 0.05 was considered significantly.

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Noor, Sumitro, Hidayat, et al                                                                                            Microarchitecture and crystal structure

RESULTS

The results of XRD characterization are
presented in Figure 1, indicating that only the
hydroxyapatite crystal peak was present and that
no other crystal phases were detected.

From search and match tests it was found
t h a t  a l l  b o n e  s a m p l e s ,  b o t h  n o r m a l  a n d
osteoporotic, had a crystal phase with a
hexagonal structure and space groups P63/m
and 176. Application of the Crystal Maker
program yielded the crystal structure depicted
in Figure 2.

Figure 1. XRD patterns of osteoporotic bone sample (red), normal bone (blue) and
standard hydroxyapatite crystals (green)

Figure 2. Crystal structure of bone
Legend: O-H = red; Ca = gray (large); P = gray (small); O = blue



33

From Figure 2 it is apparent that O-H
groups are located at the corners of the crystal
lattice, whereas Ca, P, and O atoms are located
within the crystal lattice space or volume, in a
highly regular manner. The hexagonal crystal
structure has the crystal lattice parameters a =
b ≠ c, with angles á  = â  = 900 and ã = 1200.
According to crystal geometry, this structure
possesses a relatively high stability. Crystal
sizes are presented in detail in Table 1. Anova
test concluded that there is significantly
different of crystal size in all groups (p=0.000).
Post hoc test concluded there is significantly
different of crystal size in all groups (p=0.000).

The microarchitecture of osteoporotic
bone is significantly different from that of
normal bone. Figure 3 depicts SEM images at
200x magnification, and Figure 4 depicts SEM

Table 1. Cystal sizes of standard hydroxyapatite, osteoporotic bone and normal bone

images at 3000x showing trabeculae with large
perforations surrounded by resorption cavities.
In the trabecular structure there are stump
s t r u c t u r e s  a n d  t h e  i n n e r  s u r f a c e  o f  t h e
remaining cavities appear flat and thin (A). In
normal bone the resorption cavities have not
yet formed stump structures and the trabecular
walls are still thick with a crumpled surface
(B).

DISCUSSION

In connection with the abovementioned
results, the mechanical properties of bone
should not be viewed as a table of constants,
but rather as a function of various factors.(23)

Osteoporosis is a process progressing to the
amorphic that is difficult to characterize.(24)

Figure 3. Trabeculae in osteoporotic (A) and normal (B) bone at 200x magnification

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Noor, Sumitro, Hidayat, et al                                                                                            Microarchitecture and crystal structure

Figure 4. Trabeculae in osteoporotic (A) and normal bone (B) at 3000x magnification
At 3000x magnification, osteoporotic bone consists of parallel strands of collagen fibrils with intervening

furrow-like resorption cavities, and also broken fibrils (A). Normal bone lacks strands and presents
predominantly closely packed hill-like structures (B)

In summary, the atomic mineral make-up
of the bone composite determines the crystal
size, which is the basis for the development of
bone microarchitecture. Furthermore, the results
of this study also indicate that although the
crystal structure in osteoporotic bone and
normal bone may be of similar hexagonal
geometry, at the most basic level the crystal size
and bone porosity are also important in
determining bone quality, either physical,
mechanical, or otherwise.

A limitation of this study is that SEM only
reveals bone structure in two dimensions,
necessitating further studies using micro-CT
reconstruction as a three-dimensional analytical
tool for osteoporotic bone as compared to
normal bone. The crystal size also needs to be
investigated three-dimensionally by means of
atomic force microscopy.

CONCLUSION

Crystal size and morphologic distribution
of hydroxyapatite in osteoporotic bone differs
from those of normal bone and determine the
microarchitecture of bone.

The larger crystal size in osteoporotic bone
is a result of the atomic make-up of the
composite. The porosity of bone is proportional
to the crystal size, where with a larger crystal
size there is increased porosity. This is the
reason why normal bone in this study exhibits
a smaller crystal size (has a lower porosity) in
c o m p a r i s o n  w i t h  o s t e o p r o t i c  b o n e .  T h i s
phenomenon may be explained by the fact that
within the bone structure the larger crystal size
also leads to larger-sized interstitial cavities.
The larger-sized cavities within the bone
increase the total number of voids, such that
ultimately the void ratio also increases. It is the
void ratio that increases the porosity, as shown
by the agreement between XRD and SEM
results.

According to physical fact and logic,
materials with a higher porosity level have
poorer mechanical properties, possessing
greater fragility, lower hardness, and lower
flexibility, in consequence of the weaker bonds
between the bone crystals. The present study
found a reduction in bone mass and deterioration
of its microarchitecture, which is comparable
with the findings of Shen.(18)



35

ACKNOWLEDGEMENT

The author thank Mr. Abdulloh Fuad and
Ms. Zulaikha in Malang State of University for
sample preparation.

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