Biomaterial granules used for filling bone defects constitute 3D

scaffolds: porosity, microarchitecture and molecular composition

analyzed by microCT and Raman microspectroscopy

Baptiste Arbez,1 Jean-Daniel Ku

€ n-Darbois,1,2 Thierry Convert,3 Bernard Guillaume,1,3
Philippe Mercier, Laurent Hubert,1,4 Daniel Chappard 1
1

1
Groupe Etudes Remodelage Osseux et bioMate  riaux, GEROM – LabCom NextBone, SFR 42-08, Universite
 d’Angers,
IRIS-IBS Institut de Biologie en Sante , CHU d’Angers, 49933 Angers Cedex, France
2
Service de chirurgie maxillo-faciale, CHU d’Angers, 49933 Angers Cedex, France
3
CFI, Collège Français d’Implantologie, 6 rue de Rome, 75005 Paris, France
4
 partement de chirurgie osseuse, CHU d’Angers, 49933 Angers Cedex, France
De

Received 7 February 2018; accepted 23 March 2018

Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.34133

Abstract: Biomaterials are used in the granular form to fill
small bone defects. Granules can be prepared with a grinder
from trabecular bone samples or provided as synthetic bio-
materials by industry. Granules occupy the 3D-space and cre-
ate a macroporosity allowing invasion of vascular and bone
cells when the inter-granular pores are larger than 300 mm.
We compared the 3D-porosity of granule stacks obtained or
R R
prepared with nine biomaterials OsteopureV, LubbocV, Bio-
R R R R
OssV, CopiOsV, TCP DentalV, TCP Dental HPV, KeraOsV, and
R
TCHV in comparison with that of human trabecular bone. For
had a much lower porosity than 1000–2000 mm granules and
the maximum frequency of pores was always centered at
200–250 mm. One biomaterial contained substantial amount
R
of cortical bone (Bio-OssV). The highest porosity and pore
size was obtained with TCP Dental HP. Raman spectroscopy
found differences in biomaterials of the same composition.
Stacks of granules represent 3D scaffolds resembling trabec-
ular bone with an interconnected porous microarchitecture.
R
Small granules have created pores <300 mm in diameter; this
can interfere with vascular colonization. V
C 2018 Wiley Periodi-

each biomaterial, two sizes of granules were analyzed: 250–
1000 and 1000–2000 mm. Microcomputed tomography deter-
mined porosity and microarchitectural characteristics of gran-
ular stacks and Raman microspectroscopy was used to
analyze their composition. Stacks of 250–1000 mm granules
cals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000–000,
2018.
Key Words: biomaterial granules, ceramics, Raman spectros-
copy, microCT, bone graft

€ n-Darbois J-D, Convert T, Guillaume B, Mercier P, Hubert L, Chappard D 2018. Biomaterial

How to cite this article: Arbez B, Ku
granules used for filling bone defects constitute 3D scaffolds: porosity, microarchitecture and molecular composition analyzed
by microCT and Raman microspectroscopy. J Biomed Mater Res Part B 2018:00B:000–000.

INTRODUCTION
The use of commercial biomaterial granules is a widespread
alternative to morcellized autogenous bone when a graft is
necessary to help bone repair in compromised situations.
Several types of biomaterials are now provided by a number
of companies; they can be of natural origin (i.e., allo- or
xenogenic bone) or prepared as synthetic calcium-
phosphate ceramics.1–3 Granular materials that assist in the
development of new bone in the grafted area realize in fact
a 3D scaffold that allows the penetration of fluids and cells
in a centripetal way from the surrounding tissues.4 When a
surgeon places granules within a bone defect, the voids
between the granules represent an interconnected space
available for vascular sprouts and bone cells to invade the
grafted area.5 3D geometry of the assembly of such scaffolds
has been little considered and depends on a number of
factors such as the size and shape of the granules them-
selves. Among the 3D parameters of importance, an open
porosity with a high degree of interconnectivity and a pore
size >300 mm is now considered a key factor ensuring a
rapid healing of the grafted site.6,7
X-ray microfocus computed tomography (microCT) pro-
vides a suitable solution to analyze and measures the 3D
volume, porosity and microarchitecture of granule-based
scaffolds in vitro.4,8,9 Evaluation of the volumetric and
microarchitectural parameters inside the scaffolds is very
important to have a precise idea of its biodegradation and
then bone regeneration when the granules will be implanted
in a bone defect from a patient.10–12
This study aims (i) to evaluate qualitatively and quanti-
tatively the 3D microarchitecture of different granular bio-
materials prepared and sold by several companies as bone

Additional Supporting Information may be found in the online version of this article.
Correspondence to: D. Chappard; e-mail: daniel.chappard@univ-angers.fr

C 2018 WILEY PERIODICALS, INC.

V 1

filling substitutes and to compare their characteristics with
those of human trabecular bone (ii) to verify the molecular
composition of these biomaterials by Raman microspectro-
scopy. Allogenic and xenogenic particles and synthetic Ca/P
ceramic granules commonly used in maxillo-facial surgery
were analyzed. The influence of two size of granules sold by
industry (i.e., 250–1000 mm and 1000–2000 mm) was also
considered on the 3D microarchitecture.
MATERIALS AND METHODS
Bone samples
Five bone cylinders (9 mm in diameter) were prepared
from the proximal tibial epiphysis from five human cadavers
from the Anatomical Laboratory (mean age 78.3 6 5.2 y.).
The calibrated cylinders were obtained with a diamond-
tipped coring tool (Starlite Industries, Rosemont, PA) per-
pendicular to the tibial plateau. The subjects had given their
body to science before death and had completed a form
indicating that their corpses were to be used for medical
education and research. Subjects were examined upon
arrival at the laboratory of anatomy of the Faculty of Medi-
cine. All bone samples were harvested following a strict
protocol of the laboratory of anatomy; for example, it was
impossible to know any detail which could lead to the sub-
ject’s identification (causes of death, previous medical
records). Bone cores were fixed in 10% formalin until use.
Allo- and xenogenic bone granules
Undecalcified human graft. A hemi-femoral head
R
(OsteopureV) was provided by Ost Development (France).
The sample did not contain cortical bone and the sub-
chondral bone has been removed before the cleaning pro-
cess which included defatting steps and solvent washes.13
Granules of this undecalcified purified hip were prepared
with a bone miller (Roswitha P. Quetin, Dental-Products, Lei-
men, Germany) and they were sieved to obtain two groups
of granules: 250–1000 mm (small) and 1000–2000 mm
(large).
Undecalcified bovine xenograft. Blocks (12 3 4 3 5 mm)
of bovine purified and undecalcified bone from calves were
R
prepared by Ost Development as above (LubbocV). The
blocks did not contain remnants of cortical bone. Granules
(250–1000 mm and 1000–2000 mm) were prepared with a
bone miller as above described. In order to study the influ-
ence of pyrolysis, some blocks were calcinated in a furnace
(Vecstar Furnaces, MRF1, Chesterfield, UK) during 24 h at
8008C. After cooling, they were milled and sieved as above
to produce granules of pyrolyzed calf bone.
R R
Bio-OssV. Granules of Bio-OssV (250–1000 mm and 1000–
2000 mm) were directly purchased from Geistlich Pharma
AG (Switzerland). The packaging and the leaflet package
indicates they are prepared from bovine spongy bone which
has been extensively deproteinized with a 3008C treatment
for 18 h.14 Granules of batches #81600947, #81500416 and
#81600788 were used.
2 ARBEZ ET AL.
R R
CopiOsV. Granules of CopiOsV (250–1000 mm and 1000–
2000 mm) were directly purchased from Zimmer Dental
(Germany). The packaging and the leaflet package indicates
R
they are prepared by the Tutoplast processV (Tutogen Medi-
cal GmbH, Germany) from bovine spongy bone; the process
comprises alkali washes and oxidative treatments that pre-
serve the organic and mineral parts of the bone matrix.
Granules of batches # 13517185 and #20108414 were
used.
Ca/P synthetic ceramics
Granules of sintered Ca/P ceramics were prepared by using
the polyurethane foam technology as extensively reported
elsewhere.2,15,16 Depending of the amount and nature of the
powder in the slurry, three types of materials were studied
in granules of 250–1000 mm and 1000–2000 mm in size.
R
TCP DentalV. These granules are prepared by Kasios SAS
(L’Union, France) with a slurry containing 25 g of b-TCP
powder per 100 mL of water. They are relatively compact
granules. Granules of batch 407/13.098 were used.
R
TCP Dental HPV. These granules are prepared by Kasios
SAS with a slurry containing 12.5 of b-TCP powder per
100 mL of water. Granules of batch #484/16.175 were
used. They present a high porosity making them to resem-
ble trabecular bone.
R
KeraOsV. These granules are prepared by Keramat (A
Coru~
na, Spain) Granules of batches #1115121 12007 were
used. The packaging indicates that these granules are com-
posed of b-TCP with a purity higher than 99%.
R
TCHV. These granules are prepared by Kasios SAS with a
slurry containing 25% of b-TCP and 75% of hydroxyapatite
powder per 100 mL of water. Granules of batch #404/
15.393 were used.
MicroCT analysis
MicroCT analysis was performed on a Skyscan 1172 micro-
computed tomograph (Bruker microCT, Kontich, Belgium).
Bone samples were placed in polystyrene test tubes
(10 mm in diameter) and scanned while in fixative. Particles
of the different types of biomaterials were placed in similar
tubes and gently agitated to allow the granules to settle and
optimize their 3D spatial arrangement in the dry state and
a volume of 330 mm3 was analyzed. Analysis was done in
triplicate with the different granules stacks except for Bio-
R
OssV 1000–2000 mm which was analyzed on six different
stacks. The microCT was operated at 80 kV, 100 mA with a
0.5 mm aluminum filter. The cubic voxel size was fixed at
4.95 mm and the angular rotation step at 0.258. 3D models
of bones and granules were obtained with VG Studiomax
(Volume Graphics GmbH, Heidelberg, Germany) operating in
the volume rendering mode. Microarchitecture parameters
were obtained with the CTAn software (Bruker) after
thresholding of the pores.17 They included measures of
porosity (Po, in %), mean pore diameter (Po.Diam, in mm)
3D MICROARCHITECTURE OF BIOMATERIALS

and specific surface (representing the interface between
bone or biomaterial and the porosity (Po.S/TV, in mm2/
mm3, where Po.S is the whole pore surface and TV, the vol-
ume of interest according to the ASBMR nomenclature.18).
For the bone cylinders and the granule stacks, the region
of interest (ROI) over-imposed on the 2D binarized sec-
tions was a 5 mm wide square; the height of analysis of
the samples has concerned 10 mm. The frequency distribu-
tion of pore diameter was determined by the sphere-fitting
method in the CTAn software.19 In order to further investi-
gate the porosity, the binarized stacks of 2D microCT sec-
tions of each sample were analyzed by a labmade software
(Vectopor) written in Matlab (MathWorks, Natick, MA). The
algorithm for evaluation of porosity used a vector projec-
tion on a frontal plane and has been extensively described
elsewhere.20 In the present study, the pixels of the ROI
which belonged to the same column received the same
pseudo-color according to the number of pixels superim-
posed on pores. A Look Up Table (LUT, a transfer function
which determines what screen values correspond to image
pixel intensity values at all x–y coordinates in the image)
was designed ranging from deep blue (no porosity) to red
(high porosity). Intermediate colors depend of the value of
porosity along the vector according to the LUT. The frontal
plane colored image was saved in the tif format with the
colorized LUT and analyzed by ImageJ (ImageJ 1.49c, NIH).
With this method, the large porous areas are in “hot col-
ors” (i.e., red, orange and yellow) and areas rich in bioma-
terial or bone appear in blue (“cold colors”). The method
to analyze quantitatively RGB images (Red–Green–Blue)
was proposed by several authors and is based on the three
histograms of the image.21–23 In the RGB color model, each
image can be decomposed in three planes (red, green, and
blue). The histogram of each bit plane was obtained with
ImageJ and transferred to an Excel (Microsoft) spreadsheet
(Microsoft Co). Then, only values comprised between 1
and 255 grey level (to discard irrelevant data) were
selected for computation. They were converted in percent
of the whole number of pixels and the ratio R of the
(red 1 green) pixels to the whole number of pixels was
derived. This method allows determination of the fraction
of the image containing “hot colors” because yellow is
obtained by adding red and green pixels values in the RGB
mode.
Raman spectroscopy
The chemical spectrum of the different types of granules
was analyzed by Raman spectroscopy on a Renishaw InVia
Qontor microscope (equipped with a Leica DM2700 micro-
scope, objective 203, NA 0.40, WD 0.39) with the WiRE 5.0
software (Renishaw, Champs sur Marne, France). The excita-
tion laser wavelength was 785 nm with an excitation power
of 50 mW and a grating of 1200 line/mm resolution. The
final spectrum was obtained by averaging four scans
acquired on different granules with a 10 s exposure time
for each spectrum. A concave rubberband base line correc-
tion was applied with an x11 polynomial equation.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2018 VOL 00B, ISSUE 00
ORIGINAL RESEARCH REPORT
FIGURE 1. MicroCT analysis of two bone cores harvested in subject
from the Anatomy laboratory. Note the regular arrangement of plates
and pillars creating the typical microarchitecture of trabecular bone
Statistical analysis
Statistical analysis was performed using the Systat statistical
software release 13.0 (Systat Software Inc., San Jose, CA).
All data were expressed as mean 6 standard deviation (SD).
The histogram frequency of pore diameters was computed
by pooling the data from different stacks of granules of
each formulation. Determination of polynomial equations
was computed with TableCurve (Systat Software) and the
best fit value was retained.
Differences for porosity between the groups were sought
with the Kruskall–Wallis non-parametric analysis of variance
followed by the Conover–Inman test for all pairwise com-
parisons. A difference was considered as significant when
p < 0.05.
RESULTS
MicroCT analysis
The microarchitecture of two cylinders of trabecular bone
appear on Figure 1. They are composed of trabeculae in the
form of plates connected by pillars and limiting larges
pores.
The microCT analysis of the different scaffolds of gran-
ules appears on Figure 2 for the 1000–2000 mm granules
and on Figure 3 for the 250–1000 mm granules. Granules of
R
pyrolyzed LubbocV are not imaged as they were exactly the
same than the original granules.
The largest granules of allo- or xenogenic bones were
composed of fragments of trabeculae stacked upon one
another. The original microarchitecture of trabecular bone
was disrupted but the trabeculae, which can easily be iden-
tified, maintained an interconnected porosity. There was no
morphological difference between xeno or allogenic bone
nor before or after pyrolysis/deproteinization. The only
R
noticeable difference was observed for Bio-OssV which con-
tained a substantial amount of cortical bone granules mixed
3
FIGURE 2. MicroCT analysis of stacks of 1000–2000 mm granules from different biomaterials. (A) Allogenic bone granules prepared from a
V R VR
human femoral head (Osteopure ); (B) Xenogenic bone granules prepared from bovine bone (Lubboc ); (C) Xenogenic bone granules prepared
V R
from bovine bone (Bio-Oss ), arrows indicate the presence of numerous particles of cortical bone with osteon canals; (D) Xenogenic bone gran-
VR V R R
V VR
ules prepared from bovine bone (CopiOs ); (E) granules TCP Dental HP ; (F) granules of TCP Dental ; (G) granules of KeraOs and (H) granules
RV
of TCH

with trabecular bone (Figure 2C). Cortical bone is easily rec-
ognizable by the presence of haversian canals and osteons
and more massive granules. Each different batch of this bio-
material were similar and contained cortical bone. The
respective amount of cortical/trabecular bone cannot be cal-
culated on the 3D model. For the synthetic ceramics, the
R
microarchitecture of the stacks of b-TCP HPV resembled
that of trabecular bone except the lack of anisotropy in the
blocks. The internal porosity due to the sublimations of the
polyurethane foam was easily evidenced as small connected
channels with a somewhat triangular section with concave
R R
sides. Stacks of TCP DentalV and TCHV were composed of

FIGURE 3. MicroCT analysis of stacks of 250–1000 mm granules from different biomaterials. (A) Allogenic bone granules prepared from a human
V R RV
femoral head (Osteopure ); (B) Xenogenic bone granules prepared from bovine bone (Lubboc ); (C) Xenogenic bone granules prepared from
V R
bovine bone (Bio-Oss ), arrows indicate the presence of particles of cortical bone with osteon canals; (D) Xenogenic bone granules prepared
V R VR R
V V R R
V
from bovine bone (CopiOs ); (E) granules of TCP Dental HP ; (F) granules of TCP Dental ; (G) granules of KeraOs ; and (H) granules of TCH

4 ARBEZ ET AL. 3D MICROARCHITECTURE OF BIOMATERIALS

 ORIGINAL RESEARCH REPORT

TABLE I. Morphometric Parameters Obtained by MicroCT on 3D Samples and Results Obtained From Analysis of Frontal Plane
Images Derived From Vector Analysis

Porosity (Po in %) Po.Diam (in mm) Po.S/TV (mm2/mm3) Ratio (R/(B 1 G)) %

Bone samples 83.7 6 7.8 929 6 299 2.22 6 0.45 53.8 6 4.9
250–1000 mm
R
OsteopureV 76.7 6 0.8 315 6 22 8.67 6 0.48 20.5 6 2.6
R
LubbocV 76.9 6 1.5 281 6 28 9.35 6 0.75 24.8 6 0.8
R
CopiOsV 65.3 6 1.9 222 6 15 10.31 6 0.41 16.7 6 2.1
R
LubbocV pyrolyzed 67.3 6 2.2 216 6 29 11.38 6 1.23 21.8 6 4.1
R
Bio-OssV 57.9 6 0.6 222 6 20 10.16 6 0.78 7.9 6 1.0
R
TCP Dental HPV 60.5 6 2.4 228 6 46 10.92 6 1.84 9.8 6 2.0
V R
TCP Dental 51.9 6 0.7 252 6 21 9.02 6 0.58 6.9 6 5.6
V R
KeraOs 59.3 6 0.7 281 6 3 8.83 6 0.25 4.4 6 0.9
R
TCHV 60.3 6 0.3 271 6 8 9.10 6 0.17 8.6 6 2.4
1000–2000 mm
R
OsteopureV 82.2 6 0.1 540 6 18 5.43 6 0.12 28.8 6 1.9
R
LubbocV 82.5 6 0.9 466 6 38 5.16 6 0.28 29.8 6 2.0
R
CopiosV 82.4 6 1.9 574 6 87 4.41 6 0.55 18.8 6 5.3
R
LubbocV pyrolyzed 83.0 6 1.7 499 6 43 5.11 6 0.40 25.8 6 2.0
R
Bio-OssV 71.3 6 2.7 515 6 56 5.27 6 0.41 14.5 6 3.3
R
TCP Dental HPV 79.4 6 1.7 519 6 16 5.69 6 0.11 25.5 6 3.6
R
TCP DentalV 59.6 6 2.0 474 6 21 5.61 6 0.12 5.9 6 1.3
R
KeraOsV 63.0 6 1.9 503 6 39 5.44 6 0.24 5.5 6 1.3
R
TCHV 63.7 6 0.5 497 6 14 5.61 6 1.61 10.3 6 0.6

more compact granules but they maintained an intercon-
R
nected porosity. Granules of KeraOsV also shown an internal
porosity due to polyurethane sublimation but also appear to
contain an enclosed porosity made of small round and non-
interconnected cavities.
For the 250–1000 mm granules of allo- or xenogenic
bone (Figure 3), the 3D aspect was very similar for all bony
R
materials, pyrolyzed or not, except for Bio-OssV in which
fragments of cortical bone could be also evidenced (Figure
3C). The stacks of these smaller granules appeared more
compact and the pores were smaller. For the synthetic
R
ceramics, the microarchitecture of the b-TCP HPV granules
was destroyed by crushing. Similar findings were observed
R R
for stacks of TCP DentalV and TCHV granules. Granules of
R
KeraOsV presented small non interconnected round cavities
in addition to the internal porosity due to the polyurethane
foam used to prepare the granules.
Morphometric analysis
The morphometric results appear on Table I. For each type
of biomaterial, the inter-granule porosity was reduced with
the 250–1000 mm when compared with the 1000–2000 mm
granules. This is due to a denser compaction of the smaller
granules resulting in a marked reduction of the Po.Diam.
R
Pyrolysis of the LubbocV granules had no effect on the 3D
microarchitecture of the stacks and results for Po, Po.Diam,
and Po.S/TV did not differ from the original granules. Poros-
ity obtained with the allo- or xenogenic large bone granules
was close to that of control human bone samples. However,
the Po.Diam was markedly reduced and the compaction of
the granules leads to an increase in Po.S/TV. This did not
R
apply to Bio-OssV due to the large amount of cortical bone
(identified by the presence of denser granules with osteons)
and porosity was significantly reduced when compared to
R R
LubbocV and CopiOsV which are constituted by pure frag-
ments of trabecular bone. Analysis of the frequency distri-
bution of Po.Diam confirm that all small granules had a
maximum peak centered at 250 mm when the larger gran-
ules had a more spread curve with values going up to 1250
mm. Figure 4 illustrates some distribution profiles for small
and large granules of xenogenic bone and ceramics. For
ceramic biomaterials, the curve was more irregular when
R
granules had an irregular shape (e.g., Kasios Dental HPV)
R
than when granules had smooth contours (e.g., KeraOsV).
Frontal plane analysis
The frontal plane obtained by the vector analysis software
is illustrated for some biomaterials on Figure 5. The pres-
ence of “red spots” indicates areas where porosity is maxi-
mal. The black and blue colors correspond to areas rich in
biomaterial with a limited porosity. Morphometric analysis
of these RGB images appears on Table I. The highest (R/
(B 1 G)) ratios correspond to stacks of biomaterials with a
marked 3D interconnected porosity.
Raman microspectroscopy
The Raman spectra of the main biomaterials appear on Fig-
ure 6. They were determined on the large and small gran-
ules but only minor changes were noted between the two
forms. On allo- and xenogenic bone, the presence of the
mineral and organic phases is clearly identified. The PO32
4
t1 peak appeared at 960 cm21, the PO43- t2 at 433,
PO43- t4 at 585 while the PO43- t3 and the CO32- are
combined in a band at 1070–1085. The organic phase of
the matrix is characterized by a band at 1243 for amide
III, 1672 for amide I, and 1455 for the CH2 and CH3
groups. This characteristic aspect of the calcified bone
R R R
matrix is observed for OsteopureV, LubbocV and CopiOsV. A

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2018 VOL 00B, ISSUE 00 5
FIGURE 4. Frequency distribution profiles of Po.Diam measured from microCT 3D models for granules of some xenogenic bone and ceramics.
Black curve: large granules ranging from 1000 to 2000 mm; grey curve: small granules ranging from 250 to 1000 mm

FIGURE 5. Frontal plane of stacks of granules analyzed by vector analysis. The presence of “red spots” indicates areas where porosity is maxi-
mal. The LUT used for all biomaterials appears at the bottom of the image and is used to transform the grey level of pixels to emphasize the
R
V
results. Left row: large granules ranging from 1000 to 2000 mm; right row: small granules ranging from 250 to 1000 mm. A, B: Lubboc ; C, D: TCP
R V R V
Dental HP ; E, F: KeraOs

6 ARBEZ ET AL. 3D MICROARCHITECTURE OF BIOMATERIALS


FIGURE 6. Raman microspectroscopic analysis of the biomaterials
used in the present series
higher 1070–1085 peak was noted for CopiOsV. Pyrolysis
R
suppressed the characteristic peaks from collagen pyrolyzed
LubbocV leaving only present the PO32
R
4 groups characteristic
of hydroxyapatite at 960 cm21. On the Bio-OssV biomate-
R
rial, the characteristic PO32
4 groups were also observed and
a small peak was noted in the 1250–1350 amide III band.
R
The spectrum of TCP Dental HPV granules evidenced the
typical peaks of PO32 4 t2, t3, t4, and the PO43- t1 as two
bands in the 948–970 cm21 range, narrowly separated. The
spectrum of KeraOsV was similar in the 200–1200 cm21
R
region and additional peaks were observed at 1309, 1380,
and 1475 that could be assigned to light scattering due to
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2018 VOL 00B, ISSUE 00
ORIGINAL RESEARCH REPORT
the granule size and shape (Prof. I. ur Rehman, personal
R
communication). On the TCHV granules mainly composed of
21
hydroxyapatite, the PO32
4 t1 signal appeared at 962 cm .
DISCUSSION
In the present study, a number of granular biomaterials
usable for filling bone defects were analyzed by microCT
and Raman microspectroscopy. They were either commercial
or prepared in the laboratory following classical methodol-
ogy. We prepared granules from commercial allo- and xeno-
genic bone to compare with commercial products and the
xenogenic bone was also pyrolyzed to be compared with
deproteinized granules sold by industrial companies. All dif-
ferent types of granules appeared to fill the 3D space thus
giving virtual scaffolds that mimic what happens when a
surgeon fills a bone cavity.5 The size and shape of the gran-
ules were found to considerably influence their 3D arrange-
ment. For all small granules (i.e., 250–1000 mm), porosity
and mean pore diameter were always found considerably
reduced when compared to the large granules (i.e., 1000–
2000 mm) of the same biomaterial. A careful analysis of the
frequency distribution curves of Po.Diam was much found
more informative.15 and revealed that the curves followed a
log-normal distribution for the small granules with a maxi-
mum frequency for Po.Diam near 200 mm. Clearly, a number
of papers have stressed that the optimum pore size of a
scaffold must be larger than 300 mm to allow invasion by
vascular sprouts and osteoprogenitor cells.6,24,25 When the
frequency distributions of the large granules were analyzed,
the maximum frequency was always >500 mm. Vector analy-
sis of the microCT images confirm these findings and show
that “hot spots” were considerably reduced on the frontal
plane images of the small granules with sometimes a quasi-
absence of red spots for the stacks of small granules. This
was also confirmed by the ratio of red pixels on blue and
green pixels when the frontal plane images were analyzed.
Vector analysis of porosity has been used for complex
objects in several papers of our group, including theoretical,
in vitro and ex vivo analysis of bones.20,23,26 The surface
density per volume unit Po.S/TV is an interesting parameter
to consider for biomaterial scaffolds. It represents the
amount of free surfaces of the biomaterial available to body
fluid and bone cell access. However, this parameter must be
considered in parallel with porosity. It is likely that the
small granules have an increase Po.S/TV when compared to
the large ones but the considerably reduced inter-granule
porosity of the former limits the interest of the use of these
biomaterials. On the other hand, for the large granules with
large Po.Diam, the higher Po.S/TV, the better. However, when
considering all the morphometric parameters obtained with
the different types of granules, they appear markedly different
for those of natural trabecular bone. This is because anatomi-
cal subjects used in the present study had a reduced bone
mass due to ageing.27–29 When bones from young subjects are
used for comparison, the morphometric 3D parameters of the
scaffolds of large granules are similar.24 When bone defects
are grafted granular biomaterials, a condensation on X-ray
7
images is always reported because (i) the biomaterial may
contain more calcium than the bone matrix (e.g., ceramics)30,31
and (ii) because patients candidate for a bone graft are gener-
ally aged persons.32–34 Intriguing findings were observed
R
with Bio-OssV which appeared composed of a noticeable
amount of cortical bone when the manufacturer website, the
booklet and product boxes clearly mention that the biomate-
rial is prepared from bovine spongy bone. We have bought
several samples from different batches and confirm that cor-
tical bone is always present in the different formulations ana-
lyzed here. Presence of cortical bone was confirmed
histologically after embedding granules in poly(methylmetha-
crylate) and sectioning the blocks with a diamond saw (data
not shown). Although the amount of cortical bone could not
be accurately quantified on the microCT stacks, it has some
influence on porosity and pore diameter when compared to
the other biomaterials prepared from bovine bone.
Raman microspectroscopy was also of interest in the
characterization of the granules. Raman analysis is a highly
specific and versatile technique that requires no sample
preparation for analysis of bone and bone substitutes. The
purified allo- or xenogenic bones presented the classical
peaks described for a mature bone with a carbonated
hydroxyapatite phase and a collagenic organic phase.35,36
R
The spectra for human OsteopureV and bovine bone
R R
LubbocV did not differ, however, CopiOsV was characterized
by an important peak in the phosphate m3-carbonate region
and numerous additional peaks in the 700–800 region that
could reflect the process used to clean the bovine bones or
could represent the selection of young animals to prepare
the grafts. Bones from young animals are characterized by
an a-carbonate substitution (substitution of OH2 groups in
the apatite crystal) while mature animal have a b-carbonate
substitution (substitution of PO324 groups).37 The effect of
R
pyrolysis is well observed on our LubbocV samples pyro-
lyzed at 8008C during 24 h with a complete disappearance
of the organic phase, leaving only the hydroxyapatite of the
bone matrix.38 For Bio-OssV, the spectrum was very similar
R
except the presence of a small peak at 1250–1350 cm21 in
the amide III region but not in the amide I and CH2–CH3
bands at 1450 and 1674 cm21, respectively. Traces of pro-
R
teins have been reported in Bio-OssV by SDS polyacrylamide
gel electrophoresis and Western blot analysis39 and recently
by the Kjeldahl method and thermogravimetric analysis.40
Other reports using Fourier transformed infra-red analysis
R
failed to identify any organic material in Bio-OssV and in
bone treated at 5008C for 18 h41,42 but they were still pre-
sent in bone samples heated at 3008C in the authors’ labo-
ratory.42 Raman analysis of b-TCP biomaterials was in
accordance with previously published findings reported in
the literature.43,44 In some batches of b-TCP biomaterials,
we inconsistently observe additional peaks in the 1300–
1500 cm21 region with a 785 nm laser illumination but not
with a 532 nm one. The intensity of these peaks, also
reported by others, depends on the size and shape of the
granules.44 In the TCHV biomaterial, the Raman spectrum
R
was that of hydroxyapatite with a phosphate v1 peak cen-
tered at 962 cm21 although the biomaterial contained
8 ARBEZ ET AL.
25% b-TCP. The analysis was performed at the surface of
the granules where the probability to find b-TCP areas is
low.2
CONCLUSION
We have analyzed a large series of nine granular biomateri-
als either manufactured and sold by different companies or
prepared in our laboratory to be used as controls. Two sizes
of granules from the same materials were considered when
available. All types of granular arrangements created scaf-
folds whose morphometric 3D characteristics could be ana-
lyzed by microCT. The small granules provided scaffolds
with low interconnected pores and may not be favorable for
vascular invasion in situ when grafted in a bone defect. The
large granules (i.e., 1000–2000 mm) gave 3D scaffolds with
interconnectivity well suitable for osteoconduction. Raman
spectrometry easily identified the molecular composition of
all types of biomaterials. Both methods appear interesting
to characterize accurately commercial biomaterials.
ACKNOWLEDGMENTS
This work was made possible by grants from ANR, program
LabCom “NextBone.” Mrs. N. Gaborit and S. Lemière are
thanked for their technical help with microCT analysis. Mrs F.
Pascaretti is thanked for help in Raman microspectroscopy.
We also thank the Kasios Company, 18 chemin de la Violette,
31240 L’Union, France, for providing the b-TCP-based gran-
ules, and particularly N. Guena and A. Lerch for helpful discus-
sions. The authors also thank Prof. Ihtesham ur Rehman
(University of Lahore, Pakistan and University of Sheffield, UK)
for helpful discussion on the microspectroscopic analysis of b-
TCP.
CONFLICT OF INTEREST
BA received a PhD scholarship from Kasios SAS via the
ANRT (Association Nationale Recherche Technologie) CIFRE
program.
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