CHARACTERIZATION OF STRUCTURAL, MECHANICAL AND CYTOTOXIC PROPERTIES

OF A CORAL POWDER-BASED COMPOSITE MATERIAL FOR BONE IMPLANT

APPLICATIONS
CARACTERIZACIÓN ESTRUCTURAL, MECÁNICA Y CITOTÓXICA DE UN MATERIAL COMPUESTO A BASE
DE POLVO DE CORAL PARA USO EN OSTEO-IMPLANTACIÓN

Angela Samper1, Fabio A. Rojas M.2, Diana Narváez3, Luis M. Méndez.4

1
Mechanical Engineering department, Universidad de los Andes, Bogotá, Colombia. E-mail: ang-
samp@uniandes.edu.co
2
Mechanical Engineering department, Universidad de los Andes, Bogotá, Colombia. E-mail:
farojas@uniandes.edu.co
3
Human Genetics Laboratory, Universidad de los Andes, Bogotá, Colombia. E-mail: di-narva@uniandes.edu.co
4
Mechanical engineering department, Universidad Nacional de Colombia, Bogotá, Colombia. E- mail:
lmmendezm@unal.edu.co

ABSTRACT
Different studies concerning the application of Porites asteroides coral in bone implant purposes
have demonstrated the biological viability of its use. As a complement to previous investigations
regarding the development of bone-powder based composite materials which are useful for
these applications, this study has the purpose of developing a coral powder-based composite
material, which is able to satisfy the structural, mechanical and cytotoxic properties required in
order to be considered appropriate for this use. A composite material made of coral powder,
Calcium sulfate powder and water was therefore developed, and its properties were tested upon
different compositions. General results show how the developed composite material possesses
properties that are comparable to those of human cortical bone (both at a structural and
mechanical point of view), as well as being non-toxic below a critical composite material
concentration of 0.35 mg/ml.
Key words: Porites asteroides coral, bone implants, composite material, Calcium sulfate,
characterization of biomaterials.

RESUMEN
Diferentes estudios referentes a la aplicación del coral tipo Porites asteroides en implantes
óseos han demostrado su viabilidad biológica para este fin. Como complemento a
investigaciones previas relacionadas con el desarrollo de un material compuesto a base de
polvo de hueso susceptible de osteo-implantación, el propósito de este estudio es desarrollar un
material compuesto a base de polvo de coral, que satisfaga las propiedades estructurales,
mecánicas y citotóxicas requeridas para ser considerado como un material adecuado para este
fin. A partir de esto se desarrolló un material a base de polvo de coral, polvo de sulfato de calcio
y agua, y se realizaron los respectivos ensayos en diferentes composiciones del mismo. Los
resultados generales muestran que el material compuesto desarrollado posee propiedades
comparables a las del hueso cortical humano (desde un punto de vista tanto estructural como
mecánico) y adicionalmente no muestra evidencias de citotoxicidad si se utiliza en
concentraciones menores a 0.35 mg/ml.

Palabras clave: coral Porites asteroides, implantes óseos, material compuesto, sulfato de
calcio, caracterización de biomateriales.

1. INTRODUCTION
This study is based in the development of a
Porites asteroides coral powder-based
composite material for use in future bone
implants. P. asteroides coral is made up of
99% Calcium Carbonate, known as
Aragonite mineral which grows throughout
the Pacific Coast coral reefs. Because of its
biological properties, this material has been
studied for implant purposes for more than
15 years (Vuola et al., 1996). Its structure is
similar to that of human cortical bone, and it
is considered to be “one of a limited number
of materials that will form chemical bonds
with bone and soft tissues in vivo” (Ben-
Nissan, 2003, p. 285). Through several heat
treatments, a mineral known as porous
Hydroxyapatite (HA) can be obtained from
P. asteroides coral. This substance is
structurally similar to P. asteroides coral,
and is also used in implant purposes. The
difference between HA and coral is that HA
is not biodegradable, making the implant to
last more time. (Vuola et al., 1996; Ben-
Nissan, 2003). The application of this type of
coral in bone implants has been widely
studied, in order to analyze its features
(Begley et al., 1995; Vuola et al., 1996; Ben-
Nissan, 2003: Ripamonti et al., 2008).
These studies have shown P. asteroides’s
biological viability for this purpose, as it is a
biocompatible, osteo-conductive and inert
material. Additionally, it has been stated that
this type of coral possesses osteo-inductive
properties when exposed to bone marrow
cells, but not by its self (Braye et al., 1996;
Vuola et al., 1996).
In spite of its biological properties that are
highly appropriate for implant purposes, P.
asteroides coral possesses limited
mechanical properties (Ben-Nissan, 2003),
which are equally fundamental for the
development of bone grafts and implants
that will assure an adequate level of
resistance in vivo. Therefore, in order to
develop an appropriate composite material
for bone implant purposes it is essential to
consider the mechanical strength that the in mm/min. These are followed by the tool
matrix material will need to provide. Local nose radius (r) in mm, in a smaller amount.
investigations have focused on this concern, It was found that the rest of the observed
having mainly examined the development of parameters, that is the rake and clearance
human (Rojas, 2000) and bovine bone angles ( α and β) in ° and the cutting speed
powder-based (Peñaloza, 2007; Quevedo, (v) in m/s had little or no influence in the
2004; Reina, 2005; Salazar, 2009) analyzed variables. While processing the
composite materials that satisfy implant coral specimen, a factorial experiment was
purposes requirements. Bone powder designed in order to study the effect of
showed to be an appropriate alternative for varying the specimen’s machining
developing bone implants, as it was easy to conditions on the type of powder obtained
obtain at a minimum amount of lost material. from each one, especially in terms of its size
This study describes two main phases: the and shape. Two main factors were chosen
development of a coral powder-based for analysis: the feed rate (f) in mm/s and
composite material and the characterization the depth of cut (p) in mm. Both factors were
of its structural, mechanical and cytotoxic varied according to three levels, giving as a
properties in order to determine its level of result a total of six treatment tests (see
appropriateness in bone implant Table 1).
applications. Experimental
Units
Level Level Level
factor 1 2 3
Feed rate (f) mm/s 0.16 0.24 0.37

2. MATERIALS AND METHODS Cutting depth (p) mm 1 1.5 2

Table 1. List of chosen experimental factors and their
respective levels.
2.1 Production of coral powder particles
The feed rate (f) levels were set according

Tangential milling was chosen to be the to the milling cutter’s available values; On

most appropriate machining mechanism to the other hand, the depth of cut (p) was

obtain coral powder, as the specimen’s divided into three uniform levels, each one

shape didn’t have to be modified, or separated by 0.5 mm from the other.

combined with any other substance. Factors that were held constant during the

According to previous related studies (Lugo, experiment were the speed of the cutting

2009), the most influential cutting tool (v) and the rake and clearance angles

parameters that affect superficial roughness (α and β), along with the tool nose radius (r)

and cutting force of this material, are the and the tool diameter (d). Even though

depth of cut (p) in mm, and the feed rate (f) these factors remain constant during the
experiment, they may nevertheless affect
the size of the obtained particles, according
to theoretical relations between this variable
and the different cutting parameters during a
regular milling process (Micheletti, 1980).
process of the specimen while machining
its border zones. These particles were
therefore classified as Type 4.

2.2 Characterization of produced coral

powder

Initial results showed that a mixture of

Figure 2. Appearance of type 2 P. asteroides powder
different sized particles appeared in each obtained during machining.
sample. After separating each mixture
through a sieving process, three main types
of powder were obtained, differentiated by
their size range, and grouped in the
following categories: Type 1 particles (see
Figure 1), which were smaller than 250 µm,
Type 2 particles (see Figure 2), which were
Figure 3. Appearance of type 3 P. asteroides powder
sized between 250 µm and 500 µm, and obtained during machining.

Type 3 particles (see Figure 3), which were

sized between 500 µm and 1000 µm.
Figure 1. Appearance of type 1 P. asteroides powder
obtained during machining.
Finally, particles larger than 1 mm were
discarded as they were part of a much
larger and irregular type of chip, which was
  Due to the irregularities in shape and size of the
due not to machining, but to a tearing
After having completed the factorial
experiment, each sample was statistically
analyzed in order to examine the influence
of the chosen factors in the powder’s main
features. Given that three main types of
powder were obtained per sample, three
separate analyses were carried out for each
one.1 The response variables chosen to
study each type of powder were the
following: 1) The particles’ size (s) in µm, 2)
The particles’ shape factor (sf), and 3) The
weight proportion (w) that each type of
1
Type 4 observed particles, these were not submitted
to further characterization or analysis of any type.
powder represented within the complete
sample, expressed as a percentage. The
particles’ size (s) was obtained through an
optical analysis of each type of powder’s
sample, by calculating the smallest diameter
upon which each particle could be fully
surrounded. In a similar way, the particles’
shape factor (sf) was calculated by dividing
the largest diameter that could be inscribed
inside each particle, by its previously
calculated size. For each test, 30 particle
measurements were made, in order to
acquire a statistically relevant sample of the
total population. Finally, it was necessary to
include the measurement of the weight
proportion (w) that each type of powder
represented in the sample, as to examine its
variability over the different tests carried out.
As three types of powder were analyzed per
test, a total of 18 samples were studied
(accounting for 6 tests originally made).
A two factor ANOVA (Analysis of Variance)
was built with the purpose of comparing the
averages obtained for size (s), shape factor
(sf) and weight proportion (w) in each
sample with those property averages of
samples relative to the same type of
powder.
2.3 Developing the composite material
Recent studies suggest the use of Calcium
  Type 1 coral powder wasn’t used to build the
sulfate as appropriate for implant purposes
(Cho et al., 2005). Commercially known as
gypsum, this substance has been widely
used for developing dental and orthopedic
implants (Pecora et al., 1997; Tay et al.,
1999), demonstrating its in vivo
biocompatible and osteo-conductive
properties while implanted (De Long et al.,
2007; Jung et al., 2010). An additional
advantage of using Calcium sulfate is its
easy manipulation, given that it hardens
through a chemical process at room
temperature, without the need of adding any
substance or applying any further treatment
to it. The composite material was therefore
made of coral powder, Calcium sulfate
powder and water. A factorial experiment
was designed to analyze whether different
amounts of coral powder and Calcium
sulfate added would influence the properties
of the final composite material (chosen
experimental factors are shown in Table 2).
The amount of coral powder related to the
amount of Calcium sulfate was studied in
order to examine the effects of adding more
coral powder (independently of its type) to
the composite material, whereas the amount
of Type 2 with respect to Type 3 coral
powder added was analyzed in order to
evaluate the difference between using one
or another type of coral while developing the
composite material.2
2
composite material, as its small size could potentially
affect its biocompatibility.
 Control factor Units
Level Level Level was performed in Chinese hamster ovary K1
1 2 3
Amount of coral powder cells (CHO-K1) exposed to different
% 10 30 50
used with respect to CS
concentrations of coral powder alone,
Amount of Type 2 coral
powder used with respect % 0 50 100 Calcium sulfate powder alone, and three
to Type 3
Table 2. List of chosen experimental factors and their
different samples of the composite material.3
respective levels (CS stands for Calcium Sulfate). The CHO-K1 cells were grown as
monolayers in Roswell Park Memorial
2.3.1. Mechanical properties
Institute (RPMI) 1640 medium (Sigma),
supplemented with 10% fetal bovine serum
Tests for Shore D hardness (H), apparent
(FBS), 1% penicillin/streptomycin (Gibco)
porosity (Pa), compressive strength (Sc) and
and 2% glutamine (Gibco). All samples
modulus (Mc), and flexural strength (Sf) and
tested were previously left in U.V. for 10
modulus (Mf) were carried out for each
minutes in order to sterilize them. After UV
sample in the Mechanical Properties
irradiation, the different samples were
laboratory at Universidad de los Andes.
pulverized and then prepared in RPMI
Shore D hardness test was carried upon 10
medium, as follows: a) 2mg/ml of coral
specimens of each sample following the
powder alone; b) 2mg/ml of Calcium sulfate
ASTM D695-08 norm, while compressive
alone; c) 0.05, 0.5, and 2 mg/ml of the 10,
and flexural tests were made upon five
30, and 50 percent of coral powder and
specimens per sample, as suggested in the
Calcium sulfate mixtures, respectively.
ASTM norms D790-02 and D2240-05 5
3x10 cells per sample were grown in 96-
respectively. Porosity was tested upon the
sample flat-bottomed plates, with four
same specimens used for testing flexural
replicate samples per treatment. The fifth
properties, as suggested by Peñaloza
test of each treatment corresponded to a
(2007).
blank reference, consisting of 100 µ l of
culture medium only. Cells were examined
2.3.2 Cytotoxic properties
after 24 hours of incubation and treated. The
first sample corresponded to the reference
Chronic Cytotoxicity tests were carried out in
control, consisting of 100 µl of culture
the Human Genetics Laboratory at
medium, and the remaining samples
Universidad de los Andes. They were
contained 100 µl of medium with a known
measured by a colorimetric method using 3-
concentration. The plate was then incubated
[4,5-dimethylthiazol-2-yl]-2,5-
3
diphenyltetrazolium bromide (MTT)
  These included the three different levels of amount
of coral added with respect to the amount of Calcium
according to Mosmann (1983). Each test sulfate, independently of the coral powder type.
 
 
at 37ºC in a humidified 5% CO2 atmosphere Variable Units
Type 1 Type 2 Type 3
powder powder powder
for 72 hours. After this, MTT (5 mg/ml) was Size (s) µm 211.87 474.56 936.56
Shape factor
added to each sample. Cells were incubated (sf)
none 0.51 0.49 0.48
Weight
for 4 further hours. Afterwards, dimethyl- proportion % 28.36 34.50 20.83
(w)
sulfoxide was added to dissolve Formazan Table 3. Average values obtained for each response
crystals. After 5 minutes of agitation, each variable as observed in Types 1, 2 and 3 powders.

sample was analyzed in a Bio-Rad micro

As it can be seen, each type of powder
plate reader at 595 nm, and a reference
differs considerably in size (s) but not in
wavelength of 655 nm. Results were
shape, as all shape factors (sf) results are
expressed as the percentage of living cells
fairly similar. On the other hand, the weight
as calculated from absorbance detected,
proportion (w) represented by each type
assuming 100% as the absorbance reported
shows that Type 2 powder is more abundant
by the reference control sample. The
than the other two types within each sample.
chronic cytotoxicity test was repeated twice,
and a Two-sample t-test was performed to
3.2 Structural properties
test differences between experiments, along
with a Pearson’s statistical correlation test to
A visual mechanism was used in order to
look for correlation among cell viability and
examine the samples structure and the way
the different concentrations used.
in which coral particles were agglutinated
with the [Calcium sulfate + water]
3. RESULTS
compound. Structural imperfections such as
pores, impurities or particular defects were
3.1 Characterization of produced coral
also examined. The different samples
powder
showed a high level of agglutination
between the different substances that
As the three different types of powder
formed the composite material. Coral
obtained, showed to have statistically
powder was effectively surrounded by the
equivalent analyzed properties, they could
[Calcium sulfate + water] compound, this
be grouped in three categories,
way preserving the composite’s integrity
independently of the sample they came
(see Figures 4 and 5). However, samples
from. Table 3 describes the average
showed to have a large amount of pores
obtained as statistical results for these
throughout their structure, mainly due to
groups.
Calcium sulfate’s solidifying process
occurring inside the different molds used for as the difference between the averages in
constructing specimens. each sample didn’t prove to be statistically
different.

Figure 4. Agglutination of coral powder and the

[Calcium sulfate + water] compound inside a
composite material sample (18X). Figure 6. Results for Shore D hardness (H) tested
upon every sample.

Figure 5. Agglutination of coral powder and the

Figure 7. Results for apparent porosity (Pa) tested
[Calcium sulfate + water] compound in a composite
upon every sample.
material sample (100X).

3.3 Mechanical properties

Average values for each sample tested were

recorded (see Figures 6 through 11).4 With
the aid of an ANOVA test, results show that
Figure 8. Results for compressive strength (Sc) tested
variations in apparent porosity (Pa), upon every sample.

compressive strength (Sc) and flexural

strength (Sf) values can be explained due to
variations in the amount of coral powder
used with respect to Calcium sulfate in each
sample, given that their corresponding p-
value is below the reference level of 5%.
Figure 9. Results for compressive modulus (Mc) tested
Other properties’ variations proved not be upon every sample.

caused by any of the analyzed treatments,

4
Samples labeled “0” in the graphs represent the
reference sample made of [Calcium sulfate + water]
compound only.
 shown in Figures 13 to 14 compared with
the control in Figure 15.

Figure 10. Results for flexural strength (Sf) tested

upon every sample.

Figure 11. Results for flexural modulus (Mf) tested

upon every sample.
Figure 12. Coral powder and Calcium sulfate effect on
CHO-K1 cell viability after 72 hour exposure.
3.4 Cytotoxic properties Pearson’s correlation value: r=1.00 and r=-1.00
(p<0.01), respectively.

After a 72 hour exposure to pure coral

powder (2 mg/ml), CHO-K1 cells exhibited a
dose-dependent viability increase (see
Figure 12). The Pearson’s Correlation value
suggests that the increase in pure coral
powder concentration was significantly
correlated with an increase in cell viability. Figure 13. Coral powder cytotoxic test image after 72
hour CHO-K1 cell exposure (Coral concentration: 2
On the other hand, the exposure to pure mg/ml).
Calcium sulfate powder showed a dose-
dependent viability decrease, as an increase
in Calcium sulfate’s concentration was
significantly correlated with a decrease in
cell viability according to the corresponding
Pearson’s correlation value. In this case, the
lethal concentration at which 50% of the
cells die (LC50) was approximately 1.3 mg Figure 14. Calcium sulfate cytotoxic test image after
72 hour CHO-K1 cell exposure (Calcium sulfate
/ml. Images according to these results are concentration: 2 mg/ml).
 sample, and 0.35 mg/ml for the 30% and
50% of coral powder samples. Images of
these results (for a composite material
concentration of 0.5 mg/ml) are shown in
Figures 17 to 19.

Figure 15. Cytotoxic test reference control sample,

composed by 100 µl of culture medium.

It can be observed how coral powder

represents no danger for cell viability at a
concentration of 2 mg/ml, whereas a
Calcium sulfate concentration of 2 mg/ml is Figure 17. Cytotoxic test image for 10% coral
composite material after 72 hour CHO-K1 cell
harmful for living cells (Figure 12). Only exposure (Material concentration: 0.5 mg/ml).
about 20% of them are able to survive in
these conditions.

Figure 18. Cytotoxic test image for 30% coral

composite material after 72 hour CHO-K1 cell
exposure (Material concentration: 0.5 mg/ml).

Figure 16. Different samples of composite material

effect on CHO-K1 cell viability after 72 hour exposure.
Pearson’s correlation value: r=-0.949, -0.912, and -
0.901 (p>0.05), respectively.

Additionally, cell exposure to the three

different composite material samples did not
show significant correlation with cell viability
(see Figure 16). LC50 levels were found to
be 0.5 mg/ml for the 10% of coral powder
Figure 19. Cytotoxic test image for 50% coral
composite material after 72 hour CHO-K1 cell
exposure (Material concentration: 0.5 mg/ml).
Results show that a concentration of 0.5
mg/ml of the composite material is only
adequate if using a 10% coral powder However, efforts could be made in order to
sample (Figure 17). Higher coral powder improve its compressive strength results.
proportions within the composite material
Salazar’s Peñaloza’s
Cortical Current
Property Units compound compound
show a cell viability decrease at a bone
(2009) (2007)
compound

concentration of 0.5 mg/ml, as seen in

Flexural
MPa - 1.4 - 3.2 0.31-13.64 8.36-17.56
Figures 18 and 19. strength (Sf)

Flexural
1.525-
modulus GPa 0.063-0.142 0.097-7.07 0.89-1.83
31.519
(Mf)
4. CONCLUSIONS
Compressive
MPa 33-193 22.4-31.8 0.71-16.12 13.78-34.56
strength (Sc)

A coral powder-based composite material

Shore D Shore
85-95 72.8-81.2 36.5 - 78.75 76-83.4
hardness (H) D
was effectively produced through a process
Table 4. Comparative values for flexural properties,
that allowed its reproducibility at a minimum
compressive properties and Shore D harness tested
level of material waste. Its structure was upon previously developed compounds and cortical
bone.
analyzed and tests were made for its
apparent porosity (Pa), Shore D hardness 5. NOMENCLATURE

(H), compressive strength (Sc) and modulus p: Depth of cut (mm)

(Mc), flexural strength (Sf) and modulus (Mf), f: Feed rate (mm/min)

and cytotoxic levels. General results showed r: Cutting tool nose radius (mm)

how the different properties tested varied α: Rake angle (°)

along with a larger amount of coral powder β: Clearance angle (°)
within the compound (regardless of the type
v: Cutting speed (m/s)
of powder present). As this factor increased,
d: Cutting tool diameter (mm)
the compound's compressive and flexural
s: Size of particle (µm)
properties decreased, while its apparent
sf: Shape factor of particle (µm/µm)
porosity showed a slight increase and Shore
w: Weight proportion (%)
D hardness remained stable. In comparison
with previously developed bone-implant H: Shore D hardness (Shore D)

compounds (Peñaloza, 2007; Salazar, 2009 Pa: Apparent porosity (%)

and with liofilized cortical bone) the Sc: Compressive strength (MPa)

compound possesses appropriate values for Mc: Compressive modulus (GPa)

5
most of the properties tested (see Table 4). Sf: Flexural strength (MPa)

Mf: Flexural modulus (GPa)

5
Cortical bone data was collected on November 25th,
2009 from Salazar (2009) and through the link:
www.bonesim.com/. Missing properties were those
not found for all of the specimens.
6. References pathology, Oral radiology, and Endodontology,
Vol. 84;4, 1997., pp. 424-429.
Begley, C.T. et al., Comparative study of the
Peñaloza, J., Diseño de un material compuesto para
osteoinductive properties of bioceramic, coral and
implantes óseos. Tesis presentada a la
processed bone graft substitutes. Biomaterials,
Universidad de los Andes como requisito de
Vol. 16, 1995, pp. 1181-1185.
grado del programa de pregado en Ingeniería
Ben-Nissan, B., Natural bioceramics: From coral to
Mecánica, 2007.
bone and beyond. Current opinion in solid state
Quevedo, S. M., Desarrollo de una metodología para
and materials science 7 (4-5), 2003,. pp. 283-288.
la fabricación de injertos compuestos de polvo de
Braye, F., et al., Resorption kinetics of osseous
hueso y biopolímero. Tesis presentada a la
substitute: natural coral and synthetic
Universidad de los Andes como requisito de
hydroxyapatite. Biomaterials 17, 1996., pp. 1345
grado del programa de pregado en Ingeniería
-1350.
Mecánica, 2004.
Cho, B.C. et al., The effect of chitosan bead
Reina, D. Y., Implementación de un sistema de
encapsulating Calcium sulfate as an injectable
manufactura de injertos de polvo de hueso por
bone substitute on consolidation in the
inyección. Tesis presentada a la Universidad de
mandibular distraction osteogenesis of a dog
los Andes como requisito de grado del programa
model. Journal of oral and maxillofacial surgery,
de pregado en Ingeniería Mecánica, 2005.
Vol. 63:12, 2005., pp. 1753-1764.
Ripamonti, U. et al., The induction of bone formation
De Long Jr., W.G. et al., Bone grafts and bone graft
by coral-derived Calcium
substitutes in orthopaedic trauma surgery. A
carbonate/hydroxyapatite constructs.
critical analysis. The Journal of Bone and Joint
Biomaterials, Vol. 30, 2009, pp. 1428-1439.
Surgery, 2007;89, pp. 649-658.
Rojas, F.A., Fabricación de implantes ortopédicos a
Jung, H. et al., Modulation of the resorption and
partir de hueso humano. Tesis presentada a la
osteoconductivity of α-Calcium sulfate by histone
Universidad Federal de Santa Catarina para optar
deacetylase inhibitors. Biomaterials 31, 2010., pp.
al título de Doctor of Philosophy, 2000.
29-37.
Salazar, A., Diseño de un proceso de fabricación de
Lugo S., H., Estudio de la mecánica de corte en
implantes dentales y ortopédicos de polvo de
materiales celulares: coral tipo Porites
hueso y sulfato de calcio con aplicación industrial.
Asteroides. Tesis presentada a la Universidad de
Tesis presentada a la Universidad de los Andes
los Andes, para optar al título de Magister en
como requisito de grado del programa de
Ingeniería Mecánica, 2009.
pregado en Ingeniería Mecánica, 2009.
Micheletti, G.F., Tecnología mecánica: Mecanizado
Tay, B. et al., Calcium sulfate – and Calcium
por arranque de viruta., Ed. Blume, Barcelona,
phosphate based bone substitutes: Mimicry of the
España, 1980.
mineral phase of bone. Orthopedic Clinics of
Mosmann, T. Rapid colorimetric test for cellular
North America, Vol. 30;4, 1999., pp. 615-632.
growth and survival – application to proliferation
Vuola, J., et al., Bone marrow induced osteogenesis in
and cytotoxicity tests., J. Immunol, 1983.,
hydroxyapatite and Calcium carbonate implants.
Métodos 65, 55, 63.
Biomaterials 17, 1996., pp. 1761-1766.
Pecora, G. et al., Bone regeneration with a Calcium
sulfate barrier. Oral surgery, Oral medicine, Oral