Mater. Res. Soc. Symp. Proc. Vol.

1487 © 2012 Materials Research Society

DOI: 10.1557/opl.2012.1528

Scaffolds of Collagen from Nukbone ®

Benjamín H. León 1 Miguel A. Araiza 2 M. Cristina Piña 3

1
Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria,
Coyoacán, C.P. 04510, México D.F.
2
Facultad de Odontología, División de Estudios de Posgrado e Investigación, Universidad
Nacional Autónoma de México, Cuidad Universitaria, Coyoacán, C.P. 04510, México D.F.
3
Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México,
Ciudad Universitaria, Circuito Exterior s/n, Coyoacán, C.P. 04510, México D.F.

ABSTRACT

The scaffold is obtained from acellular bovine bone: Nukbone (produced by Biocriss SAPI
de CV). This acellular bone was subjected to a demineralization process after which the
composition was found to be 10% water, 65% of collagen and 25% of hydroxyapatite.
The techniques used to characterize these natural scaffolds were: optical microscopy, scanning
electron microscopy, X-ray diffraction, energy dispersive X-ray spectroscopy, thermo
gravimetric analysis, differential scanning calorimetry and determination of pore size using
nitrogen adsorption, and physical adsorption of N2. The pore size is between 100 and 500
microns. These scaffolds have been tested in several biological tissues as urethra, trachea, blood
vessels, bone and heart successfully.

INTRODUCTION

Collagen has received increasing attention over the last years due to its excellent
biocompatibility, degradation into physiological end-products, and suitable interaction with cells
and other macromolecules. The favorable influence of collagen on cellular infiltration and
wound healing is well known. An additional benefit is that collagen can be processed on an
aqueous base. Collagen sponges have a long safety history as hemostatic agents and wound
coverings [1-3] and are under investigation as scaffolds in the emerging field of tissue
engineering [4].
The manufacturing of collagen sponge implants generally starts with the preparation of
purified collagen material into aqueous solutions or suspensions at adequate pH [5]. In this work
we obtained the collagen from bovine bone matrix after a demineralization process. The
physicochemical characterization of the collagen was done by optical microscopy (OM),
scanning electron microscopy (SEM), X-ray diffraction, spectroscopy of X-ray scattering (EDS),
thermo gravimetric analysis (TGA) and determination of pore size and specific area using BET.
One prerequisite of systems intended for parenteral application is sterility [6]. Sterile
products can be obtained with methods using heat (dry heat and autoclaving) and through ‘cold’
processes, i.e. microbicidal gases or high-energy irradiation. One alternative to obtain sterile
collagen products is treatment with ethylene oxide [7]. The results obtained from the structural
and thermal stability of the scaffolds will allow us to define the best sterilization method.
 EXPERIMENTAL DETAILS

The collagen scaffolds were obtained from blocks of 2.5 x 2.5 x 0.5 cm. of bovine bone
matrix, which were cleaned by physical, and chemical methods leaving a completely decellula-
rized matrix (Nukbone® produced by Biocriss SA de CV). The Nukbone bone matrix (BM) was
subjected to an acidic solution of 0.5M HCl [8] for 10 minutes under constant stirring. After this
time, the pieces were washed using distilled water and allowed to dry for 48 hours at room
temperature leaving only the demineralized bone matrix (DBM) used as the collagen scaffold.
The structure of the scaffold was analyzed using optical microscopy (OM) and histology
techniques, with Hematoxylin & Eosin (H&E) that stains in red the organic material, and
Masson’s trichrome staining, which stains the collagen fibers stained in blue [9,10]. For these
analyses the collagen scaffold obtained or the DBM was embedded in paraffin and cut in
sections of 3 µm thickness.
The analysis of the surface, as well as the determination of the elements contained in the
sample was performed using a JEOL JSM 7600 SEM in conjunction with EDS system [11]. The
samples were not covered with conductive layer.
X-ray diffraction patterns were obtained using a Bruker D8 Advance diffractometer, to have
information about crystal structure, chemical composition and crystalline phases of the sample
[12], if there were any.
In order to determine the collagen thermal behavior, thermo gravimetric analysis (TGA) was
performed using a TA Instruments SDTQ60, where the changed in weight as a function of the
temperature was determined in a controlled Nitrogen atmosphere [13,14].
The physical adsorption of a gas molecules on a solid surface serves as bases for obtaining
the specific surface area of a material [15], this technique is called BET and was performed with
an Quantachrome autosorb -1, the gas used was N2, the specimens were treated to a temperature
of 77° K to obtain the isotherms.

RESULTS AND DISCUSSION

In the Figure 1, it can be seen the transformations of BM from the natural stage to DBM.

a b c

Figure 1. a) Bovine bone cubes before cleaning treatment. b) Bovine bone cube after cleaning
treatment (Nukbone ). c) Collagen scaffold obtained after the demineralizing process.
 The figure 2a shows a section of trabecular DBM treated with histological techniques and H
& E staining, which, by the color it takes, shows that it is only an organic tissue. Figure 2b
corresponds to another section of the trabecular DBM treated with Masson’s trichromic staining,
the blue color corresponds to collagen while the red color corresponds to mineralized tissue,
from which we can see that the mineral (hidroxiapatite: HA) is concentrated only on the outer
edges of the trabeculae. Figure 2c corresponds to a sample observed with OM under polarized
light, this technique allowed us to observe the orientation of bundles of collagen fibers,
determining morphological characteristics of the trabecular structures.

a b c

Figure 2. a) Sample treated with H&E staining, was view with OM at 50x, where the trabecular
structure of bone without HA takes red color due to Eosinophilic pigmentation, which expressed
his affinity for organic structures. b) Sample treated with Masson’s trichromic, with OM at 50x,
the blue color corresponds to collagen tissue while the red color corresponds to mineralized
tissue. c) Sample observed with OM under polarized light at 50x, the bundles of collagen fibers
were observed.

The SEM allows observing the 3D structure of trabecular bone tissue, before and after the
demineralization process. Figure 3a shows the mineralized matrix, where one can clearly
observed a smooth surface corresponding to the mineralized trabecula, while the rough part
corresponds to the inside of the trabecula, that is organic material corresponding to the collagen.
Figure 3b corresponds to the DBM, it is clear that there is a smooth surface that corresponds to
just below the ceramic surface corresponding to the HA, however by EDS no trace of mineral
material were found, it means that there is not ceramic, see Figure 3c.

a c C

DBM
* BM DBM N

X 500
 Figure 3. a) Microstructure of bone matrix where BM is mineralized bone and DBM shows
the demineralized bone matrix. b) Microstructure of DBM, the smooth surface come into just
below to HA surface in a BM. c) EDS spectra of the samples.

Figure 4 shows the XRD patterns. Figure 4a) shows that the BM consists of a crystalline
structure which can be identified as HA crystals. On the other hand, Figure 4b) shows the XRD
pattern obtained of the DBM indicating that it is an amorphous material, with only traces of HA
crystals, this is probably because they are contained within the volume of the collagen bundles.

Figure 4. XRD patterns were compared with the file number 09-432 of the Joint Committee Powder
Difraction (JCDP). a) Corresponding to BM. b) Corresponding to DBM.

The thermogravimetric curves were obtained from room temperature until to 700 ° C
making evident at least three mass losses for the case of BM, see Figure 5a. The first one at
79.69°C that would corresponds to evaporation of the water trapped in the matrix and that is
around of 6.07% by weight and involves the denaturation of collagen that forms the bone. The
second and third occurs at 341.07 ° C, a total mass loss of 18.09% and a mass loss of 30.92% to
493.26°C which corresponds to the loss of the organic material. Finally, the remaining mass of
63.59% of the total corresponds to bone mineral.
The TGA curve for to the DBM, see Figure 5b, has two mass losses clearly visible, the first
corresponding to a mass loss of the order 15% and ending at about 150°C and another around
312 ° C corresponding to the decomposition of collagen. The remaining mass at 700°C which
corresponds to 25.64% of the total, is due to the amount of mineral remaining in the volume of
the collagen bundles which are not dissolved during the demineralization process and a small
quantity of organic material of a different nature that remains in the collagen.
The reported specific surface area by nitrogen adsorption for the BM was to 5.39 m2/g,
while the DBM was 7.34 m2/g, having a difference of 36.18%. The pore size of BM and DBM
was reported as macroporous; the isotherms obtained were of type II, according to reports from
the IUPAC [16], see Figure 6 a-b.
The reported specific surface area by nitrogen adsorption for the BM was to 5.39 m2/g,
while the DBM was 7.34 m2/g, having a difference of 36.18%. The pore size of BM and DBM
was reported as macroporous; the isotherms obtained were of type II, according to reports from
the IUPAC [16], see Figure 6 a-b.

CONCLUSIONS
The results of this study clearly show that is possible to use Nukbone as source of scaffolds
of collagen. Furthermore, it can be seen that the collagen fiber bundles are oriented following the
direction of the bone mineral (HA), which eventually results in the trabecular structure. HA
crystals are preferentially on the surface of the trabecular structure, which was verified with light
microscopy techniques.

Figure 5. a) Thermogravimetric curve for MBM and b) TGA curve for DBM. The red curves
correspond to the derivate of TGA curves

Figure 6. a) The BM and b) DBM isotherms obtained using the BET method. In both cases,
the isotherms were type II according to the IUPAC classification. The maximum volume of
nitrogen adsorbed in the BM was 30 cc/g in comparison with that required DBM 16 cc/g. This
difference is presented because DBM has a greater surface area.

Using SEM and EDS, it was demonstrated that BM consist of a structure composed
primarily of collagen volume covered by a mineral layer of HA.
By XRD, the crystalline material found in the BM was HA, while in the DBM find only
traces of HA were found.
TGA tell us that the DBM sample first loses physisorbed water, later loses water causing
structural denaturation of collagen and finally breaks down and burns leaving only organic
waste. From these results, we can conclude that for the sterilization process, it is not
recommended to use temperatures above 80ºC.
A notable difference between the specific surface area of the BM and DBM samples was
observed using the BET method, although the shape of the pore was not changed in accordance
with the isotherms obtained.

ACKNOWLEDGEMENTS

We Thank to DGAPA-UNAM for the financial support through the projects IT 104011 and
IT 114911. We also thank to Dra. Sandra Rodil by her suggestions; thanks by the technical
support to: Esteban Fregoso, Adriana Tejeda Cruz, Verónica Rodríguez Mata, Armando Pérez
Torres, Omar Novelo, Fernando Silvar, José Ocotlán Flores, Miguel Herrera, Armando Zepeda,
Francisco Pasos and Viridiana Maturano.

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