Journal of Biosciences and Medicines, 2023, 11, *-*

https://www.scirp.org/journal/jbm
ISSN Online: 2327-509X
ISSN Print: 2327-5081

Synthesis and Characterization of a Polymeric

Material Blended to Bone Forming Elements

Esther Perez-Torrero1*, Leticia Esmeralda Luna-Rodriguez1,

Gerardo Antonio Fonseca-Hernandez2, Jose Santos-Cruz3,
Eric Mauricio Rivera-Muñoz2, Maria Lucero Gomez-Herrera1
1
Facultad de Ingeniería, Investigación y Posgrado, Campus Aeropuerto, Universidad Autónoma de Querétaro, Querétaro, México
2
Centro de Física Aplicada y Tecnología Avanzada (CFATA) de la Universidad Nacional Autónoma de México
3
Facultad de Química, Investigación y Posgrado Campus Cerro de las Campanas, Universidad Autónoma de Querétaro,
Querétaro, Qro
Email: *[email protected]

How to cite this paper: Author 1, Author Abstract

2 and Author 3 (2023) Paper Title. Journal
of Biosciences and Medicines, 11, *-*. A synthetizing material blended with two distinct proteins (collagen and case-
https://doi.org/10.4236/***.2023.***** in) and mineral mixture, was developed in order to evaluate their properties
suitable for possible applications in the biomedical such as inducing the rege-
Received: **** **, ***
Accepted: **** **, ***
neration of damaged bone, either due to an accident or illness. Samples were
Published: **** **, *** evaluated by 1) Mechanical properties tests under the bending and compression
mode, 2) Scanning electronic microscopy and 3) Infrared spectroscopy and 4)
Copyright © 2023 by author(s) and
The x-ray diffraction, were carried out. The results showed that the developed
Scientific Research Publishing Inc.
This work is licensed under the Creative
material has breaking strength and structure characteristics associated with
Commons Attribution International the protein used in their composition. This fact suggests that the used protein
License (CC BY 4.0). determines the resistance of the material, in such a way according to the re-
http://creativecommons.org/licenses/by/4.0/
quired use, being able to choose appropriate strength and duration either
Open Access
short or long time. The material composition for specific use, in order to find
the most suitable mixture for bone replacement, or induce bone recovery,
according to the required properties similar to those of damaged living tissue.

Keywords
Minerals, Bone, Polymethylmethacrylate, Casein, Collagen, Mechanical
Properties, X-Ray, Infrared, Scanning Electronic Microscopy

1. Introduction
In order to find the most suitable material for tissue replacement, being two
types, soft tissue, such as the skin, tendons, ligaments and some internal organs,
and hard tissue as bone sand teeth. The bone tissue is affected by illnesses or ac-

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E. Perez-Torrero et al.

cidents, and even wars have caused many people to lose limbs or suffer bone in-
juries. The bone tissue provides support to the body and protection of the inter-
nal organs, the extremities are of great importance to lead a better quality life
and independence. To solve certain bone damage, it is of great interest to ana-
lyze the material properties for the bone replacement, according to their me-
chanical requirements, in order to choose the more adequate material [1] The
materials used as a replacement for bone tissue must have a balance between
flexibility and resistance, they must not suffer plastic deformation and they must
not have flexural memory, because the bone tissue functions for supporting the
loads. Their density is of great importance, recommended a material of similar
density to that of the tissue to be replaced, in order to prevent decubitus injuries,
related to pressure on the soft tissues [2] [3] [4]. Proteins are polymeric mole-
cules present in living organisms, performing functions of transport, structure,
catalysis, regulators of biochemical processes and in the immune system. Casein
has a great application in the production for the food industry [5]. Biomaterials
of natural origin bring different benefits, such as zero toxicity, the possibility of
having a beneficial function [5] [6]. The variety of casein types vary in amino
acid composition; their structure depends on the physic-chemical environment
[7] [8] [9] [10].
Although there are several collagen varieties, type I collagen is one of the most
abundant, can form intertwined chains that generate compact solids, form gels
of interest in the cosmetics industry. Collagen is obtained from skin, cartilage,
tendons and animal bones [11] [12] [13] [14]. The mostly applied area for pro-
tein-based materials is targeted as drug delivery, which offers a local potential,
increased dose of the drug while lowering systemic and non-specific drug ad-
ministration [15]. Taking as reference the bone composition, up about 65%
minerals and 35% organic matrix, being the 90% collagen and others proteins,
cells, water and blood vessels [16].
In order to find the most suitable material for medical replacement, the type
of tissue that needs to be repaired must be considered, such as soft tissue such as
skin, tendons, ligaments and some internal organs and hard tissue such as bones
and teeth. The experimental study is based on the organic and inorganic compo-
sition of bone tissue (Figure 1).
The goal of the current study was characterizing a synthetized material that
resembles the bone composition by mechanical and structural properties for
medical application or museum pieces’ restoration, such as art objects.

2. Material and Methods

2.1. Samples
Sol-gel synthesis was used to prepare the samples with a rectangular form
(measures), following the processes (see Figure 2), mixed the precursors and
monomer of PMMA methyl methacrylate, in the described steps such as hydro-
lysis, condensation formation de la solution (sol), and solid formation (gel-liquid),

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 E. Perez-Torrero et al.

Figure 1. Bone composition, inorganic region includes mineral composition, organic re-
gions correspond to protein [17].

as the viscosity rapidly increase, the solvent is bordered inside the gel. The
structure may change considerably along time, the gel is aging, wet gel, dried by
solvent evaporation (at environmental temperature), then the capillarity forces
will produce in contraction, the gel network will collapse and the xerogel (solid
and gas) is formed at environmental conditions. A sol consists of a liquid with
colloidal particles which are not dissolved, but do not agglomerate or sediment.
Agglomeration of small particles is due to van der Waals forces and tendency to
decrease the total surface energy. Van der Waals forces are weak, and cover only
for a few nanometers. In order to counter van der Waals interactions, repulsive
forces must be established, the liquid phase even contains sol particles and ag-
glomerates, which will continue to react and will condense as the gel dries. The
gel is originally flexible and then groups on around branches will condense,
making the gel even more viscous. This will enfold out the liquid from the inte-
rior of the gel, end shrinkage occurs, this process will continue as long as there is
flexibility in the gel [18].
Using the sol-gel method, three wo different samples of materials were obtained
(each sample contents a thirth portion of mineral mix, casein or collagen and
PMMA (organic region), 1) Mixture of collagen, a mixture of minerals (Col-Mx)
and polymethylmethacrylate (PMMA) (as reference), 2) Mixture of casein
(Cas-Mx), and 3) PMMA samples, which were used as reference points. Dried
samples, were stored in closed plastic bags in a controlled environment, Table 1.

2.2. Density
In order to carry out the calculation of the different materials density (n = 5), we
obtained the average mass of samples prepared in the containers corresponding
to the bending test, this procedure was to obtain uniform specimens, which
measurements are 79 mm long, 10 mm wide and 5 mm thick, and from which
we obtained a 3.95 cm3 volume.

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Figure 2. Synthesis process of material samples by sol-gel method [19].

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Table 1. Composition of the material used for samples elaboration. composition.

Samples Components Acronym

1 Polymethylmethacrylate PMMA
2 PMMA + Mineral mix + Collagen Col-Mx
3 PMMA + Mineral mix − Casein Cas-Mx

2.3. Mechanical Test

Flexibility is the property of a material to undergo large deformations in its elas-
tic zone, it is directly related to Young’s modulus, if this is small the material will
be flexible, however, if Young’s modulus has a high value, the material will be ri-
gid, that is, it will need a great effort to be deformed.
The samples (n = 10) were placed horizontally, on two horizontal supports
spaced; the center load point of contact with the rear surface of the midpoint of
the material, following the rules of ASTM 2007 and ISO 5833 [20] [21] (Figure
4). The accessory to carry out the bending test moves downwards at a constant
speed of 1 mm/min until it breaks in accordance with the official ISO 178:1993
standard. The specimens were made with measurements of 4 mm thick, 10 mm
wide and 79 mm long, based on 3-point bending tests [22].
Bending evaluation was carried out by three-point bending test, the samples
were placed on two supports and a force was applied in the center. The tests
were carried out at a 25.2˚C temperature of and a 46% humidity using a
Zwick/Roell model Z005 universal testing machine. The test samples were placed
with their front face downwards on two horizontal supports spaced 22 mm
apart; the central load point of contact with the rear surface of the midpoint of
the material. The accessory to carry out the bending test moves downwards at a
constant speed of 1mm/min until it breaks in accordance with the official ISO
178:2003, standard American National Standards Institute ANSI/ASAE S459
(1988) [23] [24] [25]. The tests were carried out under the established standards,
using samples 4 mm thick, 10 mm wide and 79 mm long, based on the 3-point
bending tests [25].

2.4. Infrared Spectroscopy

Infrared spectroscopy test, was used to determine the composition, as well as the
purity level. The different samples were evaluated under IR test, in a Bruker
Tensor 37 equipment, in transmittance mode, 32 scans were made with a resolu-
tion of 1 cm−1 [26].

2.5. Scanning Electron Microscopy

Scanning electron microscopy (SEM) was implemented to observe the internal
structure of material at the nanometric scale, with a resolution close to 0.5 nm.
The tests were carried out in a Carl Zeiss Scanning Electron Microscope,
model EVO-50, which works at high and low vacuum, and has SE1, BSD and
EDX (X-ray) detectors. The equipment has critical point dryers (EM CPD300,

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E. Perez-Torrero et al.

Leica; SAMDRI-PTV-3D, Tousimis) and gold, palladium and carbon coaters

(DESK II, Denton vacuum; EM ACE200, Leica; SC7620, Quorum).

2.6. Statistical Analysis

For the statistical analysis, Statistical Package for the Social Science (SPSS) ver-
sion 15 was used and the variance test was performed for the comparison be-
tween the variables studied in the different samples. All data were analyzed by
means of the using ANOVA and Pos Hoc “Tukey” test. A statistically difference
was established at “p” values less than 5% (p ≤ 0.05).3.
Experimental data were compared, to examine the score differences of com-
position among samples the following separate statistical analyses were used: (a)
density were compared with a ANOVA with repeated measures, 3 (samples
composition) × 4 (parameters evaluated. Additionally, a comparison between
samples with a one-way ANOVA was obtained analysis of variance ANOVA
with repeated measures. Additionally, a post- hoc Tukey test was conducted
when pertinent, to detect significant differences between samples throughout the
experiment.

3. Results and Discussion

3.1. Results of Density
As it can be seen in Table 2 and Figure 4(A) and Figure 4(B) PMMA (a ma-
terial for commercial uses) is taken as a reference, however, there are notable
differences between the two, the Col-Mx mixture has a significant difference
with respect to both, which shows us that it has less rigidity and greater elastici-
ty, while the Cas-Mx mixture has a performance similar to that of PMMA. Due
to the structure of the Cas-Mx sample, as for their density, there is no significant
variation. Additionally, in the bending test, the specimens, only presented a par-
tial rupture, similar to the case of the white PMMA specimens. This indicates
that this material has higher strength and lower weight, consequently makes it
ideal for applications where a strong and lightweight material is required [27].

3.2. Results of Mechanical Test

The mechanical test demonstrates as shown in Figures 3(A)-(D), Wistar rat
bone and PMMA were taken as a reference, the Col-Mx mixture has a significant
difference with respect to both, presenting us that it has less rigidity and greater

Table 2. Mean density ± error values.

Number Samples Density (g/cm3) Comparisons P values

1 PMMA 1.1712 ± 0.0103 1 vs 2 0.0022*
2 Col-Mx 1.4817 ± 0.0340 1 vs 3 0.0022*
3 Cas-Mx 1.3831 ± 0.0202 2 vs 3 0.7605 NS

ANOVA gl (1, 14) F, 5.182. Significant differences were at p ≤ 0.05 level, non-significant
differences (NS).

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elasticity, while the Cas-Mx mixture has a performance similar to that of PMMA
(Table 3 and Table 4)
Flexibility as a property of a material to present large deformations in elastic
zone, is directly related to the Young’s modulus, a small value the material will
be flexible, however, if the Young’s modulus has a high value, the material will
be rigid, that is, it will require a great effort to be deformed [20]. When exist the
necessity of recover a structure or function of bone tissue, requires a material
with similar properties, in order to prevent abrasions in the tissue to be repaired
or in the surrounding tissues. For each particular situation, the selection of the
material must be according to the type of tissue and the use, for short or long
term. In the present study, a material obtained showed similar hardness rat
bone. Therefore, to replace harder bone, it is necessary to modify its mechanical
properties, which can be more or less hard depending on the specific case.

A Maximum Force (N) B Young's modulus (N/mm²)

150 5000
4000
100 3000
2000
50
1000
0 0
PMMA Col-Mx Cas-Mx Hueso PMMA Col-Mx Cas-Mx Hueso
Bone

C D
Maximum Stress (N/mm²) Maximum deformation(%)
80 6
70
5
60
50 4
40 3
30
2
20
10 1
0 0
PMMA Col-Mx Cas-Mx Hueso
Bone
PMMA Col-Mx Cas-Mx Hueso
Bone

Figure 3. (A-D) Mechanical test comparison between the samples of different composition.

Table 3. Mean of different parameters evaluated (±EE) of mechanical test analyzed by two-way ANOVA, comparisons between
different samples composition.

Samples composition F values

Number Parameter Col-Mx PMMA Cas-Mx (1, 29) 43.17
1 Maximum force (N) 52.9129 ± 4.92 116.5367 ± 14.85 29.032 ± 9.967 (1, 29) 45.110
2 Young’s modulus (N/mm2) 2288.0086 ± 141.92 1725.53 ± 142.66 1670.166 ± 142.65 (1, 29) 5.205
3 Maximum stress (N/mm2) 19.9771 ± 2.51 50.0857 ± 4.58 12.688 ± 1.91 (1, 29) 43.17
4 Maximum deformation (%) 1.5643 ± 0.16 4.7383 ± 0.91 1.768 ± 0.62 (1, 29) 9.764

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Table 4. Comparison between different samples of different parameters evaluated of me-

chanical test, Post hoc analyzed by one-way analysis of variance.

P values
Col-Mx vs Col-Mx vs PMMA vs
Number Parameter
PMMA Casein Cas-Mx
1 Maximum force (N) 0.0273* 0.0054* 0.0016*
2 Young’s modulus (N/mm2) 0.03623* 0.01049* 0.5332 NS
3 Maximum stress (N/mm ) 2
0.0001* 0.0021* 0.0033*
4 Maximum deformation (%) 0.0001* 0.0013* 0.0654 NS

Significant differences were at p ≤ 0.05 level, non-significant differences (NS).

3.3. Results of Infrared Spectroscopy

Through IR spectroscopy, it revealed that the used materials have similar pat-
terns as those reported, showed for PMMA a small peak in the region of 2900
cm−1 that represents the C-H bonds as well as the one found at 1440 - 1450 cm−1,
also at 1730 - 1740 cm−1, we find a band corresponding to the double bonds be-
tween C=O, and between 1200 and 1300 cm−1, we observe two small characteris-
tic peaks of the C-O of the ester group as shown in, Figure 4, marked with ar-
rows and so on for collagen and casein [28]. In the IR spectroscopy it is observed
that the PMMA, collagen and casein that were used in the work have patterns
similar to those reported in the literature, specifically, the points of functional
groups coincide, which validates the use of the inputs used in this project (Table 5).

3.4. Results of Scanning Electronic Microscopy

Images from SEM microscopy, the Cas-Mx mixture generates arrangements of
the PMMA spheres, forming bridge-shaped joints between them, deposited in
the mixture of protein and minerals. The structure presented some porosity,
which could be related to a lower density of the material compared to the
Col-Mx mixture. While the PMMA spheres in the collagen and mineral mixture
do not have a structured pattern as shown in Figure 5 and Figure 6.
Bone regeneration is one of the most important and challenging tissue engi-
neering approaches considered to be an ideal strategy for treating diseases, injuries,
in regenerative medicine. The experimental material is an excellent alternative
for bone regeneration, because the lost cost in front of traditional used material
such is hydroxyapatite. In complement with bases of tissue engineering the de-
veloped material can be applied in several cases for induces bone regeneration,
because the susceptibility of natural materials to enzymatic degradation or po-
tential allergenicity it can be applied as scaffold while the bone reaches the func-
tional structure. In this case, susceptibility can be a beneficial property, in con-
trast to replace bone indefinitely, it is necessary to reinforce the material with
metallic nanoparticles, carbon nanofibers. For this reason, it is a good option to
replace existing materials, due to its low cost. Added antimicrobial substances,
are necessary it can be competitive.

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A B Collagen

→
→ ↑ ← ←
↑

C Casein

↑ ←

Figure 4. IR spectrum of samples components, PMMA (A), collagen (B) and casein (C). The peaks (arrows) corresponding to the
functional groups of mixtures components observed, see Table 5.

Table 5. Main peaks of infrared tests, correspond to the samples components functional
groups. The peaks (arrows) indicated in Figures 3(A)-(C). corresponding to the func-
tional groups observed of mixtures components [29] [30] [31].

PMMA Collagen Casein

Band Bond Band Bond Band Bond

Amina I
2900 cm−1 C-H 1657 cm−1 1750 cm−1 C=O (double)
(N-H)

1440 - 1450 cm−1 C-H 1552 cm−1 Amina II 1650 cm−1 Amida I (N-H)
Amida II
1370 - 1740 cm−1 C=O (double) 1241 cm−1 Amina III 1450 - 1550 cm−1
N=H and C=N
1200 - 1300 C-O-C (ester)

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(A)

(B)

Figure 5. Images of collagen mineral mixture an polymetylmetacrilate at different magni-

fications, obtained by SEM. At different magnification (A) and (B).

(A)

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(B)

Figure 6. Images of the casein, mineral mixture and polymethylmethacrylate, obtained by

SEM, at two different magnifications (A) 72× and (B) /00×.

4. Conclusions
The results showed that the developed material possess breaking strength and
structure characteristics associated with the protein used within their composi-
tion. Such factual result suggests that the used protein determines the resistance
of the material in such a manner according to the required medical application;
thus, being able to choose the appropriate strength and duration for the re-
placement of living damaged tissue or controlled release of pharmaceutic for a
short or long term requirement. Also, it could be used for artistic objects resto-
ration such as bone fossil remains and ceramic restoration.
We concluded that the mixtures obtained are likely to be used for biomedical
applications, such as replacement or implants. However, it is suggested that
more characterization tests may be carried out to determine the most adequate
mixture that presents the necessary mechanical properties for each type of ap-
plication. The material mixtures have a similar structure pattern, to that gener-
ated by hydroxyapatite, which is a material applied for its use for bone implants.
We suggest the tested mixtures for their use instead of hydroxyapatite, in order
to reduce the cost. The foregoing perspective, is aimed to replace the polymer
used in this material with one that is of 100% natural origin, preferably one that
can be obtained easily and at low cost. In the hybrid materials (organ-
ic-inorganic), the sol-gel method favors the creation of charges, in the presence
of a polymer with functional groups, these charges are used to join the polymer
and the inorganic phase. With these characteristics, the material could achieve
greater biocompatibility. The mixtures used preserved the integrity of the pro-
teins, collagen and casein, since the polymerization was carried out at room
temperature, which prevents their denaturalization. Because there are a variety
of possibilities for inducing bone regeneration scaffolds, studies are required, in

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order to stablish which, type of cells and growth factors are more appropriate to
specific cell culture. In order to apply the synthetized material as scaffolds, due
use it in agreement to the bone morphology and region of the bone structure
damaged.

Acknowledgements
Research was partially financially supported by Autonomous University of Que-
retaro, Mexico, [Grant number FIN-2021-23, FONDEC-UAQ]. Authors thank
to Dr. Julio G. Mendoza-Alvarez and Agustin Esparza for edition assistance. Al-
so thanks to Andrea Navarro for their technical assistance.

Conflicts of Interest
The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential con-
flict of interest.

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