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 hydrolysis, condensation formation de la solution (sol), and solid formation (gel-liquid),

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 agglomerates, 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 interior 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.

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.

Flexibility is the property of a material to undergo large deformations in its elastic 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 rigid, 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] . 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 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] .

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 resolution of 1 cm⁻¹ [26] .

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, Leica; SAMDRI-PTV-3D, Tousimis) and gold, palladium and carbon coaters (DESK II, Denton vacuum; EM ACE200, Leica; SC7620, Quorum).

For the statistical analysis, Statistical Package for the Social Science (SPSS) version 15 was used and the variance test was performed for the comparison between 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).

Experimental data were compared, to examine the score differences of composition 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.

As it can be seen in Table 2 PMMA (a material 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 elasticity, 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 partial 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] .

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

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

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.

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

Through IR spectroscopy, it revealed that the used materials have similar patterns 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 between C=O, and between 1200 and 1300 cm⁻¹, we observe two small characteristic peaks of the C-O of the ester group as shown in, Figure 4, marked with arrows 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).

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 engineering 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 developed material can be applied in several cases for induces bone regeneration, because the susceptibility of natural materials to enzymatic degradation or potential allergenicity it can be applied as scaffold while the bone reaches the functional structure. In this case, susceptibility can be a beneficial property, in contrast 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.