Table 3. Ceramic properties of the COLL-PVA 1:2 (wt) hybrid materials and COLL-PVA/HA 1:2:3 (wt) composite materials

Table 3. Ceramic properties of the COLL-PVA 1:2 (wt) hybrid materials and COLL-PVA/HA 1:2:3 (wt) composite materials

In order to understand the influence of citrate ions on the morphology of acicular and non-acicular zones, the EDS spectra of these two zones were recorded (Fig. 10). The results indicated that the ratio of Ca to P differed between these two zones, where the Ca:P ratio was 1.6 (similar to stoichiometric HA) in the non-acicular zone (Fig. 10A) and 1.1 in the acicular zone (Fig. 10B). The acicular zone occurs due to a higher content of Na+ and citrate.

Fig. 10. EDS spectra of a) a non-acicular and b) an acicular reach zone

Infrared spectroscopy (Fig. 11) is an efficient tool for the investigation of organic-inorganic interactions. The differences in morphology may be explained by these strong interactions and are evidenced in the shift of peaks or in peaks duplications. In order to understand organic-inorganic interactions, the IR spectrum of a pure collagen matrix was recorded (Fig. 11A). The most important peaks in the pure matrix were the amide peaks (Chang and Tanaka 2002; Ficai et al. 2009b). After collagen matrix mineralization with HA precursors, the main amide peak of pure collagen (1630 cm-1) was shifted to a higher wave number for the COLL/HA composite (1650 cm-1), while the phosphate peaks appeared at 1030, 609 and 564 cm-1 (Fig. 11B).

The infrared spectrum of the mineralized hydrolysate-enriched collagen matrix (Fig. 11C) was characterized by the duplication of each main collagen peak and led to the differentiation of the phosphate peak and the appearance of a shoulder at about 1110 cm-1. These results can be explained by the degree of condensation between collagen and collagen hydrolysate, which induces interactions of varying strength between mineral and organic components. As reported in the literature (Silva et al. 2001), the degradation of collagen leads to a decrease in the absorption band at 1240 cm-1. In composites obtained with hydrolysate, this band being less intense than that in pure collagen.

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Wavenumbers (cm-1)

Fig. 11. FTIR spectra of: A. a collagen matrix (cross-linked with glutaraldehyde, 1%); B. a mineralized cross-linked collagen matrix (glutaraldehyde, 1%); C. a mineralized hydrolysate-enriched collagen matrix; and D. a mineralized cross-linked collagen matrix achieved in the presence of F- (5 % (molar) reported to HA)

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Wavenumbers (cm-1)

Fig. 11. FTIR spectra of: A. a collagen matrix (cross-linked with glutaraldehyde, 1%); B. a mineralized cross-linked collagen matrix (glutaraldehyde, 1%); C. a mineralized hydrolysate-enriched collagen matrix; and D. a mineralized cross-linked collagen matrix achieved in the presence of F- (5 % (molar) reported to HA)

When the mineralization of the collagen matrix occurs in the presence of fluoride, the collagen peaks were not shifted (compared to the COLL\HA composite), but the phosphate peaks of HA were split due to the partial substitution of hydroxyl groups with fluoride (Fig. 11D).

3. The synthesis of COLL/HA composite materials with oriented structure

In this field of orientation some works were published in the last 5 years. The synthesis of COLL/HA composite materials with oriented structure can be induced by self-assembly, electric field orientation or by using a high magnetic field (Wu et al. 2007), best results being reported by self-assembly (Ficai et al. 2010c) and pulsed electric field orientation (Ficai et al. 2010a). The magnetic field orientation is possible due to the magnetic anisotropy of the hydroxyapatite (Wu et al. 2007).

3.1 Self-assembling of COLL/HA composite materials

The self-assembly is one of the most easy to realize orientation which not imply any external influences but require slow drying at pH=6.9-9 which can easily induce denaturation of the collagen.

The synthesis of COLL/HA composite materials by self-assembling consists by two successive stages but have some particularities. In the first stage, the collagen gel is treated with the desired amount of Ca(OH)2 suspension, drop-wise and magnetically stirred for 24 h and let to interact. In the second stage, the stoichiometric quantity of H3PO4 solution was added also dropwise. Ca(OH)2 was used in order to assure the necessary basic pH. During the first stage of mineralization the pH was maintained at pH - 9 by addition of HCl.

The ratio of collagen, Ca(OH)2 and H3PO4 was so chosen that the final ratio of COLL:HA to be 20:80 (wt) and at the end of synthesis the concentration of collagen to became 1.66%. After the H3PO4 solution is added, the pH was adjusted at about 9 using NaOH solution, in order to assure pure HA precipitation. During the synthesis and drying of the composite material, the temperature was kept at ~37 OC.

pH 9 was choose based on two characteristics of the collagen molecules:

a. at pH= 6.9-8 the collagen molecules are in an extended conformation (length of collagen molecule 180-200 nm) and, b. the fibrillogenesis is increasing in the range of 6.6-9.2.

The main processes which occur during the synthesis are represented in Fig. 12. The collagen molecules have different conformation, function of the pH can be linear (at pH>9) or crimpy (for pH< 7); at intermediate pH the collagen molecules being crimpy but with a more pronounced linear aspect. In order to be sure that collagen molecules are in elongated form (linear) the working pH was set at 9.

Fig. 12. Specific processes occurred in solution during the self-assembly of COLL/HA composite materials

Function of the mineralization pH, we can obtain different morphologies (Fig. 12b and d). If the pH increases, the crimpy structure of collagen molecules from gel and also from composite can be modified into the elongated one. If the drying time is slowly enough, these linear (elongated) collagen molecules can self-assembly and form cylindrical fibrils and fibres (Fig. 12e), otherwise they will form fibrils and fibres, growth tri-dimensional, with crimpy collagen molecules (for pH< 7) or with linear collagen molecules (for pH > 9). The structure presented in Fig. 12b can be transformed into the structure presented in Fig. 12d by increasing of pH at an adequate value. In this case, the pH must be higher than in case of transformation of the structure illustrated in Fig. 12a into the structure presented in Fig. 12c, probably due to the interaction between HA and collagen.

The self-assembling process was characterized especially by XRD and SEM.

XRD was used to point out the mineralization process. The XRD spectrum, in Fig. 13, shows the formation of HA.

Fig. 13. XRD pattern of COLL/HA composite material (a) before sodium chloride removal and (b) after sodium chloride removal

In the case of COLL/HA composite obtained by self-assembly comparing with the HA obtained by precipitation, the X-ray diffraction pattern exhibit a much higher intensity for the 2 1 1 peak reported to the intensity of the other peaks characteristic to HA. This result can be attributed to a preferential growth of the HA crystals in the 2 1 1 direction due to the collagen influence. As it can see, the composite material also contains NaCl (Fig. 13a). The removal of NaCl can be easily made by washing the composite materials with distilled water, following the next procedure: after drying, the COLL/HA composite material with uniaxial orientation of the constitutive fibres is crosslinked with glutaraldehyde, washed with plenty of water and dried. Following this procedure, the chloride was completely removed (Fig. 13b) without altering the mineral phase, especially from the point of view of crystallinity and preferential crystallization direction.

The self-assembling structure of collagen molecules and hydroxyapatite particles can be proved by SEM (Fig. 14). The samples were analyzed in perpendicular and parallel section reported to mineralized collagen fibres, in order to study the formation and the orientation of collagen fibrils and fibres. The sodium chloride removal not alters the composite morphology; the SEM images recorded before and after sodium chloride removal being similar. Whatever the analysis section, the recorded SEM images show the formation of collagen fibrils and fibres, which are mineralized with HA.

Fig. 14. The SEM images of collagen/hydroxyapatite composite materials, recorded at different magnification; (a) parallel view with the fibres, (b-c) perpendicular view with the fibres, (d) high resolution SEM at 80,000* magnification

The SEM image presented in Fig. 14a is recorded in a fibres perpendicular section. This image shows a homogenous arrangement of the fibres. Fig. 14b and c are recorded in a fibres parallel profile and shows, at different magnification the stratified structure of the composite materials, the fibrils and fibres being organized in layers. It can also observe highly oriented fibres and fibres bundle. The homogenous arrangement can be evidentiated also in fibres parallel profile. Analyzing the rupture profile, it can be observed a shift between the rupture point, comparing different neighbor adjacent layers. In Fig. 14c, recorded at higher magnification, it can better observe the mineral deposition. It can conclude, that at the end of collagen fibres, the HA density is higher. At broken, the rupture seems to follow the highly mineralized zone of interfibrilar gap which exist at the end of two successive fibres. Finally, at a much higher magnification (Fig. 14d) the SEM images of fibres' end allow the estimation of the dimensions of HA, these particles being in the nanometric range. At high magnification from SEM image, it can be seen that the dimensions of the HA crystals are in the nanometric range (4-40 nm), with an elongated morphology. These results are in good agreement with the observation reporting to natural HA from the natural bone.

The SEM images obtained at different magnification, in the rupture, on parallel and perpendicular direction of collagen fibres, are showing a homogenous microstructure, with fibres organized in layers, with high orientation. Taking also in consideration the way in which the rupture took place, and also the fact that we have a high concentration of HA grains in the rupture points we might conclude that the COLL/HA composite material synthesized has a very similar structure with that of the natural long bone, that have been obtained through auto-assembling process.

Fig. 15. Straight line projection onto the three dimensions and visualization of the three angles formed between the projections and the three dimensions

The orientation degree can be quantified function of the deviation angle of each collagen fibers from the direction of the applied electric field. The orientation degree of mineralized collagen fibers is highly influenced by magnification; with the increasing magnification the orientation degree increase. At a magnification of 1000* the average of deviation degree is less than 5% while the orientation degree is more than 95%. The average fiber deviation and also the orientation degree of collagen fibers can be quantified based on the following equations:

where, N is the number of fibers; DAi — correspond to deviation angle of each fiber; D (%) correspond to the average deviation of the fibers toward the applied electric field and OD(%) correspond to the orientation degree.

Obviously the orientation is difficult to quantify and only a few methods permit this kind of measurements (and usually these are indirect methods); the orientation will be quantify based on the SEM images.

Hydroxyapatite was obtained by co-precipitation in the presence of collagen gel. In fact, starting from collagen gel and hydroxyapatite precursors, in certain conditions, due to the interactions between collagen, hydroxyapatite and water it was obtained self-assembled, highly oriented composite materials. It can note that, many authors published a lot of papers dealing with COLL/HA composite materials which start from collagen gel and calcium hydroxide and ortophosphoric acid as precursors, but due to the inadequate processing conditions they do not obtained the uniaxial orientation of the constitutive fibres. In aqueous solution the collagen molecules and hydroxyapatite precursors have the capacity to induce synthetic bone formation by self-assembling. The obtained composite material is more similar with compact bones morphology; the recorded analysis being very similar with these bones.

Very important is that we also propose a new way to estimate the average deviation of fibres and we determined it, assuming a 2D model. The average deviation is 2.54+0.2% which means that the degree of orientation is 97.46+0.2%.

3.2 Electric field orientation of COLL/HA composite materials

Also, orientation can occur due to different external factors (magnetic or electric fields), most efficient and easy to realize being the electric field orientation. The electric field orientation occur really fast (less than 1h) at low electric field (<1V/cm), the best electric field being the pulsatory electric filed. The magnetic orientation can be realized but require very high magnetic field (10T). First time in the literature, the degree of orientation was mathematically quantified using a very simple equation, based on the SEM images. The synthesis of COLL/HA composite materials via electric field orientation is realized in similar conditions with the mention that, before drying different kinds of electric fields are applied. By short, the collagen mineralization was conducted in two stages, in order to mimic the in vivo osteosynthesis. First stage is the calcium deposition onto the collagen that takes 24 h. The second stage consists in phosphate addition leading to hydroxyapatite precipitation (Ficai et al. 2009a). After 24 h, at 370C and pH=9-10, the mineralization can be considered completed and the orientation process can be initiated.

For orientation purpose of the mineralized collagen gel, there were considered two types of electric field: pulsatory field (0.93 V/cm) and superposed field (direct field -0.67 V/ cm and pulsatory field -0.93 V/ cm). In order to be able to compare the obtained results, all the samples were obtained in similar hollow mold with the dimensions of WxL*H=2*3x2 cm3; if electric field is applied, the electrodes are fixed in the hollow mold at 3 cm distance. The mineralized collagen gel is maintained for 1 h in the desired electric field. After that, the samples were introduced in the freeze drier and frozen at -350C. The freezing of the material is compulsory, in order to preserve the obtained structure. After freezing, the electrodes can be removed and the freeze drying process is started.

When no electric field is applied, the collagen molecules dipoles are randomly disposed, without any orientation. If an electric field is imposed, the collagen dipoles are becoming oriented, due to the interaction between collagen and the electric field (Fig. 16).

Fig. 16. Electric field orientation of collagen dipoles

The electric field is obtained with a "home-made" device, consisting in two different electric generators, each with its own role. One is responsible for the direct current, while the other for the pulsatory current generation. These two generators can work separately, at a maximum potential of 30 V.

The SEM micrographs (Fig. 17) show relevant difference between the samples obtained in the presence and respectively in the absence of electric field. If different types of electric field are applied, different degrees of orientation are achieved. Fig. 17a, d show the microstructure of collagen/hydroxyapatite composite material obtained without electric field, Fig. 17b, e show the microstructure of collagen/ hydroxyapatite composite material, obtained by orientation in a pulsatory electric field and Fig. 17c, f show the microstructure of the composite material obtained in a combined (superposed) electric field.

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