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Carboxylate "mezorneric" Stabilized anion form carboxylate ion

I II

Fig. 2. Mezomeric form stabilization of carboxylate group due to mineralization

As a result of these interactions, hydroxyapatite particles are preferentially deposited onto the collagenous support forming so-called nucleation centers. Once formed, the nucleation centers increase and can lead even to the formation of a thin film. If the amount of deposited HA is low enough, the mineralization centers can be visualized by scanning electron microscopy (Fig. 3).

Fig. 3. The SEM micrograph of mineralized collagen fibres

In order to obtain good bone substitutes, researchers have to understand the biosynthesis of natural bone and to control some parameters such as composition, hydroxyapatite shape and size, collagen fibrils and hydroxyapatite blade orientation.

The synthesis of COLL/HA composite materials with different porosity/density is very important in order to obtain materials with tailored properties. COLL/HA composite materials with tailored ceramic properties can be easily obtained by combining the controlled air drying with freeze drying. The controlled air drying followed by freeze drying can be easily used for the synthesis of COLL/HA composite materials with any composition. In this scope, the wet composite materials are let, under controlled atmosphere for certain time (controlled temperature and humidity) followed by a final drying, realized by freeze drying. The use of mixed drying method (controlled air drying combined with freeze drying) is technically easy to control and economically sustainable. The ceramic properties of the COLL/HA composite materials can be easily controlled by the air drying step conditions (especially drying time); the longer controlled air drying time lead to composite materials with lower porosity and increasing density.

Scanning electron microscopy can be used for qualitative analysis of these materials obtained by controlled air drying followed by freeze drying (Fig. 4). Analysing the SEM images of the materials obtained by air drying (increasing drying time) followed by freeze drying it is stand out a mile that the increasing air drying lead to denser materials.

Fig. 4. SEM images of COLL/HA composite materials obtained by controlled air drying (0, 48 and 199h) followed by freeze drying

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air drying time, h

Fig. 5. Ceramic properties of the three series of COLL/HA composite materials versus air drying time

It has to mention that during the air drying process, simultaneous with the water evaporation the morpho-structural restructuring of the composite happened which leads to more compact materials, the density of the COLL/HA composite materials being 0.15-1.6g/cm3 while the open porosity 25-95% (Fig. 5). As a general rule, the density of the composite materials increases with the increase of the air drying time. This method is very useful because allow to tailor the ceramic properties of the materials without modify the composition.

The orientation of collagen/hydroxyapatite composite is induced by the mutual interaction between collagen and hydroxyapatite in aqueous solutions. For better orientation, it is possible to use electric and / or magnetic field. Cunyou Wu and co-workers (Wu et al. 2007) report unidirectional oriented hydroxyapatite/ collagen composite using 10 T magnetic field starting from calcium containing collagen solution and phosphate solution at 37 0C. If magnetic field is imposed, perpendicularly to the rotation axis, unidirectional oriented collagen fibrils and hydroxyapatite crystals have obtained due to their magnetic susceptibility anisotropy (Hellmich et al. 2004).

Due to the importance of these composite materials thousands of papers and many comprehensive reviews are published yearly. Some of the most comprehensive reviews about bones and bone grafts materials based on collagen and/or hydroxyapatite were published in the last years by Cui at al (Cui et al. 2007), Dorozhkin (Dorozhkin 2009), Murugan and Ramakrishna (Murugan and Ramakrishna 2005) and by Wahl and Czernuszka (Wahl and Czernuszka 2006).

Some of the most recent advances in the field of COLL/HA composite materials are systematically presented below.

2. The influence of organic and inorganic species on the COLL/HA composite materials

Recent studies made by Ficai et al. (Ficai et al. 2010b; Ficai et al. 2010e) reveal the influences of the PVA, denaturated collagen (hydrolysate collagen and ionic species on the collagen/hydroxyapatite composite materials. These works are essential because neither in vitro nor in vivo mineralization is readily understood, and the synthesis of materials that mimic the characteristics of bone has not been previously accomplished.

The properties of COLL/HA composite materials can be tailored by addition of different organic or inorganic components; well studied in the literature being that ions which naturally occur in natural bone: citrate, fluoride, chloride, carbonate, magnesium or some ions which accidentally occur in natural bones and, usual is responsible for different diseases (Pb2+, Sr2+ etc.).

The synthesis of complex COLL/HA composite materials which also contain PHA, collagen hydrolysate or other ionic species than calcium and phosphate were synthesized by co-precipitation of hydroxyapatite on collagenous matrices (Ficai et al. 2009b). The mineralization process consisted of two successive stages. In the first stage, a Ca(OH)2 suspension was added drop-wise and allowed to interact for 24 h. In the second stage, a stoichiometric quantity of NaH2PO4 solution was added drop-wise. The quantities of collagen matrix and HA precursors were used at a ratio of 1:4. After the hydroxyapatite precursors were added, the pH was adjusted to 9 using a solution of NaOH, in order to assure brushite-free HA precipitation. During the synthesis and drying of the composite material, the temperature was maintained at approximately 37 0C.

To study the effect of the citrate ions on the mineralization process, the collagen matrices were dipped into a dilute citrate solution (5 %o) for 30 minutes and then mineralized as presented above. Upon completion of this process, the resulting materials were analyzed.

In this chapter, two collagenous matrices that contained 5% and 70% of the hydrolysate were mineralized following the aforementioned procedure. The COLL/PVA and COLL-PVA/HA hybrid materials were obtained starting from collagen gel and PVA solution, the mixing ration being COLL:PVA = 1:2. the as obtained gel was than mineralized as presented above.

In order to obtain fluoride-substituted hydroxyapatite, a modified mineralization method was used. For this purpose, in the second stage of mineralization, a corresponding amount of NaF was added to the phosphate solution to assure the transformation of 5%hydroxyapatite into fluoroapatite.

In order to design COLL/HA composite materials and to obtain a bone-like morphology, it is important to understand the influence of each component on the morphology of the material.

The influence of the collagen hydrolysate, PVA and ionic species were analyzed by XRD, FTIR and SEM (-EDS) from the point of view of composition and morphology. The synthetic conditions and characteristics of the resulting hydroxyapatite were strongly influenced by the organic support.

Fig. 6. XRD patterns of different collagen/hydroxyapatite-based composite materials: A. mineralized cross-linked matrix (glutaraldehyde, 1%); B. mineralized cross-linked matrix (glutaraldehyde, 1%), the mineralization occurs in the presence of F- (5 % (molar) reported to HA); C. mineralized hydrolysate-enriched collagen matrix; and D. crystalline HA (ASTM 745966)

Fig. 6. XRD patterns of different collagen/hydroxyapatite-based composite materials: A. mineralized cross-linked matrix (glutaraldehyde, 1%); B. mineralized cross-linked matrix (glutaraldehyde, 1%), the mineralization occurs in the presence of F- (5 % (molar) reported to HA); C. mineralized hydrolysate-enriched collagen matrix; and D. crystalline HA (ASTM 745966)

X-ray diffraction patterns (Fig. 6) were recorded in order to confirm the synthesis of hydroxyapatite and for the qualitative estimation of preferred crystallization directions. In the XRD diffraction pattern of citrate-enriched COLL/HA composite materials, no difference was observed when compared to COLL/HA or COLL-PVA/HA (Ficai et al. 2010e). The diffraction patterns of citrate-enriched COLL/HA and COLL-PVA/HA are not presented herein, as the results were similar to the diffractogram of pure COLL/HA composite materials presented in Fig. 6A.

The results of the four diffractograms lead to the conclusion that the organic phase composition and the presence of ionic species greatly influences the mineralization process. From the comparison of the diffractograms displayed in Fig. 6A and C, it can be observed that hydrolysate induced a preferred direction for the growth of HA [210] and that the other crystalline phases exhibited approximately the same intensity.

The presence of F- had a strong influence on apatite crystallization (Fig. 6B). Among all of the crystallization directions, only a few were characteristic of partially substituted apatite (Ca^PO^OHi-xFx) and were present at a relevant intensity: [121], [100], [210] and [222]. Additionally, no other fluoride salts could be identified from the diffractograms. The main crystallization directions presented in crystalline HA (ASTM 74-5966) are presented in Fig. 6D.

Scanning electron microscopy was employed in order to study the morphology of the composite materials.

SEM images were recorded at different magnifications as a function of the morphology of each composite and the size of the deposited HA particle (Fig. 7). The morphology of the hydroxyapatite varied and was dependent on the collagenous support and on the presence of different ionic species.

In order to understand the influence of different collagen-based supports and ionic species on the morphology of COLL/HA composite materials, all data were compared to the morphology of pure COLL/HA composite materials (Fig. 7D).

After the mineralization of a pure collagen matrix, a homogenous HA deposition was observed. In mineralized hydrolysate-enriched matrices, the morphology was dependent on the hydrolysate content (Fig. 7A and B). An increase in the amount of hydrolysate induced a higher content of fine acicular HA, which is unusual in pure collagen matrix mineralization. The presence of F- during the deposition of HA onto the pure collagen matrix induced a thin lamellar deposition (Fig. 7C). The lamellar HA had a mean size of 50x20x1 pm. The presence of citrate ions induced a special kind of HA mineralization in the collagen matrices (Fig. 7E and F). The deposited HA displayed a similar morphology to those obtained in the presence of hydrolysate but at a different scale. Citrate ions appeared to induce a greater amount of organization of HA into the acicular form, as compared to hydrolysate. This finding is important because natural bones contain a small amount of citrate. In this case, the rods of HA had a mean length of ~10 ^m and a mean diameter of less than 0.4 ^m.

When PVA is present, the microstructure is strongly influenced for both COLL-PVA and COLL-PVA/HA materials.

The scanning electron microscopy has a decisive significance for the complete characterization of such materials. Therefore, the SEM images were recorded at different magnifications on all COLL-PVA/HA materials (with the component ratio of 1:2:0-blank, non-mineralised sample and 1:2:3-mineralised sample obtained by freeze drying and controlled drying).

Fig. 7. SEM images of different collagen and hydroxyapatite composite materials: A. composite obtained by mineralization of a collagen-hydrolysate matrix (5% of hydrolysate); B. composite obtained by mineralization of a collagen-hydrolysate matrix (70% of hydrolysate); C. composite obtained by mineralization of a pure collagen matrix (the mineralization was achieved in the presence of F- (5%-molar reported to HA); D. mineralized (pure) collagen matrix; E, F. composite obtained by mineralization of a collagen-citrate matrix (citrate less than 2%)

Fig. 7. SEM images of different collagen and hydroxyapatite composite materials: A. composite obtained by mineralization of a collagen-hydrolysate matrix (5% of hydrolysate); B. composite obtained by mineralization of a collagen-hydrolysate matrix (70% of hydrolysate); C. composite obtained by mineralization of a pure collagen matrix (the mineralization was achieved in the presence of F- (5%-molar reported to HA); D. mineralized (pure) collagen matrix; E, F. composite obtained by mineralization of a collagen-citrate matrix (citrate less than 2%)

The SEM images have given some morphological information about the four materials. Fig. 8a, b and Fig. 9a, b show the morphology of the two materials obtained by controlled drying in air at 30oC; they are stratified but compact materials. Fig. 8c, f and Fig. 9c, f show the morphology of the freeze dried composite materials; such materials exhibiting stratified but porous morphologies. Based on the SEM images, the freeze dried COLL-PVA/HA 1:2:0 hybrid material has the mean distance of 60-120^m between the sheets while for the freeze dried COLL-PVA/HA 1:2:3 composite, the hybrid material has the mean distance of 40-70^m between the sheets.

Fig. 8. SEM images of the COLL-PVA hybrid materials (weight ratio of 1:2) obtained by (a and b) controlled drying and (c-f) freeze drying

In fact, the two morphologies are similar, the compact materials resulted from the continuous remodelling and restructuring until it becomes compact; if the freeze drying is used, once the materials are frozen the restructuring is blocked and the materials retain their initial high porosity.

For both compositions, the controlled air drying at 30oC has led to parallel layers bounded each other by fibres of different diameters (from less than 1^m up to 10^m).

A higher magnification (Fig. 9f, for instance) has revealed the very good homogeneity and compatibility of the three components practically, no free HA agglomerates being visible.

Fig. 9. SEM images of the COLL-PVA/HA hybrid composite materials (weight ratio of 1:2:3) obtained by (a and b) controlled drying and (c-f) freeze drying

The osteointegration of the bone graft materials is induced, among the rests, by the porosity. The density, porosity and absorption (Table 1) were measured by the Arthur method. In all cases three replicates were done, the experimental error being less than 1-2%. The results are in very good agreement with the SEM observation. The highest porosities correspond to the freeze dried materials and have reached about 80 and 90%. For the materials obtained by controlled drying at 30oC in air, the porosity was less than 20%. The xylene absorption is proportional with the porosity while the density is in inverse proportion. The presence of HA has induced the density increase and a decrease in the porosity and xylene absorption. Hybrid materials with intermediary properties were obtained by a mixed drying method which involves controlled drying followed by freeze drying (not presented in this paper).

Sample

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COLL-PVA 1:2, freeze drying

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