The mineral phase of bone and teeth consists of plate-like shape nonstoichiometric carbonated-HA crystals with length of about 100nm, width of 20–30nm, thickness of 3–6nm, and different types, and foreign ions substitutions including sodium, magnesium, potassium, carbonate, and fluoride [3, 4]. There are several methods to synthesise hydroxyapatite but mainly it is possible to distinguish two types of hydroxyapatite based on the temperature of synthesis: a high-temperature sintered form (>900°C) and a low-temperature precipitate (~37°C). The compounds synthesized at low-temperature exhibit more solubility, much greater surface area, and they are inherently more active in biological systems in contrast to the biologically inert sintered versions [5]. Sintered hydroxyapatite is most commonly used in load-bearing orthopaedic implants, on the other hand it is not bioresorbable as they are stable over the lifetime of the individual. Since the bone mineralization process occurred at basic pH and physiological temperature (37°C), the synthetic procedure that uses these parameters is called biomimetic mineralization. The final HA formed, in spite of having variable composition, is predominantly carbonated apatite with low crystallinity and it is very similar to the form found in bone, a favourable factor from a tissue engineering perspective [6].

Recent studies concerning collagen coatings on Ti implants have demonstrated their effective role in stimulating cellular responses, increasing bone remodelling, and improving bone growth and bone implant contact [7, 8]. Therefore, hybrids of collagen/HA have greater potential for clinical applications than the pure HA or collagen equivalents, because they benefit from the advantageous properties of both materials. In the composite, the collagen component can act as a matrix to embed the HA particles, alleviating the brittleness of HA and promoting the adhesion of the particles to Ti substrate [9]. At the same time, the HA used as a filler of the protein could improve the mechanical strength and biological performance of the collagen.

Electrospinning process has attracted a great deal of attention in the last years as a way to reproduce the structure of natural ECM by means of producing fibers in the nanometre scale [10]. This technique is used to fabricate nanofibrous structures from natural and synthetic polymers such as collagen, chitosan, silk fibroin, poly(lactide), polyurethane, polycaprolactone, and so forth [11–14]. Several papers reported the production of nanofibrous materials made of collagen mixed with biocompatible synthetic polymers using the electrospinning technique, as well as HA-polymeric fibers [15–18]. Most of these studies have demonstrated enhanced bioactivity of the composite materials, especially in nanofibrous constructs [19, 20]. However, the data on the production of pure nano-structured collagen fibers and HA-collagen composites by electrospining, as well as the data on the deposition of them on titanium alloy or on other orthopaedic implants, are extremely scarce or have not been investigated up to now.

The self-assembly process to nucleate HA crystals depends on the negatively charged carboxyl chemical groups of collagen that can bind Ca²⁺ and on the pH of the reaction medium. The final pH value of 9.0 has been chosen in order to induce the HA crystallization in the optimal condition [25]. In order to optimize the HA crystallization, different experiments have been carried out using several calcium and phosphate salts at different concentrations by keeping the Ca/P ratio constant and equal to the HA stoichiometric value of 1.67. The use of Ca(CH₃COO)₂ 8.5mM and NH₄H₂PO₄ 5.1mM was found to be the optimal composition to precipitate HA crystals. Furthermore, these values have been chosen in order to preserve the fiber-like morphology, in fact, at the higher HA amounts, a considerable level of beads was formed instead of the development of a fibrous morphology [26]. It is reasonable that when the HA amount was high, the collagen could not effectively distribute the HA nanocrystals during the precipitation process due to the relatively low density of its amino acid chains. As a result, some of the HA nanocrystals could be precipitated in large clusters without the direct involvement of the amino acids of collagen. In order to quantify the amount of apatite in the mineralized collagen, TGA investigations were carried out (Figure 2). TGA curves of pure collagen and mineralized collagen showed similar profile with loss in the range from room temperature to 200°C due to the evaporation of physisorbed water and weight loss between 200 and 500°C associated with the decomposition of collagen molecules followed by a slight loss between 500 and 700°C resulting from the combustion of the residual organic components [27]. Considering the residue of pure collagen (~17wt%), the apatitic content in the mineralized composite was determined as about 20wt%.

The morphology of the electrospun material was studied by SEM, and the micrographs of the pure collagen and mineralized collagen scaffolds are shown in Figure 3. The electrospun fibers of pure collagen have randomly oriented features trough out the matrix, as well as the mineralized fibers. The average diameters of polymers fibers formed during electrospinning with and without HA, determined by SEM, were about 200nm. These values are well comparable with ECM fibers, which are in the range of 50–500nm [2]. Finally, the two materials appear very similar in terms of morphology and dimensions of the fibers, and the visualization of the HA crystals was not allowed by SEM probably due to their very small dimensions in the range of 20–30nm [28].

The presence of deposited HA is evidenced by the EDAX spectrum collected for the mineralized sample (inset in Figure 3(b)). This spectrum indicates that the intensity ratio of Ca and P signals was about 1.5 coherently with the value of biological HA [3].

TEM images of the reconstituted electrospun collagen and collagen-hydroxyapatite are shown in Figures 4(a) and 4(b), respectively. The average diameters of polymers fibers were very similar (about 200nm) in agreement with the SEM observations. The stained pure collagen fibers display a high degree of fibril assembly, evidencing the characteristic D-band pattern with a regular period of 67nm along the long axis of the fibril [29]. On the contrary, the mineralized fibers do not show the D-band pattern due to the presence of HA. Figure 4(b) clearly illustrates the presence of HA nanocrystals of about 20–30nm in size that completely cover the surface of collagen fibers.

To clearly identify the mineral phase developed during the mineralization of the electrospun collagen fibers, the XRD was employed and the pattern is shown in Figure 5. All of the mineral diffraction peaks were indexed to HA pure phase (JCPDS file number 9-432) and no other impurity phases were detected. The most intense peaks at 43.7° and 50.8° 2θ are due to the titanium alloy substrate (marked with * in Figure 5) [30]. The diffraction pattern of the HA exhibits not well-defined diffraction maxima, in fact the peaks corresponding to the crystallographic planes (211), (121), and (300) were all combined into one broad peak centred at about 32° 2θ, indicating a relatively low degree of crystallinity and nanosized dimensions [4]. The crystal domain sizes along the c-axis (D₀₀₂) and along the perpendicular to it (D₃₁₀), using the 2θ = 26° (002) and 2θ = 39° (310) diffraction peaks, respectively, were 25 ± 5nm and 11 ± 3nm. These values are in agreement with the TEM observations. It is worth to notice that the HA diffraction pattern was very similar to that recorded for the deproteinated bone apatite and this was also confirmed by the similarity of the crystal domain size along the c-axis (21nm) [3].

The FT-IR spectra of electrospun pure collagen and mineralized collagen are depicted in Figure 6. The spectrum of electrospun collagen shows all the characteristic bands of collagen at 1204, 1240, 1280, and 1338cm⁻¹ arising from C–OH stretching modes and 1400 and 1450cm⁻¹ associated with the asymmetric and symmetric CH₃ bending vibrations [4]. The absorbance ratio of the bands 1240/1450cm⁻¹ which is a measure of the integrity of the triple-helix structure [31] was about 1.0 suggesting that collagen triple helix secondary structure was conserved. The spectra of collagen-HA composite displays the bands of poorly crystalline carbonate-HA, a convoluted band centred at 1040cm⁻¹ (asymmetric stretching modes of the phosphate groups) and a band at 1422cm⁻¹ (antisymmetric stretching mode of C–O, consistent with a carbonate type-B-substituted apatite, where the carbonate ions replace the phosphate ions in the crystal lattice) [4], while the bands of collagen change their positions and their relative intensities. This finding could indicate that during the mineralization procedure, the collagen secondary structure has been modified by the effective interaction with HA nanocrystals. The limited amount of carbonate in the HA derived from CO₂ dissolved in the preparation media and adsorbed on the surface materials during the previous storage. The presence of carbonate in the structure of apatite was intentionally retained, in order to better mimic the biological ones [3]. In a previous FT-IR study comparing “bulk” collagen with electrospun collagen scaffolds, Stanishevsky et al. [32] reported a shift to lower wavenumbers in electrospun collagen of the major peak in the amide I band from 1667cm⁻¹ (fibers) to 1642cm⁻¹ (bulk), as well as the peak of amide II band from 1574cm⁻¹ (fibers) to 1544cm⁻¹ (bulk). Similar amide I and amide II peak shifts for our pure collagen scaffolds were observed (the wavelengths of the bands related to amide I and amide II are 1642 and 1547cm⁻¹, resp.). It was suggested that changes in the triple-helix structure of collagen fibrils during the fiber drawing in the electrified jet may account for this infrared shift. Other changes in the infrared spectra were reported (such as a blue shift of the amide I band) by the addition of apatite nanoparticles to the electrospun collagen fibers [32]. Similar peak shifts in the amide I and amide II of collagen-apatite scaffolds were clearly observed (1642 to 1658cm⁻¹ and 1547 to 1555cm⁻¹, resp.). The observed changes in the infrared spectra are associated with the chemical interaction between the apatite nanoparticles and collagen, in particular they are due to the chemical link between the collagen carboxyl groups and the calcium of HA.

FT-IR microspectroscopy was used to analyze the electrospun coatings with the aim to map and characterize the chemical distribution of the sample. FT-IR microspectroscopy allows to define the structural interactions between HA nanocrystals and reconstituted collagen fibers. Moreover, this technique is able to verify the topographic and quantitative distribution of the components in the sample.

In Figures 7(a) and 7(b), the correlation maps obtained by loading the representative spectrum of collagen (Figure 6, red spectra) in the chemical maps of electrospun pure collagen (1300 × 1900μm) and mineralized collagen (1200 × 1100μm) samples, respectively, are shown. The predominance of a dark area in the Figure 7(b) is related to the absence of the pure collagen, due to the mineralization of all the collagen fibers. On the contrary, Figure 7(c) shows the correlation map obtained by loading the spectrum of collagen-HA (Figure 6, black spectra) in the same chemical map of mineralized collagen sample (1200 × 1100μm) represented in Figure 7(b). In this case, the representative bands of HA prevail on those of pure collagen and the large white area allows to visualize the crystallization of HA over the protein.