In order to maintain the fibrous structure of the polymer, electrospun collagen in the form of nanofibrous scaffolds was produced. The spinning conditions to obtain collagen scaffolds were optimized by manipulating the experimental parameters, such as concentration of solution, distance between the spinneret and the collector, and the flow rate, in order to produce fibrous structure without any bead formation. By optimizing so (see Section 2
), uniform fibers with three-dimensional pore structure with high porosity, appropriate for self-assembling mineralization, have been formed. These findings are comparable to the results reported by Boland et al. [23
] for electrospun type I collagen fibers with average fiber diameters ranging from about 100
nm to 4.6μ
m depending on the concentration. In line with previous reports on other polymeric materials [24
], we conclude that electrospinning of collagenous proteins at concentrations above 2.5
wt% will yield smooth and uniform fibers of several hundred nanometers in diameters. By contrast, electrospinning at lower concentrations will result in small fibers, but with beads.
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(CH3
mM and NH4
mM 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 (). 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 (~17
wt%), the apatitic content in the mineralized composite was determined as about 20
TGA curves of electrospun pure collagen (straight line) and collagen-hydroxyapatite composite (dotted line).
The morphology of the electrospun material was studied by SEM, and the micrographs of the pure collagen and mineralized collagen scaffolds are shown in . 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 200
nm. These values are well comparable with ECM fibers, which are in the range of 50–500
]. 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 20–30
Scanning electron microscopy (SEM) images and energy dispersive X-rays (EDAX) analysis of electrospun pure collagen (a) and collagen-hydroxyapatite composite (b).
The presence of deposited HA is evidenced by the EDAX spectrum collected for the mineralized sample (inset in ). 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 and , respectively. The average diameters of polymers fibers were very similar (about 200
nm) 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 67
nm 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. clearly illustrates the presence of HA nanocrystals of about 20–30
nm in size that completely cover the surface of collagen fibers.
Transmission electron microscopy (TEM) images of electrospun pure collagen (a) and collagen-hydroxyapatite composite (b).
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 . 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 ) [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
) and along the perpendicular to it (D310
), using the 2θ
= 26° (002) and 2θ
= 39° (310) diffraction peaks, respectively, were 25 ± 5
nm and 11 ± 3
nm. 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
X-Ray diffraction (XRD) pattern of collagen-hydroxyapatite composite; *indicates the Ti alloy diffraction peaks.
The FT-IR spectra of electrospun pure collagen and mineralized collagen are depicted in . The spectrum of electrospun collagen shows all the characteristic bands of collagen at 1204, 1240, 1280, and 1338
arising from C–OH stretching modes and 1400 and 1450
associated with the asymmetric and symmetric CH3
bending vibrations [4
]. The absorbance ratio of the bands 1240/1450
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 1040
(asymmetric stretching modes of the phosphate groups) and a band at 1422
(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 1667
(fibers) to 1642
(bulk), as well as the peak of amide II band from 1574
(fibers) to 1544
(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 1547
, 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 1658
and 1547 to 1555
, 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.
Figure 6 FT-IR spectra of electrospun pure collagen (red) and collagen-hydroxyapatite composite (black) in the region 1800–700cm−1.
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 and , the correlation maps obtained by loading the representative spectrum of collagen (, red spectra) in the chemical maps of electrospun pure collagen (1300 × 1900μ
m) and mineralized collagen (1200 × 1100μ
m) samples, respectively, are shown. The predominance of a dark area in the is related to the absence of the pure collagen, due to the mineralization of all the collagen fibers. On the contrary, shows the correlation map obtained by loading the spectrum of collagen-HA (, black spectra) in the same chemical map of mineralized collagen sample (1200 × 1100μ
m) represented in . 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.
Figure 7 Correlation maps obtained by loading the representative FT-IR spectrum of collagen (, red spectrum) in the chemical maps of electrospun pure collagen (a) and mineralized collagen (b) and correlation map obtained by loading the FT-IR spectrum of (more ...)
It is well accepted that the formation of HA on the surfaces of electrospun fibers was controlled by the charged density of chemical groups on the fiber surface. Previous investigations about the nucleation sites of HA crystals on collagen fibers have suggested that the binding of calcium ions on the negatively charged carboxylate groups of collagen is one of the key factors for the first-step nucleation of HA crystals [33
]. Collagen has large number of negatively charged carboxyl chemical groups that can bind Ca2+
ions to nucleate the HA crystal growth. The carboxyl groups are present in about 11% of the amino acid residues of collagen molecules. Moreover, in a neutral solution, more than 99% of the carboxyl groups of aspartyl and glutamyl ionize favouring the chelation of calcium ions. The carboxyl groups on the outside of the collagen threefold spiral are one kind of site for collagen mineralization [34
]. In this way, higher content and smaller crystal size of HA could be formed on fibers with higher densities of carboxyl groups. Carboxyl groups were initially combined with calcium ions through electrostatic attraction, and then phosphoric ions, which were considered to promote the nucleation of HA, until the whole coverage of the fiber with the primary layer of HA was completed. This hypothesis is well supported in this work by the FTIR and TEM investigations and, in particular, by the blue shift of the amide I and amide II bands in the spectra of mineralized collagen. Moreover, by using FT-IR microspectroscopy, we found that part of the HA crystallized outer the fibers, coherently with the fact that the electrospinning technique allows the formation of the collagen fibers and in this case, the carboxyl groups outside the fibers are responsible for the HA nucleation.