Fabrication and Characterization
In  HA nanorods that were ~28 nm in length and ~8 nm in diameter were added to a gelatin solution prepared and were extensively sonicated in order to achieve a homogeneous dispersion. After the pH was adjusted tanic acid (TA) powder was added and stirred in order for cross-linking to occur. In addition to TA being a natural phenolic compound, it reduces the solubility of gelatin in liquid environments. The mineralization process was then performed using the layer-by-layer deposition .
A first step in examining the applicability of such scaffolds in bone regeneration was to perform scanning electron microscopy to reveal their microstructure, so as to examine if the porosity of the composite was sufficient and the distribution of the nanoparticles uniform. Figure 4.1a, b depicts the gelatin-nanoparticle composite.
Fig. 4.1 Scanning electron microscopy images in the (a) longitudinal and (b) transverse direction of the lyophilized gelatin-hydroxyapatite; the scale bars in each image are: (a) 100 pm (b) 20 pm. (c) Pore wall morphology of lyophilized gelatin-HA scaffold, scale bar: 2 pm. These samples contained 0.80 g/mL gelatin and 0.85 mg/mL HA (Reproduced with permission from )
The scale bar in Fig. 4.1 is in the microscale, hence it was not expected to see the individual particles, and the absence of microscale aggregates indicates that the HA nanoparticles were indeed dispersed uniformly throughout the gelatin, which had a pore size between 35 and 288 ^m, and was similar to the promising porosity of HA foams . In Fig. 4.1 it can be seen that the HA particles were within the pore walls and aggregation did not occur. In order to examine the effect that the crosslinking concentration has on the graft morphology two different concentrations were used: 12.4 mg TA/g and 33.3 mg TA/g. Increasing the TA concentration resulted in a similar porous morphology, however, the fiber diameter decreased (~73 ^m) while the interconnections increased, decreasing hence the overall porosity .
In addition to the internal structure of the scaffold, their surface structure, particularly their roughness also plays a significant role in determining how the native tissue will adhere to it. In Fig. 4.1c it is seen that the surfaces were rough as they consisted of aligned fibers . When such rough implanted surfaces come in contact with the bone, which has a lower elastic modulus, stress concentrations occur at their interface , and it has been shown that high stress concentrations at the implant-bone interface increase bone resorption . The magnitude of these stress concentrations is proportional to the sharpness of the implant , and therefore it is important to be able and control the scaffold roughness.
In addition to microstructure the strength of the scaffolds was examined, by uniaxial compression tests, in order to ensure that they could withstand the internal stresses exerted by the body after implantation. It was found that increasing the concentration of the HA nanoparticles and cross-linking agent resulted in an increase of the ultimate tensile strength and yield strength. However, for increasing bone ingrowth a high open porosity and pore size are preferred [3, 15], which dictates the use of low cross-linking concentrations in fabricating appropriate microstructures. Low contents of TA, such as 12.4 mg TA/g in GE, were therefore necessary in order to obtain highly porous microstructures, however, they had a poor mechanical strength, since only 650 kPa were enough to induce a 14% strain . Addition of HA nanorods, however, in this polymer network required twice the stress level (~1.2 MPa) in order to reach such strains; it is interesting to note that similar stress-strain curves were obtained for a TA concentration of 33.3 mg/g in GE without addition of HA. Hence, by reinforcing gelatin biopolymers with HA allowed to retain an open porous structure while increasing their strength. It follows that adding HA-nanorods to the 33.3 mg TA/g GE resulted in brittle biopolymer fibers that would collapse easily. Hence low TA concentrations in GE reinforced with HA resulted in the preferred properties for scaffold engineering.
Fig. 4.2 Formation of the calcium-phosphate layer on the surface of gelatin-HA scaffolds to examine their biocompatibility and biodegradability. (a-b) after 7 days of immersion in SBF; (c-d) after 14 days of immersion in SBF (Reproduced with permission from )