Protein Hydrogels Reinforced with a Nano-HAp/PHB Fiber Network
Another type of hybrid scaffold similar to the one described previously is that of using an electrospun fiber mesh containing nano-HAp but with the biodegradable polymer polyhydroxybutyrate (PHB), and a gelatin-based hydrogel . Such a hybrid scaffold can increase the functionality of the system, over other studies which use synthetic hydrogels.
The gelatin was treated with methacrylate making it photocrosslinkable, while PHB was chosen as it is the simplest member of microbial polyhydroxyalkanoates (PHAs), which are recently being considered for tissue engineering. The HA nanoparticles allowed to further enhance the mechanical properties of the scaffold, as well as its osteoconductivity.
Fabrication and Characterization
The HA nanoparticles and methacrylated gelatin were prepared according to standard procedures. In order to distinguish the gelatin from the PHB nanofibers, fluorescein isothiocyanate (FITC) was added to it during fabrication. The hydrogel consisted of a photoinitiator, 0.1 mg/mL HA and 5% (w/v) of the as prepared gelatin .
To prepare the fiber mats, 15 wt% HA nanoparticles were dispersed in chloroform and then Poly(3-hydroxybutyric) acid was added to the solution as described in , after which the solution was placed in the electrospinning syringe to produce the desired fiber network.
In fabricating the 3D layered scaffolds, the following procedure was followed: (i) a 5 ^L droplet was placed on a glass slide; (ii) the 7 mm diameter electrospun fiber mats were soaked in the hydrogel solution and then placed on the hydrogel droplet of step (i); (iii) a 5 ^L droplet was placed on the soaked fiber mat and then a glass slide on the top. The composite structure was then exposed to UV light and incubated in PBS for 2 h. While in the PBS the glass was detached and therefore the gelatin/PHB/HA scaffolds were obtained. For comparison purposes a similar process was followed to make control scaffolds, without the fiber mat reinforcement, using only gelatin and HA.
Scanning electron microscopy images of the fiber mats indicated that they had a random microstructure and the fiber diameter was ~2 ^m. By performing electron- dispersive spectroscopy (EDS) it was possible to obtain a map of the HA distribution, which indicated that it was evenly distributed throughout. The nanoparticles were ~25 nm along the (002) plane and their crystallinity is 60%. The SEM and EDS mapping indicated that the HA did not attach on the surface of the fibers but were fully encapsulated within the fiber. Such encapsulation is preferred from a mechanical point of view, although surface adhesion increases bioactivity .
Furthermore, performing fluorescent microscopy on the cross section of a fiber indicated that the gel solution diffused to the center of the fibers, as they also appeared to be fluorescent. In sectioning no deformation took place at the fiber-gel interface, suggesting that the interface between the fibers and hydrogels was very strong. SEM was also performed on freeze-dried samples, in order to get a higher resolution of the resulting microstructure and it was observed that the surface and material volume had a high porosity. Particularly, the layers of the gelatin consisted of pores between 30 and 70 ^m, which can allow for preferred adhesion and proliferation of osteoblasts. The use of electron dispersive spectroscopy indicated a uniform distribution of HA nanoparticles within the hydrogel. In order to determine the thermal stability, thermogravimmetry analysis was performed, indicating that the electrospun fibers began to degrade at 250 °C, due to thermal decomposition of PHB. The hydrogel layers began to decompose at ~325 °C .
Tension tests were performed to determine the mechanical strength of the hybrid PHB/hydrogel/HA scaffolds. Their elastic modulus was calculated as 7.0 ± 1.2 MPa, while their tensile strength as 329 ± 18 kPa. Such tests could not be performed on the control hydrogel/HA materials as they were very soft; the modulus and strength of such pure hydrogels has been estimated to be ~0.1 MPa and 100 kPa , respectively, hence the addition of the fiber mat allowed for a significant increase in mechanical properties.