**3. Results and Discussions**

All of the electrospun nanofibers were calcined at three different temperatures, namely, 600, 800 and 1000 ◦C, to study the crystal phase structure. Figure 1 shows the XRD pattern for the nanofibers calcined at different temperatures. The electrospun fibers were mostly crystalline calcium carbonate at 600 ◦C. The amount of CaO increased when the calcination temperature increased, which was due to the breakdown of calcium carbonate that formed calcium oxide, accompanied by the evolution of CO2. The HAp phase formed at 800 ◦C.

**Figure 1.** XRD pattern of the fibers calcined at different temperatures under an air atmosphere. ( : CaO, PDF 70-4068; : hydroxyapatite, PDF 84-1998; : CaCO3, PDF 85-1108).

Figure 2 shows the XRD pattern for the nanofibers calcined under air and nitrogen atmosphere at 800 ◦C. The main crystal phase comprised HAp and CaO. However, the amount of HAp was higher under the nitrogen than under the air atmosphere. The amount of dissolved ambient CO2 can be limited by performing the reaction under a nitrogen atmosphere [16]. Hatzistavrou [17] fabricated hydroxyapatite-CaO composites using the sol-gel method and found that the presence of CaO accelerated the formation of carbonate hydroxyapatite in simulated body fluid. This was due to the dissolution of the CaO phase that rapidly formed carbonate hydroxyapatite. Hence, HAp/CaO composites have been used as scaffolds with tuneable properties by varying the composition.

**Figure 2.** XRD pattern of the fibers calcined at 800 ◦C under various calcination atmospheres. ( : CaO, PDF 70-4068; : hydroxyapatite, PDF 84-1998; : CaCO3, PDF 85-1108).

Figure 3 shows a Fourier transform infrared (FTIR) characteristic spectrum for the nanofibers that were calcined at 800 ◦C under a nitrogen atmosphere. Peaks at approximately 566 and 609 cm−<sup>1</sup> are due to the bending vibration of the P-O bond in PO4 <sup>3</sup><sup>−</sup> [18]. The bands at approximately 960 and 1000~1100 cm−<sup>1</sup> are associated with the stretching modes of the PO4 <sup>3</sup><sup>−</sup> bonds in Hap [19]. The characteristic bands at approximately 873 and 1440~1470 cm−<sup>1</sup> are attributable to the CO3 −2 group. Taherian et al. [20] pointed out CO3 <sup>2</sup><sup>−</sup> ions may form due to the incomplete pyrolysis of organic compounds, or the absorption of CO2 from the atmosphere. Bilton et al. [21] pointed out that the evaporation of unreacted triethyl phosphite from the sol or gel could constitute to the presence of CaCO3. CaCO3 decomposes to CaO after calcination at 800 ◦C

**Figure 3.** FTIR spectra of the fibers calcined at 800 ◦C under an N2 atmosphere.

The dissolution of CO2 from the atmosphere occurs by the following reaction:

$$\text{CO}\_2\text{ (g)} + 2\text{ OH}^-\text{ (aq)} \rightarrow \text{CO}\_3^{2-} \text{ (aq)} + \text{H}\_2\text{O (l)}$$

The characteristic peaks observed at 632 and 3571 cm−<sup>1</sup> are attributable to the respective hydroxyl functional group (-OH) deformation vibration and stretching vibrations of HAp [22]. Additionally, a sharp peak was observed at 3642 cm<sup>−</sup>1, which confirms the formation of the CaO phase [23].

The morphology and structure of the p-HApFs were observed under SEM and TEM. Figure 4a shows the SEM image of the p-HApFs. The average diameter of the p-HApFs was 461 ± 186 nm. Figure 4b shows the TEM image of the p-HApFs. The TEM image showed that the nanofiber was composed of a number of nanocrystals and clearly revealed the existence of mesopores within the nanocrystals. The p-HApFs did not exhibit an ordered orientation of the mesopores, which generally had a random arrangement.

The nitrogen adsorption–desorption isotherms of the p-HApFs were type IV hysteresis loops, which are typical for mesoporous materials (shown in Figure 5a). The specific surface areas of the p-HApFs calculated from the BET equation were 7.2 m2/g. The pore size distribution was plotted according to the BJH nitrogen desorption model as shown in Figure 5b. The average pore size of the p-HApFs calculated from the BJH equation was approximately 28 nm. Figure 5b shows a broad peak ranging from 10 to 130 nm, centered at approximately 50 nm, which suggests that most pores had a size of approximately 50 nm; however, the pore sizes were not uniform.

**Figure 4.** (**a**) SEM and (**b**) TEM micrographs of the nanofibers after heat treatment at 800 ◦C under an N2 atmosphere. (**c**) an enlarged graph in red square of (**b**). The red arrows in (**c**) indicates mesopores within the nanocrystals.

**Figure 5.** (**a**) N2 adsorption–desorption isotherm and (**b**) pore size distribution curve of p-HApFs.

The amount of TC loaded within the p-HApFs was 8.83 ± 0.09 mg/10 mg (TC/p-HApFs). The loading efficiency of TC was 88.34 ± 0.89% (*w*/*w*). The cumulative drug-release curve as a function of time for TC release from the TC-loaded p-HApFs was analyzed in triplicate (shown in Figure 6). The drug-release data indicated that a no-burst release phenomenon occurred at the initial step. The TC release from the TC-loaded p-HApFs was steady and slow over a period of 14 days. The drug-release data were analyzed using different kinetic models: zero-order, first-order, Higuchi, and Korsmeyer-Peppas. The best fit with the highest correlation coefficient (*r*2) was obtained using the first-order equation (*r*<sup>2</sup> = 0.996), followed by the Higuchi model (*r*<sup>2</sup> = 0.941), and zero-order equation (*r*<sup>2</sup> = 0.983). The diffusion exponent *n*-value of the Korsmeyer–Peppas model was 0.75, which indicates anomalous diffusion or both diffusion- and erosion-controlled drug release [24].

**Figure 6.** In vitro cumulative tetracycline (TC) release from TC-loaded p-HApFs.

To clarify the antibacterial activity of TC released from the TC-loaded p-HApFs, the release solution was used to cultivate bacterial strains. As shown in Figure 7, the release solution had a strong ability to inhibit bacterial growth, even on day 7.

**Figure 7.** Susceptibility trend of the released tetracycline against *Staphylococcus aureus* and *Pseudomonas aeruginosa*.
