Fabrication and Characteristics of Porous Hydroxyapatite-CaO ... - MDPI

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Fabrication and Characteristics of Porous Hydroxyapatite-CaO Composite Nanofibers for Biomedical Applications Shiao-Wen Tsai 1,2,3 , Sheng-Siang Huang 4 , Wen-Xin Yu 4 , Yu-Wei Hsu 4 and Fu-Yin Hsu 4, * 1 2 3 4

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ID

Graduate Institute of Biomedical Engineering, Chang Gung University, Taoyuan City 33302, Taiwan; [email protected] Department of Orthopedic Surgery, Chang Gung Memorial Hospital, Linkou 33305, Taiwan Department of Periodontics, Chang Gung Memorial Hospital, Taipei 10507, Taiwan Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung City 20224, Taiwan; [email protected] (S.-S.H.); [email protected] (W.-X.Y.); [email protected] (Y.W.H.) Correspondence: [email protected]; Tel.: +886-02-2462-2192

Received: 9 July 2018; Accepted: 24 July 2018; Published: 26 July 2018

Abstract: Hydroxyapatite (HAp), a major inorganic and essential component of normal bone and teeth, is a promising biomaterial due to its excellent biocompatibility, bioactivity, and osteoconductivity. Therefore, synthetic HAp has been widely used as a bone substitute, cell carrier, and delivery carrier of therapeutic genes or drugs. Mesoporous materials have attracted considerable attention due to their relatively high surface area, large pore volume, high porosity, and tunable pore size. Recently, mesoporous HAp has also been successfully synthesized by the traditional template-based process and has been demonstrated to possess better drug-loading and release efficiencies than traditional HAp. It is widely accepted that cell adhesion and most cellular activities, including spreading, migration, proliferation, gene expression, surface antigen display, and cytoskeletal functioning, are sensitive to the topography and molecular composition of the matrix. The native extracellular matrix is a porous, nanofibrous structure. The major focus of this study is the fabrication of porous hydroxyapatite-CaO composite nanofibers (p-HApFs) and the investigation of its drug-release property. In this study, nanofibers were prepared by the sol-gel route and an electrospinning technique to mimic the three-dimensional structure of the natural extracellular matrix. We analyzed the components of fibers using X-ray diffraction and determined the morphology of fibers using scanning and transmission electron microscopy. The average diameter of the nanofibers was approximately 461 ± 186 nm. The N2 adsorption–desorption isotherms were type IV isotherms. Moreover, p-HApFs had better drug-loading efficiency and could retard the burst release of tetracycline and maintain antibacterial activity for a period of 7 days. Hence, p-HApFs have the potential to become a new bone graft material. Keywords: sol-gel; electrospinning; hydroxyapatite; nanofiber; antibacterial

1. Introduction Bone is a natural inorganic–organic composite consisting of collagen fibrils containing well-arrayed apatite nanocrystals. Hydroxyapatite [HAp, Ca10 (PO4 )6 (OH)2 ] is chemically similar to the inorganic component of the bone matrix, which has led to extensive research efforts to use this material as a bone substitute in both orthopedic and dental fields. Recently, HAp has been used in range of biomedical applications, such as matrices for drug release and bone tissue engineering scaffold materials.

Nanomaterials 2018, 8, 0; doi:10.3390/nano8080570

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HAp can be synthesized through a variety of well-developed techniques, such as the sol-gel process [1], the wet-chemical reaction [2], the solid-state reaction [3] and the chemical vapor deposition [4]. The synthesis of HAp using the sol-gel method consists of the molecular-level mixing of calcium and phosphorus precursors under significantly milder conditions. Hence, a sol-gel synthesis of HAp has recently attracted considerable attention. The sol-gel synthesis technique is also used in conjunction with various spinning techniques to fabricate HAp fibers [5]. Electrospinning is an easy and simple method that utilizes electrical fields to fabricate nano- to microfibers from a polymer solution. The topological structure of the electrospun matrix closely mimics the dimensions of the natural extracellular matrix (ECM). The ECM plays a key role in triggering intracellular signaling cascades for tissue regeneration. Thus, the development of new ECM substitutes has attracted a wide attention as a scaffold for tissue engineering. Franco et al. successfully fabricated HAp nanofibers by combining electrospinning and a non-alkoxide sol-gel system [6]. Pasuri noted that electrospun HAp nanofibers do not activate macrophages in vitro and can be resorbed by human osteoclasts [7]. Recently, much attention has been attracted by HAp as a drug delivery carrier for the loading and delivery of therapeutic agents, such as proteins, growth factors, genes, and drugs [8–10]. Mesoporous HA nanoparticles possess a higher drug-loading capacity and drug-release efficiency than HAp nanoparticles as a result of their large surface areas and high pore volumes [11]. The synthesis of mesoporous HAp nanoparticles using template reagents, such as Pluronic P123 [12], cetyltrimethylammonium bromide (CTAB) [13], and Pluronic F127 [14], is the most well-known method. In the synthesis of mesoporous HAp, the self-assembly of inorganic HAp phases and template reagents followed by template removal can produce the mesoporous structure. Münchow [15] incorporated CaO nanoparticles into electrospun matrices and demonstrated improved cell viability and osteogenic differentiation. Hence, the aims of this study were to fabricate and characterize a nanofibrous structure of porous hydroxyapatite-CaO (p-HApFs) by utilizing an electrospinning process based on a sol-gel precursor and to evaluate the release profiles and antibacterial activity of tetracycline from p-HApFs. 2. Materials and Methods 2.1. Synthesis and Characterization of Porous Hydroxyapatite Nanofibers Mesoporous hydroxyapatite nanofibers were synthesized using poly(ethylene glycol)-block-poly (propylene glycol)-block-poly(ethylene glycol) (P123, Sigma-Aldrich, St. Louis, MO, USA) as the structure-directing agent. Briefly, 1.0 g of P123 and 6.172 mL of triethyl phosphite (TEP, Merck, Germany) were mixed in a 10 mL ethanol aqueous solution (50% v/v), which was then continuously stirred to form a clear solution. Ca(NO3 )2 (14 g, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 10 mL of absolute ethanol under magnetic stirring at room temperature. The calcium nitrate solution was slowly added under stirring to the above P123/TEP solution to form a precursor solution. The precursor solution was tightly capped and placed in an oven at 60 ◦ C for 12 h. Then, 1.5 g poly(vinyl pyrrolidone) (PVP, Sigma-Aldrich, St. Louis, MO, USA), 0.45 g P123 and 7 mL absolute ethanol were prepared and incorporated into the 3 mL precursor solution to obtain a transparent mixture solution. The mixture solution was placed inside a plastic syringe fitted with a stainless needle (18 G, inner diameter = 0.838 mm) and then inserted into a syringe pump to supply a steady flow rate (1.27 mL/h). An electrical field (1.3 kV/cm) was applied between the needle and an aluminum substrate (grounded collector) using a high-voltage power supply (SL 60, Spellman, New York, NY, USA). The polymer solution, which formed a Taylor cone upon exit, was collected on an aluminum substrate in the form of nonwoven nanofiber structures and was then calcined at different temperatures (600 ◦ C, 800 ◦ C and 1000 ◦ C) and environments (air and nitrogen) to obtain the calcined p-HApFs nonwoven structures.


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2.2. Characterization of the p-HApF The p-HApFs morphology was observed using scanning electron microscopy (SEM, Hitachi S-4800, Tokyo, Japan), which operated at an accelerating voltage of 15 kV The average fiber diameter of the p-HApFs was analyzed by image analysis software (Image-Pro Express Version 6.0, Media Cybernetics, Rockville, MD, USA) based on the SEM images. Moreover, the p-HApFs pore structure was observed using transmission electron microscopy (TEM, JEOL JEM-2100, Tokyo, Japan) at an accelerating voltage of 100 kV. Nitrogen adsorption–desorption measurements were used to obtain the Brunauer-Emmett-Teller (BET) specific surface area, and the pore size was also analyzed (Micromeritic ASAP 2020 instrument, Norcross, GA, USA). The phase of p-HApFs was characterized by X-ray diffraction (XRD, Bruker D2-Phaser, Madison, WI, USA) with a copper target. Powder diffraction patterns were acquired over 2-theta, ranging from 20 to 60◦ with a step size of 0.04◦ . The functional groups of p-HApFs were analysed with Fourier transitioned infrared spectroscopy (FTIR, Bruker tensor II, Madison, WI, USA). The spectra were recorded from 4000 to 400 cm−1 wave number with a resolution of 2 cm−1 . 2.3. In Vitro Study of Drug Loading and Release Tetracycline (TC) was selected as the model drug for the evaluation of drug loading and release. p-HApFs (10 mg) was added to a TC aqueous solution (5 mg/mL, 10 mL) and stirred for 24 h. After the loading procedure, the amount of TC adsorbed onto p-HApFs, which calcined at N2 atmosphere 800 ◦ C, was calculated by determining the difference in the TC concentration before and after loading. The TC concentration was analyzed at a wavelength of 360 nm using a UV-Vis spectrophotometer (Ultrospec 1100 Pro, Amersham Biosciences, Piscataway, NJ, USA). The drug contents and loading efficiency were calculated according to the following formulas: Drug content (w/w) = weight of the TC in the p-HApFs/weight of the p-HApFs Loading efficiency (%) = (weight of the TC in the p-HApFs/initial weight of the p-HApFs) × 100% After loading, the p-HApFs were dried using a lyophilization process. TC-loaded p-HApFs (20 mg) were added to a phosphate-buffered saline (PBS) solution (2 mL) and agitated in a horizontally shaking bath at 37 ◦ C. The release medium was withdrawn and replaced with fresh PBS at each measurement. The mechanism of drug release was analyzed by fitting the experimental data to equations describing different kinetic orders. Linear regression analyses were performed for zero-order [Mt /M0 = K0 ·t] and first-order [ln(M0 − Mt ) = K1 ·t] kinetics, as well as the Higuchi [Mt /M0 = KH ·t1/2 ] model, where K is the kinetic constant and Mt /M0 is the fraction of TC released at time t. The best-fitted model was assessed on the basis of the correlation coefficient (r2 ). The drug release data were further analyzed with the Ritger-Peppas equation [Mt /M∞ = kr ·tn ], where Mt is the amount of drug released at time t, M∞ is the amount of drug released at time ∞, and kr and n represent the release rate constant and release exponent, respectively. 2.4. Antibiotic Activity against Staphylococcus aureus and Pseudomonas aeruginosa The antibiotic activity of TC in the in vitro release study was assessed against a Gram-positive bacterial strain, Staphylococcus aureus, and a Gram-negative bacterial strain, Pseudomonas aeruginosa, using the antibiotic elution samples from a turbidity assay. TC (500 µL) eluated during the in vitro release study or from PBS (as the control group) was added to the inoculum containing 500 µL of the 2× nutrient broth solution and 100 µL of bacteria in 5-mL test tubes. After 24 h of incubation, the optical density (OD) of the culture medium was measured using a spectrophotometer at a wavelength of 600 nm. The relative antibacterial activity (R%) was calculated as follows: R% = [(ODgrowth medium − ODreleased tetracycline )/ODgrowth medium ] × 100%

Figure Figure 1. 1. XRD XRD pattern pattern of of the the fibers fibers calcined calcined at at different different temperatures temperatures under under an an air air atmosphere. atmosphere. (( :: CaO, :: CaCO CaO, PDF PDF 70-4068; 70-4068; :: hydroxyapatite, hydroxyapatite, PDF PDF 84-1998; 84-1998; CaCO33,, PDF PDF 85-1108). 85-1108).

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Figure 22 shows the Figure shows the XRD XRD pattern pattern for for the the nanofibers nanofibers calcined calcined under under air air and and nitrogen nitrogen atmosphere atmosphere Nanomaterials 8, x FOR PEER REVIEW 4 of 9 3. Discussions 3. Results Results3.and and Discussions Results and2018, Discussions at at 800 800 °C. °C. The The main main crystal crystal phase phase comprised comprised HAp HAp and and CaO. CaO. However, However, the the amount amount of of HAp HAp was was higher higher 3. Results and Discussions nanofibers were calcined at three temperatures, namely, 600, 800 All the electrospun nanofibers were calcined at three different different temperatures, namely, 600, 800 All of of the electrospun All ofnitrogen the electrospun nanofibers calcined at three different temperatures, namely, under the the air atmosphere. The amount of ambient CO be 3.Nanomaterials Results and Discussions under the nitrogen than under the airwere atmosphere. The amount of dissolved dissolved ambient CO22600, can be 2018, 8,than x FORunder PEER REVIEW 4can of 9 800 and °C, study the crystal phase structure. Figure 11 shows the XRD pattern for the and 1000 1000limited °C, to to study the crystal phase structure. Figure shows the XRD pattern for the nanofibers nanofibers All of°C, the nanofibers were calcined atatmosphere three different temperatures, namely, 600, 800 and 1000 toelectrospun study the crystal phase structure. Figure 1 shows the XRD pattern for the nanofibers by performing the reaction under a nitrogen [16]. Hatzistavrou [17] fabricated limited by performing the reaction under a nitrogen atmosphere [16]. Hatzistavrou [17] fabricated All◦ of the electrospun nanofibers were calcined at three different temperatures, namely, 600, 800 3. Results and Discussions calcined different temperatures. fibers were mostly crystalline calcium carbonate calcined at at different temperatures. The electrospun electrospun fibers were mostly crystalline calcium carbonate and 1000 to study the The crystal phase structure. Figure 1were shows the XRD pattern for the nanofibers calcined atC, different temperatures. The electrospun fibers mostly crystalline calcium carbonate hydroxyapatite-CaO composites the sol-gel method and that the of hydroxyapatite-CaO composites using the sol-gel method and found that the presence of CaO CaO and 1000 °C, to study the crystal using phase structure. Figure 1 shows thefound XRD pattern for presence the nanofibers at The amount of CaO increased when the calcination temperature increased, which was due at 600 600 °C. °C.accelerated The amount of CaO increased when the calcination temperature increased, which was due calcined at different temperatures. The electrospun fibers were mostly crystalline calcium carbonate at at 600 °C. The amount of CaO increased when the calcination temperature increased, which was the formation of carbonate hydroxyapatite in simulated body fluid. This was due to the accelerated the formation of carbonate hydroxyapatite in simulated body fluid. This was due to the All of the electrospun nanofibers were calcined at three different temperatures, namely, 600, 800 calcined at different temperatures. The electrospun fibers were mostly crystalline calcium carbonate due ◦ C. The to of calcium carbonate that formed calcium oxide, accompanied by the evolution of to the the breakdown breakdown of calcium carbonate that formed calcium oxide, accompanied by the evolution of 600the amount of CaO increased when the calcination temperature increased, which was due to to breakdown of calcium carbonate that formed calcium oxide, accompanied by the evolution of and 1000 °C, to study the crystal phase structure. Figure 1 shows the XRD pattern for the nanofibers dissolution of the CaO phase that rapidly formed carbonate hydroxyapatite. Hence, HAp/CaO dissolution of the CaO phase that rapidly formed carbonate hydroxyapatite. Hence, HAp/CaO at 600 °C. The amount of CaO increased when the calcination temperature increased, which was due CO HAp phase formed at 800 °C. CO22.. The Thecomposites HAp phase formed atformed 800 °C. the ofbeen calcium carbonate that formed calcium oxide, accompanied by of CO 2breakdown .calcined The HAp phase 800 °C. at different temperatures. Thewith electrospun fibers were mostly crystalline calcium carbonate have used as scaffolds tuneable properties by varying the composition. composites have been used asat scaffolds with tuneable properties by varying the composition. to the breakdown of calcium carbonate that formed calcium oxide, accompanied bythe theevolution evolution of CO2 . ◦ at 2600 °C. Theformed amount CaO increased TheCO HAp phase atof800 . The HAp phase formed atC. 800 °C. 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 XRD pattern the fibers calcined at different temperatures under an air atmosphere. Figure pattern of the fibers calcined at different under an air atmosphere. Figure 1. 1. XRD XRD pattern ofpattern the fibers calcined atcalcined different temperatures under an air atmosphere. Figure 1.XRD pattern of the fibers calcined at °C different temperatures under an air atmosphere. 1. XRD pattern ofof the fibers calcined attemperatures different temperatures under an atmospheres. air atmosphere. Figure 2. of the fibers at 800 under various calcination Figure 2. XRD pattern of the fibers calcined at 800 °C under various calcination atmospheres. Nanomaterials 2018, 8, x FOR PEER REVIEW ( 70-4068; : CaO, PDF:: 70-4068; : hydroxyapatite, PDF 84-1998; :PDF CaCO 3, PDF 85-1108). (( :: CaO, hydroxyapatite, PDF 84-1998; : CaCO 3 , 85-1108). CaO, PDF PDF 70-4068; hydroxyapatite, PDF 84-1998; : CaCO 3 , PDF 85-1108). CaO, PDF 70-4068; hydroxyapatite,PDF PDF84-1998; 84-1998; :: :CaCO CaCO33,,3,,PDF PDF85-1108). 85-1108). (((( ::::CaO, CaO, PDF 70-4068; hydroxyapatite, PDF 84-1998; PDF 85-1108). CaO,PDF PDF70-4068; 70-4068; :::hydroxyapatite, hydroxyapatite, CaCO PDF 85-1108). Figure 1. XRD pattern of the fibers calcined at different temperatures3 under an air atmosphere.

3. atmosphere Results and Discussions Figure 2 shows the XRD pattern for the nanofibers calcined under air85-1108). and nitrogen ( : CaO, PDF 70-4068; : hydroxyapatite, PDF 84-1998; : CaCO 3, PDF Figure XRD the calcined air and Figure 22 shows shows the XRD pattern pattern for the nanofibers nanofibers calcined under under airunder and nitrogen nitrogen atmosphere Figurethe 2 shows the XRDfor pattern for the nanofibers calcined air and atmosphere nitrogen atmosphere atFigure 800 °C. 2The main crystal phase comprised HAp and CaO. However, the amount of HAp wasatmosphere higher shows the XRD pattern for the nanofibers calcined under air and nitrogen Allwas of the electrospun nanofibe at The main crystal phase comprised HAp and CaO. However, the of HAp was higher at 800 800 °C. °C.at The main crystal phase comprised HApfor and CaO. However, the amount amount ofamount HAp was higher 800 °C. The main crystal phase comprised HAp and CaO. However, the of HAp higher ◦ Figure 2 shows the XRD pattern the nanofibers calcined under air and nitrogen atmosphere underC.the nitrogen than under thecomprised air atmosphere. of dissolvedthe ambient COof 2 can be at 800 The main crystal phase HApThe andamount CaO. However, amount HAp was and 1000 °C, to study the crystal ph under the nitrogen than under the air atmosphere. The amount of dissolved ambient CO 2 can be under theunder nitrogen than under the air atmosphere. The amount of dissolved ambient CO 2 can be the °C. nitrogen than under thecomprised air atmosphere. The amount ofthe dissolved ambient CO 2 can be at 800 The crystal phase HAp andatmosphere CaO. However, amount of HAp was higher limited by performing the reaction under nitrogen Hatzistavrou [17] fabricated higher under themain nitrogen than under the aair atmosphere. The [16]. amount of dissolved ambient CO2 temperatures. T calcined at different limited performing the reaction nitrogen [16]. Hatzistavrou [17] fabricated limited by by performing the reaction under nitrogen atmosphere [16]. Hatzistavrou [17] fabricated limited by the performing the under reaction under a atmosphere nitrogen atmosphere Hatzistavrou [17] under nitrogen than underaathe air the atmosphere. The amount of[16]. dissolved ambient COof 2 fabricated can composites sol-gel and found that the presence CaObe[17] canhydroxyapatite-CaO be limited by performing theusing reaction under method a nitrogen atmosphere [16]. Hatzistavrou at 600 °C. amount of CaO incre hydroxyapatite-CaO composites using the sol-gel method and found that the presence of CaO hydroxyapatite-CaO composites using the sol-gel method and found that the presence of CaO limited by performing the reaction under a nitrogen atmosphere [16]. Hatzistavrou [17] fabricated hydroxyapatite-CaO composites usinghydroxyapatite the sol-gel method and body foundfluid. thatThis the was presence of CaO accelerated the formation of carbonate in simulated due to The the fabricated hydroxyapatite-CaO composites using the sol-gel method and found that the breakdown presence of of calcium carbon to the hydroxyapatite-CaO the formed sol-gel method and found that theHence, presence of CaO accelerated the formation of hydroxyapatite in body fluid. This was due the accelerated the formation of carbonate hydroxyapatite in simulated simulated body fluid. This was due to to the dissolution thecarbonate CaOcomposites phase thatusing rapidly carbonate hydroxyapatite. HAp/CaO accelerated theofformation of carbonate hydroxyapatite in simulated body fluid. This was due to the CaOaccelerated acceleratedthe theformation formation of carbonate hydroxyapatite in simulated body fluid. This was due to CO The HAp of hydroxyapatite in simulated body Hence, fluid. This was2. due to the phase formed at 800 dissolution of the phase that rapidly carbonate hydroxyapatite. HAp/CaO dissolution of the CaO CaO phase that rapidly formed carbonate hydroxyapatite. Hence, HAp/CaO composites been used as carbonate scaffolds with tuneable byhydroxyapatite. varying the composition. dissolution ofhave the CaO phase thatformed rapidly formed properties carbonate Hence, HAp/CaO the dissolution of the CaO phase that rapidly formed carbonatehydroxyapatite. hydroxyapatite.Hence, Hence, HAp/CaO dissolution ofbeen the CaO that rapidly formed carbonate HAp/CaO composites have been used as tuneable by composition. composites have beenhave used as scaffolds scaffolds with tuneable properties by varying varying the composition. composites used phase as with scaffolds with properties tuneable properties bythe varying the composition. composites have been used as scaffolds with tuneable properties by varying the composition. 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 1. XRD pattern of the fibers Figure 2. XRD pattern of the fibers calcined at 800 °C under various calcination atmospheres. ◦various ( :: CaO, : hydrox Figure 2. XRD pattern of the fibers calcined at 800 C under various calcination atmospheres. ( CaO, PDF 70-4068; Figure pattern of the fibers calcined at 800 °C under calcination atmospheres. Figure 2. 2. XRD XRD pattern of the fibers calcined at 800 °C under various calcination atmospheres. pattern of the :fibers calcined atPDF 80084-1998; °C under :various atmospheres. ( : CaO, PDF 70-4068; hydroxyapatite, CaCO3, calcination PDF 85-1108).

(( :: CaO, 70-4068; :: hydroxyapatite, PDF :: CaCO 85-1108). CaO, PDF PDF 70-4068; hydroxyapatite, PDF 84-1998; 84-1998; PDF 85-1108). 70-4068; hydroxyapatite, CaCO333,,,PDF PDF 85-1108). (PDF : CaO, PDF 70-4068; : hydroxyapatite, PDF 84-1998; : CaCO 3, PDF 85-1108).

Figure 2 shows the XRD patter at 800 °C. The main crystal phase com under the nitrogen than under the limited by performing the reaction hydroxyapatite-CaO composites u

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−1 are due to the bending vibration of the P-O bond in PO43− [18]. The bands at approximately 960 and 1000~1100 cm−1 are associated with the stretching modes of the PO43− bonds in Hap [19]. The Nanomaterials 2018,bands 8, 0 5 of 9 characteristic at approximately 873 and 1440~1470 cm−1 are attributable to the CO3−2 group. 2− Taherian et. al [20] pointed out CO3 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 Figure 3 shows a Fourier transform infrared (FTIR) characteristic spectrum for the nanofibers evaporation of unreacted triethyl phosphite from the sol or gel could constitute to the presence of that were calcined at 800 ◦ C under a nitrogen atmosphere. Peaks at approximately 566 and 609 cm−1 CaCO3. CaCO3 decomposes to CaO after calcination at 800 °C are due to the bending vibration of the P-O bond in PO4 3− [18]. The bands at approximately 960 The dissolution of CO2 from the atmosphere occurs by the following reaction: and 1000~1100 cm−1 are associated with the stretching modes of the PO4 3− bonds in Hap [19]. 1 are attributable to the CO −2 − (aq) The characteristic bands at approximately and → 1440~1470 CO32− (aq) +cm H− 2O (l) CO2 (g) + 2 OH873 3 2 − group. Taherian et al. [20] pointed out CO3 ions may form due to the incomplete pyrolysis of organic The characteristic peaks observed at 632 and 3571 cm−1 are attributable to the respective hydroxyl compounds, or the absorption of CO2 from the atmosphere. Bilton et al. [21] pointed out that the functional group (-OH) deformation vibration and stretching vibrations of HAp [22]. Additionally, a evaporation of unreacted triethyl phosphite from the sol or gel could constitute to the presence of sharp peak was observed at 3642 cm−1, which confirms the formation of the CaO phase [23]. CaCO3 . CaCO3 decomposes to CaO after calcination at 800 ◦ C

Figure3.3.FTIR FTIRspectra spectraof ofthe thefibers fiberscalcined calcinedatat800 800◦°C underan anN N2 atmosphere. atmosphere. Figure C under 2

The morphology and structure of the p-HApFs were observed under SEM and TEM. Figure 4a The dissolution of CO from the atmosphere occurs by the following reaction: shows the SEM image of 2the 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 CO2 (g) + 2 OH− (aq) → CO3 2− (aq) + H2 O (l) composed of a number of nanocrystals and clearly revealed the existence of mesopores within the nanocrystals. The p-HApFs not exhibit ordered orientation of the mesopores, which hydroxyl generally −1 are attributable The characteristic peaks did observed at 632an and 3571 cm to the respective had a random arrangement. functional group (-OH) deformation vibration and stretching vibrations of HAp [22]. Additionally, The nitrogen adsorption–desorption isotherms of the were type IVphase hysteresis a sharp peak was observed at 3642 cm−1 , which confirms thep-HApFs formation of the CaO [23]. loops, which are typical for mesoporous materials (shown in Figure 5a). The specific surface areas of the4a pThe morphology and structure of the p-HApFs were observed under SEM and TEM. Figure 2 HApFsthe calculated from equation 7.2 m /g. Theofpore size distribution plotted shows SEM image of the the BET p-HApFs. Thewere average diameter the p-HApFs was 461was ± 186 nm. according to the BJH nitrogen desorption model as shown in Figure 5b. The average pore size the Figure 4b shows the TEM image of the p-HApFs. The TEM image showed that the nanofiberofwas p-HApFs calculated from the BJH equation was approximately 28 nm. Figure 5b shows a broad peak composed of a number of nanocrystals and clearly revealed the existence of mesopores within the ranging fromThe 10 to 130 nm, did centered at approximately 50 nm, which suggests that most pores had a nanocrystals. p-HApFs not exhibit an ordered orientation of the mesopores, which generally size of approximately 50 nm; however, the pore sizes were not uniform. 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.

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Figure 4.(a) (a) SEMand and (b)TEM TEM micrographsof of thenanofibers nanofibers afterheat heat treatmentatat800 800◦°C under an Figure C under Figure4. 4. (a) SEM SEM and (b) (b) TEM micrographs micrographs of the the nanofibersafter after heattreatment treatment at 800 °C underan an atmosphere. (c) (c) an an enlarged enlarged graph graph in in red red square squareof of(b). (b). The The red redarrows arrowsin in(c) (c)indicates indicatesmesopores mesopores N2 atmosphere. N 2 N2 atmosphere. (c) an enlarged graph in red square of (b). The red arrows in (c) indicates mesopores withinthe thenanocrystals. nanocrystals. within within the nanocrystals.

Figure 5. (a) N2 adsorption–desorption isotherm and (b) pore size distribution curve of p-HApFs. Figure5. 5. (a) (a) N N2 adsorption–desorption adsorption–desorption isotherm isotherm and and (b) (b) pore pore size size distribution distribution curve curve of of p-HApFs. p-HApFs. Figure 2

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The amount of TC 0.09 mg/10 mg mg (TC/p-HApFs). The The TC loaded loaded within withinthe thep-HApFs p-HApFswas was8.83 8.83± ± 0.09 mg/10 (TC/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 TCofwas 0.89% cumulative drug-release curve as a function The loading efficiency TC88.34 was ±88.34 ±(w/w). 0.89%The (w/w). The cumulative drug-release curve as of a loading efficiency of TC was 88.34 ± 0.89% (w/w). The cumulative drug-release curve as a function of time for TC release the TC-loaded wasp-HApFs analyzedwas in triplicate in Figure 6). The function of time forfrom TC release from thep-HApFs TC-loaded analyzed(shown in triplicate (shown in time for TC release from the TC-loaded p-HApFs was analyzed in triplicate (shown in Figure 6). The drug-release indicateddata thatindicated a no-burstthat release phenomenon occurred at the initial step. TC Figure 6). Thedata drug-release a no-burst release phenomenon occurred at theThe initial drug-release data indicated that a no-burst release phenomenon occurred at the initial step. The TC release from TC-loaded p-HApFs was steady andwas slowsteady over aand period of over 14 days. The drug-release step. The TCthe release from the TC-loaded p-HApFs slow a period of 14 days. release from the TC-loaded p-HApFs was steady and slow over a period of 14 days. The drug-release datadrug-release were analyzed using kinetic first-order, Higuchi, and KorsmeyerThe data weredifferent analyzed usingmodels: differentzero-order, kinetic models: zero-order, first-order, Higuchi, data were analyzed using different kinetic models: zero-order, 2first-order, Higuchi, and Korsmeyer2 ) was Peppas. The best fit with the highest correlation ) was obtained using the first-order and Korsmeyer-Peppas. The best fit with the highestcoefficient correlation(rcoefficient (r obtained using the Peppas. The best fit with the highest correlation coefficient (r2) 2was obtained using the first-order 2 2 2 2 equation (requation = 0.996),(rfollowed the Higuchi model (r =model 0.941),(rand (r equation = 0.983). first-order = 0.996),byfollowed by the Higuchi = zero-order 0.941), and equation zero-order equation (r2 = 0.996), followed by the Higuchi model (r2 = 0.941), and zero-order equation (r2 = 0.983). 2 = 0.983). The diffusion n-value n-value of the of Korsmeyer–Peppas model waswas 0.75, indicates (r The exponent diffusion exponent the Korsmeyer–Peppas model 0.75,which which indicates The diffusion exponent n-value of the Korsmeyer–Peppas model was 0.75, which indicates anomalous diffusion diffusion or or both both diffusiondiffusion- and anderosion-controlled erosion-controlleddrug drugrelease release[24]. [24]. anomalous anomalous diffusion or both diffusion- and erosion-controlled drug release [24].

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

To clarify the antibacterial activity of TC released from the TC-loaded p-HApFs, the release To clarify clarify the the antibacterial antibacterial activity activity of of TC TC released released from from the the TC-loaded TC-loaded p-HApFs, p-HApFs, the the release release To solution was used to cultivate bacterial strains. As shown in Figure 7, the release solution had a strong solutionwas wasused usedto to cultivate cultivatebacterial bacterialstrains. strains. As As shown shown in in Figure Figure 7, 7, the the release release solution solution had had aa strong strong solution ability to inhibit bacterial growth, even on day 7. ability to to inhibit inhibit bacterial bacterial growth, growth,even evenon onday day7.7. ability

Figure 7. Susceptibility trend of the released tetracycline against Staphylococcus aureus and Figure 7.7.Susceptibility Susceptibility trend of the released tetracycline against Staphylococcus aureus and Figure Pseudomonas aeruginosa.trend of the released tetracycline against Staphylococcus aureus and Pseudomonas Pseudomonas aeruginosa. aeruginosa.


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4. Conclusions A mesoporous hydroxyapatite nanofibrous matrix to mimic the three-dimensional structure of the natural extracellular matrix was successfully fabricated by an electrospinning process based on a sol-gel precursor. p-HApFs exhibited a high drug-loading efficiency and could retard the burst release of TC, as well as maintain antibacterial activity over a period of 7 days. Hence, it is anticipated that p-HApFs have the potential to be used as drug delivery carriers and as bone graft substitutes. Author Contributions: Data curation, S.-W.T. and F.-Y.H.; Formal analysis, S.-W.T., S.-S.H. and F.-Y.H.; Methodology, S.-S.H., W.-X.Y. and Y.-W.H.; Project administration, F.-Y.H.; Writing—original draft, S.-W.T. and F.-Y.H. Funding: This research was funded by the Ministry of Science and Technology of Taiwan, grant number MOST 102-2221-E-019-003-MY3 and MOST 105-2221-E-019-008. Conflicts of Interest: The authors declare no conflict of interests.

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