Electrospun Polyhydroxybutyrate/Hydroxyapatite Nanohybrids: Microstructure and Bone Cell Response

doi:10.1016/j.jmst.2016.07.007

Abstract

Abstract:

A substantial amount of nanosized hydroxyapatite (HAp) was tried to incorporate into the electrospun polyhydroxybutyrate (PHB), and electrospinning parameters were optimized to fabricate defect-free PHB/HAp fibers with the smallest possible diameter. According to the results, while the needle inner diameter could not be an effective factor for controlling the fiber morphology, the fiber diameter was observed to increase with increasing the applied voltage and solution concentration. More importantly, it was found that nanoparticles could be successfully encapsulated and distributed inside the ultrafine fibers fabricated under relatively optimum conditions. In vitro cell assays demonstrated that while preosteoblasts had high cell viability and cell spreading on the fibrous nanohybrids, cell metabolic activity can also increase with increasing incubation time. Accordingly, encapsulation of HAp nanoparticles within the PHB in the fibrous form may be promising for bone regeneration applications.

Key words: Electrospinning, Bone tissue engineering, Polymer nanocomposites, Hydroxyapatite, Nanohybrids

Sadat-Shojai Mehdi. Electrospun Polyhydroxybutyrate/Hydroxyapatite Nanohybrids: Microstructure and Bone Cell Response[J]. J. Mater. Sci. Technol., 2016, 32(10): 1013-1020.

Figures/Tables 9

Fig. 1. Schematic of the steps required to fabricate PHB/HAp nanohybrids and to culture cells in vitro.

Table 1 Electrospinning parameters optimized in this study along with the sample codes

Sample code	Electrospinning parameter (x)
Sample code	Needle inner diameter (mm)	Voltage (kV)	Polymer conc. (% w/v)
El-4-V15-N(x)	0.16-0.84	15	4
El-4-V(x)-N0.34	0.34	10-30	4
El-(x)-V25-N0.34	0.34	25	3-7

Fig. 2. Structural and biological characterization of nanosized HAp: (a) SEM micrograph along with EDX analysis (inset); (b) XRD pattern (inset shows the ICDD standard pattern for HAp); (c) FTIR spectrum showing the absorption peaks corresponding to the phosphate and hydroxyl groups; (d) SEM micrographs with different magnifications for HAp powder soaked in SBF at 37 °C for 20 d; (e) representative fluorescence images of Live/Dead staining (day 4) of MC3T3-E1 cells cultured in the presence of nanosized HAp in comparison with the control (tissue culture plastic surface).

Fig. 3. Optical microscopy images of PHB/HAp fibers electrospun under different processing conditions: (a) polymer concentration of 4%, voltage of 15 kV, needle inner diameter of 0.16-0.84 mm; (b) polymer concentration of 4%, voltage of 10-30 kV, needle inner diameter of 0.34 mm; and (c) polymer concentration of 3%-7%, voltage of 25 kV, needle inner diameter of 0.34 mm.

Table 2 Results for morphology (bead defects) and mean diameter of PHB/HAp fibers electrospun under different processing conditions

Sample code	Bead/diameter (μm)
El-4-V15-N(x)	x = 0.84 mm	x = 0.51 mm	x = 0.34 mm	x = 0.26 mm	x = 0.16 mm
El-4-V15-N(x)	Yes/2.3 ± 0.4	Yes/2.2 ± 0.4	Yes/1.9 ± 0.2	Yes/1.8 ± 0.1	Yes/1.8 ± 0.1
El-4-V(x)-N0.34	x = 10 kV	x = 15 kV	x = 20 kV	x = 25 kV	x = 30 kV
El-4-V(x)-N0.34	Yes/1.7 ± 0.2	Yes/1.9 ± 0.2	No/1.9 ± 0.3	No/2.0 ± 0.2	No/2.5 ± 0.3
El-(x)-V25-N0.34	x = 3%	x = 4%	x = 5%	x = 6%	x = 7%
El-(x)-V25-N0.34	Yes/1.9 ± 0.2	No/2.0 ± 0.2	No/2.7 ± 0.3	No/2.9 ± 0.3	No/3.2 ± 0.2

Fig. 4. Representative SEM micrographs of electrospun fibers having a large number of bead defects (a) in comparison with defect-free nanohybrids electrospun under relatively optimum conditions (b). (c) EDX-mapping of HAp crystals encapsulated within PHB fibers shown in (b). (d) Schematic representation of a single PHB fiber embedded with HAp nanoparticles. (e) SEM micrographs of optimum fibers in regions with some agglomerated particles. (f) EDX-mapping of HAp crystals inside PHB fibers shown in (e); circles indicate the location of particle agglomerates.

Fig. 5. TGA thermogram of PHB/HAp fibers showing two weight losses during the thermal scan.

Fig. 6. Representative fluorescence images of Live/Dead staining of MC3T3-E1 cells after 1 and 4 days of culture on the surface of PHB/HAp nanohybrids compared with the control (tissue culture plastic surface).

Fig. 7. Cell metabolic activity (a) and ALP level (b) of MC3T3-E1 cells cultured on the surface of PHB/HAp nanohybrids in comparison with the control (tissue culture plastic surface); results are mean ± SD of nine measurements and asterisks (*) indicate significant difference (p < 0.05) between time points. (c) Representative fluorescence images of F-actin microfilaments (red) and nucleus (blue) of MC3T3-E1 cells after 4 d of culture on the surface of PHB/HAp nanohybrids, indicating the elongated morphology of cells at two different magnifications.

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