Nanofibrous electrospun scaffold doped with hydroxyapatite derived from sand lobster shell (Panulirus homarus) for bone tissue engineering

Abstract

Healing of significant segmental bone defects remains a challenge, and various studies attempt to make materials that mimic bone structures and have biocompatibility, bioactivity, biodegradability, and osteoconductivity to native bone tissues. In this work, a nanofiber scaffold membrane of polyvinyl alcohol (PVA)/polyvinylpyrrolidone (PVP)/chitosan (CS) combined with hydroxyapatite (HAp) from sand lobster (SL; Panulirus homarus) shells, as a calcium source, was successfully synthesized to mimic the nanoscale extracellular matrix (ECM) in the native bone. The HAp from SL shells was synthesized by co-precipitation method with Ca/P of 1.67 and incorporated into the nanofiber membrane PVA/PVP/CS synthesized by the electrospinning method with varying concentrations, i.e. 0, 1, 3, and 5% (w/v). Based on the morphological and physicochemical analysis, the addition of HAp into the nanofiber successfully showed incorporation into the nanofiber with small agglomeration at HAp concentrations of 1, 3, and 5% (w/v). This led to a smaller fiber diameter with higher concentration of Hap, and incorporating HAp into the nanofiber could improve the mechanical properties of the nanofiber closer to the trabecula bone. Moreover, in general, swelling due to water absorption increases due to higher hydrophilicity at higher HAp concentrations and leads to the improvement of the degradation process and protein adsorption of the nanofiber. Biomineralization in a simulated body fluid (SBF) solution confirms that the HAp in the nanofiber increases bioactivity, and it can be seen that more apatite is formed during longer immersion in the SBF solution. The nanofiber PVA/PVP/CS HAp 5% has the most potential for osteoblast (MC3T3E1) cell viability after being incubated for 24 h, and it allowed the cell to attach and proliferate. Additionally, the higher HAp concentration in the nanofiber scaffold membrane can significantly promote the osteogenic differentiation of MC3T3E1 cells. Overall, the PVA/PVP/CS/HAp 5% nanofiber scaffold membrane has the most potential for bone tissue engineering.

Fig. 1. Schematics of the experimental procedure; (A) synthesis of HAp and (B) preparation of the electrospinning solution and experimental setup of the electrospinning process.

Fig. 2. Characteristics of the sand lobster shell powder before and after calcination treatment (A) Fourier transform infrared, (B) X-ray diffraction, and (C) X-ray fluorescence.

Fig. 3. Characteristics of HAp based on (A) FTIR, (B) XRD, (C) SEM and (D) EDX analysis and diameter of HAp particles.

Fig. 4. Characteristic (A) XRD and (B) FTIR of nanofiber PVA/PVP/CS/HAp 0, 1, 3 and 5%.

Fig. 5. Morphology of nanofiber membrane PVA/PVP/CS/HAp with HAp concentrations of (A1) 0, (B1) 1, (C1) 3, (D1) 5%, and diameter of nanofiber PVA/PVP/CS/HAp with HAp concentrations of (A2) 0, (B2) 1, (C2) 3, (D2) 5%.

Fig. 6. Diameter of the nanofiber.

Fig. 7. TEM images of nanofiber PVA/PVP/CS/HAp (A) 0% and (B) 5%.

Fig. 8. (A) The stress and strain curve and mechanical property analysis of nanofiber PVA/PVP/CS/HAp: (B) elongation break, (C) tensile strength, and (D) Young's modulus.

Fig. 9. (A) Protein adsorption (B) swelling ratio and (C) biodegradability analysis of PVA/PVP/CS nanofiber with various concentrations of HAp.

Fig. 10. The effect of HAp on the nanofiber PVA/PVP/CS bioactivity immersed for 1, 3, and 5 days in SBF solution.

Fig. 11. SEM image of nanofiber scaffold PVA/PVP/CS/HAp 5% after immersion in SBF solution for 5 days with (A) low magnification and (B) high magnification.

Fig. 12. FTIR analysis of nanofiber PVA/PVP/CS/HAp 5% after soaking for 1, 3, and 5 days in SBF solution.

Fig. 13. EDX spectra of apatite from nanofiber PVA/PVP/CS/HAp 5% after immersion for (A) 1, (B) 3, and (C) 5 days in SBF solution.

Fig. 14. Antibacterial activity against P. aeruginosa and S. aureus based on diameter zone of inhibition (A) semi-quantitative and (B) qualitative.

Fig. 15. (A) Cell viability of nanofiber PVA/PVP/CS with HAp various concentrations and morphology of MC3T3E1 cells after being incubated for 24 h in (B) control and PVA/PVP/CS/HAp (C) 5, (D) 3, and (E) 1%.

Fig. 16. (A) The ALP osteogenic differentiation of MC3T3E1 cells after being incubated for 7 days in the nanofiber; the ALP staining image of (B) control, PVA/PVP/CS/HAp (C) 0, (D) 1, (E) 3, and (F) 5%.