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In this study, we have successfully fabricated electrospun bead-free silk fibroin [SF]/chitosan [CS] composite nanofibers [NFs] covering the whole range of CS content (0%, 25%, 50%, 75%, and 100%). SF/CS spinning solutions were prepared in a mixed solvent system of trifluoroacetic acid [TFA] and dichloromethane. The morphology of the NFs was observed by scanning electron microscope, and the average fiber diameter ranges from 215 to 478 nm. Confocal laser scanning microscopy confirms the uniform distribution of SF and CS within the composite NFs. To increase biocompatibility and preserve nanostructure when seeded with cells in culture medium, NFs were treated with an ethanol/ammonia aqueous solution to remove residual TFA and to change SF protein conformation. After the chemical treatment, SF/CS NFs could maintain the original structure for up to 54 days in culture medium. Properties of pristine and chemically treated SF/CS NFs were investigated by Fourier transform infrared spectroscopy [FT-IR], X-ray diffraction [XRD], and thermogravimetry/differential scanning calorimetry [TG/DSC]. Shift of absorption peaks in FT-IR spectra confirms the conformation change of SF from random coil to β-sheet by the action of ethanol, which is also consistent with the SF crystalline diffraction patterns measured by XRD. From TG/DSC analysis, the decomposition temperature peaks due to salt formation from TFA and protonated amines disappeared after chemical treatment, indicating complete removal of TFA by binding with ammonium ions during the treatment. This was also confirmed with the disappearance of F1s peak in X-ray photoelectron spectroscopy spectra and disappearance of TFA salt peaks in FT-IR spectra. The composite NFs could support the growth and osteogenic differentiation of human fetal osteoblastic [hFOB] cells, but each component in the composite NF shows distinct effect on cell behavior. SF promotes hFOB proliferation while CS enhances hFOB differentiation. The composite SF/CS NFs will be suitable for bone tissue engineering applications by choosing a suitable blend composition.PACS: 87.85.jf; 87.85.Rs; 68.37.Hk.

The presence of both osteoconductive and osteoinductive factors is important in promoting stem cell differentiation toward the osteogenic lineage. In this study, we prepared silk fibroin/chitosan/nanohydroxyapatite/bone morphogenetic protein-2 (SF/CS/nHAP/BMP-2, SCHB2) nanofibrous membranes (NFMs) by incorporating BMP-2 in the core and SF/CS/nHAP as the shell layer of a nanofiber with two different shell thicknesses (SCHB2-thick and SCHB-thin). The physicochemical properties of SCHB2 membranes were characterized and compared with those of SF/CS and SF/CS/nHAP NFMs. When tested in release studies, the release rate of BMP-2 and the concentration of BMP-2 in the release medium were higher for SCHB2-thin NFMs because of reduced shell thickness. The BMP-2 released from the nanofiber retained its osteoinductive activity toward human-bone-marrow-derived mesenchymal stem cells (hMSCs). Compared with SF/CS and SF/CS/nHAP NFMs, the incorporation of BMP-2-promoted osteogenic differentiation of hMSCs and the SCHB-thin NFM is the best scaffold during in vitro cell culture. Gene expression analysis by real-time quantitative polymerase chain reaction detected the evolution of both early and late marker genes of bone formation. The relative mRNA expression is in accordance with the effect of BMP-2 incorporation and shell thickness, while the same was reconfirmed through the quantification of bone marker protein osteocalcin. In vivo experiments were carried out by subcutaneously implanting hMSC-seeded SCHB2-thin NFMs and acellular controls on the back sides of nude mice. Immunohistochemical and histological staining confirmed ectopic bone formation and osteogenesis of hMSCs in SCHB2-thin NFMs. In conclusion, the SCHB2-thin NFM could be suggested as a promising scaffold for bone tissue engineering.

Incorporation of nanohydroxyapatite (nHAP) within a chitosan (CS)/silk fibroin (SF) nanofibrous membrane scaffold (NMS) may provide a favorable microenvironment that more closely mimics the natural bone tissue physiology and facilitates enhanced osteogensis of the implanted cell population. In this study, we prepared pristine CS/SF NMS, composite CS/SF/nHAP NMS containing intrafibrillar nHAP by in situ blending of 10% or 30% nHAP before the electrospinning step, and composite CS/SF/nHAP NMS containing extrafibrillar nHAP by depositing 30% nHAP through alternative soaking surface mineralization. We investigated the effect of the incorporation of HAP nanoparticles on the physicochemical properties of pristine and composite NMS. We confirmed the presence of ~30 nm nHAP in the composite nanofibrous membranes by thermogravimetry analysis (TGA), X-ray diffraction (XRD), and scanning electron microscopy (SEM), either embedded in or exposed on the nanofiber. Nonetheless, the alternative soaking surface mineralization method drastically influenced the mechanical properties of the NMS with 88% and 94% drop in Young’s modulus and ultimate maximum stress. Using in vitro cell culture experiments, we investigated the effects of nHAP content and location on proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). The proliferation of hMSCs showed no significant difference among pristine and composite NMS. However, the extent of osteogenic differentiation of hMSCs was found to be positively correlated with the content of nHAP in the NMS, while its location within the nanofiber played a less significant role. In vivo experiments were carried out with hMSCs seeded in CS/SF/30%nHAP NMS prepared by in situ blending and subcutaneous implantation in nude mice. Micro-computed tomography images as well as histological and immunohistochemical analysis of the retrieved hMSCs/NMS construct 1 and 2 months postimplantation indicated that NMS had the potential for bone regeneration and can be suggested as a promising scaffold for bone tissue engineering.

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