| In nerve tissue engineering, regeneration and reparation of injured nervous tissues still remains to be one of the most intensively investigated topics. Compared with the direct restoration, autogenous nerve transplantation and allogenic nerve transplantation, the nerve scaffold provides a new method to solve the problems, such as the limited sources of nerve, the low success rate of transplantation, severe rejection and small range of reparation. As the subject of application, the nerve scaffold plays a decisive role in nerve tissue engineering. It is not only provides the best biological micro environment, but also can influence cell adhesion, migration, proliferation and differentiation. This process is aimed to promote nerve cell regeneration and nerve tissue repair. Keratin is a kind of important functional materials, has a good prospect for clinical application due to its good biocompatibility, absorbability, biodegradability and low immunogenicity. But keratin structure lead to its spinnability of poorer, less should be used in the manufacture of mono-component nanofibers scaffold. In previous research on tissue engineering, some attempts have already been done by mixing the keratin and many biomaterials to be electrospun. But with the incorporation of keratin, the mechanical property of composite fiber decreased while the surfaces of fibers were not completely covered by keratin. Furthermore, these properties could also greatly affect biological performance. This paper is aim to produce keratose(KOS) nanoparticles-coated-PVA fibers scaffold by electrospinning, thereby to culture of nerve cell. The KOS/PVA composite fibers scaffold was also studied as a comparison. Kos NPs/PVA scaffold does this by using PVA fibers as a skeleton that covered with keratose nanoparticles. By comparison with KOS/PVA scaffold, Kos NPs/PVA scaffold can enhance the potential of cytocompatibility.The main contents and results of present study are as follows:1. Hair keratin was extracted with oxidation method. The extraction ratio, qualitative analysis and quantitative research of keratose were completed.(1)SDS-PAGE showed that the molecular weight of the protein was about 40 k Da.(2)Keratose, containing at least 17 kinds of amino acid. The content of serine, glutamic and proline were very high.(3)The total content of amino acids was determined to be 50%. As an good oxidant, peracetic acid had a good effect of extracting keratose.2. A combination of electrospinning and electrospraying was used to prepare the keratose composite scaffold. The morphologies and characteristics of the composite scaffold were analyzed using comprehensive methods such as SEM, XRD and physical and chemical properties testing.(1)KOS Nanoparticles-coated-PVA fibers 1:2.5, 1:5, 1:7.5 and 1:10 scaffolds were generated by electrospinning and electrospraying. KOS/PVA composite fibers 1:2.5, 1:5, 1:7.5 and 1:10 scaffolds were generated by electrospinning.(2) SEM: Better dispersed KOS nanoparticles with even size was attached to the surface of the fibers. This result showed the scaffolds had typical structure.(3)FTIR: KOS Nanoparticles did not bring about a complete change to the secondary structure of keratose, it still had characteristics of the peptid. This result showed the component of scaffolds was stable. This keratose added causes the diffraction peak was broadened, the crystallinity of PVA was decreased. The structure of keratose and PVA remain almost unchanged during the process of mixing. The keratose scaffold was amorphous.(4)The keratose scaffolds had strong hydrophilic. The water contact angle had a downtrend with the increasing of keratose content. The contact angles of Kos NPs/PVA 1:10, 1:7.5, 1:5 and 1:2.5 scaffold were 25.1±1.8o, 23.2±1.3o, 17.1±1.9oand 15.7±2.0o respectively. This result showed that keratose were bioactive molecules and possess various groups on their surfaces, which could enhance the surface of hydrophilicity.(5)There was almost no change in Kos NPs/PVA scaffold and PVA scaffold. But the addition of keratose could obviously improve the physical mechanics performances of KOS/PVA scaffold such as tensile strength, Young's modulus and elongation at break.3. So, Kos NPs/PVA 1:7.5 and 1:5 scaffold were experimental groups, KOS/PVA 1:7.5 and 1:5 scaffold as positive control groups. The cytocompatibility of keratose scaffold was investigated.C6 cells and PC12 cells were used to simulate the neurons cell model of development and growth in vitro cultivation. We focused on the cell responses such as morphology, adhesion, proliferation and gene expression and investigated the cytocompatibility of the Kos NPs/PVA scaffold.(1)Cell morphology: Studies found that all keratose scaffolds could support spreading. Kos NPs/PVA scaffold allow more adhesion, which was favorable for PC12 cells and C6 cells growth and proliferation.(2)Cell adhesion: The order of cell adhesion of PC12 cells was as follows: KOS/PVA>Kos NPs/PVA>PVA scaffold, and C6 cells was as follows: Kos NPs/PVA>KOS/PVA>PVA scaffold. This showed that the KOS Nanoparticles increased specific surface area, the ability of adhesion of cell was improved, but C6 cell was sensitive to KOS.(3)Cell proliferation: Within 24 hours, the cells proliferation reaction was not notable. From 24 hours to 48 hours, the Kos NPs/PVA scaffold could promote the increase of PC12 cells and C6 cells number and proliferation. These phenomenon indicated that the Kos NPs/PVA scaffold played a supporting role in cell growth and proliferation.(4)Gene expression: Kos NPs/PVA scaffold could up-regulate the expression of NCAM, GAP-43, MAP-2 genes in PC12 cells, thus enhanced the adhesion of cells and the structure of microtubules stability. Kos NPs/PVA scaffold also could activate the NCAM, GAP-43 gene expression in C6 cell. These properties study benefited the application of Kos NPs/PVA scaffold in a good external environment for cell adhesion, growth and proliferation. |
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