Biocompatibility study of a silk fibroin-chitosan scaffold with adipose tissue-derived stem cells in vitro

Abstract

The use of tissue engineering technology in the repair of spinal cord injury (SCI) is a topic of current interest. The success of the repair of the SCI is directly affected by the selection of suitable seed cells and scaffold materials with an acceptable biocompatibility. In this study, adipose tissue-derived stem cells (ADSCs) were incorporated into a silk fibroin-chitosan scaffold (SFCS), which was constructed using a freeze-drying method, in order to assess the biocompatibility of the ADSCs and the SFCS and to provide a foundation for the use of tissue engineering technology in the repair of SCI. Following the seeding of the cells onto the scaffold, the adhesion characteristics of the ADSCs and the SFCS were assessed. A significant difference was observed between the experimental group (a composite of the ADSCs with the SFCS) and the control group (ADSCs without the scaffold) following a culture period of 6 h (P<0.05). The differences in the results at the following time-points were statistically insignificant (P>0.05). The use of an MTT assay to assess the proliferation of the cells on the scaffold revealed that there were significant differences between the experimental and control groups following culture periods of 2 and 4 days (P<0.05). However, the results at the subsequent time-points were not statistically significantly different (P>0.05). Scanning electron microscopy (SEM), using hematoxylin and eosin (H&E) staining, was used to observe the cellular morphology following seeding, and this revealed that the cells displayed the desired morphology. The results indicate that ADSCs have a good biocompatibility with a SFCS and thus provide a foundation for further studies using tissue engineering methods for the repair of SCI.

Keywords: adipose stem cells; biocompatibility; silk fibroin-chitosan scaffold.

Figure 1.

Morphology of the adipose tissue-derived stem cells (ADSCs). Morphology of: (A) ADSCs at the six-hour time point; (B) the primary culture at three days; (C) the primary culture at seven days; (D) the seventeenth generation. Magnification, ×100.

Figure 2.

Osteogenic and adipogenic induction demonstrated by staining. Osteogenic induction at 2 weeks in the (A) experimental and (B) control groups [alkaline phosphatase (ALP) staining; magnification, ×100]. Osteogenic induction at 4 weeks, in the (C) experimental and (D) control groups (Alizarin Red staining; magnification, ×100). The arrow indicates calcified nodules. (E) Adipogenic induction at 2 weeks in the experimental group (Oil Red O staining; magnification, ×400). The arrow indicates fat droplets. (F) Adipogenic induction at 2 weeks in the control group (magnification, ×200).

Figure 3.

Observation of cell morphology through scanning electron microscopy (SEM). (A) Silk fibroin-chitosan scaffold (SFCS); (B) observation of the scaffold through SEM (magnification, ×600); (C) adipose tissue-derived stem cell (ADSC) morphology following 2 days of culture with the SFCS (magnification, ×3,000); (D) ADSC morphology following 6 days of culture with the SFCS (magnification, ×1,000).

Figure 4.

Observation of cellular morphology through hematoxylin and eosin (H&E) staining. (A and B) Cell morphology at two days (magnification, ×200 and ×400 respectively). The arrow indicates the cells on the silk fibroin-chitosan scaffold (SFCS) surface. (C) Morphology at six days (magnification, ×200); (D) morphology at eight days (magnification, ×200). The arrow indicates the cells moving into the interior of the scaffold. (E and F) Cell morphology at ten days [magnification, ×200 and ×400, respectively). The arrow indicates the cell distribution in the porous scaffold.