Tissue Engineered Silk-fibroin Scaffolds for Meniscus Regeneration

INTRODUCTION

Tissue engineering (TE) of meniscus aims to restore the loss of meniscus tissue both anatomically and functionally, by transplanting a substitute that should be able to mimic the properties of native tissue¹. In this study, novel silk fibroin scaffolds² combined with human meniscus fibrochondrocytes were evaluated in vitro for their performance as meniscus substitutes.

EXPERIMENTAL METHODS

Silk-based scaffolds (10 and 12 wt%) were produced in the form of discs (4 mm in diameter and ~3 mm in height) by means of combining salt leaching and lyophilization methods. Human meniscus cells (HMC’s) were isolated from macroscopically intact human fresh menisci using an enzymatic digestion-based technique and expanded using standard culture conditions. HMC’s were then seeded at a cell density of 5x10⁴ cells/scaffold and the cell-laden constructs were cultured in static conditions, up to 21d. Viability (Calcein-AM staining), metabolic activity (MTS assay) and proliferation (DNA quantification) were evaluated until 21d of culturing. HMC’s adhesion was investigated by scanning electron microscopy (SEM). Expression of typical cell markers was observed by histology, immunocytochemistry and RT-PCR. Dynamic mechanical analyses (DMA) were also performed. Results are presented as mean ± SEM, independently of the population used.

RESULTS AND DISCUSSION

Fibroblast-like (elongated form) and chondrocyte-like (rounded morphology) cells were observed in culture after HMC’s isolation (Figure 1A). Calcein-AM staining showed that HMC´s were viable up to 21d after culturing onto the silk-10% (Figure 1B for 7d) and silk-12% scaffolds. SEM analysis revealed that HMC’s effectively adhered to scaffold’s surface after 7d (Figure 1C). MTS assay (Figure 1D) and DNA quantification (Figure 1E) analysis showed that HMC’s were metabolically activeand proliferated throughoutthe period of culturing in both types of scaffolds.

Table 1 shows the influence of seeding HMC’s on scaffolds’ biomechanical properties studied by DMA analysis. It was observed that the E’ value at 1 Hz of the cell-laden constructs increased with culturing time, reaching a maximum after 14d of culturing, i.e. 4.22 ± 1.74 x 10⁵ Pa and 11.67 ± 1.10 x 10⁵ Pa, for silk-10 and silk-12, respectively. Moreover, it was observed significantly higher values for the modulus of the cell-laden constructs when compared to the acellular scaffolds after 7 and 14d of culturing.

CONCLUSION

Silk scaffolds supported HMC’s adhesion, proliferation and viability, while improving the biomechanical features of acelular scaffolds, and thus showed great promise for meniscus TE applications.

REFERENCES

1. Pereira H. et al., Arthroscopy 27:1706-1719, 2011

2. Yan L.P., et al., Acta Biomater. 8:289-301, 2012