Success and efficiency of cell seeding in Avian Tendon Xenografts - A promising alternative for tendon and ligament reconstruction

Abstract

Decellularized tendon xenografts offer a promising alternative for reconstruction by using ubiquitously available material. This study compares static and centrifugal seeding of avian tendon scaffolds with NIH 3T3 fibroblasts. Incorporation of viable cells was achievable with both techniques, represented by DNA content. Proliferation rate and viability assay showed neither damage by centrifugal force nor superiority of the technique. Cell proliferation after 10 days of culture demonstrated that the scaffold did not hinder 3-D culturing. Confocal laser microscopy revealed structural details as formation of focal adhesions, to provide deeper insight into the process of cell attachment and growth in xenografts.

Keywords: Cell adhesion; Cell seeding efficiency; Confocal laser microscopy; Tendon scaffold; Tissue engineering.

Fig. 1

Steps of scaffold preparation. 1a: Chicken foot with excised FDP tendon. 1b: Isolated FDP tendon prior to decellularization. 1c: Freeze-dried tendon scaffold.

Fig. 2

DNA content of xenografts after 24 h (n=10) and 10 days of culturing (n = 16). Values already zeroed to unseeded constructs; Measured in ng DNA/mg tissue. The asterisk marks the significant difference between 1 × 10min and 5 × 2min centrifugation after 24 h of culturing (p < 0.05). Two asterisks mark the significant increase of DNA content after 10 days of culturing in all groups (p < 0.05).

Fig. 3

Final outcome of seeding and culturing. Displayed values show content of cells in 10³ cells/mg tissue. Calculated by DNA content of xenografts after 10 days of 3-D-culture compared to a standard curve of NIH 3T3 fibroblasts in known concentrations; corrected by the inter-species-comparison-factor of 1.4 (Mus musculus vs. Gallus gallus) and compared to the cell content of native avian FDP tendons.

Fig. 4

Cell viability 24 h after seeding. Measured by MTS conversion and displayed in viable cells per mm² surface area; n = 4; p < 0.05. The asterisk marks the significant difference between 1 × 10min and 5 × 2min centrifugation.

Fig. 5

Histological comparison of native tendon tissue with a decellularized scaffold. 5a: Native tendon tissue overview, DAPI staining, 2x magnification, 5b: Decellularized scaffold overview, DAPI staining, 2x magnification, 5c: Decellularized scaffold details, H&E staining, 20x magnification.

Fig. 6

Transversal sections of avian FDP xenografts: GFP signal of seeded cells. a: Static Pipetting, 24 h incubation. b: Static Pipetting, 10 days of culturing. c: 5 × 2min Centrifugal Seeding, 24 h of incubation. d: 5 × 2min Centrifugal Seeding, 10 days of culturing. e: 1 × 10min Centrifugal Seeding, 24 h of incubation. f: 1 × 10min Centrifugal Seeding, 10 days of culturing.

Fig. 7

Confocal laser microscopy: Xenograft seeded by Static Pipetting after 10 days of culturing. Legend: Vinculin stain = pink, Actin stain = red, GFP = green. a, b, c: Single Cell @ 63x magnification. d, e, f: Line of Cells on Graft Surface @ 20x magnification, Images of Vinculin (a&d), Actin (b&e) and Merge of DIC and Fluorescent Images (c&f). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)