IN VITRO TRANSPLANTATION OF GENETICALLY MODIFIED CELLS TO THE TENDON SURFACE

Abstract

The objective of this paper was to study in vitro transfection of tendon cells and adherence of transfected cells to different tendon surfaces. Achilles tendon fibroblasts from 2-month-old New Zealand white rabbits were cultured to confluence, after which the cells were transfected by an adenovirus carrying either the β-galactosidase reporter gene or the green fluorescent protein (GFP) gene at multiplicities of infection (MOIs) of 50, 100, or 500. Two days later, the cells were transplanted onto the surfaces of rabbit Achilles, peroneus brevis, flexor profundus, and extensor longus tendons. The tendons were assessed by X-gal staining after 9 days, and by GFP fluorescence at 7, 14, and 21 days. Twenty percent to 50% of the treated cells stained for β-galactosidase at an MOI of 500. The GFP-labeled cells showed nearly 100% fluorescence at an MOI of 50. No positive cells were visible in the control group. The β-galactosidase and GFP-expressing cells remained viable for as long as 3 weeks. It is possible to introduce foreign genes into rabbit tendon cells, transplant the cells onto tendon surfaces, and maintain viability of the cell/tendon construct for several weeks.

Fig. 1

β-galactosidase staining on tendon surface (×40). Transfected cells stain blue.

Fig. 2

Roller device method.

Fig. 3

Roller bank cells with GFP staining after 1, 7, and 21 days (confocal fluorescent microscopy, ×100). Transfected cells fluoresce green. A: Achilles tendon; FDP: flexor digitorum profundus tendon.

Fig. 4

Hyaluronan synthase transfection as demonstrated by red blood cell (RBC) exclusion. (a) Control. Note the RBC cover tendon fibroblasts. (b) RBC exclusion in a halo around transfected cells. (c) Halo effect abolished by treatment with hyaluronidase. (All ×400.)