Evidence that translocation of collagen fibril segments plays a role in early intrinsic tendon repair

Abstract

Background: Severed tendon repair advances with either a scar through extrinsic repair or regeneration through intrinsic repair. The authors examined whether intrinsic tendon repair reintroduces embryonic fibrillogenesis, whereby preformed collagen fibril segments are incorporated into growing collagen fibers at wound edges.

Methods: Isolated tendons from 10-day-old chicken embryos were suspended in 1 mg/ml of the antibiotic gentamicin for 90 days, which released fibril segments that were fluorescently tagged with rhodamine. Tendons isolated from 14-day-old chicken embryos were wounded to half their diameter and then maintained as explants in stationary organ culture. Fluorescent-tagged fibril segments were introduced to wounded tendon explants in the presence of high concentrations of neomycin, an antibiotic; cycloheximide, a protein synthesis inhibitor; cytochalasin D, a disruptor of microfilaments; and colchicine, a disruptor of microtubules. At 24 hours, explants were viewed by means of fluorescent microscopy.

Results: Untreated, wounded tendon explants showed the translocation of fluorescent-tagged fibril segments from the explant surface to accumulation at wound edges. In the presence of high concentrations of neomycin, cytochalasin D, or colchicine, fluorescent-tagged fibril segments failed to accumulate at wound edges and were retained on the explant surface. Inhibition of protein synthesis by cycloheximide did not alter the accumulation of fluorescent-tagged fibril segments at wound edges.

Conclusions: Inhibiting fluorescent-tagged fibril segment accumulation by antibiotics is consistent with their role in releasing fibril segments. Experimental findings show fibril segment translocation and accumulation at wound edges involves microfilaments and microtubules, but not protein synthesis. The experiments support the hypothesis that intrinsic tendon repair advances through the incorporation of fibril segments at wound edges.

Fig. 1

The organ culture tendon explant setup is presented. A cut section of 14-day-old chicken embryo tendon is located on the surface of Gelman Sciences filter membrane that is placed on a stainless steel mesh screen suspended over the central well of a 60 mm Falcon #3037 organ culture dish. The dish’s central well is filled with 1.0 mL of complete DMEM, and the surrounding well or outer moat has 2 mL of sterile water. Magnification 12×

Fig. 2

A TEM of negatively stained collagen fibril segment. A typical fibril segment is shown from the supernatant of a 10-day-old chicken embryo tendon subjected to 1 mg/mL of gentamicin antibiotic for 90 days at 4°C. Please note a collagen banding pattern within the central region of the FS as well as one end is tapered and the other blunted. Also included in the figure is negatively stained debris. Magnification 62,000×.

Fig. 3

Changes in the fluorescence pattern of nick wounded tendon explants over time. FTFS were incubated with nick wounded tendon explants. (Above left) at 4 hours fluorescence intensity was uniform along the length of the explant with minimal increased intensity at wound edges. (Above right) at 18 hours there was a continued increase in intensity at wound edges with a loss of intensity along the length of the tendon explant. (Below left) at 24 hours fluorescence intensity along the length of the explant was minimal, while fluorescence intensity was mostly restricted to wound edges. The 24 hour fluorescence pattern (Below left) represents the standard fluorescence pattern for nick wounded tendon explants accumulation of FTFS at wound edges. (Below right) was the typical fluorescence pattern that appeared at the ends of untreated tendon explants at 24 hours, which showed the accumulation of FTFS was at all wound edges regardless of their location at the nick wound or the end of the tendon explant. Magnification 10×.

Fig. 4

High doses of antibiotic effect upon fluorescence with nick wounded tendon explants. (Above) an untreated, control nick wounded explant shows fluorescence intensity restricted to wound edges. (Below) compared to control tendon explants, tendon nick wounded explants that received 100 μg/mL of neomycin, failed to accumulate FTFS at wound edges. Magnification 10×.

Fig. 5

Pattern of fluorescence in nick wounded tendon explants treated with an inhibitor of microtubules, microfilaments or protein synthesis. (Above) at 24 hrs a typical explant treated with the microfilament actin polymerization inhibitor cytochalasin D, failed to accumulate fluorescence at wound edges. (Center) a typical explant treated with the microtubule inhibitor colchicine, failed to accumulate fluorescence at wound edges. (Below) a typical explant treated with the protein synthesis inhibitor cyclohexamide showed a fluorescence pattern similar to controls with accumulation of fluorescence exclusively at wound edges. Magnification 10×.