Carbodiimide-derivatized hyaluronic acid surface modification of lyophilized flexor tendon: a biomechanical study in a canine in vitro model

Abstract

Background: Intrasynovial grafts are the ideal solution to replace defects in intrasynovial flexor tendons, but autologous graft sources are rarely available. The purpose of the present study was to test the hypotheses that an intrasynovial tendon prepared with repetitive freeze-thaw cycles and lyophilization (as a means of reducing immunogenicity) has increased frictional force (gliding resistance) in comparison with fresh intrasynovial tendons and that a lyophilized intrasynovial flexor tendon that is modified with carbodiimide-derivatized hyaluronic acid and gelatin has decreased frictional force in comparison with untreated lyophilized tendons.

Methods: Thirty-six flexor digitorum profundus tendons from the second and fifth digits of canine hind paws were randomly assigned to three groups. Twelve tendons were immediately assessed both mechanically and morphologically and served as the normal tendon group. The other twenty-four tendons were prepared with repetitive freeze-thaw cycles and lyophilization and were randomly assigned to two groups, including one group in which the tendons were treated with carbodiimide-derivatized hyaluronic acid and gelatin and one group in which the tendons were not treated. The frictional force was measured during 1000 cycles of simulated flexion-extension motion in all tendons, and the mean frictional forces were compared. The tendons were then observed with use of transmitted light microscopy for residual hyaluronic acid on the tendon surface, and the smoothness of the surface was evaluated with use of scanning electron microscopy.

Results: The frictional force after lyophilization was significantly increased by 104.9% after the first cycle and by 99.5% after 1000 cycles in comparison with the normal tendon (p < 0.05). The frictional force of the lyophilized tendons after treatment with carbodiimide-derivatized hyaluronic acid and gelatin was not significantly different from that of normal tendons. The untreated lyophilized tendon surfaces were observed on scanning electron microscopy to be rough in appearance, whereas the normal surface and the surface treated with carbodiimide-derivatized hyaluronic acid and gelatin were smooth, with residual hyaluronic acid present on the gliding surface.

Conclusions: Lyophilization alters tendon surface morphology and increases tendon frictional force. Surface modification with carbodiimide-derivatized hyaluronic acid and gelatin can mitigate this adverse effect.

Clinical relevance: Tendon surface modification with carbodiimide-derivatized hyaluronic acid and gelatin can improve the gliding ability of lyophilized flexor tendons and therefore may improve the utility of lyophilized tendon allografts as a tendon graft substitute.

Fig. 1

Illustration depicting the testing device used to measure frictional force (gliding resistance). The actuator was positioned at an angle of 30° and the distal load-transducer was positioned at an angle of 20°. F1 = force 1, F2 = force 2, PIP = proximal interphalangeal, and K-wire = Kirschner wire.

Fig. 2-A Fig. 2-B

Figs. 2-A and 2-B Illustrations depicting the lateral view (Fig. 2-A) and top view (Fig. 2-B) of the pulley unit. The proximal phalanx with an intact A2 pulley was mounted in the device and was fixed in full proximal interphalangeal (PIP) joint extension with a longitudinal Kirschner wire (K-wire). FDS = flexor digitorum superficialis.

Fig. 2-A Fig. 2-B

Figs. 2-A and 2-B Illustrations depicting the lateral view (Fig. 2-A) and top view (Fig. 2-B) of the pulley unit. The proximal phalanx with an intact A2 pulley was mounted in the device and was fixed in full proximal interphalangeal (PIP) joint extension with a longitudinal Kirschner wire (K-wire). FDS = flexor digitorum superficialis.

Fig. 3

Bar graph illustrating the frictional force (gliding resistance) for the groups of normal and untreated lyophilized tendons. The values are expressed as the mean and the standard deviation.

Fig. 4

Bar graph illustrating the frictional force (gliding resistance) for the groups of normal tendons, untreated lyophilized tendons, and lyophilized tendons treated with cd-HA-gelatin. The values are expressed as the mean and the standard deviation.

Fig. 5

Transmitted light microscopy image showing the appearance of residual hyaluronic acid binding on the tendon surface. The tendon surface was well covered with a thin layer of cd-HA-gelatin even after 1000 cycles of motion. An area that was well coated with cd-HA-gelatin (arrows) was identified on the surface of the transversely sectioned cd-HA-gelatin-treated tendon (scale bar = 100 μm).

Fig. 6

Selected scanning electron microscopic images, made after 1000 cycles of tendon excursion, showing normal flexor digitorum profundus tendon, untreated lyophilized tendon, and tendon treated with cd-HA-gelatin. The top row of images was made at low magnification (×25), and the bottom row of images was made at high magnification (×5000). The surface of the lyophilized tendons appeared to be rough, whereas the surface of the lyophilized tendon that had been treated with cd-HA-gelatin appeared to be smoother.