Effects of trypsinization and mineralization on intrasynovial tendon allograft healing to bone

Abstract

The purpose of the current study was to develop a novel technology to enhance tendon-to-bone interface healing by trypsinizing and mineralizing (TM) an intrasynovial tendon allograft in a rabbit bone tunnel model. Eight rabbit flexor digitorum profundus (FDP) tendons were used to optimize the trypsinization process. An additional 24 FDP tendons were stratified into control and TM groups; in each group, 4 tendons were used for in vitro evaluation of TM and 8 were transplanted into proximal tibial bone tunnels in rabbits. The samples were evaluated histologically and with mechanical testing at postoperative week 8. Maximum failure strength and linear stiffness were not significantly different between the control and TM tendons. A thin fibrous band of scar tissue formed at the graft-to-bone interface in the control group. However, only the TM group showed obvious new bone formation inside the tendon graft and a visible fibrocartilage layer at the bone tunnel entrance. This study is the first to explore effects of TM on the intrasynovial allograft healing to a bone tunnel. TM showed beneficial effects on chondrogenesis, osteogenesis, and integration of the intrasynovial tendon graft, but mechanical strength was the same as the control tendons in this short-term in vivo study.

Keywords: intrasynovial tendon; mineralization; tendon-to-bone healing; trypsinization.

Figure 1

Two tibial tunnels were created, with a horizontal tunnel for tendon transplantation and an oblique tunnel for synovialization of the tendon-transplant tunnel. A, After the horizontal tendon transplant tunnel was created with a horizontal Kirschner wire, a second Kirschner wire was inserted obliquely to meet the first wire. B, The horizontal Kirschner wire was withdrawn and the oblique Kirschner wire was further advanced into the knee joint. Thus, the horizontal tunnel was interconnected with the knee joint to form an intrasynovial environment.

Figure 2

Optimizing duration of trypsinization for rabbit flexor digitorum profundus tendons. A, Samples were stained for lubricin. Boxes indicate lesions magnified in figure parts B–E. B, Lubricin was observed on the surface and substance of control (untreated) tendons. C–E, Lubricin expression decreased with trypsinization time (C, 10 minutes; D, 30 minutes; E, 60 minutes).

Figure 3

In vitro evaluation of the trypsinized and mineralized (TM) tendon. A, Gross observation of tendon grafts (alizarin red S stain) shows obvious mineralization (dark purple staining) at the end of the TM tendon (arrow). No dark purple staining was observed on the control (CT) tendon. B, Von Kossa staining shows the junction between mineralized and unmineralized portions of a TM tendon. Boxes indicate regions magnified in figure parts C–D. C, Mineral deposition was visible inside the tendon in a graded manner. D, No mineral was deposited outside the mineral-treated portion of the tendon. E, Lubricin immunohistochemical staining. Boxes indicate regions magnified in figure parts F–H. F, No positive staining in the treated portion of the tendon. G, Positive staining in the tendon substance at the junction between treated and untreated regions. H, Obvious positive staining of the untreated tendon portion.

Figure 4

Histology of the tendon graft inside the bone tunnel. All samples were stained with hematoxylin-eosin. A, Control tendon. Inside the bone tunnel, a clear gap is observed between the tendon graft (TG) and tibial bone (TB). B, Rectangular region from figure part A shows mononuclear cell infiltration (arrow) inside the graft. C, Circular region from figure part A shows interrupted fibrous bands of scar tissue at the interface (arrow). D, The trypsinized-mineralized tendon had thicker fibrous scar tissue at the graft-to-bone interface. E, Rectangular region from figure part D also shows mononuclear cell infiltration (arrow) inside the graft. F, Circular region from figure part D shows obvious new bone formation (arrow) inside the graft.

Figure 5

Histology of the tendon graft at the entrance of the bone tunnel. Samples were stained with hematoxylin-eosin unless otherwise indicated. A, Control tendon graft. B, Rectangular region from figure part A shows the tendon graft at the bone tunnel entrance. C–D, Circular region from figure part A shows fibrous scar tissue forming at the interface in the control group (D, Masson trichrome stain). E, Trypsinized-mineralized graft. F, Rectangular region from figure part E shows the tendon graft. G–H, Circular region from figure part E shows fusion with the bone tunnel, with a visible fibrocartilage zone forming at the interface (H, Masson trichrome stain).

Figure 6

Histology of the tendon graft outside the bone tunnel. Samples were stained with hematoxylin-eosin. A and D, Control and trypsinized-mineralized tendon grafts, respectively. B and E, Rectangular regions from figure parts A and D, respectively, show no cell infiltration inside the tendon grafts. C and F, Circular regions from figure parts A and D, respectively, show a distinct layer of mononuclear cells on the graft surface.