Enhanced tendon–bone healing with acidic fibroblast growth factor delivered in collagen in a rabbit anterior cruciate ligament reconstruction model

Eight-four healthy New Zealand rabbits (44 females and 40 males) with an average age of 6 months and an average weight of 4.5 kg were provided by the Experimental Animal Center of The Fourth Affiliated Hospital of Harbin Medical University. This double-blinded study was approved by the Institutional Animal Care and Use committee at the The Fourth Affiliated Hospital of Harbin Medical University and performed under the Guidelines for the Care and Use of Animals in Research. A statistical random number generator was used to generate a randomization list, and with stratification by gender, equal numbers of female and male rabbits were assigned to each group. This was a double-blinded study in that the individuals collecting, recording, and analyzing the data were blinded to group assignment, and data were stored by a third party until the experiments were completed.

The rabbits were randomly divided into four groups (n = 21/group) as follows: (1) ACL reconstruction with an autograft, and 4 μg aFGF incorporated into 15 ml collagen applied in the bone tunnel [20]; (2) ACL reconstruction with an autograft and or 1 μg FGF incorporated into 15 ml collagen in the bone tunnel; (3) ACL reconstruction with an autograft, and 15 ml collagen applied in the bone tunnel; (4) ACL reconstruction with an autograft only. Rabbits in each group were sacrificed at postoperative 4 (n = 7), 8 (n = 7), and 12 weeks (n = 7). For each group and time point, seven specimens were used for biomechanical analysis and then used for histological evaluation.

The fat and fascia were removed from the fresh beef Achilles tendon, cleaned, and hardened in a − 20 °C refrigerator; the tendon was sliced into uniform pieces. About 100 g tendon was washed with deionized water and disinfected for 30 min using 75% alcohol. Then, the tendon was repeatedly washed using double distilled water, placed into 1 L 0.5 M glacial acetic acid, and kept at 4 °C for over 24 h with intermittent stirring. The expanded collagen was mixed in a high-speed tissue crusher with 4 L 0.5 M acetic acid. Then, 1 g pepsin was added and dissolved at 4 °C for more than 24 h. The supernatant was collected after centrifugation at 4000 r/min for 30 min at 4 °C. An equal volume of 20% NaCl solution was added to the supernatant for salting for over 48 h. The precipitated flocculent was dissolved in 2 L 0.5 M glacial acetic acid and filtered using the double-layer gauze. The resulting collagen solution was dialyzed with double distilled water for 5 days or more. The water was changed twice daily to produce the collagen solution. Finally, 1 μg or 4 μg aFGF (provided by Jinan University) was added into 15 ml of collagen solution for the two test treatments. After freeze-drying, aFGF/collagen complex with two concentrations of aFGF were obtained for the low-dose aFGF (0.067 μg/ ml) and high-dose aFGF groups (0.267 μg/ ml). All test samples were sterilized using ⁶⁰Co.

The long digital extensor tendon was isolated from the left hind limb of the rabbit, and then, a medial parapatellar arthrotomy was made on the right hind limb to expose the ACL. The ACL was excised from the top and bottom end points. The bone tunnel was created using a 1.2-mm-diameter drill bit as the bottom end point. Similarly, bone tunnel was made on the femoral condyle bone as the upper end point. Both ends of the sutures attached to the graft were pulled out of the joint cavity and passed the ends of the graft through the bone tunnel. For fixation of the ends, a Kirschner wire was used to drill in the cortical bone slightly lower than the tibial tubercle, and one surgical thread was passed through the tunnel and tied into a knot with the end of another suture. Then, two single cortical bone tunnels were drilled on the femoral stem ligament end from outside to inside laterally using a Kirschner wire with a 0.3-cm interval. The two ends of the graft were passed through the bone tunnel with the knee bent at 90° and then secured with a tightened ligament knot.

Collagen was applied according to the following steps. One milliliter of collagen suspension and 0.5 ml thrombin were prepared in separate 1-ml syringes. Using a three-way valve, they were injected into the bone tunnel sequentially, and the incision was sutured layer-by-layer once the collagen had coagulated and attached within the bone tunnels at both ends of the graft. Ropivacaine was injected in the incision postoperatively, and rabbits were caged individually. Penicillin was injected intramuscularly for 3 days to prevent infection.

At 4, 8, and 12 weeks postoperatively, seven animals from each group were sacrificed. The soft tissues surrounding the ACL and sutures were removed, and 5 cm residues of the femur and tibia were spared for fixation. The ACL graft was tested in a single arm-type electronic stretching machine (Instron 3342 Series Single Column System, Instron USA) and adjusted so that the axis of distraction was parallel to the long axis of the graft. The specimens were preconditioned with preload of 5 N; then, a load-to-failure test was performed with an elongation rate of 5 mm/min on the tibial tendon–bone interface. The machine recorded the load and displacement data and reported a load–displacement curve from which the ultimate stress value was retrieved. The stiffness and elastic modulus were calculated according to the curve. Saline solution spray was used to maintain specimen hydration throughout the testing procedure, and all tests were performed at room temperature.

At 4, 8, and 12 weeks postoperatively, specimens were harvested and the biomechanical analysis was carried out. Then, all specimens were fixed in 95 ml of 12% formaldehyde mixed with 5 ml glacial acetic acid at room temperature for 24 h. After fixation, the specimens were washed for 15 min and then decalcified using a 30% hydrochloric acid formaldehyde solution (30 ml hydrochloric acid + 10 ml formaldehyde + 60 ml water) for 24 h. The specimens were rinsed with water for 3 h and then rinsed with distilled water. The femoral tunnel and tibial tunnel were cut open along the longitudinal axis and trimmed to specimens 1 cm × 0.5 cm × 0.5 cm in size. The specimens were dehydrated in a gradient series of 75%, 85%, 95%, and 100% alcohol for 1.5 h, cleared twice in xylene for 1 h at room temperature, and embedded in paraffin heated to 60 °C. Five-micrometer-thick sections were stained with hematoxylin for 10 min and rinsed with water followed by hydrochloric acid alcohol and blue solution. Finally, the sections were co-stained with eosin for 20 s before observation of the tendon–bone interface under a microscope (BX43 Olympus Japan Olympus Co., Ltd.). The evaluation of histology was double-blinded to evaluators.

Histological sections of the ACL graft tunnel demonstrated differences in histological appearances between grafts that treated with different amount of aFGF/collagen at different time points. At 4 weeks postoperatively, fibrous connective tissue was observed at the tendon–bone interface in all groups. Organized, tightly packed collagen fibers and mature new bone-forming were observed at the tendon–bone interface in the aFGF/collagen treatment groups, especially in the high aFGF concentration group. By comparison, loose and disordered tissues were observed at the tendon–bone interface in the collagen alone and control group with indication of only minimal osteogenesis (Fig. 1).

In the high aFGF concentration group, densely arranged Sharpey-like fibers were observed vertical to the tendon–bone interface at 8 weeks postoperatively with new bone ingrowth incorporated into the tendon. In the low aFGF concentration group, six out of seven (85.7%) specimens showed Sharpey-like fibers, whereas in the control group, only one out of seven (14.3%) specimens showed Sharpey-like fibers with new bone growth into the tendon and a loose tendon–bone interface (Fig. 2).

At 12 weeks postoperatively, continuous and calcified fibrocartilage had replaced the tendon–bone interface in six out of the seven specimens (85.7%) in the high aFGF concentration group, similar to fibrocartilage enthesis, with fewer Sharpey-like fibers forming a “tide mark”-like structure. In the low aFGF concentration group, a direct enthesis had formed at tendon–bone interface in five of the seven specimens (71.4%); calcified fibrous cartilage was not observed in the other two specimens. The tendon–bone interface gradually matured in the control group, with formation of Sharpey-like fibers and a small amount of poorly differentiated fibrocartilage. No “tide mark” structure was observed in the narrow tendon–bone tissue transition in the collagen alone and control group (Fig. 3). Our quantification of the histological evaluation of all groups also confirmed that the high and low aFGF groups had significantly more tendon–bone interface and “tide mark” structure compared to the control group at both 8 and 12 weeks (Table 1).

The percentage of collagen degradation was measured by histological evaluation at all time points (Table 2). As the percentage of collagen degradation increased with time, no significant differences were observed among the different groups at any of the time points. At 4 and 8 weeks, the percentages of collagen degradation were around 25% and 52%, respectively, for all groups. Collagen degradation reached around 76% at 12 weeks, suggesting that approximately 24% of the collagen remained at 12 weeks.

At 4 weeks, the average load-to-failure, elastic modulus, and stiffness of harvested specimens differed significantly among the four groups. The graft tunnel load-to-failure, elastic modulus, and stiffness were higher in the high and low aFGF/collagen treatment groups compared with the collagen alone and the control group, respectively, with the high aFGF/collagen being the highest. There was no significant difference between the collagen alone and control group (Fig. 4a).

At 8 weeks, the average load-to-failure and elastic modulus in the high aFGF/collagen concentration group remained the highest compared with the other three groups, and the load-to-failure of the low aFGF/collagen concentration group was significantly higher than that of the control group and the collagen alone group, respectively. There was no significant difference between high and low aFGF/collagen concentration group in stiffness (Fig. 4b).

Similarly, at 12 weeks, the high aFGF/collagen concentration group had the highest load-to-failure, elastic modulus, and stiffness. Compared with those of the control group and collagen alone group, the load-to-failure, elastic modulus, and stiffness of the low aFGF concentration group were significantly higher (Fig. 4c).