Ligamentum teres reconstruction using autogenous semitendinosus tendon with toggle technique in rabbits

Subjects

Twenty-six female Norfolk rabbits, approximately 3 months of age, weighing from 1.6 to 2.9 kg were used. The animals were obtained from the experimental animal center (EAC) from Sao Paulo State University (UNESP), Botucatu Campus. The EAC is associated with the department of animal science—rabbits and bioterium animals from the same institution.

The rabbits were randomly divided into two equal groups after a blood cell count (BCC) exam to evaluated previous health status: G1—no reconstruction of LT and capsulorrhaphy, G2—reconstruction of the LT using semitendinosus tendon autograft. Each rabbit was placed in an individual cage (60 × 60 × 60 cm) clear of the ground and with no restriction of movement, in an environment of approximately 27 °C temperature, controlled by exhaust fans and illumination for 12 h daily. The animals and the cages were identified with the number and the group of the rabbits. Environment was enriched with nipple-piped water, clay feed pot and banana tree leaf. Pinecone was given once a week. All the procedures of this study were executed in the Veterinary Hospital of UNESP.

Anesthesia and surgical procedures

Rabbits were given ketamine (30 mg/kg, Dopalen; Lab. CEVA, Rockville, MD, USA) in combination with morphine (2 mg/kg, Dimorf; Cristalia, Itapira, Brazil) and midazolam (2 mg/kg, Dormire; Cristalia, Itapira, Brazil) intramuscularly. Approximately 10 min after, epidural anesthesia was performed with 2% lidocaine (Xylestesin; Cristalia, Itapira, Brazil). Then, rabbits were intubated for maintainance of anesthesia with isoflurane (Isoforine; Cristalia, Itapira, Brazil) by a certified anesthetist. Anesthesia monitorization comprised heart and respiratory rate, peripheral hemoglobin oxygen saturation, expired CO₂ fraction (EtCO₂) (multi-parameter monitor), non-invasive systolic arterial blood pressure (Doppler ultrasound), and rectal temperature (digital thermometer).

In G1, a craniolateral approach was used to approach the right hip joint. A partial tenotomy of the deep gluteal was done. The joint capsule was identified and incised along the ventral acetabular margin, taking care not to injure the cartilage. The LT was excised using a number 11 scalpel blade, and the femoral head was dislocated. After repositioning the femoral head in the acetabulum, the joint capsule was closed with a simple interrupted suture using polyglactin 910, in 5-0 size. A cruciate suture pattern was used to reattach the tendon of the deep gluteal muscle. The remaining closure was performed in a routine fashion.

In G2, a 5 cm longitudinal incision was made in the medial aspect of the left hind limb, from the medial condyle of the femur to the medial malleolus. The semitendinosus tendon was identified, carefully dissected from the surroundings, and harvested for its whole length (File S1). The fascia and soft tissue in the harvest site were closed in a simple continuous pattern using a 3-0 monofilament nylon suture. Skin apposition was done with a simple continuous intradermal pattern using the same suture material.

After harvesting, the tendon was folded into two equal parts (Fig. 1A). A loop with 2-0 nylon suture was applied at the folded end of the graft (Fig. 1B). The free ends of the nylon thread were passed through the loop and tensioned (Fig. 1C) in order to use posteriorly in the toggle rod. The two free ends of the graft were maintained together with other 2-0 nylon sutures (Figs. 1D and 1E). Two loops were made around the ends of the graft (Fig. 1F) and one of the free ends of the suture was passed inside the loops (Fig. 1G). Then, both ends of the suture were pulled and tied with the surgeon’s knot (Fig. 1H) in order to use posteriorly in the suture button. At the same time that the graft was prepared by an assistant, the right hip joint was approached as used in G1. The graft had a mean of 5.5 cm in length and 2 to 4 mm in width. A 3.2 mm hole was drilled through the acetabular fossa in the area of LT origin to accommodate the toggle rod and graft, and a bone tunnel was drilled (2.5 mm drill) starting at the level of the third trochanter, centering on the femoral neck and towards the fovea capitis with aid of C-shaped targeting guide. The graft was attached to the toggle rod (2.5 × 8 mm) that was inserted through the acetabular fossa. After the toggle rod was positioned to the medial aspect of the acetabulum, the graft was passed through the bone tunnel from the femoral head to the trochanter guided using a suture passer. Then, tension was placed on the tendon, which was tied to a button to the lateral aspect of the femur. The illustrative scheme of the preparation of semitendinosus tendon graft using a double-bundle technique is in Fig. 1 and File S2.

In both groups, on induction of anesthesia and for 5 consecutive days after surgery enrofloxacin (10 mg/kg sc q24h, Baytril; Bayer, Leverkusen, Germany), tramadol chlorhydrate (10 mg/kg q12h, Tramadol; John Martin, Chicago, IL, USA) and meloxicam (0.5 mg/kg q24, Maxicam; Ourofino, Cravinhos, Brazil) were administered subcutaneously.

Clinical evaluations and imaging studies

The rabbits were evaluated before surgery (M1), 48 h (M2), 15 days (M3), 30 days (M4) and at 90 days (M5) after surgery by using visual gait analysis (presence or absence of lameness and/or hopping motion) (Miwa & Carrasco, 2019; McClure, 2020). For visual gait analysis, each animal was removed from the cage and allowed to walk freely in a quiet area with a concrete floor.

Except for M2, radiographic and ultrasound exams of the right hip joint were performed at the same time points. Radiographs were taken on extended ventrodorsal and right mediolateral projections. A digital radiography system (SR 8100; SIUI, Shantou, China) was used to obtain the images. The presence of hip dislocation or subluxation, toggle rod position, and signs of infection were assessed. Ultrasound (LOGIQ, GE Healthcare, Chicago, IL, USA) was used to assess joint capsule thickness, joint congruence, regularity of the articular surface, presence of osteophytes, and muscle organization. The images were obtained with an 8–13 MHz linear transducer. An experienced examiner evaluated the radiographic images and another one the ultrasound images.

Gross and histological examination

At 90 days after surgery, all rabbits were euthanized. For euthanasia, the rabbits were submitted to dissociate anesthesia with xylazine hydrochloride (1 mg/kg, Xilazin; Syntec, London, England) and ketamine (50 mg/kg) administered in the same syringe intramuscularly. Subsequently, propofol (Propovan; Cristalia, Itapira, Brazil) was used intravenously until the respiratory arrest, followed by 19.1% potassium chloride (Cloreto de potassio; Samtec, New Albany, IN, USA) intravenously until the cardiac arrest. All the animals from both groups were euthanized to achieve the comparison from both treatments in gross and histological examination, following the relevant statistic number of samples.

In both groups the right hip joint was evaluated macroscopically for joint capsule thickness (mild, moderate, or intense); articular cartilage lesion of the femoral head (Grade 0—surface intact, Grade 1—superficial defect, Grade 2—deep defect, Grade 3—erosion with exposed subchondral bone) (Yoshioka et al., 1996); periarticular osteophytes and bone remodeling of the femoral head (mild or intense).

The hip joint of the G2 was fixed in a 10% buffered formalin solution for 48 h followed by nitric acid solution until decalcification (Bancroft & Gamble, 2008). Neutralization was accomplished by immersing decalcified bone in a 10% buffered formalin solution for 3 h. Specimens were cut into 4-μm sections and stained with hematoxylin and eosin. The organization and thickness of the conjunctive fibers from the joint capsule and tendon-bone interface were evaluated using light microscopy.

Statistical analysis

The body mass values were analyzed using Student’s t-test for independent samples.

Statistical significance was set at p < 0.05. Data were analyzed using Minitab statistical software.

Clinical evaluations and imaging studies

There was no statistically significant difference in body mass between G1 (2 ± 0.4 kg) and G2 (2.2 ± 0.4 kg) (File S3). One rabbit from G2 had a femur fracture at M3, and euthanasia was performed. Lameness was not recognized in both groups, but the rabbits from G2 showed more limitations for hopping motion until M3 compared to G1. Two rabbits from G2 with hip luxation showed the greatest limitation for hopping motion.

In the radiographic evaluation of G1, nine rabbits showed no complications (Figs. 2A and 2B). However, hip joint subluxations in four rabbits were seen; in three rabbits at M4 and one rabbit showed subluxation at M5 (Figs. 2C and 2D). In G2, eight rabbits did not show any complication with the implant (Figs. 3A and 3B). Hip joint luxations in two rabbits at M3 and in one case subluxation at M4 were seen. As previously reported, one rabbit had a fracture of the right femur detected at M3. In addition, lytic lesions in the acetabulum, and periarticular tissue proliferation was observed in one rabbit at M4. In all cases of subluxation and luxation, the X-rays showed that the toggle rod had displaced toward the acetabular hole (Figs. 3C and 3D).

The sonographic findings in G1 revealed that the joint surface had no irregularities in 69.2% of the rabbits. The irregular articular surface was seen in 30.8% of the rabbits that had subluxated hips. LT was not seen in M3, M4, and M5. Disorganization of the orientation of the muscle fibers was seen at M3 that improved at M4 and M5. Three rabbits with hip subluxation at M4 and one rabbit with hip luxation at M5 had thickening of the joint capsule at M5. In G2, the articular surface had no irregularities on ultrasound at M1. Disorganization of the orientation of the muscle fibers and two hip luxations were seen at M3. One hip subluxation with severe signs of osteoarthritis was observed at M4. Thickening of the joint capsule was detected in the two hip luxations, and in the one hip subluxation. Improved muscle fiber organization was seen at M5. The two hip luxations and the hip with osteoarthritis had irregular articular surfaces.

Gross and histological examination

The gross evaluation of G1 showed mild capsular thickness and mild remodeling of the femoral head in 30% and no alteration in 70% of rabbits. Articular cartilage lesions were classified as 23.1% of rabbits in Grade 0, 15.4% in Grade 1, 30.8% in Grade 2, and 30.8% in Grade 3, which also presented joint subluxation.

The gross evaluation of G2 showed 53.8% mild, 46.2% moderate, and 15.4% intense capsular thickness. In addition, articular cartilage lesions were classified 30.8% rabbits in Grade 1, 38.4% Grade 2, and 30.8% Grade 3. Two rabbits with Grade 3 (15.4%) had hip luxation due to graft failure (resorption or toggle rod displacement). The integrity of the tendon graft (Fig. 4) was verified in 76.92% of the rabbits including the rabbit euthanatized 15 days after surgery (M3) due to femur fracture. Two rabbits (15.4%) showed rupture of the tendon graft in its proximal anchorage (acetabulum) associated with hip luxation. One rabbit (7.7%) showed partial rupture of the intra-articular tendon graft.

Histological analysis revealed characteristics of the early phase of tendon graft ligamentization with widely diffuse and homogeneous multifocal areas of collagen fibers, consistent with sharpey fibers at the tendon-bone interface (Fig. 5). The fibers were in the early phase to bind the periosteum accompanying the presence of organized connective tissue. Blood vessels were seen in the graft concomitant to 1–3 chondrocyte layers bordering the bone graft. G2 had a higher rate of fissures and irregularity in the cartilage surface compared to G1. Joint capsule thickness was observed. However, joint capsule fibers had preserved architecture.