The Potential of Using an Autogenous Tendon Graft by Injecting Bone Marrow Aspirate in a Rabbit Meniscectomy Model

Abstract

Bone marrow aspirate (BMA) is an excellent source of cells and growth factors and has been used successfully for bone, cartilage, and soft-tissue healing. This study aimed to investigate the histological and biomechanical properties of autogenous tendon graft by injecting BMA and its protective effect against degenerative changes in a rabbit model of meniscal defects. Adult white rabbits were divided into untreated, tendon, and tendon + BMA groups, and meniscal defects were created in the knees. The tendon graft and articular cartilage status were evaluated by macroscopic and histological analysis at 4, 12, and 24 weeks postoperatively among the three groups. The tendon graft in the tendon and tendon + BMA groups were used for biomechanical evaluation at 4, 12, and 24 weeks postoperatively. The meniscal covering ratio in the tendon + BMA group was better than that in the tendon and untreated groups at 12 and 24 weeks postoperatively. The matrix around the central portion of cells in the tendon + BMA group was positively stained by safranin O and toluidine blue staining with metachromasia at 24 weeks. The histological score of the tendon graft in the tendon + BMA group was significantly higher than that in the untreated and tendon groups at 12 and 24 weeks postoperatively. In the tendon + BMA group, cartilage erosion was not shown at 4 weeks, developed slowly, and was better preserved at 12 and 24 weeks compared to the untreated and tendon groups. Histological scores for the articular cartilage were significantly better in the tendon + BMA group at 24 weeks. The compressive stress on the tendon graft in the tendon + BMA group was significantly higher than that in the tendon group at 12 weeks postoperatively. Transplantation of autogenous tendon grafts by injecting BMA improved the histologic score of the regenerated meniscal tissue and was more effective than the tendon and untreated group for preventing cartilage degeneration in a rabbit model of massive meniscal defects.

Keywords: autogenous tendon graft; bone marrow aspirate; meniscus.

Figure 1

Macroscopic analyses for regenerated meniscus. (A) Macroscopic observation. Scale bar = 5 mm. (B) Meniscus cover ratio. Bars show the mean ± SD (n = 6). *, p < 0.05; At 12 and 24 weeks, the meniscus cover ratios in the tendon + BMA group were larger than those in the tendon group and untreated group. (C) Explanation for meniscus cover ratio, defined as the ratio of medial meniscus area (red line of Area A) to medial plateau area (red line of Area B).

Figure 2

Radiographic analysis of the regenerated meniscus. No calcified change in the anteroposterior radiographs was noted in the tendon + BMA, tendon, or untreated groups over the experimental period of 24 weeks.

Figure 3

Hematoxylin and eosin (H&E) staining, toluidine blue staining, and modified Pauli’s scoring of the native meniscus and regenerated meniscus at 4, 12, and 24 weeks postoperative of three groups. (A) H&E staining of the regenerated meniscus revealed oval- or round-shaped fibrochondrocytes in the center of the regenerated meniscus in the tendon + BMA group. (black arrows). (B) Toluidine blue staining revealed metachromasia in the tendon + BMA group at 12 and 24 weeks postoperatively (black arrows). (C) Modified Pauli’s scoring; the tendon + BMA group had greater regeneration than the untreated and tendon groups at 12 and 24 weeks postoperatively (n = 6, * p < 0.05). (D) H&E staining and toluidine blue staining of the native meniscus.

Figure 4

Safranin O staining and type II collagen immunostaining of native and regenerated menisci in the three groups at 4, 12, and 24 weeks postoperatively. (A) Safranin O staining. Positive safranin O staining was consistently noted at the central margin of the grafts in the tendon + BMA group at 12 and 24 weeks postoperatively (black arrows). (B) Type II collagen immunostaining. Immunohistochemistry exhibited positive staining of type II collagen in the cells at 24 weeks postoperatively (black arrows). (C,D) Safranin O staining and type II collagen immunostaining of the native meniscus.

Figure 5

Histological analyses for articular cartilage at the medial femoral condyle. (A) Toluidine blue staining of medial femoral condylar cartilage. (B) Mankin score. (n = 6, * p < 0.05). The Mankin score in the tendon and tendon + BMA groups was significantly lower than that in the untreated group.

Figure 6

Histological analyses for articular cartilage at the medial tibial plateau. (A) Toluidine blue staining of medial tibial plateau cartilage. (B) Mankin score. (n = 6, * p < 0.05). The Mankin score in the tendon + BMA groups was significantly lower than that in the untreated and tendon groups at 24 weeks postoperatively.

Figure 7

Biomechanical analysis of regenerated meniscus at 4, 12, and 24 weeks post-transplantation in tendon and tendon + BMA groups (n = 6, * p < 0.05). Values are presented as mean ± SD. The compressive stress of the regenerated meniscus in the tendon + BMA group was significantly higher than that in the tendon group at 12 weeks postoperatively.

Figure 8

Method of injection into the semitendinosus tendon and the replacement of the meniscus tissue. (A) Bone marrow aspirate (BMA) was obtained from the lateral side of the tibial of the knee joint using a 15-gauge Bone Marrow Harvest Needle. (B) BMA was injected using a 26-gauge needle (black arrowhead) that was inserted into the center of the rabbit’s semitendinosus tendon (black arrow). Abbreviations: MCL, medial collateral ligament. (C) The tendon graft was sutured using 5-0 nylon sutures (black arrowhead) to the posterior cruciate ligament (PCL), the posteromedial corner of the tibia, the anteromedial capsule, and the anterior stump of the original medial meniscus. Abbreviations: ACL; anterior cruciate ligament.

Figure 9

Study protocol. Rabbits were sacrificed at 4, 12, and 24 weeks after surgery (n = 6 at each time point), and the macroscopic and histological findings among the three groups were compared. Meanwhile, biomechanical comparisons between the tendon and tendon + BMA groups were performed at 4, 12, and 24 weeks postoperatively (n = 6 at each time point).