Overgrowth of long bone in rabbits by growth stimulation through metaphyseal hole creation

Abstract

Overgrowth of long bones was noted in pediatric patients who underwent anterior cruciate ligament reconstruction. Hyperaemia during creating a metaphyseal hole and the microinstability made by the drill hole may induce overgrowth. This study aimed to determine whether metaphyseal hole creation accelerates growth and increases bone length and compare the effects of growth stimulation between metaphyseal hole creation and periosteal resection. We selected 7- to 8-week-old male New Zealand white rabbits. Periosteal resection (N = 7) and metaphyseal hole creation (N = 7) were performed on the tibiae of skeletally immature rabbits. Seven additional sham controls were included as age-matched controls. In the metaphyseal hole group, the hole was made using a Steinman pin at the same level of periosteal resection, and the cancellous bone beneath the physis was removed by curettage. The vacant space in the metaphysis below the physis was filled with bone wax. Tibiae were collected 6 weeks after surgery. The operated tibia was longer in the metaphyseal hole group (10.43 ± 0.29 cm vs. 10.65 ± 0.35 cm, P = 0.002). Overgrowth was higher in the metaphyseal hole group (3.17 ± 1.16 mm) than in the sham group (- 0.17 ± 0.39 mm, P < 0.001). The overgrowth in the metaphyseal hole group was comparable to that in the periosteal resection group (2.23 ± 1.52 mm, P = 0.287). In rabbits, metaphyseal hole creation and interposition with bone wax can stimulate long bone overgrowth, and the amount of overgrowth is similar to that seen in periosteal resection.

Figure 1

Overgrowth of rabbit tibiae in sham and metaphyseal hole groups. (a) Quantification analysis of the overgrowth lengths of sham and metaphyseal hole groups using radiographic images of rabbit tibiae (n = 7 per group). Overgrowth lengths were measured by Image J software. (b) Quantification analysis of mineral apposition rate (MAR) sham and metaphyseal hole groups using the double calcein labeling images (n = 3 per group). MAR (µm/day) = double fluorescence line spacing/days between the two lines. *P < 0.05, **P < 0.01, ***P < 0.001 compared to sham.

Figure 2

Radiographic images of rabbit tibiae at 6 weeks after surgery. Surgery was performed on the left tibiae.

Figure 3

Histological analysis of bone growth after surgery. (a) Haematoxylin and eosin staining for overgrowth bone using decalcified bone tissues. The distance between red and black dotted lines represents a new bone area scale bar, 2 mm. (b) Bone dynamic histomorphometry analysis for assessment of growth rate on tibia using un-decalcified bone specimens. For dynamic histomorphometric analysis, calcein (15 mg/kg) was subcutaneously injected into the rabbits 3 and 7 days before sacrifice. Calcein double labels were measured using fluorescence microscopy. The distance between the red and yellow dotted lines represents a new bone area.

Figure 4

Surgery was performed on the left tibiae of rabbits weighing 2.5–3 kg. A longitudinal medial skin incision was made over the left proximal tibial regions, approximately 1–2 cm in length, in each group. During the creation of the metaphyseal hole, a Steinman pin was used instead of a drill to avoid thermal damage. The vacant space was filled with bone wax.