Optimal timing for intermittent administration of parathyroid hormone (1-34) for distraction osteogenesis in rabbits

Abstract

Background: To date, the usefulness of parathyroid hormone [PTH (1-34)] in distraction osteogenesis has been reported in several studies. We aimed to determine the optimal timing of PTH (1-34) administration in a rabbit distraction osteogenesis model.

Methods: The lower hind leg of a Japanese white rabbit was externally fixed, and tibial osteotomy was performed. One week after the osteotomy, bone lengthening was carried out at 0.375 mm/12 h for 2 weeks. After 5 weeks, the lower leg bone was collected. Bone mineral density (BMD), peripheral quantitative computed tomography (pQCT), micro-computed tomography (micro-CT), and mechanical tests were performed on the distracted callus. The rabbits were divided into three groups according to the timing of PTH (1-34) administration: 4 weeks during the distraction and consolidation phases (group D + C), 2 weeks of the distraction phase (group D), and the first 2 weeks of the consolidation phase (group C). A control group (group N) was administered saline for 4 weeks during the distraction and consolidation phases. Furthermore, to obtain histological findings, lower leg bones were collected from each rabbit at 2, 3, and 4 weeks after osteotomy, and tissue sections of the distracted callus were examined histologically.

Results: The BMD was highest in group C and was significantly higher than group D. In pQCT, the total cross-sectional area was significantly higher in groups D + C, D, and C than group N, and the cortical bone area was highest in group C and was significantly higher than group D. In micro-CT, group C had the highest bone mass and number of trabeculae. Regarding the mechanical test, group C had the highest callus failure strength, and this value was significantly higher compared to group N. There was no significant difference between groups D and N. The histological findings revealed that the distracted callus mainly consisted of endochondral ossification in the distraction phase. In the consolidation phase, the chondrocytes were almost absent, and intramembranous ossification was the main type of ossification.

Conclusion: We found that the optimal timing of PTH (1-34) administration is during the consolidation phase, which is mainly characterized by intramembranous ossification.

Keywords: Administration timing; Bone mineral density; Callus strength; Distraction osteogenesis; Parathyroid hormone.

Fig. 1

Three phases in distraction osteogenesis. There are three phases in the course of distraction osteogenesis (latency, distraction, and consolidation). The latency phase is the waiting period from the time of osteotomy to the start of distraction. The distraction phase is the period in which tension is applied to the callus at a constant rate and rhythm to extend it to the desired length. The consolidation phase is the period in which the callus undergoes mineralization and finally stabilizes

Fig. 2

Position of osteotomy and pin insertion for the external fixator. The Orthofix M-100 was fixed to the tibia with four 2.0-mm half pins. The middle two pins were set 20 mm apart and drill hole osteotomy was performed between the two pins, i.e., 10 mm distal to the inferior tibiofibular junction

Fig. 3

Experimental protocol. The 1-week latency phase was followed by a 2-week distraction phase and a 5-week consolidation phase. Group D + C received PTH (1–34) for 4 weeks from the start of distraction. Group D received PTH (1–34) for 2 weeks from the start of distraction and saline for the subsequent 2 weeks. Group C received saline from the start of distraction and PTH (1–34) for the subsequent 2 weeks, and group N received saline solution for 4 weeks from the start of distraction. The experimental animals were euthanized at 8 weeks after osteotomy

Fig. 4

Three-dimensional micro-computed tomography images of the distracted callus. Longitudinal (left side) and transverse (right side) sections for a representative specimen. A Group D + C; B Group D; C Group C; D Group N

Fig. 5

Mechanical properties by three-point bending analysis. The measurement parameter was absorption energy until the distracted callus fracture. The work to failure (mean ± SD) was highest in group C. Compared with group N, the work to failure for groups C and D + C increased by 90% and 50%, respectively, with statistical significance. There was no significant difference between groups D and N and between groups C and D + C. *p < 0.05, among four groups

Fig. 6

Histological analysis. Representative histological sections at the distracted callus stained with hematoxylin and eosin. a The histology of the distraction phase by the protocol in group N. b The histology of the consolidation phase by the protocol in group N. c The histology of the distraction phase by the protocol in group D + C. d The histology of the consolidation phase by the protocol in group D + C. Each of histological images a–d was high-power fields of the area around the distal part of the osteotomy where new bone is beginning to appear. Chondrocytes were abundant in the distracted callus 2 weeks after the osteotomy (distraction phase), and a new bone was formed by endochondral ossification. The woven bone with some cartilage is predominant. Almost no chondrocytes were observed in the distracted callus 4 weeks after osteotomy (consolidation phase), and a new bone was formed by intramembranous ossification. The mature lamellar bone is predominant. Car cartilage cell, Nb new bone, Cb cortical bone, 2w 2 weeks after the osteotomy, 3w 3 weeks after the osteotomy, 4w 4 weeks after the osteotomy