Session VI - Fracture Repair
Recombinant Growth Hormone Increases Bone Tissue Formation in the Early Phase of Distraction Osteogenesis
Hermann Bail; M. Raschke; S. Kolbeck; A. Weiler; N.P. Haas, Humboldt University, Berlin, Germany
Purpose of the study: While there exist no comparable, quantitative histomorphometrical data about the effects of growth hormone (GH) on the content and structure of callus in distraction osteogenesis, we performed a histomorphometrical analysis of the regenerates of distracted micropig tibiae in order to determine whether administration of recombinant porcine GH leads to changes in callus structure and amount and whether these changes are time-dependent.
Methods: 20 mature female Yucatan micropigs were divided into two treatment groups, one receiving a daily injection of recombinant porcine GH (100 µg / kg) and the other receiving sodium-chloride as placebo. The left tibiae of the micropigs were osteotomized and stabilized with an external fixator. They were distracted twice daily (2 mm / day) for 10 days and then allowed to consolidate for 10 days. At the end of the distraction period each of the animals received 30 mg/kg body weight tetracycline as in-vivo labeling. All procedures were carried out with the ethical permission from the animal rights protection authorities. The pigs were sacrificed and the tibiae harvested on day 25 of the experiment. Three animals were removed from the study due to bone infection or surgery failure. The regenerate and 2 cm of the adjacent cortical bone were divided into 3-mm thick sagittal sections using a precision grinding saw and then embedded in methylmethacrylate. Six-µm serial slices were then produced using a hard-cutting microtome. The sections were stained using the modified von Kossa method or a Safranine-O stain. At the same time, 80 µm sagittally oriented precision grinds for fluorescence microscopy were produced. The regions of interest were digitized by a 3-Chip CCD colour camera and processed using the LEICA Quantimet image anlysis work station. With specially developed algorithms, the bone tissue area (bta) in mm_, and bone tissue perimeter (btp) in mm were measured using the 6-µm slices. The fluorescent area (fla) within the distraction gap was measured using the 80-µm grinds. These data reflect the callus area at the end of the distraction period (at day 15 of the experiment). Due to inter-animal differences in the dimensions of the tibiae, the parameters were normalized with the sagittal section area (ssa) of the distraction gap which was computed in mm_ from the width of the bone ends and the distraction distance. The following parameters were calculated: normalized bone area (nba) = [bta/ssa], normalized fluorescent area (nfla) = [fla/ssa] and bone area / bone perimeter ratio (bapr) = [bta/btp]. T-tests were used to determine differences between the treatment groups.
Results: The nba was significantly higher in the GH-treated group (n=8), than in the placebo group (n=9) (GH: 1,15 ± 0,36; placebo: 0,85 ± 0,27; p = 0.043). The structure of the callus, measured with the bapr, was not significantly different between the two groups (GH: 15,3 ± 2,8 placebo: 14,9 ± 2,5; n.s.). The nfla was more than two-fold higher in the GH-treated group than in the controls (GH: 0,98 ± 0,21; placebo: 0,41 ± 0,14; p = 0.001).
Discussion: It has recently been demonstrated that application of growth hormone (GH) results in biomechanically stronger regenerates both in secondary fracture healing (1) and in distraction osteogenesis (2). However, a histological investigation has shown that the callus formed in secondary fracture healing in GH-treated rats was larger and had a looser, less dense structure than that formed in a control group (3). The same study also found that GH had an initial stimulatory effect on callus formation. Our results demonstrate that GH increases bone tissue formation in the regenerate, which may explain the biomechanically stronger regenerates found in GH-treated animals in recent experiments (1,2). This increased bone formation in GH-treated micropigs appears to be achieved without structural changes in callus microstructure since the bapr was not significantly different in the two groups. This finding indicates that the loose callus structure in the late stages of fracture healing reported by Mosekilde et al. (3) is not present in the early stages of regenerate consolidation in distraction osteogenesis. These results suggest either that callus structure changes substantially as healing progresses or that GH acts differently during distraction osteogenesis than it does during secondary fracture healing. According to Mosekilde et al. (3) we found; that the stimulatory effect of GH acts in the early phase of bone healing. In the present study, in the GH-treated group, the bone formation was more than doubled throughout the distraction phase. Clinically, there exists no relevant bone formation in the regenerate during the distraction procedure. This indicates that GH may have a stimulatory effect on the transformational pathway from the mesenchymal cell to the osteoblastic cell. Our results suggest that systemic administration of homologous GH causes bone formation in regenerate in the very early phase in distraction osteogenesis without changing the callus microstructure. Given these results, GH might be used clinically in the future to improve healing in distraction osteogenesis patients.
Table 1: Histomorphometrical parameters
nba bone area |
nfla fluorescent area |
bapr (mm) area/perimeter ratio |
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| Control (n=9) |
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| GH-treated (n=8) |
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References: (1) Bak et al., Bone 11: 233, 1990. (2) Raschke et al., Proc Orthop Res Soc, 43rd Annual Meeting (3) Mosekilde et al., Bone 14: 19, 1993.
This work was supported by AO/ASIF Foundation, Davos, Switzerland and by the Novo Nordisk A/S, Gentofte, Denmark