Effect of Intermittent Treatment of Low Dose Human Parathyroid Hormone (1-34) on Fracture Healing in Rats

Introduction: Recent reports have shown that intermittent treatment with PTH(1-34) increases callus formation and mechanical strength in experimental fracture healing (1). Thus far, however, the optimal dose for enhancement of fracture healing has not been determined. Furthermore, little is known about the molecular mechanisms by which PTH regulates the fracture healing process. In this study, we tested the hypothesis that intermittent low dose treatment of fractures with human PTH(1-34) can increase the callus formation and mechanical strength. In addition, we analyzed the molecular mechanisms by which such an effect may enhance the healing process. Materials and Methods: Animals and hormone administration: 7-weekold male SD rats were divided into two groups: A PTH group in which rats were injected daily with 10 μg of PTH(1-34)/kg, and a control group in which rats received a daily injection of vehicle solution. The injections were given subctaneously beginning on the day fractures were produced until the time of sacrifice. Fracture model: Closed mid-diaphysial fractures were created in the right femora (2). Samples harvested at 4, 6, and 8 weeks after fracture were used for dual-energy X-ray absorptiometry (DXA) and mechanical testing, and those at 2, 4, 7, 14, and 21 days were used for molecular and histological analyses. DXA: Bone mineral content (BMC) and bone mineral density (BMD) of the fracture calluses were measured. Mechanical testing: The mechanical strength of healing fractures was measured by a destructive three-point bending procedure. Immunohistochemistry (IHC): To evaluate cell growth activity, sections were reacted with an antibody for PCNA. The percentage of PCNApositive cells over total cells in the periosteal calluses was determined as a PCNA score (3). Tartarate resistant acid phosphatase (TRAP) assay: For the identification of osteoclasts, TRAP staining was performed. The number of osteoclasts (N.Oc) at days 7, 14, and 21 was counted, and the osteoclast index (OI; N.Oc/callus perimeter in mm) was also calculated (4). Northern blot analysis: After the isolation of total RNA from fracture calluses, Northern blotting was carried out with cDNA probes for collagen types I (COL1), II (COL2), X (COL10), alkaline phosphatase (ALP), osteonectin (ON), osteocalcin (OCN), insulin-like growth factor-I (IGF-I), osteoprotegelin (OPG), and osteoprotegelin-ligand (OPGL). Results: DXA and mechanical testing: BMC, BMD and mechanical strength were significantly increased in the PTH group compared with the control group at weeks 4, 6, and 8 (p<0.01) (Fig. 1A, 1B). Molecular and histological analyses: TRAP assay demonstrated that the OI of the PTH group increased to approximately 2.5-fold that of the control group at day 7 (Fig. 2A). The maximal increase of the PTH group was found at day 7, whereas that of the control group was at day 14 (Fig. 2A). IHC analysis showed that, from day 2 to day 7, osteoprogenitor cells under the periosteum were positive for PCNA. The PCNA score was significantly increased in the PTH group at day 2 (p<0.01); the score of the PTH group and the control group was 39% and 28%, respectively (Fig. 2B). However, no significant difference was seen in the score between the two groups after day 4 (Fig. 2B). Northern blot analysis demonstrated that expression levels of COL1, ALP, and ON, which are markers for immature osteoblasts, were elevated in the PTH group in the later stages of fracture healing (from day 14 to day 21) (Fig. 3). OCN, which is a marker of mature osteoblasts, was also elevated in the later stages (Fig. 3). Interestingly, IGF-I mRNA was up-regulated only in the early stages (from day 4 to day 7) in the PTH group (Fig. 3). The expression patterns of COL2 and COL10 showed similar profiles for the two groups. Moreover, expression of OPG was upregulated at day 21 in the PTH group. Discussion: The results demonstrate that PTH stimulates the proliferation of osteoprogenitor cells and increases the number of osteoclasts in the periosteal calluses. This suggests that PTH modulates not only periosteal callus formation but also callus resorption, accelerating the remodeling of calluses. Previous reports have shown that the anabolic effect of PTH on bone formation is mediated by IGF-I (5). In the present study, however, PTH did not increase the expression of IGF-I mRNA in the later stages of fracture healing, in spite of the up-regulation of the marker genes for immature and mature osteoblasts. We speculate that, in the later stages, PTH directly acts on osteoblasts through a protein kinase A pathway, or indirectly acts on them by mediating molecules other than IGF-I. The molecular mechanisms observed in the present study may account for the measured increases in BMC, BMD, and mechanical strength of fracture calluses during the healing process. We conclude that intermittent low dose human PTH (1-34) could be an effective strategy for the enhancement of fracture healing and become the first systemic intervention for the repair of skeletal injuries.