Intermittent PTH Treatment Rescues Impaired Fracture and Bone Graft Healing with Aging

INTRODUCTION Aging decreases chondrogenesis, bone formation, callus vascularization and remodeling during facture healing in mice, which is associated with reduced cyclooxygenase-2 (COX-2) expression, and is restored by administration of an EP4 agonist. Activation of the EP4 receptor stimulates cAMP and activation of the protein kinase A (PKA) signaling pathway. These findings suggest that enhanced PKA signaling compensates for deficient fracture healing associated with aging. The parathyroid hormone receptor (PTH1R), similar to the EP receptors, is a G-coupled 7-transmembrane receptor that activates PKA signaling. PTH1R mediates the anabolic effect of Forteo (PTH 1-34) in osteoporosis patients via PKA signaling. Because of the similar signaling effects of PGE2/EP4 and PTH/PTH1R, we hypothesized that PTH 1-34 would rescue the reduced fracture healing that observed in aging. Here we tested this using stabilized tibia fractures in mice, and a complementary uni-cortical defect-bone-graft model. MATERIALS AND METHODS Experimental Animals: C57/B6, 7to 8-week-old (young) and 52to 53-week-old (aged) female mice were subjected to tibia fracture and unicortical tibia bone grafting. After surgery, mice from both models were divided into two treatment groups: (1) PTH 1-34 (Forteo; 40ug/kg); and (2) normal saline. Daily subcutaneous drug administration was delivered for either 2 weeks (grafting) or 3 weeks (tibia fracture). Animal Models: (1) Tibia fracture: An intramedullary pin was inserted into the tibia at the knee and an open fracture in the proximal tibia diaphysis was performed. (2) Tibia uni-cortical defect-graft model: A 2mm x 1mm segment of the anterior cortex was removed and was used to graft a similarly sized tibial defect. Young/aged grafts were transplanted into defects in young or aged mice, and harvested at days 5, 7, 10, 14, and 21 for micro-CT, histology, and RNA extraction. Radiographs and μCT: Weekly radiographs (Faxitron X-ray, Wheeling, IL) were obtained to monitor bone healing. Specimens were scanned at 10.5-micron isotropic resolution using a Scanco VivaCT 40 (Scanco Medical AG, Switzerland) at indicated time points. Mineralized callus volume, callus bone mineral density (BMD) and total mineral content (BMC) were determined. Biomechanical Torsion Testing: Fracture specimens were mounted on an EnduraTec TestBench system with a 200 N.mm torque cell (Bose Corp., Minnetonka, MN) and tested in torsion at a rate of 1/sec until failure to determine the torsional stiffness, ultimate torque, ultimate rotation, and strain energy to failure. Quantitative Real-time RT-PCR: The fracture callus and 1mm of adjacent bone was excised and total RNA extracted using the QIAGEN RNeasy kit. cDNA was synthesized from 1μg of RNA per callus using a first-strand cDNA synthesis kit (Invitrogen). Real-time RT-PCR analyses were performed using murine specific primers for chondrogenesis and osteogenesis related genes (col2a1, colX, sox9, ihh, osterix, runx2 and osteocalcin). Histology & Histomorphometry: Specimens were harvested at 5, 7, 10, 14, and 21 days and paraffin embedded after 3 days of fixation in 10% NB-Formalin. 4-m sections underwent H&E staining and histomorphometric analyses using Osteometrics software (Decatur, GA). Statistics: Results are shown as the mean +/standard deviation. Statistical tests included Student’s t-tests and two-way ANOVA followed by Tukey-Kramer test. P <0.05 was considered significant.