title: Effect of combined treatment with zoledronic acid and parathyroid hormone on mouse bone callus structure and composition.
abstract: In recent years, great interest in combined treatment of parathyroid hormone (PTH) with anti-resorptive therapy has emerged. PTH has been suggested to aid bridging of atrophic fractures and improve strength in closed fracture models. Bisphosphonate treatments typically result in a larger woven bone callus that is slower to remodel. The combination of both drugs has been demonstrated to be effective for the treatment of osteoporotic bone loss in many preclinical studies. However, the effect of combined treatment on fracture repair is still largely unexplored. In this study, we aimed to compare these drugs as single-agent and in combination in a murine closed fracture model. We wanted to assess potential differences in material properties, morphometry and in the development of the lacuno-canalicular network. A total of 40 female, 11-week-old wild type mice underwent a closed fracture on the midshaft of the tibia and were assigned to four groups (n=8-10 per group). Beginning on post-operative day 8, animals received different subcutaneous injections. Group 1 received a single injection of saline solution and Group 2 of zoledronic acid (ZA). Group 3 received daily dosing of PTH. Group 4 received a dual treatment, starting with a single dose of ZA followed by daily injection of PTH. Three weeks after fracture, all animals were euthanized and tibiae were assessed using micro-computed tomography (micro-CT), high-resolution micro-CT (HR micro-CT), Raman spectroscopy, quantitative histomorphometry, and deconvolution microscopy (DV microscopy). Combined treatment showed a significant increase of 41% in bone volume fraction and a significant decrease of 61% in the standard deviation of the trabecular spacing compared to vehicle, both known to be strong predictors of callus strength. An analysis via HR micro-CT showed similar results on all groups for lacunar numerical density, whereas mean lacuna volume was found to be higher compared to vehicle in treated groups, but only PTH mono-treatment showed a significant increase compared to vehicle (+45%). Raman spectroscopy did not reveal detectable changes in material properties of the bone calluses. Sclerostin staining, tartrate resistant acid phosphatase (TRAP) staining and canalicular analysis with DV microscopy on a subset of samples did not display distinctive difference in any of the treatments. We therefore consider PTH+ZA treatment beneficial for bone healing. No clear negative effect on bone quality was detected during this study.
authors.affiliation: Institute for Biomechanics, HCP H 22.1, Leopold-Ruzicka-Weg 4, ETH Zurich, 8093 Zurich, Switzerland. Electronic address: mcasanova@ethz.ch.
authors.forename: Michele
authors.initials: M
authors.lastname: Casanova
authors.affiliation: Institute for Biomechanics, HCP H 22.1, Leopold-Ruzicka-Weg 4, ETH Zurich, 8093 Zurich, Switzerland. Electronic address: herellej@alum.mit.edu.
authors.forename: Janelle
authors.initials: J
authors.lastname: Herelle
authors.affiliation: Institute for Biomechanics, HCP H 22.1, Leopold-Ruzicka-Weg 4, ETH Zurich, 8093 Zurich, Switzerland. Electronic address: thomamar@ethz.ch.
authors.forename: Marcel
authors.initials: M
authors.lastname: Thomas
authors.affiliation: Institute for Biomechanics, HCP H 22.1, Leopold-Ruzicka-Weg 4, ETH Zurich, 8093 Zurich, Switzerland. Electronic address: rsoftley1@gmail.com.
authors.forename: Rowan
authors.initials: R
authors.lastname: Softley
authors.affiliation: Orthopaedic Research and Biotechnology, The Children's Hospital at Westmead, Westmead, NSW 2145, Australia; Discipline of Paediatrics and Child Health, University of Sydney, Camperdown, Australia. Electronic address: aaron.schindeler@sydney.edu.au.
authors.forename: Aaron
authors.initials: A
authors.lastname: Schindeler
authors.affiliation: Orthopaedic Research and Biotechnology, The Children's Hospital at Westmead, Westmead, NSW 2145, Australia; Discipline of Paediatrics and Child Health, University of Sydney, Camperdown, Australia. Electronic address: david.little@health.nsw.gov.au.
authors.forename: David
authors.initials: D
authors.lastname: Little
authors.affiliation: Institute for Biomechanics, HCP H 22.1, Leopold-Ruzicka-Weg 4, ETH Zurich, 8093 Zurich, Switzerland; Bioengineering Science Research Group, Faculty of Engineering and the Environment, University of Southampton, Southampton, UK. Electronic address: p.schneider@soton.ac.uk.
authors.forename: Philipp
authors.initials: P
authors.lastname: Schneider
authors.affiliation: Institute for Biomechanics, HCP H 22.1, Leopold-Ruzicka-Weg 4, ETH Zurich, 8093 Zurich, Switzerland. Electronic address: ram@ethz.ch.
authors.forename: Ralph
authors.initials: R
authors.lastname: Müller
mesh_terms: D000818:Animals; D050071:Bone Density Conservation Agents / Q000008:administration & dosage*; D002146:Bony Callus / Q000000981:diagnostic imaging / Q000187:drug effects*; D004164:Diphosphonates / Q000008:administration & dosage*; D004359:Drug Therapy, Combination; D005260:Female; D017102:Fracture Healing / Q000187:drug effects*; D007093:Imidazoles / Q000008:administration & dosage*; D051379:Mice; D008810:Mice, Inbred C57BL; D010281:Parathyroid Hormone / Q000008:administration & dosage*; D013059:Spectrum Analysis, Raman / Q000379:methods; D013978:Tibial Fractures / Q000000981:diagnostic imaging / Q000188:drug therapy*; D016896:Treatment Outcome; D055114:X-Ray Microtomography / Q000379:methods; D000077211:Zoledronic Acid
chemical_list: D050071:Bone Density Conservation Agents; D004164:Diphosphonates; D007093:Imidazoles; D010281:Parathyroid Hormone; D000077211:Zoledronic Acid
keywords: Bone healing; Bone quality; Micro-computed tomography; Parathyroid hormone; Raman spectroscopy; Zoledronic acid