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Characterizing the Composition of Bone Formed During Fracture Healing Using Scanning Electron Microscopy Techniques

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Abstract

About 5–10 % of all bone fractures suffer from delayed healing, which may lead to non-union. Bone morphogenetic proteins (BMPs) can be used to induce differentiation of osteoblasts and enhance the formation of the bony callus, and bisphosphonates help to retain the newly formed callus. The aim of this study was to investigate if scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) can identify differences in the mineral composition of the newly formed bone compared to cortical bone from a non-fractured control. Moreover, we investigate whether the use of BMPs and bisphosphonates—alone or combined—may have an effect on bone mineralization and composition. Twelve male Sprague–Dawley rats at 9 weeks of age were randomly divided into four groups and treated with (A) saline, (B) BMP-7, (C) bisphosphonates (Zoledronate), and (D) BMP-7 + Zoledronate. The rats were sacrificed after 6 weeks. All samples were imaged using SEM and chemically analyzed with EDS to quantify the amount of C, N, Ca, P, O, Na, and Mg. The Ca/P ratio was the primary outcome. In the fractured samples, two areas of interest were chosen for chemical analysis with EDS: the callus and the cortical bone. In the non-fractured samples, only the cortex was analyzed. Our results showed that the element composition varied to a small extent between the callus and the cortical bone in the fractured bones. However, the Ca/P ratio did not differ significantly, suggesting that the mineralization at all sites is similar 6 weeks post-fracture in this rat model.

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Acknowledgments

The Project was funded from the European Commission (FRACQUAL-293434), the Swedish Agency for Innovation Systems, Vinnova, Carl Trygger foundation (CTS 12:200, CTS 13:185), and the foundations of Greta and Johan Kock. The authors would like to thank Prof. Reine Wallenberg and Ms. Gunnel Karlsson (Centre for Analysis and Synthesis, LU) for the technical support and scientific discussions. The authors would also like to thank Ms. Mea Pelkonen, MSc (Department of Orthopaedics, Lund University) for her valuable assistance with the sample preparation.

Conflict of interest

Christina Perdikouri, Magnus Tägil, and Hanna Isaksson state that they have no conflicts of interest.

Human and Animal Rights and Informed Consent

The study was approved by the local animal ethics committee at Lund University (ethical permit number: M216-08).

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Authors and Affiliations

  1. Division of Solid Mechanics, Lund University, Lund, Sweden

    Christina Perdikouri & Hanna Isaksson

  2. Department of Biomedical Engineering, Lund University, BMC-D13, 221 84, Lund, Sweden

    Christina Perdikouri & Hanna Isaksson

  3. Department of Orthopedics, Clinical Sciences, Lund University and Skåne University Hospital, Lund, Sweden

    Magnus Tägil & Hanna Isaksson

Authors
  1. Christina Perdikouri
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  2. Magnus Tägil
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  3. Hanna Isaksson
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Correspondence to Christina Perdikouri.

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Perdikouri, C., Tägil, M. & Isaksson, H. Characterizing the Composition of Bone Formed During Fracture Healing Using Scanning Electron Microscopy Techniques. Calcif Tissue Int 96, 11–17 (2015). https://doi.org/10.1007/s00223-014-9930-z

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  • DOI: https://doi.org/10.1007/s00223-014-9930-z

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