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Orthopedic and Rehabilitation Engineering

Orthopedic and Rehabilitation Engineering - Poster Session E

Y25 - Impact of Iron Deficiency and Intravenous Iron Treatment on Bone Healing in a Mouse Fracture Model

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

QN

Quynh Truc Nguyen

Student
George Mason University
Fairfax, Virginia, United States

Co-Author(s)

VD

Victoria R Duke, BS

Ph.D. Student
Oregon Health & Science University
Portland, Oregon, United States
KM

Kaia Matthias

Highschool Intern
Oregon Health & Science University, United States
Cynthia A. Alcazar, BS

Research Associate
Oregon Health & Science University
Tigard, Oregon, United States
ZW

Zachary Working

Associate Professor, Director of Orthopaedic Trauma
Oregon Health & Science University, United States
NW

Nick J. Willet, PhD

Associate Professor
University of Oregon, United States

Last Author(s)

NW

Nick J. Willet, PhD

Associate Professor
University of Oregon, United States

Co-Author(s)

Karina Nakayama

Associate Professor
Oregon Health & Science University
Portland, Oregon, United States

Primary Investigator(s)

Karina Nakayama

Associate Professor
Oregon Health & Science University
Portland, Oregon, United States

Introduction: : Iron homeostasis plays a critical role in bone repair, as both iron deficiency and iron overload can impair collagen synthesis, osteoblast activity, and bone mineralization. Iron deficiency anemia (IDA), which affects over 1.6 billion people worldwide, is the most common nutritional disorder globally. In 2005, the global prevalence of anemia was estimated at 24.8%, with exceptionally high rates among women of reproductive age (41.8%) and preschool-aged children (47.4%) [1]. IDA is also highly prevalent among orthopedic trauma patients due to blood loss from fractures and surgical interventions. Despite this, the direct effects of IDA on fracture healing remain poorly defined.
Intravenous (IV) iron treatment is frequently used to correct IDA; however, its effects on bone regeneration are not well understood. The goal of this study is to evaluate how IDA and subsequent IV iron treatment influence bone healing following fracture in a mouse model. We hypothesize that IDA will impair fracture repair, while systemic iron repletion via IV iron treatment will improve healing outcomes.

Materials and

Methods: : Male mice with and without IDA were subjected to a stabilized, closed 1-mm femur osteotomy. Experimental groups with IDA received a retro-orbital injection of either a saline placebo, standard dose (15 mg/kg), or high dose (25 mg/kg) of IV iron treatment one day post-fracture. At 28 days post-injury, animals were euthanized for tissue collection. To validate iron deficiency, systemic iron status was evaluated by quantifying non-heme iron content in the liver, spleen, and kidney using a bathophenanthroline-based colorimetric assay. Tissues (n = 3-8/group) were collected, dried, weighed, and digested with acid. A pilot assay was first conducted to determine the optimal dilution factor for accurate quantification across all tissue types. Bone healing was assessed using microcomputed tomography (microCT) and histology. Ex vivo microCT scans of femurs (n = 3/group) were performed using a Bruker Skyscan 1276 system at 4 µm voxel resolution. Reconstruction and artifact correction protocols were refined to improve consistency across samples. Bone volume and bone mineral density (BMD) were measured within a standardized region. For qualitative assessment of healing, representative femurs (n=1/group) were fixed, decalcified in 19% ethylenediaminetetraacetic acid, embedded in paraffin, sectioned, and stained with Hall’s Brundt Quadruple (HBQ) stain for histological evaluation. Group differences were assessed using one-way ANOVA with Tukey’s post hoc test. Outliers were identified using the ROUT method (Q = 5%) and removed prior to analysis.

Results, Conclusions, and Discussions:: Mice with untreated IDA exhibited significantly reduced tissue iron content in the spleen (p < 0.001) and kidney (p < 0.001) compared to control group, confirming successful induction of iron deficiency (Figure 1A-B). Mice treated with the standard dose of IV iron showed significantly elevated tissue iron content in the spleen (p < 0.001) and kidney (p < 0.05) relative to the IDA group (Figure 1A-B). Spleen, kidney, and liver iron content were comparable between IDA mice treated with a standard dose of IVIT and the control group, suggesting standard dose of IVIT systemically repletes iron. High dose of IVIT – treated IDA showed significantly elevated tissue iron content in the spleen (p < 0.0001) and liver (p < 0.01) compared to controls (Figure 1B-C), indicating high dose IVIT may lead to supraphysiological iron accumulation.
MicroCT analysis of femurs 28 days post-fracture showed mice with untreated IDA exhibited significantly reduced BMD compared to the control group (p < 0.05) (Figure 2A). Treatment with a standard dose of IVIT increased BMD relative to the IDA group, there was no significant difference. In contrast, a high dose IVIT did not improve BMD compared to untreated IDA and remained significantly lower than control group (p < 0.05) (Figure 2A). While bone volume trended higher in untreated IDA mice versus control group, there were no significant differences between groups (Figure 2B).
Histological analysis of HBQ-stained femur sections showed limited callus in control mice (Figure 3A), which may suggest more advanced remodeling and resolution of the fracture site. In contrast, untreated IDA mice (Figure 3B) appeared to have extensive callus formation, potentially indicating a prolonged or dysregulated healing response. The standard dose IVIT group (Figure 3C) showed an intermediate amount of callus, possibly reflecting partial restoration of healing. This study validated iron deficiency status in a mouse model of IDA with concurrent orthopedic fracture. MicroCT analysis suggests that both IDA and IVIT treatment may impact bone mineralization during healing, yet further studies are needed to confirm these trends. Overall, this model provides a valuable platform for studying how IDA impacts bone healing and optimizing treatment strategies for orthopedic trauma patients with IDA.

Acknowledgements and/or References (Optional): : WHO, “Worldwide prevalence of anaemia 1993-2005,” www.who.int, 2008. https://www.who.int/publications/i/item/9789241596657