Title: Effect of Mg and Mg-10Gd immune response in 28d longitudinal study in rat femur fracture model

Authors: Sana Riyaz¹, Yu Sun¹, Heike Helmholz¹, Olga Will², Tuula Penate Medina², Oula Penate Medina², Regine Willumeit Römer¹

Affiliations

1. Institute of Metallic Biomaterials, Helmholtz-Zentrum Hereon, Geesthacht 21502, Germany.

2. Section Biomedical Imaging, Molecular Imaging North Competence Center (MOIN CC), University Hospital Schleswig-Holstein (UKSH), Kiel University, Kiel 24118, Germany.

Background: Using biodegradable magnesium (Mg) implants in surgery would allow avoiding the implant removal surgery after successful healing. For optimal degradation speed, there are also Mg implants with alloying metals. Gadolinium (Gd) as an alloying element could satisfy the improvement in degradation and mechanical properties (1,2). In addition, in vitro studies suggest an influence of Mg and Mg-10Gd on bone inflammation that could be beneficial (3) but supporting evidence from in vivo studies is still required.

Aims: The aim of this study is to evaluate the effect of Mg and Mg-10Gd immune reactions in vivo microenvironment in a femur fracture model with external fixation. This data can be used for evaluation for future clinical applications.

Methods: Three groups of animals were analyzed in SHAM (no implant), Mg, and Mg-10Gd implants using the following techniques to evaluate the inflammatory reactions from early immune cells like neutrophils, and macrophages using:

• Fluorescence Molecular Tomography (FMT): in vivo imaging using established Indocyanine green dye (ICG) conjugated sphingomyelin (SM) liposome treatment (5) for imaging and evaluation of the inflammation at different time points in a 28d study at the site of osteotomy.

• Flow cytometry: ex vivo cell population analysis of the immune cells at the site of osteotomy

• Histology: ex vivo analysis of the healing status progression and presence of immune cells at the site of osteotomy.

Results: The in vivo imaging indicates a linear increase in inflammation in SHAM group in comparison to implant groups (Mg and Mg-10Gd). In flow cytometry, data we observed an increase in neutrophil cells in the Mg group among all three groups. In histology, we did not observed the presence of neutrophils at the site of osteotomy.

Conclusion: The 28d longitudinal study shows that the presence of Mg implants did not increase the inflammation at the site of osteotomy. This indicated a promising application of the material for implant development.

Reference

1. M.P. Staiger, A.M. Pietak, J. Huadmai, G. Dias, Magnesium and its alloys as orthopedic biomaterials: a review, Biomaterials 27(9) (2006) 1728-1734.

2. N. Hort, Y. Huang, D. Fechner, M. Störmer, C. Blawert, F. Witte, C. Vogt, H. Drücker, R. Willumeit, K.U. Kainer, F. Feyerabend, Magnesium alloys as implant materials--principles of property design for Mg-RE alloys, Acta Biomater. 6(5) (2010) 1714-1725.

3. M.D. Costantino, A. Schuster, H. Helmholz, A. Meyer-Rachner, R. Willumeit-Römer, B.J.C. Luthringer-Feyerabend. Inflammatory response to magnesium-based biodegradable implant materials, Acta Biomaterialia (101) (2020) 598-608

4. Sun, Y.; Helmholz, H.; Will, O.; Damm, T.; Wiese, B.; Luczak, M.; Peschke, E.; Luthringer-Feyerabend, B.; Ebel, T.; Hövener, J.; Glüer, C.; Willumeit-Römer, R. , Dynamic in vivo monitoring of fracture healing process in response to magnesium implant with multimodal imaging: Pilot longitudinal study in a rat external fixation model. (2022) Biomaterials Science, Vol. 10, 6, 1532 - 1543

5. Helmholz H, Will O, Penate-Medina T, Humbert J, Damm T, Luthringer-Feyerabend B, Willumeit-Römer R, Glüer CC, Penate-Medina O. Tissue responses after implantation of biodegradable Mg alloys evaluated by multimodality 3D micro-bioimaging in vivo. J Biomed Mater Res A. 2021 Aug;109(8):1521-1529. doi: 10.1002/jbm.a.37148. Epub 2021 Feb 16. PMID: 33590952.

Sana Riyaz (M.Sc.)