Acute in vivo inflammatory response towards engineered bioabsorbable Magnesium-Aluminum subcutaneous implants.

Introduction: Bioabsorbable metal implants have gained traction as alternatives to classic permanent implant materials for various cardiovascular and orthopedic medical devices. While the foreign body reaction for traditional implant materials has been thoroughly described, there is a general lack of information regarding the dynamic inflammatory response towards bioabsorbable metals. Complicating the matter further, studies have demonstrated that the microstructure of absorbable metals plays a key role in regulating immune reactions, however emphasis has been mostly placed on the long-term response, without much consideration to the acute phase reaction. The goal of this study is to investigate the impact of microstructurally distinct and clinically relevant magnesium-aluminum (Mg-Al) implants and its impact on acute inflammation in vivo. Here, we use leukocyte derived myeloperoxidase (MPO) activity as a biomarker for assessing acute inflammation, using a well characterized luminol based bioluminescent imaging assay. MPO activity indicates the presence of activated neutrophils and macrophages at the implant site. These activated immune cells release reactive oxygen species and other inflammatory mediators which can significantly impact the long-term performance and biocompatibility of the implant. By investigating MPO activity post subcutaneous implantation in a mouse implant model, we examine how microstructural differences between stoichiometry equivalent Mg-Al alloys affects corrosion rate and impacts inflammation and tissue regeneration.

Materials and

Methods: Magnesium – 9% wt. aluminum alloys underwent two different thermal aging procedures. The first solution treatment (ST, 450°C for 24 hrs.) homogenizes the Al rich phases into the Mg bulk matrix. Following ST, alloys were peak aged (STPA, 150°C for 100 hrs.) to produce secondary Mg17Al12 precipitates throughout the microstructure. Sample dimensions were discs at ST (Ø4.90 mm, t 0.33mm) and STPA (Ø4.91 mm, t 0.5mm). After sterilization, ST and STPA samples were implanted subcutaneously on either side of a male mouse’s supine midline for 1-5 days. Luminol sodium salt saline solution (50mg/ml) was injected intraperitoneally, and imaging was performed using a IVIS Spectrum CT system. Serial bioluminescence images were taken at 1 min intervals for 1 hr. CT acquisition was completed before luminal injection with machine default settings. Images were analyzed and presented in total flux (photon/second) with maximum and minimum thresholds indicated. Two ROI selections were created over the implantation site and normalized by background noise to calculate the total bioluminescent emission signal. Histological analysis was performed at endpoints throughout study.

Results, Conclusions, and Discussions: In a previous study, the in vitro corrosion behavior of the ST and STPA implants were evaluated. The STPA implant generally experiences faster corrosion than the ST implant (> 2 days immersion), due to its inhomogeneous microstructure which contains Mg17Al12 precipitates, ultimately accelerating the micro galvanic corrosion reaction at the surface. Due to this, we hypothesized that the acute phase inflammatory reaction between the two materials would not be equivalent, and the long term pro-healing response would favor the slower degrading ST implant. Fig. 1A &B shows representative CT scans of a mouse at day 1 and 5 containing both implants. At day 1, void space is seen around both implants, indicating H2 gas buildup, a common occurrence with Mg degradation which is proportional to the amount Mg corrosion. At day 5, H2 pockets surrounding the ST implant have largely diffused/ regressed, while the STPA presents a persistent gas pocket. The ST implant is still visualized at day 5 (Fig.1C), while the STPA implant is mostly degraded with remaining product still visible; this corroborates the previous findings in vitro showing STPA has a faster corrosion rate than ST.
Fig. 2A-C shows the MPO activity indirectly measured using the luminol assay at day 1 and 5. In this study, initially high amounts of signal is seen for the ST implant at day 1. However, at day 5 the highest luminol signal is colocalized with the STPA implant. Although MPO activity is not solely specific to neutrophils in vivo, histology performed on implants at day 8 confirm large amounts of polymorphonuclear cells still present within the STPA implant footprint. This indicates a persistent acute phase inflammatory reaction, possibly exacerbated by residual Mg17Al12 particles which are more noble and likely undegraded. Future work will determine the extent of neutrophil/macrophage presence in both implants using immunostaining, and differential gene regulation via RNAseq analysis. This work will directly aid in future efforts to identify appropriate Mg microstructures not only for manipulating corrosion and mechanical performance, but also for tuning the inflammatory response towards more regenerative reactions.

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