Spatially Resolved Molecular Analysis of Host Response to Medical Device Implantation Using the 3D OrbiSIMS Highlights a Critical Role for Lipids

Abstract A key goal for implanted medical devices is that they do not elicit a detrimental immune response. Macrophages play critical roles in the modulation of the host immune response and are the cells responsible for persistent inflammatory reactions to implanted biomaterials. Two novel immune‐instructive polymers that stimulate pro‐ or anti‐inflammatory responses from macrophages in vitro are investigated. These also modulate in vivo foreign body responses (FBR) when implanted subcutaneously in mice. Immunofluorescent staining of tissue abutting the polymer reveals responses consistent with pro‐ or anti‐inflammatory responses previously described for these polymers. Three Dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) analysis to spatially characterize the metabolites in the tissue surrounding the implant, providing molecular histology insight into the metabolite response in the host is applied. For the pro‐inflammatory polymer, monoacylglycerols (MG) and diacylglycerols (DG) are observed at increased intensity, while for the anti‐inflammatory coating, the number of phospholipid species detected decreased, and pyridine and pyrimidine levels are elevated. Small molecule signatures from single‐cell studies of M2 macrophages in vitro correlate with the in vivo observations, suggesting potential for prediction. Metabolite characterization by the 3D OrbiSIMS is shown to provide insight into the mechanism of bio‐instructive materials as medical devices and to inform on the FBR to biomaterials.


Figure. S1 .
Figure.S1.Histological analysis of tissue sections following 28 days implantation of polymer coated catheter sections in a murine model of foreign body response.Representative H&E staining images, showing a well-defined inflammatory reaction fibroblast (F), blood vessels (BV), macrophage (M) and catheter (C) captured at × 40 magnification the scale bar = 20 µm.MTC staining image of each tissue section slide for identifying collagen thickness, captured at × 10 magnification.Scale bar = 100 μm.(A-C) show the infiltration count of macrophages, neutrophils and collagen thickness from the site surrounding the foreign body.All data are presented as the mean with ±s.d (N=2, n=3).Significance was calculated by one-way ANOVA with Tukey's post-hoc analysis: *p<0.05,** p<0.01, *** p<0.001.(D) Representative images show tissue section stained for the M1 marker iNOS in green and M2 marker arginase1 in magenta and C represents the catheter site.(Images were acquired on confocal).Scale bar = 25 µm.(D) The ratio of M2-like macrophages to M1-like macrophages for each polymer.All data are presented as the mean with ±s.d (N = 2 and n = 5).Significance was calculated by one-way ANOVA with Tukey's post-hoc analysis: *** <0.0001

Figure. S3 .
Figure.S3.Chemical imaging of tissue sample.(A) View of the area where OrbiSIMS optical ion images were acquired (area of 450 µm × 450 µm).(B-D) 3D OrbiSIMS ion images were recorded in the negative ion mode, (B) total ion image, (C) ion image of the sum of phospholipid specie ions including PA, PS, PE and PI which are divided by total intensity.(D) The main ion of PI (38:4), showing the contribution of PI (38:4) ion are the signature fragments of PI head group, two fatty acids fragments (FA 18:0 and FA 20:4) and (E) RGB ion images showing nuclear marker (blue) and PA 34:4 (red).

Table S2 .
Show unique lipids signature for each phenotype; 8 unique for PDMS, 16 unique for M1-polymer and 4 unique for M2-polymer.

Table S4 .
Characteristic molecular ion and fragments of amino acid in 3D OrbiSIMS spectra (positive polarity)

Table S5 .
Peak exported from SurfaceLab positive mode from each tissue sample, consisting of ions detected in the spectrum and assigned as RG sequences of lysozyme [M-H] + C8H16N5O2 + , m/z 124.1298.

Table S6 .
Other small molecules in each sample and search by human metabolome data base.

Table S9 .
Masson Trichrome Stain Kit (Light Green) Masson 1929 schedule.Mounting media onto tissue slide and covered with a thin coverslip