Immunological Profiling of Macrophage Phenotype Response to Cranial Dural Reconstruction Graft Materials

Introduction: : The dura mater encloses cerebrospinal fluid (CSF) protecting the brain, spinal cord, and associated vasculature. Dural reconstruction materials are used to repair traumatic injury and prevent CSF egress, a postoperative neurosurgical complication with reported rates ranging from 0.9% to 42% [1]. Although material selection focuses on achieving watertight closure of the CSF, identifying immunological factors that promote optimal graft performance and reduce adverse inflammation outcomes is critical for improving long-term outcomes.
Current dural substitutes include homologous, acellular, and synthetic materials. Homologous grafts are non-immunogenic but limited by donor availability and disease transmission risks. Acellular grafts retain native architecture and enhanced biocompatibility, while synthetic materials offer precise fabrication but lack biological cues for cell adhesion and tissue remodeling [1].
Preclinical studies suggest that macrophage phenotype is a strong predictor of graft remodeling and long-term functional outcomes [2]. Macrophages differentiate into pro-inflammatory or anti-inflammatory phenotypes in response to local microenvironmental cues, which guide the production of cytokines and matrix metalloproteinases (MMPs). Anti-inflammatory macrophages play a critical role in tissue remodeling by fostering a regenerative environment that promotes wound healing. Research shows that an increased number of these macrophages is associated with more favorable graft remodeling outcomes [3].
We used an in vitro model to characterize the macrophage response to acellular porcine dura and commercially available Lyoplant® grafts. We hypothesized that acellular porcine grafts, with their maintained extracellular matrix (ECM) architecture, would promote increased macrophage attachment and tissue remodeling, activating more anti-inflammatory macrophages compared to the purely collagen Lyoplant® grafts.

Materials and

Methods: : Primary rat bone marrow macrophages (rBMDMs) were seeded and cultured on acellular porcine and Lyoplant® grafts and cultured for 7 days then fixed with 4% paraformaldehyde. (Figure 1A).
Immunocytochemistry (ICC) was conducted to quantify functional immune markers, macrophage mannose receptor CD206 and inducible nitric oxide synthase (iNOS). Z-stack images of 2 µm height at 10X were acquired with a confocal microscope. Max intensity z-projects were generated and ‘Gaussian Blur’ and ‘Despeckle’ were applied in ImageJ. Cellular segmentation for colocalized CD206 signal was performed in Cellpose, and mean fluorescence intensity (MFI) was measured in ImageJ. The MFI for iNOS was measured in ImageJ with ‘Analyze Particles’. The upper limit of the accepted MFI range was defined by the measured MFI of the secondary-only controls, while the lower limit was set at two standard deviations below the mean. Kolmogorov-Smirnov (KS) test and Cohen’s d were performed to assess mean differences and effect size of the distribution between the two graft types for iNOS and CD206 MFIs.
Media was collected from rBMDMs cultured on acellular porcine and Lyoplant® grafts on days 5 and 7 and stored at –80°C until analysis. Protein concentration was determined using a bicinchoninic acid protein assay. Sodium dodecyl sulfate polyacrylamide gel electrophoresis technique was used to run 20 mg of sample protein in 10% gelatin zymogram gels. Imperial™ Protein Stain was used to stain the gels. Gels were imaged on LI-COR Odyssey CLx and analyzed in ImageJ to measure the area under the curve of the unstained bands.

Results, Conclusions, and Discussions:: To profile the immunological responses of macrophages to acellular porcine and Lyoplant® graft, we quantified MMP-9 activity. MMP-9 mediates tissue remodeling and healing, assisting in the breakdown of ECM, removal of damaged tissue, and formation of new tissue during wound healing. Zymogram analysis showed that rBMDMs cultured on acellular porcine grafts had trends of increased MMP‑9 activity from day 5 to day 7, while Lyoplant® grafts showed no measurable MMP-9 activity (Figure 1B).
Next, expressions of iNOS and CD206 of rBMDMs cultured on the two grafts were assessed with ICC. iNOS is a marker of pro-inflammatory differentiated macrophages, and its expression is associated with promotion of inflammatory response and nitric oxide mediated pathogen killing. CD206 is a marker of anti-inflammatory differentiated macrophages, and its expression correlates to promotion of tissue remodeling and secretion of associated cytokines. MFI quantification from acquired images (Figure 1D) revealed increased CD206 expression for acellular porcine grafts compared to Lyoplant® grafts while iNOS expression remained relatively similar between the two graft types (Figure 1C).
For iNOS MFI, KS d and Cohen’s d were 0.09 and 0.06 respectively, suggesting minimal differences in both the distribution and means between the two graft types. In contrast, KS d and Cohen’s d for CD206 MFI were 0.51 and 0.70 respectively, indicating a moderate difference in both the shape and means of the distributions between the two graft types. These findings suggest a potential shift in macrophage phenotype associated with graft composition skewed towards anti-inflammatory.
This dual elevation in MMP-9 and CD206 is characteristic of anti-inflammatory like macrophage in the context of ECM degradation and remodeling. This observed macrophage phenotype is consistent with current understanding of acellular materials and their increased biocompatibility, skewing macrophages towards anti-inflammatory like phenotypes [4]. This may be attributed to preserved ECM architecture, which provides biological cues that favor macrophage-mediated remodeling and integration.
In contrast, Lyoplant® grafts lack native ECM, which could contribute to the lower expression of anti-inflammatory macrophage, like CD206, suggesting a more neutral macrophage profile.

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