(H-306) Electrospun Fiber Surface Roughness Modulates Macrophage Polarization for Soft-Tissue Injury Regeneration

Introduction:: Electrospun materials are used for tissue engineering applications due to their ability to mimic features of the extracellular matrix (ECM) such as fiber diameter and alignment [1]. This is significant, as materials which mimic the ECM have been shown to promote a wound-healing response after injury [2]. Previous studies have shown that surface roughness (SR) may be an important feature of the ECM that promotes healing. The d-band, which is the major source of roughness on the collagen fibril, is 67 nm – a value achievable on nano to micron scale electrospun fibers [3]. Studies in two-dimensions have shown an impact of SR on macrophage polarization, but the observed trends have been contradictory [4-6]. In three dimensional studies, it has been seen that rougher breast and dental implants led to increased inflammation [7, 8]. However, the impact that SR plays in the macrophage polarization response to fibrous, non-woven scaffolds used for tissue engineering and the resulting mechanism is unknown. Thus, there is a critical need to study the impact that increasing SR plays in driving the response of macrophages to scaffolds used for treatment of fibrous, soft-tissue injuries. The objective of this study is to utilize a tunable, PCL-based electrospun fiber system with fiber diameters on the scale of native human collagen fibrils to study the impact of surface roughness on macrophage polarization. We hypothesize that a material with a surface roughness on the same length-scale as the d-band of collagen will minimize inflammation and promote regeneration.

Materials and Methods:: Mesh Fabrication. Scaffolds with increasing SR were fabricated by electrospinning 20% w/v% poly-caprolactone (PCL; MW = 50,000 g/mol) in chloroform at 12 kV with a tip-to-collector distance of 30 cm. Humidity was increased (RH = 30 ± 2, 50 ± 2, 70 ± 2) to capitalize on Vapor-Induced Phase Separation (VIPS). SR was analyzed via atomic force microscopy (AFM). Fiber diameter and morphology was assessed via scanning electron microscopy (SEM). Analysis was performed on three mesh per group. Quantifying Protein Adsorption. 3T3 fibroblasts were lysed to produce the damage-associated molecular patterns (DAMPs). Five mesh per group were incubated in either DAMPs or 40% fetal bovine serum (FBS) (ten mesh total) and adsorbed protein was analyzed using a BCA assay. Characterization of Macrophage Phenotype. Primary macrophages were seeded onto the mesh for 3 days then stained for Phalloidin Alexa Fluor 488, DAPI, CD80 and CD206 to assess morphology and phenotype. Assessing Cytokine Release. Media from the previous study was analyzed using ELISAs to quantify the release of pro- (TNF-α and IL-1β) and anti-inflammatory (IL-10 and TGF-β) cytokines over time with increasing SR. Characterizing Macrophage Response in-vivo. Mesh with increasing SR and a non-fibrous, PCL film control were implanted subcutaneously in the flank of MacGreen mice (4 mesh per mouse). Five mice were used. After three days, the mesh were explanted and stained as in the previous study. GraphPad Prism 9 was used to perform 2-way ANOVA statistical analysis with a Bonferroni post-hoc test on all data in this study.


Results, Conclusions, and Discussions:: Results: SR was modulated by controlling relative humidity (Figure A) without affecting diameter (Figure B) with high SR on the scale of collagen d-band. DAMP and protein adsorption was significantly higher on mesh from the high roughness group (Figure C). An increase in SR led to decreased staining for CD80 (pro-inflammatory) and increased staining for CD206 (anti-inflammatory) (Figure D). ELISA results showed a significant increase in release of pro-inflammatory cytokines in the low roughness group. However, there was not a clear trend for the release of anti-inflammatory cytokines at timepoints tested (Figure D). These results corresponded with our hypothesis that mesh with roughness on the scale of the d-band would be the least inflammatory. Mesh was successfully implanted in MacGreen mice (Figure F) and immunohistological analysis to assess number and phenotype of migrated macrophages is ongoing.

Conclusion and Future Work: Our results show that the SR of fibrous electrospun mesh can modulate the release of inflammatory cytokines from macrophages. Furthermore, the results of our DAMP and protein adsorption studies provided a possible mechanism by which changes in SR could illicit this effect. This study has provided valuable insight into the interaction between macrophages and soft, fibrous biomaterial scaffolds which will help to guide the design of future materials which promote healing. Future studies will complete our in-vivo experiment and determine the macrophage polarization response to our material in mice. Additionally, we will repeat our in-vitro experiment with longer timepoints to capture the anti-inflammatory response and the temporal progression of phenotypic switch. Finally, we will investigate the mechanism behind these results by quantifying changes in mechanosensitive proteins in response to increasing SR.


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