Liver‐Inspired Polyetherketoneketone Scaffolds Simulate Regenerative Signals and Mobilize Anti‐Inflammatory Reserves to Reprogram Macrophage Metabolism for Boosted Osteoporotic Osseointegration

Abstract Tissue regeneration is regulated by morphological clues of implants in bone defect repair. Engineered morphology can boost regenerative biocascades that conquer challenges such as material bioinertness and pathological microenvironments. Herein, a correlation between the liver extracellular skeleton morphology and the regenerative signaling, namely hepatocyte growth factor receptor (MET), is found to explain the mystery of rapid liver regeneration. Inspired by this unique structure, a biomimetic morphology is prepared on polyetherketoneketone (PEKK) via femtosecond laser etching and sulfonation. The morphology reproduces MET signaling in macrophages, causing positive immunoregulation and optimized osteogenesis. Moreover, the morphological clue activates an anti‐inflammatory reserve (arginase‐2) to translocate retrogradely from mitochondria to the cytoplasm due to the difference in spatial binding of heat shock protein 70. This translocation enhances oxidative respiration and complex II activity, reprogramming the metabolism of energy and arginine. The importance of MET signaling and arginase‐2 in the anti‐inflammatory repair of biomimetic scaffolds is also verified via chemical inhibition and gene knockout. Altogether, this study not only provides a novel biomimetic scaffold for osteoporotic bone defect repair that can simulate regenerative signals, but also reveals the significance and feasibility of strategies to mobilize anti‐inflammatory reserves in bone regeneration.


Figure S1 .
Figure S1.Preparation and characterization of the liver extracellular skeleton.A) Hematoxylin and eosin (HE) staining, B) DAPI staining and C) DNA quantification of samples of the Liver Tissue group, the Skeleton group and the Skeleton-Flat group confirming nuclei depletion (error bars, means ± SD; n = 3).D) Raman spectra of the Skeleton sample and the Skeleton-Flat sample.E) The content of HGF in the two liver extracellular skeleton samples detected by ELISA (error bars, means ± SD; n = 4).Data were analyzed by C) ordinary one-way ANOVA with Tukey's post-hoc test, E) two-tailed t-test with Welch's correction and respective P values are provided.

Figure S2 .
Figure S2.Characterization of various PEKK scaffolds.A) SEM images of the surface of PEKK scaffolds modified by femtosecond laser etching.B) SEM images of cross section of the biomimetic scaffolds.C) The element compositions of various PEKK scaffolds (error bars, means ± SD; n = 3).D) Contact angles of water droplets on the surface of various PEKK scaffolds (error bars, means ± SD; n = 3).E) The changes in pH value of the solution after various scaffolds soaking in simulated body fluid for different lengths of time (error bars, means ± SD; n = 3).Statistical significance was analyzed by ordinary one-way ANOVA with Tukey's post-hoc test and respective P values are provided.

Figure S3 .
Figure S3.Evaluation of antioxidative system of macrophages regulated by various PEKK scaffolds.A) Immunofluorescent images staining with DCFH for intracellular reactive oxygen species (ROS) of RAW264.7 cells cultured on scaffolds for 3 days.B) Representative fluorescent images staining with MitoSOX for mitochondrial ROS of RAW264.7 cells cultured on various scaffolds for 3 days.Quantification of C) DCFH staining and D) MitoSOX staining (error bars, means ± SD; n = 3).E) Expression of antioxidase gene Sod1 in RAW264.7 cells cultured on various scaffolds (error bars, means ± SD; n = 3).F) Comparison of RAW264.7 cells for levels of lactate regulated by various scaffolds (lower and upper box boundaries, line inside box and lower and upper lines represent 25th and 75th percentiles, median, minimum and maximum respectively; n = 5).Statistical significance was analyzed by ordinary one-way ANOVA with Tukey's post-hoc test and respective P values are provided.

Figure S4 .
Figure S4.The effect of MET signaling suppression on Arg2 translocation and metabolic reprogramming.A) Heat map depicting the expression of Ass1 and Asl in RAW264.7 cells on scaffolds for 3 days (n = 3; * and # represent PEKK-L versus PEKK-SW and PEKK-L versus PEKK respectively).B) Protein expression of ASS1 and ASL.C) The expression of genes in the obtained cluster in Figure 4A.D) Heat

Figure
Figure S5.OVX-BMDM polarization and immune sensitization regulated by PEKK-L in vitro.A) Representative dot images of surface markers (CCR7 and CD206) of RAW264.7 analyzed by flow cytometry in Figure 7D.Heat maps depicting the fold changes B) in the expression of polarization genes (n = 3; * and # represent PEKK-L versus PEKK-SW and PEKK-L versus PEKK respectively) and C) in concentrations of secreted cytokines detected by Luminex technology (n = 3) of OVX-BMDMs cultured on various scaffolds for 4 days relative to that of the Control group.D) Heat maps depicting the fold changes in the expression of selected genes of OVX-BMDMs on scaffolds and gelatin after stimulation for 1, 3, 5, and 7 hours respectively relative to that of 1 h stimulation (n = 3; dotted lines mark the 1.5 fold change).E) The concentrations of TLR4 and IL4R of OVX-BMDMs on various scaffolds and gelatin after stimulation for 12 hours detected by ELISA (error bars, means ± SD; n = 3).Protease K of different concentrations (0, 0.1, 1 and 10 μg/mL) was used to remove membrane receptors to determine the content of intracellular receptor proteins.Statistical significance was analyzed by ordinary one-way ANOVA with Tukey's posthoc test and respective P values are provided.

Figure S6 .
Figure S6.Evaluation of osteogenic microenvironment created by macrophages on various PEKK scaffolds.A) A schematic diagram showing constructed co-cultured macrophage-conditioned microenvironment by Transwell and the concentrations of BMP2 and TGF-β of the co-culture microenvironment regulated by RAW264.7 cells on various PEKK scaffolds (error bars, means ± SD; n = 3).B) Biological process enrichment analysis of differentially expressed genes and gene set variation analysis (GSVA) of C) biological process and D) signaling pathways related to osteogenesis with enrichment of differential genes in Figure 8H.E) Quantitative analysis of bone surface density and trabecular number according to micro-CT data in Figure 8L (error bars, means ± SD; n = 3).Statistical significance was analyzed by ordinary one-way ANOVA with Tukey's post-hoc test and respective P values are provided.