Biomaterial-mediated modulation of macrophage behavior to promote bone regeneration

摘要

Introduction: The inflammatory response plays a key role in bone repair, but prolonged inflammation can impair healing. Macrophages, the primary cells of the inflammatory response, are considered to be crucial regulators of healing. Previous work has shown that sequential M1 (classically activated) and M2 (alternatively activated) macrophages are required to support tissue regeneration. Still, their phenotypic contributions to biomaterial-mediated bone repair are unclear. Therefore, we investigated the interactions in vitro between macrophages and ceramic scaffolds Baghdadite (Ca_3ZrSi_2O_9) and Sr-HT Gahnite (Sr-Ca_2ZnSi_2O_7-ZnAl_2O_4), which have been shown in vivo to regenerate large bone defects under load, compared to clinically utilized tricalcium phosphate-hydroxyapatite (TCP-HA) scaffolds. We hypothesized that interactions with macrophages contribute to the success of these scaffolds to promote bone regeneration. In this work, we characterized the temporal response of macrophages to the scaffolds and their corresponding soluble factors via gene expression and protein secretion for markers indicative of phenotype and bone regeneration, and we probed the mechanism of macrophage modulation by varying scaffold grain size. Materials and Methods: Human monocytes were isolated from blood and differentiated into macrophages as described previously. Unactivated macrophages (MO) were seeded either directly onto Baghdadite, Sr-HT Gahnite and TCP-HA scaffolds, or in transwell co-culture with the scaffolds using ultra-low attachment plates and incubated for 6 days with a media change on day 3. On days 2 and 6 post-cell seeding, conditioned media was collected for ELISA assays and the scaffolds were transferred into TRIzol for RNA extraction. RT-PCR was conducted as described previously. The data are shown as fold change over GAPDH(±)SEM (n≥4, see figure captions for details of statistical analysis). These data were then converted into a combinatorial score indicative of the M1-M2 character of the macrophages based on a similar algorithm that accurately predicted healing of human diabetic wounds. The score was defined as the ratio of expression of multiple M1 markers over M2 markers, such that higher scores represent increased proinflammatory (M1-like) behavior with respect to M2 behavior. To visualize patterns in gene expression and assess differences among samples, principal component analysis (PCA) was implemented using Matlab? software. Results and Discussion: Gene expression analysis revealed sequential upregulation of M1 and M2c-related genes by Baghdadite scaffolds (Fig. 1), while Sr-HT Gahnite suppressed late M1-like behavior and TCP-HA promoted chronic inflammation. Soluble factors had marginal effects on macrophage behavior (Fig. 2A), which was corroborated via PCA (Fig. 2B). Increasing grain size promoted enhanced TNF secretion, but had only modest effects for all other proteins tested (Fig. 3). Conclusion: Overall, these results suggest that Baghdadite and Sr-HT Gahnite scaffolds may directly modulate macrophage behavior to promote healing, while TCP-HA scaffolds cause prolonged inflammation. Understanding cell-scaffold interactions may aid in biomaterial design to enhance bone regeneration.