(A-38) Adoptive Transfer of Anti-Inflammatory Macrophages Improves Inflammatory Response and Increases Bone Formation Around Titanium Implants

Introduction:: Macrophages play a central role in orchestrating the inflammatory response and are sensitive to changes in the chemical and physical characteristics of the implanted biomaterials. Moreover, macrophages respond to biological, chemical, and physical cues by polarizing into several described phenotypes, generally divided into pro-inflammatory and anti-inflammatory states. Pro-inflammatory macrophage activation triggers the release of pro-inflammatory interleukins, chemokines, and reactive oxygen species to promote inflammatory cell recruitment and prolong the response. Conversely, anti-inflammatory macrophages release high levels of immunomodulatory factors such as IL-10 to attenuate the inflammatory response. We previously showed that rough-hydrophilic titanium (Ti) implants skew macrophage polarization towards an anti-inflammatory phenotype and increase mesenchymal stem cell (MSCs) recruitment and bone formation around the implant when compared to smooth or rough Ti implants. Previous studies have investigated immune cell therapies in cancer and autoimmune disorders, but it remains unclear if anti-inflammatory macrophage adoptive transfer can improve biomaterial implantation outcomes. Therefore, this study aimed to investigate if adoptive transfer of macrophages in different polarization states can reduce the inflammatory microenvironment and improve biomaterial integration.

Materials and Methods:: First, to elucidate the role of macrophages in the inflammatory response to biomaterial implantation, Ti implants (1 mm diameter, 4 mm length) of varying wettability (rough-hydrophobic and rough-hydrophilic) were placed in the femur of 12-week-old male C57BL/6J or macrophage-ablated (Macrophage Fas-induced Apoptosis (MaFIA) mice (n=6). Macrophages were ablated by injecting the dimerization drug AP20187, which induces apoptosis in macrophages, for five consecutive days before the surgical procedure. The immune cell phenotypes and MSCs surrounding the implants were assessed by flow cytometry at 3- and 7-days post-implantation. Second, we performed adoptive transfer experiments into macrophage-competent (C57BL/6J) or macrophage-ablated (MaFIA) mice to determine how macrophage polarization affected the peri-implant microenvironment. Bone marrow-derived macrophages from C57BL/6 mice were activated into two distinct macrophage populations: pro-inflammatory macrophages with high production of IL-1β and TNF-α or anti-inflammatory macrophages with high production of IL-10. PBS was used as a control. Then, we performed adoptive transfer of fluorescently-tagged “naïve” (PBS), pro-inflammatory (INF-γ + LPS), or anti-inflammatory (IL-4 + IL-10) macrophages (1 x10⁶ cells/animal, injected via tail vein) into 12-week-old male macrophage competent (C57BL/6J) or macrophage ablated (MaFIA) mice (n=6) before implantation of rough Ti rods, which are the most commonly used for craniofacial or orthopaedic applications. Assessment of the innate immune response and MSC recruitment in the peri-implant tissue was performed by flow cytometry at 3- and 7-days post-implantation. Bone formation around the implants was assessed by micro computed tomography (microCT). Data were analyzed by ANOVA with Tukey HSD assessment of differences.

Results, Conclusions, and Discussions:: Macrophage ablation in MaFIA mice resulted in increased neutrophil levels and decreased pro- and anti-inflammatory macrophages, CD4⁺, CD8⁺ T cells at day 3 post-implantation when compared to macrophage competent C57BL/6J mice. By day 7 post-implantation, neutrophils remained elevated in MaFIA mice in all implant modifications tested. Interestingly, macrophage abundance was higher in MaFIA mice than in C57BL/6J mice by 7 days due to the rebound in macrophage production after initial macrophage depletion. At this time point, a reduction of pro-inflammatory macrophages in C57BL/6J mice was observed in rough-hydrophilic implants when compared to smooth and rough Ti implants, and an increase in anti-inflammatory macrophages observed in smooth and rough Ti implants compared to rough-hydrophilic Ti implants. These changes in macrophage phenotype indicate the beginning of the resolution of the inflammatory stimuli. However, these changes were not observed in MaFIA mice with high levels of pro-inflammatory macrophages and reduced levels of anti-inflammatory macrophages and T cells. Macrophage ablation also dramatically reduced MSC recruitment to the implantation site. Adoptive transfer of pro-inflammatory macrophages resulted in increased levels of neutrophils, macrophages, CD4⁺, and CD8⁺ T cells in both C57BL/6J and MaFIA mice at 3 days post-implantation when compared to PBS control groups. At 7 days post-implantation, innate immune cells remained elevated and MSC recruitment was reduced in mice administered pro-inflammatory macrophages compared to control. Anti-inflammatory macrophages reduced levels of neutrophils, pro-inflammatory macrophages, and CD8⁺ T cells and increased recruitment of MSCs in C57BL/6J. Finally, bone formation around the Ti implants was increased in C57BL/6J mice receiving anti-inflammatory macrophages, and bone formation was reduced in mice receiving pro-inflammatory macrophages compared to PBS control. Bone formation around the Ti implant was increased in MaFIA mice following adoptive transfer, regardless of macrophage phenotype, compared to PBS control; however, levels for all MaFIA groups were significantly lower when compared to macrophage-competent C57BL/6J mice. Together, our results show the importance of macrophages during the healing response after biomaterial implantation and that delivery of anti-inflammatory macrophages may be a potent modulator of the inflammatory response during biomaterial integration.

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