Objective: Advances in the field of biomaterials have positively affected implant acceptance and nowadays nanostructured surfaces are known to have a positive effect on bone regeneration. The development of the field of osteoimmunology has contributed to a steadily increasing interest towards the investigation of the immunomodulatory effect of nanopatterned surfaces. Luckily, advances in nanofabrication methods have resulted in fabricating relatively large nanostructured areas with high resolution. This study is taking advantage of the 3D printing technique called two-photon polymerization (2PP) for the generation of 3D submicron pillar structures (patterns) to systematically investigate their effect on murine macrophages regarding cell migration, viability, cytoskeletal organisation and phenotype polarization. Methods: Six different pillar patterns were fabricated using a Photonic Professional GT (Nanoscribe, Germany) system. The pillars were characterized with scanning electron microscopy (SEM). Cell viability of J774A.1 macrophages on the nanofabricated patters was assessed by live/dead staining with calcein AM and ethidium homodimer-1 and fluorescently analyzed. Cell migration to the patterns was studied using DAPI staining. The cytoskeletal organization of the macrophages was investigated by actin staining and analyzed both through fluorescent microscopy as well as SEM. Macrophage expression of nitric oxide (NO) was tested with Griess assay. The phenotype polarization of the macrophages on the patterns was explored by dual immunofluorescent staining with iNOS/ARG-1 and CCR7/CD206.
Results: It was demonstrated that different pillar heights and pillar pitches induce different cell attachment mechanisms (lamellipodia, filopodial extensions) and directly affect cytoskeletal reorganization of the macrophages after one day of seeding. Patterns with a height of 1000 nm and a pitch of 700 nm cause an increase in the number of elongated cells. Round cells are experiencing larger area growth when adherent on patterns with a pitch of 1000 nm. Elongated macrophages are strongly affected by patterns of 1000 nm height and 700 nm pitch. Moreover, patterns with a pillar height and pitch of 1000 nm are shown to be the more potent leading to a M1/M2 macrophage phenotype switch.