52. Calcium phosphates with submicron topography enhance human macrophage M2 polarization in vitro

Abstract

BACKGROUND CONTEXT Synthetic bone grafts are a commonly used alternative to autograft in grafting procedures, such as spinal fusions. In pre-clinical spinal fusion models, it has been recently shown that calcium phosphate grafts with a submicron surface topography enhance bone healing potential when compared to conventional calcium phosphate grafts. Immune cells such as macrophages, are pivotal to the (osteo)immunological response upon implantation of a graft as they can polarize into a proinflammatory M1 phenotype or anti-inflammatory M2 phenotype. Therefore, it is hypothesized that calcium phosphates with submicron topography upregulate M2 macrophages, resulting in enhanced bone healing and tissue integration. PURPOSE The purpose of this study was to evaluate M1/M2 polarization of primary human macrophages after in vitro culture on two commercially available calcium phosphate bone grafts, one with a submicron surface topography and the other with a conventional surface. STUDY DESIGN/SETTING Primary monocytes were isolated from buffy coats and cultured on granules of a biphasic calcium phosphate with submicron topography (BCP PATIENT SAMPLE Not applicable. OUTCOME MEASURES To determine macrophage phenotype, enzyme-linked immunosorbent assays (ELISAs) for chemokine ligand 5 (CCL5) as a marker for M1 macrophages and chemokine ligand 18 (CCL18) as a marker for M2 macrophages, were performed on the cell culture supernatants. Protein concentrations were normalized to DNA content. Macrophage morphology on the surface of the bone graft materials was observed by scanning electron microscopy (SEM). METHODS For both materials, 0.4 cc/well of granules were transferred to well plates and preincubated in RPMI culture medium with 10% fetal bovine serum for at least 2 hours. CD14+ monocytes were isolated from buffy coats (n=2) and subsequent CD14 magnetic-activated cell sorting. CD14+ monocytes were seeded at 4.5×105 cells/well on the calcium phosphate granules and supplemented with 10 ng/ml macrophage colony-stimulating factor. After 24h and after 72h of culture, the culture medium was collected for ELISAs. The macrophages were lysed for DNA quantification. For visualization by SEM, macrophages on granules were fixed in glutaraldehyde. RESULTS After 24h and 72h of culture, CCL18 concentrations were 3-3.5 times higher when macrophages were cultured on BCP CONCLUSIONS Higher levels of M2 macrophage marker CCL18 after culture on calcium phosphate with submicron topography, indicate M2 polarization. The initial presence of CCL5 and its subsequent abatement at 72h in both materials suggests early M1 macrophage activity that is reduced thereafter. The stretched-out, elongated macrophage shape on the material with submicron topography may suggest a higher affinity to the submicron surface topography than the conventional material and is consistent with M2 morphology described in literature. These results indicate that upregulation of M2 macrophages at the surface of calcium phosphate with submicron topography, may play a role in its enhanced performance in bone healing compared to that of conventional calcium phosphate bone grafts. FDA DEVICE/DRUG STATUS MagnetOs Granules (Not approved for this indication), Vitoss Granules (Not approved for this indication).