The initial inflammatory response to bioactive implants is characterized by NETosis

Abstract

Implants trigger an inflammatory response, which is important for osseointegration. Here we studied neutrophil extracellular trap (NET) release of human neutrophils in response to sandblasted large-grit acid etched (SLA) implants using fluorescent, confocal laser scanning and scanning electron microscopy. Our studies demonstrate that human neutrophils rapidly adhered to SLA surfaces, which triggered histone citrullination and NET release. Further studies showed that albumin or acetylsalicylic acid had no significant effects on the inflammatory response to SLA surfaces. In contrast to bioinert materials, which do not osseointegrate, the bioactivity of SLA surfaces is coupled with the ability to release NETs. Further investigations are necessary for clarifying the role of NETosis for osseointegration.

Fig 1. Cell adhesion to SLA surfaces from whole peripheral blood with different pre-treatments.

(A) Representative images of the three pre-treatment groups (blue: DAPI, green: NE, red: CitH3), scale bar: 20μm. (B-F) Boxplots (interquartile range; line: median, whiskers: 1.5 x interquartile range) showing absolute (B-D) and relative (E) cell numbers and the areas covered by NETs (F) for the three groups. Superscript letters indicate groups of statistically significant differences. The three types of treatment were compared by univariate analysis of variance and by Tukey HSD post hoc test with subject and sampling session as covariates,. Superscript letters (a, b) indicate groups of statistically significant differences at the P<0.05 level (similar letters: no significant differences, different letters: significant difference).

Fig 2. Characteristics of cells adhered to SLA surface from whole peripheral blood as detected by immunofluorescence.

(A) NE-postitive PMNs with their typically lobulated nuclei, and other NE-negative nucleated cells. (B) A single PMN committed to the NETotic cascade as clearly shown by decompensated chromatin, the swollen, partly disrupted nucleus and NE and citH3 staining co-located with chromatin and at cytoplasmic locations. (C) Chromatin extrusion from PMN. (D) Fully spread NETs between PMNs of different activation states. Scale bars: 10μm.

Fig 3. Immunostaining of neutrophils.

(A) 4h SLA sample. PMNs (white arrows) und others, not further determined, cells (black arrows). Blue—DNA staining with DAPI, (B) red—immunostaining for CitH3, (C) immunostaining for neutrophil elastase, (D) merged. The other cells lack both NE and CitH3. Scale bars: 50μm.

Fig 4. Immunostaining of neutrophils with NETs.

4h SLA sample. Staining as in Fig. 3. Overview: ripe NETs (arrows) between the adhered cells. Scale bars: 50μm.

Fig 5. Immunostaining of platelets (A) Detection of platelets adhered from whole peripheral blood to PDL-coated cover slips incubated for 5 min by immunolabelling against CD41 (green).

Large clusters of cells can be found accompanied by few individual nucleated cells (red—PI). Scale bar 10 μm. (B) Platelets staining for CD41 (green) on SLA surface incubated for 5 min with whole peripheral blood are scarcely present between adhered PMNs as shown by lobulated nuclei (blue—DAPI). Scale bar 20 μm. (C) Platelets shown by SEM on uncoated glass cover slips incubated for 4 h with whole peripheral blood. Scale bar 5 μm. (D): SLA incubated for 4 h with whole peripheral blood show no platelets, but numerous erythrocytes as well as fine fibres with fibrin-like and NET-like morphology. Scale bar 10 μm.