Time resolved 3D live-cell imaging on implants

Abstract

It is estimated that two million new dental implants are inserted worldwide each year. Innovative implant materials are developed in order to minimize the risk of peri-implant inflammations. The broad range of material testing is conducted using standard 2D, terminal, and invasive methods. The methods that have been applied are not sufficient to monitor the whole implant surface and temporal progress. Therefore, we built a 3D peri-implant model using a cylindrical implant colonized by human gingival fibroblasts. In order to monitor the cell response over time, a non-toxic LIVE/DEAD staining was established and applied to the new 3D model. Our LIVE/DEAD staining method in combination with the time resolved 3D visualization using Scanning Laser Optical Tomography (SLOT), allowed us to monitor the cell death path along the implant in the 3D peri-implant model. The differentiation of living and dead gingival fibroblasts in response to toxicity was effectively supported by the LIVE/DEAD staining. Furthermore, it was possible to visualize the whole cell-colonized implant in 3D and up to 63 hours. This new methodology offers the opportunity to record the long-term cell response on external stress factors, along the dental implant and thus to evaluate the performance of novel materials/surfaces.

Fig 1. LIVE/DEAD staining of chlorhexidine treated gingival fibroblasts.

CYTO-ID Red labeled human gingival fibroblasts were treated with or without chlorhexidine. CYTO-ID Red labeled gingival fibroblasts, fixed with 4% w/v PFA in PBS and subsequently stained with DRAQ7 served as a positive control for the LIVE/DEAD staining: (A) CYTO-ID Red, (B) DRAQ7, and (C) their overlay. Labeled gingival fibroblasts without addition of chlorhexidine served as a negative control after DRAQ7 staining: (D) CYTO-ID Red, (E) DRAQ7, and (F) their overlay. CYTO-ID Red labeled gingival fibroblasts were treated with 132 mM chlorhexidine for 2 hours prior DRAQ7 staining: (G) CYTO-ID Red, (H) DRAQ7, and (I) their overlay. Arrows show live cells without DRAQ7 nucleus staining. The samples were examined under the CLSM (Leica TCS SP2). Scale bars: 100 μm.

Fig 2. LIVE/DEAD staining of gingival fibroblasts treated with different chlorhexidine concentrations.

CYTO-ID Red labeled human gingival fibroblasts were treated with different concentrations of chlorhexidine for 8 hours prior DRAQ7 staining. LIVE/DEAD stained gingival fibroblasts after treatment with 18 μM chlorhexidine: (A) CYTO-ID Red, (B) DRAQ7, and (C) their overlay. LIVE/DEAD stained gingival fibroblasts after treatment with 36 μM chlorhexidine: (D) CYTO-ID Red, (E) DRAQ7, and (F) their overlay. LIVE/DEAD stained gingival fibroblasts after treatment with 72 μM chlorhexidine: (G) CYTO-ID Red, (H) DRAQ7, and (I) their overlay. LIVE/DEAD stained gingival fibroblasts after treatment with 181 μM chlorhexidine: (J) CYTO-ID Red, (K) DRAQ7, and (L) their overlay. The samples were examined under the CLSM (Leica TCS SP2). Scale bars: 200 μm.

Fig 3. 3D peri-implant model for SLOT.

(A) Schematic representation of the 3D peri-implant model consisting of a colonized titanium implant by CYTO-ID Red labeled human gingival fibroblasts embedded in a PEGylated fibrin hydrogel. The fibroblast-colonized titanium cylinder was placed vertically in the middle of a flat bottom glass tube filled with the hydrogel. A gas permeable sterile filter was used to seal the glass tube. (B) Titanium implant colonized by CYTO-ID Red labeled human gingival fibroblasts (green). Scale bar: 200 μm.

Fig 4. Cytocompatibility of tested hydrogels.

(A) Metabolic activity of gingival fibroblasts grown on various hydrogels for 24 and 72 hours measured by the Cell Proliferation Kit I (MTT). The metabolic activity is shown as percentage relative to control, tissue culture plastic, which was set to 100%. (B) Cytotoxicity of the PEGylated fibrin hydrogel on gingival fibroblasts after 24 and 72 hours measured by the Cell Cytotoxicity Assay (LDH). The percentage of cytotoxicity was calculated in relation to the low (spontaneous LDH release from fibroblasts grown on tissue culture plastic) and high (fibroblasts grown on tissue culture plastic treated with 2% v/v triton-X-100) toxicity controls.

Fig 5. Maximum intensity projections (MIP) for the individual time points and corresponding fluorescence intensity profile along the titanium implant after the addition of chlorhexidine.

(A) MIP of the live cell stain with CYTO-ID Red (green) and dead cell stain with DRAQ7 (red) at the different time points. Rectangles indicate area of zoomed in versions of each MIP. (B) Fluorescence intensity profile of the MIPs (see corresponding image above in A). The profile was measured top-down and averaged for the full width of the titanium implant. (C) The difference spectrum for consecutive profiles in B.

Fig 6. The intensity increase location on the titanium implant after chlorhexidine treatment plotted versus the time.

The location of the peak in Fig 5C was determined and plotted versus the time (black dots). A linear fit was performed (red line), resulting in a slope of 0.2 mm/h and a coefficient of determination of R² = 0.988.