3.3.1. Live/Dead Assay

Human osteoblastic MG-63 cells were counted using Image-J (Fiji, Version 1.52 h), through which the cell number/mm<sup>2</sup> of living and dead cells was determined. Figure 13 shows representative samples with live/dead staining of the inner surface of the 500 μm scaffold as compared to the Curasan control after 3, 7 and 10 days. Long-term studies (>4 weeks) were not assessed.

**Figure 13.** *Cont*.

**Figure 13.** Living/dead cells on the inner surface of the ceramic; 500 μm scaffold after three days ((**a**): Hybrid foam; (**b**): Curasan), seven days ((**c**): Hybrid foam; (**d**): Curasan) and 10 days ((**e**): Hybrid foam; (**f**): Curasan); (**g**)**:** Auto-fluorescence of the ceramics; (**h**)**:** Thermanox membrane (pos. control, 10 days); white bar = 200 μm. Green indicates living cells; red indicates dead cells.

Quantitative results of the number of living and dead cells per mm<sup>2</sup> is shown in Figure 14a,b, respectively. The number of living cells increased over the course of the experiment in both the scaffold and the Curasan control, with no significant differences between our scaffold and the Curasan control.

**Figure 14.** Overview of the biocompatibility tests: cell counts of (**a**) living cell numbers and (**b**) dead cell numbers per mm<sup>2</sup> on the materials after 3, 7 and 10 days; (**c**) WST assay to demonstrate proliferation of MG 63 on the samples. Means and controls were statistically compared to assess the material effect. Significances set at *p* < 0.05 are assigned the same symbol. (**d**) Cytotoxicity of hybrid foams compared to the Curasan control; pos. Control = cells, neg. Control = TritonX. 162

## 3.3.2. Cell Proliferation Assay

Figure 14c shows that the growth rate of the cells on the scaffold and Curasan control compared to the growth of cells on a Thermanox cover slip as a positive control. The growth rate of the cells on the scaffolds in the cell culture plates increased only up to seven days and stagnated thereafter, while the cells on the Thermanox cover slip continuously proliferated. No significant difference in cell proliferation was observed between the hybrid foam and the Curasan control.

#### 3.3.3. LDH Assay

The cytotoxicity for both the hybrid foam and the Curasan control was slightly above the positive control (cells on the Thermanox cover slip) and very clearly below the negative control (Triton X), with no significant differences between our scaffold, the Curasan control and the positive control noted. The graphs of cytotoxicity over time were nearly congruent for the Curasan control and the Hybrid Foam (see Figure 14d).

#### 3.3.4. GIEMSA Staining

In the GIEMSA staining, it was evident that the MG-63 cells only colonized the inner sponge area of the hybrid foams after 3 and 10 days, but only sporadically on the surface of the CerAM VPP shell (see Figure 15). Once again we saw mainly complete material and a form fit but also a gap between the ring structures and the Freeze Foam (Figure 15, upper right-hand side.)

**Figure 15.** GIEMSA staining after (**a**) 3 and (**b**) 10 days; top and side view.
