mean ± standard deviation (SD), n = 5.

#### *3.2. Colonization of Scaffolds*

In order to enable colonization within porous scaffolds, it is important to equilibrate the scaffolds for 24 h in a cell culture medium before seeding. Following the equilibration, the scaffolds were seeded with hMSCs and incubated for up to 12 weeks in order to study

colonization. Determination of DNA and LDH was performed to evaluate the proliferation of cells cultivated on the scaffolds (Figure 3). After four weeks, cells completely covered the CPC strands of the topmost layer, but the cell number of the CPC scaffolds was significantly lower in comparison to the control (BioOss®). After 12 weeks, the cell number of scaffolds A, B and C was significantly lower in comparison to the control, while scaffolds D, E and F showed a colonization comparable to that of BioOss®.

≤ ≤ ≤ ≤ **Figure 3.** Proliferation of MSCs on/in the scaffolds depending on the various porosities after 4, 8 and 12 weeks in comparison to BioOss® (strand-to-strand-distance µm/pore size µm: A: 430/100; B: 560/230; C: 690/360; D: 820/490; E: 950/620; F: 1080/750; \*\* *p* ≤ 0.01, \*\*\*\* *p* ≤ 0.0001, mean ± standard deviation, n = 5). ≤ ≤ ≤ ≤

Live/Dead staining was carried out to assess the viability of the cells. Through the culture period, the density of living cells (stained green) increased. Furthermore, microscopically, a widespread colonization of scaffolds was observed earlier in those with a higher pore size in comparison to those with a smaller pore size. The cells covered the superficial cement strands and also those in subjacent layers. Scaffold D, with a strand-to strand-distance of 820 µm and a pore size of 490 µm, exhibited a colonization similar to that of the control (BioOss®) (Figure 4). In all cases, no dead cells (stained red) were detected.

**Figure 4.** Colonization of cubic scaffolds with MSCs: Scaffold B (**a**), Scaffold D (**b**) and BioOss® (**c**) after 28 days. (Live/Dead-staining).

Microscopic SEM evaluation of colonized scaffolds after 28 days revealed that, similar to the Life/Dead staining, cells completely covered the scaffold strands. For Scaffold D especially, cell clusters bridging the interspaces between strands were observed (Figure 5).

**Figure 5.** SEM imaging of CPC scaffold D non-colonized (**a**) and after 28 days (**b**) in comparison to BioOss® (**c**).

#### *3.3. Scaffold Design for Intraoral Applications*

In addition to the above mentioned scaffolds with strands laying 90◦ in relation to the scaffold's edges, we printed scaffolds with strands laying 45◦ related to the scaffold's edges to enhance the surface area (Figure 6).

**Figure 6.** Three dimensional (3D) plotted scaffold with defined pore size (0.49 mm) and strand distance (0.82 mm): (**a**) strand orientation 90◦ related to edge, (**b**) strand orientation 45◦ related to edge, (**c**) BioOss®; scale bars: 1 mm.

For this purpose, we used a pore size of 0.49 mm and a strand-to-strand-distance of 0.82 mm. Since the chewing process causes the application of forces from different directions, we investigated both strand orientations applying uniaxial strength from above and laterally. Young's modulus (Figure 7b) was estimated from the initial slope of the stress–strain curves (Figure 7a) in the elastic region. Compressive strength (Figure 7c) was evaluated from the stress–strain curves (Figure 7a). Data are presented in Table 2.

The results have shown that the energy absorption of the different scaffold types varies not only depending on strand orientation but also on the direction that the strength is applied from. Scaffolds with a 90◦ strand orientation seem to be more stable compared to 45◦ scaffolds (Figure 7c).

≤ ≤ ≤ ≤ **Figure 7.** Mechanical properties of CPC scaffolds with different strand orientations in comparison with BioOss®. (**a**) Representative compressive stress–strain curves. (**b**) Young's modulus and (**c**) compressive strength determined from the curves (\*\* *p* ≤ 0.01, \*\*\*\* *p* ≤ 0.0001, mean ± standard deviation, n = 5).


**Table 2.** Mechanical properties of scaffolds with different printing directions in comparison to the control.
