**4. Silicon nitride–titanium nitride**

#### **4.1. Background**

**Figure 5.** Morphology of MC3T3 cells seeded on different surfaces and stained with phalloidin–rhodamine and DAPI to visualize, respectively, the cytoskeleton and nucleus (see Methods). (A) Quantification of focal adhesion density measured by normalizing the number of focal adhesions on cell area (see Methods). (B) The rough surfaces significant‐

**Figure 6.** Colorimetric quantification of ALP activity (A) and calcium deposition (B) (see Methods). The rough surfaces

Although surface chemistry is known to play a role in cueing the biological systems [81], the present experimental data showed that roughened surfaces were more efficient in inducing an osteogenic response in vitro independently of the application of the chemical treatment. In other terms, roughness per se seemed to overpower the effect of the chemical treatment which was deemed bioactive on the ground of the Kokubo tests previously performed (i.e., the capacity to induce hydroxyapatite precipitation) [85]. Within the obvious limits of this

significantly increase the level of either ALP activity (A) and calcium deposition (B).

ly increase the density of focal adhesion.

74 Dental Implantology and Biomaterial

Silicon nitride (Si3N4) is a high-strength and tough ceramic used as a viable implant material [93–95]. Since the first clinical trial in 1986 [96], over two decades have passed before the introduction of Si3N4 to the biomedical market of the US and EU. Since 2008, it has been used as a fusion cage for arthrodesis of the cervical and thoracolumbar spine [97], with few adverse reported events [98]. Silicon nitride has been shown to possess favorable cell interaction characteristics [94, 95, 99–104], along with bacteriostatic properties [105, 106]. Also, porous or unpolished Si3N4 osseointegrates with adjacent bone [104, 105, 107–109].

Silicon nitride derives its strength and toughness through its microstructure, which is com‐ posed of asymmetric needle-like interlocking grains surrounded by a thin (<2 nm) refractory grain-boundary glass [110]. Unlike other ceramics, no phase transformation is involved. Thus, similar to alumina, Si3N4 exists as an irreversibly stable phase at room temperature, but an advancing crack must navigate a high energy path through the ceramic, and bridging grains within the crack wake restrict its continued propagation [111–113].

Industrial standards have been adopted for Si3N4 composition, processing, and properties [114, 115]. However, sintered Si3N4 is usually machined by hard grinding with diamond tools and the high hardness of Si3N4 makes the production of complex shapes through conventional mechanical machining difficult and expensive. To address this issue, electrically conductive reinforcements, such as TiN, TiC, TiB2, ZrB2, were added to the Si3N4 matrix, generating composites suitable to be wrought by electrical discharge machining (EDM) [116]. The EDM has been introduced with encouraging results, achieving complex shapes from dense electro‐ conductive bulks with high densification [94]. Accurate semi-finished Si3N4–TiN surfaces may be either used as they are, or further finished through diamond polishing [116]. Some pre‐ liminary data comparing in vitro the osteogenic behavior of two different surface modifica‐ tions of a silicon nitride–titanium nitride (Si3N4–TiN) composite are here presented. The two surfaces were, respectively, the very product of the EDM process (henceforth Si3N4–TiN\_A) and the result of partial polishing with diamond suspensions (henceforth called Si3N4–TiN\_B). For material and methods please refer to Sections 3.2.2 and 3.2.3.

#### **4.2. Results and discussion**

A detail of the two silicon nitride–titanium nitride surfaces is reported in **Figure 7**.

**Figure 7.** Surface electron micrographs of Si3N4–TiN\_A (A) and Si3N4–TiN\_B (B) at 10.000 magnifications.

Si3N4–TiN\_A showed an interesting coalesced structure derived from the melting generated during the manufacturing process, whilst, in Si3N4–TiN\_B the microstructure of silicon nitride– titanium nitride is clearly appreciable along with the remnants of the peaks after polishing. The tridimensional analysis of Si3N4–TiN\_A and Si3N4–TiN\_B is graphically depicted in **Figure 8**, whilst Sa values were, respectively, 2.92 ± 0.07 and 0.88 ± 0.06 μm. Thus, Si3N4–TiN\_A resulted rougher than Si3N4–TiN\_B.

**Figure 8.** Tridimensional graphical representation of Si3N4–TiN\_A (A) and Si3N4–TiN\_B (B).

MC3T3 cells grew well on both samples. Notably, fluorescent images of adherent cells at 24 h (**Figure 9A**) clearly show that Si3N4–TiN\_A induced a more complex morphology with more tapered shape cells than Si3N4–TiN S, as expected for rougher surfaces. Consistently, a higher density of focal adhesions was quantified on the Si3N4–TiN R surface [117] (**Figure 9B**).

**Figure 9.** Morphology of MC3T3 cells seeded on Si3N4–TiN\_A and Si3N4–TiN\_B and stained with phalloidin–rhoda‐ mine and DAPI to visualize, respectively, the cytoskeleton and nucleus (see Methods). MC3T3 cells seeded on Si3N4– TiN\_B display a more complex shape with a lower spreading level than Si3N4–TiN\_A (A). Quantification of focal adhe‐ sion density measured by normalizing the number of focal adhesions on cell area (see Methods) (B). Si3N4–TiN\_B sig‐ nificantly increase the density of focal adhesion.

**Figure 7.** Surface electron micrographs of Si3N4–TiN\_A (A) and Si3N4–TiN\_B (B) at 10.000 magnifications.

**Figure 8.** Tridimensional graphical representation of Si3N4–TiN\_A (A) and Si3N4–TiN\_B (B).

MC3T3 cells grew well on both samples. Notably, fluorescent images of adherent cells at 24 h (**Figure 9A**) clearly show that Si3N4–TiN\_A induced a more complex morphology with more tapered shape cells than Si3N4–TiN S, as expected for rougher surfaces. Consistently, a higher density of focal adhesions was quantified on the Si3N4–TiN R surface [117] (**Figure 9B**).

resulted rougher than Si3N4–TiN\_B.

76 Dental Implantology and Biomaterial

Si3N4–TiN\_A showed an interesting coalesced structure derived from the melting generated during the manufacturing process, whilst, in Si3N4–TiN\_B the microstructure of silicon nitride– titanium nitride is clearly appreciable along with the remnants of the peaks after polishing. The tridimensional analysis of Si3N4–TiN\_A and Si3N4–TiN\_B is graphically depicted in **Figure 8**, whilst Sa values were, respectively, 2.92 ± 0.07 and 0.88 ± 0.06 μm. Thus, Si3N4–TiN\_A

The osteogenic differentiation was evaluated based on the alkaline phosphatase activity as well as the deposition of bone matrix on the specimens. A statistically significant difference between Si3N4–TiN\_A and Si3N4–TiN\_B was determined in favor the former, when ALP activity was determined (**Figure 10A**).

**Figure 10.** Colorimetric quantification of ALP activity (A) and calcium deposition (B) (see Methods). Si3N4–TiN\_B sur‐ face significantly increase the level of either ALP activity (A) and calcium deposition (B).

The rougher surface promoted a greater osteogenic response than the smooth surface in terms of calcium deposition (**Figure 10B**).

The biological responses induced in MC3T3 cells, a widely diffused osteoblast model, were correlated with the surface roughness, even in this case. The effect of roughness on osteoblast adhesion has been mainly attributed to an increased surface-to-volume ratio that may provide more sites for cell attachment [118]. Consistently, the rougher surface tested (Si3N4–TiN\_A) could promote better cell viability, higher density of focal adhesions and more pronounced calcium deposition than the smoother one (Si3N4–TiN\_B). Taken together, these data confirmed the biocompatibility of silicon nitride–titanium nitride composites in accordance with the literature, which has indeed so far explored preferably the pristine Si3N4 material [93, 94, 99, 119]. The possible application of surfaces directly obtained by EDM to Si3N4–TiN is therefore noteworthy. Further research should be oriented at investigating the in vivo effects of such surface finishing, as well as the importance of the texture in the pattern recognition operated by cells.
