3.1.8. Film Wettability

Water contact angle testing was performed to establish the surface hydrophobicity or hydrophilicity of thin film samples. Figure 9 shows measured contact angles for HA and SiHA thin films. All silicon doped samples exhibited lower contact angles than the pure HA films whether in an as deposited or annealed state. The as deposited HA film had a water contact angle of 67◦, which decreased with increasing silicon content down to an angle of 27◦ for the SiHA3 samples. Following heat treatments at 600 ◦C, the contact angle for all samples decreased when compared to the as deposited samples to values of 54, 41, 31 and 26◦ for the HA, SiHA1, SiHA2 and SiHA3, respectively. After heat treatments at 700 ◦C, an increase in contact angle was seen for all samples when compared to either the as deposited or samples heat treated at 600 ◦C, measuring 69, 56, 42 and 36◦ for the HA to the SiHA3 samples. Figure 9 also shows optical images of water droplets on as deposited HA and SiHA surfaces. It can therefore be seen that the hydrophilicity increases with increasing silicon content.

**Figure 9.** Measured contact angles of water droplets on HA and SiHA thin sputtered films. Mean ± standard error of the mean where n ≥ 6. Also shown are digitally enhanced representative grayscale photographs of water droplets on (**A**) HA (**B**) SiHA1 (**C**) SiHA2 and (**D**) SiHA3 thin sputtered films, showing the effect of silicon doping on the contact angle of water. Photographs taken from as deposited sample set.

### *3.2. In Vitro Cytocompatibility Testing*

### 3.2.1. Elusion Testing – Metabolic Activity, DNA Content and Morphology

Figure 10A1 shows no significant difference (*p* > 0.05) between the metabolic activity of either the control thermanox samples or the samples in HA and SiHA dissolution media. This was confirmed by the Hoest DNA staining assay (Figure 10A2), in which there was no significant difference (p > 0.05) in DNA content of HOBs grown in the dissolution products of any of the coatings.

Cell morphologies for the control (Figure 10B insert) and all samples (Figure 10B–D) appeared to be similar, showing a monolayer over the thermanox surface. Cells were well spread, appearing to cover similar cell areas. Filopodia and lamellapodia were also observed, indicating cell signalling was occurring successfully and did not demonstrate significant cytotoxicity.

**Figure 10.** Combined elusion testing results showing: (**A1**) Metabolic activity and (**A2**) DNA content of pre-seeded HOB cells exposed to dissolution media of HA and SiHA samples for 24 h (Both expressed as mean ± standard error; n = 6); (**B**–**D**) SEM micrographs of HOB cell morphology on thermanox slides after 24 h of culture in media containing the dissolution products of (**B**) HA, (**C**) SiHA1, (**D**) SiHA2 and (**E**) SiHA3 thin films (insert image in B shows cells culture in fresh media for reference).

### 3.2.2. Initial Attachment

Figure 11 shows the adhesion of HOB cells as a percentage of a TCP control. As deposited and heat treated HA thin films showed good attachment after 90 min with values of 51 and 91% of the control, and were found to be significantly different (p < 0.05). As deposited and heat treated SiHA1 and SiHA2 samples showed poor cell attachment which was less than 20% of the control. All values for SiHA1 and SiHA2 samples were significantly lower than the heat treated HA thin film (p < 0.05). The as deposited SiHA3 samples showed good cell attachment compared to the as deposited and annealed SiHA1 and SiHA2 samples, and were not significantly different from the heat treated HA samples (p < 0.05). Following heat treatments at 600 ◦C for 2 h, the SiHA3 sample showed poor cell adhesion; 7% of the attachment seen on the control surface.

**Figure 11.** The 90 min attachment of HOB cells to as deposited and heat treated HA and SiHA films at 600 ◦C. Values are mean ± standard error where n = 6.

### 3.2.3. Proliferation and Differentiation—Cellular Activity, ALP, and Morphology

The AlamarBlue™ assay results of all sample types annealed at 600 ◦C (Figure 12A) demonstrate that the HA surface exhibited the highest cellular metabolic activity at every time point. The lowest activity was recorded on the SiHA1 samples. There appeared to be no increase in metabolic activity until day 14, where cell activity significantly increased. Conversely, metabolic activity increased with increasing silicon content of SiHA samples. The SiHA2 and SiHA3 samples showed higher cellular activity compared to the SiHA1 samples, and their metabolic activity increased gradually over time. However, HA and SiHA1 surfaces annealed at 700 ◦C (Figure 12B) demonstrated similar metabolic activity at all time points, but the differences were not statistically significant (p > 0.05). Overall proliferation of both samples was seen to increase gradually up to the 10 d time point, followed by a slight reduction at day 14.

DNA measurements (Figure 12C,D) confirmed the trends shown by the Alamar Blue™ assay, with HA annealed at 600 ◦C (Figure 12C) exhibiting the highest DNA content, followed by the SiHA3, SiHA2 and then the lowest, exhibited by the SiHA1 samples. HA consistently showed higher DNA values than all other samples. Annealing at 700 ◦C showed no significant difference between the HA and SiHA1 samples at any time point (Figure 12D).

ALP activity was negligible for all 600 ◦C annealed surfaces after seven days of culture (Figure 12E). After a time period of 10 days significant ALP activity was seen for the HA samples at approximately 35% of the TCP control. Negligible values were recorded for all silicon containing coatings. For the 700 ◦C annealed samples (Figure 12F), ALP was not expressed until day 10 for the HA sample, recording a value at 45% of the control with no significant difference at day 14. The SiHA1 samples arguably exhibited ALP production at day seven at approximately 10% of control. This was seen to significantly increase (p < 0.05) at day 10 to a value of 50%, which increased further to approximately 55% of the control at day 14.

The morphology of cells on thermanox slides (Figure 12G), HA (Figure 12H) and SiHA3 (Figure 12I) samples annealed at 600 ◦C at day 14 are shown in Figure 12G–I. SiHA3 was used as a representative for SiHA samples. HOBs on all surfaces appeared to be multi-layered, indicating desirable osteoblast cell growth. Both HA (Figure 12J) and SiHA1 (Figure 12M) surfaces (one day incubation) annealed at 700 ◦C appeared to be more textured than samples treated at 600 ◦C. Cells appeared to react to

this topography in the case of both samples by larger numbers of extending extra cellular processes compared to the 600 ◦C sample. After seven days of cell culture (Figure 12K,N), cells on both samples covered the sample surfaces and multilayering had occurred, and no differences were seen between the two samples. Cracks were also seen in both cell samples, which was due to the dehydration protocol adopted. Fourteen-day samples (Figure 12L,O) exhibited cracks again, but no difference in morphologies was seen. Some directional growth can be seen in both samples.

**Figure 12.** Combined cellular proliferation data showing: AlamarBlue™ assay of HOBs on HA and SiHA surfaces heat treated at (**A**) 600 ◦C and (**B**) 700 ◦C; DNA content of HOBs on HA and SiHA surfaces heat treated at (**C**) 600 ◦C and (**D**) 700 ◦C; ALP activity of HOBs on HA and SiHA surfaces heat treated at (**E**) 600 ◦C and (**F**) 700 ◦C (All graphs are plotted with mean ± standard error of the mean where n = 6); SEM micrographs of cellular morphology showing 14 day incubation on 600 ◦C annealed (**G**) Thermanox (Control), (**H**) HA, and (**I**) SiHA3; Further SEM micrographs of cell morphology on 700 ◦C annealed HA and SiHA1 samples incubated at day (**J**),(**M**) 1, (**K**),(**N**) 7 and (**L)**,(**O**) 14, respectively.
