*3.6. Cell Studies*

The micrographs presented in Figure 8a–l show the attachment of human osteosarcoma cells (HOS) on the coated and uncoated Ti6Al4V surfaces taken at days 5 and 7 of incubation. HOS showed consistent growth on all of the surfaces, except for the Ti/pDA surface decorated with the peptide KR12/32-5911 shown in Figure 8k–l, where cells were observed to be detached on day 7 of culture. On the Ti/pDA KR12/32 sample shown Figure 8g–h, the surface cells showed a flatter morphology compared to the other surfaces. On all of the other surfaces, although minor differences in the densities of cells can be seen, there is little change between the growth of cells and there is clear evidence of an increase in the number of cells from day 5 to day 7 of culture.

**Figure 8.** *Cont.*

**Figure 8.** *Cont.*

**Figure 8.** *Cont.*

**Figure 8.** SEM micrographs showing the growth of the seeded human osteosarcoma cells (HOS) on day 5 and 7 for (**a**,**b**) Ti6Al4V, (**c**,**d**) Ti6Al4V/pDA, (**e**,**f**) Ti6Al4V/pDA-KR12, (**g**,**h**) Ti6Al4V/pDA-KR12/32, (**i**,**j**) Ti6Al4V/pDA-KR12/5911, and (**k**,**l**) Ti6Al4V/pDA-KR12/32-5911 at 100× and 500× magnification, respectively.

#### **4. Discussion**

#### *4.1. Peptide MIC Characterisation*

Infections caused by the investigated bacteria are some of the most commonly occurring incidences during orthopaedic joint replacement surgeries [21]. Factors such as prolonged antibiotic treatment or administration below the MIC value have been shown to increase bacterial resistance towards antibiotics [22]. Thus, it is important to design antimicrobial peptides with low MIC values that are effective against orthopaedic bacteria. When comparing the MIC values of the human cathelicidin LL-37 with the designed peptides, as summarised in Table 3, the KR12 peptide showed promising MIC values with a higher antimicrobial tendency towards Gram-negative bacteria.

LL-37 was shown to have antimicrobial activity against fungi [23], bacteria [24], and viruses [23,25]. Additionally, it has immunostimulatory and immunomodulatory functions against infections [26] and stimulates angiogenesis [27] during the wound healing process [28]. LL-37 immobilised on polymer surfaces has also been proven to retain its antimicrobial activity [29]. Therefore, the designed KR12 analogous peptide may share similar properties with LL-37, but this remains to be investigated.

#### *4.2. Polydopamine Coating of Ti6Al4V*

Dissolving dopamine in a solution of alkaline pH changes the colour of the solution from translucent white to brown, then eventually to black, as shown in Figure 2. This colour change can be explained by the structure of dopamine. Dopamine contains phenol groups, and when polymerised it creates polyphenols structures that contain many aromatic rings joined together [30]. Multiple aromatic rings in close proximity in one molecule are known as charge transfer complexes, whereby the charge is delocalised between the rings [31]. These complexes promote absorption of visible light, causing electron transition from a lower electron state to higher electron state, as a result of which a dark colour is observed [32]. Immersing Ti6Al4V plates in a solution of dopamine resulted in the

formation of a thin film on the surface of the metallic plate, which agrees with Messersmith et al. [33], who reported that pDA was able to adhere to virtually any surface and that its polymerisation in slightly basic solution resulted in a film formation on the surface. The SEM morphology and topology studies showed that the surface of the pDA coating was built from small round particles of ≈100 nm diameter. It was proposed by Jiang et al. that the pDA first polymerises in nanoaggregates, which over time attach themselves on the surface, creating a uniform coating [34,35].

The attachment of the nanoaggregates to the surface could potentially have been driven by the sedimentation of the particles towards the bottom of the container where the substrate to be coated was located. The coating then accumulated with more beads and increased the thickness of the coating, which varied depending on the size of the beads accumulated. This could explain the ≈10 nm coating thickness after the initial 24 h, and the increases of the thickness to 32 nm after 48 h and to 57 nm after 52 h of pDA polymerisation. Similar film thickness growth was reported by Jiang et al. [33,35]. In this study, a thickness plateau was reached after 72 h of polymerisation. This pattern of growth was also previously reported by Bensmann et al. [36], where the pDA film reached 62.8 nm in thickness after 72 h and did not increase in thickness afterwards. Formation of the plateau can be explained by the lack of oxygen after the formation of the first pDA layer. Since oxidation is a common reaction of polyphenols [37], it can be assumed that the polymerisation required oxygen to continue and the presence of the film limited the accessibility to oxygen. In addition, as oxidation on the surface will continue as oxygen is available on the surface, polymerisation will eventually stop when dopamine is consumed.
