*3.3. Fluorescence Microscopy*

Fluorescence microscopy images of the various coated Ti6Al4V surfaces are shown in Figure 4a–f, while the subsequent fluorescence intensities are shown below in Table 5. From the fluorescence intensities of the peptide coating without the polydopamine, it can be observed that the peptide KR12 (Figure 4a) was present on the surface in the smallest quantities, while the lines presented in the image originated from the surface roughness and defects of the unpolished Ti6Al4V surface prior to coating. For the Ti6Al4V surfaces with a pDA linking substrate (Figure 4b–e), the green colour of the carboxyfluorescein (5(6)-FAM)-labelled peptides was generally distributed uniformly across the surface of Ti6Al4V, indicating a uniform peptide coating.

**Figure 4.** *Cont.*

**Figure 4.** Fluorescence microscopy images of the Ti6Al4V surface coated for 24 h with 5(6)-FAM-labelled (**a**) KR12, (**b**) pDA-KR12, (**c**) pDA-KR12/32, (**d**) pDA-KR12-5911, and (**e**) pDA-KR12/32-5911; and (**f**) for Ti6Al4V without coating.



#### *3.4. Peptide Release Studies*

The peptide release profiles were all studied in simulated body fluid (SBF) over a total period of 30 days. The fluorescence-based release profiles of the pDA-attached peptides on the Ti6Al4V surface are shown in Figure 5. Roughly 30% of the peptides were released from the surface in the initial 6 h and 40–50% was released after 30 days. To verify the release profiles based on fluorescence, the studies were repeated with no labelled peptides and analysed by HPLC. The release profiles based on cumulative absorbance measured by HPLC are shown in Figure 6, which indicated that roughly 70% of the cumulative peptides were released in the first 6 h.

**Figure 5.** Release profile of 5(6)-FAM-labelled peptides that were released from the Ti6Al4V-coated surface based on fluorescence measurements; x-axis displayed in logarithmic scale (base = 12).

**Figure 6.** Release profile showing the percentage of the total peptides released in SBF from the pDA-coated Ti6Al4V surface based on HPLC; x-axis displayed in logarithmic scale (base = 12).
