## 4 Surface-Immobilized Peptides on Medical Implants
### Antimicrobial Peptides Released from Titanium  
Coatings aimed to release antimicrobial compounds are often considered preferable for orthopedic implants due to release of the antimicrobial agent at the peri-implant area. However, a suitable drug-loading system has to be developed that can effectively deliver a sustained amount of antimicrobials on the surface of implants, without impairing peri-implant bone growth. That means a depot of the antimicrobial molecule has to be stored on the surface of the implant and released in a controlled manner over a preferentially prolonged period. Among several reported systems that store antimicrobial agents on the implant, calcium phosphate (CaP) coatings, titanium dioxide (TiO\({}_{2}\)) nanotubes, layer-by-layer assembly and hydrogels are discussed below (Figure 4). CaP is a porous ceramic which can be coated on titanium implants, as it improves the integration of the implant to the bone and enhances bone growth [151; 152]. Thus, the combination of CaP and an antimicrobial peptide has potential to promote osteointegration of the implant and provide antimicrobial protection at the same time. AMPs can be successfully loaded to the CaP-coated titanium substrates through a simple soaking method, that has been reported for loading Tet213 (Table 4) [131]. This implant had broad-spectrum antimicrobial activity in vitro against both Gram-positive and Gram-negative pathogens, and retained its potency after repeated uses, while remaining biocompatible to osteoblast-like cells. Another technique for the delivery of AMPs is the formation of TiO\({}_{2}\) nanotubes on the surface of titanium implants. These nanotubes can significantly accelerate osteoblast growth and adhesion in vitro [153] and improve bone formation and bonding strength in vivo [154; 155; 156]. Even though the design of TiO\({}_{2}\) nanotubes was reported in other studies [157; 158; 159; 160; 161], it was [PERSON] and colleagues, who first tested loading self-organized, vertically oriented TiO\({}_{2}\) nanotubes as local delivery system of AMPs, using HHC-36 [162]. As a result, peptide loaded TiO\({}_{2}\) nanotube surface effectively killed _S. aureus_ in vitro, and the release of the AMP from the nanotubes was verified via liquid chromatography coupled mass spectrometry (LC-MS) analysis [134]. Furthermore, GL13K peptide loaded in TiO\({}_{2}\) nanotubes, on titanium surfaces, by a simple soaking technique demonstrated a sustained and slow drug release profile in vitro and eradicated the growth of _Fusarium nucleatum_ and _P. gingivalis_ within 5 days of exposure [133]. Due to the fact that both CaP coating and TiO\({}_{2}\) nanotubes adsorb peptide, the release rate in both systems is fairly rapid, with previous studies suggesting a maximum cumulative release amount only within a few hours [131]. Therefore, these AMP delivery methods are unsuitable for long-term peri-implant infection, where sustained release is required for a longer period. Hence, in order to prolong the drug release, layer-by-layer (LBL) antimicrobial coatings on titanium implants have been studied, using both CaP and vertically aligned TiO\({}_{2}\) nanotubes, both loaded with HHC-36 (Table 4) [132]. These films were topped with a thin palmitoyl-oleoyl phosphatidylcholine-choline phospholipid (POPC) film to control the release of the peptide [163; 164]. POPC is found naturally  
Figure 4: AMP release coating strategies on titanium surfaces. CaP: calcium phosphate coating; NT: TiO\({}_{2}\) nanotubes. in eukaryotic cell membranes, has antimicrobial properties, and also is suitable for bone cell attachment [165] and osteointegration in vivo [166]. Other LBL strategies, involve electrostatic interactions between charged polyanions and polycations, in a number of layers, producing a film. Each layer enters antimicrobial agent, and through degradation of each layer, continuous release is achieved over time.