Influence of a Composite Polylysine-Polydopamine-Quaternary Ammonium Salt Coating on Titanium on Its Ostogenic and Antibacterial Performance

Thin oxide layers form easily on the surfaces of titanium (Ti) components, with thicknesses of <100 nm. These layers have excellent corrosion resistance and good biocompatibility. Ti is susceptible to bacterial development on its surface when used as an implant material, which reduces the biocompatibility between the implant and the bone tissue, resulting in reduced osseointegration. In the present study, Ti specimens were surface-negatively ionized using a hot alkali activation method, after which polylysine and polydopamine layers were deposited on them using a layer-by-layer self-assembly method, then a quaternary ammonium salt (QAS) (EPTAC, DEQAS, MPA-N+) was grafted onto the surface of the coating. In all, 17 such composite coatings were prepared. Against Escherichia coli and Staphylococcus aureus, the bacteriostatic rates of the coated specimens were 97.6 ± 2.0% and 98.4 ± 1.0%, respectively. Thus, this composite coating has the potential to increase the osseointegration and antibacterial performance of implantable Ti devices.


Introduction
When Ti is used as an implant material, the inert oxide coating on its surface prevents it from having an "active repair function," thus, rendering the surface vulnerable to bacterial growth. This decreases the biocompatibility between the implant material and the bone tissue, resulting in a decreased rate of osseointegration [1][2][3][4]. Ti has high corrosion resistance in most alkali or salt solutions, although its surface can corrode in an acidic environment. Additionally, Ti has good wettability, and its surface easily adsorbs pollutants [5,6]. Surface modification technology is widely used to address the above problems of rapid breeding of bacteria and surface pollution [7,8]. There are several methods currently available for Ti surface modification, such as self-assembly or layer-by-layer (LBL) technology, anodic oxidation technology, pulse laser method, magnetron sputtering, ion beam assisted deposition, sol-gel, thermal spraying, dip coating, and plasma spraying [9]. LBL technology may be used to produce nanoscale multifunctional composite coatings that optimize the surface properties of Ti [10,11]. A single-layer film or a multi-layer film could be deposited using this technology, which fixes required functional molecules as proteins, peptides, enzymes, drugs, and organic polymers, on the surface [12].
Polylysine (PLL) has strong biocompatibility, and its interaction with cells could be strengthened by using electrostatic principles to promote cell adhesion [13][14][15]. Additionally, some cells can digest and absorb PPL because of its strong penetration into the

Wettability of Surfaces
An optical contact angle measuring instrument was used to measure the Water Contact Angle (WCA) of the Ti surface after various stages in the deposition of the composite coating ( Figure 2). The surface of the as-received and uncoated Ti is hydrophobic, with a WCA of 100 ± 2.0 • . The surface of Ti activated by hot alkali has super hydrophilic properties porous Ti (pTi), with a WCA of 7 ± 1.2 • . This is because the microstructure of the Ti surface is changed (porous) by hot alkali treatment by introducing a large number of hydroxyl groups. After the multilayer PLL and PDA were deposited, the WCA of the surface increased markedly, with the surface WCA of pTi-(PLL-PDA) 1 and pTi-(PLL-PDA) 2 being 22 ± 1.1 • and 27 ± 1.2 • , respectively, as a result of the joint action of PLL and PDA. The WCA of the surface of the coated Ti increased slightly as the number of layers in the composite coating increased, with pTi-(PLL-PDA) 10 and pTi-(PLL-PDA) 15

Wettability of Surfaces
An optical contact angle measuring instrument was used to measure the Water Contact Angle (WCA) of the Ti surface after various stages in the deposition of the composite coating ( Figure 2). The surface of the as-received and uncoated Ti is hydrophobic, with a WCA of 100 ± 2.0°. The surface of Ti activated by hot alkali has super hydrophilic properties porous Ti (pTi), with a WCA of 7 ± 1.2°. This is because the microstructure of the Ti surface is changed (porous) by hot alkali treatment by introducing a large number of hydroxyl groups. After the multilayer PLL and PDA were deposited, the WCA of the surface increased markedly, with the surface WCA of pTi-(PLL-PDA)1 and pTi-(PLL-PDA)2 being 22 ± 1.1° and 27 ± 1.2°, respectively, as a result of the joint action of PLL and PDA. The WCA of the surface of the coated Ti increased slightly as the number of layers in the composite coating increased, with pTi-(PLL-PDA)10 and pTi-(PLL-PDA)15 being 33 ± 1.1° and 34 ± 1.0°, respectively, indicating that the coating was stable. The above results show that PLL and PDA fully covered the porous Ti surface and formed a hydrophilic surface.

Surface Roughness
As shown in Figure 3, the uncovered Ti surface roughness (Ra) is 2.31 nm, and Ra is 14.52 nm when the number of assembled PLL and PDA layers is n = 1. The results show that as n increases, the peak on the material surface gradually becomes smooth, and the Ra of the Ti surface exhibits an increasing trend ( Figure 4). Ra is 67.87 nm when n = 15 assembled layers because both PLL and PDA are long macromolecular chain structures; they coat and wind on the peaks on the surface of pTi after being assembled on the surface of pTi using LBL technology, and the pores are gradually filled. The above results show that PLL and PDA were successfully assembled on the surface of pTi, and Ra increases

Surface Roughness
As shown in Figure 3, the uncovered Ti surface roughness (Ra) is 2.31 nm, and Ra is 14.52 nm when the number of assembled PLL and PDA layers is n = 1. The results show that as n increases, the peak on the material surface gradually becomes smooth, and the Ra of the Ti surface exhibits an increasing trend ( Figure 4). Ra is 67.87 nm when n = 15 assembled layers because both PLL and PDA are long macromolecular chain structures; they coat and wind on the peaks on the surface of pTi after being assembled on the surface of pTi using LBL technology, and the pores are gradually filled. The above results show that PLL and PDA were successfully assembled on the surface of pTi, and Ra increases with the increase in n. The rough biomolecular coating on the nano level can stimulate osteogenesis around the implant, which increases bone anchoring and biomechanical stability, thereby reducing the bone-bonding period.

Surface Roughness
As shown in Figure 3, the uncovered Ti surface roughness (Ra) is 2.31 nm, and Ra is 14.52 nm when the number of assembled PLL and PDA layers is n = 1. The results show that as n increases, the peak on the material surface gradually becomes smooth, and the Ra of the Ti surface exhibits an increasing trend ( Figure 4). Ra is 67.87 nm when n = 15 assembled layers because both PLL and PDA are long macromolecular chain structures; they coat and wind on the peaks on the surface of pTi after being assembled on the surface of pTi using LBL technology, and the pores are gradually filled. The above results show that PLL and PDA were successfully assembled on the surface of pTi, and Ra increases with the increase in n. The rough biomolecular coating on the nano level can stimulate osteogenesis around the implant, which increases bone anchoring and biomechanical stability, thereby reducing the bone-bonding period.    Figure 5 shows the XPS test results for the Ti surface covered with various composite coatings. The Ti substrate is gradually covered by the self-assembly of PLL and PDA, the peak of Ti 2p (458.5 eV) is reduced to zero, and the peaks of N 1s (400.5 eV) and O 1s (532.5 eV) are gradually enhanced [36][37][38], which confirms that PLL and PDA were successfully   Figure 5 shows the XPS test results for the Ti surface covered with various composite coatings. The Ti substrate is gradually covered by the self-assembly of PLL and PDA, the peak of Ti 2p (458.5 eV) is reduced to zero, and the peaks of N 1s (400.5 eV) and O 1s (532.5 eV) are gradually enhanced [36][37][38], which confirms that PLL and PDA were successfully modified on the Ti surface.  Figure 5 shows the XPS test results for the Ti surface covered with various composite coatings. The Ti substrate is gradually covered by the self-assembly of PLL and PDA, the peak of Ti 2p (458.5 eV) is reduced to zero, and the peaks of N 1s (400.5 eV) and O 1s (532.5 eV) are gradually enhanced [36][37][38], which confirms that PLL and PDA were successfully modified on the Ti surface.

Molar Grafting Rate
As shown in Table 1 (according to the mathematical formula in 3.8), (2,3-Epoxypropyl) trimethylammonium chloride (EPTAC) has a higher grafting rate than the other two quaternary ammonium salts because its molecular chain is shorter, allowing more molecules to be grafted on the surface of the self-assembled layer. Additionally, the molar grafting rate increases gradually after grafting the quaternary ammonium salt with an increase in the number of self-assembled layers. It was shown that the catechol group in PDA is easily oxidized to a quinone structure under alkaline conditions, and the amino group can be grafted with an epoxy group or carboxyl group.

Cell Compatibility
Cytotoxicity was determined in terms of optical density (OD). For a given specimen, the higher the OD the more cells have proliferated and differentiated on it, and the higher the biological activity of the cells. Figure 6 shows that the cytotoxicity performance is: pTi-(PLL-PDA) 15 -EPTAC > pTi-(PLL-PDA) 15 -DEQAS > pTi-(PLL-PDA) 15 -(MPA-N + )/DEQAS > pTi-(PLL-PDA) 15 -(MPA-N + ). When the cells were cultured for 24 h, compared with the blank specimens, the existence of quaternary ammonium salt as a functionally modified surface had no effect on cell viability and growth. Among the coated specimens, the ones on which MPA-N + grafted had been onto self-assembled multilayers showed the highest cell vitality, exhibiting excellent cell adhesion and proliferation. This shows that the polylysine-polydopamine-quaternary ammonium salt composite coating promotes cell proliferation. The above results show that the composite coating is non-toxic to cells and favorable to the biocompatibility of cells.

Cell Compatibility
Cytotoxicity was determined in terms of optical density (OD). For a given specimen, the higher the OD the more cells have proliferated and differentiated on it, and the higher the biological activity of the cells. Figure 6 shows that the cytotoxicity performance is: pTi-(PLL-PDA)15-EPTAC > pTi-(PLL-PDA)15-DEQAS > pTi-(PLL-PDA)15-(MPA-N + )/DEQAS > pTi-(PLL-PDA)15-(MPA-N + ). When the cells were cultured for 24 h, compared with the blank specimens, the existence of quaternary ammonium salt as a functionally modified surface had no effect on cell viability and growth. Among the coated specimens, the ones on which MPA-N + grafted had been onto self-assembled multilayers showed the highest cell vitality, exhibiting excellent cell adhesion and proliferation. This shows that the polylysine-polydopamine-quaternary ammonium salt composite coating promotes cell proliferation. The above results show that the composite coating is non-toxic to cells and favorable to the biocompatibility of cells.

Antibacterial Performance
Antibacterial performance was determined using the plate colony counting method. Plain or uncoated Ti (pTi) attracted a large number of bacteria after 24 h of culture in E. coli and S. aureus ( Figure 7) and, as such, had very poor antibacterial properties. In contrast, coated Ti specimens in which the coating consisted of a grafted quaternary ammonium salt had excellent antibacterial properties ( Figure 7). The index of antibacterial performance is expressed as the kill ratio (defined as % of bacteria that remained on the specimen surface 24 h after cultivation with the test bacterium) ( Figure 8). The kill ratios against E. coli and S. aureus after coating that included grafting with DEQAS were 70.8 ± 3.0% and 92.9 ± 2.0%, respectively, whereas the kill ratios of specimens on which the coating included grafted MPA-N + to E. coli and S. aureus were 93.1 ± 2.0% and 94.7 ± 2.0%, respectively. The kill ratios of specimens coated with DEQAS and MPA-N + to E. coli and S. aureus were 97.6 ± 2.0% and 98.4 ± 1.0%, respectively. The kill ratio of specimens whose coating comprised a mixture of grafted DEQAS and MPA-N + was higher than that of those whose coating comprised a single grafted salt of quaternary ammonium. This is because the raw material for MPA-N + synthesis had anti-inflammatory properties, and the antibacterial property of two quaternary ammonium salts was higher than that of one quaternary ammonium salt.
Based on the composite coating as an implant material, we only used a single HU-VECs cell to detect biocompatibility during the study and did not select multiple cells. The selected antibacterial species are not perfect, and the flora affecting peri-implantitis should be mainly anaerobic bacteria. For example, corresponding antibacterial experiments should be performed on Porphyromonas gingivalis, Streptococcus, etc. Although there are few biological experiments tested, the above data show that the polylysine-polydopaminequaternary ammonium salt composite coating has good biocompatibility and excellent antibacterial properties.
respectively. The kill ratios of specimens coated with DEQAS and MPA-N + to E. coli and S. aureus were 97.6 ± 2.0% and 98.4 ± 1.0%, respectively. The kill ratio of specimens whose coating comprised a mixture of grafted DEQAS and MPA-N + was higher than that of those whose coating comprised a single grafted salt of quaternary ammonium. This is because the raw material for MPA-N + synthesis had anti-inflammatory properties, and the antibacterial property of two quaternary ammonium salts was higher than that of one quaternary ammonium salt.  Based on the composite coating as an implant material, we only used a single HU-VECs cell to detect biocompatibility during the study and did not select multiple cells. The selected antibacterial species are not perfect, and the flora affecting peri-implantitis should be mainly anaerobic bacteria. For example, corresponding antibacterial experiments should be performed on Porphyromonas gingivalis, Streptococcus, etc. Although there are few biological experiments tested, the above data show that the polylysine-polydopamine-quaternary ammonium salt composite coating has good biocompatibility and excellent antibacterial properties.

Preparation of Collagen Peptide Solution
Synthesis of DEQAS [39]: The 250.0 mL three-port flask was taken, and distilled water (22.0 mL), K 2 SO 4 (0.2 g), methanol (17.0 mL), and epichlorohydrin (EC, 9.5 g) were added to it in turn. Then the three-port flask was heated to 50 ± 1 • C and stirred at a constant temperature for 0.5 h. During this time, tetramethylethylenediamine (TMEDM) was added to the flask at a rate of 12 drops/min. The reaction was stopped after stirring for 1.5 h. The solution was poured into a 250 mL round-bottom flask and distilled under reduced pressure to obtain a yellowish liquid (DEQAS) (Figure 9).  Synthesis of MPA-N + : The rosin acid-derived quaternary ammonium maleic acid cation (MPA-N + ) was prepared using a method detailed in the literature [40], and with the reaction route shown in Figure 10. The Abietic acid (100.0 g, 0.28 mol) was heated to 180 °C and refluxed for 3 h under a nitrogen atmosphere, and the heated rosin acid was cooled to 120 °C, after which maleic anhydride (27.5 g, 0.28 mol) and acetic acid (400.0 mL) were added to the above reaction system. The reaction was refluxed at 120 °C for 12 h. Then the reaction was cooled to room temperature and left for another 2 h. The crude maleic pine acid was recrystallized twice in acetic acid to obtain pure maleic pine acid (MPA, 91.0 g, 97% purity, 79% yield).
MPA-N (1.0 g, 0.0021 mol) and bromoethane (3.1 mL, 0.043 mol) were dissolved in redistilled tetrahydrofuran (THF, 30.0 mL) and reacted at 40 °C for 48 h. Then the product MPA-N + (0.94 g, purity: 90%, yield: 70%) was crystallized, filtered, and dried.   Synthesis of MPA-N + : The rosin acid-derived quaternary ammonium maleic acid cation (MPA-N + ) was prepared using a method detailed in the literature [40], and with the reaction route shown in Figure 10. The Abietic acid (100.0 g, 0.28 mol) was heated to 180 • C and refluxed for 3 h under a nitrogen atmosphere, and the heated rosin acid was cooled to 120 • C, after which maleic anhydride (27.5 g, 0.28 mol) and acetic acid (400.0 mL) were added to the above reaction system. The reaction was refluxed at 120 • C for 12 h. Then the reaction was cooled to room temperature and left for another 2 h. The crude maleic pine acid was recrystallized twice in acetic acid to obtain pure maleic pine acid (MPA, 91.0 g, 97% purity, 79% yield).
Molecules 2023, 28, x FOR PEER REVIEW 9 of 18 added to the flask at a rate of 12 drops/min. The reaction was stopped after stirring for 1.5 h. The solution was poured into a 250 mL round-bottom flask and distilled under reduced pressure to obtain a yellowish liquid (DEQAS) (Figure 9). Synthesis of MPA-N + : The rosin acid-derived quaternary ammonium maleic acid cation (MPA-N + ) was prepared using a method detailed in the literature [40], and with the reaction route shown in Figure 10. The Abietic acid (100.0 g, 0.28 mol) was heated to 180 °C and refluxed for 3 h under a nitrogen atmosphere, and the heated rosin acid was cooled to 120 °C, after which maleic anhydride (27.5 g, 0.28 mol) and acetic acid (400.0 mL) were added to the above reaction system. The reaction was refluxed at 120 °C for 12 h. Then the reaction was cooled to room temperature and left for another 2 h. The crude maleic pine acid was recrystallized twice in acetic acid to obtain pure maleic pine acid (MPA, 91.0 g, 97% purity, 79% yield).

Preparation of Polylysine-Polydopamine-Quaternary Ammonium Salt Composite Coating
Preparation of polylysine-polydopamine coating: High-purity Ti sheets were polished, cleaned, and dried in a constant temperature drying oven at 60 °C for 12 h, and the sheets were designated uncoated or pure Ti (pTi). The Ti sheets were then put into 5 mol/L NaOH solution, activated at 60 °C for 5 h, washed with distilled water to neutral, dried with high purity nitrogen, and dried in a constant temperature drying oven at 60 °C for 12 h. Next, we accurately weighed 250 mg of PLL in a 25 mL small beaker, added an appropriate amount of distilled water to dissolve it, stirred it with a glass rod to accelerate

Preparation of Polylysine-Polydopamine-Quaternary Ammonium Salt Composite Coating
Preparation of polylysine-polydopamine coating: High-purity Ti sheets were polished, cleaned, and dried in a constant temperature drying oven at 60 • C for 12 h, and the sheets were designated uncoated or pure Ti (pTi). The Ti sheets were then put into 5 mol/L NaOH solution, activated at 60 • C for 5 h, washed with distilled water to neutral, dried with high purity nitrogen, and dried in a constant temperature drying oven at 60 • C for 12 h. Next, we accurately weighed 250 mg of PLL in a 25 mL small beaker, added an appropriate amount of distilled water to dissolve it, stirred it with a glass rod to accelerate the dissolution, and then transferred it to a 50 mL volumetric flask. The Tris-HCl buffer with a pH of 8.5 ± 0.1 and a concentration of 0.01 mol/L was prepared, and then DA was added to prepare a 2 mg/mL DA solution. The pTi sheet was immersed in a PLL solution with a concentration of 5 mg/mL after undergoing a reaction over a period of 24 h in a dark environment to allow PLL to form a uniform coating. Then, the sheet was immersed in a PDA solution with a concentration of 2 mg/mL after undergoing a reaction over a period of 24 h in a dark environment to allow PDA to form a uniform coating. After assembly, the pTi sheets were washed five times with distilled water, dried with high-purity nitrogen, and stored in nitrogen. The above process was repeated; the last layer was PDA, and the sheet was designated pTi-(PLL-PDA) n .
Preparation of polylysine-polydopamine-EPTAC composite coating: Firstly, a Na 2 CO 3 /NaHCO 3 buffer solution with a pH of 9.6 was prepared, and 5 mL of buffer solution was added to the reaction bottle. Ten drops of EPTAC were added to a 1 mL syringe, and the reaction bottle was ultrasonically treated for 10 min to make EPTAC completely dissolve in the buffer solution. The sample pTi-(PLL-PDA) n was placed in a reaction bottle at a constant temperature of 50 • C for 12 h and then uniformly pulled up and down in distilled water 10 times to remove weakly bound or unbound quaternary ammonium salts. The samples were dried with high-purity nitrogen and stored in nitrogen. The obtained coating was labeled as pTi-(PLL-PDA) n -EPTAC.
Preparation of polylysine-polydopamine-DEQAS composite coating: Na 2 CO 3 /NaHCO 3 buffer solution (pH = 9.6) was prepared, 5 mL buffer solution were poured into the reaction bottle, 10 drops of DEQAS were added to a 1 mL syringe, and the reaction bottle was ultrasonically treated for 10 min to make DEQAS completely dissolve in the buffer solution. , the reaction bottle was ultrasonically treated for 10 min so that MPA-N + could be completely dissolved in the buffer (the concentration of MPA-N + was 0.0144 mol/L (8.4 mg/mL)); then, the prepared self-assembled composite coating was placed in the above reaction bottle, and the reaction was carried out at 50 • C for 12 h. After that, it was pulled up and down at a constant speed in distilled water 10 times to remove the weakly bound or unbound quaternary ammonium salt, dried with high-purity nitrogen, and stored in nitrogen. The obtained coating was labeled as pTi-(PLL-PDA) n -MPA-N + .
Preparation of polylysine-polydopamine-DEQAS/MPA-N + composite coating: The pH = 9.6 Na 2 CO 3 /NaHCO 3 buffer solution was prepared, and 5 mL buffer solution and DE-QAS (21.3 mg) were added to the reaction bottle. The above reaction bottle was placed in an ultrasonic cleaner for 10 min so that DEQAS could be completely dissolved in the buffer solution (the concentration of DEQAS was 0.0142 mol/L). Then, the prepared self-assembled composite coating was placed in the above reaction bottle, and the reaction was carried out at a constant temperature of 50 • C for 12 h. After that, it was uniformly pulled up and down 10 times in distilled water to remove the weakly bound or unbound quaternary ammonium salt. The coating was dried with high-purity nitrogen and stored in nitrogen. The obtained coating was labeled as pTi-(PLL-PDA) n -DEQAS. Then another reaction bottle was taken, and 5 mL buffer solution was added, EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxythiosucc-inimide) and MPA-N + (20.30 mg) (n (MPA-N + ):n (EDC) = 1:427.35); the n (MPA-N + ):n (NHS) = 1:854.70), the above reaction bottle was placed in an ultrasonic cleaner for 10 min, so that MPA-N + could be completely dissolved in the buffer solution (the concentration of MPA-N + was 0.00173 mol/L); then, the prepared self-assembled coating pTi-(PLL-PDA) n -DEQAS was placed in the above reaction flask, and the reaction was carried out at a constant temperature of 50 • C for 12 h. The coating was uniformly pulled up and down 10 times in distilled water, and the weakly bound or unbound quaternary ammonium salt was removed, dried with high-purity nitrogen, and stored in nitrogen. The obtained coating was labeled as pTi-(PLL-PDA) n -DEQAS/(MPA-N + ).

Determination of UV-Visible Spectra
Eight 30 mL sample bottles were used to configure an equal volume and equal concentration of DA solution (concentration of 2 mg/mL), which were irradiated under an ultraviolet lamp for 0 min, 15 min, 30 min, 60 min, 120 min, 180 min, 240 min, and 300 min, respectively. Then, 3 mL were taken out with a pipette and put into a cuvette, and the ultraviolet test was carried out by using a UV spectrophotometer (UV-1800PC).
Visible UV Characterization of DA Solution: PDA may be applied to any substrate, and when exposed to ultraviolet radiation, the PDA layer can produce free radicals, thus initiating the polymerization process. The substrate was immersed in DA solution (2 mg/mL, 10 mM Tris HCl, pH = 8.5), and PDA deposition was triggered by ultraviolet irradiation (36 W lamp), allowing spontaneous deposition of PDA film, which was very important for a well-controlled deposition process. This process was different from the slow process of early dynamics, and PDA was triggered to produce free radicals for the polymerization of various monomers under further sunlight irradiation. Figure 12 shows the color change of the DA solution at different reaction times under ultraviolet and dark conditions. After 2 h, the UV-irradiated solution became darker, but the non-irradiated solution showed a gradual color change. Figure 13 shows the absorption spectrum of DA solution under ultraviolet irradiation, indicating that the absorption peak increased around 410 nm and increased progressively with the polymerization time of PDA.

Determination of UV-Visible Spectra
Eight 30 mL sample bottles were used to configure an equal volume and equal concentration of DA solution (concentration of 2 mg/mL), which were irradiated under an ultraviolet lamp for 0 min, 15 min, 30 min, 60 min, 120 min, 180 min, 240 min, and 300 min, respectively. Then, 3 mL were taken out with a pipette and put into a cuvette, and the ultraviolet test was carried out by using a UV spectrophotometer (UV-1800PC).
Visible UV Characterization of DA Solution: PDA may be applied to any substrate, and when exposed to ultraviolet radiation, the PDA layer can produce free radicals, thus initiating the polymerization process. The substrate was immersed in DA solution (2 mg/mL, 10 mM Tris HCl, pH = 8.5), and PDA deposition was triggered by ultraviolet irradiation (36 W lamp), allowing spontaneous deposition of PDA film, which was very important for a well-controlled deposition process. This process was different from the slow process of early dynamics, and PDA was triggered to produce free radicals for the polymerization of various monomers under further sunlight irradiation. Figure 12 shows the color change of the DA solution at different reaction times under ultraviolet and dark conditions. After 2 h, the UV-irradiated solution became darker, but the non-irradiated solution showed a gradual color change. Figure 13 shows the absorption spectrum of DA solution under ultraviolet irradiation, indicating that the absorption peak increased around 410 nm and increased progressively with the polymerization time of PDA.  In this paper, different coatings were tested differently, as shown in Table 2. The specific experimental steps are as follows :  In this paper, different coatings were tested differently, as shown in Table 2. The specific experimental steps are as follows:

Determination of WCA
The water contact angle (WCA (θ)) value can be used to directly determine the hydrophilicity of the surface. When θ is less than 90 • , the surface of the material can be wetted by liquid (Figure 14a), which is hydrophilic. The smaller the θ angle, the better the wettability of the surface. When θ is greater than 90 • , the surface of the material cannot be wetted by liquid (Figure 14b), which is hydrophobic. The larger the θ angle, the higher the hydrophobicity of the surface [41][42][43][44][45].

Determination of WCA
The water contact angle (WCA (θ)) value can be used to directly determine the hydrophilicity of the surface. When θ is less than 90°, the surface of the material can be wetted by liquid (Figure 14a), which is hydrophilic. The smaller the θ angle, the better the wettability of the surface. When θ is greater than 90°, the surface of the material cannot be wetted by liquid (Figure 14b), which is hydrophobic. The larger the θ angle, the higher the hydrophobicity of the surface [41][42][43][44][45]. At room temperature, WCAs of the above prepared Ti specimens were measured using an optical contact angle measuring instrument (DSA-100, Kruss, Germany). The distilled water of the automatic distribution controller was dripped onto the sample to be tested (~5 µL), and five different positions were dripped on each sample. After 20 s (the contact angle decreases with time, and the optimum measurement time is 20 s), the Laplace-Young fitting algorithm was used to determine the mean value of WCA, click on the photo, and observe and record the WCA of the image.

AFM Characterization
The surface morphology of the prepared samples was characterized by AFM (Multi-mode8, Bruker, Germany). The Ra of the coating surface was measured by AFM, and the specimens were characterized using the Peak Force mode. The scanning range of AFM was set to 1 µm, and the scanning speed was set to 0.977 Hz. Five different regions were selected for a sample to preserve the data with uniform distribution of surface morphology changes. A software package (NanoScope Analysis) was used to process the acquired data.

X-ray Photoelectron Spectroscopy (XPS)
The prepared specimens were examined using X-ray photoelectron spectroscopy (ESCALABXi+, Thermo, Waltham, MA,, USA, Microfocusing monochromatic (Al Kα) Xray source (the best sensitivity of monochromatic light source: 1600 kcps (Al Kα 0.60 eV Ag 3d5/2 peak)) at an angle of incidence of 30° (measured from the surface) and an emission angle normal to the surface. The power used was 150 W, and the maximum resolution depth was 10 nm. Survey spectra (Binding Energy (BE) in the range of 0-5000 eV) were used for element identification and quantification. The middle position of the specimen was scanned using a high magnification mode. The specimens pTi, pTi-(PLL-PDA)1, pTi-(PLL-PDA)5, pTi-(PLL-PDA)10, and pTi-(PLL-PDA)15 were analyzed by composition (0-5000 eV) and high-resolution energy spectra (N 1s, O 1s). All reported spectra are averages of five scans taken at a resolution of 0.1 eV and referenced to the C 1s peak of hydrocarbons at 284.8 eV. Data acquisition and processing were performed using Thermo Advantage software. The XPS spectra were fitted with Voigt profiles obtained by convolving Lorentzian and Gaussian functions. The analyzer transmission function, Scofield sensitivity At room temperature, WCAs of the above prepared Ti specimens were measured using an optical contact angle measuring instrument (DSA-100, Kruss, Germany). The distilled water of the automatic distribution controller was dripped onto the sample to be tested (~5 µL), and five different positions were dripped on each sample. After 20 s (the contact angle decreases with time, and the optimum measurement time is 20 s), the Laplace-Young fitting algorithm was used to determine the mean value of WCA, click on the photo, and observe and record the WCA of the image.

AFM Characterization
The surface morphology of the prepared samples was characterized by AFM (Multi-mode8, Bruker, Germany). The Ra of the coating surface was measured by AFM, and the specimens were characterized using the Peak Force mode. The scanning range of AFM was set to 1 µm, and the scanning speed was set to 0.977 Hz. Five different regions were selected for a sample to preserve the data with uniform distribution of surface morphology changes. A software package (NanoScope Analysis) was used to process the acquired data.

X-ray Photoelectron Spectroscopy (XPS)
The prepared specimens were examined using X-ray photoelectron spectroscopy (ESCALABXi+, Thermo, Waltham, MA, USA, Microfocusing monochromatic (Al Kα) X-ray source (the best sensitivity of monochromatic light source: 1600 kcps (Al Kα 0.60 eV Ag 3d5/2 peak)) at an angle of incidence of 30 • (measured from the surface) and an emission angle normal to the surface. The power used was 150 W, and the maximum resolution depth was 10 nm. Survey spectra (Binding Energy (BE) in the range of 0-5000 eV) were used for element identification and quantification. The middle position of the specimen was scanned using a high magnification mode. The specimens pTi, pTi-(PLL-PDA) 1 , pTi-(PLL-PDA) 5 , pTi-(PLL-PDA) 10 , and pTi-(PLL-PDA) 15 were analyzed by composition (0-5000 eV) and high-resolution energy spectra (N 1s, O 1s). All reported spectra are averages of five scans taken at a resolution of 0.1 eV and referenced to the C 1s peak of hydrocarbons at 284.8 eV. Data acquisition and processing were performed using Thermo Advantage software. The XPS spectra were fitted with Voigt profiles obtained by convolving Lorentzian and Gaussian functions. The analyzer transmission function, Scofield sensitivity factors, and effective attenuation lengths (EALs) for photoelectrons were applied for quantification. EALs were calculated using the standard TPP-2M formalism.

Molar Grafting Rate
In order to simulate a Ti sheet, pure Ti powder was pressed into a 75 mm diameter disk. A thin layer of Ti was deposited on a quartz disk (diameter: 25 mm) by reactive magnetron sputtering using a radio-frequency magnetron sputtering system (CFS-4ES-231). The magnetron sputtering chamber was evacuated to a certain pressure and kept at 6.7 × 10 −1 Pa with argon, resulting in a Ti layer. The Ti layers were cleaned with sodium dodecyl sulfate and UV-ozone cleaner before the QCM measurements. In QCM, ∆F depends on the adsorbed mass following Sauerbrey's equation: where F 0 is the fundamental frequency of the crystal (27 × 10 6 Hz), A is the electrode area (0.049 cm 2 ), ρ q is the quartz density (2.65 g/cm 3 ), and µ q is the shear modulus of quartz (2.95 × 10 11 dyn/cm). The measurements were taken in triplicate. The molar grafting rate of the quaternary ammonium salt was calculated by the following formula [46].
where W D is the mass after grafting quaternary ammonium salt, W 0 is the mass before grafting quaternary ammonium salt, and M W is the molecular mass of the quaternary ammonium salt. The standard deviation is ±0.001.

Cell Compatibility Determination
Cytotoxicity was obtained using the 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide (MTT) assay. Pure Ti, pTi-(PLL-PDA) 15 -DEQAS, pTi-(PLL-PDA) 15 -MPA-N + , pTi-(PLL-PDA) 15 -DEQAS/MPA-N + , and pTi-(PLL-PDA) 15 -EPTAC were placed in the pores of the cell culture plate. HUVECs (2 × 10 5 cells/well) were seeded in the wells of the cell culture plate. The specimens were placed in RPMI 1640 medium with a temperature of 37 • C, a CO 2 concentration of 5%, and a fetal bovine serum (FBS) content of 10% for 24 h. The cells were washed twice with the essential medium Eagle (MEM) (serum-free) after 24 h, and then 15 mL of MTT solution was added to the wells of the cell culture plate. The cells were cultured at a temperature of 37 • C and a CO 2 concentration of 5% for 1 h. The samples were clipped out with tweezers and placed in the wells of the new cell culture plate. A pipette was used to add 200 µL of DMSO to each well. The well plate was shaken manually in an even manner. After waiting for 10 min, the optical density (OD) value of the mixed product was measured by a microplate reader at a wavelength of 490 nm. Finally, the cell viability was calculated by the following formula. Viability = (Specimen abs/Control abs) × 100 The OD value of various liquids was measured by a microplate reader because MTT can be reduced to blue crystalline formazan by succinate dehydrogenase in the mitochondria of cells, which can be dissolved by DMSO. Therefore, the larger the OD value of the mixed solution measured by the microplate reader, the more the number of cells and the more the proliferation. All measurements were taken in triplicate.

Antibacterial Performance Determination
When E. coli and S. aureus grew to the mid-log phase, the bacterial suspension was diluted to 10 6 CFU/mL. Pure Ti, pTi-(PLL-PDA) 15 -DEQAS, pTi-(PLL-PDA) 15 -MPA-N + , and pTi-(PLL-PDA) 15 -DEQAS/MPA-N + were cultured in 5 mL bacterial suspension at 37 • C for 24 h. After incubation, the specimens were washed twice with PBS. The specimens were soaked in 5 mL of PBS for 5 min, and then 3 mL of the soaked bacterial solution was