Surface-adhesive layer-by-layer assembled hydroxyapatite for bioinspired functionalization of titanium surfaces

Abstract

A novel and versatile fabrication route for titanium-based biocomposites has been realized by mussel adhesion chemistry through layer-by-layer assembly of polydopamine (PDOA) and hydroxyapatite (HA). The assembly process was monitored by XPS analysis and weight shifts. The chemical structure and the surface morphologies of the assembly layers were analyzed by FTIR, XPS and SEM, respectively. It was found that the assembly between PDOA and HA can be repeated indefinitely, and that a continued growth of this multilayer is possible. The as-prepared hybrid biocomposite materials present a rough surface, and such a structure is beneficial for bone cell adhesion. Primary cell proliferation experiments indicated that PDOA–HA-modified Ti substrates exhibit a good bioactivity. The hybrid assembly layers displayed an outstanding adhesion stability, which suggests that PDOA can be employed as an on-demand robust glue for creating functional biomaterials. These new materials can find widespread use in the field of orthopedic and dental implants due to their excellent mechanical properties and biocompatibility.

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1. Introduction

A biocompatible interface is very important when implanting biomaterials.^1,2 When a biomaterial implant is in contact with body fluids, the surfaces at the boundary between the bulk of the biomaterial and the outer environment are the first to interact with the body environment and thus play a more important role than the bulk.^3,4 For this reason, it is effective to directly immobilize various biocompatible substances, such as poly (ethylene glycol),^5–10 zwitterionic materials,¹¹ amino acids,^12–15 heparin,¹⁶ and hydroxyapatite (HA)^17–19 onto the surface of a hydrophobic biomaterial in order to obtain a good biocompatibility.

Titanium and its alloys are widely used as orthopedic and dental implants due to their excellent mechanical properties and corrosion resistance. However, a major concern with titanium implants is their integration into existing tissue. When bone cells cannot adhere to the surface of the implanted material, the implant cannot be integrated and will eventually detach from the body in the long term.^20,21 One approach to resolving the interface problem involves coating titanium surfaces with HA, the principal mineral in bones and teeth. Methods such as plasma spraying,^22–24 sol–gel techniques,^25,26 electrophoretic deposition,^27,28 and even solution phase apatite growth,^29,30 have all been explored. But, at the bonding interface, it is difficult to chemically combine a HA coating with a Ti substrate due to their different lattice structures. Moreover, there is a large difference between their Young moduli and heat expansion coefficient.

Bone tissue is a complex biocomposite material with a variety of organic (e.g., proteins, cells) and inorganic (e.g., HA crystals) components hierarchically organized with nano/microscale precision. Based on the understanding of such a hierarchical organization of bone tissue and its unique mechanical properties, efforts are being made to mimic these organic–inorganic hybrid biocomposites. A key factor for the successful designing of complex, hybrid biomaterials is the facilitation and control of adhesion at the interfaces, as many current synthetic biomaterials are inert, lacking interfacial bioactivity. Although novel bioactive interface materials enhancing biocompatibility and durability of the implant are emerging, most of the available materials suffer from insufficient stability in an aqueous environment.

As an alternative and feasible approach for the fabrication of bio-inspired structural materials, the hierarchical assembly of micro/nano-sized building blocks into bulk materials has become more attractive. Many assembly techniques such as evaporation-induced self-assembly,³¹ Langmuir–Blodgett (LB) assembly,³² and even higher-order spatial self-assembly³³ methods have been developed. These procedures for the assembly of micro/nano-particles have shown great potential for the fabrication of bio-inspired functional materials.

This work has explored a novel and versatile route for fabricating a titanium-based biocomposite material by mussel adhesion chemistry. Inspiration was taken from a magic compound of dopamine, a functional molecule containing catechol and amine groups, both of which are crucial for the highly adhesive property of Mytilus edulis foot protein 5 (Mefp-5).^34,35 It has been demonstrated that catechol and its derivatives can efficiently and facilely modify almost all kinds of inorganic and organic materials and yield functional surface materials.³⁶ However, there have been no reports on layer-by-layer assembly of polydopamine (PDOA) and HA to construct organic–inorganic hierarchical hybrid biocomposites. In this paper, layer-by-layer assembly of PDOA and HA was used to modify Titanium foil surface, which will endow with biocompatible interface and excellent mechanical stability.

Consequently, titanium was here immersed into a dilute dopamine aqueous solution after which the spontaneously formed thin polymer film was anticipated to provide a versatile interfacial adhesion for assembling HA films through a layer-by-layer assembly method.³⁷ The mechanical stability of the as-prepared biocomposites was also evaluated.

2. Materials and methods

2.1. Materials

Titanium foil was obtained from Goodfellow, Inc. (Berwyn, PA). Dopamine and tris-(hydroxymethyl) aminomethane (Tris) were purchased from Sigma-Aldrich. All reagents were of analytical grade and used without further purification. Deionized water was used through out the experiments. Neutral red staining solution was provided by Beyotime Institute of Biotechnology (China). Dulbecco's modified Eagle's medium (DMEM) and 0.25% trypsin–EDTA were purchased from Beijing Solarbio Science & Technology Co., Ltd. (China). Sterile filtered fetal bovine serum (FBS) was supplied by Beijing Yuanhengjinma Biotechnology Co., Ltd. (China). Other reagents were AR grade chemicals and were used without further purification.

2.2. Preparation of 1.5 SBF incubation solution

In the present study, 1.5 SBF was used as an incubation solution for HA formation on the surface of titanium foil. 1.5 SBFs were prepared by dissolving reagent-grade NaCl, NaHCO₃, Na₂SO₄, KCl, K₂HPO₄, MgCl₂·6H₂O, and CaCl₂·2H₂O in distilled deionized water, as described previously.³⁸ Noticeably, before the addition of CaCl₂, a certain amount of HCl was needed to maintain the pH of the solution at a low value (<7.00) to prevent precipitation of HA in the solution. 1.5 SBF was adjusted to a final pH of 7.20 at 36.5 °C with HCl and NaOH. The obtained incubation solution was used immediately to deposit HA onto titanium foil.

2.3. Preparation of PDOA coating

The PDOA coating was prepared as described by Lee et al.³⁴ Briefly, a thin PDOA layer was created on the surface of titanium foil by oxidative polymerization of dopamine–hydrochloride dissolved in 10 mM Tris buffer and the pH was adjusted to 8.5. The PDOA coating procedure was carried out for 24 h. After this time, the PDOA-coated substrates were extensively rinsed with deionized water and dried with a stream of N₂ gas.

2.4. PDOA-assisted self-assembly of HA (Method A, denoted as HA–PDOA-Ti)

The PDOA-coated titanium samples were kept in polypropylene tubes (one per tube) containing 50 mL of incubation solution. After incubation for 3 days at 36.5 °C, the samples were taken out, rinsed with distilled water and dried with a stream of N₂ gas.

2.5. Layer-by-layer deposit of PDOA and HA on titanium foils (Method B, denote as LBL-HA–PDOA-Ti)

The titanium foil was alternately immersed in the dopamine buffer solution (pH ∼ 8.5) and the 1.5 SBF solution (pH ∼ 7.2) at room temperature for 1 day and 14 days, respectively. After each immersion step, the Ti sample was extensively rinsed with deionized water and dried with a stream of N₂ gas. After five repeat cycles, the sample was dried under vacuum for characterization. The weight of each PDOA bilayer and HA bilayer were recorded on UMX2 Automated-S, the accuracy of the balance is 0.0001 g.

2.6. Cell adhesion assay

The affinity of murine osteoblastic cell line MC-3T3-E1 for the modified Ti foil surfaces was assessed according to the literature.^3,4 MC-3T3-E1 cells were cultured in DMEM supplemented with 10 vol% FBS, 4.5 g L⁻¹ Glucose, 100 units per mL penicillin, 5958 mL L⁻¹ N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) and 100 g mL⁻¹ streptomycin. The modified Ti foils with a diameter of 5 mm were sterilized in 70 vol% ethanol aqueous solution for 1 h, followed by extensive rinsing in PBS. Then the Ti foils were put into individual wells of a 24 well plate. Subsequently, each film was seeded with a suspension containing 5000 cells in 150 L of medium at room temperature.

After 1 h, the cells had adhered to the surface, 850 L of medium was added into each well. Then plates were incubated at 37 °C with 5% CO₂. All the cell culture procedures were carried out under sterile conditions, and the cells were dyed by neutral red staining solution before photographed. To count the number of cells on each surface, the sample was transferred to a new well, and 200 L of trypsin–EDTA was added into each well for removing the cells from the samples. Then the density of the cell suspension was calculated.

2.7. Characterization

Fourier transform infrared (FT-IR) spectra were recorded in the wave number range 4000–500 cm⁻¹ with a Bio-Rad digilab Division FTS-80 spectrometer using a KBr wafer. The surface morphologies of the samples were observed by an XL30 ESEM FEG field emission scanning electron microscope (FE-SEM, FEI Company with 20 kV operating voltage), and elemental analysis was performed by X-ray energy dispersive spectroscopy. X-ray photoelectron spectroscopy (XPS) analysis was used to confirm the presence of PDOA and HA on the surface of Ti. The bonding strength of the composite HA coating was evaluated by a standard tensile adhesion test.

3. Results and discussion

3.1. Preparation of PDOA coating on titanium foil

Commercially pure titanium (Ti) foil was cut into rectangular sections with dimensions 5 × 5 mm². The foils were then cleaned ultrasonically for 15 min each in reagent-grade dichloromethane, acetone, and deionized water. Subsequently, the Ti foils were immersed without drying in a 2 mg mL⁻¹ freshly prepared solution of dopamine (10 mM Tris buffer, pH 8.5) at room temperature in the dark. After 24 h, the substrates were sonicated for 10 min in water thrice to remove the nonattached dopamine followed by drying under nitrogen.

The X-ray photoelectron spectroscopy (XPS) analysis showed that the PDOA-coated substrates exhibited C(1s), N(1s), and O(1s) peaks without a substrate peak (i.e., Ti(2p)), thus indicating the formation of a polymer coating of more than 20 nm in thickness. The nitrogen-to-carbon signal ratio (N/C) of 0.12 was similar to that of the theoretical value for dopamine (N/C = 0.125), implying that the coating was derived from the dopamine polymerization (Fig. 1).

Fig. 1 XPS spectra of (bottom to top) PODA-coated titanium, HA- and PODA-coated titanium surfaces.

Contact angle measurements with water droplets were also conducted to investigate the effect of the PDOA coating on the surface properties. The PDOA coating was found to significantly decrease the contact angle from 68° ± 1.2° to 39° ± 1.5° and thereby confirmed that the Ti surface had become far more hydrophilic, which will facilitate soakage with SBF solution for deposition of HA.

3.2. PDOA-assisted self-assembly of HA

The untreated (control experiment, denoted as HA-Ti) or PDOA-treated titanium samples were kept in 1.5 SBF incubation solution that was renewed each day. The SEM images of the HA coating after soaking in 1.5 SBF are shown in Fig. 2. It can be seen that the PDOA coating facilitated the HA nucleation. The HA formed larger spherical shape agglomerates on the surface of the PDOA-coated Ti substrate in 3 days (Fig. 2a), whereas only a tiny HA particles formed on the unmodified substrate at the same time (Fig. 2b). This result implies that the free catechols on the surface of the substrate were essential for HA nucleation. According to EDX analysis, the Ca/P ratio for the agglomerates was 1.63, which is close to the theoretical ratio of HA (Ca/P = 1.67). structure.

Fig. 2 SEM images of HA deposited on the (a) PDOA-modified titanium and (b) unmodified titanium surfaces, and EDX analysis of HA deposited on the PDOA-modified titanium.

The chemical structure of the newly formed minerals was examined by FTIR analysis and X-ray photoelectron spectroscopy (XPS) after incubation of the Ti foil in 1.5 SBF for 10 days, as shown in Fig. 1 and 3. The FTIR spectra in Fig. 3 confirmed this to be an apatitic structure with characteristic bands at 567, 605, 964, and 1037 cm⁻¹. The broad band at 1454.0 cm⁻¹ was assigned to the CO₃²⁻ group. X-ray photoelectron spectroscopy (XPS) also confirmed the presence of HA on the surface of the PDOA (Fig. 1).

Fig. 3 FTIR spectrum of HA–PDOA-modified titanium surfaces.

3.3. Layer-by-layer deposition of PDOA and HA on titanium foils

Based on the successful deposition of HA on the dopamine-modified Ti foil, an improved method, i.e., a layer-by-layer assembly, was used to construct organic–inorganic hierarchical hybrid biocomposites.

The assembly technique is illustrated in Scheme 1. The first layer of PDOA was built based on the self-polymerization of dopamine to produce an adherent polydopamine coating on the Ti substrates. The as-formed PDOA acted as an effective surface anchor via catechol groups on the exterior layer and facilitated HA nucleation and the formation of an HA assembly layer on the PDOA-modified Ti surface. The assembly process was monitored by XPS analysis and weight shifts (Fig. 4 and 5).

Scheme 1 Assembly mechanism of HA and PDOA on Ti substrate.

Fig. 4 XPS analysis of the PDOA–HA assembly process.

Fig. 5 Weight shifts for the alternating assembly of PDOA and HA. The even layers (n = 2, 4, 6, 8 and 10) were HA layers.

Fig. 4 shows the results from the XPS analysis of PDOA–HA assembled on the Ti substrate. With an increasing number of PDOA and HA bi-layers, the counts of the peaks at 287 eV and 349 eV changed alternately after each assembling cycle of PDOA and HA. For example, the counts of the C1s and Ca2p peaks decreased or disappeared after assembling the second HA layer and the third PDOA layer, respectively. These observations indicated that the assembly of PDOA and HA on the Ti substrate leads to the formation of stable organic–inorganic hybrid biocomposites.

In order to monitor the assembly process, the weight shifts of the Ti foil was measured after each assembly process. All samples were prepared and tested in at least duplicate with a total of 3 samples. At least 3 specimens of each sample were measured, results of which were averaged and standard deviation from the mean as ±10%.

Fig. 5 shows the weight shifts of the assembly process. The weight of each PDOA bilayer and HA bilayer is approximately in the range of 2.7–3.6 μg and 0.132–0.135 g, corresponding to a layer thickness of about 55 nm and 5.2 μm, respectively. The regular increase in weight with the number of the deposited bilayers indicates that the assembly between PDOA and HA can be repeated indefinitely, and that a continued growth of this multilayer is possible.

The weight of the first PDOA bilayers was about 2.7 μg, which was much lower than the other four PDOA bilayers (about 3.6 μg each), as indicated in Fig. 6. This interesting observation is believed to be due to the availability of the substrate surface area for the assembly of PDOA. The SEM image shows that the surface of the Ti foil is much smoother than that of the assembled HA, which indicates that the surface area of the HA layer is much larger than that of the Ti substrate (Fig. 7). The weight of the PDOA deposited on the rough surface is thus higher than that on the smooth surface for an equivalent assembly time.

Fig. 6 The weight shifts for each PDOA bilayers.

Fig. 7 The SEM image of the smooth Ti surface and the rough HA surface (coating condition: Method A).

The HA formed on the surface of Ti foil is a spherical shape structure. Most of the spherical HA is alone adhered to the surface of titanium foil after incubation for 3 days (Fig. 8a). With an increasing time of incubation, the spherical HA connected together to form a closely packed surface topography (Fig. 8b and c). After incubation for two weeks, the Ti substrate was fully and uniformly covered by the HA minerals (Fig. 8d). High-magnification scanning electron microscopy (SEM) images reveal that the HA sphere have a lath-like surface structure (Fig. 8e and f), suggesting that the microsphere has a rough surface. Such a structure is beneficial for the adhesion of bone cells, such as osteoblasts.

Fig. 8 The SEM images of the HA formed on the titanium surfaces with different incubation time. Incubation for 3 days (a), incubation for 7 days (b), incubation for 10 days (c), incubation for 14 days (d), the surface morphology of single HA particle (e and f).

Recently, professor W. B. Tsai developed a facile and effective “one-pot” deposition method based on dopamine polymerization for the development of cell-adhesive, osteoconductive, and osteoinductive titanium implants.³⁹ HA was immobilized on titanium surfaces together with PDOA. The researchers suggested that the immobilization of HA nanoparticles on titanium surfaces was achieved by entrapment and/or conjugation with PDOA. The conjugation of HA nanoparticles greatly enhanced the adhesion, proliferation and the mineralization of osteoblasts on the modified titanium surfaces.⁴⁰ These results prompted us to investigate the cell adhesion and proliferation on our bio-composite material.

Herein, murine osteoblastic cell line MC-3T3-E1 was used to assess the cytocompatibility and the adhesion of the modified Ti surfaces. Interactions between MC-3T3-E1 cells and the Ti surfaces were analyzed by optical microscopy. As shown in Fig. 9, the osteoblastic cell readily adhered, spread and grew very well and there was no significant difference in cell viability between the PDOA–HA-modified Ti group and its unmodified counterpart. But the cell adhesion of the unmodified Ti group was lower as compared with the PDOA–HA-modified Ti group. As shown in Fig. 9a1 and b1, just a few cells resided on the virgin Ti surfaces, and the density of the cells is ∼1742 cells per cm² within the 1 days incubation time. On the contrary, the PDOA–HA-modified Ti surfaces illustrated good cytocompatibility due to the moderate HA of their surfaces. A large amount of cells with cell densities up to ∼1.4 × 10⁴ cells per cm² were adhered on the PDOA–HA-modified Ti substrates Fig. 9a2 and b2. This result indicates that HA–PDOA-modified Ti substrates have a good bioactivity, further bioactivity experiments are currently ongoing.

Fig. 9 Fluorescent photomicrographs of the cell adhesion of osteoblasts grown on pristine Ti (a1 and b1) and an LBL-HA–DOPA-Ti surface (a2 and b2) after 1 day of incubation. Size of the scale bars: 100 μm.

The adhesion stability of the coating layer is critical for practical applications. We thus investigated the adhesion stability of the HA layer using ultrasonication tests. We selected the same size of the Ti foil and LBL-HA–PDOA-Ti, and obtained the weight of the sample on UMX2 Automated-S precision balance before and after ultrasonication test. The mechanical stability of the coating layer was evaluated by the weight shift before and after ultrasonication test. Two kinds of HA-coated Ti samples, fabricated by different methods, were evaluated. After strong ultrasonication (42 kHz, 135 W) for 1 h, there remained only 6.8 wt% of HA on the unmodified Ti surface (sample HA-Ti). However, there was great increase of adhesion on the LBL assembly of PDOA and HA-modified Ti surface (sample LBL-HA–PDOA-Ti), the LBL-HA–PDOA-Ti remained stable after strong ultrasonication for 1 h with about 98.2 wt% of the HA still firmly attached to the Ti surface. The SEM images of the HA-Ti foils before and after ultrasonication were used to monitor the stability of the coating layer. Fig. 10a and b show that most of the HA on the unmodified Ti surface peeled off after ultrasonication test for 1 h, whereas there was no apparent change of the surface morphology for the sample LBL-HA–PDOA-Ti, and the HA attached to the Ti surface firmly.

Fig. 10 SEM images of the HA modified Ti surface before and after ultrasonication test and peel test. SEM images of HA on the unmodified Ti surface (a and b), SEM images of LBL-HA–PDOA modified Ti surface (b–d).

Fig. 10e SEM images of HA minerals grown on Ti substrate by LBL assembly of PDOA and HA after the peeling test. The samples were prepared by modifying the Ti substrates with a polydopamine coating and incubating in 1.5 SBF for 10 days. A peeling test was carried out by pressing a piece of Scotch tape (KS T1046, adhesive strength higher than 1.23 N cm⁻¹) down firmly on the samples and removing it quickly. Although a slight disruption of the lath-like structure of the hydroxyapatite minerals was observed after the peeling test, about 96.5 wt% of hydroxyapatite minerals remained attached to the underlying Ti substrate.

A number of recent reports have demonstrated the good interfacial stability of PDOA under various conditions.^41,42 For example, PDOA modification was found to be useful as an anti-corrosive lubricant coating,^43,44 and a single-molecule adhesion study revealed that the catechol-Ti adhesion force was four times stronger than biotin–streptavidin interactions.⁴⁵ These results suggest that PDOA can work as an on-demand robust glue for creating functional biomaterials.

The outstanding adhesion stability of LBL-HA–PDOA-Ti can be ascribed to the organic–inorganic hybrid microstructures. Presumably, if some voids were generated in the HA deposition process, the adjacent PDOA could serve as a barrier and repair the layer to minimize the defects. In other words, the unexpected voids on the surface of the HA layer could be covered by PDOA, which would serve as the active layer to boost the HA deposition. So, the voids may be healed, or at least controlled, on the HA layer.⁴⁶ Such a hierarchical organic–inorganic hybrid structure is similar to bone tissue, and endowed with its unique mechanical properties.⁴⁷

4. Conclusions

In summary, a novel and versatile route for fabricating a titanium-based biocomposite material has been realized by mussel adhesion chemistry through layer-by-layer assembly of polydopamine and hydroxyapatite to construct organic–inorganic hierarchical hybrid biocomposites. These hierarchical hybrid biocomposites exhibited excellent stability compared with the control experiment, the results suggest that PDOA can function as an on-demand robust glue for creating functional biomaterials. Primary cell proliferation experiments indicated that HA–PDOA-modified Ti substrates had good bioactivity, and further bioactivity experiments are currently ongoing.

Acknowledgements

The authors acknowledge the financial support of the National Natural Science Foundation of China (Project no. 81371184).

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