Micropatterned Polypyrrole/Hydroxyapatite Composite Coatings Promoting Osteoinductive Activity by Electrical Stimulation

: Conductive polypyrrole (PPy) has excellent biocompatibility and structural stability. It is an ideal electroactive biomaterial that can apply exogenous electrical stimulation to promote osteoblast differentiation. However, PPy is a kind of bio-inert material, which does not have osteoinductive capacity. Therefore, we have introduced a kind of bioactive material, hydroxyapatite (HA), to construct PPy/HA composite to enhance bioactivity and osteoinduction. In addition, micron-topological morphology of scattered grid pattern has been designed and introduced to the PPy/HA coatings, which can further enhance the regulation ability of the coatings to the adhesion, proliferation and differentiation of MC3T3-E1 cells. In vitro simulated body ﬂuids (SBFs) immersion test results have demonstrated that the fabricated micropatterned PPy/HA composite coatings perform bioactivity well and can promote the mineral deposition of HA on the surface. Moreover, it can also beneﬁt the proliferation and osteognetic differentiation of MC3T3-E1 cells, when accompanied by external electrical stimulation (ES). In this study, we have successfully constructed electroactive and bioactive coatings, the method of which can potentially be applied to the surface functional modiﬁcation of traditional bone repair metals.


Introduction
The growth and remodeling of bone tissue are accompanied by the generation of endogenous electrical signals [1][2][3][4]; therefore, the electrical environment is one of the important microenvironments for bone cells. In fact, external electrical stimulation (ES) has been used in the clinical treatment of bone diseases, such as fractures and osteoporosis [5][6][7]. Till now, many studies have also confirmed that ES can promote the proliferation and differentiation of osteoblasts [8], so as to promote the formation and regeneration of new bone [9,10]. In addition, the chemical composition and topological morphology of the material surface in contact with cells, and the application of external stimulation signals (electricity, light, magnetism, etc.), have significant impact on the metabolism of cells [11,12]. Therefore, appropriate morphological design, chemical component improvement and external applied stimulation can have important effects on the adhesion, spreading, proliferation and differentiation of cells [13][14][15]. With the help of some high-precision processing methods, it is possible to design and construct composite structures with different physical and chemical properties and topological morphologies. The micro electro mechanical system (MEMS) integrates many high-precision machining methods, such as lithography, corrosion and so on [16]. At present, it is widely used to manufacture advanced micro-integrated intelligent packing devices. Application of lithography technology can help to fabricate some patterned structures with micro-or nano-scale graphics. The self-assembled monolayer (SAM) technique could achieve functional modification of materials by adjusting the end groups at both ends of long-chain organic molecules [17].
Conductive polypyrrole (PPy) is an ideal electroactive biomaterial with excellent biocompatibility [18,19], and it can be used as conductive coatings to directly apply ES to cells with electrical response, including osteoblasts, so as to regulate cell behavior and metabolism [20,21]. Many related studies, and our previous works, have also confirmed that PPy, as a kind of conductive coating, can promote the proliferation and differentiation of MECT3-E1 cells with ES [22][23][24][25][26]. However, PPy is a kind of bio-inert material without bioactivity, and cannot induce cell differentiation [27]. Therefore, there is a need to prepare new composite materials to improve the bioactivity of PPy, on the premise of preserving the conductivity [28,29].
Hydroxyapatite (HA, Ca 10 (PO 4 ) 6 (OH) 2 ) is the main inorganic component of human bone [30,31]. It has excellent biocompatibility, bioactivity, and osteoinductivity [32][33][34][35][36]. Therefore, HA has been applied in clinical bone repair to provide a functional coating, or scaffold, for new bone formation [37][38][39][40][41]. However, the poor mechanical capacity of HA greatly restricts its application. At present, HA is mainly used as the surface modification coating of metal implants [42][43][44]. Based on the huge differences in physical properties, the interaction is not close [45]. In addition, long-term erosion from immersing in a biologically liquid environment might lead to peeling, dissolution and erosion of HA coatings from the metal surface, thus leading to the failure of the material implantation and tissue repair [46,47].
At present, many researchers are investigating the combination of PPy and HA so as to improve the application of these two materials. Wu et al. chose a pulse electrochemical method and used PPy as a regulator to manufacture a dexamethasone/hydroxyapatite composite coating on a titanium surface, achieving a stable and slow release of HA and dexamethasone, as well as sustained osteoinductivity [26]. Chakraborty et al. synthesized HA-PPy composite coating by pulsed reverse electrochemical deposition, which improved the corrosion protection performance behavior of SS316 [48]. Song et al. fabricated a threedimensional reduced-graphene oxide/polypyrrole/hydroxyapatite composite scaffold through an electrostatic layer-by-layer assembly strategy, followed by an electrochemical deposition process [49], which had good mechanical properties and good osteogenic performance. However, these studies did not concern themselves with the effect of ES on osteogenesis, and, also, they did not take advantage of two-dimensional topological structuring.
In this work, bioactive and osteoinductive micropatterned PPy/HA composite coatings with micron-scale topological morphology were fabricated, which possess both the electroactivity of PPy and the bioactivity of HA. The electroactive composite coatings were constructed by a two-step method, in which conductive PPy, with micro-scale pattern, was first electrochemically deposited on a micropatterned substrate processed by MEMS technology. Subsequently, HA was further selectively mineral deposited and filled the checkered area with the assistance of SAMs. Characterizations showed that the micropatterned PPy/HA composite coatings maintained good conductivity and electrochemical activity, and could accelerate the deposition of HA on the surface. Moreover, combined with ES, the adhesion, proliferation and differentiation of MC3T3-E1 cells were significantly promoted, and the osteogenesis of MC3T3-E1 cells was accelerated.
Pyrrole was purified by distillation under the protection of nitrogen and seal preserved at −20 • C for subsequent use. Other reagents were used directly without further treatment. The water used in all experiments was ultrapure water (18 MΩ).
Monocrystal silicon wafers were cleaned using the RCA standard cleaning method. Then, they were placed in an oxidation diffusion furnace at 950 • C until the thickness of the oxide layer reached 500 nm. Finally, 500 Å Ti and 1000 Å Au were sputtered on the surface of the oxidized silicon wafer. The Au/Ti/Si wafer was used for further experimentation (Figure 1 1 ).  1 The Au/Ti/Si wafer; 2 Micropatterned photoresist substrate; 3 The micropatterned PPy; 4 SAM-modified micropatterned PPy coatings; 5 The SAM-modified micropatterned PPy coatings soaked in 10× SBF solution; 6 The micropatterned PPy/HA composite coatings.

Fabrication of Micropatterned PPy Coatings
Firstly, micropatterned photoresist substrate (Figure 1 2 ) was prepared by conventional ultraviolet lithography. The photoresist was applied with homogenizer at 2000 rpm/min for 30 s, then put in the oven at 92 • C and baked for 10-12 min. After baking, the photoresist was exposed for 12 s and developed for 90 s. Finally, after cleaning with deionized water and drying with N 2 , the micropatterned photoresist was obtained.
Then, PPy was deposited on the prepared micropatterned photoresist substrate by electrochemical polymerization. The process was carried out in a three-electrode system, in which the photoresist Au/Ti/Si wafer was the working electrode, a platinum wire was the counter electrode and a saturated calomel electrode (SCE, INESA Scientific Instrument Co., Ltd. Shanghai, China) was the reference electrode. The 0.1 M pyrrole monomer was dissolved in the electrolyte of aqueous solution (pH = 7.4) containing 0.1 M DBS. The micropatterned PPy coatings were directly electrochemically polymerized on the Au/Ti/Si wafer under a constant current density of 0.1 mA/cm 2 for 1500 s with CHI660D electrochemical workstation (Chenhua Instrument Company, Shanghai, China). Finally, the micropatterned PPy (Figure 1 3 ) was ultrasonically cleaned with acetone and ethanol to remove the photoresist. After washing with ultrapure water, the sample was blow dried with N 2 for further use.
PPy coatings with common morphology were electrochemically deposited on the Au/Ti/Si substrate in the same electrolyte and deposition conditions.

Fabrication of Micropatterned PPy/HA Composite Coatings
The micropatterned PPy was immersed in 2 mM of fresh MDA ethanol solution for 12-16 h. Based on the interaction between sulfhydryl and Au, MDA molecules would self-assemble in the area uncovered by PPy to form a closely arranged SAM (Figure 1 4 ). Then, the SAM-functionalized micropatterned PPy coatings could be obtained by washing with ultrapure water and drying with N 2 . Finally, the SAM-modified micropatterned PPy coatings were soaked in 10× SBF solution for 2 h (Figure 1 5 ). Based on the electrostatic attraction of the active carboxyl groups at the end of the SAMs to Ca 2+ in solution, Ca 2+ and PO 4 3− ions were selectively aggregated and deposited on the SAMs to induce HA nucleation and growth, so as to obtain micropatterned PPy/HA composite coatings (Figure 1 6 ).

Characterization
The morphologies of micropatterned photoresist on the Au/Ti/Si substrate and micropatterned PPy coatings were observed by metallographic microscopy (L150, Nikon, Germany). The morphologies of micropatterned PPy coatings, micropatterned PPy/HA composite coatings and HA in micropatterned PPy/HA composite coatings were observed by scanning electron microscopy (SEM, LEO1530, Zeiss, Germany) with an accelerating voltage of 20.0 kV. The FTIR spectra of micropatterned PPy and micropatterned PPy/HA composite coatings were analyzed using Fourier transform infrared spectrometer (FTIR, Avatar 360, Nocalet Instrument Corporation, Waltham, MA, USA) with the KBr pellet method. The element composition and crystal structure of deposited HA was further characterized by X-ray energy spectrum (EDS, EDS7426, Oxford Instruments, Oxford, Britain) and X-ray diffraction (XRD, X-pert, Panalytical, Almelo, The Netherlands). The static contact angle of different samples was measured by contact angle meter (JC2000A, Shanghai Zhongchen Digital Technic Apparatus Co., Ltd., Shanghai, China) with water dropped at three different positions of each sample, and each sample was randomly tested in five parallel specimens. The electrochemical activity and stability were characterized by cyclic voltammetry (CV), tested in 0.9% NaCl using a CHI660D electrochemical workstation. The scanning voltage range was −0.9~0.2 V, and the scanning speed was 50 mV/s.

Bioactivity Assessment
The micropatterned PPy/HA composite coatings were immersed into fresh 1× SBF solution under constant temperature at 36.5 • C, and the 1× SBF solution was replaced every 48 h. The soaked samples were taken out at 2 and 4 days respectively, and the micropatterned PPy/HA composite coatings were washed with deionized water and dried naturally at room temperature, then characterized by SEM, EDS and XRD.

Cell Culture
The mouse embryonic osteoblast precursor cells (MC3T3-E1) were purchased from the Peking Union Cell Center. MC3T3-E1 cells were cultured in the alpha-modified minimum essential medium (α-MEM, Hyclone, Logan, UT, USA), supplemented with 10% FBS (Gibco, Waltham, MA, USA) and 1% penicillin and streptomycin (Gibco, Waltham, MA, USA) in a humidified 5% CO 2 incubator at 37 • C. The medium was changed every 2 days during the normal subculture of cells.

Effect of Micropatterned PPy/HA Composite Coatings with ES on MC3T3-E1 Cell Adhesion, Proliferation and Differentiation
The self-made device to exert electrical stimulation was soaked in chromic acid washing solution for more than 12 h, then washed with ultra-pure water and sterilized by high pressure steam sterilization for 20 min. Micropatterned PPy/HA composite coatings, non-patterned PPy, silicon film, copper rods, screws and wires were soaked in 75% alcohol overnight then cleaned with ultra-pure water. Before samples were installed into the electrical stimulation devices, all materials and equipment were irradiated under UV radiation for 1 h.
The sterilized coatings were placed in the self-made electric stimulation devices and the exposed area of coatings in contact with cells was 1.0 × 0.5 cm 2 . Suspension of MC3T3-E1 cells with 2 × 10 4 cells/mL was seeded into each well and cultured in a cell incubator at 5% CO 2 and 37 • C. The medium was changed every 24 h.
The ES was applied to cells from the day of inoculation with a constant current of 10 µA for 4 h every day for 14 days. The electrical signal was output by an electrochemical workstation using the multi-current steps mode. Each ES chamber (as shown in Figure 2) was connected in a series to the electrochemical workstation through a wire connecting electrode rod.

Cell Morphology
The morphology of cells on different coatings was observed by fluorescence microscope (Leica DM2500, Leica Camera, Wetzlar, Germany) and SEM. After MC3T3-E1 cells were cultured for 24 h, cells were stained with 200 µL Calcein-AM staining solution (2 µmol/L) at 37 • C for 15-30 min, then the staining solution was discarded and they were washed with PBS 3 times. Finally, samples were observed by fluorescence microscope.
The cells on different coatings for SEM detection needed to undergo gradient dehydration with ethanol solutions and further dried in a 37 • C oven. Before observation, the sample needed to be sprayed with Au.

MTT Assay
The cell viability of MC3T3-E1 cells growing on different coatings was investigated via MTT assay. MC3T3-E1 cells were cultured with different coatings. At different tested time points (4, 24 and 48 h), the medium was removed and washed twice with PBS, 500 µL of fresh culture medium, without FBS, was mixed with 100 µL MTT working solution (5 mg/mL) and added to each well and co-incubated at 37 • C for 4 h. Finally, the solution was completely removed and 500 µL DMSO was added to fully dissolve the formazan crystals. The testing solution was transferred into a 96-well plate, and the optical density (OD) values at 570 nm were measured by enzyme standardizer (Infinity 200pro, Tecan, Männedorf, Switzerland). This experiment was repeated 4 times and each sample was set in 6 parallel samples.

Determination of the BSA Protein Content, Alkaline Phosphatase Activity (ALP) and Inorganic Calcium Content
The effect of different coatings and ES on MC3T3-E1 cell biosynthesis was evaluated by testing the BSA protein content, ALP activity and inorganic calcium content on the 3rd, 7th and 14th days, respectively. MC3T3-E1 cells in each group were washed with PBS and lysed with 200 µL 0.1% TritonX-100 for 30 min at 4 • C, then the lysate was centrifuged at 4 • C with 3000 rpm/min for 2 min. The supernatant was collected for further detection of protein content and ALP activity. After this, 500 µL HCl (0.1 M) was added to the supernatant, and it was incubated at 4 • C for 4 h to fully dissolve the calcium. Then, the solution was centrifuged at 4 • C with 1000 rpm/min for 2 min. The supernatant was collected for further inorganic calcium content detection.
The total protein content was measured by a commercial BCA protein Assay Kit (Pulley Genetic Technology, Beijing, China) by testing the absorption values at 570 nm. The ALP activity was measured using the commercial ALP Assay Kit (Jiancheng technology Co., Ltd, Nanjing, China) by testing the absorption values at 405 nm. Inorganic calcium content was measured by using a Calcium Assay Kit (Jiancheng technology Co., Ltd, Nanjing, China) by testing the absorption values at 610 nm. These experiments were repeated 3 times. In each experiment, 4 parallel samples of each coating were set, and the absorption values were measured with enzyme standardizer.

Preparation and Characterization of Micropatterned PPy/HA Composite Coatings
The process of micropatterned PPy/HA composite coating preparation is shown in Figure 1. In brief, evenly distributed square micropattern was first fabricated on an/a Au/Ti/Si substrate by photolithography. Metallographic microscope images showed that the micropatterned photoresist was distributed in a regular lattice on the Au/Ti/Si substrate and consisted of squares with side length and spacing of 10 µm (Figure 2a). Subsequently, the photoresist micropatterned Au/Ti/Si substrate was used as the template, and PPy was selectively electrochemically polymerized on the conductive areas uncovered by photoresist. After the photoresist was removed, it could be seen that the continuous grid PPy coatings with smooth and flat surface were successfully deposited and the boundary of the pattern was clear (Figure 2b,c). Next, based on the strong interaction between the sulfhydryl group and Au, functional 16-mercaptohexadecanoic acid (MDA) SAMs were constructed on the exposed Au regions. Meanwhile, the carboxyl group at the exposed end of the MDA SAMs could attract Ca 2+ by electrostatic interaction, and then adsorbed Ca 2+ continued to attract anions, including phosphate and hydroxyl, thus inducing the nucleation and mineralization growth of HA. Under the environment of high concentration SBF (10×), SAMs could more quickly induce the rapid and localized growth of HA in the SAMs' covered areas. By soaking in 10× SBF for only 2 h, HA could highly selectively fill the grid pattern without invading other PPy covered areas (Figure 2d), and the growing HA showed a typical chrysanthemum-like crystal morphology (Figure 2e). Thus, we obtained micropatterned PPy/HA composite coatings. The PPy with ordinary morphology coatings, as the control group, had the same micro-morphology as the PPy part in micropatterned PPy/HA composite coatings, showing a typical cauliflower shape (Figure 2f).
The chemical structures of the PPy and HA parts in PPy/HA composites were characterized by FTIR, respectively. From the FTIR spectra of PPy and HA, it could be seen that characteristic peaks of PPy appeared at 3346 cm −1 , corresponding to N-H stretching vibration and 1465 cm −1 ascribing to C=N/N-H stretching vibrations in the pyrrole ring. In addition, peaks at 1090, 604, 563 and 436 cm −1 were basically consistent with the characteristic peaks of PO 4 3− in HA. Moreover, the peaks at 1456 and 1405 cm −1 , attributed to CO 3 2− , indicated CaCO 3 existed in the mineralized HA (Figure 3a). The EDS was carried out to semi-quantitatively analyze elements in the HA part, and Ca, P, O, C, etc. elements were detected. Among them, the Ca/P ratio was about 1.1-1.4 at different regions, which was between the theoretical Ca/P ratio of HA (1.67) and CaHPO 4 ·2H 2 O (DCPD, 1.0) [50] ( Figure 3b). Therefore, it could be inferred that a variety of minerals composed of apatite, dominated by HA, were deposited in 10× SBF. Therefore, it was speculated that the deposition obtained from 10× SBF was a mixture mineral, including HA and other apatites. At the same time, the crystal diffraction peak analysis of the deposited mineral was obtained by XRD and also confirmed that there were at least two types of crystals, including DCPD and HA (Figure 3c). The contact angle tests showed that the micropatterned PPy/HA composite coatings had good surface hydrophilicity (Figure 3d). On the PPy/HA composite coatings, the micropatterned PPy still existed as a continuous structure, which ensured the conductivity of the composite coatings. The conductivity of micropatterned PPy coatings was about 10 S/cm, which made the feasibility of subsequent ES on cells valid. In addition, considering the composite coatings needed to be immersed in medium with applied electrical signals for a long time, in subsequent experiments we further evaluated the bonding and electrical stabilities of the micropatterned PPy/HA composite coatings. It was noted that the PPy could uphold a relatively stable redox property after multiple cycles of a CV scanning test (Figure 3e). Furthermore, the electrochemical activity of the micropatterned PPy/HA composite coatings did not change significantly even after soaking in solution for 1 month, which also demonstrated that the micropatterned PPy coatings could maintain stable adhesion with the substrate under the applied ES for a long time (Figure 3f).

Bioactivity Assessment of Micropatterned PPy/HA Composite Coatings
An ideal bone repair material should not only have excellent biocompatibility, but also possess biological activity. Bioactive materials can quickly exchange ions with human body fluid in the body, and form HA layers similar to the composition of natural bone tissue on the surface through a series of reactions, which is beneficial to the forming of a stable chemical interaction with bone to further induce tissue regeneration. Hence, HA was introduced into the micropatterned PPy coatings to enhance the bioactivity. The bioactivity of micropatterned PPy/HA composite coatings to promote HA deposition in vitro was evaluated with simulated body fluid (1× SBF).
Immersing micropatterned PPy/HA composite coatings in SBF (×1) with standard concentration, it was found that the HA part acted as nucleation sites to promote further mineralized deposition of HA. From the SEM images, it could be seen that newly formed HA covered the original HA region and continued to epitaxially grow after soaking in 1× SBF for only 2 d. Till the 4th d, HA almost enclosed the whole substrate and formed a new layer of HA (Figure 4a,b). However, no HA crystals were observed on pure PPy coatings under the same conditions (Figure 4c). These results indicated that the PPy/HA composite coatings had excellent biological activity and could quickly induce the mineral deposition of HA. The EDS and XRD tests of the newly deposited HA layer displayed that the Ca/P ratio of the induced formed HA was about 1.70, which was very close to that of natural HA (Figure 4d,e). Combined with the results of XRD (Figure 4e), these results both demonstrated that the bioactive composite coatings could induce the deposition of HA with similar chemical structure to natural HA.

Effect of Micropatterned PPy/HA Composite Coatings with ES on Cell Activity
To investigate the positive effect of micropatterned PPy/HA composite coatings and the synergistic effect with ES on adhesion, spreading and proliferation of MC3T3-E1 cells, the cell activity and morphology of the contact between MC3T3-E1 cells with different materials were assessed. The attachment and spreading of cells on the substrates are the prerequisites for subsequent osteogenic differentiation. The interaction between the cell and substrate is intuitively reflected in the spreading morphology of cells. From the fluorescent images, it could be directly observed that there were significantly more MC3T3-E1 cells when the cells were co-cultured with different coating materials for 24 h on two different substrates with ES, than were the cases with the other two groups without ES. Moreover, whether ES was applied or not, cells preferred to grow on the micropatterned PPy/HA composite coatings, compared to the PPy coatings with ordinary morphology (Figure 5a-d). In total, micropatterned PPy/HA composite coatings together with ES had a synergistic effect on MC3T3-E1 cells. SEM images more intuitively showed the adhesion and spreading behavior of cells on different substrates (Figure 5e-h). Actually, it was noted that MC3T3-E1cells presented a slender spindle shape on the surface of unpatterned PPy coatings, while a more stretched polygon shape, with many cell bumps, were observed on the micropatterned PPy/HA composite coatings. This indicated that MC3T3-E1 cells had a more thorough spreading shape and larger spreading area on the micropatterned PPy/HA composite coatings surface than on the ordinary PPy coatings, and that the micropatterned PPy/HA composite coatings surface was conductive for cells to contact each other and transmit relevant signals. Furthermore, MC3T3-E1 cells growing on micropatterned PPy/HA composite coatings with ES stretched out more pseudopodia and cell synapses, which suggested that cells had closer interaction and tighter adhesion with the substrate. The proliferation of MC3T3-E1 cells cultured on different material surfaces and with or without ES was evaluated by MTT assay. During the first 4-6 h after cells were inoculated on different substrate materials, the main behavior of cells was to adhere to the surface of the materials. As shown in Figure 5i, the adhesion of MC3T3-E1 cells on different materials displayed significant disparity after 4 h, when more cells adhered to the two kinds of coatings with ES than to those without ES, and micropatterned coatings accompanied with ES had the most positive effect on promoting cell adhesion. Cell proliferation continued to be detected on different materials, and it was noted that the proliferation rate of ME3T3-E1 cells with ES was obviously faster than without ES. Moreover, the advantage of promoting cell proliferation by coatings with micropatterned morphology appeared, and the synergistic effect of micropatterned PPy/HA composite coatings together with ES resulted in the most efficient performance and the promotion of proliferation of MC3T3-E1 cells.

Effect of Micropatterned PPy/HA Composite Coatings with ES on Cell Functional Characteristics
When osteoblasts adhere to the surface of the material and proliferate rapidly, they initiate the synthesis of related proteins [51]. Therefore, the assay of the total cellular protein content could reflect the cell survival and proliferation. Based on the total protein content of MC3T3-E1 cells grown on different materials (Figure 6a), it could be seen that the total protein content increased with time in all groups, indicating that all groups of materials could promote the secretion and synthesis of proteins. Protein synthesis in cells with high proliferative activity was more abundant and metabolically vigorous, which could disperse, spread and multiply on the material surface in a short period of time. The protein content of cells on the micropatterned PPy/HA composite coatings was higher than that on the un-patterned PPy, which indicated that the modification of HA and micropatterning can promote cell adhesion, spreading and proliferation, thus, further promoting the increase of total cellular protein content. The cellular protein content on all materials of the ES groups was significantly higher than that of the non-ES groups, and the discrepancy was greatest at 14 d. This indicated that the cellular growth state was exuberant with ES, resulting in a significant increase in cellular protein synthesis. The highest protein content was found in the group wherein cells grew on micropatterned PPy/HA composite coatings with ES, demonstrating that MC3T3-E1 cells under this culture condition had more vigorous metabolic and proliferative activities. ALP is an early symbol of osteoblast differentiation and functional maturation. ALP could enhance the local concentration of phosphorus in vivo to promote the nucleation and mineralization of HA in bone tissue, and thus start the process of ECM mineralization. By detecting the changes of ALP activity, we could intuitively show the differentiation, maturity and bone mineralization ability of MC3T3-E1 cells. The results of the ALP activity assay of MC3T3-E1 cells on different coatings are shown in Figure 6b. After culturing for 3 d, the ALP activity of MC3T3-E1 cells on all coatings was low and did not show significant differences. This indicated that the cells were in the proliferation stage at this time, and the cells had not started the differentiation process. With the extension of time, the ALP activity in all groups increased significantly, and the differences between different groups were gradually obvious. Till the 7th d, the ALP activity of the ES groups was significantly higher than that of the non-ES groups and the ALP activity of the cells on the micropatterned PPy/HA composite coatings with ES was the highest, which was almost 2.2-times higher than that of the PPy with ordinary morphology, without ES (the control group). The ALP activity in the ES or micropatterned factors alone groups only increased 1.6 and 1.3 folds, respectively. At the 14th d, since no effective induction conditions were applied, the ALP activity in the control group still had no obvious change, but the ALP activity of MC3T3-E1 cells in the other three groups continued to increase. Among them, the ALP activity of MC3T3-E1 cells on the micropatterned PPy/HA composite coatings together with ES still maintained the advantage over the other groups, being almost twice as high as the control group. The above results further confirmed that micropatterned PPy/HA composite coatings with ES have synergetic effects on the functional differentiation of cells and can significantly promote the mineralization and osteogenesis of MC3T3-E1 cells.
Their calcification ability is an important biological characteristic of osteoblasts. The inorganic calcium content of the cells was measured to investigate the calcification ability in osteoblasts. As seen in Figure 6c, the calcium content in all groups was very close and at a relatively low concentration level at the 3rd d. This was because cellular calcium ion deposition occurs mainly during the matrix calcification phase after the increase of ALP activity, so the cellular calcium content of the micropatterned PPy/HA composite coatings with ES group started to be significantly higher than the rest of groups until the 7th d, and increased almost 1.8-fold more than that in control group. When cells were cultured until the 14th d, most MC3T3-E1 cells had matured and entered the matrix calcification phase, which led to the calcium content constant increasing significantly. The calcium content of the cells on the micropatterned PPy/HA composite coatings was significantly higher than that on other groups. The result was consistent with the results of the BSA protein content and ALP activity assay experiments, and demonstrated again that micropatterned PPy/HA composite coatings with ES can promote the cellular matrix mineralization of osteoblasts, thus promoting new osteogenesis.

Conclusions
In summary, we designed and fabricated bioactive micropatterned PPy/HA composite coatings. The composite coatings had both the advantages of excellent conductivity and electrochemical activity from PPy and good biological activity from HA, and further constructed topological microstructures with scattered square morphology by MEMS and SAM technology. In vitro experimentation demonstrated that micropatterned PPy/HA composite coatings had excellent mineralization inducing activity, which could induce and shorten the mineral deposition time of HA in SBF solution. Moreover, the results of cell experiments further verified that the micropatterned PPy/HA composite coatings performed excellent bioactivity and osteoinductivity. When combined with externally applied ES, the composite coatings could promote the adhesion, proliferation and osteogenic differentiation of MC3T3-E1 cells growing on them. Therefore, the micropatterned PPy/HA composite coatings are functional coatings with excellent biocompatibility and bioactivity, and can be regarded as an ideal strategy to improve the surface bioactivity and osteoinductivity of bone transplantation and to repair metal materials.