Mussel-inspired bioactive ceramics with improved bioactivity, cell proliferation, differentiation and bone-related gene expression of MC3T3 cells

Abstract

Mussels possess the ability to attach to virtually any type of inorganic and organic surfaces due to the existence of phenylalamine and lysine amino acids. Inspired by the property of mussels, polydopamine has been used for modifying bioinert materials, such as metals, semiconductors and plastics to improve their surface hydrophilicity. However, there are no reports about the effect of a polydopamine modification on apatite mineralization and the biological response of bioactive ceramics (not bioinert materials) for bone regeneration applications. Akermanite bioceramics (AKT, Ca₂MgSi₂O₇) are a typical bioactive material with osteostimulation properties for bone tissue regeneration. The aim of this study is to systematically investigate the effect of a polydopamine modification on the physicochemical and biological properties of AKT bioceramics, including attachment, proliferation, ALP activity and bone-related gene expression of tissue cells. The results show that a self-assembled polydopamine layer on the surface of AKT bioceramics was formed by incubating AKT bioceramics in a dopamine/Tris–HCl solution. Polydopamine-modified AKT (PDB-AKT) bioceramics showed significantly improved surface roughness, hydrophilicity and apatite-mineralization ability compared to AKT bioceramics. In addition, the polydopamine modification distinctively enhanced the attachment, proliferation, alkaline phosphate activity and bone-related gene expression of MC3T3 cells on AKT bioceramics. The possible reason for the improved cytocompatibility may be related to the improved surface roughness and apatite mineralization as well as the ionic environment at an early stage of cell culture. Our results suggest that the polydopamine modification is a viable method to further improve the apatite mineralization and biological response of bioactive ceramics for better bone regeneration applications, indicating that the polydopamine modification is a universal method to enhance the bioactivity for both bioinert and bioactive materials.

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1. Introduction

Previous studies have showed that the mussel's adhesive versatility might lie in the amino acid composition of proteins found near the plaque–substrate interface, which is rich in 3,4-dihydroxy-L-phenylalamine (DOPA) and lysine amino acids.^1,2 Inspired by this property in mussels, polydopamine was prepared by the polymerization of dopamine to mimic the chemical compositions of the plaque–substrate interface in mussels.³ Polydopamine contains a large number of bioactive groups, such as catechol moieties, OH⁻ and NH₂⁻, and therefore can bind strongly to different kinds of materials.^1,4,5 Previous studies have reported that many materials, such as metals, semiconductors and plastics, can induce a self-assembled attached layer of polydopamine on their surface after soaking in a dopamine solution.^1,4,6–8 The formed polydopamine layer is of great importance to induce apatite mineralization in SBF. Therefore, the polydopamine modification offers a bioactive surface for these traditional bioinert materials, such as metals, semiconductors and plastics.^6,8 However, although the polydopamine modification improves the apatite-mineralization ability for these bioinert materials, there are still lots of issues for the application of bioinert materials for tissue regeneration, as bioinert materials may lack sufficient degradation and bioactivity.^9,10 Inspired by the improved apatite-mineralization ability for polydopamine-modified bioinert materials, we assume that the polydopamine modification may further improve the bioactivity of bioactive ceramics. To the best of our knowledge, there are no reports about polydopamine-modified bioceramics for bone regeneration applications.

Bioceramics play an important role in bone regeneration.¹¹ Akermanite (AKT, Ca₂MgSi₂O₇) is a Ca, Mg and Si-containing bioceramic, which possesses an apatite-mineralization ability and a moderate degradation rate in SBF as well as generally good mechanical properties.^12–14 AKT bioceramics support the attachment of osteoblasts, bone marrow stromal cells (BMSCs) and periodontal ligament cells (PDLCs), and the Ca, Mg and Si-containing ionic products from AKT bioceramics significantly stimulate the proliferation and osteogenic differentiation of several kinds of stem cells, including dental pulp cells, bone marrow stromal cells, adipose-derived stem cells and periodontal ligament cells.^15–18 Therefore, it is suggested that AKT bioceramics possess significant osteostimulation properties in vitro and in vivo for bone regeneration applications.¹⁹ As one of the typical bioactive ceramics, it is therefore interesting to modify AKT bioceramics and elucidate whether the polydopamine modification can further improve their bioactivity, which may provide important evidence that the polydopamine modification is a universal method to improve the bioactivity for both bioinert and bioactive materials. There are no reports about the study of polydopamine-modified bioactive ceramics for bone regeneration applications. It is unclear what the effect of the polydopamine modification is on the apatite-mineralization, physicochemical and biological properties of bioactive ceramics. Therefore, the aim of this study is to systematically investigate the effect of the polydopamine modification on the physicochemical and biological properties of AKT bioceramics, including surface roughness, hydrophilicity and apatite mineralization as well as cell attachment, proliferation, ALP activity and bone-related gene expression of 3T3 cells on AKT bioceramics.

2. Experimental section

2.1 Preparation and characterization of PDB-AKT bioceramics

AKT powders were synthesized by the sol–gel process with the use of tetraethyl orthosilicate [(C₂H₅O)₄Si,TEOS], magnesium nitrate hexahydrate [Mg(NO₃)₂·6H₂O] and calcium nitrate tetrahydrate [Ca(NO₃)₂·4H₂O] as raw materials according to our previous publication.²⁰ The AKT bioceramic discs with dimensions of 12 mm Ø × 2.5 mm were prepared by uniaxial pressing of the AKT powders under 10 MPa and sintering at 1350 °C for 3 h. The sintered ceramics were characterized by X-ray diffraction (XRD, D8ADVANCE, Bruker, Germany) and scanning electron microscopy (SEM, JSM-6700F, JEOL, Japan).

To prepare polydopamine-modified AKT (PDB-AKT) bioceramic discs, dopamine hydrochloride was firstly dissolved in 10 mM Tris–HCl (pH 8.5) with the concentrations of 2, 4 and 6 mg mL⁻¹. Then, the prepared AKT bioceramic discs were soaked in a dopamine/Tris–HCl solution for 24 h. The color of the dopamine/Tris–HCl solution becomes dark due to the pH-induced oxidation of dopamine. After soaking, PDB-AKTs were rinsed in water and dried at 60 °C overnight.

The surface morphology and surface composition of AKT and PDB-AKT as well as the cross section of PDB-AKT bioceramic discs were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and XRD. The effect of the polydopamine modification with varied concentrations on the surface roughness of AKT bioceramics was investigated by atomic force microscopy (AFM) analysis. The peak value of roughness refers to the height of the highest peak in the tested area. The average roughness refers to the average value of all peaks in the tested area. The hydrophilicity of both AKT and PDB-AKT bioceramics was investigated by measuring the water contact angle.

2.2 The apatite mineralization of PDB-AKT bioceramics

To investigate apatite mineralization of PDB-AKT bioceramics, simulated body fluids (SBF) containing ion concentrations similar to those in human blood plasma were prepared according to the method described by Kokubo.²¹ The PDB-AKT discs were soaked in SBF (pH 7.40) at 37 °C for 1, 3 and 7 days. After the set soaking time, the PDB-AKT discs were removed from the SBF, rinsed with distilled water, dried at 60 °C and characterized by SEM, EDS and FTIR. Pure AKT bioceramic discs without the polydopamine modification were used as the control.

2.3 The attachment and proliferation of 3T3 cells on PDB-AKT bioceramics

MC3T3 (MC3T3-E1 Subclone 14) cells were purchased from cell bank, Chinese Academy of Sciences. The 4th passage of MC3T3 was used for the evaluation of the interaction of cells with PDB-AKT bioceramic discs, including the attachment, proliferation, alkaline phosphate (ALP) activity and bone-related gene expression (ALP, RUNX2 and COL 1). Pure AKT bioceramic discs without polydopamine modification were used for the control.

For the evaluation of cell attachment, 3T3 cells were cultured on PDB-AKT and AKT bioceramics placed in 48-well culture plate at an initial density of 1 × 10⁴ cells per cm². The cells were then incubated for 1 and 7 days in α-MEM culture medium supplemented with 10% FCS in humidified culture conditions. At the completion of culture, the disks were removed from the culture wells, rinsed with PBS and fixed with 1.25% glutaraldehyde. The fixative was removed by washing with buffer containing 4% (w/v) sucrose in PBS and post fixed in 1% osmium tetroxide in PBS followed by sequential dehydration in graded ethanol. The specimens were dried in hexamethyldisilazane (HMDS) for 30 min before coating with gold for SEM analysis. The morphological characteristics of the attached cells on the coating disks were determined using SEM.

For the investigation of the proliferation of 3T3 cells on PDB-AKT, an MTT assay was performed in triplicate according to our previous study protocol.²² This assay is based on the cleavage of MTT into insoluble formazan crystals by the mitochondrial enzymes of the viable cells. Briefly, 3T3 cells were seeded on PDB-AKT or AKT bioceramic discs and cultured in growth medium for 1, 3 and 7 days. 40 μL of a 0.5 mg mL⁻¹ MTT solution (Sigma-Aldrich) was added to 360 μL growth medium at each time point. After incubation for 4 h, the medium was removed and the formazan product was solubilized in 200 μL of dimethyl sulfoxide (DMSO). An aliquot of 100 μL was taken from each well and transferred to a fresh 96-well plate. The absorbance was measured at λ = 495 nm on a microplate reader (SpectraMax, Plus 384, Molecular Devices, Inc., USA). A cell culture plate without materials was used as the blank control. All the results were demonstrated as the optical density values minus the absorbance of the blank wells.

2.4 Alkaline phosphatase (ALP) activity of 3T3 cells on PDB-AKT bioceramics

The ALP activity assay was performed on day 3 and 7 to access the effect of the polydopamine modification on the early osteogenic differentiation of 3T3 cells on AKT bioceramics. Cells were seeded at a concentration of 1 × 10⁴ cells onto each ceramic disc placed individually in a 48 well plate. The cells were left to grow for 3 and 7 days at 37 °C in a humidified atmosphere of 5% CO₂. ALP activity was assayed by our previous method.²³ Aliquots of cell lysates were incubated with the reaction solution (containing 2-amino-2-methyl-1-propanol, MgCl₂, and p-nitrophenylphosphate) at 37 °C for 30 min. The conversion of p-nitrophenylphosphate to p-nitrophenol was stopped by adding NaOH, and the absorbance at 405 nm was measured with a spectrophotometer (UV-vis 8500, Shanghai). The ALP activity was normalized by the total intracellular protein contents. The ALP activity of the cells cultured on AKT bioceramics was used as the control.

2.5 Bone-related gene expression of 3T3 cells on PDB-AKT bioceramics

The osteogenic differentiation of 3T3 on PDB-AKT and pure AKT bioceramic discs was further assessed by real-time quantitative RT-PCR (RT-qPCR) to measure the mRNA expression of ALP (alkaline phosphatase), Runx2 (Runt-related transcription factor 2) and Col 1 (collagen type 1). Discs were transferred into 24-well plastic culture plates and a total of 1 × 10⁵ 3T3 were placed onto each disc. The cells were incubated at 37 °C in 5% CO₂ for 7 and 14 days. On day 7 and 14, the samples were removed and the total RNA was isolated using Trizol Reagent® (Invitrogen) according to the manufacturer's instructions. Relative expression levels for each gene were normalized against the Ct value of the house keeping gene and determined by using the delta Ct method. Three samples were used for this test.

2.6 Ionic concentration analysis

After cell culture, the ionic concentrations of Si, Ca, P and Mg in the cell culture medium were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Varian Co., USA).

2.7 Statistic analysis

All data were expressed as means standard deviation (SD) and were analyzed using one-way ANOVA with a post hoc test. A p-value < 0.05 was considered statistically significant.

3. Results

3.1 Characterization of PDB-AKT bioceramics

The SEM micrographs of AKT bioceramics before and after the polydopamine modification are shown in Fig. 1. It can be seen that most of the crystal grains of the AKT bioceramics are sintered with a clear crystal boundary (Fig. 1a). After modification by polydopamine, a thin polydopamine layer covered the surface of the AKT bioceramics and the crystal boundary could not be observed (Fig. 1b). From the micrograph of the cross section, an obvious layer of polydopamine can been found on the surface of the PDB-AKT ceramic discs and the thickness of this layer is about 20 nm (Fig. 1c).

Fig. 1 The surface morphology of AKT (a) and PDB-AKT (b). The cross section of PDB-AKT (c).

The XRD patterns of the AKT bioceramics before and after the polydopamine modification are shown in Fig. 2. It was obvious that only the AKT characteristic peaks exist in the patterns for both the AKT and PDB-AKT bioceramic discs. The intensity of the characteristic peaks for PDB-AKT is much lower than that for AKT (Fig. 2).

Fig. 2 XRD analysis for AKT and PDB-AKT bioceramics.

AFM analysis shows that the concentration of polydopamine greatly influences the surface roughness of the AKT bioceramics (Fig. 3). It is shown that both the peak roughness and average roughness reach a peak value at the concentration of 2 mg mL⁻¹ and then decrease as the concentration of polydopamine increases (Fig. 3a and b).

Fig. 3 The effect of the PDB modification on the surface roughness of AKT bioceramics by AFM analysis. (a) Average roughness and (b) Peak value.

The water contact angle of AKT and PDB-AKT bioceramics is 50.4 ± 3.0° and 37.8 ± 7.0°, respectively. The hydrophilicity of PDB-AKT is significantly higher than that of AKT bioceramics (P < 0.05) (Fig. 4).

Fig. 4 The contact angle of PDB-AKT and AKT bioceramics. *: significant difference (p < 0.05) between the PDB-AKT group and the AKT group.

3.2 The apatite mineralization of PDB-AKT bioceramics

The SEM micrographs of the ceramics soaked in SBF for various time periods are presented in Fig. 5. Before the ceramics were soaked in SBF, both PDB-AKT and AKT bioceramics show smooth surfaces (Fig. 5a and b). After soaking for 1 day in SBF, there is no obvious apatite mineralization on the surface of AKT bioceramics (Fig. 5c). In contrast, PDB-AKT bioceramics induce obvious apatite mineralization (Fig. 5d). After soaking for 3 days, there are a few apatite particles deposited on the surface of the AKT bioceramic discs (Fig. 5e); however, there are a great number of apatite clusters with a diameter of 500 nm on the surface of PDB-AKT bioceramics (Fig. 5f). After soaking for 7 days, AKT bioceramics induce nano-apatite mineralization and the size of the formed apatite particles is around 100 nm (Fig. 5g); however, PDB-AKT bioceramics induce lath-like apatite crystals with the size of several hundred nanometers in length and several tens of nanometers in diameter (Fig. 5h). EDS analysis shows that there is no P element detected in the patterns of the PDB-AKT bioceramics before soaking in SBF (Fig. 5i); however, after soaking in SBF for 1, 3 and 7 days, P signals are distinct in the EDS patterns, where the Ca/P ratio for the formed apatite is 8.01, 1.96 and 1.61, respectively (Fig. 5j–l).

Fig. 5 The surface morphology of AKT (a, c, e and g) and PDB-AKT (b, d, f and h) bioceramics soaked in SBF for different time periods: (a, b) 0 day, (c, d) 1 day, (e, f) 3 days and (g, h) 7 days. PDB-AKT induced quicker apatite mineralization than AKT. (i), (j), (k) and (l) are the EDS analysis for the PDB-AKT bioceramics soaked in SBF for 0, 1, 3 and 7 days, respectively.

FTIR analysis confirms that polydopamine was coated on the AKT bioceramics and apatite mineralization was formed on the surface of the PDB-AKT bioceramics (Fig. 6). There are two obvious P–O peaks at 562 and 601 cm⁻¹ in the pattern of the PDB-AKT bioceramics after soaking in SBF; however, there are no distinct P–O peaks at 562 and 601 cm⁻¹ in the AKT pattern (Fig. 6).

Fig. 6 FTIR analysis for AKT and PDB-AKT bioceramics before and after soaking in SBF.

3.3 Attachment, proliferation, ALP activity and bone-related gene expression of 3T3 cells on PDB-AKT bioceramics

3T3 cell attachment and morphology on AKT and PDB-AKT bioceramic discs are examined by SEM (Fig. 7). It is shown that after 1 and 7 days of culture, both the AKT and PDB-AKT bioceramic discs support 3T3 cell attachment; however, there are more cells on the surface of the PDB-AKT bioceramics discs than on the AKT bioceramics at both day 1 and day 7 (see white arrows, Fig. 7a, b, e and f). High magnification images show that cells spread well and have a close attachment in both discs types (Fig. 7c, d, g and h).

Fig. 7 3T3 cell attached on AKT (a, c, e and g) and PDB-AKT (b, d, f and h) bioceramics at day 1 and day 7. (a, b, c, d) For day 1, (e, f, g h) for day 7, (c), (d), (g), (h) are the higher magnification images. White arrows point to cells.

MTT analysis shows that cell proliferation on the AKT and PDB-AKT bioceramics increases distinctively with increasing culture time (Fig. 8). The cell attachment rate of AKT and PDB-AKT is (89.1 ± 10.2)% and (91.1 ± 11.6)%, respectively. There are no obvious differences for cell proliferation between the two types of disc after incubation for 1 and 3 days. However, at day 7, the proliferation of 3T3 cells on PDB-AKT bioceramics is significantly higher than that on the pure AKT bioceramics (p < 0.05) (Fig. 8).

Fig. 8 The proliferation of 3T3 cells after culture on AKT and PDB-AKT ceramics. *: significant difference (p < 0.05) between the PDB-AKT group and the AKT group.

There is no significant difference for the ALP activity of 3T3 cells on the two ceramic discs at day 3; however, the ALP activity of 3T3 cells on PDB-AKT bioceramic discs is significantly higher than that of the AKT bioceramic discs at day 7 (Fig. 9).

Fig. 9 The ALP activity of 3T3 cells after culture on AKT and PDB-AKT ceramics. *: significant difference (p < 0.05) between the PDB-AKT group and the AKT group.

RT-qPCR analysis shows that the polydopamine modification on the surface of the AKT bioceramics can enhance the bone-related gene expression of 3T3 cells. The 3T3 cells cultured on PDB-AKT bioceramics have a higher ALP gene expression than those on AKT bioceramics at both day 7 and 14 (Fig. 10a). Runx2 expression at day 7 and Col 1 expression at day 14 for 3T3 cells on PDB-AKT bioceramics are significantly higher than those on AKT bioceramics (Fig. 10b and c).

Fig. 10 Bone-related gene expression of 3T3 cells on AKT and PDB-AKT bioceramics. (a) ALP, (b) RUNX 2 and (c) COL. *: significant difference (p < 0.05) between the PDB-AKT group and the AKT group at day 7. **: significant difference (p < 0.05) between the PDB-AKT group and the AKT group at day 14.

The ionic concentrations of Si, Ca, P and Mg in the cell culture medium are also tested (Fig. 11). It is found that after 1 day of culture, the ionic concentrations of Ca²⁺, Mg²⁺, PO₄³⁻ and SiO₄⁴⁻ ions in the AKT and PDB-AKT culture media are significantly different and there is no obvious difference for the ion concentrations at both days 3 and 7.

Fig. 11 The ionic concentrations of Si, Mg, Ca and P in the medium cultured with the PDB-AKT and AKT bioceramics.

4. Discussion

Previous studies have reported that many bioinert materials, such as metals and plastics can induce a self-assembled layer of polydopamine on their surface after soaking in a dopamine solution.¹ This process may involve the oxidation of the catechol moiety of dopamine to a quinone, followed by polymerization in a manner reminiscent of melanin formation.¹ Polydopamine may then form strong covalent and noncovalent interactions with the substrates.¹ In this study, we, for the first time, applied polydopamine to modify bioactive ceramics and tried to generate a layer of polydopamine on the surface of AKT bioceramic discs to improve their bioactivity and cytocompatibility. SEM, XRD and FTIR analysis confirmed that the polydopamine layer formed on the surface of AKT bioceramic discs. The result suggests that the facile incubation of AKT bioceramics in a dopamine solution is a viable method to form a self-assembled polydopamine layer on their surface, further indicating that polydopamine has universal adhesion characteristics on both bioactive and bioinert materials.

We further investigated the effect of the polydopamine modification of AKT bioceramic discs on physicochemical and biological properties. AFM analysis suggests that the polydopamine modification can obviously increase the surface roughness of AKT bioceramic discs and further tailors the roughness by adjusting the initial concentration of the dopamine solution. Before soaking in the dopamine solution, the surface of the original ceramics is generally smooth. In contrast, the value of the roughness increases rapidly due to the formation of a loose polydopamine layer after soaking in the low concentration dopamine solution. With the increase of dopamine concentration, more and more polydopamine molecules may adhere on the surface and the polydopamine layer becomes dense and therefore the surface roughness decreases. Our study further showed that PDB-AKT bioceramics had an improved hydrophilicity than pure AKT bioceramics. Therefore, the polydopamine modification also has an obvious effect on the hydrophilicity of AKT bioceramics. It is generally accepted that the formation, growth and maintenance of the tissue–biomaterials interface is closely related to apatite mineralization.^24–27 Furthermore, a layer of mineralized apatite is probably able to adsorb serum proteins and growth factors, which then stimulate cell proliferation and activate cell differentiation according to previous studies.^28,29 Therefore, this kind of apatite layers on the surface of biomaterials possesses the capacity to enhance osteoblastic activity. A recent study showed that the polydopamine modification can offer bioinert materials (e.g. plastic, mental and nylon) an apatite mineralization ability.⁶ However, there is no report about the exact effect of the polydopamine modification of bioactive ceramics on their apatite mineralization. In this study, the results of SEM, EDS and FTIR analysis have indicated that the polydopamine modification significantly promotes apatite mineralization on the surface of AKT bioceramics in SBF. Previous studies showed that the typical mechanism of apatite mineralization on biomaterials is that bioactive materials firstly release Na⁺ and/or Ca²⁺ ions and induce the formation of a negative surface with OH⁻ groups, which provides the nucleation sites for apatite mineralization.^30,31 In this study, AKT bioceramics themselves can release parts of Ca²⁺ ions, which can promote apatite mineralization. At the same time, the polydopamine modification provides a great number of bioactive groups, such as OH⁻ and NH₂⁻ groups, which further induces apatite mineralization in SBF. Therefore, it is reasonable to speculate that the combination of Ca²⁺ ions released from AKT bioceramics and bioactive groups from polydopamine significantly promote their apatite mineralization. In addition, previous studies showed that the surface roughness of biomaterials played an important role in apatite mineralization.^32,33 In this study, we found that the surface roughness of AKT bioceramics increased obviously after the polydopamine modification. The enhanced surface roughness may be another important factor for enhancing the apatite formation of AKT bioceramics by providing more nucleation sites for apatite mineralization.

Most interestingly, our study has found that the polydopamine modification of AKT bioceramics significantly promoted the attachment, proliferation, ALP activity and bone-related gene expression of 3T3 cells. It is well known that the surface chemistry, hydrophilicity and surface roughness of biomaterials are the main factors influencing the interaction between cells and biomaterials.^34–38 There are three possible reasons that the polydopamine modification enhances the biological response of cells on AKT bioceramics. Firstly, both the improved surface roughness,³⁷ hydrophilicity, and bioactive functional groups (e.g. OH⁻ and NH₂⁻)⁸ for PDB-AKT bioceramics are probably responsible for the improvement of the attachment, proliferation and differentiation of the cells. Secondly, previous studies indicated that apatite mineralization on the surface of biomaterials benefits protein adsorption and therefore contributes to the proliferation and differentiation of cells.^28,29 The improvement of apatite mineralization of PDB-AKT bioceramics may be the another important reason to enhance the adsorption of serum proteins and further improve attachment, proliferation, differentiation and bone-related gene expression of 3T3 cells. Thirdly, it is found that the ionic concentrations of Ca²⁺, Mg²⁺, PO₄³⁻ and SiO₄⁴⁻ ions in the AKT and PDB-AKT culture media are significantly different after 1 day of culture despite the fact that there is no obvious difference for the ions concentrations at both day 3 and 7. The difference of ionic environment at an early stage of cell culture (e.g. day 1) may influence cell response to biomaterials.^39,40 AKT showed higher concentrations of both Si and Mg than PDB-AKT in the medium at the early stage (day 1) of cell culture, which may be due to the inhibitory effect of polydopamine on the ion release from AKT ceramics. Previous studies showed that Si ions from AKT bioceramics play an important role in influencing cell proliferation, when the concentrations of Si ions were lower than 2.25 mM (around 63 ppm) Si ions could stimulate the proliferation of osteoblast; however, if Si concentrations were higher than 63 ppm, Si ions inhibited cell proliferation.¹³ In this study, the Si concentrations in AKT and AKT-PDB are 100 and 55 ppm, respectively. The high Si concentrations of the AKT group may have an inhibitory effect on cell proliferation. However, this inhibitory effect of high Si concentrations may not immediately affect cell proliferation at day 1, a relatively early stage, leading to no significant difference in cell proliferation between the AKT and AKT-PDB groups; however, with an increasing culture time, more and more Si ions from AKT might be uptaken by cells and lead to an inhibitory effecton cell proliferation due to high the concentration of Si ions. Therefore, cell proliferation on AKT ceramics is lower than that on AKT-PDB ceramics at day 7. Besides this, it is known that cellular responses to biomaterials are influenced by several factors, including ionic compositions, ionic concentrations, pH value and the surface physicochemistry of biomaterials. In our study, after modification by polydopamine, the surface composition and microstructure, including hydrophilicity and surface roughness of the AKT bioceramics changed. The improved hydrophilicity and functional groups on the AKT-PDB ceramics could be the other important factor to improve cell proliferation on their surface, compared to pure AKT ceramics.

In addition, it is known that ALP is a marker for indicating the early-stage osteogenic differentiation of bone-forming cells. Collagen is the major matrix component of the bone extracellular matrix and is expressed at the beginning of osteoid matrix deposition. Previous studies showed that if the cells were induced toward osteogenic differentiation, it did not mean that all bone-related genes would have the corresponding improved expression. Since cell differentiation is a complex process, sometimes the corresponding genes may have a competitive effect, which may influence their gene expression.³⁹ Therefore, in this study, although most genes had been improved by the polydopamine modification, some genes, such as Col 1, decreased at day 7. It is interesting to find that the phosphate concentrations for the blank control are higher than those for AKT and PDB-AKT. Both AKT and PDB-AKT bioceramics possess an apatite-mineralization ability, which will consume parts of the phosphate elements in the medium, leading to the decrease of phosphate concentration, compared to blank control.

5. Conclusions

Polydopamine-modified AKT ceramics were successfully prepared by incubating AKT bioceramics in a dopamine/Tris–HCl solution through a self-assembly processes. Polydopamine modification significantly enhanced the surface roughness, hydrophilicity, apatite-mineralization ability, cell attachment, proliferation, ALP activity and bone-related gene expression of 3T3 cells on AKT ceramics. This study indicates that the polydopamine modification can be also applied to bioactive ceramics for better bone regeneration applications, suggesting that the polydopamine modification is a universal method to enhance bioactivity for both bioinert and bioactive materials.

Acknowledgements

Funding for this study was provided by the Recruitment Program of Global Young Talent, China (Dr Wu) and the Natural Science Foundation of China (Grant 81201202 and 81190132).

References

1. H. Lee, S. M. Dellatore, W. M. Miller and P. B. Messersmith, Science, 2007, 318, 426–430.
2. J. H. Waite and X. Qin, Biochemistry, 2001, 40, 2887–2893.
3. S. C. Nicklisch and J. H. Waite, Biofouling, 2012, 28, 865–877.
4. H. Lee, B. P. Lee and P. B. Messersmith, Nature, 2007, 448, 338–341.
5. H. Lee, N. F. Scherer and P. B. Messersmith, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 12999–13003.
6. J. Ryu, S. H. Ku, H. Lee and C. B. Park, Adv. Funct. Mater., 2010, 20, 2132–2139.
7. P. B. Messersmith, H. Lee and J. Rho, Adv. Mater., 2009, 21, 431–434.
8. C. Wu, W. Fan, J. Chang and Y. Xiao, J. Mater. Chem., 2011, 21, 18300–18307.
9. L. L. Hench and J. M. Polak, Science, 2002, 295, 1014–1017.
10. L. L. Hench and I. Thompson, J. R. Soc. Interface, 2010, 7(Suppl_4), S379–S391.
11. A. J. Salinas, P. Esbrit and M. Vallet-Regi, Biomater. Sci., 2013, 1, 40–51.
12. C. Wu, J. Chang, W. Zhai, S. Ni and J. Wang, J. Biomed. Mater. Res., Part B, 2006, 78, 47–55.
13. C. Wu, J. Chang, S. Ni and J. Wang, J. Biomed. Mater. Res., A, 2006, 76, 73–80.
14. C. Wu and J. Chang, J. Biomater. Appl., 2006, 21, 119–129.
15. H. Sun, C. Wu, K. Dai, J. Chang and T. Tang, Biomaterials, 2006, 27, 5651–5657.
16. S. W. Choi, Y. Zhang and Y. Xia, Langmuir, 2010, 26, 19001–19006.
17. G. Liu, C. T. Wu, W. Fan, X. G. Miao, D. C. Sin, R. Crawford and Y. Xiao, J. Biomed. Mater. Res., Part B, 2011, 96, 360–368.
18. Q. H. Liu, L. Cen, S. Yin, L. Chen, G. P. Liu, J. Chang and L. Cui, Biomaterials, 2008, 29, 4792–4799.
19. Y. Huang, X. Jin, X. Zhang, H. Sun, J. Tu, T. Tang, J. Chang and K. Dai, Biomaterials, 2009, 30, 5041–5048.
20. C. T. Wu and J. Chang, Mater. Lett., 2004, 58, 2415–2417.
21. T. Kokubo and H. Takadama, Biomaterials, 2006, 27, 2907–2915.
22. C. Wu, Y. Zhou, W. Fan, P. Han, J. Chang, J. Yuen, M. Zhang and Y. Xiao, Biomaterials, 2012, 33, 2076–2085.
23. C. Wu, Y. Ramaswamy and H. Zreiqat, Acta Biomater., 2010, 6, 2237–2245.
24. L. L. Hench, Biomaterials, 1998, 19, 1419–1423.
25. L. L. Hench, J. Am. Ceram. Soc., 1991, 74, 1487–1510.
26. C. Wu, W. Fan, M. Gelinsky, Y. Xiao, J. Chang, T. Friis and G. Cuniberti, J. R. Soc. Interface, 2011, 8, 1804–1814.
27. C. Wu, Y. Ramaswamy, D. Gale, W. Yang, K. Xiao, L. Zhang, Y. Yin and H. Zreiqat, Acta Biomater., 2008, 4, 569–576.
28. C. Wu, J. Chang, W. Zhai and S. Ni, J. Mater. Sci.: Mater. Med., 2007, 18, 857–864.
29. N. Olmo, A. I. Martin, A. J. Salinas, J. Turnay, M. Vallet-Regi and M. A. Lizarbe, Biomaterials, 2003, 24, 3383–3393.
30. L. L. Hench, R. J. Splinter and T. K. Greenlee, J. Biomed. Mater. Res., 1971, 5, 117–141.
31. L. L. Hench and J. Wilson, Science, 1984, 226, 630–636.
32. D. O. Costa, B. A. Allo, R. Klassen, J. L. Hutter, S. J. Dixon and A. S. Rizkalla, Langmuir, 2012, 28, 3871–3880.
33. V. Mourino, P. Newby, F. Pishbin, J. P. Cattalini, S. Lucangioli and A. R. Boccaccini, Soft Matter, 2011, 7, 6705–6712.
34. C. Wu, Y. Zhou, M. Xu, P. Han, L. Chen, J. Chang and Y. Xiao, Biomaterials, 2013, 34, 422–433.
35. F. Zhang, S. W. Liu, Y. Zhang, Z. G. Chi, J. R. Xu and Y. Wei, J. Mater. Chem., 2012, 22, 17159–17166.
36. C. T. Wu, W. Fan, Y. H. Zhou, Y. X. Luo, M. Gelinsky, J. Chang and Y. Xiao, J. Mater. Chem., 2012, 22, 12288–12295.
37. C. Luo, L. Li, J. R. Li, G. Yang, S. Ding, W. Zhi, J. Weng and S. B. Zhou, J. Mater. Chem., 2012, 22, 15654–15664.
38. C. Wu, Y. Ramaswamy, X. Liu, G. Wang and H. Zreiqat, J. R. Soc. Interface, 2009, 6, 159–168.
39. P. Han, C. Wu and Y. Xiao, Biomater. Sci., 2013, 1, 379–392.
40. Y. Zhou, C. Wu and Y. Xiao, Acta Biomater., 2012, 8, 2307–2316.

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