Biomineralization and osteogenic differentiation modulated by substrate stiffness

European Polymer Journal xxx (xxxx) xxxx

Contents lists available at ScienceDirect

European Polymer Journal journal homepage: www.elsevier.com/locate/europolj

Biomineralization and osteogenic differentiation modulated by substrate stiffness ⁎

Ruitao Jina, Yuting Wanga, Yanpeng Liub, Chunmei Dinga, Jing Xiea, , Jianshu Lia,

⁎

a

College of Polymer Science and Engineering, Sichuan University, Chengdu 610065, PR China Zhejiang Provincial Key Laboratory for Drug Evaluation and Clinical Research, Research Center for Clinical Pharmacy, First Affiliated Hospital, Zhejiang University, NO. 79 Qingchun Road, Hangzhou 310003, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Copolymer Stiffness Biomineralization Osteogenic differentiation Coatings

It is well known that extracellular matrix stiffness can affect cell proliferation and differentiation. However, when substrates of different stiffness are transferred into organisms, they affect cell behavior, and at the same time, the substrates themselves also undergo biomineralization owing to the extracellular matrix, this process of biomineralization during osteogenic differentiation adjusted by substrate stiffness has seldom been investigated. In this paper, we copolymerized 2-vinyl-terephthalic acid bis-[4-(2-carboxy-ethyl)-phenyl] ester (LCPE) as rigid chains and butyl acrylate (BA) as flexible chains to prepare a series of polymer materials. Films based on these copolymers are prepared by dip-coating method with controllable stiffness. Vickers indentation hardness tester was used to verify the stiffness of the films (199.9–5.3 kPa) with different ratio of LCPE and BA. Polymer coated substrates with different stiffness on biomimetic mineralization demonstrated that a moderate stiffness substrate (14.9 kPa) can induce the best biomimetic mineralized hydroxyapatite (HA), and further promote the proliferation and differentiation behavior of osteoblasts.

1. Introduction In natural tissues, the extracellular matrix (ECM) constitutes most of the microenvironment of cells. The complex physical and biochemical clues of ECM play an important role in guiding the fate of cells [1–3]. In particular, extracellular matrix microenvironment regulates cellular forces, adhesion, migration, and polarization through mechanical signaling pathway [4–6]. Mooney et al. demonstrated that substrate stress relaxation and stiffness as the fundamental physical properties of model ECM, have a substantial impact on cell behavior and function [7]. Furthermore, stiffness, as a typical factor, regulates the role of cells, have been widely investigated. For example, relatively soft substrates (< 1 kPa) promote the differentiation of mesenchymal stem cells (MSCs) into adipocytes, while harder substrates (34 kPa) promote osteogenic differentiation of cells [8–10]. Moreover, Lee et al. cultured osteoblasts on controllable stiffness substrates constructed with engineered bacteriophages, it was found that the higher the stiffness of the matrix (97.9 kPa) achieve the better extension and osteogenic differentiation of MC3T3 cells [11]. However, osteoblasts spontaneously secrete and synthesize collagen and osteoprotein fibers in vivo, no matter there is implants or not; meanwhile, calcium and phosphorus are absorbed into the pore of the fibers to precipitate and crystallize,

⁎

resulting in the biomineralization process and the formation of new bone tissue [12,13]. As we all know, the coating of CaP-based on the surface of implant material can improve the biocompatibility and bone conductivity of the material, and promote the osteogenic differentiation of osteopoietic progenitor cells [14–19]. In addition, CaP-based mineral layer can improve the osseointegration of implants [20–24]. As the transferred implanted materials into the matrix can influence the behavior of osteoblasts, at the same time, there is a certain biomineralization process on the substrate of implanted materials. Thus, it′s vital important to study the biomineralization behavior of implant materials induced by the ECM microenvironment. To the best of our knowledge, the process of biomineralization during osteogenic differentiation adjusted by substrate stiffness has seldom been investigated. Nowadays, the preparation of CaP coatings includes physical method (plasma spraying), chemical method (sol-gel method, alternating immersion method, etc.), and biomimetic mineralization, [25–29] and factors influencing the deposition of CaP-based minerals like biological organic matter, crystal growth mechanism and external environment are also investigated [30–33]. Whereas, whether substrate stiffness affects mineralization has been rarely studied. Here, we expect to prepare a stiffness-adjustable 2-dimensional (2D) polymer thin layers, investigate the mineralization behavior in the simulated

Corresponding authors. E-mail addresses: [email protected] (J. Xie), [email protected] (J. Li).

https://doi.org/10.1016/j.eurpolymj.2019.109395 Received 23 October 2019; Received in revised form 25 November 2019; Accepted 29 November 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Ruitao Jin, et al., European Polymer Journal, https://doi.org/10.1016/j.eurpolymj.2019.109395

European Polymer Journal xxx (xxxx) xxxx

R. Jin, et al.

adjusting the ratio of LCPE to BA (1:0.5, 1:1, 1:2, 1:3, 1:5). AABA copolymer were synthesized in the same way above, and by controlling the ratio of AA to BA the copolymer have the corresponding carboxyl content with LCBA.

physiological conditions and further osteoblast′s behavior based on it. There are many kinds of methods to prepare materials with tunable mechanical properties in previous studies, [34–39] and copolymerizing a rigid monomer with a soft monomer to obtain copolymer materials with different stiffness by adjusting the ratio of the two monomers was adopted in this work. Vinyl terephthalic acid (VPTA) is chosen as raw material, which has rigid side chains and mineralizable carboxyl groups [40]. Based on the effect of “side-chain jacketed effect” on the rigidity enhancement of the main chain, [41–43] chemical modification was used to enhance the rigidity of the main chain while maintaining the carboxyl units of its side chain, so that the copolymer with mineralization ability and gradient mechanical properties could be obtained. Butyl acrylate (BA) is chosen as it′s an ideal materials in the preparation of block polymers and self-healing materials due to its flexible chains and high adhesion and good film-forming property, [44,45] and PBA networks are cell compatible for murine and human cells [46]. In this paper, a rigid monomers (2-vinylterephthalic acid bis [4-(2carboxyethyl) -phenyl] esters, LCPE) with mineralizing activity were chemically modified, and then copolymerized with a soft monomer (butyl acrylate, BA) in different proportions. A series of polymer films were obtained by dip-coating method with controllable stiffness. The stiffness of the films was characterized by Vickers hardness. The biomimetic mineralization experiments in vitro were carried out using these films as substrates to simulate the biomineralization. Scanning electron microscope (SEM) and X-ray diffraction (XRD) were adopted to study the content and purity of HA in the mineral layer. The mineralization behavior under different stiffness, and the effects of different stiffness on osteoblasts′ behavior before and after mineralization were investigated.

2.4. Characterization of chemical and thermal properties of materials 1

H NMR was carried out on a Bruker ARX400 spectrometer, where deuterated dimethyl sulfoxide (DMSO-d6) was used as the solvent and tetramethylsilane (TMS) as the internal standard. The characteristic peaks of polymer was analyzed by fourier transform infrared spectroscopy (FTIR) spectra (Nicolet 6700, Thermo ScientificTM, USA). The samples were prepared by KBr pressing method and scanned by the transmission mode among the wavelength ranged from 500 to 4000 cm−1. The molecular weight of the synthesized monomer was measured by liquid chromatography-mass spectrometry (LCMS-8080, Japan). Gel permeation chromatography (PL-GPC 220) was utilized to determine the number-average molecular weight (Mn) and molecular weight distribution (PDI, Mw/Mn). 2.5. Preparation and characterization of copolymer films The dip-coating method was used in this experiment. A suitable amount of copolymer solid powder was dissolved in THF with the concentration of 100 mg/mL. After complete dissolution, the solution was dip-coated on the substrate (glass/titanium sheet), the films were obtained after drying in an oven at 60 °C for 5 h. Thickness gauge was used to measure film thickness, and Vickers indentation hardness tester was used to estimate the stiffness.

2. Materials and methods

2.6. In vitro biomimetic mineralization experiment

2.1. Materials

Titanium sheet is used as the substrate for copolymer film mineralization. The standard 1.5 times simulated body fluid (1.5 × SBF) was prepared according to the conventional method at the constant temperature of 37 °C and pH 7.4. The copolymer films coated titanium sheets was immersed and upside down in the SBF solution. Induced biomimetic mineralization was carried out at 37 °C for 3 weeks, and the SBF solution were changed daily.

Vinyl terephthalic acid (VTPA) as a key monomer was purchased from Sigma-Aldrich. P-Hydroxybenzene propanoic acid (PHPA), butyl acrylate (BA), acrylic acid (AA), oxalyl chloride (COCl)2, azodiisobutyronitrile (AIBN), and N-methylmorpholine were purchased from TCI. Dichloromethane (CH2Cl2), N,N-dimethylformamide (DMF), and tetrahydrofuran (THF) as solvents were purchased from Energy Chemistry. L (+)-Ascorbic acid, alpha modified Eagle medium (α-MEM), phosphate buffer saline (PBS), fetal bovine serum (FBS), rhodamine-labeled phalloidin, 4′,6-diamidino-2-phenylindole (DAPI), and penicillinstreptomycin solution were used in cell experiment. Deionized water was used throughout the experiment.

2.7. Structural and compositional analysis of in situ mineralized coating Scanning electron microscopy (SEM; JEOL, JSM-7500F, Japan) was employed to observe the surface micromorphology and distribution of the in situ mineralized hydroxyapatite crystals. Energy dispersive spectroscopy (EDS) was used to determine the calcium-phosphorus ratio of hydroxyapatite formed in situ mineralization. X-ray diffraction (XRD; Dmax = 1400, 40 kV, 110 mA, Japan) was employed to analyze the crystal orientation and mineral phase of the mineralized coating, the diffraction patterns were obtained over the 2θ range of 5–90°.

2.2. Synthesis of rigid monomer 576 mg vinyl terephthalic acid was dissolved in 5 mL CH2Cl2, stirred in ice bath, rapidly 3 mL (COCl)2 was added, and reacted with 35 °C for 6 h. Then unreacted solvent was removed by rotary evaporation, and the solid was obtained. 1195 mg PHPA (1.2/1) was dissolved in 5 mL THF and 1.74 mL N-methylmorpholine was added as acid dressing agent to obtain PHPA solution. The solid obtained from the previous step was dissolved in THF, with PHPA solution slowly dripped to this solution, and reacted at room temperature for 24 h. Then, a rigid monomer, 2-Vinyl-terephthalic acid bis-[4-(2-carboxy-ethyl)-phenyl] ester (LCPE) was prepared.

2.8. MC3T3-E1 cell culture The MC3T3-E1 Subclone 24 cell line (Procell, China) derived from mouse osteoblasts was cultured to biocompatibility of the mineralized polymer materials. The osteoblastic cells were inoculated into 24-well cell culture plates with unmineralized and mineralized coatings, and the cell density was about 3000 cells per hole (α-MEM medium with 10% FBS and 1% antibiotics). The orifice plate was placed in an incubator containing 5% CO2 at 37 °C and incubated for 24 h to allow the cells fully adhere to the wall and spread out.

2.3. Synthesis of copolymer LCBA and AABA The appropriate amount of LCPE was dissolved in DMF, 300 mg BA, 5 mg AIBN (1 mol%) were added successively, deoxidized by N2 for 30 min. Free radical copolymerization was carried out for 24 h at 65 °C, then the reaction solution was dripped into methanol to precipitate the product. The copolymer LCBA with different rigidity were obtained by

2.9. Cytotoxicity and cell proliferation The cytotoxicity and cell proliferative capacity of the mineralized 2

European Polymer Journal xxx (xxxx) xxxx

R. Jin, et al.

The mass spectra of esterification products also confirmed this result (Fig. S2). Then, LCPE and BA in different mass proportions (2:1, 1:2, 1:3, 1:5) were copolymerized by free radical copolymerization to obtain polymer materials. As shown in Table 1, the actual polymerization ratio of LCPE to BA was calculated by the integral ratio of hydrogen on the benzene ring to hydrogen on the methylene group. According to the 1H NMR spectra of the copolymer (Fig. 1A), the disappearance of the characteristic hydrogen peaks of carbon-carbon double bonds (σ = 5–6) indicated that the monomers were polymerized. The coexistence of the characteristic hydrogen peaks a on the benzene ring of LCPE and the characteristic hydrogen peaks b and c on the methylene of BA confirmed that the two monomers were copolymerized. The chemical structure of the copolymer was further determined by FTIR (Fig. 1B). The broad peak at 3500–3200 cm−1 corresponds to the –OH association peak on the carboxyl group. Fig. S8 shown the typical Fourier transform infrared (FTIR) spectra for different polymerization ratio of copolymer. Strong bands related to the carbonyl stretching mode of copolymer were easily identified at 1725 cm−1. The characteristic peaks at 1600 cm−1–1450 cm−1 attributed to the skeleton vibration of benzene ring were clearly observed, which further confirmed the successful copolymerization of two monomers. As the composition of LCPE in the copolymer increased, the intensity of the –OH characteristic peak increased gradually, indicating that there is a gradient in the –COOH content of the copolymer with different proportions. The molecular weight of the copolymer was characterized by GPC. The actual Mn, Mw and PDI of the copolymers in different proportions were shown in Table 1. With the increased BA ratio, the molecular weight of the copolymer increased. According to the GPC spectra (Fig. S3), all copolymers′ curves showed a single peak, indicating that the two monomers realized the copolymerization rather than homopolymerization.

materials were tested by Cell Counting Kit-8 (CCK-8) assay kit (MedChemExpress, USA). For CCK-8 assay, cells were cultured on a 96well plate (Nest Biotechnology Co. Ltd., China) at a density of 3 × 104 cells mL−1 for 1, 3, 7 days. At each time point, 110 μL of complete medium with 10 μL of CCK-8 solution were added in each well, incubated for additional 3 h. Eventually, the absorbance at 450 nm was measured using Victor 3 microplate reader (KHB ST-360, China). Cytotoxicity of the material was calculated by one day′s optical density (OD), and cell proliferation was analyzed based on 1, 3 and 7 days′ optical density. 2.10. Cytofluorescence staining To observe the morphology and spreading ability of MC3T3-E1 cells, cells′ F-actin and nucleus were stained by rhodamine-labeled phalloidin and DAPI, respectively, according to the product instructions. First, cells were fixed with 4% paraformaldehyde for 20 min and lysed at room temperature for 10 min with 0.5% Triton X-100, then cytoskeleton were stained for 30 min with rhodamine-labeled phalloidin. Eventually, nuclei were stained for 10 min with DAPI. Fluorescence images were visualized with Fluorescence microscope (IX71, Olympus). 2.11. Alkaline phosphatase (ALP) assay After culturing in the medium for 7 and 14 days, the promotion of osteogenic differentiation was examined by alkaline phosphatase (ALP) assay kit (P0321, Beyotime) and enhanced BCA protein assay kit (P0010, Beyotime). The activity of ALP was determined by following the product instruction. BCA protein assay was utilized to determine the content of the total intracellular protein, which was adopted to normalize the ALP activity.

3.2. Fabrication of copolymer films and stiffness investigation

2.12. Calcium concentration assay

In order to prepare the stiffness controllable substrate, the copolymer solid powder was dissolved in the volatile organic solvent THF at the same concentration (100 mg/mL). Then, the solution was dripped onto the slide and the film area was controlled at 2*2 cm2. Finally, the polymer film could be obtained after solvent evaporation. Thickness gauges were used to test the thickness of different groups of copolymer films. As shown in Fig. S4, all films thickness ranged from 100 to 250 μm. The difference of film thickness between different groups was mainly due to the difficulty in accurately controlling the film area because of the different surface tension of the solution during the process of solution film formation. However, this is within the height range where the stiffness can be accurately measured, and this diversity of thickness would not affect the characterization of stiffness [47]. Then, Vickers indentation hardness tester was used to characterize the stiffness of the films [48,49]. The diagonal length of the indentation on the film surface was measured after the diagonal indentation was maintained for 10 s. The stiffness value HV of the film is calculated according to the following formula:

Intracellular calcium concentration was detected by methyl thymol blue (MTB) colorimetric method (Calcium Assay Kit, C004-2-1, Nanjing Jiancheng Bioenginerring Institute), according to instruction manual. The ability of mineralized materials to promote osteogenic differentiation was indirectly analyzed by the experimental data of calcium concentration. 2.13. Statistical analysis The experimental data were expressed as mean ± standard deviation (S.D.). The values represent the means ± SD of at least three independent experimental data. Graphpad Prism 6 software was used to test the one-way analysis of variance (ANOVA) and double-tailed T test. A P value < 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Characterization of synthesized materials

HV = 0.102 ×

First, hard and soft monomers in different proportions were copolymerized to obtain polymer materials with controllable stiffness. Vinyl terephthalate (VPTA) with rigid chains and butyl acrylate (BA) with flexible chains were used as the hard and soft monomers, respectively. Before polymerization, VPTA was modified to increase rigidity by strengthening its side chain, while retaining the carboxyl unit of the end chain. 2-Vinyl-terephthalic acid bis-[4-(2-carboxy-ethyl)-phenyl] ester (LCPE) with stronger rigidity was obtained by esterification of VPTA and p-hydroxyphenylpropionic acid (PHPA) (Scheme 1). The occurrence of methylene hydrogen peak (σ = 2–3) and the increase of the ratio of hydrogen on the benzene ring (σ = 7–8) can observed in 1H NMR spectrum (Fig. S1), which proved the successful synthesis of LCPE.

2F sin

α 2

d2

F – load (N) α – angle of the opposite side of the indenter α = 136° d – average indentation diagonal length (mm). The indentation length of different groups were measured by the instrument indenter (Fig. 2A). The stiffness values of the copolymer films of each group after calculation are shown in Fig. 2B (For easy comparison, the unit of stiffness converted from N·mm−2 to kPa). It was found that the stiffness of copolymer films showed a non-linear gradient change trend (L2B1-199.9 kPa, L1B2-36.1 kPa, L1B3-14.9 kPa, L1B55.3 kPa) with the change of the ratio of L monomer to B monomer. This 3

European Polymer Journal xxx (xxxx) xxxx

R. Jin, et al.

Scheme 1. (A) The synthetic process of rigid monomer 2-Vinyl-terephthalic acid bis-[4-(2-carboxy-ethyl)-phenyl] ester (LCPE) and copolymer of LCPE/BA. (B) Copolymer films formation and application in biomineralization and osteoblast culture.

polymer coatings. A polymer film was obtained on a titanium sheet by a solution forming method. Then, the coating was inverted and immersed in a 1.5 × SBF solution at 37 °C for 3 weeks. After 3 weeks of mineralization, the micro-morphology of mineralized surface was observed by SEM. The images of mineralized layers on different stiffness substrates are shown in Fig. 3. The mineral deposited on the surface of the L2B1 coating were the least and irregularly aggregated, even though it possessed the highest stiffness. While there are many mineral deposited on the surface of L1B2 and L1B3 coatings, which covering the whole polymer coatings. And there is also a layer of minerals deposited on the surface of L1B5 coating, but most mineral were wrapped by polymer layers, bringing about reduced mineral content on the surface. Overall, the L1B2 and L1B3 coatings with medium stiffness had better mineralization layer. Therefore, it can be concluded that the substrate with moderate stiffness is more conducive to biomimetic mineralization. Furthermore, the component of mineralized coatings was analyzed by EDS. According to the EDS spectrum (Fig. 3), the main elements of each group were C, O, Ca and P, so it could be determined that all mineral layers are CaP materials. The Ca/P ratio of mineral layers in majority groups was lower than that of standard hydroxyapatite (1.67), which may be due to a small amount of other phosphates in minerals. There may be calcium chloride deposited on the surface of L1B5 film, so its Ca/P (1.85) was higher than the standard. Comparing Ca/P of different groups, Ca/P (1.61) of L1B3 group was more close to HA than other groups, indicating that the inorganic mineral structure of L1B3 after 3 weeks of mineralization is

Table 1 The feed ratio, actual ratio, Mn, Mw, PDI of LCPE (L) and BA (B) in different copolymers. Group

Feed Ratio

Actual Ratio

–COOH (mol, %)

Mn (g/mol)

Mw (g/ mol)

PDI

L2B1 L1B2 L1B3 L1B5

1:0.5 1:2 1:3 1:5

1:0.56 1:2.4 1:5.0 1:9.1

47 17 9 5

3936 11,957 11,761 55,311

4934 32,948 29,487 97,204

1.25 2.76 2.51 1.76

confirms the original experimental hypothesis that the stiffness of polymer materials can be improved by increasing the content of rigid monomer. It is proved that the materials stiffness can be changed by simply adjusting the ratio of L monomer to B monomer. 3.3. Biomimetic mineralization research Some studies have demonstrated that hard substrate can promote the proliferation and differentiation of osteoblasts. However, in the actual environment of bone implant repair, the first step in the process of osteogenesis is the deposition of minerals on the substrate surface. In fact, the relationship between biomimetic mineralization and the substrate stiffness has not been well studied. Therefore, we investigated the effect of polymer substrates with different stiffness on biomimetic mineralization. In order to easier cutting and better experimental characterization, titanium sheets (1 × 1 cm2) were used as the substrate of

Fig. 1. Chemical characterization of copolymers. (A) 1H NMR and (B) FTIR spectrum of LCPE/BA copolymers with different proportions (L2B1, L1B2, L1B3, L1B5). 4

European Polymer Journal xxx (xxxx) xxxx

R. Jin, et al.

Fig. 2. Characterization of the properties of copolymer films. (A) The indentation length of indenter in Vickers hardness tester experiment of different copolymer films. (B) Actual stiffness of different copolymer films.

groups, the gap between Ca/P ratio of AA/BA group and standard value was larger, which demonstrated that LC/BA copolymer films induced better mineralization effect. It can be found that the differences among A2B1, A1B2, A1B3, A1B5 groups were very small, indicating that the number of carboxyl units has little effect on the mineralization for 3 weeks. As reported, the number of carboxyl groups in the complexing agent plays a major role in crystallization, stoichiometry and morphological changes in HA, while has no significant effect on the crystal size, [51] and the mineralized layer in this system cover the whole surface after long term biomineralization under enough carboxyl group. On this basis, the effect of carboxyl content on biomineralization can be excluded in this system, so stiffness can be studied as the only influencing factor on the biomineralization. In order to further determine the structure of the mineralized layer obtained by biomimetic mineralization, the mineral was analyzed by XRD, the diffraction pattern of the mineral crystal was shown in Fig. 5. Due to the presence of an organic coating at the bottom of the mineral

more similar to hydroxyapatite. In addition, it should be noted that carboxyl group act as the mineralization site, its content also affect mineralization in short-term mineralization [38]. In order to verify the influence of the content of carboxyl units in this research, soft copolymers with different proportions of acrylic acid and butyl acrylate were copolymerized to obtain copolymers (A2B1, A1B2, A1B3, A1B5) with same ratios of carboxyl groups compared to L2B1, L1B2, L1B3, L1B5 (Table S1, Figure S5, Figure S6). These polymers were prepared and mineralized in the same way as before. As AA and BA possess similar flexible chain structure, and PAA films usually show low Young′s modulus (~1.32 kPa), [50] their polymer membranes with different proportions show relatively soft and viscous properties. After 3 weeks of mineralization, the AA/BA copolymer films were also characterized by SEM-EDS (Fig. 4). The Ca/P ratio of mineral layers on different AA/BA groups was shown in Fig. 4, the values were lower than the standard Ca/P ratio of hydroxyapatite expect A2B1. Comparing with the Ca/P ratio of LC/BA mineralized

Fig. 3. SEM images and EDS spectra of mineralized coatings on polymer surface after 3 weeks′ mineralization (L2B1, L1B2, L1B3, L1B5). 5

European Polymer Journal xxx (xxxx) xxxx

R. Jin, et al.

Fig. 4. SEM images and EDS spectra of mineralized coatings on AA/BA copolymers surface after 3 weeks′ mineralization (A2B1, A1B2, A1B3, A1B5).

mineralization has also been studied, taking the L1B3 group as an example. With the extension of mineralization time, diffraction peak of HA increased gradually, which means more minerals on the coating surface (Fig. 5B).While for the HA diffraction peak on 3 or 4 weeks, there isn′t big difference, verifying the mineralization can be completed in 3 weeks. In addition, there is only peak strength change between different groups without peak displacement, which indicates that the change of stiffness will not affect the crystal form of minerals [54]. On the whole, the results confirmed that these copolymer films can modulate biomimetic mineralization by controlling stiffness.

layer, the baseline at the front of the diffraction curve was higher, but it would not affect the diffraction peak position and peak intensity of the mineral crystal. The broad peak at 19.8° (Fig. 5A) was attributed to the b-sheet crystalline domain of the LCPE chain. The (2 1 1), (0 0 2), (2 2 0), (2 1 3), (0 0 4) and (3 2 3) diffraction peaks are characteristic peaks of HA, [52,53] which appeared on the copolymer coating. By comparison with the XRD peaks of standard HA, the mineral diffraction peaks of these groups except L2B1 group can correspond to the diffraction peaks of HA, confirming the formation of HA. By comparing the HA peaks of L1B2 group and L1B3 group (Fig. 5A), it could be found that the intensity of L1B3 group was slightly higher, indicating that the content of HA of L1B3 was higher. Therefore, the moderate stiffness can induce the best biomimetic mineralization effect. The effect of time on

Fig. 5. XRD diffraction patterns of HA from mineralization of polymer films. (A) XRD of HA from 4 groups (L2B1, L1B2, L1B3, L1B5). (B) XRD of HA from L1B3 group at different mineralization times (1–4 weeks). 6

European Polymer Journal xxx (xxxx) xxxx

R. Jin, et al.

M−L1B3 group. As shown in Fig. 7, there were more and larger osteogenic cells on M−L1B3 coating compared with the other two mineralized groups, which indicated that the excellent HA mineral layer could promote the adhesion and expansion of osteoblasts. After 7 d of culture, the cells proliferated sharply, and there was no significant difference among the mineralized groups. Overall, mineralized coatings showed a much better effect on cells′ proliferation and differentiation, demonstrated that the HA mineral play an important role in cells′ adhesion and spreading. Fluorescent staining of MC3T-E1 cells further confirmed the good cytocompatibility and cell adhesion of the mineralized coating.

3.4. Cellular compatibility of mineralized and unmineralized copolymer films to MC3T3-E1 cells As demonstrated above, the stiffness of the bioactive substrate can affect the content and purity of deposited mineral. During the process of bone repair, osteogenic progenitor cells adhere and proliferate on the implant surface, at the same time, calcium and phosphorus are absorbed into the implants surface to precipitate and crystallize for biomineralization, leading to the formation of new bone tissue [11]. In order to determine whether such changes will further affect the proliferation and differentiation of osteoblasts in the biological environment or not, the proliferation and differentiation of MC3T3-E1 cells on the unmineralized and mineralized substrate is carried out. Firstly, the cytotoxicity and cytocompatibility of the material were verified. MC3T3-E1 cells were cultured on the surface of the coating material after mineralization(named as M−L2B1, M−L1B2, M−L1B3, M−L1B5). Because of the poor mineralization effect and easy cracking after drying of M−L2B1 group, the M−L2B1 group was abandoned in the cell experimental group, and the pore plate group and pure titanium plate group were set as the control group. After 1 d culture, CCK-8 kit was used to test the cytotoxicity. The cell viability calculated by OD value was shown in Fig. 6A. The cell viability of the experimental group with mineralized coating was higher than that of the control group and the Ti group, which indicated that the mineralized polymer coating materials had better biocompatibility. As shown in Fig. 6B, the proliferation of mineralized M−L1B3 group was better than that of M−L1B2 and M−L1B5 group on 3 d and 7 d, which indicated that HA on the M−L1B3 group with better mineralized layer played a pivotal role to promote the proliferation of MC3T3-E1 cells. Besides, the cell compatibility of copolymer materials before mineralization was also tested (named as L1B2, L1B3, L1B5) for comparison. As showed, the polymer materials themselves had no cytotoxicity as well. Furthermore, without mineralization, the cell proliferation on the polymer coating was proportional to the stiffness of the material itself. That is, the higher stiffness of the substrate promote better proliferation of osteoblasts, which is consistent with the conclusion of previous studies [11]. Compared with the proliferation of osteoblasts cultured before and after the mineralization of copolymer film, as shown in Fig. 6B, it is obvious that the mineralized coating has better cell compatibility, which is attributed to the effect of HA on the surface. The adhesion and spreading of MC3T3-E1 cells were observed by fluorescence staining. DAPI and rhodamine-labeled phalloidin were used for staining, it′s clear to see the nucleus (blue) and cytoskeleton (red) of MC3T3-E1 cells under fluorescence microscope (Fig. 7). There were fewer cells in all groups on 1 d than other days, and all the MC3T3-E1 cells had not spread as little red cytoskeleton can be seen in 1 d. The cytoskeleton of the mineralized group expanded more than that of the titanium plate control group on 3 d, especially in the

3.5. Effect of polymer-HA composite on osteoblast function Because cell adhesion and CaP mineral deposition occur simultaneously when restorative materials are implanted in vivo, it is necessary to study the interaction between mineralized materials and cells. Therefore, the ability of different mineralized coatings to promote the differentiation of osteocytes was emphatically studied. Finally, the effects of HA mineral coatings induced by different stiffness substrates on the osteogenic differentiation of MC3T3-E1 cells was investigated. After 7 d and 14 d of culture, the differentiation degree of osteoblasts was determined by testing the ALP activity and the Ca2+ concentration, and BCA kit was used to measure the total protein concentration of cells to accurately calculate the actual ALP content (Fig. S7). It is shown that the ALP activity and intracellular Ca concentration in the experimental group are significantly higher than those in the Ti control group (Fig. 8), as there are a lot of HA crystals on the coating of the experimental group. As the most abundant CaP material in bone, HA has a good promoting effect on the differentiation of osteoblasts [12,14]. It is worth noting that the cells on M−L1B3 coating with the best mineralization effect showed the highest ALP activity at 7 d and 14 d (Fig. 8A). The change trend of intracellular Ca concentration was similar to that of ALP, and M−L1B3 group showed more obvious numerical advantages over M−L1B2 group and M−L1B5 group (Fig. 8B). The mineral layer can further affect the differentiation behavior of osteoblasts, which indicates that the differentiation degree of osteoblasts can be indirectly regulated by adjusting the stiffness of mineralizable base materials. Based on this, the stiffness of implants suitable for different bone repair environments can be designed. By adjusting the stiffness of the polymer film, controllable biomimetic mineralization can be achieved, and the most suitable surface for the growth of preosteoblast can be obtained. 4. Conclusion In summary, we prepared a series of stiffness controllable coatings with mineralizable ability by copolymerization of hard monomer LCPE

Fig. 6. Cellular Compatibility of Composites to MC3T3-E1 cells. (A) The cytotoxicity of mineralized copolymer films (M−L1B2, M−L1B3, M−L1B5) and unmineralized copolymer films (L1B2, L1B3, L1B5). (B) Comparison of proliferative ability among different copolymer coatings. (n = 6) (*P < 0.5, **P < 0.01, ***P < 0.001.) 7

European Polymer Journal xxx (xxxx) xxxx

R. Jin, et al.

Fig. 7. Fluorescence images of MC3T3-E1 cells cultured for 1 d, 3 d, 7 d. cells′ F-actin (red) and nucleus (blue) were stained by rhodamine-labeled phalloidin and DAPI, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

and soft monomer BA. The copolymer material was formed into a film by dip-coating method, and the stiffness of the film can be adjusted from 199.9 to 5.3 kPa by changing the monomer feed ratio. The followed data of biomimetic mineralization show that all polymer films can induce HA mineral deposition, but the stiffness difference will directly affect the mineralization effect. The hardest film is not conducive to the adhesion of HA crystals, and the softest film wrap HA deposited on the surface, only the film with moderate stiffness can induce the most HA mineral formation. The results showed that the mineral layer of L1B3 group with 14.9 kPa stiffness had the highest HA content and had a closer Ca/P ratio to HA. In vitro cell experiments, the composite coatings can better promote cell adhesion, proliferation and osteogenic differentiation compared to the non-mineralized coatings. Most importantly, the optimal mineralization group M−L1B3 modulated by stiffness can promote best osteogenic differentiation. To summarize, the mineralized base controlled by different stiffness after mineralization can lead to different degrees of cell proliferation and differentiation, and a substrate with moderate stiffness can conduce the best biomineralization effect and osteoblast differentiation. This result provides a

rational basis that when designing new implant materials in tissue engineering, it is necessary to take into account the stiffness change induced biomineralization in order to achieve the best repair effect. Author contributions J.L. and J.X. designed the concepts. R.J., Y.W. and Y.L. carried out the experiments. C.D. discussed the results. R.J. and Y.W. designed and carried out the cell experiments. All the authors contributed to the interpretation of the data. J.L. and J.X. supervised the manuscript. Funding sources This work was supported by the National Natural Science Foundation of China (51703138, 51773128, 51925304), Department of Science and Technology of Sichuan Province (2019JDRC0090), China Postdoctoral Science Foundation (Grant No. 2018M633368), and the Fundamental Research Funds for Central Universities.

Fig. 8. The osteogenic differentiation of MC3T3-E1 cells culture on different substrates. (A) ALP activity of different groups was examined by alkaline phosphatase (ALP) assay kit. (B) Intracellular calcium concentration of different groups was detected by methyl thymol blue (MTB) colorimetric method (Calcium Assay Kit). (n = 6) (**P < 0.01, ***P < 0.001.) 8

European Polymer Journal xxx (xxxx) xxxx

R. Jin, et al.

CRediT authorship contribution statement [17]

Ruitao Jin: Conceptualization, Methodology. Yuting Wang: Data curation, Writing - original draft. Yanpeng Liu: Visualization, Investigation. Chunmei Ding: Software. Jing Xie: Supervision, Validation. Jianshu Li: Writing - review & editing.

[18]

[19]

Declaration of Competing Interest We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[20] [21]

Acknowledgement

[22]

P.Y., J.M.L., J.Q.X. and A.Q.L. are gratefully acknowledged for their help during the research. Data availability The raw data and processed data required to reproduce these findings are available to download from https://doi.org/10.17632/ 8h3nyjm5fg.2.

[23]

[24]

[25]

Appendix A. Supplementary material [26]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2019.109395.

[27]

References

[28]

[1] A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, Matrix elasticity directs stem cell lineage specification, Cell 126 (2006) 677–689. [2] A. Colak, B. Li, J. Blass, K. Koynov, A. Del Campo, R. Bennewitz, The mechanics of single cross-links which mediate cell attachment at a hydrogel surface, Nanoscale 11 (2019) 11596–11604. [3] A.S. Mao, J.W. Shin, S. Utech, H. Wang, O. Uzun, W. Li, M. Cooper, Y. Hu, L. Zhang, D.A. Weitz, D.J. Mooney, Deterministic encapsulation of single cells in thin tunable microgels for niche modelling and therapeutic delivery, Nat. Mater. 16 (2017) 236–243. [4] S. Nasrollahi, C. Walter, A.J. Loza, G.V. Schimizzi, G.D. Longmore, A. Pathak, Past matrix stiffness primes epithelial cells and regulates their future collective migration through a mechanical memory, Biomaterials 146 (2017) 146–153. [5] P.H. Xue, W.D. Liu, Z.Y. Gu, X.C. Chen, J.J. Nan, J.H. Zhang, H.C. Sun, Z.C. Cui, B. Yang, Graded protein/PEG nanopattern arrays: well-defined gradient biomaterials to induce basic cellular behaviors, ACS Appl. Mater. Interfaces 11 (2018) 1595–1603. [6] B.X. Tian, X.L. Ding, Y. Song, W.C. Chen, J.Q. Liang, L. Yang, Y.B. Fan, S. Li, Y. Zhou, Matrix stiffness regulates SMC functions via TGF-β signaling pathway, Biomaterial 221 (2018) 119407. [7] O. Chaudhuri, L. Gu, M. Darnell, D. Klumpers, S.A. Bencherif, J.C. Weaver, N. Huebsch, D.J. Mooney, Substrate stress relaxation regulates cell spreading, Nat. Commun. 6 (2015) 6364. [8] J.P. Winer, P.A. Janmey, M.E. McCormick, M. Funaki, Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli, Tissue Eng. Part A 15 (2009) 147–154. [9] M. Bao, J. Xie, N. Katoele, X. Hu, B. Wang, A. Piruska, W.T.S. Huck, Cellular volume and matrix stiffness direct stem cell behavior in a 3d microniche, ACS Appl. Mater. Interfaces 11 (2018) 1754–1759. [10] Y. Peng, Q.-J. Liu, T. He, K. Ye, X. Yao, J. Ding, Degradation rate affords a dynamic cue to regulate stem cells beyond varied matrix stiffness, Biomaterials 178 (2018) 467–480. [11] H.-S. Lee, J.-I. Kang, W.-J. Chung, D.H. Lee, B.Y. Lee, S.-W. Lee, S.Y. Yoo, Engineered phage matrix stiffness-modulating osteogenic differentiation, ACS Appl. Mater. Interfaces 10 (2018) 4349–4358. [12] R.A. Surmenev, M.A. Surmeneva, A.A. Ivanova, Significance of calcium phosphate coatings for the enhancement of new bone osteogenesis – a review, Acta Biomater. 10 (2014) 557–579. [13] S. Hosseini, H. Naderi-Manesh, H. Vali, M. Baghaban Eslaminejad, F. Azam Sayahpour, S. Sheibani, S. Faghihi, Contribution of osteocalcin-mimetic peptide enhances osteogenic activity and extracellular matrix mineralization of human osteoblast-like cells, Colloids Surf. B Biointerfaces 173 (2019) 662–671. [14] T. Qu, J. Jing, Y. Ren, C. Ma, J.Q. Feng, Q. Yu, X. Liu, Complete pulpodentin complex regeneration by modulating the stiffness of biomimetic matrix, Acta Biomater. 16 (2015) 60–70. [15] J. Zhao, Y. Zhao, Q. Guan, G. Tang, Y. Zhao, X. Yuan, K. Yao, Crosslinking of electrospun fibrous gelatin scaffolds for apatite mineralization, J. Appl. Polym. Sci. 119 (2011) 786–793. [16] J. Fang, P. Li, X. Lu, L. Fang, X. Lu, F. Ren, A strong, tough, and osteoconductive

[29]

[30]

[31]

[32]

[33] [34]

[35]

[36]

[37]

[38] [39]

[40]

[41]

[42]

[43]

[44]

9

hydroxyapatite mineralized polyacrylamide/dextran hydrogel for bone tissue regeneration, Acta Biomater. 88 (2019) 503–513. X. Yang, Z. Li, H. Xiao, N. Wang, Y. Li, X. Xu, Z. Chen, H. Tan, J. Li, A universal and ultrastable mineralization coating bioinspired from biofilms, Adv. Funct. Mater. 28 (2018) 1802730. S. Jin, F. Sun, Q. Zou, J. Huang, Y. Zuo, Y. Li, S. Wang, L. Cheng, Y. Man, F. Yang, J. Li, Fish collagen and hydroxyapatite reinforced poly(lactide- co-glycolide) fibrous membrane for guided bone regeneration, Biomacromolecules 20 (2019) 2058–2067. J.M. Sadowska, F. Wei, J. Guo, J. Guillem-Marti, M.-P. Ginebra, Y. Xiao, Effect of nano-structural properties of biomimetic hydroxyapatite on osteoimmunomodulation, Biomaterials 181 (2018) 318–332. F.A. Shah, P. Thomsen, A. Palmquist, Osseointegration and current interpretations of the bone-implant interface, Acta Biomater. 84 (2019) 1–15. R.E. Abouzeid, R. Khiari, D. Beneventi, A. Dufresne, Biomimetic mineralization of three-dimensional printed alginate/TEMPO-oxidized cellulose nanofibril scaffolds for bone tissue engineering, Biomacromolecules 19 (2018) 4442–4452. D. Wang, J. Jang, K. Kim, J. Kim, C.B. Park, “Tree to bone”: lignin/polycaprolactone nanofibers for hydroxyapatite biomineralization, Biomacromolecules 20 (2019) 2684–2693. A. Nishiguchi, T. Taguchi, Osteoclast-responsive, injectable bone of biophosphonated-nanocellulose that regulates osteoclast/osteoblast activity for bone regeneration, Biomacromolecules 20 (2019) 1385–1393. A. Patelli, F. Mussano, P. Brun, T. Genova, E. Ambrosi, N. Michieli, G. Mattei, P. Scopece, L. Moroni, Nanoroughness, surface chemistry, and drug delivery control by atmospheric plasma jet on implantable devices, ACS Appl. Mater. Interfaces 10 (2018) 39512–39523. T. Wang, Y. Zhai, M. Nuzzo, X. Yang, Y. Yang, X. Zhang, Layer-by-layer nanofiberenabled engineering of biomimetic periosteum for bone repair and reconstruction, Biomaterials 182 (2018) 279–288. L.E. Rustom, M.J. Poellmann, A.J. Wagoner Johnson, Mineralization in micropores of calcium phosphate scaffolds, Acta Biomater. 83 (2019) 435–455. A. Katsumura, K. Sugimura, Y. Nishio, Calcium carbonate mineralization in chiral mesomorphic order-retaining ethyl cellulose/poly(acrylic acid) composite films, Polymer 139 (2018) 26–35. Y. Shasha, J. Biao, L. Zhaoming, S. Changyu, Z. Ruibo, W. Xiaoyu, T. Ruikang, Biomineralization: from material tactics to biological strategy, Adv. Mater. 29 (2017) 1605903. X. Zhao, L. Zhou, Q. Li, Q. Zou, C. Du, Biomimetic mineralization of carboxymethyl chitosan nanofibers with improved osteogenic activity in vitro and in vivo, Carbohydr. Polym. 195 (2018) 225–234. N. Rauner, M. Meuris, S. Dech, J. Godde, J.C. Tiller, Urease-induced calcification of segmented polymer hydrogels - a step towards artificial biomineralization, Acta Biomater. 10 (2014) 3942–3951. M. Farhadi-Khouzania, C. Schützb, G.y.M. Duraka, J. Fornellc, J. Sortd, G.N.S.-A.L. Bergströmb, D. Gebauer, A CaCO3/nanocellulose-based bioinspired nacre-like material, J. Mater. Chem. A 13 (2017) 45–53. C. Shao, R. Zhao, S. Jiang, S. Yao, Z. Wu, B. Jin, Y. Yang, H. Pan, R. Tang, Citrate improves collagen mineralization via interface wetting: a physicochemical understanding of biomineralization control, Adv. Mater. 30 (2018) 1704876. Y. Wang, C. Liu, J. Du, J. Huang, S. Zhang, R. Zhang, The microstructure, proteomics and crystallization of the limpet teeth, Proteomics 18 (2018) 1800194. P. Feng, P. Wu, C. Gao, Y. Yang, W. Guo, W. Yang, C. Shuai, A multimaterial scaffold with tunable properties: toward bone tissue repair, Adv. Sci. 5 (2018) 1700817. H.Y. Mi, X. Jing, G. Yilmaz, B.S. Hagerty, E. Enriquez, L.S. Turng, In situ synthesis of polyurethane scaffolds with tunable properties by controlled crosslinking of triblock copolymer and polycaprolactone triol for tissue regeneration, Chem. Eng. J. 348 (2018) 786–798. C.H. Kuo, J. Xian, J.D. Brenton, K. Franze, E. Sivaniah, Complex stiffness gradient substrates for studying mechanotactic cell migration, Adv. Mater. 24 (2012) 6059–6064. E. George, A. Barai, P. Shirke, A. Majumder, S. Sen, Engineering interfacial migration by collective tuning of adhesion anisotropy and stiffness, Acta Biomater. 72 (2018) 82–93. A. Stolzing, H. Colley, A. Scutt, Effect of age and diabetes on the response of mesenchymal progenitor cells to fibrin matrices, Int. J. Biomater. 2011 (2011) 378034. Y. Yang, X. Wang, F. Yang, L. Wang, D. Wu, Highly elastic and ultratough hybrid ionic-covalent hydrogels with tunable structures and mechanics, Adv. Mater. 30 (2018) e1707071. L. Yu, Y. Zhang, X. Dai, L. Zhang, J. Tan, Monodisperse poly(methyl methacrylate) microspheres with tunable carboxyl groups on the surface obtained by photoinitiated RAFT dispersion polymerization, Chem. Commun. 55 (2019) 7848–7851. H. Luo, C. Ma, X. Zhou, S. Chen, D. Zhang, Interfacial design in dielectric nanocomposite using liquid-crystalline polymers, Macromolecules 50 (2017) 5132–5137. J.-C. Zhu, T. Han, Y. Guo, P. Wang, H.-L. Xie, Z.-G. Meng, Z.-Q. Yu, B.Z. Tang, Design and synthesis of luminescent liquid crystalline polymers with “jacketing” effect and luminescent patterning applications, Macromolecules 52 (2019) 3668–3679. Z.-Y. Zhang, Q. Wang, P.-P. Hou, Z. Shen, X.-H. Fan, Effects of rigid cores and flexible tails on the phase behaviors of polynorbornene-based mesogen-jacketed liquid crystalline polymers, Polym. Chem. 6 (2015) 7701–7710. Z. Wang, W. Fan, R. Tong, X. Lu, H. Xia, Thermal-healable and shape memory metallosupramolecular poly(n-butyl acrylate-co-methyl methacrylate) materials, RSC Adv. 4 (2014) 25486–25493.

European Polymer Journal xxx (xxxx) xxxx

R. Jin, et al.

[50] P.C. Nalam, H.S. Lee, N. Bhatt, R.W. Carpick, D.M. Eckmann, R.J. Composto, Nanomechanics of pH-Responsive, Drug-Loaded, bilayered polymer grafts, ACS Appl. Mater. Interfaces 9 (2017) 12936–12948. [51] M.P. Neupane, S.J. Lee, I.S. Park, H.H. Park, M.H. Lee, T.S. Bae, M.K. Yu, K.W. Lee, H.S. Kim, Influence of the number of carboxyl groups on the nucleation of hydroxyapatite, Met. Mater. Int. 16 (2010) 333–338. [52] Z. Xu, L. Shi, D. Hu, B. Hu, M. Yang, L. Zhu, Formation of hierarchical bone-like apatites on silk microfiber templates via biomineralization, RSC Adv. 6 (2016) 76426–76433. [53] M. Chen, J. Yang, J. Li, K. Liang, L. He, Z. Lin, X. Chen, X. Ren, J. Li, Modulated regeneration of acid-etched human tooth enamel by a functionalized dendrimer that is an analog of amelogenin, Acta Biomater. 10 (2014) 4437–4446. [54] M.M. Elmi, F. Elmi, P. Sekineh Khazaee, Preparation and characterization of marine mineralized ASC nanocomposites through biomineralization, Int. J. Biol. Macromol. 123 (2019) 817–825.

[45] G. Li, T. Wang, X. Fu, T.M. Gao, M.F. Huang, Study of core-shell structure poly(vinyl acetate-butyl acrylate) emulsion particles modification, Adv. Mater. Res. 1061 (2014) 277–282. [46] A. Kruger, S. Braune, K. Kratz, A. Lendlein, F. Jung, The influence of poly(n-butyl acrylate) networks on viability and function of smooth muscle cells and vascular fibroblasts, Clin. Hemorheol. Microcirc. 52 (2012) 283–294. [47] X. Rao, F. Zhang, X. Luo, F. Ding, Characterization of hardness, elastic modulus and fracture toughness of RB-SiC ceramics at elevated temperature by Vickers test, Mater. Sci. Eng., A 744 (2019) 426–435. [48] Z. Yin, S. Tao, X. Zhou, C. Ding, Evaluating microhardness of plasma sprayed Al2O3coatings using Vickers indentation technique, J. Physics D Appl. Physics 40 (2007) 7090–7096. [49] A. Nastic, A. Merati, M. Bielawski, M. Bolduc, O. Fakolujo, M. Nganbe, Instrumented and vickers indentation for the characterization of stiffness, hardness and toughness of zirconia toughened Al2O3 and SiC armor, J. Mater. Sci. Technol. 31 (2015) 773–783.

10