In Vitro Osteocompatibility and Enhanced Biocorrosion Resistance of

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Biological and Medical Applications of Materials and Interfaces

In vitro Osteocompatibility and Enhanced Biocorrosion Resistance of Di-ammonium Hydrogen Phosphate-Pretreated/Poly Ether Imide Coatings on Magnesium for Orthopedic Application Yuyun Yang, Juncen Zhou, Qiang Chen, Rainer Detsch, Xiufang Cui, Guo Jin, Sannakaisa Virtanen, and Aldo R. Boccaccini ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b11073 • Publication Date (Web): 23 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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ACS Applied Materials & Interfaces

In vitro Osteocompatibility and Enhanced Biocorrosion Resistance of Di-ammonium Hydrogen Phosphate-Pretreated/Poly Ether Imide Coatings on Magnesium for Orthopedic Application Yuyun Yang a,b,c, Juncen Zhou c, Qiang Chen d, Rainer Detsch b, Xiufang Cui a, Guo Jin a, Sannakaisa Virtanen c,*, and Aldo R. Boccaccini b,* a

Institute of Surface/Interface Science and Technology, Department of Material Science and Chemical Engineering, Harbin Engineering University, 150001 Harbin, China

b

Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, 91058 Erlangen, Germany

c

Institute for Surface Science and Corrosion, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, 91058 Erlangen, Germany d

State Key Laboratory of Solidification Processing, Northwestern Polytechnical University, Xi’an, 710072 Shaanxi, China

KEYWORDS: Osteocompatibility, Coatings, Magnesium, Cytocompatibility, Corrosion, Poly Ether Imide, Di-ammonium Hydrogen Phosphate, Stem Cells

ACS Paragon Plus Environment

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ABSTRACT

Magnesium, as a biodegradable metal, is a promising candidate for biomedical applications. To modify the degradation behavior of magnesium and improve its osteo-compatibility, chemical conversion and spin coating methods were combined to develop a di-ammonium hydrogen phosphate-pretreated/poly ether imide (DAHP/PEI) co-coating system. The di-ammonium hydrogen phosphate pretreatment was employed to enhance the attachment between PEI coatings and the magnesium substrate, meanwhile it could serve as another bioactive and anti-corrosion layer when PEI coatings break down. Surface characterization, electrochemical tests, and shortterm immersion tests in DMEM were performed to evaluate DAHP/PEI coatings. Electrochemical measurements showed that DAHP/PEI coatings significantly improved the corrosion resistance of pure magnesium. No obvious changes of the chemical compositions of DAHP/PEI coatings occurred after 72 hours of immersion in DMEM. An in vitro cytocompatibility study confirmed that viability and LDH activity of MG-63 cells on DAHP/PEI coatings showed higher values than those on DAHP-pretreated layer and pure magnesium. DAHP pre-treated layer could still enhance the ALP activity of MG-63 cells after the degradation of PEI in DAHP/PEI coatings. Besides that, the in vitro cellular response to the treated magnesium was investigated to gain knowledge on the differentiation and proliferation of human adipose-derived stem cells (hADSCs). Cell distribution and morphology were observed by fluorescent and SEM images, which demonstrated that DAHP/PEI coatings facilitated cell differentiation and proliferation. The high level of C-terminals of collagen type Ӏ production of hADSCs on DAHP/PEI coatings indicated the great potential of the coating for promoting


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osteogenic differentiation. Positive results from long-term cytocompatibility and proliferation tests indicate that DAHP/PEI coatings can offer an excellent surface for hADSCs cells.

1. Introduction Magnesium and its alloys are attractive metallic biodegradable materials to be used in the biomedical field because of their suitable strength and biodegradability. The elastic modulus of magnesium (45GPa) is much lower than the one of other metallic materials, such as stainless steel and titanium, making Mg more suitable for orthopedic applications

1–3.

comparable elastic modulus with that of bone, the stress shielding effect

4

Indeed, due to

is minimized by

applying magnesium-based biomaterials. However, the main obstacle for the application of magnesium is its unfavorable corrosion resistance under physiological conditions, i.e., even if corrosion in the human body is desired for biodegradable implants, the corrosion rate of Mg is too high, especially in the initial stages of implantation. Moreover, corrosion of Mg leads to formation of H2 gas and alkalization of the surrounding. This behavior leads to many detrimental effects on implants surfaces and in adjacent tissues 5,6. Many efforts have been made to improve the corrosion resistance of magnesium, such as decreasing impurity level, alloying, and altering the microstructure by heat treatments, etc.

7,8.

However, modification of the magnesium surface with protective coatings represents an efficient and cost-effective approach to enhance Mg/Mg alloy corrosion resistance

9–13.

Materials and

coating techniques that have been applied to enhance the corrosion resistance of Mg substrates include polymeric coatings including anodization

24,

12,14–19,

chemical conversion coatings

electrophoretic deposition

25,

CVD

20–23,

26,

and surface treatments

PVD

27,

flame or plasma

spraying 28,29.


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Due to their effective protective ability, polymer coating systems are raising great attention in this field. Poly ether imide (PEI) is regarded as an hydrophobic thermal polymer with stable mechanical properties

12,30

and defect-free coatings can be achieved by solving PEI into

particular solvents at certain drying temperature on magnesium

15,31.

A mono-PEI layer offers

less pronounced protection, since minor defects lead to H2 production and release, which may weaken the binding between the PEI coating and the Mg substrate. Therefore, inorganic pre-coatings on magnesium should be designed to improve the bonding between top polymer coatings and magnesium substrates. Di-ammonium hydrogen phosphate is a key compound to synthesize hydroxyapatite in hydrothermal methods

32,33.

It is well known

that hydroxyapatite interacts with bone cells being an osteoconductive material 34. Di-ammonium hydrogen phosphate pretreatment on magnesium can be thus considered not only as a bonding layer between the Mg substrate and polymeric coatings, but also to facilitate calcium phosphate deposition, and to induce hydroxyapatite formation, after the degradation or potential fracture of the polymeric coating. Besides the functions mentioned above, Di-ammonium hydrogen phosphate pre-treated layer could serve as a corrosion-resistant barrier as well to slow down the degradation rate of magnesium. Thus, di-ammonium hydrogen phosphate pre-treatment can be considered to “multi-functionalize” the surface of magnesium as investigated in this study. After the fabrication of a DAHP/PEI coating system on magnesium substrate, the cytocompatibility was tested. Two different types of bone-tissue related cell lines were selected in this study, including human osteoblast-like MG-63 cells and human ADSCs (hADSCs). MG63 cell line, with osteoblast-like traits, serves as a typical model to evaluate the osteocompatibility, in term of cell attachment, proliferation, and osteo-conductivity of biomaterials 35,36.

Human ADSCs are considered with highly multi-lineage potential, including osteogenic,


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chondrogenic, and adipogenic lineages

37.

The primary objective of this study is therefore to

develop DAHP/PEI coatings to impart a favorable and multi-functionalized surface on magnesium. Besides that, the growth of MG-63 cells and hADSCs, including attachment, proliferation and differentiation, are methodically characterized to generate a deeper understanding of the cell response and cell interaction with the biodegradable coating system on magnesium. 2. Experimental Work 2.1 Substrate Preparation Pure Magnesium (99.99% Mg) samples of 24.5 mm in diameter and 2-4 mm in thickness were prepared from magnesium rods. The samples were ground up to 2000 grit SiC paper followed by fine polishing with diamond paste in 6 μm, 3 μm and 1 μm, respectively. Cleaning for 3 min was carried out in an ultrasonic bath with ethanol, samples were then dried with air steam. 2.2 Di-ammonium hydrogen phosphate pre-treatment The samples were immersed in 0.05 mol/L di-ammonium hydrogen phosphate solution for 60 min at 80 °C. The following reactions take place during the procedure: (NH4)2HPO4 → 2NH4+ + HPO42- → 2NH4+ + H+ + PO43Mg + 2H+ → Mg2+ + H2↑ Mg2+ + HPO42- + xH2O → MgHPO4•xH2O 3Mg2+ + 2PO43- + xH2O → Mg3(PO4)2•xH2O Mg2+ + NH4+ + PO43- + xH2O → Mg(NH4+)(PO4)2•xH2O The prepared samples were dried in warm air and kept in dry condition for further treatments.


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2.3 DAHP/PEI composite coatings PEI solution was prepared by dissolving PEI pellets with a concentration of 15% (w/v) in N,Ndimethylacetamide (DMAc) solvent

under magnetic stirring at 60°C for 60 min. These

parameters were selected and refined based on literature results

12,14,38.

Di-ammonium hydrogen

phosphate pretreated Mg samples were spin-coated with PEI solution for 45 seconds at 3000 rpm to achieve a uniform layer. After spin coating, the samples were dried at 60 °C (PEI60). Diammonium hydrogen phosphate pretreated layers were denoted as DAHP coatings in the following sections. The composite coatings were denoted as DAHP/PEI coatings. 2.4 Characterization methods Scanning electron microscopy (SEM, model Auriga, Zeiss) equipped with energy-dispersive X-ray analysis (EDX) was used to examine the micro-morphology and to estimate chemical composition of coatings, which was further investigated by Fourier transform infrared spectroscopy (FTIR, Nicolet 6700, Thermo Scientific). FTIR was carried out directly on the prepared samples and the collected spectra were analyzed with OPUS software. X-ray diffraction (XRD) measurements were carried out on an X’pert Pro diffractometer using Cu-K radiation. Electrochemical properties were measured via an electrochemical workstation “IM6eX” (Zahner-Elektrik GmbH & Co. KG, Kronach, Germany). The treated Mg samples were employed as the working electrode, a platinum plate as the auxiliary electrode and an Ag/AgCl electrode in 3-molar KCl as the reference electrode. Impedance measurements were carried out at the corrosion potential after 10 min stabilization of the open circuit potential in the frequency range of 10 mHz – 10000 Hz. The electrochemical impedance spectroscopy (EIS) results will be presented as Nyquist plots. Afterwards, potentiodynamic polarization was conducted with a scan rate of 1 mV/s starting from -300 mV related to the corrosion potential to -500 mV. Nyquist plots


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and potentiodynamic polarization tests were performed in Dulbecco’s modified Eagle’s medium (DMEM, Biochrome AG) at 37 ± 2 °C. Every experiment was repeated at least three times. Immersion tests were performed for 72 hours in DMEM to investigate the short-term degradation behavior of coatings. 2.5 In vitro biocompatibility Human osteoblast cells (MG-63; Sigma-Aldrich) and human adipose-derived stem cells (hADSCs, Lonza Group US) were selected to investigate the cell response to the coated magnesium in a direct contacting method. Since intended applications of magnesium include bone implants and scaffolds, MG-63 cells were used for in vitro biocompatibility assessment of treated magnesium as a model cell line to reveal the cell behavior in short term. The detailed MG-63 cell culture procedures and measurements including cell viability, cell proliferation, and osteoblast differentiation can be found in a previous study

39.

Cell morphology and cell

distribution were observed by labeling with Vybrant® Dil cell tracers (Life Technologies, Darmstadt, Germany) and bone mineralization was assessed by labeling cells with OsteoImageTM fluorescent staining kit (Lonza, Germany), which is based on the specific binding to the hydroxyapatite portion of bone-like nodules deposited by cells. Long-term cell biocompatibility tests were carried out by using hADSCs to evaluate the cytocompatibility and differentiation on the coated magnesium samples. The thawed hADSCs were sub-cultured for at least 1 passage prior to the seeding procedure. 10000 cells·well-1 were seeded in a 12-well plate with the sterilized samples in growth medium for cell attachment as following the instruction. The growth medium was changed by osteogenic induction medium when hADSCs attached on the surface of the samples, around 18-24h after the initial seeding. The plates were kept in the incubator at 37.5°C with 5% CO2 and 90% humidity. Afterwards, the induction medium was completely


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changed every 3 days to mimic the buffer environment in in vivo conditions. In order to reveal the cell proliferation and differentiation during the cultivation, the samples were taken out after 3, 9, and 15 days of incubation. Collagen expression of hADSCs on the treated samples at different days of incubation in induction medium was assessed to investigate the production of C-terminals of collagen Type Ӏ (Sigma-Aldrich, US) (CICP). Collagen is synthesized as procollagen, which consists of mature collagen with extension peptides. The release level of these peptides into the circulation delivers an indication of the collagen production and subsequently bone formation

40.

In this study, the

MicroVueBone CICP Assay was used to quantitatively measure CICP levels in an enzyme immunoassay. The MicroVue CICP assay is a sandwich enzyme immunoassay in a microtiter plate format utilizing a monoclonal anti-CICP antibody coated on the plate, a rabbit anti-CICP antiserum, a goat anti-rabbit alkaline phosphatase conjugate, and a pNPP substrate to quantify CICP. Levels of CICP are indicative of collagen production in vivo and have a close link to osteogenic differentiation. The experiment was carried out by following the instruction of the supplier, CICP EIA kit (Quidel). In brief, the reagents and specimen solution were prepared in the well plates and incubated at 18-25 °C for 120 min. Washing with Wash Buffer for 3 minutes was carried out after every incubation. Rabbit anti-CICP was added and kept at 18-25 °C for 45 min. Enzyme Conjugate Solution, consisted of lyophilized goat anti-rabbit antibody conjugating to alkaline phosphates, was pipette into wells and the plates were incubated for 45-50 min at 1825 °C. Substrate Solution was added for 30-35 min of incubation. At last step, Stop Solution, containing 0.5 N NaOH, was used to stop the reaction and the optical density was read by a UVVis spectrophotometer. All the solution preparation followed the instruction provided by the


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supplier. CICP concentration released by hADSCs on the samples was normalized with that of references, which was considered as 100%, at different incubation time accordingly. Vybrant® Dil, DAPI (Invitrogen), and OsteoImage staining were performed to visualize hADSCs on samples by labeling the cytoskeleton, cell nuclei, and bone mineralization. The images were collected under a fluorescence microscopy (FM) (Axio Scope A.1, Carl Zeiss Microimaging GmbH). To observe cell morphology and distribution, samples seeded with hADSCs were prepared for SEM imaging. The attached cells on the samples were fixed with 2.5% glutaraldehyde in PBS for 2h. After the fixation step, the samples were thoroughly washed with PBS 3 times, 5 min each time. The dehydration step was carried out in graded series of ethanol, namely 30%, 50%, 70%, 80%, 85%, 90%, 93%, 97%, and 99%, for 30min each. All the operations were conducted in the working hood. Critical point drying method was applied to dry the samples. The samples with dried cells were sputtered with Au for SEM observation to avoid the charging effect. 2.6 Statistical analysis All data were collected from at least three replicates and were reported as mean values ± standard deviation (SD). Statistical analysis of cytocompatibility was accomplished by using one-way analysis of variance (ANOVA) with applying Turkey post-hoc test. The value p DAHP > bare magnesium, whereas the values of the corrosion current density (icorr) could be ranked in an opposite way as: DAHP/PEI60 < DAHP < bare magnesium. Therefore, the anticorrosion properties of all samples could be ranked as followings: DAHP/PEI60 > DAHP > bare magnesium. The polarization results reach in same agreement with impedance results. Table 1. Values of charge transfer resistance (Rct), corrosion potentials (Ecorr), and corrosion current density (icorr) for DAHP/PEI60, DAHP, and bare magnesium. 　

Rct (KΩ•cm2)

±

Bare Mg

1.32E+00

0.18

DAHP

5.32E+04

DAHP/PEI60 1.10E+06

icorr (µA/cm2) 5.26E-03

±

Ecorr (V)

±

1.17E-04

-1.65

0.01

2.11E+04 2.70E-7

1.63E-7

-0.83

0.03

4.69E+04 7.11E-8

3.12E-8

-0.71

0.06

3.3 Immersion tests of the coatings


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Figure 3 shows FTIR, XRD and SEM data for the different samples before and after 72 h of immersion. FTIR spectra for fresh samples and after immersion were collected to reveal possible composition changes of DAHP (Figure. 3(A)) and DAHP/PEI60 (Figure. 3(B)). (FTIR results of bare Mg have been discussed in a previous study 41). No obvious changes of the characteristic bands were detected after 72 hours of immersion for both DAHP and DAHP/PEI60. The bands assigned to HPO43- are observed in the range of 950-1100 cm-1

42,43.

O-N=P binding group was

found at 760 cm-1 indicating the occurrence of ammonium hydrogen phosphate

44,45.

The

stretching mode of P-O band can be recognized at 630 cm-1 and 568 cm-1. The chemical composition of DAHP thus shown no obvious changes after immersion and is mainly composed of phosphate and ammonium phosphate. In Figure 3(B), the main characteristic bands of PEI can be observed in the curve of DAHP/PEI60. The presence of the bands at 2400-2250 cm-1 may indicate the presence of DAHP, which can also be observed in Figure 3(A). The remaining characteristic bands of DAHP are largely overlapped with that of PEI in the range of 1100-500 cm-1, where the characteristic bands of DAHP appeared. The disappearance of DAHP bands in DAHP/PEI60 could also due to the limited detection depth of FTIR spectra, which cannot penetrate through the PEI coating to reach the buried DAHP coating beneath. Relative intensity of peaks declined after 72 hours of immersion in DMEM, which may indicate dissolution of the coatings and/or formation of corrosion products that would weaken the signals from PEI coatings. Since the bands corresponding to precipitates were less pronounced than those corresponding to PEI, they were hardly distinguished from PEI peaks in DAHP/PEI60. XRD patterns of bare Mg, DAHP, and DAHP/PEI60 samples are plotted in Fig. 3(C) to demonstrate the composition change after immersion tests. The main characteristic peaks were assigned to different compositions by comparing with the standard XRD database. Besides the


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peaks derived from bare Mg, a new broad peak can be found which is related to the formation of Mg(OH)2, indicating that the corrosion products are mainly composed of Mg(OH)2 after 72h immersion in the medium. The sharp peaks assigned to Mg(NO4) (PO4), MgH (PO4), and Mg(OH)2 can be found in the plot of the DAHP coating. The corresponding peaks are still present at respective angular location in the XRD plot of the immersed DAHP sample, only the intensity of the peaks is decreased. It suggests that no changes in the composition of DAHP can be observed after the immersion. Furthermore, besides the peaks associated with DAHP, two reflection humps between 2 θ = 10 ° and 30 ° appear in DAHP/PEI60 and the immersed DAHP/PEI60 indicating that PEI is amorphous

46.

Therefore, the composition of the coatings

derived from XRD results is in agreement with the FTIR data. The surface micro-morphology results of bare Mg, DAHP, and DAHP/PEI60 after 72h immersion in DMEM are presented in Fig. 3 (D)(E)(F). A cracked corrosion product layer is observed on the bare Mg sample, this is a typical morphology of corrosion product layers on Mg after immersion in simulated body fluids. When comparing the surface structure of the freshly prepared DAHP in Fig. 1, it can be clearly observed that the laminate structure still remained after 72h immersion. The major change is a slight decrease in the dimension of the laminates, or an onset of degradation of the laminates. However, the characteristic feature of DAHP is kept and no obvious surface structure changing occurred after 72 h of immersion. Also for the DAHP/PEI60 coating, no changes of the surface morphology are observed after 72 h of immersion. The immersion test up to 72 hours was carried out to evaluate the initial protection of the DAHP and DAHP/PEI60 coatings to the magnesium substrate. As there were no obvious changes during the 72 hour of immersion test, as indicated by FTIR and SEM results, the


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confirmed stable surface of the coatings would offer cells a suitable platform to attach and grow in the initial stage. Consequently, the short-term immersion test was designed to examine the primary stability of the coatings and the adaptability for the following cell tests.

Figure. 3 FTIR spectra (A)(B), XRD patterns (C), and SEM images (D)(E)(F) of DAHP and DAHP/PEI60 after 72 hours of immersion in DMEM. All relevant peaks have been identified in FTIR spectra and XRD patterns. 3.4 In vitro assessment


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The short term and long term in vitro evaluation of the coatings was carried out to reveal the interaction between cells and the substrate by direct contact assays with two types of cells, MG63 and hADSCs, separately. The short-term experiments were carried out by using MG-63 cells to inspect cell attachment and distribution after 72 hours of co-incubation with the treated samples. Cell attachment and proliferation of MG-63 were systematically examined by using Vybrant™ Dil staining, OsteoImage staining, WST-8, LDH, and ALP activity. On the basis of the short-term evaluation, long-term tests were conducted to investigate the differentiation and proliferation of hADSCs up to 15 days.

Figure. 4 Vybrant® Dil staining (in red) and OsteoImage staining (in green) of MG-63 cells seeded on bare Mg(A)(a), DAHP (B)(b), and DAHP/PEI60(C)(c) in DMEM after 72 hours of cultivation. OsteoImage and Vybrant Dil staining were fluorescently imaged in the same spot. Cell attachment and distribution on bare magnesium and coated samples were observed by staining MG-63 cells with Vybrant™ Dil after 72 hours of culture and the results are shown in Figure.4 (A)(B)(C). Few living cells were observed on bare magnesium surfaces, while the density of living cells on DAHP increased obviously compared to that on bare magnesium. In


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addition, DAHP/PEI60 samples present the highest cell density, which is possible because the smooth surface likely facilitates cell attachment and the integrity of coatings inhibits the negative influence from dissolution to cell attachment. OsteoImage was performed to assess the biomineralization behavior on samples and the fluorescence images are shown in Figure. 4(a)(b)(c) accordingly to the same spots where Dil staining was observed. The intensity of the green fluorescence is proportional to the amount of calcium phosphate formed on the surface of the sample 47,48. Comparing the fluorescence images of DAHP to those of bare magnesium, it has been found that a higher proportion of calcium phosphates was formed on DAHP after 72 hours of cultivation with MG-63 cells. The typical morphology of phosphate formation in fluorescence images was associated with the rough surface structure of DAHP (Figure. 1(A)(a)). The OsteoImage fluorescence morphology of DAHP/PEI60 indicates the mineralization process that occurred during cell proliferation. Altogether, DAHP had a relatively high proportion of green area in OsteoImage, while DAHP/PEI60 exhibited a slight decrease of the green area compared with bare magnesium and DAHP.


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Figure. 5 Relative cell viability (A), ALP activities (B), and LDH (C) of MG-63 cell on bare magnesium, DAHP, and DAHP/PEI60 coatings after 72 hours of cultivation in DMEM. The data were normalized to control values (well plate only, without any sample), which was set as 100% cell viability. Asterisks denote significant difference, *p < 0.05, **p < 0.01 and ***p < 0.001. Figure. 5(A)(B)(C) showed the relative cell viability (WST-8), LDH activity and ALP activity of the adhered MG-63 cells on bare magnesium, DAHP, and DAHP/PEI60 coatings, respectively. The WST-8 result in Figure. 5(A) is in agreement with cell viability results as Dil staining shown in Figure. 4. Especially the relative cell viability of DAHP/PEI60 has even a higher value than the control, which indicates the increased amount of viable cells on the coating after 72 hours’ culture. Relative viability of MG-63 cells on DAHP exhibited gradually decreased values compared with that on DAHP/PEI60, while both of them were higher than the values for bare


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magnesium, this result demonstrates that the cytocompatibility of bare magnesium could be significantly improved after the coating process. ALP activity is closely related to the biomineralization process and osteoblastogenesis in the early state

49.

The evaluation of ALP could be cooperatively assessed with OsteoImage results.

DAHP/PEI60 had the lowest activity in Figure. 5(C), as the same trend was found in OsteoImage results. LDH assessment was performed to evaluate the metabolic activity of the adhered cells on the treated samples. Similar outcome as for WST-8 results can be found, as shown in Figure. 5(C). LDH activity of MG-63 on bare magnesium was less than 5%, which was a rather low activity compared with that of the coated samples. The low LDH activity indicates that few cells adhered on bare magnesium samples during the experiment, which was also observed in Figure. 4(A). DAHP pre-layer showed higher LDH activity, with the value at 38.43%, in comparison for bare magnesium. Moreover, DAHP/PEI coatings presented the highest LDH activity, at 72.65%. LDH activity of MG-63 cells on all samples can be graded in the following order: DAHP/PEI60 > DAHP > bare magnesium. Since the short-term cytocompatibilty experiments exhibited favorable cell attachment and proliferation of MG-63, a long-term assessment was carried out to investigate the differentiation and proliferation behavior of hADSCs on the coated specimens.


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Figure. 6 Light microscope images, Vybrant® Dil staining (in red) and Dapi staining (in blue), and OsteoImage staining (in green) of hADSCs on bare Mg, DAHP, and DAHP/PEI60 in osteogenic induction media at 3, 9, and 15 days of cultivation. Human ADSCs morphology images and fluorescent images were collected respectively by light and fluorescent microscopy after different incubating durations and are shown in Figure. 6. In an overall comparison of the light microscope images, and Vybrant and Dil staining images, the attached cell density of both DAHP and DAHP/PEI60 is clearly higher than that on uncoated magnesium in each day of cell culture. When comparing the same sample at different days, the density of hADSCs is seen to have a significant increase with longer cultivation period, which can be observed in all samples. Besides that, the cells were evenly spread on both DAHP and DAHP/PEI60, while locally scattered on magnesium. Interestingly, the proportion of green area in OsteoImage in magnesium and DAHP is larger than in DAHP/PEI60. However, the green area of magnesium mismatched with the location where cells were attached. A similar situation was concurrently observed in DAHP. On the other hand, the green dots in DAHP/PEI60 perfectly match the corresponding spots occupied by hADSCs.


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Figure. 7 Derived CICP concentrations at 3, 9, and 15 days of incubation in osteogenic induction media. Asterisks denote significant difference, * p < 0.05, ** p < 0.01. The derived CICP concentrations of hADSCs on Mg, DAHP, and DAHP/PEI60 at different days of incubation are shown in Figure. 7 to demonstrate the time-dependent variation of CICP levels. The value of the CICP concentration was normalized to the value of the reference, as shown in the Supporting Information. CICP levels of magnesium samples present an irregular fluctuation with the incubation period. Furthermore, a slight and gradual rise in CICP concentrations of DAHP is detected with longer incubation periods. It is interesting that a decrease trend of CICP concentrations on DAHP/PEI60 was displayed, unlike on either magnesium or DAHP.


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Figure. 8 SEM images of hADSCs on bare Mg, DAHP, and DAHP/PEI60 in osteogenic induction media at 3, 9, and 15 days of cultivation.


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Figure. 8 shows SEM images of the seeded hADSCs on magnesium, DAHP, and DAHP/PEI60 after different days of cultivation. The representative areas on each sample were captured under SEM at different magnifications to demonstrate the features of hADSCs. The cell density of all samples was increased with the longer cultivation period and it can be ranked as: Mg < DAHP < DAHP/PEI60, which is the same trend shown in Figure. 6, To take a close observation of each sample, hADSCs were shown to distribute in an uneven status on the magnesium surface, and in a homogeneous fashion on DAHP and DAHP/PEI60. hADSCs were only observed on partial areas of the magnesium surface, even after 15 days of incubation. The crack feature of the magnesium surface after immersion was captured and shown in images a1, a2, and a3. Moreover, hADSCs stuck on the top of the randomly oriented laminates in DAHP and the cells are seen to be largely stretched to communicate with each other via extension of filopodia, as clearly shown in images B1 and b1. As shown in images B2 and B3, the whole surface of DHAP was gradually covered by muti-layers of hADSCs, indicating the differentiation and proliferation of cells. Particularly, the globular accretions, indicate by the blue arrows, could be obviously observed in the higher magnification images of DAHP, while to a less extent in image A3 (magnesium). Globular accretions are considered as a secreting form of extra cellular matrix indicating the formation of the primary bio apatities and calfication 50,51. In addition, the multi-layed hADSCs crawled on the whole surface of DAHP/PEI60 since the first 3 days of cell culture. At longer periods, the bulges formed by the buried and unburied DAHP laminates present under PEI became less distinctive by comparing with that in Figure. 1. The disappeared bulges can be related to the multi-layer hADSCs that have grown to a rather thick layer to cover overall the surface. The globular accretions could also be found on DAHP/PEI60, while not as many as on DAHP. This observation may be due to the fact that the globular


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accretions formed on the smooth DAHP/PEI60 surface were more easily washed away during the series of dehydration steps for SEM observation.

Figure. 9 EDX results of hADSCs on bare Mg, DAHP, and DAHP/PEI60 at 3, 9, and 15 days of cultivation. EDX results were obtained on the samples with different days of incubation and are shown in Figure. 9. The relevant elements C, O, Na, Mg, P, K, and Ca were analyzed to discover the elemental variation during incubation. C was mostly derived from the cells because of the attached hADSCs on the top of the substrate, and partially from the corrosion products. On the contrary, most O was considered from the corrosion products and only a small amount from the cells. The rest of elements were mostly derived from the corrosion products.


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In order to compare the amount of C with and without hADSCs, EDX results at 3 days of incubation were collected at the spot where no hADSCs were observed. It is shown that C content in both magnesium and DAHP without hADSCs dropped approximately down to half and one third values, respectively, in comparison for samples with hADSCs. The decline of C in samples without hADSCs spots was caused largely by the non-existence of hADSCs. Since the whole surface was covered by the cells at 3 days, EDX results of DAHP/PEI60 were not included in the cell-free graph. Besides that, C content in magnesium and DAHP increased gradually from less than 30 wt% to more than 30 wt% with longer incubation periods, indicating the differentiation and proliferation of hADSCs. Especially, C content in DAHP has a faster rise up to 44 wt% at 15 days than that in pure magnesium. C content in DAHP/PEI60 was relatively steady at around 77 wt% and O content was at around 22 wt% in all days of incubation. No other elements in DAHP/PEI60 can be detected, especially no magnesium was found, demonstrating that DAHP/PEI60 offered an ideal platform for the growth of hADSCs and the coatings remained protecting the magnesium matrix even after 15 days. In addition, the detected Mg could be regarded as the corroded or dissolved magnesium matrix into the corrosion products deposited on the surface. Higher Mg content indicates faster dissolution of the magnesium substrate. The amount of Mg was less than 15 wt% in all samples with hADSCs up to 15 days of incubation. After 15 days of incubation, approximately 9 wt% of Mg was found in DAHP while 14 wt% of Mg was present in the bare magnesium sample. The less content of Mg in DAHP indicates that the substrate dissolving rate is retarded by both DAHP coatings and by the fully covered hADSCs on the substrates, which might prevent the penetration of the corroding medium into the substrate. Besides that, the content of P in DAHP was always higher than that in bare magnesium. The main corrosion products of Mg and DAHP, therefore, were made up by


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magnesium oxide/hydroxide, magnesium phosphate, and small amount of other corrosion products contained Na, K, and Ca since very small amounts of Na, K, and Ca were found in the samples. 4. Discussion 4.1 Corrosion behavior The corrosion behavior was assessed by electrochemical measurements and SEM observation after different days of incubation with hADSCs. The electrochemical measurements were used to evaluate the anti-corrosion properties of the coatings and the surface of the incubated coatings were directly observed under SEM to show the micro-morphology characteristics with the growth of hADSCs. The integrity of the DAHP/PEI60 was perfectly maintained after 15 days of incubation with cells. The electrochemical results demonstrate that DAHP/PEI60 coatings provide excellent protection to the substrate from the surrounding medium, in agreement with the SEM images after immersion in DMEM with cells (Figure. 8). Besides that, the electrochemical results of DAHP show an adequate protection of the substrate. The surface structure of DAHP coating indicates almost no altering after 3 days of immersion with and without cells in the culture medium, in agreement with the electrochemical results. Especially, FTIR, XRD, and SEM results confirmed the sustained main composition and surface structure of both DAHP and DAHP/PEI60 after the immersion test up to 72h in culture medium. After 9 days of incubation with cells, a different micro-structure can be observed in the area where no cells were attaching and spreading. The changing surface structure of DAHP may be due to a slow dissolution of the coatings, however it had no negative effect on cell proliferation and differentiation. The cell growth proceeded smoothly from 9 days of incubation to 15 days. Therefore, as the pretreatment of DAHP/PEI60, DAHP coatings can still protect the substrate offering a favorable platform for


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the differentiation and proliferation of stem cells, even after the break down of PEI coatings on top of DAHP/PEI60 with longer incubation periods. 4.2 Biomineralization OsteoImage and ALP results reveal the cell biomineralization process and they are relevant to assess the osteogenic potential and biomineralization activity of the coatings 50. Both of them are related with the existing phosphates in the solution during the measurement. OsteoImage is based on the specific binding to the hydroxyapatite portion of bone-like nodules deposited by cells. As it is well-known, hydroxyapatite with the formula Ca10(PO4)6(OH)2 has groups of phosphates in it. The ALP result is mainly based on the amount of alkaline phosphate in the testing solution. As the corrosion products composition of bare Mg contains abundant unstable phosphates, it is anticipated that the unstable phosphate components would have great influence on the results of OsteoImage and ALP, while, the unstable corrosion products of bare Mg have no impact on the results of WST-8, LDH, cell staining, and SEM. The influence of phosphates on OsteoImage and ALP results of DAHP can also be traced, but not in DAHP/PEI60. In this study, the impact of the phosphates formed during the corroding or degrading process on OsteoImage and ALP results was relevant. Nonetheless, the prime target of the research is focused on the osteocompatibility of DAHP/PEI60, and the corrosion products up to 15 days of immersion should have nondetectable influence on the results of DAHP/PEI60. OsteoImage and ALP results on MG-63 cells on pure magnesium, DAHP, and DAHP/PEI60 showed a different trend to cell viability (WST-8) and metabolic activity (LDH) after 72 hours incubation (Figure. 4(a), (b)and Figure. 5). In general, OsteoImage and ALP results should only be derived from the alkaline phosphates produced by living MG-63 cells on the samples. In our study, besides the phosphates produced by the cells, additional alkaline phosphates were formed by the dissolution of Mg substrates or


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DAHP coatings during the incubation period. The extra phosphates formed during magnesium and DAHP dissolution would also react with the reagent in OsteoImage and ALP arrays. Therefore, extra alkaline phosphates were detected during both measurements of OsteoImage and ALP. Higher values were reflected in the respective results, as shown in OsteoImage (Figure. 4(a)(b)) and ALP (Figure. 5(B)) results. When combining the results of cell staining images in Figure. 4A, B, it can also be confirmed that the OsteoImage and ALP results of pure magnesium were contributed by the deposited phosphate during substrate dissolution and only partial results were from the phosphates produced by cells since very few live cells were found on pure magnesium (in Figure. 4(A)). By contrast, higher cell density can be observed on DAHP, shown in Figure. 4 (B), which could demonstrate that the OsteoImage and ALP results of DAHP were contributed by MG-63 cells. Therefore, it is reasonable to suggest that OsteoImage and ALP measurements should be conducted in combination with other measurements to reveal the actual biomineralization on the investigated substrates. Moreover, the globular accretions found by SEM on DAHP and DAHP/PEI60 (in Figure. 8) also indicates the biomineralization of hADSCs. In addition, it is considered that only the green area (in OsteoImage results), where the red spots are present in corresponding position to the Vybrant™ Dil staining results, should be regarded as the valid results about biomineralization activity of the samples. Only few living cells (in Figure 4(A) Vybrant™ Dil staining) were observed, while the extra green was captured in Figure 4a (OsteoImage) close to the adjacent area of living cells. In addition, it is also found that the green area of the bare Mg sample without cells (in Figure S2(A) OsteoImage) takes a large proportion in the image, indicating that the corrosion products of bare Mg in DMEM after 72h have an influence on the results of OsteoImage assay. Therefore, it can be concluded that the


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influence of the substrates to the results of OsteoImage staining could be evaluated by correspondingly comparing the green area in OsteoImage of samples with and without cell seeding. By comparing with OsteoImage results on DAHP-pretreated coatings, less green area can be observed (Figure S2(B)) demonstrating a lower outcome induced by the dissolution or degradation of DAHP-pretreated coatings. As for DAHP/PEI, only few spots at lower fluorescent intensity were observed (Fig. S2(C)) confirming that the OsteoImage results of the DAHP/PEI sample will have been very little influenced by the degradation of coatings. The same comparison of ALP and CICP results can be drawn between tests carried out with (in Figure 5(B) and Figure 7) and without cell seeding (in Figure S3). The influence exerted by the substrates on the results of ALP and CICP can be observed in Figure S2. Namely, DAHP/PEI sample has little effect on the results of ALP and CICP, DAHP-pretreated sample after several days of immersion has certain influence on the results of ALP and CICP, and the degradation or dissolution of bare Mg has significant impact on the results of ALP and CICP. 4.3 CICP concentration The MicroVue CICP assay is a sandwich enzyme immunoassay utilizing a monoclonal antiCICP antibody, a rabbit anti-CICP antiserum, a goat anti-rabbit alkaline phosphatase conjugate, and a pNPP substrate to quantify CICP. The goat anti-rabbit alkaline phosphatase conjugate is called Enzyme Conjugate Solution in the kit. Therefore, if extra phosphates, except the ones produced by the cell, exist in the testing solution during CICP measurements, the results would be influenced. CICP levels of hADSCs on Mg, DAHP, and DAHP/PEI60 were evaluated by using the kit. CICP results of Mg, DAHP, and DAHP/PEI60 have different trends in the days of incubation (in Figure.7). Trends are, namely, an irregular fluctuation on Mg substrate, an upward trend on DAHP, and a downward trend on DAHP/PEI60 coatings. CICP concentration


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of hADSCs on Mg might be influenced by the presence of unstable magnesium phosphates, which may have conjugated to the Enzyme Conjugate Solution (a goat anti-rabbit alkaline phosphatase conjugate) during the measurement. Due to the constantly non-stable magnesium phosphates deposition, it is assumed that the CICP results for magnesium were largely affected by the phosphates. Since only few hADSCs can be observed on Mg (in Figure. 6 and in Figure. 8), it is expected that hADSCs contributed little to the result of CICP level on Mg, mostly due to sequentially formed magnesium phosphates. Unlike the vulnerable magnesium substrate, DAHP is relatively stable during the first 3 days of incubation with hADSCs, which was confirmed by the electrochemical assessment (Figure. 2) and the remaining laminate structure (Figure. 8(b2)). After that, the attached hADSCs gradually covered the whole surface of DAHP, which would gradually decrease the exposed areas of the substrates from the culture medium and it will further slowdown the dissolution or degradation of DAHP. Though it is very challenging to evaluate the amount of phosphates released from DAHP coatings to the medium, we believe the release process will be hindered and the release amount of phosphates will be restricted down to a very low level with the rapid cell expansion on the surface. Therefore, the CICP concentration of hADSCs on DAHP is mostly derived from the differentiation of cells. CICP level of hADSCs on DAHP increased with longer incubation time, which indicates the enhanced potential for osteogenetic differentiation. Moreover, the very stable and smooth surface of DAHP/PEI60 offered hADSCs an ideal platform to differentiate and proliferate. CICP levels of hADSCs at different days exhibited a downward trend on DAHP/PEI60. The highest CICP concentration is found at 3 days, which indicates that the osteogenic differentiation potential of hADSCs was gradually weakened with longer incubation time. Even though the declined proliferating and differentiating capabilities of DAHP/PEI60 has been discovered, the osteogenic proliferation and


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differentiation of DAHP pre-coating beneath PEI in DAHP/PEI60 can be considered quite promising. It is indicating that the “second round” of derivation of hADSCs on DAHP/PEI60 can be promoted by the DAHP after the degradation of PEI coatings.

5. Conclusions DAHP/PEI coatings were successfully fabricated on magnesium substrates with a combination of chemical conversion and spin coating methods. The electrochemical tests indicated that DAHP/PEI coatings significantly improved the corrosion resistance of the magnesium substrate. Immersion test (72 hours) showed that no changes occurred to the chemical composition of DAHP/PEI coatings. In vitro cytocompatibility assessment demonstrated that MG-63 cells on DAHP/PEI coatings possess the highest viability, proliferation rate, and LDH activity by comparing with DAHP and pure magnesium samples. However, the biomineralization process, demonstrated by OsteoImage and ALP assessments, was less pronounced on DAHP/PEI coatings than on pure magnesium and DAHP, it has been proposed that the reason could be the deposition of extra phosphates during pure magnesium dissolution and the formation of extra phosphates during the coating fabrication which clearly impacted the measurements. The results of CICP levels of hADSCs gave a similar trend as that of OsteoImage and ALP measurements. When we summarize all results, especially considering fluorescent and SEM images, it can be stated that DAHP/PEI largely facilitated the osteogenic differentiation and proliferation of hADSCs. Besides that, the osteocompatibility and corrosion protection of DAHP could also enhance the potential of magnesium as orthopedic material. Thereafter, the DAHP/PEI coating system is a promising candidate for magnesium surface modification. In future, in vivo studies should be


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carried out, especially the in vivo degradation mechanism of DAHP/PEI coatings and the longterm cell behavior during the degradation must be investigated.


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ASSOCIATED CONTENT Supporting Information Available: Data about derived CICP concentrations (reference) at 3, 9, and 15 days of incubation, OsteoImage staining and ALP values of samples without MG-63 cell seeding after 72 hours in DMEM, and CICP concentrations of samples at 3, 9, and 15 days without cell co-culturing can be found in the supporting information.

Corresponding Authors (*) [email protected] [email protected]

ACKNOWLEDGMENT The authors would like to thank Alina Grünewald (Institute of Biomaterials), Kai Zheng (Institute of Biomaterials), Xiaoyan Du and Yicheng Zhao (Institute of Materials for Electronics and Energy Technology (i-MEET)) from University of Erlangen-Nuremberg, Germany, for the experimental supports of cell tests, SEM, and XRD measurements. Yuyun Yang acknowledges the China Scholarship Council (CSC) (scholarship No. 201308230074) and the support from the Fundamental Research Funds for Central Universities (HEUCFJ181004, 3072019CF1013) and the Scientific Research Foundation for the Returned Overseas Scholars in Heilongjiang Province


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(159100130004). Qiang Chen would like to acknowledge the financial support by the National Natural Science Foundation of China (31800802) and Shaanxi Provincial Key R&D Program (2018KW-031).

ABBREVIATIONS CICP, C-terminals of collagen Type Ӏ; DAHP, di-ammonium hydrogen phosphate; DMEM, Dulbecco’s modified Eagle’s medium; EDX, energy-dispersive X-ray; FTIR, fourier transform infrared spectroscopy; hADSCs, human adipose-derived stem cells; MG-63, Human osteoblast cells; PEI, poly ether imide; SEM, scanning electron microscopy; XRD, X-ray diffraction.

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In Vitro Osteocompatibility and Enhanced Biocorrosion Resistance of

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