Article pubs.acs.org/journal/abseba
Adhesive Prebiotic Chemistry Inspired Coatings for Bone Contacting Applications Donna J. Menzies,† Andrew Ang,‡ Helmut Thissen,*,† and Richard A. Evans*,† †
Commonwealth Scientific and Industrial Research Organisation (CSIRO) Manufacturing, Clayton, VIC 3169, Australia Faculty of Science, Engineering and Technology, Swinburne University, Hawthorn, VIC 3122, Australia
‡
S Supporting Information *
ABSTRACT: New and improved bone-contacting medical devices are required to provide excellent bioactivity at the biointerface. Here, we have used coatings based on prebiotic chemistry inspired polymerization of aminomalonitrile (AMN) in combination with comonomers 3,4-di- and 3,4,5-trihydroxybenzaldehyde (DHBA and THBA). The comonomers were incorporated into the AMN coatings to enhance polymerization kinetics, adhesive properties, metal binding efficacy, and human mesenchymal stem cell (hMSC) response. Incorporation of DHBA and THBA as separate comonomers enhanced the polymerization kinetics compared to that of AMN polymerization alone, with 30 mol % THBA (30T) resulting in a 6-fold increase in thickness over 24 h. Furthermore, the adhesion of AMN coatings to silicon was enhanced when copolymerized with the HBA monomers, where the interfacial adhesion of the 30T coating was increased 20-fold. The ability of the coatings to incorporate zinc ions was investigated, and X-ray photoelectron spectroscopy (XPS) analysis demonstrated that incorporating 30T increased the binding efficiency 4-fold compared to that of AMN alone. The attachment, proliferation, and morphology of human mesenchymal stem cells (hMSC) on these coatings was investigated and reported. Finally, the utility of the coatings as osteogenic support matrices via the induced osteogenic differentiation of hMSCs is reported. The AMN and 30T coatings resulted in the greatest efficiency of osteogenic differentiation, as measured by intracellular ALP activity and mineralization. Incorporation of zinc had a stimulatory effect on hMSC proliferation with 30T coatings, while enhanced mineralization was observed with the zinc functionalized AMN and 30T coatings. This study highlights the potential of prebiotic chemistry inspired coatings in biomedical applications. KEYWORDS: prebiotic chemistry, universal coatings, stem cells, bioactive coatings, osteogenic differentiation, adhesive coating
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INTRODUCTION Coatings are critical to the successful application of a broad number of biomedical devices, including implants, which may possess the integrity, load-bearing capabilities, and threedimensional structure required for its performance but often lack the ability to control the biological response at the biointerface, for effective integration and function. Some examples include orthopedic and dental implants, which are often made from inert polymeric materials, ceramics, or metals such as titanium. In the case of bone-contacting materials, the osseointegration of the bulk materials can be enhanced by the application of a coating that facilitates biomineralization, an essential process for the formation of functional bone whereby minerals are deposited intra- and extracellularly. During stem cell and osteoblast differentiation, the early upregulation and expression of alkaline phosphatase (ALP) serves to generate an excess of free phosphate ions to bind with calcium ions for the formation of hydroxyapatite. Matrix vesicles are extracellular nanoparticles of hydroxyapatite which are located around the cell membrane, and the initial formation of hydroxyapatite © XXXX American Chemical Society
crystals are stimulated by the expression of ALP. Physiologically or in the case of laboratory simulating conditions, the presence of sufficient calcium and phosphate ions in the local environment continues the cascade of the biomineralization process, with the matrix vesicles serving as nucleation sites.1 The process, which takes osteoblast progenitor cells, hMSC, from an osteogenic lineage commitment to mature osteoblasts is a biologically complex process with multiple steps. Therefore, researchers are generating synthetic materials which possess bioinstructive compositions.2 For example, various growth factors such as bone morphogenic protein (BMP-2),3 osteoinductive peptide cues,2,3 as well as various metal ions such as Zn2+, Mg2+, Co2+, and Sr2+,4−7 have been used for enhanced osteogenic and biomineralization effects. Zinc in particular is an essential trace element showing a stimulatory effect on bone formation and mineralization both in vivo and in Received: January 18, 2017 Accepted: March 20, 2017 Published: March 20, 2017 A
DOI: 10.1021/acsbiomaterials.7b00038 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
surfactant (Pierce) in 2% ethanol in Milli-Q water, followed by a further 15 min of ultrasonication in Milli-Q water. Substrates were then dried in a high pressure stream of high purity nitrogen. For XPS and SEM analysis of the AMN based coatings, deposition was performed onto silicon wafers (Ultraflat single crystal silicon wafers (⟨100⟩, 1 cm2 × 0.5 mm thick, M.M.R.C P/L). For specular reflectance FTIR analysis of the polymeric coatings, indium tin oxide coated aluminosilicate glass (Delta Technologies, Corning 1737, CB50IN) was used to provide a reflective substrate. For the analysis of the interactions and behavior of hMSCs on the AMN based coatings, the wells of TCPS plates (Nunc) were coated directly and used as received. AMN Coating Procedure. AMN coatings were prepared from a stock solution (20 mg/mL, 7.90 × 10−2 M) of aminomalonitrile ptoluenesulfonate (Sigma-Aldrich, 98%) in 100 mM pure phosphate buffer (pH 8.5) and the pH increased to 8.5 with NaOH (1 M). The concentration of the AMN solution was then adjusted to 1% w/v (3.95 × 10−2 M) using 100 mM phosphate buffer (pH 8.5) for polymerization. For incorporation of the hydroxybenzaldehyde (HBA) comonomer films, stock solutions of 3,4-dihydroxybenzaldehyde (DHBA, Merck, 98%) and 3,4,5-trihydroxybenzaldehyde monohydrate (THBA, 99% Sigma-Aldrich) were prepared at 5 mg/mL, and solubilized by increasing the pH to 8.5 with 1 M NaOH. The 10, 20, and 30D and T samples were prepared by substituting 10, 20, or 30 mol % of AMN (20 mg/mL stock) for the desired HBA monomer. For example, to prepare 6 mL of 30T, 2.1 mL of 20 mg/mL AMN stock solution (1.7 × 10−4 mol) was mixed with 2.45 mL of the preprepared THBA stock solution (7.1 × 10−5 mol), and an additional 1.45 mL of buffer added, resulting in final concentrations of 2.76 × 10−2 and 1.18 × 10−2 M for the AMN and THBA, respectively. To coat the wells of 96 well TCPS plates, 350 μL of each solution was added to the wells and left to polymerize for 24 h at 25 °C, before removing the solution and rinsing the wells with Milli-Q water 6 times, followed by a further 24 h rinse in Milli-Q water before being left to dry in the laminar flow cabinet for 24 h. For the coating of Si wafers and ITO-coated glass, 6 mL of the desired solution was added to a 15 mL centrifuge tube. Precut substrates with a size of approximately 0.9 × 0.8 cm2 were placed upside down and horizontally approximately midway in the solution of the tube. This was left to react at 25 °C for 24 h, before the solution was decanted and rinsed 6 times with Milli-Q water and left for a further 24 h in fresh Milli-Q water at room temperature. Finally, the samples were rinsed once more with Milli-Q water, then dried using a stream of high purity nitrogen. Zinc Functionalization. AMN and AMN-co-HBA films were functionalized with zinc by incubating the films in 100 mM zinc nitrate hexahydrate (Sigma-Aldrich, 98%) solution for 24 h. Samples were then rinsed 6 times with Milli-Q water before being dried under a high pressure stream of high purity nitrogen. Masking and Film Thickness Analysis. Film thickness was measured on premasked Si wafers using stylus profilometry. The masking procedure was done by solvent casting a 10% w/v solution of poly(D,L-lactide) in acetone onto a section of a cleaned Si wafer and letting it dry in air. Once the AMN-based polymer was deposited over the top of the mask, the mask was gently peeled away using tweezers, enabling the measurement of the well-defined step heights for film thickness analysis. A Veeco Dektak 6 M stylus profilometer was used for the thickness measurements. Briefly, the force of the stylus (having a diameter of 12.5 μm) was set to 3 mg and traced across a distance of 700 μm over 10 s. Average values are reported from 5 repeats per sample. X-ray Photoelectron Spectroscopy (XPS). X-ray photoelectron spectroscopy (XPS) analysis was performed using either an AXIS Ultra DLD or an AXIS Nova spectrometer (Kratos Analytical Inc., Manchester, UK) with a monochromated Al Kα source at a power of 180 W (15 kV × 12 mA), a hemispherical analyzer operating in the fixed analyzer transmission mode, and the standard aperture (analysis area: 0.3 mm × 0.7 mm). The total pressure in the main vacuum chamber during analysis was typically between 10 and 9 and 10−8 mbar. Survey spectra were acquired at a pass energy of 160 eV. To
vitro, while a deficiency can result in retardation of bone growth, development, and maintenance. The exact mechanism is not well understood; however, it has been hypothesized that the presentation of Zn2+ results in enhanced phosphate binding and hence calcification, as well as having the ability to inhibit osteoclastic bone resorption in vitro.8 Zn2+ is also present on the active site of ALP and has been shown to stimulate and enhance the ALP activity of osteoblast cells.9,10 In order to improve the biointerfacial interactions of bonecontacting devices, simple, water-based universal coatings would ideally be used, which have the ability to coat and adhere to almost any substrate material. However, very few such coatings exist, with polydopamine coatings introduced by Messersmith et al.11 being the exception. The outstanding interest in such simple coating chemistries is demonstrated by the number of applications that have been reported in a short amount of time, ranging from the control of cell attachment,12 the direction of stem cells,3 antifouling and antibacterial properties,13 drug delivery,14 membrane filtration for water detoxification, and energy storage devices.15 Other polyphenolic and catecholamine based materials have also been investigated for their film forming abilities and biocompatibility, for example, tannic acid.16−18 In contrast, we recently reported a novel approach to the fabrication of water based universal polymeric coatings, which was inspired by prebiotic chemistry.19 In this approach, the spontaneous polymerization of aminomalonitrile (AMN) can be induced by neutralizing the commercially available salt in simple aqueous solutions. The coating chemistry, which has been demonstrated to provide the same result over a broad range of substrate materials and shapes, provides highly biocompatible and cell adhesive coatings. In addition, the rich chemistry has been demonstrated to allow for the copolymerization and subsequent reaction with a range of compounds containing functional groups such as aldehydes and amines. In this article, we present a physicochemical analysis of AMNbased coatings, with and without the comonomers 3,4-di- and 3,4,5-trihydroxy benzaldehyde (DHBA and THBA). The copolymer components were incorporated into the AMN coatings at concentrations ranging from 10 to 30 mol % with the aim to further enhance the polymerization kinetics, adhesive properties, metal binding efficacy, and human mesenchymal stem cell (hMSC) response. Subsequent to the incorporation of zinc ions, we investigated the biological activity of all of these surfaces toward human mesenchymal stem cells (hMSCs) and tested the utility of these coatings as osteogenic support matrices. While the incorporation of HBA comonomers enhanced the attachment, proliferation, and cytoskeletal arrangement of hMSCs, it was found that the osteogenic differentiation on the pure AMN surface was facilitated to levels equivalent to hMSCs on tissue culture polystyrene (TCPS), and this was further enhanced by the incorporation of zinc ions. The study demonstrates that tunable physicochemical properties of robust coatings can be generated from AMN based copolymers and that these bioactive surface coatings are capable of influencing the phenotype of hMSCs toward an osteogenic lineage.
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MATERIALS AND METHODS
Substrate Materials and Cleaning. All substrates were cleaned prior to deposition of the aminomalonitrile p-toluenesulfonate (AMN, Sigma) based coatings. Substrate cleaning was performed by ultrasonication for 45 min in a solution containing 2% RBS-35 B
DOI: 10.1021/acsbiomaterials.7b00038 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering obtain more detailed information about chemical structure, oxidation states, etc., high resolution spectra were recorded from individual peaks at 40 eV pass energy (yielding a typical peak width for polymers of
