Inkjet printing of laminin gradient to investigate endothelial cellular alignment

https://doi.org/10.1016/j.colsurfb.2009.04.008Get rights and content

Abstract

To investigate the influence of the protein surface-density gradient on endothelial cell alignment, a novel approach for the fabrication of a laminin gradient on gold-coated substrates has been developed in this study. Our approach involves programmed inkjet printing of an alkanethiol (11-mercaptoundecanoic acid, C10COOH, MUA) gradient onto gold-coated substrates, followed by backfilling with 11-mercapto-1-undecanol (C11OH, MUD). The –COOH moieties were activated and then covalently linked with laminin. This treatment led to a surface-density gradient of laminin. Contact angle measurement, X-ray photoelectron spectroscopy (XPS) and fluorescence microscopy were employed to characterize the self-assembled monolayers (SAMs) and protein gradient, respectively. Results proved the feasibility of the fabrication of a protein gradient by using the inkjet printing technique. The self-assembled monolayer gradients displayed a high packing density, as indicated by dynamic contact angle measurement. More importantly, the gradient slope was easily tunable over a significant distance from 20 to 30 mm. The laminin gradient was clearly visible by fluorescence microscopy observation. Endothelial cells cultured on the surface-density gradient of laminin demonstrated a strong alignment tendency in parallel to the gradient. The higher the laminin density the more cells were observed. The result indicates that cell attachment is dependent on the surface density of laminin. This work broadens our methodology to investigate chemical stimuli-induced cell directional alignment. It is potentially important for understanding cell alignment/ingrowth behavior for angiogenesis and implant technology including tissue-engineered structures.

Introduction

Although tissue engineering has achieved great successes in the past decades, many challenges remain. One of the challenges is the formation of blood vessels (angiogenesis) of microcirculation [1]. Angiogenesis is essential for the successful implantation for biomaterials and tissue-engineered structures because newly formed blood vessels provide the necessary nutrition and oxygen for their enduring performance. Endothelial cells (ECs) migration and alignment are essentially important for vascular remodeling during angiogenesis. In principle, ECs secrete specific protease to degrade extracellular matrix, and then migrate into the prevascular space to proliferate, align to form new blood vessels [2].

ECs alignment could be regulated by multiple factors through various mechanisms, such as mechanotaxis [3], hepatotaxis [4] and chemotaxis [5]. Chemotaxis refers to the characteristic movement or orientation of a cell along with a chemical concentration gradient. Concentration gradient of soluble growth factors, such as vascular endothelial growth factor (VEGF) [6], fibroblast growth factor 2 (FGF2) [7] and fN’LFN’YK peptide [8], was well documented to direct ECs alignment. For tissue engineering application, however, materials surface-bound proteins or peptide gradients are particularly important to mimic the biological microenvironment. It is beneficial for producing biocompatible or bioconductive scaffold materials. Surface modification with extracellular matrix proteins is an alternative to fabricate materials surface-bounded protein gradients, which may in turn induce cell alignment.

Many studies have reported that gradients of surface-bound proteins or growth factors affected cell behaviors, such as attachment and alignment. For example, Liu et al. demonstrated that EC alignment was regulated by surface-density gradients of fibronectin, VEGF, or both proteins [9]. Hydrogel surface of covalently immobilized gradient arginine–glycine–aspartic acid (RGD) peptide was shown to direct the alignment of fibroblasts [10]. Meanwhile, the migration of human microvascular endothelial cells (hMECs) was confirmed to be sensitive to the gradient slope of fibronectin [11]. Dertinger et al. showed that orientation and axonal specification of neurons were affected by surface-bound laminin gradients [12]. Gunawan et al. found that the protein expression of cells was region-specifically controlled by two-component counter gradients of collagen and laminin [13]. However, no work has investigated the influence of the surface-density gradient of laminin on the alignment of endothelial cells. In this current study, laminin was employed to create a protein gradient. Laminin is one of the proteins within extracellular matrix with potential to promote angiogenesis [14].

Different techniques have been developed for generating gradients on substrate surfaces, including microfluidics [15], controlled diffusion [16], micropattern [17], and photo-linking immobilization [18]. However, these methods share common disadvantages, such as high cost and difficulty in operation. In this study, we employed the inkjet printing technique to fabricate surface-density gradient of laminin due to its several advantages. An obvious advantage of inkjet printing is its low cost. Meanwhile, inkjet printing is highly automated and controlled by a computer program. It ensures the high reproducibility. In addition, inkjet printing is a non-contact method. In operation, the nozzle of printer does not come into the contact with the printed surface, reducing the risk of cross contamination. Inkjet printer was initially utilized to printing 11-mercaptoundecanoic acid on gold-coated substrate for creating gradients of –COOH moieties. Laminin was then covalently immobilized on the printed substrates, resulting in a laminin gradient.

To the best of our knowledge, this is the first study to employ the inkjet printing technique to fabricate a laminin gradient on gold-coated substrates. The objective of this study was to fabricate and characterize the surface-density gradient of laminin. The alignment behavior of endothelial cells in response to the laminin gradient was investigated in vitro as well.

Section snippets

Materials

Silicon wafer substrates were purchased from Hongkong Chujie Co. (China). The 11-mercaptoundecanoic acid (MUA), 11-mercapto-1-undecanol (MUD), 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma Co. (USA). Silicon wafer substrates were cleaned with a “Piranha” solution (7/3 of concentrated sulfuric acid and hydrogen peroxide. CAUTION, it is highly corrosive) for 60 min, followed by rinsing with copious amounts of distilled water and

Laminin gradient design

In this study, we utilized the inkjet printing technique to fabricate laminin gradient due to its advantage of accurate deposition of liquids [22], [23]. The laminin gradient was fabricated in three steps (Fig. 1): first, inkjet printing of gradient density of C10COOH onto gold-coated substrates; second, backfilling with the second thiol of C11OH, leading to form a C10COOH/C11OH counter-propagating SAMs gradient. The initial inkjet printed C10COOH surfaces displayed an order-gradient, i.e.,

Conclusions

In this study, we reported a novel approach for the fabrication of a laminin gradient on gold-coated substrates via inkjet printing. The SAM gradients and laminin gradient were well characterized with combined techniques of contact angle measurement, X-ray photoelectron spectroscopy (XPS) and fluorescence microscopy. The in vitro cell culture analysis demonstrated that laminin gradient substrates positively influence the endothelial cells’ alignment and cytoskeletal organization. This work

Acknowledgments

This work was financially supported by Natural Science Foundation of China (50603032), China Ministry of Science and Technology (973 Project no. 2009CB930000), Program for New Century Excellent Talents in University (NCET-07-0904), and“111 project” (B06023). KDJ gratefully acknowledges the partial financial support of the BMBF for this work within the project “Innovations- und Gründerlabor für neue Werkstoffe (Biomaterialien) und Verfahren (IGWV) an der Friedrich-Schiller-Universität Jena”

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