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Biological and Medical Applications of Materials and Interfaces
Mechanically stable C2-Phenylalanine hybrid hydrogels for manipulating cell adhesion Auphedeous Y. Dang-i, Ayesha Kousar, Jinying Liu, Vincent Mukwaya, Changli Zhao, Fang Wang, Lei Hou, and Chuan-Liang Feng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b08655 • Publication Date (Web): 19 Jul 2019 Downloaded from pubs.acs.org on July 20, 2019
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Mechanically stable C2-Phenylalanine hybrid hydrogels for manipulating cell adhesion Auphedeous Y. Dang-i, † Ayesha Kousar, † Jinying Liu,† Vincent Mukwaya, † Changli Zhao,† Fang Wang, † Lei Hou,*§ and ChuanLiang Feng*†, AUTHOR ADDRESS † State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiaotong University, Dongchuan Rd 800, 200240, Shanghai § Department
of Cardiology, Tongren Hospital, Shanghai Jiaotong University, School of Medicine, 200336, Shanghai
KEYWORDS: Amino dextran, carboxylmethyl dextran, cell culture, supramolecular hydrogels, self-assembly ABSTRACT: The tuning of viscoelastic properties of supramolecular hydrogel to be used as biological materials substrates in tissue engineering has become significantly relevant in recent years due to their ability to influence cell fate. In the quest to enhance the stability and mechanical properties of a derived C2-phenylalanine gelator (LPF), derivatives of the polysaccharide dextran were incorporated as additives in order to promote hydrogen bonding and - stacking with the gelator. Dextran was esterified to yield carboxylmethyl dextran (CMDH), which was subsequently amidated to furnish aminodextran (AD), the resulting hybrid hydrogels were denoted as LPF-ADx and LPF-CMDHx, where x represents the amount of AD and CMDH (mg). The LPF gelator interacted with the carboxyl and amino functional groups of the CMDH and AD respectively through hydrogen bonding and π-π stacking, resulting in mechanically stable hydrogels. Morphological studies revealed that the hybrid hydrogels were formed as a result of dense highly-branched thin and broad fibers for LPF-AD and LPF-CMDH respectively. Rheological studies confirmed the superiority of the hybrid hydrogels over the neat hydrogel, where LPFCMDH3 exhibited the best mechanical properties with an improved elastic modulus of 11654 Pa over 1518 Pa and 140 Pa for LPF-AD4.5 and LPF respectively. The adhesion and spreading behavior of NIH 3T3 fibroblast cells was significantly improved on LPF-CMDH3 substrate owing to its enhanced mechanical properties. The tuning of mechanical properties of therein hydrogels via facile incorporation of biodegradable and biocompatible functionalized additives opens up avenues for strengthening the supposed weak supramolecular gelators, and hence increasing their potential of being employed largely in the field of tissue engineering.
INTRODUCTION The nature of interactions present between multicellular cells and substrate matrix at the interface in the field of tissue engineering is substantially crucial for the development and growth of healthy cell clusters.1-2 These interactions are not solely determined by the extracellular matrix (ECM) proteins,3 but regulated along with the physical and chemical attributes of the substrate being used.4-5 The ability of the cells to exhibit better interactions with substrate materials possessing certain features are given special focus in order to manipulate the fate of the cells.6-9 The boosting effect of substrate stiffness on the ability of cells to adhere to substrate surface has become a desirable feature in addition to low toxicity and biocompatibility. Supramolecular hydrogels (SMH) have been successfully demonstrated as suitable substrate material in the field of tissue engineering.10-13 This is probably due to the fact that SMH are able to self-assemble into nano-fibrous components that closely resemble the extracellular matrix (ECM). Furthermore, the base materials used for the synthesis of SMHs have tunable material properties and enhanced biological functions, making them desirable candidates for tissue engineering applications.14-18 Regardless of the various desirable attributes that SHMs substrate matrix may possess, their intrinsically poor mechanical properties limit their utilization as scaffolds in the field of tissue engineering. This undesirable trait of SMHs has however put them at a disadvantage compared to their polymeric hydrogel counterparts, as
they possess less endurance to large forms of compression or crack propagations, making them undesirable for applications as soft tissues.19 In principle, the mechanical properties of SMHs are largely dependent on the concentration of the gelator and the degree of entanglement of the 3D nano-fibrous network, which presents difficulties in tuning the mechanical properties of these hydrogels once they are formed. In order to tackle this issue, multiple strategies have been developed to tune the viscoelastic properties of SMH by enhancing hydrophobic interactions, hydrogen bonding, ionic crosslinking etc.20 The addition of biopolymers (i.e. polysaccharides) can impart desirable properties to SMH, making them ideal to be used for tissue engineering purposes. They play a structural role and additionally serve as biological ligands due to their biocompatibility. For instance, previous works from our group have shown that the incorporation of sodium-alginate in one case and sodium-hyaluronate in another, did not only enhance the mechanical properties of C2-phenylalanine derived gelators, but the reinforced hydrogel exhibited significant improvements in cell adhesion and spreading.21-22 However, hydrogels formed via the facile incorporation of biomolecules still lack the required physical properties, for they drastically disintegrate in water when left uncross-linked.23-24 One possible way to remedy this predicament is via the functionalization
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of biomolecules, which affords them functional moieties that allow more effective interactions with gelator molecules resulting in adequate cross-linking and subsequently furnishing more stable hydrogels. Over the last decade and counting, it has been shown that integral properties such as molecular flexibility, nanotopography, chemical functionality, degradability and most importantly the inherent stiffness of scaffold materials influences a variety of cell parameters (i.e. adhesion, viability differentiation and proliferation).25-26 For example, Prager-Khoutorsky et. al. demonstrated, that human cultured fibroblast cells are easily polarized when cultured on rigid substrates as compared to softer ones. Cells plated on rigid substrates formed large and uniform focal adhesion, whereas those on softer substrates formed small numerous rounded orientation leading to the conclusion that focal orientation may be a
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contributing factor for cell polarization.27 Ji and colleagues also developed an electrochemical redox multi-layer stimuli-responsive film with controllable surface stiffness. The application of their film in the culture of NIH 3T3 fibroblast cells on stiffer substrate revealed that cells adhered properly, spreading to cover a high area with many elongated observable filopodia. However, in the case of softer substrates, lower cell population was recorded and the cells were mostly rounded in nature.28 The challenge with most polymeric substrate used in cell studies stems from their poor biocompatibility. As a result, they have to be laminated with cell adhesive ligands and/or peptides such as collagen or fibronectin. This aggravates to the already tedious synthetic routes of these polymeric substrates.
Figure 1. Schematic representation of molecular structures of the gelator (LPF), Amino dextran (AD) and carboxylmethyl dextran (CMDH), the structures of their self-assembled nanofibers and the illustration of their effect on NIH 3T3 cell adhesion and proliferation.
In this study, we report two kinds of hybrid hydrogels prepared via a straightforward route that entails the blending of a C2-phenylalanine gelator (LPF) and two derivatives of dextran; Amino dextran (AD) and carboxymethyl dextran (CMDH) (Figure.1). The LPF gelator interacted with the carboxyl and amino functional groups of the CMDH and AD respectively through hydrogen bonding and - stacking, resulting in mechanically stable hydrogels. The procedure for obtaining these hydrogels present a facile approach which can be adopted for obtaining mechanically stable SMH since it does not require any complex chemical procedure or further purification of resultant precursors. The influence of substrate mechanical properties on cell adhesion and proliferation was probed by employment of the therein fabricated hybrid hydrogels as cell culturing scaffolds. EXPERIMENTAL SECTION
Materials and reagents. All chemicals and reagents used in this study were obtained from Aladdin chemicals and were used without any further purification. The gelator (LPF), amino dextran and carboxymethyl dextran were synthesized using the conventional liquid phase reaction as shown in Figure S1 and S2. Synthesis of phenylalanine derived gelator (LPF). 6g of Lphenylalanine methyl ester hydrochloride (26.1mmol) was suspended in dry dichloromethane (DCM) (50ml) and triethylamine (Et3N 8.0ml, 58.3mmol) was added dropwise, the mixture was stirred for 30 minutes. A suspension of 1,4-benzenedicarbonyl dichloride (6.0g, 26.1mmol) in DCM was then added to the stirring solution dropwise and allowed to stir at room temperature for 24 hours. All solvents were then removed under vacuum and the resulting residue dissolved in ethanol (100ml). This was followed by the filtration and drying of the undissolved residue to give the dimethyl ester of the gelator (5.3g, 10.9 mmol, 84%). An aqueous
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solution of NaOH (10ml, 2.0 M) was adding to a cooled (0 °C) suspension of the obtained product (3.0g, 6.14mmol) in methanol (20ml). The mixture was allowed to stir for 24 hours at room temperature and then acidified with 2.0 M HCl to a pH
