Photopatterning of hyaluronic acid hydrogels for cell culture scaffolds

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1 Photopatterning of hyaluronic acid hydrogels for cell culture scaffolds Ana Maria Porras 1, Frida Sjögren 1, Liyang Shi 2, Dmitri Ossipov 2, Maria Tenje* 1,3,4 1 Dept. of Engineering Sciences Uppsala University, Uppsala, Sweden. 2 Dept. of Chemistry, Uppsala University, Uppsala Sweden. 3 Dept. of Biomedical Engineering, Lund University, Lund, Sweden. 4 Science for Life Laboratory, Uppsala, Sweden. *maria.tenje@angstrom.uu.se Abstract. Organs-on-chips technologies require the development of micro engineered devices to represent functional units of human organs. These devices use cell culture scaffolds to give support and structure for the cultured cells. Hydrogels are attractive scaffold materials, due to their high water content and because they are derived from natural polymers found in the extracellular matrix of different tissues in the human body. Hyaluronic acid can form hydrogels when functionalized with chemo-selective groups. These chemically cross-linked hydrogels can be modified with adhesion motifs, such as RGD peptides, to increase their biocompatibility and promote cell adhesion. In this work we use a photolithographic method to pattern the RGD peptide into distinct areas of the hyaluronic acid hydrogel with the aim to spatially control the cell attachment on the HA hydrogel scaffolds. 1. Introduction The emerging research field organs-on-chips relies on material scaffolds to serve as the basis for cell cultures. A scaffold is defined as a temporary supporting structure for growing cells and tissue. It can also be referred as a synthetic extracellular matrix (ECM). The scaffold organizes cells into a threedimensional architecture, which will stimulate and direct the growth and formation of the desired tissue [1][2]. Hydrogels are attractive scaffold materials, due to their high water content and ideal mechanical and chemical properties similar to those in body tissues [2][3][4][5]. The mechanical properties of the hydrogel structures depend on the crosslinking density between the polymer chains. The chemical properties of the hydrogel are determined by the functional groups of the polymers. There are a variety of synthetic and natural polymers used to form hydrogels. Naturally derived polymers are preferred in tissue engineering applications since they are derived from the natural ECM and therefore, have properties similar to the natural ECM [2]. Hyaluronic acid (HA) is a polysaccharide formed by repetitive D-glucuronic acid and N-acetyl-Dglucosamine monosaccharide (Figure 1), and it is found in the ECM of different tissues [6][7]. The

2 unmodified polysaccharide has a molecular weight between 10 and 10 4 kda [4][8]. HA is negatively charged, which attracts positive ions resulting in an osmotic balance that brings in water. This characteristic allows the polymer to form hydrogels [4]. However, the functionalization of HA with chemo-selective groups is required for the formation of chemically cross-liked HA hydrogel materials [9]. Figure 1. Hyaluronic acid. HA is a polysaccharide formed by repetitive D-glucuronic acid and N-acetyl-D-glucosamine monosaccharide. The carboxylic acid and hydroxyl groups are the most common sites used for covalent modification of hyaluronic acid. By Edgar181 - Own work, Public Domain, Hyaluronic acid cell culture scaffolds have been achieved by several chemical modifications, resulting in mechanically and chemically robust materials. The most common modification of HA is crosslinking to form hydrogels [9][10][11]. Hyaluronic acid contains groups on its backbone that are available for functionalization [3][12]. The two most common sites used for covalent modification are the carboxylic acids and the hydroxyl groups (Figure 1). Also, thiol or alkene groups can be attached into the HA backbone to allow thiol-ene addition reactions for further attachment of bioactive molecules into the cross-linked HA hydrogels [7][13]. Thiol-ene reactions occur between a thiol and an alkene (Figure 2). Thiol groups present weak sulfurhydrogen bonds, which results in a large amount of chemical reactions with an ability to initiate by mild conditions [14][15]. Figure 2. Thiol-ene reaction. The reaction involves the addition of a thiol (-SH) to across a double bond (-C=C) in the presence of light. Exposure to thiol to UV light generates thiyl radical which is addet to the double bond with localization of unpaired electron on the carbon atom. HA hydrogels were synthesized from high molecular weight hyaluronic acid. High and low molecular weight hyaluronic acid possess opposing effects on cell functions [4]. It has been shown that high molecular weight HA inhibits cell proliferation and angiogenesis [16]. HA can be modified with cell adhesion ligands to make the hydrogel biocompatible. These ligands can be adhesion motifs, antibodies, growth factors and gene-encoding nucleic acids, or other bioactive molecules [4]. Integrins are transmembrane proteins that act as cell adhesion receptors. RGD- binding integrins receptors recognize the RGD motif, a sequence of amino acids Arg-Gly-Asp [17]. This motif is found in many ECM molecules such as fibronectin, collagen and osteopontin [17][18]. Such motif can be included in the cell culture scaffold to increase its biocompatibility. In this work we use photolithographic method to pattern the RGD peptide into distinct areas of the HA hydrogel with the aim to spatially control cell attachment on the HA hydrogel scaffold. In order to incorporate RGD peptides into the hyaluronic acid derivatives used for this work, a RGD peptide was designed to contain thiol groups (Figure 3). The RGD peptides are chemically immobilized into the HA-acrylamide hydrogel by UV light triggered thiol-ene addition reaction.

3 Figure 3. RGD containing peptide. A RGD containing peptide is designed to have a fluorescent tag and a thiol group. The cysteine amino acid contains a thiol group, which will be used for the peptide linkage into the gel matrix. 5-FAM is a fluorophore that will allow detecting the presence of the peptide in the hydrogel by fluorescence microscopy. 2. Synthesis of hyaluronic acid acrylamide derivative Sodium hyaluronate was purchased from Lifecore Biomedical. N-(3-Dimethylam)inopropyl)-N'- enthylcarbodiimide hydrochloride (EDC), 1-Hydroxybenzotriazole hydrate (HOBt), acetonitrile and Irgacure 2959 were purchased from Sigma Aldrich. Hydrochloric acid 37% (HCl:H 2 O) and sodium chloride (NaCl) were purchased from Fischer Scientific. Dialysis membranes Spectra/Por were purchased from SpectrumLabs and filter paper was purchased from Munktell. Sodium hyaluronate (200 mg, MW=135 kda) was dissolved in deionized water at a concentration of 8 mg/ml. Acrylamide containing linker (56 mg) was added to the mixture, and maintained in the dark. HOBt, (72 mg) was separately dissolved in a 1:1 (v/v) mixture acetonitrile and deionized H 2 O at a concentration 27 mg/ml (3.0 ml). The mixture was heated to solubilize HOBt. Once the HOBt solution was cooled to room temperature, it was added to the HA solution. ph of the reaction solution was adjusted to ph 6. The coupling reaction was initiated by the addition of EDC (155 mg). The reaction was stirred at room temperature overnight in the dark. The reaction solution was transferred to a dialyzing membrane (MW cut off 3.5 kda,) and dialyzed against distilled H 2 O and 5% w/v NaCl for 24 hours. Few drops of concentrated HCL were added to adjust the ph to 3.5. The solution was filtered to give rise to a clear and transparent solution. The solution was freeze-dried and kept at -20 ºC until further used. 3. Preparation and photopatterning of HA-Am hydrogels with RGDSC peptides Irgacure 2959 initiator was dissolved in degassed water at a concentration of 0.4% (w/v). HA modified with acrylamide groups (HA-am) was dissolved in the initiator solution to a concentration of 2% (w/v). The solution was placed between two glass slides, one of them covered with parafilm, separated by an adhesive spacer. The system was then irradiated with UV light for 4 s at an intensity of 1700 mw/cm 2. The gels were placed in PBS solution (0.1 mm). The RGDSC-5-FAM peptide solution (0.3 mm) was deposited (20 µl) onto the HA-am hydrogels. The gel was covered with a plastic photomask and then the system was exposed to UV light for 4 s at an intensity of 1700 mw/cm 2. The unreacted peptides were removed by a washing step with PBS (0.1 mm) overnight, placed on a shaker (Figure 4).

4 Figure 4. Preparation and photopatterning of HA-Am hydrogels with RGDSC peptides. HA-acrylamide liquid solution is placed between two glass slides. The system is irradiated by UV light to start the photopolymerization of the acrylamide groups. After the hydrogel is formed, it is placed in a RGDSC solution. The gel is then again exposed to UV light to immobilize RGDSC peptides through thiol-ene addition reaction. The irradiated hydrogel is washed in PBS solution overnight

5 Hyaluronic acid acrylamide derivative formed a gel after exposure to UV light. After the gel was formed the hydrogel was cover by a photomask and placed in a RGD solution. The hydrogel was irradiated with UV light for another 4 s. After washing the gel in PBS overnight it was possible to observe a pattern, where the RGD had been selectively linked to the HA hydrogel in the doubleexposed areas (Figure 5). a 500 µm b 500 µm Figure 5. Patterned HA-am hydrogels. a. The picture was taken using a FITC filter (Ex , BA 605/55). b. The picture was taken under bright field. Scale bars equal to 500 µm. The photomask used was printed on transparencies. The design of the photomask is 800 µm squares with a separation distance of 800 µm. The hydrogels were visualized using a Nikon Eclipse TE200-U microscope. The pictures were taken using an embedded camera (Diagnostic Instruments Inc., 7.0 Monochrome w/o IR). The patterned area was observed under FITC filter (Fig. 5a) and under bright field (Fig. 5b). The patterned area was first exposed for 4 s to form the hydrogel (without a mask). Then, it was exposed for other 4 s (through the mask) to attach the RGD peptides into the matrix, resulting in a higher crosslinking than the rest of the gel, making it possible to observe the area under bright field. 4. Conclusions In this work we show a method to photopattern RGD peptides in hyaluronic acid hydrogels. The use of photolithography shows to be a successful technique for the patterning of bioactive molecules where spatially well-defined areas can be functionalized. Here it is shown that by using a photomask one can achieve different patterns designs. Photolithography shows to be a straightforward promising technology for the patterning of hyaluronic acid hydrogels. Acknowledgments This work is financed by the Swedish Research Council Formas and Uppsala University. References [1] M. N. Collins and C. Birkinshaw, Morphology of crosslinked hyaluronic acid porous hydrogels, J. Appl. Polym. Sci., vol. 120, no. 2, pp , Apr [2] J. L. Drury and D. J. Mooney, Hydrogels for tissue engineering: scaffold design variables and applications, Biomaterials, vol. 24, no. 24, pp , Nov [3] C. A. Goubko and X. Cao, Patterning multiple cell types in co-cultures: A review, Mater. Sci. Eng. C, vol. 29, no. 6, pp , Aug [4] J. Lam, N. F. Truong, and T. Segura, Design of cell-matrix interactions in hyaluronic acid hydrogel scaffolds, Acta Biomater., vol. 10, no. 4, pp , Apr [5] I. Tomatsu, K. Peng, and A. Kros, Photoresponsive hydrogels for biomedical applications, Adv. Drug Deliv. Rev., vol. 63, no , pp , Nov

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