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Physicochemical properties of ionic and non-ionic biocompatible hydrogels in water and cell culture conditions: Relation with type of morphologies of bovine fetal fibroblasts in contact with the surfaces

https://doi.org/10.1016/j.colsurfb.2017.07.032Get rights and content

Highlights

  • Ionic and non-ionic hydrogel surfaces are studied as scaffolds of BFF cells.

  • Physicochemical and superficial properties of hydrogel change with medium conditions.

  • Flattened and spindle-shaped and spheroid cell morphologies are formed on different surfaces.

  • Non-ionic and hydrophilic surfaces with moderated wettability induce the formation of cell spheroids.

Abstract

Cationic, anionic and non-ionic hydrogels having acrylamide polymer backbones were synthesized via free radical polymerization with N,N-methylenebisacrylamide (BIS) as crosslinker. The chemical structures of the hydrogels were characterized by Fourier Transform Infrared Spectroscopy (FTIR). Physicochemical properties such as swelling kinetic, maximum swelling capacity, volume phase transition temperature (VPTT) and wettability (static water contact angle) of hydrogels swollen in aqueous and cell culture medium, at room and cell culture temperatures were studied. In order to correlate the surface properties of the hydrogels and cellular adhesivity of bovine fetal fibroblasts (BFFs), cellular behaviour was analyzed by inverted fluorescence optical microscopy and atomic force microscopy (AFM). MTT assay demonstrated that the number of viable cells in contact with hydrogels does not significantly change in comparison to a control surface. Flattened and spindle-shaped cells and cell spheroids were the adopted morphologies during first days of culture on different hydrogels. Cell spheroids were easily obtained during the first 5 days of culture in contact with PNIPAM-co-20%HMA (poly (N-isopropylacrylamide-co-20%N-acryloyl-tris-(hydroxymethyl)aminomethane)) hydrogel surface. After 15 days of culture all hydrogels showed high adhesion and visual proliferation. According to obtained results, non-ionic and hydrophilic surfaces with moderated wettability induce the formation of BFFs cell spheroids. These hydrogel surfaces could be used in clinical and biochemical treatments at laboratory level to cell growth and will allow generating the base for future biotechnologic platform.

Introduction

Biomaterials are being used in cell culture technique as scaffolds to create tissue-like structures that simulate the mechanical and physiological features of in-vivo tissues. Biomaterials based on hydrogels have great potential for tissue reconstruction as either temporary or stable scaffolds or cell vehicles for tissue engineering [1]. In addition, the cell–cell and cell–extracellular matrix (ECM) interactions play a significant role in cell growth. Hydrogels are crosslinked polymers (covalent or physic bond) with high content of hydrophilic groups allowing high uptake of biological fluids. For that reason, these are one of the most commonly used materials in biomedicine since they could emulate ECM structure providing sites for adhesion, proliferation and even more cell differentiation [2].

Nowadays, synthetic and natural hydrogels are also being widely investigated for biotechnological applications, for example the immobilization of Saccharomyces cerevisiae in hydrogel matrix for bioethanol production [3]. In this case, the biocompatibility and protective character of poly-acrylamide hydrogel on yeast were demonstrated. Gelatin is a kind of natural polymer with low cost and good biocompatibility but this could be quickly denatured [4] which may be a disadvantage depending on the applications.

Poly(N-isopropylacrylamide) (PNIPAM) is the most extensively studied synthetic polymer for therapeutic purposes. PNIPAM has a low critical solution temperature (LCST) value at 32 °C [5], [6]. When the environment temperature exceeds the PNIPAM LCST value, the hydrogel undergoes a reversible volume phase transition, collapses and expels the internal liquid. The phase transition produces a drastic change on hydrophilic/hydrophobic properties of the material [7] and consequently this change could be altering the cell adhesion/detachment mechanisms [8], [9]. These changes were observed when the chemical composition of scaffold material was modified by copolymerization with hydrophilic or hydrophobic co-monomers [10]. Moreover, interpenetrated or semi-interpenetrated systems based on thermosensitive hydrogel and a second polymer [11], polymeric nanocomposites based on hydrogel matrix [7], [12] and thermosensitive surfaces [7] showed great changes on their physicochemical, superficial and mechanical properties.

Citotoxicity, viability and cellular damage have been studied in contact with PNIPAM for Caco-2 and Calu-3 cells [6], mesenchymal stem cells derived from rat bone marrow (BM-MSCs), human adipose tissue (AT-MSCs) [13], smooth muscle (SMC) [14] and other biological systems. However, there is scarce information about physicochemical properties of hydrogels exposed to cell culture medium. We believe that it is important to know the physicochemical behavior of hydrogels under cell culture conditions, in order to understand and predict how the cell will respond to environment changes [15].

In this work, we propose to study the physicochemical properties of polyacrylamide (PAAM), PNIPAM hydrogel and copolymers functionalized with anionic, cationic and non-ionic monomers in both aqueous and cell culture medium. Material properties as swelling kinetics, volume phase transition temperatures (VPTT) and wettability (static contact angle) were characterized. The physicochemical properties of the hydrogel surfaces in culture conditions were related to the adhesion and morphology kinds adopted by bovine fetal fibroblasts (BFFs).

It was demonstrated that flattened and spindle or spheroids cells can be formed on hydrogel surfaces depending on chemical composition. These surfaces kinds will allow applying medical and biomedical in-vitro treatments and quickly selecting the best one to be given to the sick patient. The formation of spheroids on these surfaces has the advantage that these are exposed to a direct treatment avoiding that the drug passes through a barrier material. The use of these surfaces for in-vitro studies at laboratory level would help to define more quickly which of the medical treatments will be more suitable to apply in-vivo (e.g. antibiotic selection, anti-carcinogenic treatments, etc).

Follow-up studies will lead to design scaffolds based on hydrogels for other cell lines and future biomedical applications.

Section snippets

Materials and experimental methods

Hydrogels were synthesized via free radical polymerization of acrylamide (AAM), N-isopropylacrylamide (NIPAM) (Scientific Polymer Products) and copolymerized with N-acryloyl-tris-(hydroxymethyl)aminomethane (HMA) (Sigma-Aldrich), (3-acrylamidopropyl)trimethylammonium chloride (APTA) (Sigma-Aldrich) and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) (Scientific Polymer Products). N,N-methylenebisacrylamide (BIS) (Sigma-Aldrich) was used as crosslinker agent. Ammonium persulfate (APS)

FTIR measurement

Hydrogels were washed and dried to analyze the presence of characteristic functional groups in polymers and copolymers by FTIR. It is known that amide spectra shows particularly the carbonyl stretching vibration typically known as the amide I band (1670–1650 cm−1, state solid) and an intense Nsingle bondH stretching vibration at 3400 cm−1. In solid state and in the presence of hydrogen bonding, these bands could be shifted to 3350–3200 cm−1 because the carbonyl band is dependent on the amount of hydrogen

Conclusions

It has been demonstrated how the physicochemical characteristics of hydrogels change considerably when they are swollen in cell culture conditions (complete DMEM with 10% v/v FBS) regarded to water, being even more notably on thermosensitive hydrogel surface. For the made assays, all materials synthesized proved to be biocompatible and showed high cellular proliferation after 15 days of culture. Furthermore, different cellular morphologies are observed according to superficial properties. High

Acknowledgements

The authors gratefully acknowledge the financial support provide by FONCYT, CONICET and SECyT-UNRC. R. Rivero, F. Alustiza, C. Liaudat and V. Capella thank CONICET for graduate research fellowships. P. Bosch, C. Barbero and C. Rivarola are permanent research fellows of CONICET.

References (36)

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1

Present address: Marcos Juarez Agricultural Experimental Station INTA, Animal Health. Marcos Juarez (Córdoba), Argentina.

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