The effect of photopolymerization on stem cells embedded in hydrogels
Introduction
Hydrogels are attractive materials for various tissue engineering (TE) applications as they can be used to homogeneously incorporate cells, growth factors and other bioactive compounds. Overall, hydrogels show a good biocompatibility, and provide the embedded cells with a highly hydrated environment that is amenable to rapid diffusion of nutrients and metabolites [1], [2], [3]. Hydrogels generally cause little irritation in vivo due to their high water content and the resulting low quantity of degradation products. Hydrogel-forming polymers can be tailored to present biochemical, cellular and physical stimuli to guide cellular processes such as migration, proliferation and differentiation [4], [5], [6].
Some types of hydrogels are obtained by photopolymerization of liquid photosensitive polymer solution in the presence of photoinitiators using visible or ultraviolet (UV) light either in vitro or in vivo [7], [8], [9]. These gels are stable and mechanically strong, because the polymer networks are held together by covalent crosslinks. Furthermore, photopolymerization is an attractive technique as the conversion of liquid polymer solution to a gel occurs rapidly, under physiological temperature, with minimal heat production, and can be controlled in time and space [7]. Photopolymerization has been widely employed for drug delivery [10], [11], for controlled release of DNA and growth factors [12], [13], [14], [15] and to create layered matrix devices for investigation of drug-release in non-uniform concentration profiles [16].
In orthopaedic TE applications, photosensitive polymers are used as fillers for bone restoration [17] and for encapsulation of chondrocytes and osteogenic progenitors [12], [18], [19], [20], [21]. Photopolymerization provides the ability to gellify photosensitive polymers in situ, with a possibility to form complex shapes that adhere and conform to tissue structures [7]. Chondrocytes have already been encapsulated in vivo via transdermal photopolymerization as a minimally invasive technique [19]. Organ- or tissue printing (OP), based on layered deposition of (cell-laden) hydrogels is a novel technology suitable for application of photopolymerizable hydrogels [3]. This OP approach allows building of 3D organized scaffolds with multiple cell types at predetermined locations.
During the photopolymerization process (UV) light homolytically splits photoinitiator molecules into radicals, which initiates the formation of a polymer network. Free radicals can however also directly react with cellular components such as cell membranes, proteins and DNA, thereby directly inducing unwanted cellular damage, or indirectly via formation of reactive oxygen species (ROS). Despite the use of the exogenous defenses against oxidative damage and intracellular anti-oxidants to quench ROS, exposure to ultraviolet A (UVA) radiation can induce the formation of ROS. This can cause base lesions that could potentially lead to malignant transformation of photo-exposed cells, although UVA-induced lesions have low mutagenicity [22]. While many different methods exist to test for various DNA damage, most of the lesions are repaired quickly with little impact on cell homeostasis. Therefore, although these are unlikely to result from UVA irradiation [23], it is desirable to detect the rare, not easily repaired damage such as double strand DNA breaks that can induce deleterious mutations and initiate carcinogenesis. To do this, detection of p53 binding protein-1 (p53BP1) is a useful tool. This mediator of a DNA damage checkpoint responds to, amongst others, DNA double strand breaks by quickly localizing to discrete nuclear foci [24], [25]. Furthermore, exit from the cell cycle as a result of photoexposure-induced stress could lead to apoptosis or senescence, and is undesirable in a tissue engineering approach.
As further development of photopolymerizable hydrogels for TE has the potential to produce structurally organized, cell-laden implants designed to repair or augment tissues, detailed studies are needed on the effect of photopolymerization on the exposed cells. Most studies in the literature provide a comparison between photoinitiator components with regard to their cytotoxicity, with [26] or without UV-exposure [27], [28]. Several photoinitiators exhibit a good toxicological profile, with Irgacure 2959 providing the best results [28]. Photoinitiator concentrations and UV light intensity should be minimized to prevent adverse effects on the viability of the cells and their proliferation and/or differentiation potential [7], [29]. Detailed studies describing the effect of UV-irradiation on cells in the presence of a photoinitiator and the hydrogel building blocks are lacking.
In our work we are interested in encapsulating bone marrow derived multipotent stromal cells (MSCs) in a photopolymerizable hydrogel for the development of printed bone grafts. The aim of this study was to analyze the effect of photopolymerization on the fate of photoexposed goat MSC monolayers, with regard to viability, DNA damage and subsequent cell cycle progression and differentiation. Examining these outcomes in the absence of hydrogel enables us to make general statements on effects of photoexposure, because additional interference from the hydrogel is excluded. Additionally, we have studied the behavior of these primary photoencapsulated cells in their microenvironment when entrapped inside two different hydrogels – a methacrylated synthetic and a methacrylated natural hydrogel – focusing on cell-survival and osteogenic differentiation.
Section snippets
Cells
Multipotent stromal cells (MSC) were obtained from iliac bone marrow aspirates of Dutch milk goats, and isolated by adherence to tissue culture plastic. The cells were culture-expanded as described previously [30], in expansion medium consisting of αMEM (Gibco) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco), 2 mm l-glutamine (Glutamax, Gibco) and 15% v/v Fetal Calf Serum (Cambrex). For osteogenic differentiation, expansion medium was supplemented with 10 nm dexamethasone
Viability of monolayers
In order to assess to which degree the survival of photoexposed cells is affected by the variables of photoexposure conditions, namely the time of UV exposure (and thus the irradiation dose) and presence of the photoinitiator molecules, the survival of MSC monolayers was studied after exposure to 6 mW/cm2 of 362 nm UV light for varying periods of time (0–5 min) and various Irgacure 2959 concentrations (from 0 to 0.1% (w/v)). Irgacure 2959 was chosen for UV photoinitiation because it generally
General discussion
The current study evaluates the toxicity of a photopolymerization process to monolayers and encapsulated MSCs, and assesses the proliferation and differentiation of these cells upon exposure to UV-light and photoinitiator Irgacure 2959, under conditions regularly applied in numerous cell-based TE applications [36], [37], [38]. MSCs are widely used cells in TE research due to their multilineage potential and are often embedded in photopolymerizable hydrogels to study their differentiation
Conclusion
Photopolymerization is an attractive method to crosslink hydrogel-forming polymers, resulting in mechanically strong, stable matrices suitable for cell-encapsulation. We demonstrate adverse effects of photopolymerization on viability and cell cycle progression of exposed MSC monolayers, while their differentiation potential remains unchanged. We also show that the viability of encapsulated cells is not adversely affected by photopolymerization of the surrounding hydrogel, which is likely the
Acknowledgements
We would like to thank the department of Immunopathology of University Medical Center Utrecht, The Netherlands and Hugo Alves from the Department of Tissue Regeneration, University of Twente for their assistance with immunocytochemical stainings. We would like to acknowledge the financial support of the Mosaic scheme 2004 of the Netherlands Organisation for Scientific Research (NWO; grant number: 017.001.181), the Foundation De Drie Lichten and the Dutch Platform for Tissue Engineering (project
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