The adaptation of lipid profile of human fibroblasts to alginate 2D films and 3D printed scaffolds
Introduction
In the recent years the development of innovative active materials in combination with new production technologies (i.e. micro- and nano-systems, 3D printing, electrospinning, etc.) are of growing interest in several research and application fields (i.e. drug delivery, regenerative medicine, gene therapy, in vitro diagnostic tests, etc.) [[1], [2], [3]]. The driving idea is to overcome essential limitations of traditional approaches (i.e. introduction of more reliable diagnostic tests, improvement of drug delivery systems, bio-fabrication of tissues or organ-like systems etc.), and many efforts are made to evaluate the effectiveness of these systems through in vitro and in vivo studies.
In such a context, the investigation of the interactions between cells and biomaterials is pivotal to obtain a more comprehensive mechanistic understanding of the cause-effect relationships of the whole system. The capability to offer thorough pictures of these complex living processes allow to drive new development strategies in the huge application landscape of biomaterials. However, many challenges still remain open, as it is well known that cell behavior depends upon several factors, including microenvironment dimensions, structure and chemico-physical composition.
Biomaterials interact with the cells at various length scales, from that of individual cells (micrometers) to the that (nanometers) of single molecules (i.e. proteins, lipids). These interactions are based on both physical contact and chemical binding and depend over time as a function of the dimension of the system. Individual cells interact via integrin with a biomaterial for days or weeks, while individual proteins, or lipids or glycosaminoglycans interact through secondary bonds and hydrophobic interactions on time scales as short as seconds and minutes [4,5]. A complex network of non-covalent kinetically rapid interactions such as hydrogen bonds, van der Waals and hydrophobic interactions can affect the more driving and thermodynamically stable ionic interactions. The spatial architecture, surface area, interstitial pore distribution and dimensions of native extracellular matrix (ECM) strongly influence the cell migration, proliferation and differentiation [6]. Biomaterial surfaces can induce changes either in the cell membrane fluidity and permeability, which in turn regulate cellular and tissue functions, or in cell phenotype, including morphology, proliferation and biochemical properties [7,8]. For example, the surface and inner pore size of a scaffold influence the migration speed of cells: in collagen-GAG scaffolds the smaller is the average pore size, the lower is fibroblast migration speed [[9], [10], [11]]. Nonetheless, it has been shown that prostate cancer cells migrate faster than fibroblasts through the same scaffold [12]. It is clear that the knowledge of these effects at the molecular level can help to develop and tune optimal biomaterials and scaffolds as a function of the application.
Alginate (ALG) is a widely investigated biomaterial used in drug delivery and in many biomedical applications thanks to its excellent properties, such as biocompatibility, low toxicity, low cost and ability to undergo spontaneous gelation under mild conditions [[13], [14], [15], [16]]. ALG is a natural occurring anionic polymer extracted from the brown algae cell wall. It is an unbranched binary copolymer consisting of the repetition of the monomer units D-mannuronic acid (M) and L-glucuronic acid (G), held together by β1-4 bonds [17]. ALG, as cross-linked hydrogel presents structure, flexibility, porosity and diffusive transport characteristics similar to the ECM of human native tissues and is used in regenerative medicine as dressings to keep a wound moist, minimize battery infection and accelerate the healing process [18].
As for hydrogel formation, during ionotropic gelation a network of interactions involving different junction zones (i.e. OH, COO− and O− groups) of consecutive G-residues of the ALG chains and the cation occurs. Calcium chloride (CaCl2) represents one of the most used crosslinking agent for the formation of the ALG ionotropic hydrogel. The divalent cations bind only to the glucuronic acid blocks of the ALG chains forming a cross-linking model called “egg-box”, resulting in a gel structure. Conversely, chelation with the trivalent Fe3+ ions allows for more spherical shapes [18]. In general, the speed of gelation is an important parameter to control the uniformity and shape of the gel. Slower gelation leads to the production of more uniform structures and greater mechanical integrity [19]. Furthermore, better cell adhesion and proliferation have been observed on ALG matrices when the latter is gelled with trivalent ions, such as Fe3+ [20]. Among applications ALG is widely exploited for the controlled release of drugs and proteins, for the transport of cells to a specific site [[21], [22], [23]] and to perform two-dimensional (2D) and three dimensional (3D) cell studies to understand cell-matrix interactions. Although cells in a living system are exposed to complex 3D biological environments, biological phenomena is still extensively investigated by means of 2D substrates. 2D assays present major limitations to accurately describe the space constraints of cells in vivo and can induce different cell activities and/or loss of the original cell phenotype. 2D films are still widely used in several cell culture experiments, but 3D printing manufacturing process is rapidly gaining a prominent role as innovative technology in the medical or diagnostic fields to shape biopolymers in a variety of architectures to progressively replace two dimensional systems [[24], [25], [26], [27], [28], [29]].
With the aim to improve the basic knowledge available in the cell-biomaterial interaction field, in this study the effects of different architectures and gelling media used for the preparation of ALG hydrogels were investigated for the first time on the targeted lipid profile of human fibroblasts. Controlled and reproducible 2D films (2D-ALG) and 3D printed ALG scaffolds (3D-ALG) were produced by gelation with CaCl2 or iron chloride (FeCl3), and a selected targeted lipid profile of human fibroblasts seeded on them was evaluated. Cell biochemistry can be studied at different complementary levels (i.e. the transcriptome, proteome, lipidome or metabolome) to gain information useful to frame their behavior [30,31]. Here we decide to focus our attention on lipids, as they play a crucial role in the physiology of cells, tissues and organs as demonstrated by a large number of genetic studies [32]. The deregulation of lipid metabolic pathways therefore leads to the onset of diseases, including cardiovascular disorders, cancer and diabetes [[33], [34], [35]].
Lipidomics is nowadays a consolidated field capable of a comprehensive analysis of lipids in complex biological systems. Lipidomics aims to profile the lipid structures and quantity in a biological sample, to assess their metabolic functions and transformations that occur in different physiological and pathological conditions [36]. The birth of lipidomics has been possible thanks to technological advances in the field of analytical instrumentation such as mass spectrometry (MS) [[37], [38], [39]]. This set of techniques is the golden standard approach for the investigation of the lipids in cells by virtue of their ability to perform the simultaneous identification and quantification of thousands of analytes in the same biological sample. Here a liquid chromatography-electrospray-tandem mass spectrometer, with a triple quadrupole mass analyzer, was used for the identification and relative quantitative detection of lipids belonging to the following classes, selected as powerful mediators of cell functions: ceramides (CER), fatty acids (FFA), lysophosphatidic acids (LPA) and lysophosphatidylcholines (LPC). CER and FFA are lipid species that modulate membrane rigidity, creating micro-domains, and altering membrane permeability, thus regulating cell membrane functions [40]. Moreover, CER enhance the bioavailability of drugs by acting as lipid delivery systems, they play a structural role in liposome formulations and enhance the cellular uptake of amphiphilic drugs, such as chemotherapics [41]. In a recent study FFA have been chemically linked with biological drug molecules to enhance oral absorption of therapeutic peptides and to provide a platform for oral peptide drug development [42]. LPA derivatives are bioactive phospholipids present in biological fluids that regulate many important fibroblast functions, including proliferation, migration and contraction. Alteration in normal LPA signaling may contribute to a range of diseases, including neurodevelopmental and neuropsychiatric disorders, pain, cardiovascular disease, bone disorders, fibrosis, cancer, infertility, and obesity [43]. Therefore, therapies targeting LPA biosynthesis and signaling may be feasible for the treatment of devastating human diseases [44]. LPC are present as minor phospholipids in the cell membrane and blood plasma, promote inflammatory effects [45] and play a role in the pathway of fibrotic injury in human cardiac fibroblasts [46].
Section snippets
Reagents
Acetonitrile, methanol, hexane and isopropanol were supplied from Sigma-Aldrich (Taufkirchen, Germany). Water was purified (0.055 uS/cm, TOC 1 ppb) with a Purelab pulse + Flex ultra-pure water system (Elga Veolia, Milan, Italy).
Scaffold preparation
ALG formulation (Ph.Eur. grade; molecular weight by gel filtration chromatography (GFC) 180–300 kDa; slowly soluble in water, Carlo Erba, Italy) was prepared by dissolving the sodium ALG 6% (w/v) in deionized water. The formulation was left under stirring overnight on a
2D-ALGand 3D-ALG preparation and characterization
In the first part of the work the preparation and characterization of the 2D-ALG and 3D-ALG (Fig. 1) were addressed in order to study the effects of the different gelling media on the final hydrogel properties. In general, ALG presents different chelating affinity for its cross-linking cations, as a function of their charge and dimensions, resulting in hydrogels having different properties such as swelling, elasticity and stability.
The determination of the water content, a parameter that allows
Conclusions
3D cell cultures can add unknown physiologically relevant aspects compared to 2D.
Here the behavior of fibroblasts as well as their lipid profile in contact with ALG systems was demonstrated to be influenced both by the architecture (2D or 3D) and the type of gelling agent. 2D- and 3D-ALG were prepared by using two gelling agents (CaCl2 or FeCl3), and characterized from a chemical-physical point of view through determination of the water content, swelling tests and mechanical resistance tests
Author statement
Lisa Elviri: Conceptualization, project administration, methodology, data curation, draft revision, manuscript revision, funding acquisition. Giulia Remaggi: Writing-original draft and investigation. Carlo Bergonzi: Investigation. Ruggero Bettini: Review. Ilaria Zanotti: Supervision and Investigation, manuscript revision. Franco Bernini: Review, funding aquisition. Silvia Marando: Investigation, formal analysis.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The Authors thank Dr. Ivana Lavota, Dr. Giorgia D'Andrea and Mr. Davide Dallatana for the excellent technical assistance.
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