Bi-compartmental 3D scaffolds for the co-culture of intervertebral disk cells and mesenchymal stem cells
Graphical abstract
Introduction
In native tissue, numerous homotypic and/or heterotypic cell–cell interactions typically occur through direct contacts or the exchange of soluble factors, which greatly influence cellular behavior such as survival, apoptosis, migration, proliferation, and differentiation [1], [2]. A significant number of studies have been aimed at developing highly efficient cell co-culture platforms (or scaffolds) to better understand the various cell–cell interactions and mimic the cellular microenvironments where various cell types are optimally positioned relative to one another [3], [4], [5], [6]. Most of the recent progress in co-culture platforms is based on the surface chemistry of the material in conjunction with microfabrication techniques that allowed the spatial patterning of multiple cell types, where the cells exist on two-dimensional (2D) flat surfaces and formed an outspread 2D monolayer [7], [8], [9], [10], [11], [12], [13]. With the emphasis on the importance and advantages of 3D culture systems over 2D cultures systems, many studies reported the use of 3D scaffolds such as hydrogel and nanofiber matrices, for the co-culture of different cell types [14], [15], [16], [17], [18], [19], [20]. However, most of those co-culture systems were prepared by simply mixing two or more cell types and seeding them simultaneously onto the same scaffolds, making it very difficult to localize each cell type into a specific region within the scaffold, and, therefore, precise control of the cell–cell interactions is impossible.
On the other hand, the degeneration of intervertebral disk (IVD), which is composed of a peripheral annulus fibrosus (AF) and a central nucleus pulposus (NP), is a major cause debilitating neck and/or back pain [21], [22]. Although the exact pathological mechanisms are not fully understood, the decreased production of the extracellular matrix (ECM) in aging IVD cells is thought be one of the major causes of IVD degeneration [23]. In addition to the current treatments for IVD degeneration, such as palliative therapies and surgical intervention, cell-based tissue engineering approaches have received great attention for the treatment of IVD disease [24], [25], [26]. For a successful tissue engineering-based IVD therapy, sufficient amount of IVD cells as well as polymeric scaffolds are required. However, clinically, it is very difficult to quickly obtain a sufficient number of fresh cells due to the hypocellularity of the IVD [27], [28]. One strategy to overcome this problem is to use mesenchymal stem cells (MSCs) that are capable of differentiating into a number of lineages, including both chondrocytes and NP-like cells [29], [30], [31], [32], [33]. Among the various methods to increase the efficacy of differentiation induction, co-culture of MSCs with IVD cells is a viable option, because the co-culture system not only enhances the MSC differentiation into IVD cells, but also significantly activates the biological properties of the NP cells such as cell proliferation, DNA synthesis and extracellular matrix (ECM) production [34], [35], [36], [37]. Similar to other co-culture studies, most co-cultures of MSCs and IVD cells have been carried out using the 2D monolayer model [37], [38], spherical pellet culture model [39], [40], or a random mixture of MSCs and IVD cells within a 3D scaffold [41], [42], [43], [44], [45]. However, to our knowledge, there have been no reports regarding the construction of 3D scaffolds that are able to localize MSCs and IVD cells into distinct scaffold domains.
In order to develop the 3D co-culture scaffolds that allow the localization of each cell type into a specific domain, we fabricated bi-compartmental scaffolds consisting of a PCL nanofiber-incorporated hydrogel matrix and bare alginate hydrogel. In this system, the cells can be located in vertically different domains; one is within the bare hydrogel region and the other is within the nanofiber-incorporated region. A co-culture of IVD cells and hMSCs was constructed as a model system by placing the intervertebral disk (IVD) cells and hMSCs within the bare alginate hydrogel and nanofiber regions, respectively. After construction of the co-culture system, we investigated the various biological activities of the co-cultured cells, such as cell proliferation, gene expression, protein production, and GAG content.
Section snippets
Materials
Polycaprolactone (PCL, MW: 80,000), calcium chloride (CaCl2), 2,2,2-trifluoroethanol (TFE) and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) were purchased from Sigma–Aldrich (Milwaukee, WI, USA). Sodium alginate (80–120 cP) was purchased from Wako Pure Chemical Industries (Osaka, Japan).
Preparation of electrospun PCL fibrous scaffolds
Conventional electrospinning was used to prepare the PCL nanofiber scaffolds. After PCL was dissolved in TFE to form a 20 wt% polymer solution, a 7.5 kV positive voltage was applied to the
Preparation of bi-compartmental scaffolds
Fig. 2 shows the cross section SEM and fluorescence images of bi-compartmental scaffold consisting of a nanofiber-incorporated hydrogel matrix as the bottom part and a bare alginate hydrogel as the upper part. For the fluorescence images, red dye (Alexa Fluor 510) and green dye (Alexa Fluor 610) were incorporated into fibers and hydrogel, respectively. The thickness of nanofiber matrix was controlled by the electrospinning time. The electrospinning of a PCL solution produced ultrathin polymer
Discussion
We developed a highly efficient 3D co-culture system of different cell types by fabricating bi-compartmental scaffolds using a combination of electrospinning and alginate hydrogel synthesis. The resulting scaffolds could have two vertically distinct domains consisting of a PCL nanofiber-incorporated hydrogel matrix and a bare alginate hydrogel by controlling the electrospinning collection time and the volume of the hydrogel precursor solution. From a cell culture perspective, the
Conclusion
We fabricated bi-compartmental scaffolds consisting of a PCL nanofiber-incorporated hydrogel matrix and a bare alginate hydrogel for the purpose of developing a 3D co-culture system. As a model system, a co-culture of IVD cells and hMSCs was constructed by placing IVD cells and hMSCs within the bare alginate hydrogel and nanofiber regions, respectively. The co-culture of IVD cells and hMSCs resulted in increased GAG content and accelerated upregulation of discogenic markers, such as collagen
Acknowledgments
This work was supported by the National Research Foundation (NRF) grant funded by the Ministry of Science, ICT and Future Planning (MSIP) (2015R1A2A1A15054532 and 2015R1D1A1A01060444) and by a grant (HI15C1744) from the Korean Health Technology R&D Project through the Korean Health Industry Development Institute (KHIDI).
This work was supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University.
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These authors contributed equally to this work and are the co-first authors.