Several natural bioactive molecules have been used in the development of scaffolds to enhance biocompatibility or biodegradability and macroalgae contain many bioactive compounds that regulate the physiological activities of cells. In this study, extrapolymeric substances (EPS) from brown algae,
Skin protects the inner organs of an organism from a variety of stresses. Skin is primarily composed of three layers, the epidermis, dermis, and hyperdermis, each of which contains several types of cells. The dermis layer is made almost completely of fibroblasts, which are known to have an effect on keratinocyte proliferation in the epidermis and to be related to some immune responses of skin [
Artificial skin can be made from skin cells cultured in/on three-dimensional biomaterials. Artificial skin requires a scaffold that is able to offer an environment that enables cells to grow well to establish a cellular community as well as to control their proliferation. Considering that all natural tissues make their complete functions via incorporation with extracellular matrix (ECM), scaffolds should be equivalent to ECM and provide cells with sufficient chances to attach to and spread along scaffolds and activate cellular metabolism normally [
To mimic ECM, microenvironment for activating cellular metabolism, nanofibrous scaffold has been intensively applied in tissue engineering. Each nanofiber strand supplies cell attachable point such as collagen fibrous strand of
For forming this structure, there have been several developing processes of materialization such as electrospinning, self-assembled nanofiber, and so on. These processes have already been studied in tissue engineering to replace specific parts of body such as bone [
Among those processes, electrospinning is a simple and versatile technique to fabricate well-organized nanofibrous structure, and electrospun nanofibers have frequently been used in the fields of medical and tissue engineering. This is because electrospun nanofibers reproduce almost the same structure as natural ECM, which has a diameter between 50 and 500 nm [
Natural ECM also has several growth factors and cytokines that influence the physiological activities and metabolism of cells. Therefore, scaffolds for tissue engineering are generally fabricated with bioactive components such as natural peptides, proteins, and polysaccharides.
Primary fibroblasts were isolated from the dermal layer of SD rats aged 1 day (SAMTACO) as previously described [
Fibroblasts were seeded in a 96-well plate at a concentration of 1 × 103 cells/well as low cell density and 5 × 103 cells/well as high cell density cultured for one day, after which EPS was dissolved in culture medium and loaded at 1, 10, 25, or 50
Each 15% (v/v) PCL and 15% (v/v) PCL with 1% (v/v) EPS were dissolved in a solvent of tetrahydrofuran (THF, Junsei, Japan) and NN-dimethylformanide (DMF, Junsei, Japan) (7 : 3). After each solution was mixed well by a stirrer overnight, it was poured into a syringe with an 18 gauge needle, 15 cm distant from a collector. Then, the solution was ejected at a flow rate of 2 mL/hr under the electric filed at 15 kV which is sufficient to fabricate the nanofibers.
To measure the cytotoxicity of the nanofibers toward rat primary fibroblasts, PCL and EPS-PCL nanofibers were incubated in culture medium for 24 hr, after which the old medium was loaded with precultured fibroblasts and then seeded in a 96-well plate with a final concentration of 3 × 103 cells/well. After incubation at 37°C for 1 day, the cells were subjected to an MTT assay. For the adhesion assay, fibroblasts were cultured on nanofibers with a concentration of 3 × 104 cells per fiber sheet. At 2, 8, and 24 hr after cell-seeding, an MTT assay was performed.
Fibroblasts of 1 × 104 cells/well were cultured for 5 days in the PCL and EPS-PCL nanofiber mat in a 24-well plate. To obtain images using SEM (Hitachi S-4300), the cells were washed with PBS and then incubated with 3% glutaraldehyde for 30 min at 37°C, after which the samples were dehydrated with ethanol with serially increasing concentrations of 25, 50, 75, 90, and 100%. Dehydrated samples were then reacted with three drops of HMDS (Sigma, USA) for 3 min and evaporated for 30 min.
Fibroblasts were seeded into PCL and EPS-PCL nanofiber mats at 1 × 104 cells per mat and cultured for 5 days. After incubation, the cells were fixed with 4% paraformaldehyde for 20 min, subjected to H&E staining, and then washed with PBS. Dehydration was accomplished by serial treatment with 10, 20, and 30% ethanol/deionized water, after which the samples were frozen with Tissue-Tek O.C.T compound (Sakura Finetek, USA) at −20°C. The samples were next cut into sections with a width of 50
We investigated the effects of EPS on the activity and proliferation of fibroblasts by MTT assay and BrdU assay, respectively. It is known that soluble MTT is changed into insoluble formazan within the mitochondria of viable cells. As shown in Figure
BrdU and MTT assay for cellular activities: (a) proliferation, (b) graph of viability at low cell density inoculum and (c) graph of viability at high cell density inoculum.
To successfully produce a tissue-like scaffold, cellular metabolism must be increased or at least maintained, even under confluent conditions, because ECM must be continuously produced by the cells to make tissue compact. In this study, cells on nanofibers were found to metabolize less MTT than those on EPS nanofibers (Figure
It is well known that cells usually use oxygen for metabolism but that cells exposed to higher concentrations of O2 show decreased proliferation and cellular metabolism as a result of (reactive oxygen species) ROS [
To confirm whether EPS affects cellular behavior in a structural way, electrospun scaffold was fabricated to give a three-dimensional structure similar to that of tissue in humans. The morphologies of PCL and EPS-PCL nanofibers were then observed by SEM (Figures
SEM images of (a) PCL and (b) EPS-PCL nanofibers.
To determine if toxic substances were released from the nanofibers, each nanofiber was immersed in medium for 1 day. A
As shown in Figure
Analyses of fibroblast adhesion by (a) SEM images and (b) MTT assay.
Moreover, the cellular adhesion was indirectly studied by MTT assay. To determine how many cells adhered to the nanofibers before cellular division, ELISA was used to measure the OD of formazan-containing media. As seen in Figure
As shown in Figure
Considering that scaffolds offer spaces for cells to grow, it was necessary to investigate the distribution of cells in the newly formed biomaterials after infiltration. To accomplish this, fibroblasts were seeded onto the top of the nanofibers, of which thickness is about 200
Cross-section assay using H&E staining. (a) Image of PCL nanofiber mat and (b) EPS-PCL nanofiber mat.
In addition, EPS showed no cytotoxicity during fibroblast infiltration and fibroblast infiltration was not different irrespective of the incorporation of EPS (Figure
In order for there to be a cooperative effect between EPS and nanofiber structure, the sensitivity of nanofiber structure to cellular infiltration should first be balanced with that of EPS by controlling the pore size or diameter of the nanofibers. However, such an investigation is beyond the scope of this study. Nevertheless, the results of the present study indicate that the developed nanofibers have the potential for use as a biomaterial in the production of artificial skin by demonstrating that they can enhance fibroblast proliferation without cytotoxicity and support fibroblast infiltration without any critical problems. Fibroblasts could be integrated into EPS nanofibers, indicating that EPS-PCL nanofibers have the potential for use in the manufacture of artificial skin despite showing no obvious difference to nanofibers composed of PCL alone.
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This research was a part of the project titled “Technology Development of Marine Industrial Biomaterials,” funded by the Ministry of Oceans and Fisheries, Korea.