Poster N1 - Near-field electrospinning nanofibers on top of hydrogels using a 3D bioprinter to create composite scaffolds

Introduction: Hydrogels are one of the most common biomaterials used in tissue engineering applications due to the similarities to human tissue extracellular matrix (ECM). Hydrogels have a high-water content, support 3D cell culture, and can mimic the chemical composition of native ECM. Mechanical properties can vary between hydrogel type and concentration, where mechanical strength tends to increase with increasing chemical concentration. Unfortunately, increased concentration can have a negative impact on cellular activity, where decreased pore size limits cell migration. An alternative strategy to increasing the mechanical strength of hydrogels would be to incorporate nanofibers. Nanofiber scaffolds, most often produced via electrospinning, can also mimicking the extracellular matrix of native tissue with diameters at the same scale as many proteins and proteoglycans in the ECM. Additionally, nanofibers can be used to direct stem cell differentiation and cell activity, inducing cells to produce their own ECM content. Near-field electrospinning (NFES) offers a means to produce organized and aligned nanofibers, rather than random fibers produced via traditional (far-field) electrospinning methods. Further, using a 3D bioprinter to deposit the nanofibers via NFES would allow for more control over the alignment of the fibers. Using this technique, fibers can be incorporated into hydrogel scaffolds and into complex orientations (other than linearly aligned). Integrating NFES nanofibers into hydrogels would improve existing hydrogel scaffolds by combining the benefits of each component. Specifically, the nanofibers would improve the mechanical properties of hydrogels without limiting cell migration and provide stimulation for incorporated cells to direct cell activity.

Materials and

Methods: The Ender-5 Pro (Creality) 3D plastic printer was purchased and assembled following the guidebook. A custom extruder plate was designed in SolidWorks and printed using the printer. Modifications were made to the printer to allow for syringe-pump deposition of biomaterials using the custom extruder plate. After modifying the printer, a syringe pump was used to push solution from a syringe, through tubing, and out of the needle attached to the extruder plate on the printer. A voltage source was used connecting the needle and a copper plate at the collection site of the printer to generate an electric current for electrospinning. Poly-L-lactic acid (PLLA) was dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at 10% concentration. PLLA nanofibers were NFES using the printer and custom setup to produce aligned fibers on top of agarose gel that was placed on the copper collection plate. Unconfined compression testing was conducted on the scaffolds using a Mark-10 compression machine. Following fabrication of the nanofiber/hydrogel scaffolds, human mesenchymal stem cells (hMSCs) were seeded on the scaffolds and grown in standard culture medium (alpha-MEM with fetal bovine serum and penicillin-streptomycin supplementation) and standard conditions (incubator at 37 C with CO2). After 3, 5, and 10 days of culture, PicoGreen was used to quantify cellular activity, and immunofluorescence staining was used to visualize the cell morphology.

Results, Conclusions, and Discussions: PLLA nanofibers have been successfully NFES via a 3D bioprinter onto an agarose hydrogel. Stacks of nanofiber/hydrogel composites were layered to generate full-thickness scaffolds containing nanofibers throughout the thickness in various directions. Unconfined compression testing revealed an increase in modulus when nanofibers were incorporated into the agarose gel compared to an agarose gel without nanofibers. When hMSCs were seeded on top of the scaffold, they were seen to migrate throughout the thickness of the gel at 3, 5, and 10 days. Additionally, the cells within the scaffold were aligned along the direction of the nanofibers. Therefore, NFES offers a strategy to combine nanofibers and hydrogels where the alignment of the nanofibers is highly controllable.
There are numerous tissue engineering applications with such a scaffold. An example is the meniscus, a fibrocartilaginous tissue composed mainly of ECM. Collagen type I makes up most of the dry weight of the ECM and is predominantly arranged circumferentially and radially within the crescent-shaped meniscus. Self-healing of injured menisci is generally inhibited by the lack of vasculature, influencing tissue engineering strategies for meniscus repair. A crescent-shaped hydrogel composed of nanofibers arranged circumferentially and radially to mimic the natural ECM of the meniscus offers a new tissue engineering strategy for meniscus repair. Such a meniscal scaffold would have the physical cues to direct cellular alignment and activity for matrix deposition.

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