BIOCOMPATIBLE MICRON-SCALE SILK FIBERS FABRICATED BY MICROFLUIDIC WET SPINNING

This short review article is based on the research paper titled “Biocompatible Micron-Scale Silk Fibers Fabricated by Microfluidic Wet Spinning”, authored by Arne Lüken, Matthias Geiger, Lea Steinbeck, Anna-Christin Joel, Angelika Lampert, John Linkhorst, Matthias Wessling.

The research paper was published in The Journal Advanced Healthcare Materials on the 31st of July, 2021. In the study, the authors developed, tested and validated a method that applies a pressure-driven flow controller to fabricate biocompatible micron-scale silk fibers by microfluidic wet spinning.

You can also read our application note about microfluidic pressure-driven flow control!

ABSTRACT

In this study, the authors manufacture a microfluidic spinneret to produce single micron-sized wet-spun silk fibers. The fibers are beneficial for tissue engineering applications because cells interact predominantly with similar sized geometrical structures. Additionally, the method allows to tailor the fiber’s cross section geometry and is applicable for various solidification mechanisms.

The micro-spinneret is 3D-printed in a microfluidic PDMS (polydimethylsiloxane) chip using two-photon lithography with nano-scale resolution, applying an innovative surface treatment that permits a tight print-channel sealing.

The spinning process relies on digital pressure pumps for achieving a steady flow.

Watch the universal flow control webinar by Robin Oliveres that introduces microfluidic flow control!

INTRODUCTION

Fiber-based tissues have great biomedical applications based on their large surface to volume ratio and their ability to guide neuronal growth [1]. Additionally, fibrous scaffolds enable 3D cell culture, which mimics the natural extracellular matrix in the artificial environment [2-5]. Small fibers with single micron diameters are manufactured using electrospinning and microfluidic spinning. Electrospinning allows the fabrication of unstructured fibers sheets, however, is not suitable for single fiber processing.

Microfluidic spinning using a pressure-driven flow controller utilizes flow-focusing geometries to produce stable, concentric co-flows of pre-polymer or dissolved protein solution and shear fluid. The pre-polymer or protein solidifies by precipitation in the shear flow or by an initiated cross linking reaction to form a fiber. The surrounding shear fluid permits to avoid contact of the solidified polymer to the channel walls, preventing adhesion, and thus reduces clogging. [6, 7]

The method developed by Arne Lüken et al. combines the versatility and adaptability of additive manufacturing and microfluidic spinning. This method creates new opportunities for the production of smaller and complex-shaped fibers adjusted towards specific applications in tissue engineering.

AIM & OBJECTIVES

In short, the authors in this paper:

• leverage in-chip DLW to design microfluidic spinnerets, combining the scalability and reproducibility of 3D-printing technology with the versatility of capillary devices

• showcase the function of the spinnerets by producing fibers from polyacrylonitrile and alginate via non-solvent-induced phase separation and ionic crosslinking, respectively

• spun mechanically strong fibers from regenerated silkworm silk with two different cross-section shapes

• analyzed the strength of the created fibers based on their fiber diameter, and tested their biocompatibility through a 3D cell-culture

MATERIALS & METHODS: Microfluidic Wet Spinning to fabricate Biocompatible Micron-Scale Silk Fibers

The microfluidic channels and the spinnerets are designed in 3D CAD and manufactured 2-photon lithography based on the method suggested by Loelsberg et al. [8]. First, a mold is printed on a glass slide, which is afterwards molded using PDMS. After bonding the PDMS slap a thin glass slide, the microfluidic channels are filled with the photoresin and the microscopic spinneret is fabricated in-chip (Video 1). The oxygen inhibition layer of the PDMS is covered by a photo-reactive Sol-Gel coating, which increases adhesion of the print to the chip and allows liquid tight sealing of the printed spinneret with the PDMS device (Figure 1). The spinning process is performed by applying fluid pumps, the microfluidic PDMS chip and a harvesting device (Figure 2).

Video 1: Fabrication process of the micronozzle in the microfluidic channel. The first seconds are in real time, afterward the video is time-lapsed to get an impression about the emerging structure. The whole print takes approximately 20 min (courtesy of the author).

The spinning dope and shear fluid are each pumped by a digital constant pressure pump and flushed into the microfluidic chip with the nozzle in the center part (Figure 2). At the nozzle tip, the dissolved protein spinning fluid is injected into the center of the shear fluid without contacting the outlet channel wall, flowing tangentially in the center of the outlet channel along the stream-lines while being hydrodynamically elongated. The spinning dope solidifies in the outlet channel of the device, and the fiber is harvested by spooling on a digitally controlled harvesting coil (Figure 2).

The process does not require a phase boundary between the spinning solution and the shear fluid. Therefore, the shape of the fibers are tailored by the spinnerets geometry and versatile materials can be applied. The authors chose three different fiber materials to demonstrate possible solidification mechanisms with the in-chip DLW nozzle.

A stable spinning process without nozzle blocking requires the adaption of the solidification kinetics by changing the salt concentration and the non-solvent concentration of the shear fluid for the alginate fibers, PAN fibers and silk fibers, respectively.

KEY FINDINGS: Microfluidic wet spinning process

Tensile strength of silk fibers

The authors analyzed the tensile strength of the microfluidic spun silk fibers by measuring the tensile force and strain using a self-built tensile test setup. By analyzing the diameter of the fibers using field emission scanning electron microscopy (FESEM) images, they were able to estimate the stress–strain diagram, the tensile strength at the breaking point, and the Young’s modulus.

Comparing the stress–strain measurements with literature data, two distinct observations were noted. On one hand, the average breaking stress and Young’s modulus of the fibers seems to be significantly smaller than reported fibers.[6, 7, 9] On the other hand, the authors’ data reveal that the smaller the diameter is, the tougher the fiber is. Based on Griffith’s criterion on fracture mechanics, it is known that smaller fiber diameters increase strength.[10]

Cell culture on silk fibers

Applied as tissue for cells, the fiber’s mechanical properties, such as the Young’s modulus, affect the cell adhesion and stem cell differentiation. Therefore, tailoring the mechanical properties by the spinning process is of major importance in the field. Additionally, applied as nerve regeneration tissue, the fibers might be applied for connecting disrupted structures and guide nerve growth, where a high tissue stability is of significant importance.

As a first step towards application as tissue, the study applies CaCo2 cells on the fibers and evaluates their tendency to grow on the fibers (Figure 4). This study proves biocompatibility and serves as a first step to show that silk fibers manufactured by the developed process are suitable for tissue engineering applications. The result encourages further the influence of the material properties, shape, and size on its performance as tissue.

CONCLUSION

The study highlighted here presents an in-chip fabricated microfluidic fiber spinning system. Encapsulation of two-photon printed micron-sized structures in microfluidic PDMS chips allows the fabrication of versatile 3D geometries in microfluidic channels. The presented spinneret is capable of fabricating the smallest reported microfluidic spun fibers and allows the processing of various materials and the synthesis of different fiber cross-sections. The method boosts microfluidic fibers spinning processes by applying the versatility and accuracy of 3D-printing technology. The silk fibers’ strengths are analyzed depending on their size by stress-strain measurements. As a first step towards tissue engineering applications, cell-culture experiments prove biocompatibility of the fibers. The presented study pushes the boundaries of fiber shapes and the reproducibility in microfluidic fiber fabrication and will substantially promote future scaffold materials for healthcare applications.

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References