The development of bio-inks capable of being 3D-printed into cell-containing bio-fabricates with sufficient shape fidelity is highly demanding. Structural integrity and favorable mechanical properties can be achieved by applying high polymer concentrations in hydrogels. Unfortunately, this often comes at the expense of cell performance since cells may become entrapped in the dense matrix. This drawback can be addressed by incorporating fibers as reinforcing fillers that strengthen the overall bio-ink structure and provide a second hierarchical micro-structure to which cells can adhere and align, resulting in enhanced cell activity. In this work, the potential impact of collagen-coated short polycaprolactone-fibers on cells after being printed in a hydrogel is systematically studied. The matrix is composed of eADF4(C16), a recombinant spider silk protein that is cytocompatible but non-adhesive for cells. Consequently, the impact of fibers could be exclusively examined, excluding secondary effects induced by the matrix. Applying this model system, a significant impact of such fillers on rheology and cell behavior is observed. Strikingly, it could be shown that fibers reduce cell viability upon printing but subsequently promote cell performance in the printed construct, emphasizing the need to distinguish between in-print and post-print impact of fillers in bio-inks.

Besides their long history in the traditional textile industry, silk materials provide various attractive properties such as biocompatibility and mechanical stability, arousing constantly increasing scientific interest within the last two decades. This book chapter will review the predominant classes of regenerated Bombyx mori silk and recombinant spider silk with respect to the most relevant features of the natural fiber spinning system, which is considered the role model when technically reproducing silk fiber characteristics. Furthermore, it provides an overview of the diversity of different spinning techniques that were recently applied to produce artificial silk fibers aiming for various high-performance applications.

When using medical-grade poly(ε-caprolactone) (PCL) for melt electrowriting (MEW), one notable feature is that the deposited fiber is typically continuous.1 Even when the collector speed is increased to 165 mm/s, the direct-written jet is only further stretched but rarely breaks.2 Such fast speeds were recently taken advantage of to suspend MEW fibers over millimeter wide gaps without sagging3 in order to form well-defined oriented fiber arrays for neurons.4 However, there are several applications where discontinuous microfibers with defined morphology are relevant including the reinforcement of hydrogels in the field of bioprinting, or the use as injectables for drug delivery. Short fibers can induce biological functionality by generating anisotropy,5, 6 improve printability of extrusion-based bioinks7-10 or alter the mechanical properties of three-dimensional (3D) printed constructs11, 12 while still enabling simple processing through a cannula. Recently the high-speed formation of PCL-dumbbell-like morphologies via precise parametrization of solution electrospinning was demonstrated.13 Entropy elastic forces in the deposited, not fully dried fibers were found to induce polymer creep which resulted in fiber breakage. The molecular weight of PCL was identified as a critical parameter to tailor the aspect ratio of such fibers in a restricted window. In this study a MEW process was conducted to produce fragmented microfibers with perfect control on fiber length and without the need of organic solvents. A 20 μm deep microrelief substrate was fabricated with microscopic air gaps and was placed upon an aluminum collector for MEW (Figure 1A). The ambient conditions could be controlled to a level where defined microfiber fragments could be produced. Therefore an elevated temperature environment of 36°C and 60% humidity was used to favor the breakage of the molten jet. Figure 1B is a photograph of the fragmented fibers on the microrelief substrate, while a top view perspective is provided in Figure S1. In a related study that suspended MEW fibers, a lower temperature of 19.7°C enabled multi-millimeter spanning of fiber walls, while at 23.7°C fibers tended to sag and collapse across air gaps.3 Here, we increase the ambient temperature to above 36°C, to the level that the molten jet breaks apart due to Plateau–Rayleigh instabilities in the air gap instead of sagging or spanning. Figure 1C shows the approximate polymer melt and ambient temperatures that decide the jet breaking probability. Interestingly, a slower collector speed slightly above the critical translation speed (CTS) favored jet breaking to form fiber fragments. Figure 1D shows that more fiber fragments were obtained for a collector speed of 1.2 × CTS, while the sample printed with 2.0 × CTS showed suspended rather than broken fibers (Figure 1E and Figure S2). Such breaking at lower collector speeds can be partly attributed to the decreased overall distance that the jet travels to the collector which reduces cooling time1 and partly due to the latent heat of a larger jet diameter and therefore a higher mass of melt, sustaining the viscous behavior that is required for breaking.2 Using the smallest air-gap achievable (20 μm), the MEW fiber could still be broken at 1.2 × CTS (Figure S3) on the microrelief substrate. The fragment length was primarily defined by the platform width, which ranged from 15 μm up to 80 μm, with greater lengths achievable. The distance between the adjacent fibers does not seem to have an effect on the breakage. The fiber fragments have different morphologies due to breaking effects. When the air gap is small, the fiber fragment has an abrupt ending; by printing across larger air gaps, a dumbbell-shaped fragment (Figure 1F) would occur. These dumbbell fragments are a result of the broken fiber still being in a molten state and contracting into spheres where it solidifies. Larger gaps result in increased diameter of the spherical end since there is more material in the gap available for the contraction. The dumbbell-like morphology shown here is similar to another approach that uses solution electrospinning.13 The driving forces of the fragmentation, elastic forces and intrinsic entropy of the polymer chains, consequently counterbalance within such highly anisotropic stretching of the jet or deposited fiber, irrespective whether it is a polymer solution or melt. Moreover, fiber breaking requires sufficient contact of the fibers on a substrate to avoid sliding and restrict homogeneous shrinkage as previously shown for thermally-induced shape recovery and shrinkage of electrospun regular and chemically crosslinked PCL fibers.14, 15 Another common observation was the occurrence of fiber fragments with a clear spherical front part and an opposing rod-like end, specific to the direct-writing direction. Furthermore, fiber fragmentation is also observed on a PDMS microrelief substrate (Figure 1E). Further images of fiber fragments are shown in Figures S4–S6. In summary, MEW onto a microrelief substrate regularly broke the fiber in a controlled manner at lower collector speeds, when the temperature was elevated. This study identified the combination of melt and ambient temperatures required for producing defined fiber fragments with well-tailored monomodal lengths. Working without any organic solvents and at moderate melting temperatures of usually less than 100°C, renders this approach of making fragmented fibers attractive for medical applications that include bioink reinforcement, aligned tissue engineering scaffolds16 or electroactive fibers. Medical grade poly(ε-caprolactone) (PCL) (Corbion Inc, Netherlands, PURASORB PC 12, Lot# 1712002224, 05/2018) was used as received and stored as described elsewhere.1 All experiments were performed with a custom-built, climate-controlled MEW printer. This chamber allows the control of both temperature and humidity, controlled from 32 to 36°C and 60% relative humidity respectively. A polymer melt temperature ranged from 80 to 110°C, while the remaining conditions are typical for MEW of PCL.17 This includes an applied voltage of 6.0 kV, a 25G nozzle, a 3.5 mm collector distance and an applied air pressure of 100 kPa. The CTS for MEW at these ambient and melt temperatures are shown in Figure S7. The dimensions of the microrelief substrate were 1.1 cm × 1.2 cm. A digital mask was used to make a microrelief substrate on a silicon-wafer of various platform widths and air gaps to a z-depth of 20 μm. The wafer was cut into squares and coated with an adhesion promoter monolayer and a positive photo-resin (Microchemicals GmbH, AZ 4562, Germany). Smart print rapid prototyping technology (Smart Force Technologies S.A.S., Smart Print, France) was used to directly lithograph an image with a micrometric resolution. After lithography, a developer (Microchemicals GmbH, AZ 400 K, Germany) was used to dissolve the exposed parts of the photoresist. A separate PDMS chip was produced, fabricated by casting a thin layer of onto the resin chip. After detaching the solidified PDMS layer, the corresponding negative shape of the resin chip is obtained. Samples were imaged with a Leica DMS1000 digital microscope, or a Crossbeam 340 SEM (Carl Zeiss Microscopy, Göttingen, Germany) equipped with a Zeiss Gemini column to determine the fiber fragment diameter, length, and morphology. An example of imaging the array to count suspended fibers is shown in Figure S3. Prior to SEM analysis all samples were platinum-coated using a Leica EM ACE600 high vacuum sputter coater. The length of 15 fiber fragments were measured at ambient temperatures of 33 and 34°C, and 90°C melt temperatures. An average and standard deviation of 115.0 and 22.6 μm for 33°C ambient temperature, and 83.7 and 9.2 μm for 34°C ambient temperature, respectively. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Project number 326998133—TRR 225 (sub-projects A04 and A07). We appreciate the German Research Foundation (DFG) State Major Instrumentation Program for funding the Zeiss Crossbeam CB 340 SEM (INST 105022/58-1 FUGG). Proof-reading by B. Tandon and I. Liashenko is appreciated. Open access funding enabled and organized by Projekt DEAL. Data available on request from the authors. Appendix S1: Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Biopolymeric particles can be fibrous or spherical, highly determining their areas of medical applications such as biomimetic fibers for tissue engineering or injectable capsules. In this work, we present a completely novel morphology, combining structural features of fibers and spheres in dumbbell-shaped Poly-epsilon-Caprolactone (PCL) micro-particles. Strikingly, such complex structures could be achieved by simply balancing out the parameters of a standard electrospinning setup together with fine-tuned spinning dope compositions. In-situ entropy-elastic snapping of deposited fibers could be triggered by combining PCL with different molecular weights, allowing for control over the aspect ratio of generated dumbbells in an accurate manner. With a single electrospinning nozzle, high production rates in the range of 80.000 dumbbells per second were achieved. To assess morphologically induced biofunctionality, dumbbell suspending and surface modification with collagen was performed and in vitro-testing with U87 reporter cells demonstrated significantly enhanced integrin-based cell adhesion on PCL-dumbbells as compared to continuous PCL-fibers. The results clearly unravel the remarkable potential of such structures in the field of tissue engineering. Revealing this new class of polymeric particles will also open the door to various new approaches including injectables, biofabrication and cosmetics, moreover, defining a novel type of filler material. (C) 2021 The Authors. Published by Elsevier Ltd.

A crucial property for the evaluation of bioinks, besides biocompatibility, is printability, which is determined by resolution and shape fidelity. Recently, fiber reinforcement was used to overcome rheological limitations and introduce biomimetic structuring.This study provides a systematic approach to evaluate the printability of fiber reinforced hydrogels. Alginate and Pluronic hydrogels were blended with cellulose nanofibers (CeNF) and polycaprolactone (PCL) microfibers. SEM imaging revealed fiber-induced structural changes. Oscillatory rheological experiments showed that the addition of fiber fragments significantly altered the complex viscosity. A customized setup was utilized to determine strut spreading behavior in a real extrusion printing process. Strikingly, the data displayed excellent correlation with viscoelastic model-based predictions. CeNF increased the shape fidelity of both hydrogels, while PCL microfibers increased the viscosity but resulted in a time dependent loss of structural integrity in Pluronic. The results emphasize the need to complement shear-rheological analysis of bioinks by print-related customized analytical tools.

The discovery of penicillin started a new era of health care since it allowed the effective treatment of formerly deadly infections. As a drawback, its overuse led to a growing number of multi-drug resistant pathogens. Challenging this arising threat, material research focuses on the development of microbe-killing or microbe repellent agents implementing such functions directly into materials. Due to their biocompatibility, non-immunogenicity and mechanical strength, silk-based materials are attractive candidates for applications in the biomedical field. Furthermore, it has been observed that silks display high persistency in their natural environment giving reason to suspect that they might be attractive candidates to prevent microbial infestation. The current review describes the process of biofilm formation on medical devices and the most common strategies to prevent it, divided into effects of surface topography, material modification and integrated additives. In this context, recent state of the art developments in the field of natural and artificial silk-based materials with microbe-repellant or antimicrobial properties are addressed. These silk properties are controversially discussed and conclusions are drawn as to which parameters will be decisive for the successful design of new bio-functional materials based on the blueprint of silk proteins.

When characterizing the viscoelastic properties of polymers, shear rheological measurements are commonly the method of choice. These properties are known to affect extrusion and nozzle-based processes such as fiber melt spinning, cast film extrusion and 3D-printing. However, an adequate characterization of shear thinning polymers can be challenging and still insufficient to not only describe but predict process relevant influences. Furthermore, the evaluation of rheological model systems in literature is mostly based on stress–relaxation experiments, which are rarely available for various polymeric materials. Therefore, a simple approach is presented, that can be used to evaluate and benchmark a wide range of rheological model systems based on commonly accessible frequency sweep data. The approach is validated by analyzing alginate PH176 solutions of various concentrations, a thermoplastic poly-urethane (TPU) Elastollan 1180A melt, the liquid silicon rubber Elastosil 7670 and a polycaprolactone (PCL) fiber-alginate composite system. The used rheological model systems, consisting of simple springs and dashpots, are suitable for the description of complex, viscoelastic material properties that can be observed for polymer solutions and gel-like systems. After revealing a suitable model system for describing those material properties, the determination and evaluation of relevant model parameters can take place. We present a detailed guideline for the systematic parameter revelation using alginate solutions of different concentrations as example. Furthermore, a starting point for future correlations of strut spreading in 3D-bioprinting and model parameters is revealed. This work establishes the basis for a better understanding and potential predictability of key parameters for various fabrication techniques.